Post on 04-Jul-2018
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 1
Thomas Jefferson University
Annual Progress Report: 2012 Formula Grant
Reporting Period
January 1, 2013 – June 30, 2013
Formula Grant Overview
Thomas Jefferson University received $2,776,880 in formula funds for the grant award period
January 1, 2013 through December 31, 2016. Accomplishments for the reporting period are
described below.
Research Project 1: Project Title and Purpose
The BRM (brahma) Atpase as a Gatekeeper for Prostate Cancer Development and Progression –
There is a critical need to discern the mechanisms that govern prostate cancer development and
progression. Based on our published and preliminary data, it is evident that the Brm ATPase
plays a significant role in both processes. Our findings strongly support a model wherein loss of
Brm functions as a tumor suppressor by modulating cell cycle progression and checkpoints.
Moreover, “weakened” BRM activity cooperates with loss of the p27 tumor suppressor to induce
hyperplastic phenotypes and castrate-resistance. Studies will determine the molecular
mechanisms by which p27kip1 is induced by Brm loss (Aim 1), the impact of p27kip1 as a
barrier to Brm-loss induced tumorigenesis and ADT-resistance in vivo (Aim 2), and the impact
of coordinate p27kip1 and Brm loss in human disease (Aim 3).
Anticipated Duration of Project
1/1/2013 – 12/31/2016
Project Overview
Prostate cancer (PCa) is the most frequently diagnosed malignancy and second leading cause of
cancer death of men in the USA. While local disease can be effectively treated through radical
prostatectomy or radiation therapy, no durable means of treatment has been identified for non-
organ confined PCa. First line therapeutic intervention for this stage relies on chemical
castration, referred to as androgen deprivation therapy (ADT), as PCa is exquisitely dependent
on the action of the androgen receptor for cell survival and proliferation. However, ADT-
resistant, lethal tumors form within 2-3 years for which no effective therapeutic option has been
identified. Thus, there is a significant need to understand the mechanisms that lead to prostate
cancer development and promote castration-resistant tumor growth.
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 2
The hypothesis is that Brm serves as a barrier to prostate tumorigenesis and lethal tumor
phenotype progression, but that the consequence of Brm loss could be exploited for therapeutic
gain.
Aim 1: Determine the mechanisms of Brm-loss induced p27kip1 induction. Our data indicate
that p27kip1 serves as a critical barrier to tumorigenesis associated with Brm loss. Here, the
underlying mechanisms of p27kip1 regulation will be determined.
Aim 2: Impact of p27 as a barrier to Brm-loss induced tumorigenesis and ADT-resistance. Aim 1
will identify the underlying mechanisms by which the p27kip1 checkpoint is induced as a barrier
to tumorigenesis after Brm loss. Here, the consequence of p27 induction will be assessed, by
determining the impact of this event on tumorigenesis and progression to castrate-resistant, lethal
disease.
Aim 3: Determine the impact of coordinate p27 and Brm loss in human disease.. Given the
strong correlation between these events in human disease, here we will examine clinical
correlates of dual p27kip1 and Brm loss.
Principal Investigator
Karen Knudsen, PhD
Professor
Thomas Jefferson University
125 S. 9th Street
Philadelphia, PA 19107
Other Participating Researchers
William Ostrander – employed by Thomas Jefferson University
Expected Research Outcomes and Benefits
It is expected that these studies will provide a monumental leap in the understanding of BRM
and tumor suppressor function in prostate cancer. If successful, the outlined strategies will
provide the justification for developing future pre-clinical strategies to effectively manage
tumors with loss of BRM. In summary, the studies outlined in this project will have translatable
outcomes and completion of this work will:
1. Lead to the development of new tumor markers.
2. Reveal insight for novel therapeutic design.
3. Uncover new areas of prostate cancer research.
4. Define the role of chromatin remodeling components in a therapeutic model.
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 3
Summary of Research Completed
Aim 1
Goal: Determine the mechanisms of Brm-loss induced p27kip1 induction. Our data indicate that
p27kip1 serves as a critical barrier to tumorigenesis associated with Brm loss. Here, the
underlying mechanisms of p27kip1 regulation will be determined.
During this reporting period, we made significant progress toward understanding Brm regulation
in prostate cancer. First, we showed that Brm and its requisite cofactor in prostate cancer, the
BAF57 SWI/SNF subunit, controls genes associated with acquisition of the metastatic
phenotype. These data reveal for the first time that Brm and SWI/SNF alterations (especially
BAF57) impinge on cell migration and invasion.
Purpose:
BAF57, a component of the switching-defective and sucrose nonfermenting (SWI/SNF)
chromatin-remodeling complex conglomerate, modulates androgen receptor activity to promote
prostate cancer. However, the molecular consequences of tumor-associated BAF57 expression
have remained undefined in advanced disease such as castration-resistant prostate cancer and/or
metastasis.
Experimental design:
Clinical human specimens of primary and metastatic prostate cancer were
immunohistochemically examined for tumor-grade association of BAF57 expression. Global
gene expression analyses were conducted in models mimicking tumor-associated BAF57
expression. Aberrant BAF57-dependent gene expression changes, bypass of androgen-mediated
signaling, and chromatin-specific SWI/SNF complex alterations with respect to cytoskeletal
remodelers such as integrins were validated. Cell migration assays were used to profile the
biologic phenotypes conferred under conditions simulating tumor-derived BAF57 expression.
Results:
Immunohistochemical quantitation of primary human specimens revealed that BAF57 was
significantly and aberrantly elevated as a function of tumor grade. Critically, gene expression
analyses showed that BAF57 deregulation circumvented androgen-mediated signaling, elicited
α2 integrin upregulation, and altered other SWI/SNF complex components at the α2 integrin
locus. BAF57-dependent α2 integrin induction conferred a prometastatic migratory advantage,
which was attenuated by anti-α2 integrin antibody blockade. Furthermore, BAF57 was found to
be markedly upregulated in human prostate cancer metastases of the lung, lymph node, and dura.
Conclusion:
The findings herein, identifying tumor-associated BAF57 perturbation as a means to bypass
androgen-signaling events that facilitate novel prometastatic phenotypes, link BAF57
upregulation to tumor dissemination. These data thereby establish BAF57 as a putative marker of
metastatic potential that could be leveraged for therapeutic intervention.
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 4
Aim 2
Goal: Impact of p27 as a barrier to Brm-loss induced tumorigenesis and ADT-resistance.
Studies for this aim have commenced which will assess the consequence of p27 and SWI/SNF
(Brm) alterations on tumorigenesis and progression to castrate-resistant, lethal disease. Major
findings to date are:
As shown below in Figure 1, we have made excellent progress towards this end by first assessing
the incidence of Brm alterations in human disease. Nuclear Brm and SWI/SNF expression is lost
as a function of tumor development and progression, and frequently co-occur with loss of p27.
These studies lend further importance to testing the impact of p27 and SWI/SNF alterations in
models of human disease.
As shown in Figure 2, analyses of a second cohort by immunohistochemistry strongly supports
the contention that nuclear Brm function is lost in advanced disease, thus showing for the first
time that not only is Brm expression reduced as a function of disease progression, but that
subcellular localization is altered. These new findings shed significant new light into Brm
alterations and the clinical ramifications of these events.
Aim 3
Goal: Determine the impact of coordinate p27 and Brm loss.
Progress has begun for this sub-aim by generating the double null p27-/- Brm-/- animals.
Multiple lines have been successfully generated and male mice are now being aged to assess
phenotypes.
Figure 1: p27 and SWI/SNF alterations frequently co-occur in advanced disease. As shown,
genomic analyses of human metastatic prostatic adenocarcinoma co-occur with high frequency.
The Odds Radio for co-occurrence is 18.75, with a 95% confidence interval of 2.756966 -
127.517905, and a p-value of p-value: 0.001994 [Fisher's Exact Test].
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 5
Figure 2: Brm expression and subcellular localization is altered as a function of disease
progression. As shown, examination of Brm expression by immunohistochemistry strongly
demonstrates that not only is Brm expression reduced in advanced disease, but that the nuclear
function of this SWI/SNF ATPase is lost through mislocalization to the cytoplasm as a function
of disease progression.
Research Project 2: Project Title and Purpose
Modulation of Human Breast Cancer Growth In Vivo by Activated Human Breast Fibroblasts –
The work is part of a long-term objective of providing new experimental models for more
accurate drug response testing of human breast cancer in mice. Many drugs work well when
tested on human breast cancer xenotransplant lines in mice, but fail in patients. The focus of this
proposal is to characterize growth and drug-responsiveness of human breast cancer
xenotransplants in mice with or without humanized tumor stroma. We hypothesize that novel
human mammary fibroblast lines will humanize the stromal microenvironment and modulate
tumor growth and drug responsiveness more accurately.
Anticipated Duration of Project
1/1/2013 – 12/31/2013
Project Overview
Despite enormous expenditures and efforts by academic, government, and pharmaceutical
institutions, most drugs that show promise against human breast cancer in preclinical testing in
mice, fail to cure breast cancer in the clinic. The inability to predict whether candidate drugs
will work in patients is very costly, and key resources are spent on clinical trials of drugs that
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 6
most often fail. There is a great need for more accurate predictive preclinical testing to identify
the best drug candidates for human breast cancer.
Consistent with the national initiatives to stimulate translational and interdisciplinary efforts to
accelerate medical discovery, our laboratory’s long range goal is to facilitate faster development
of effective breast cancer therapies. To this end, we have genetically engineered mice to more
accurately mimic the hormonal environment in patients. We will now extend these efforts to
employ novel strategies to humanize the stroma of human tumor xenotransplants by testing a
panel of five previously undescribed immortalized human mammary fibroblast lines.
The primary objective is to establish new experimental xenotransplant models to achieve more
accurate predictive drug response testing of human breast cancer xenotransplants. A secondary
objective is to explore the consequences of a humanized tumor stroma on breast tumor biology,
including growth, angiogenesis and progression. We hypothesize that human mammary
fibroblasts with myofibroblast characteristics will humanize the stromal microenvironment and
modulate tumor growth and drug responsiveness to better mimic conditions in patients.
Aim 1) Characterize a panel of human mammary fibroblast lines established from discarded
tissue from reduction mammoplasties for features common to activated fibroblasts.
Aim 2) Test the ability of select human mammary fibroblast lines to modulate tumor biology and
drug responsiveness of xenotransplanted human breast cancer lines in prolactin-humanized mice.
Principal Investigator
Hallgeir Rui, MD, PhD
Professor
Thomas Jefferson University
125 S. 9th
Street
Philadelphia, PA 19107
Other Participating Researchers
Alicia Yanac, MS – employed by Thomas Jefferson University
Expected Research Outcomes and Benefits
By the end of this project we expect to have accomplished the following outcomes.
Outcome 1 – Characterization of five novel human mammary breast fibroblast lines, with
emphasis on identifying lines with features of activated fibroblasts/myofibroblasts. We expect
based on preliminary data that the cell line HMF1 will be validated to display key features of
activated human fibroblasts. Such a cell line may then be used for co-culture or co-implantation
studies by our laboratory and shared with other scientists.
Outcome 2 – Validation of the ability of human mammary fibroblasts to modulate tumor growth,
biology and drug responsiveness of human breast cancer xenotransplants in mice. We expect
that the data will support the concept that a humanized stroma provides a more representative
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 7
model system for biology and drug response testing in vivo. Human breast cancer xenotransplant
models with humanized stroma may become a standard for preclinical testing of drugs against
breast cancer.
Summary of Research Completed
We are pleased to report solid progress during the first six months of this project.
Novel Progress related to Aim 1
Purpose: Characterize a panel of human mammary fibroblast lines established from discarded
tissue from reduction mammoplasties for features common to activated fibroblasts.
Screening primary human mammary fibroblasts.(HMFs). Primary HMFs were isolated from
surgically excised non-malignant mammary tissues, which were obtained from reduction
mammoplasty or mastectomy. HMFs were then cultured in vitro. The purity of fibroblasts was
confirmed by universal expression of vimentin and lack of cytokeratin expression using
immunofluorescent microscopy (data not shown). We then examined the expression of alpha
smooth muscle actin (α-SMA), a marker for activated fibroblasts, in a panel of five isolated
primary HMFs by immunofluorescent staining (Fig1A). Most HMF-1 cells had medium to high
level of α-SMA, while almost none of HMF-2 cells expressed α-SMA at detectable levels. Low
percentage of HMF-3, 4 and 5 expressed α-SMA at detectable levels.
To immortalize HMF-1 for long-term use, we introduced a lenti-viral vector (pLoxP-hTERT-
IRED-TK, Addgene, MA, USA) in HMF-1. We then compared the immortalized line of HMF-1
(hTERT HMF-1) to the primary HMFs for α-SMA expression by western blotting (Fig1B).
hTERT HMF-1 and HMF-1 expresses similar levels of α-SMA. Consistent with the staining
results in Fig1A, HMF-2, 3 and 4 had very low levels of α-SMA, and HMF-5 expressed
intermediate levels of α-SMA. Further examination by α-SMA staining showed immortalized
line of HMF-1 maintained the high expression level of this marker as in the primary HMF-
1(Fig1C). Therefore, the line of hTERT HMF-1 was a good candidate to be used as activated
mammary fibroblasts.
Characterization of HMFs in vitro. We then characterized the hTERT HMF1 to determine
whether it shared the functional characteristics of activated fibroblasts. HMF-2 was also
included since it had much lower expression levels of α-SMA. We first examined the fibroblasts
for mRNA expression of a group of molecules that were reported to be up-regulated in activated
fibroblasts and are important mediators for their cancer-promoting function. A luminal breast
cancer cell line, MCF-7, was included in gene expression assays as baseline (Fig 2). We found
both hTERT HMF-1 and HMF2 had higher levels of SDF1α, SDF1β, VEGF, IL6, podoplanin
and HGF than MCF7. Between the two HMFs, hTERT HMF-1 had significantly higher levels
than HMF-2 for SDF-1 α (A) and β (B), VEGF (C), IL6 (D) and podoplanin (E) (Fig. 2). HMF-2
had higher HGF mRNA expression than hTERT HMF1 (Fig 2F). We also examined TGFβ gene
expression, but the levels in all three cells were not detectable by our assay.
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 8
Next, we examined the function of HMFs by soft agar colony assays, to see whether they could
promote anchorage-independent growth of breast cancer cells in vitro. The fibroblasts were first
plated in the wells and grown to confluence. Then MCF7 cells were suspended in soft agar and
plated on top of with the fibroblasts. After 5 weeks, there were significantly more colonies with
HMFs than MCF cells alone. These result suggested both HMFs strongly promote anchorage-
independent growth of breast cancer cells in vitro (Fig. 3).
Examining function of HMFs by tumor xenotransplant in vivo. To examine whether the HMFs
promote tumor growth as activated fibroblasts would do in vivo, we tested them in a breast
cancer xenograft mouse model. hTERT HMF1 or HMF2 were admixed with MCF7 cells before
injected into the mouse mammary gland orthotopically. MCF7 cells alone were also injected as
control. The growth of xenograft tumors by each group were plotted in Fig 4A, the average
tumor sizes in both groups with HMFs were consistently bigger than the group with MCF7
alone, suggesting hTERT HMF-1 and HMF-2 both promoted tumor growth to some degree.
However, only tumors in the group of MCF7 + hTERT HMF-1 became statistically different
than MCF7 alone after day 60. In the groups of MCF7 alone or MCF7 + HMF-2 fibroblasts, the
growth of the tumors plateaued around day 40 and even started to regress thereafter. In contrast,
the group of MCF7+hTERTHMF1 tumors continued to grow throughout the experiment.
Therefore, hTERT HMF-1 indeed acted as activated fibroblasts and promoted the growth of
MCF7 tumors in vivo.
To better understand the mechanism(s), through which hTERT HMF1 promoted tumor growth,
the xenograft tumors were further studied. H&E staining of the xenograft tumors from all three
groups were shown in Fig4B. Tumors from the group of MCF7 alone comprised many big
tumor nests with granular features. In contrast, tumors from the groups with HMFs showed that
the cancer cells were more uniformly dispersed in the stroma in much smaller clusters,
resembling higher grade cancer. SMA staining demonstrated the presence of activated
fibroblasts in all the tumors. While SMA positive fibroblasts surrounded the tumor nests in the
group of MCF7 alone, the distribution of these cells intermingled with cancer cells in the groups
with HMFs. Staining of collagen in these tumors again confirmed the distinct distribution of
tumor and stromal fibroblasts in tumors arose from MCF7 alone or MCF and HMF admixture.
To examine the contribution of HMFs in the tumors, we also stained for human vimentin, a
marker for HMFs in mouse xenograft tumors. The presence of HMFs was confirmed in the
tumors arose from MCF7 and HMF admixture after 85 days. But they only accounted for a
small portion of the tumor and their number did not differ significantly between the two groups
(data not shown), suggesting they did not contribute to the size difference of the tumors.
To find the mechanism underlying the growth difference of the xenograft tumors, we examined
the xenograft tumors for their proliferation and angiogenesis. Ki67 staining revealed
significantly higher percentage of Ki67 positive cancer cells in the groups with HMFs, indicating
both HMFs promoted proliferation of MCF7 in vivo (Fig4C). The group with hTERTHMF-1 had
only slightly higher Ki67positive cancer cells than the group with HMF-2 (83% vs 79%;
p<0.05). The tumors were also stained with anti-CD31 antibody to determine the number of
micro blood vessels present, an index for angiogenesis. Fig4D shows that the group with hTERT
HMF-1 had significantly more micro vessels than the groups with HMF-2 or MCF7 alone, while
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 9
there was no difference between MCF-7 alone and the group with HMF-2.These data suggested
hTERT HMF-1 may facilitate tumor growth mainly by enhancing angiogenesis and promoting
proliferation.
Conclusions: Once fibroblasts get in contact with tumor cells, they could become activated and
then, in turn, aid in the development, progression, and angiogenesis of breast carcinomas.
Therefore, activated fibroblasts are an important component of tumor microenvironment. When
modeling invasive breast cancer in preclinical animal models, the presence of human activated
fibroblasts would better recapitulate the cancer stroma in xenograft studies of human breast
cancer in mice. Despite of recent efforts of generating CAFs through series of procedures of in
vitro and in vivo manipulation, the availability of activated mammary fibroblasts remains limited
and may hinder the progress of research and new therapy discovery. In this study, we aimed to
establish and characterize activated HMFs for future breast cancer studies.
Aim 2. – No progress during this reporting period.
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 10
HMF-3
Control
HMF-4 HMF-5
HMF-1 HMF-2A
Figure 1. Screening of HMFs for activated fibroblasts. A. a panel of primary HMFs isolated from
human mammary tissues was stained for α-SMA. Control was stained with only secondary
antibody. B. hTERT-immortalized HMF-1 and the panel of primary HMFs were examined for α-
SMA expression by western blotting. GAPDH, a housekeeping protein, was used to demonstrate
loading. C. hTERT HMF-1 has similar expression levels of α-SMA as HMF-1 by
immunofluorescent staining.
B
SMA
GAPDH
C
HMF-1 hTERT HMF-1
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 11
Figure 2. Expression of secreted factors and membrane proteins by HMFs. HMFs have elevated
mRNA expression levels of proteins associated with activated fibroblast specific. MCF7 cells,
hTERT HMF-1, and HMF-2 were analyzed for relative mRNA expression levels of SDF-1 α (A)
and β (B), VEGF (C), IL-6 (D), podoplanin (E) and HGF (F). One-way ANOVA was used for
statistics, then followed by Bonferroni's Multiple Comparison Test. *, p<0.05. **, p<0.01.***,
p<0.001.
Figure 3: HMFs promote anchorage-independent growth of MCF7 cells. A. Representative
pictures of colony growth in soft agar for MCF7 alone, MCF7 + HMF-2 or MCF7 + hTERT
HMF-1are shown. B. Colonies were counted in each field and an average of all the fields (n=8)
were calculated. For comparison among the three groups, one-way ANOVA was used, then
followed by Bonferroni's Multiple Comparison Test. ***, p<0.001.
A
MCF7 MCF7 +
HMF-2
MCF7 +
hTERT
HMF-1
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 12
Figure 4: HMFs promote tumor growth of MCF7 cells in vivo. hTERT HMF-1 promoted breast
cancer growth, angiogenesis and progression in vivo. A. The growth of MCF7 alone, or MCF7 +
HMF-2 or MCF7 + hTERT HMF-1 xenografts in vivo was followed over time (n≥8). Two-way
ANOVA was used to compare the size of xenograft tumors. B. H&E, α-SMA and collagen
staining of xenograft tumors at 100x magnification. C. The xenografts were stained for Ki67 and
percentages of positive MCF7 cells in each group were compared. D. the xenografts were stained
for CD31 to label micro vessels. The vessels counts were plotted and compared among the three
groups. Three random fields of each tumor were used for analyses. For comparison among the
three groups, one-way ANOVA was used, then followed by Bonferroni's Multiple Comparison
Test. *, p<0.05; **, p<0.01; ***, p<0.001.
B MCF7
alone
MCF7 +
HMF-2MCF7 +
hTERT HMF-1
H&E
α-SMA
collagen
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 13
Research Project 3: Project Title and Purpose
Creating and Phenotyping New Mouse Models of Cancer Cachexia – Cachexia, or
dysmetabolism leading to muscle and fat wasting, diminishes quality of life and response to
therapy in cancer patients. Cachexia afflicts most patients with tobacco-related cancers,
including up to 80% of those with lung cancer. Cachexia itself, distinct from other tumor effects,
causes ~1/3 of cancer deaths. Despite this, few animal models of cancer cachexia have been well
characterized and ~4 are commonly used. The purpose of this project is to create and phenotype
new mouse models of cancer cachexia. Mice with tumors of mouse and human origin will be
subjected to clinical, pathological, histological, and molecular profiling in order to expand the
models available to the research community.
Anticipated Duration of Project
1/1/2013 – 6/30/2014
Project Overview
The long-term goal of my research is to define the key regulatory pathways that mediate muscle
wasting in cancer, for the purpose of developing therapeutics to combat cachexia. Cancer
cachexia is driven by the host response to the tumor, and by factors produced by the tumor which
effect changes in central and peripheral pathways modulating activity, food intake, energy
expenditure and metabolism. Experimental analysis of cachexia has centered on a handful of
mouse models, with a candidate cytokine or single gene/pathway approach to analysis. While
this approach has led to the identification of several pathways as peripheral mediators of
cachexia, many other pathways are likely involved. Identifying those is essential to find
therapeutic targets for this currently incurable condition. The application of modern molecular
techniques to existing models of cancer cachexia will enable the discovery of new cachexia
drivers. Moreover, the existing mouse models cover only colon cancer, melanoma and a form of
lung cancer. Substantially greater numbers of models across different tumor types are necessary
to determine the relevance of known and novel pathways across the spectrum of cancer types.
The objective of this project is to perform molecular phenotyping of models of cancer cachexia
to stimulate discovery of new drivers and targets. While physiological data including muscle
weights and piecemeal gene/protein expression data are available in about 12 rodent models of
cachexia, there is a paucity of large-scale data molecular data. Indeed, across all models, there
are publicly available data on multiplex serum cytokines and muscle gene expression for only a
single model, the C26 model, from my laboratory. Specific Aims are:
Aim 1: Establish models of cancer cachexia.
Aim 2: Characterize the systemic and tumor pathophysiology associated with the models.
Aim 3: Quantify cytokine levels and muscle wasting activity in these models.
Aim 4: Perform muscle microarray analysis to identify genes and pathways regulated in common
across models or uniquely within models.
Aim 5: Collect serum, cell lines, tumors and tissues to build a resource for future analysis and
sharing with the research community.
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 14
Principal Investigator
Teresa A. Zimmers, PhD
Associate Professor
Thomas Jefferson University
125 S. 9th Street
Philadelphia, PA 19107
Other Participating Researchers
Zongxiu Zhang – employed by Thomas Jefferson University
Expected Research Outcomes and Benefits
The project objective is to perform molecular and pathophysiological profiling of existing and
new mouse models of cancer cachexia in order to stimulate discovery. There are five clear
outcomes, all of which will benefit the research community and promote understanding of the
molecular underpinnings of cachexia. First, this work will expand the number of well-
characterized cachexia models by systematically evaluating the cachexiogenic potential of
different cell lines representing a spectrum of cancer types and sub-types. This is important to
promote research beyond the 3 most-oft used models and expand upon the total of 12 models in
the literature. Our published data on these new models will permit others to use them with
confidence. Second, this work will provide large, quantitative datasets to be deposited in public
databanks, including multiplex cytokine profiling and muscle gene expression. Analysis of those
data will permit hypothesis testing (e.g., Is increased IL-6 associated with STAT3 target gene
expression across models of cachexia?), as well as discovery. Investigators in other laboratories
will be able to query their genes/pathways of interest in these well-characterized datasets. Third,
class prediction analysis on the collected datasets across all the models and time-points analyzed
will determine whether there are particular cachexia phenotypes that are similar within or across
models. Identification of such independent phenotypes would represent a paradigm shift in the
field of cachexia research, which collective assumes all cancer cachexia is alike. Fourth, the
work will result in identification of murine cachexia gene signatures. Fifth, the banking of the
serum, cell lines, tumors and tissues will permit future analysis when funds are available. In all,
the project will be a major leap forward for the world-wide cancer cachexia research effort.
Summary of Research Completed
This progress report details results obtained from January 2013 through June 2013. Overall, we
have established one new mouse model of ovarian cancer cachexia, two of colon cancer
cachexia, and six of pancreatic cancer (Aim 1). We have characterized the systemic and tumor
pathophysiology for all (Aim 2). We have begun to quantify serum cytokine levels and muscle
protein expression (Aim 3). In the murine pancreatic cancer model, we have observed that gene
expression will cluster by cell line (Aim 4). Finally, we have banked the serum, cell lines, tumors
and tissues obtained in these models for further analysis of gene expression and for sharing of
data and tissues (Aims 5).
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 15
SPECIFIC AIMS 1-3: Establish models of cancer cachexia. Characterize the systemic and tumor
pathophysiology associated with these models. Quantify cytokine levels and muscle wasting
activity. Hypothesis/need: Cachexia is generally discussed as a single entity, while patient
characteristics and physician clinical intuition indicate that there are likely multiple cachexia
drivers and phenotypes even within a single tumor type. The paucity of data available on murine
models limits the scope of cachexia research. Approach: Cell lines of human and murine origin
were engrafted into mice, either subcutaneously or orthotopically and the cachectic phenotype
was evaluated.
Results to date:
Ovarian cancer: Ovarian cancer often presents at late stage with disseminated disease, ascites and
evidence of inflammation and muscle wasting. To model this, female athymic nu/nu mice were
injected i.p. with 107 OVCAR-3, SK-OV-3 or ES-2 human ovarian cancer cells to mimic
disseminated abdominal disease. By 60 days, the OVCAR-3 and SK-OV-3 cell lines had not
resulted in detectable tumors. In contrast, beginning 8 days after inoculation, ES-2 cells caused
severe cachexia with marked loss of body weight and massive ascites accumulation (Fig. 1). ES-
2 cells infiltrated the pancreas, diaphragm and other abdominal organs, forming multiple solid
tumors of histology consistent with clear cell carcinoma. Muscles, including tibialis,
gastrocnemius, quadriceps and heart of tumor-bearing mice appeared markedly smaller than
normal controls (~-20% vs. control; p<0.01). Fat pads and spleen were reduced in size, while the
liver was unchanged. Piximus imaging revealed decreased bone mineral density (-16% vs.
control; p<0.05). Muscle loss was due generally to fiber atrophy, as evidenced by reduced
average myofiber cross sectional area in tibialis, although some fibers were hypertrophic. In
quadriceps muscle, ES-2 cachexia was associated with high levels of phosphorylated STAT3
(pSTAT3), whose causal role in tumor-associated muscle wasting was previously reported in our
laboratory, and also high levels of pSTAT5, which might be associated with the hypertophic
fibers.
In order to understand whether tumor-derived factors were directly responsible for the wasting
effects observed in vivo, differentiated C2C12 murine myotubes were exposed to conditioned
medium from ES-2 cell cultures and evaluated for effects on myotube size. After 48 hours, the
average diameter of C2C12 myofibers was severely reduced (-28% vs. control; p<0.001). These
effects were evident at a low concentration of supernatant and were associated with elevated
pSTAT3 levels. Consistent with reports of human ovarian cancer cachexia, Interleukin (IL)-6
levels were dramatically elevated in both blood and ascites, as was IL-11 (blood not shown).
While IL-6 was detectable only in ascites and not in cultured ES-2 cells, IL-11 was also
expressed by cultured ES-2 cells, suggesting at least some of the IL-11 was of tumor origin.
Neutralizing antibodies against IL-6 and IL-11 together could block ES-2 conditioned medium
induced atrophy of C2C12 myotubes. Taken together, the data indicate that the ES-2 cachexia
model shares features of inflammatory cachexia with the established C26 colon adenocarcinoma
model, with unique features of ascites production, disseminated metastases and complex effects
on myofiber size.
Colon cancer (data not shown): Cachexia is frequent in colon cancer but only one model is
routinely available to investigators, C26 adenocarcinoma in Balb/c or CD2F1 mice. Thus male
athymic nude mice were injected with HT-29, CaCo-2, T-84, RKO, HCT-116 or Colo205 human
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 16
colorectal adenocarcinoma cells. Initial studies using orthotopic implantation of 106 cells into the
cecal wall resulted in bowel obstruction, which caused morbidity and complicated the cachexia
analysis. Subsequent studies using subcutaneous implantation of 5x106 cells gave rise to
reproducible localized tumors. Of these, HCT-116 and Colo205 resulted in robust cachexia, as
indicated by reduced body mass, muscle mass and muscle fiber diameter. As with ES-2 ovarian
cancer and C26 colon cancer, HCT-116 and Colo205 cachexia were associated with
inflammation, high levels of IL-6 in serum, and elevated pSTAT3 in skeletal muscle. Culture of
C2C12 myotubes with HCT-116 or Colo205 conditioned medium or plasma from the
corresponding tumor-bearing mice resulted in myotube atrophy, indicating that tumor cells
secrete cachexiogenic factors, one of which is IL-6. Tissues were collected for Aims 4 and 5,
which are underway.
Pancreatic cancer: Some 80% of patients with pancreatic cancer develop cachexia and most will
die within 5 years of diagnosis, but no models of pancreatic cancer cachexia are in common use.
We modeled pancreatic cancer cachexia using lines of murine and human origin, implanted by
injection into the pancreas of syngeneic or athymic nude mice, respectively. Three lines were
given by David Tuveson, who derived the clonal cell lines from tumors arising in mice
engineered to express mutant Kras on a background of pancreas-specific deletion of p53.
Although they possess the same transgenes, in our hands the three lines (32043, 32047, 32908)
gave rise to tumors of different histological and systemic pathophysiology in C57BL/6 mice
(Fig. 2). At a dose of 5x106 cells injected, line 32908 was the most aggressive, causing near-fatal
cachexia in 8 days, while 32047 and 32043 were lethal within 14 days. All lines induced
anorexia, likely contributing to weight loss.
Histologically, tumors from line 32908 were the least differentiated and revealed a neurogenic
phenotype, while lines 32047 and 32043 had features consistent with adenocarcinoma. All three
lines led to loss of body mass, including muscle and fat mass, with variable effects on organ size.
Tumor cells metastasized outside the pancreas to infiltrate colon, mesenteric fat, and other
abdominal organs. All lines resulted in muscle atrophy as determined from reduced tibialis
myofiber diameter. Muscles from all mice showed elevated pSTAT3, while only line 32047
induced phosphorylation of SMAD2 in muscle. This result suggested that the drivers of muscle
wasting in the 3 lines might be biochemically distinct. Western blotting analysis of tumor lysates
showed that all lines expressed the cachexia inducing factors IL-6, myostatin and Activin,
however Activin levels in blood were highest for 32043 and 32908 (not shown). Plasma from
tumor bearing mice induced atrophy in cultured C2C12 myotubes, indicating that the tumors
induced a muscle wasting activity in the blood. Moreover, in vitro adipose wasting activity was
observed using plasma from tumor-bearing mice on cultured 3T3L1 adipocytes. Analysis is
ongoing, however thus far results indicate that the individual cachexia phenotypes are
reproducible and characteristic of each of these three tumor cell lines, even though they are
derived from identical genetic backgrounds.
A similar approach was used with cell lines of human origin in athymic nude mice (Figure 3). In
this comparison, MIA-PaCA-2 cells were the most cachexiogenic, followed by BxPC-3 then
PANC-1. MIA-PaCa-2 cells induced hepatosplenomegaly and cachexia, consistent with its high
levels of expression of IL-6 and myostatin, while BxPC-3 induced wasting without
organomegaly. Muscle wasting was proportionate to muscle pSTAT3. Cell lysates showed
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 17
expression of the cachexiogenic cytokines IL-6 and myostatin/GDF-8, with MIA-PaCa-2 also
showing expression of Activin.
SPECIFIC AIMS 4, 5: Perform muscle microarray analysis to identify genes and pathways
regulated in common across models or uniquely within models. Collect serum, cell lines, tumors
and tissues to build a resource for future analysis and sharing with the research community.
Hypothesis: Phenotypic classification using molecular profiling will enable identification of
cachexia drivers. Approach: Blood and muscle from different models of cachexia will be
subjected to hypothesis-driven and unsupervised clustering to identify patterns associated with
cachexia.
Results to date:
In a proof-of-concept study, muscle from mice with Kras/p53 mutation driven orthotopic
pancreatic cancer cachexia were subjected to Affymetrix Gene 2.0 arrays. Unsupervised
hierarchical clustering separated 32908 from 32043, while 32047 was intermediate. A
framework for data analysis has been generated in R and pilot studies using Weighted Gene
Centered Cluster Analysis for clustering of clinical characteristics have been performed on data
from each mouse (not shown). We have banked cells, tumors, tissues and plasma from all of the
models above and have prepared samples for Affymetrix Gene arrays for RNA from quadriceps
and multiplex cytokine arrays for plasma.
METHODS:
Mice. C57BL/6J mice (Jackson Laboratories) or athymic nu/nu mice (Harlan) were used at 8-10
weeks. Ovarian cancer cells were injected i.p. Colon cancer cell lines were injected
subcutaneously. Pancreatic cancer cell lines were injected into the pancreas after laparotomy
under general anesthesia, followed by analgesia and frequent monitoring. All mice were
observed at least daily for body weight, condition and tumor growth. All mouse experiments
were approved by the Thomas Jefferson University Institutional Animal Care and Use
Committee. Cell culture and reagents. Ovarian, colon and pancreatic cancer cell lines from
ATCC or for pancreatic KPC lines, from David Tuveson, were cultured then trypsinized and
injected into mice. Murine C2C12 myoblasts (ATCC, Manassas, VA) were grown in DMEM
with 20% FBS then switched to DMEM with 2% horse serum for differentiation. C2C12
myotubes were treated for 48h with tumor cell conditioned medium or DMEM plus 2% murine
plasma from tumor bearing or control mice, then were fixed, stained for myosin heavy chain and
photographed. Myotube diameters were measured to quantify muscle wasting activity in the
plasma samples. Cytokine and protein assays. IL-6, IL-11 and Activin A levels were assayed by
ELISA. Tumor production of IL-6, myostatin or Activin A was determined by
immunohistochemistry of formaldehyde fixed frozen sections. As well, we performed Western
blotting of cultured tumor cell lysates and quadriceps muscle protein to determine the relative
contribution of circulating cytokines.
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 18
FIGURE 1: ES-2 ovarian cancer cachexia. (A) Mice with ES-2 tumors showed reduced final body weight
(FBW) despite higher initial body weight (IBW), due in part to loss of carcass and muscle mass, fat mass,
bone mineral density and bone mineral content. (B) Hematoxylin and eosin stained sections of tibialis
muscle showed abnormal muscle histology, with overall reduction in cross sectional area despite a
spreading across the histogram (inset and C). (D) Western blotting of quadriceps muscle showed
increased pSTAT3, consistent with our establishment of a causal role of STAT3 in muscle wasting.
Phosphorylated STAT5 was also increased in vivo. (E-G) ES-2 conditioned medium caused atrophy of
C2C12 myotubes and increased pSTAT3 but reduced pSTAT5 in vitro. (H) Addition of neutralizing
antibodies against IL-11 and IL-6 blocked ES-2 conditioned medium-induced C2C12 myotube atrophy.
(I) Consistent with a causal role in ES-2 cachexia, IL-6 levels were high in ascites from ES-2 mice,
whereas IL-11 was present even in ES-2 cell lysates, suggesting it was tumor-derived.
Body weights
IBW
FBW
Car
cass
0
5
10
15
20
25
Gra
ms
(g)
Control
ES-2
+10%
*
-7%
-2%
Muscles
GSN
Tibia
lis
Qua
dricep
s
Hea
rt0.0
0.2
0.4
0.6
0.8
Weig
ht /
100m
g IB
W
Control
ES-2
-20%
*
-23%
*
-26%
*
-12%
*
Organs
Live
r
Splee
nFat
0
2
4
6
8
Weig
ht /
100m
g IB
W
Control
ES-2
+5%
-24%
*
+83%
*
CSA Tibialis ES-2 July 2012
Control
ES-20
1000
2000
3000
CS
A (m
m2 )
-11% ***
200400
600800
10001200
14001600
18002000
22002400
26002800
30003200
34003600
38004000
42004400
46004800
50005200
54005600
58000
5
10
15
Freq
uenc
y di
strib
utio
n (%
)
CSA (mm2)
Control
ES-2
DEXA scan ES-2
BMD (g
/cm
2)
BMC(g
)
Are
a (c
m2)
Lean
(g)
Fat (g)
Total (
g)
Fat (%
)0.00.10.20.30.4
2
4
6
8
10
2025303540
Control
ES-2-16%
*
DEXA scan SOCS3 baseline
BM
D (g
/cm
2)
BMC(g
)
Are
a (c
m2)
Lean
(g)
Fat (g)
Total (
g)
Fat (%
)0.0
0.2
0.4
0.6
5
10
253035404550
WT
SOCS3
-27%
**
DEXA scan Colo205/HCT116
BMD (g
/cm
2)
BMC(g
)
Area
(cm
2)
Lean
(g)
Fat (g
)
Total (
g)
Fat (%
)0.0
0.2
0.4
0.6
2
4
6
8
10
253035404550
Control
Colo205
HCT116
-26%
*
-15%
*
-37%
*
-16%
*
DEXA scan RKO
BMD (g
/cm
2)
BMC(g
)
Area (c
m2)
Lean
(g)
Fat (g)
Total (
g)
Fat (%
)0.00.10.20.30.4
2
4
6
8
10
2025303540
Control
RKO
DEXA scan carcasses (James Carson, USC) 02-2013
Organs
Live
r
Splee
nFat
0
2
4
6
8
Weig
ht /
100m
g IB
W
Control
ES-2
+5%
-24%
*
+83%
*Bon
CSA Tibialis ES-2 July 2012
Control
ES-2
0
1000
2000
3000
CS
A (mm
2)
-11% ***
200400600800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
3800
4000
4200
4400
4600
4800
5000
5200
5400
5600
5800
0
5
10
15
Freq
uen
cy d
istrib
utio
n (%
)
CSA (mm2)
Control
ES-2
Fiber size, C2C12 exposed to ES-2 ovarian cancer conditioned medium, 24-48h, 11/2012
24h
48h
0
5
10
15
20
Fib
er
dia
mete
r (m
m)
Control medium25% ES-2 medium50% ES-2 medium100% ES-2 medium
Control medium: 100% ES-2 unconditioned medium
-8%
**
-10%
***
-19%
***
-5%
*
-12%
***
-28%
***
Fiber size, C2C12 exposed to ES-2 ovarian cancer unconditioned medium, 24-48h, 11/2012
24h
48h
0
5
10
15
20
Fib
er dia
mete
r (m
m)
DM25%50%100%
A
B C
D E
F G
H I
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 19
FIGURE 2: Murine pancreatic cancer cachexia from three independent Kras/p53-driven tumor lines is
phenotypically separable. All three lines induced muscle wasting (shown here as QUAD for quadriceps)
and loss of adipose tissue or white fat (W-FAT) (A). Muscle wasting was associated with loss of tibialis
myofiber cross sectional area (CSA) (B). Western blotting analysis of quadriceps revealed uniformly high
pSTAT3 levels, but elevated pSMAD2 was observed only with 32047 tumor bearing mice, indicating a
biochemical difference in cachexia drivers in muscle (C). Plasma from tumor bearing mice caused C2C12
myotube wasting (D), likely in part due to the expression of pro-cachectic IL-6, myostatin/GDF-8 and
Inhibin-beta A in all cell lines as detected by Western blotting of cell lysates (E). ELISA of plasma from
tumor-bearing mice showed highest levels of Activin in 32908 tumor-bearing mice (not shown).
Microarray analysis revealed that while many genes are regulated in common across the three cachexia
types, each has a unique profile of gene expression, suggesting that these pathways are phenotypically
distinct.
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 20
FIGURE 3: Characterization of cachexia induced by human pancreatic cancer cells implanted into
athymic nude mice. Analysis of spleen, liver, white fat and muscle (quad=quadriceps) weights indicates
that MIA-PaCa-2 cells induce the most muscle and fat loss with simultaneous organomegaly (A-D). The
next most cachectic mice were those bearing BxPC-3 tumors, which showed muscle and fat loss without
organomegaly. Western blotting analysis revealed that pSTAT3 was most elevated in muscle of MIA-
PaCa-2 tumor-bearing mice, followed by BxPC-3 mice. In contrast to murine pancreatic cancer cachexia,
pSMAD2 levels were not altered in muscle (E). Western blotting analysis of cell extracts shows that all
three human pancreatic cancer lines express IL-6 and myostatin/GDF-8, with detectable Activin A
expressed by MIA-PaCa-2 cells.
Research Project 4: Project Title and Purpose
Liganded Glucocorticoid Receptor Functionally Interacts with Transcription Factor Stat5 in
Human Prostate Cancer Cells – Glucocorticoids are the standard treatment of advanced prostate
cancer. Recent preclinical and clinical studies have challenged the benefits of glucocorticoids
because of potential induction of therapy resistance in malignant solid tumors. Stat5a/b is highly
critical for the viability of human prostate cancer cells in vitro and for prostate tumor growth in
vivo, and active Stat5a/b predicts poor clinical outcome of prostate cancer. We hypothesize here
that the glucocorticoid receptor (GR) functionally interacts with transcription factor Stat5a/b in
prostate cancer cells. The purpose of this work is to establish whether Stat5a/b interacts with GR
signaling in human prostate cancer cells, and if this functional co-action promotes prostate
cancer cell survival.
Anticipated Duration of Project
1/1/2013 – 12/31/2013
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 21
Project Overview
Recent data from preclinical and clinical studies have challenged the benefits of glucocorticoids
for patients with solid tumors, including prostate cancer, because glucocorticoids may induce
therapy resistance in malignant solid tumors irrespective of the tumor origin. The cellular
responses to glucocorticoids are mediated through the glucocorticoid receptor (GR). There are
two major mechanisms of gene regulation by GR. The direct transcriptional regulation
(transactivation) occurs via binding of the GR to glucocorticoid-response elements. The indirect
regulation is mediated via functional interaction with other transcription factors.
Stat5a/b is highly critical for the viability of human prostate cancer cells in vitro and prostate
xenograft tumor growth in nude mice. This laboratory has shown that active Stat5a/b in primary
prostate cancer predicts early disease recurrence, and our recent work demonstrated that Stat5a/b
is in the active state in the majority of hormone-refractory prostate cancers.
We hypothesize that liganded GR increases transcriptional activity of Stat5 in prostate cancer
cells and promotes Stat5-induced prostate cancer cell survival. The objectives of this project are:
1) Establish whether liganded GR increases transcriptional activity of Stat5a/b, and Stat5a/b in
human prostate cancer cells. This will be achieved by determining whether liganded GR
increases transcriptional activity of Stat5 in reporter gene assays.
2) Determine molecular mechanisms underlying the functional co-action of GR and Stat5 in
human prostate cancer cells. This will be achieved by testing whether liganded GR increases
nuclear translocation of Stat5 by immunocytochemistry and physical interaction with Stat5 by
co-immunoprecipitation experiments.
Principal Investigator
Marja T Nevalainen, MD, PhD
Associate Professor
Thomas Jefferson University
125 S. 9th Street
Philadelphia, PA 19107
Other Participating Researchers
Lei Gu, MD – employed by Thomas Jefferson University
Expected Research Outcomes and Benefits
It is expected that liganded GR promotes Stat5 signaling in human prostate cancer cells. We will
determine if liganded GR increases transcriptional activity of Stat5a/b cells, and if Stat5a/b
physically interacts with GR in prostate cancer. In addition, we will determine if liganded GR
enhances tyrosine phosphorylation and nuclear translocation of Stat5a/b in prostate cancer cells.
Future studies should determine whether liganded GR alters the function of the PrlR-Jak2
complex, the Jak2 activity, or the rate of Stat5 dephosphorylation. We have shown previously
that Stat5a/b is critical for the viability of human prostate cancer cells in culture and for prostate
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 22
xenograft tumor growth in nude mice. We have further shown that Stat5a/b is activated in high-
grade prostate cancer but not in normal human prostate epithelium, and that Stat5a/b is activated
in the majority of prostate cancer hormone-refractory and metastases. Importantly, activation of
Stat5a/b in primary prostate cancer predicted early recurrence. The results of the work proposed
here will determine if liganded GR promotes Stat5-induced transcription and prostate cancer cell
survival. Future work will determine whether activation of Stat5a/b combined with positivity for
nuclear GR would provide a more powerful marker of poor clinical outcome than active Stat5a/b
alone. In addition, it will be important to establish whether activation of Stat5a/b in advanced
prostate cancer predicts unfavorable response to glucocorticoid treatment. In conclusion, this
work is important since the co-action of active Stat5a/b with liganded GR may contribute to the
induction of survival signals in prostate cancer cells.
Summary of Research Completed
Glucocorticoids have been extensively used for the treatment of advanced prostate cancer, and
the combination of paclitaxel and dexamethasone is a standard treatment for prostate cancer
patients with castrate-resistant and/or disseminated disease. Although known for their anti-
inflammatory activity, the use of corticosteroids in prostate cancer patients does not aim to
destroy malignant cells, but rather to alleviate symptoms caused either by the treatment or by the
tumor, such as edema, inflammation, pain, lack of appetite, and electrolyte imbalance.
Glucocorticoids may also exert anti-tumor effects on castrate-resistant prostate cancer by
negative feedback on the hypothalamic-pituitary-adrenal axis, leading to a decrease in both
testicular and adrenal androgens. However, recent data from preclinical and clinical studies have
challenged the benefits of glucocorticoids for patients with solid tumors, including prostate
cancer, because glucocorticoids may induce therapy resistance in malignant solid tumors
irrespective of the tumor origin. Tissue-specific differences in glucocorticoid-induced apoptosis
versus promotion of tumor growth are thought to be due to cell-type-specific transcriptional
regulation. It has been suggested that the inhibition of cell cycle progression by glucocorticoids
may be crucial in shifting the balance of several interacting pathways towards survival.
The cellular responses to glucocorticoids are mediated through the glucocorticoid receptor (GR).
In the absence of glucocorticoids, GR is sequestered in an inactive complex with chaperone
proteins in the cytoplasm. Following ligand binding, the GR dissociates from the chaperones and
forms homodimers, which enter the nucleus. There are two major mechanisms of gene regulation
by GR. The direct transcriptional regulation (transactivation) occurs via binding of the GR to
glucocorticoid-response elements. The indirect regulation is mediated via functional interaction
with other transcription factors.
Stat5a/b is highly critical for the viability of human prostate cancer cells in vitro and prostate
xenograft tumor growth in nude mice (16). Stat5a and Stat5b, encoded by separate genes, belong
to the seven member family of Stat transcription factors. Stat5a/b is activated via
phosphorylation of a specific tyrosine residue in the C-terminus by a tyrosine kinase, such as
Jak2. Tyrosine phosphorylated Stat5a/b homo- or heterodimerizes, translocates to the nucleus,
and activates transcription by binding to specific Stat5a/b response elements of target gene
promoters. Stat5a/b is the principal signaling protein downstream of the prolactin/prolactin
receptor/Jak2 (Prl/PrlR/Jak2) pathway in normal and malignant prostate cells. Prl, in turn, is a
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 23
mitogen and survival factor for prostate cells, and Prl protein and its receptor are expressed in
prostate cancer cells. In addition, both Prl protein expression and Stat5a/b activation, are
associated with high histological grade of human prostate cancer. We further showed that active
Stat5a/b in primary prostate cancer predicts early disease recurrence, and our recent work
demonstrated that Stat5a/b is in the active state in the majority of hormone-refractory prostate
cancers.
Here it is reported that GR is expressed in CWR22Rv1, CWR22Pc, DU145 and PC-3 human
prostate cancer cell lines. Liganded GR increases transcriptional activity of Stat5a and Stat5b.
Prostate cancer cell lines
DU145, PC-3, LNCaP, CWR22Rv1 and CWR22Pc prostate cancer cells were cultured in RPMI-
1640 (Mediatech, Manassas, VA) with 10% fetal bovine serum (Atlanta Biologicals, Norcross,
GA), 2 mM L-glutamine (Mediatech), 5 mM HEPES, pH 7.3 (Mediatech), 50 IU/ml penicillin
and 50 µg/ml streptomycin (Mediatech). LNCaP and CWR22Pc (26) cells were cultured with 0.5
nM and 0.8 nM dihydrotestosterone (DHT) from Sigma-Aldrich (St. Louis, MO), respectively.
Luciferase reporter gene assays
PC-3 cells and DU145 cells were transiently co-transfected using FuGENE6 transfection reagent
(Roche, Indianapolis, IN) with 0.25 µg of pStat5a or pStat5b, and/or pProlactin Receptor (pPrlR)
(0.25 µg), and/or human pGlucocorticoid Receptor (pGR) (0.25 µg), and either 0.5 µg of
pGASx4T109-Luc (pNTCP-Luc), pBeta-Casein-Luc, pCIS-Luc or pCyclin-D1-Luc, and pRL-
TK (Renilla) (0.025 µg) as an internal transfection control. The total amount of plasmid DNA
was normalized by the empty vector (pDNR). The cells were starved in serum-free medium and
stimulated with 10 nM human prolactin (hPrl) (NIDDK, Bethesda, MD) and/or 100 nM
dexamethasone (Dex) (Sigma-Aldrich) and/or vehicle for additional 16 h. The cells were lysed
and assayed for firefly and renilla luciferase activities using the Dual-Luciferase reporter assay
system (Promega, Madison, WI) with an automatic POLARstar OPTIMA (BMG LABTECH,
Germany). Three independent experiments with triplicate measurements were carried out using
at least two sets of plasmids prepared separately. Relative luciferase activity was obtained, and
from the mean values of each independent run, the overall mean and its standard error (S.E.)
were calculated.
Liganded GR increases transcriptional activity of Stat5a/b in prostate cancer cells.
Stat5a/b is constitutively active in prostate cancers of high histological grade and in the majority
of castrate-resistant prostate cancers. Given that the GR is expressed in clinical prostate cancers
and corticosteroids are a standard treatment for metastatic prostate cancer, the investigators
tested the hypothesis that GR and Stat5a/b signaling pathways interact in human prostate cancer
cells. Transcriptional activity of Stat5a/b was determined using luciferase reporter gene driven
by promoters regulated by Stat5a/b. Beta-Casein and Cyclin-D1 promoters both contain a
Stat5a/b binding site flanked by a non-consensus Stat5a/b site, and therefore constitute strong
tetrameric Stat5a/b binding promoters. The promoter of CIS (Cytokine Inducible SH2 containing
protein) contains four consensus Stat5a/b recognition sequences, whereas the NTCP
(4×GASpT109, sodium taurocholate cotransporting protein GAS-like elements)-luciferase
reporter gene construct contains an artificial Stat5 promoter of four Stat5a/b response elements
upstream of HSV thymidine kinase promoter. In the first set of experiments, PC-3 cells (Fig.
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 24
1A) and DU145 cells (Fig.1B) were co-transfected with Beta-casein-luciferase (luc), Prl receptor
(PrlR), Stat5a, or Stat5b, and/or GR, serum-starved and stimulated with human Prl (hPrl) (10
nM) and/or dexamethasone (Dex) at indicated concentrations for 16 h. Upon co-expression of
active Stat5a/b with liganded GR, Prl-induced transcriptional activity of Stat5a and Stat5b was
increased by 2-4-fold in PC-3 cells (Fig. 1A) and by 2-15 fold in DU145 cells (Fig. 1B) (p<0.03)
over a wide concentration range of Dex (from 1 nM to 1000 nM). When assayed with CIS-luc,
Cyclin-D1-luc, or NTCP-luc, Prl-induced transcriptional activity of Stat5a or Stat5b was
increased by 2-3-fold in cells expressing liganded GR and Stat5a or Stat5b compared to cells
negative for liganded GR (Fig. 1C) (p<0.005).
To extend the findings to the protein level, the investigators employed Bcl-xL protein expression
as a quantifiable indicator of Stat5 transcriptional activity in CWR22Rv1 cells. The cells were
infected with adenoviruses expressing wild-type Stat5b (AdWTStat5b) and treated with hPrl or
Dex for 16 h (Fig. 1D). In line with the results obtained from the Stat5-regulated reporter gene
assays, the levels of Bcl-xL protein were increased if prostate cancer cells expressed both active
WTStat5b and liganded GR (Fig. 1D, lane 4) compared to cells expressing Dex-stimulated GR
only (lane 3) or cells expressing Prl-activated Stat5a/b only (lane 2). In summary, the data
presented here indicate that liganded GR increases transcriptional activity of Stat5a/b in prostate
cancer cells.
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 25
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 26
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 27
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 28
Research Project 5: Project Title and Purpose
Increasing Patient-Centered Care for Patients with Early Prostate Cancer – More than 90% of
patients with newly diagnosed prostate cancer have localized disease, and therefore face the
difficult decision of whether to initiate active surveillance (AS) or active treatment (AT). Given
that AT may have serious side effects, and mortality rates are comparable for men who have AS
and those who have AT, it is important for men to make an informed decision about treatment.
We propose to conduct a pilot study to test the impact of a novel patient decision support
intervention, on AS versus AT uptake. The study will involve early-stage prostate cancer
patients who have a scheduled visit for consultation at the Jefferson Prostate Cancer
Multidisciplinary Clinic (JMDC) in Philadelphia.
Anticipated Duration of Project
1/1/2013 – 12/31/2014
Project Overview
The primary objective of the study is to pilot test a decision support intervention related to
treatment decisions [(active surveillance (AS) versus active treatment (AT)] among men with
low-risk prostate cancer seen in Thomas Jefferson’s Prostate Cancer Multidisciplinary Clinic
(JMDC). We propose to pilot test the intervention with 25 newly diagnosed patients. Because
the intervention is a pilot test, we propose a non-randomized design, such that all enrolled
patients are exposed to the intervention. More specifically, we plan to meet eligible patients in
the JMDC, immediately prior to their scheduled appointment with the clinical team. A nurse
educator will educate the patient about his treatment options and guide him through decision
counseling, which involves the patient identifying factors that are likely to influence him when
deciding between AS or AT. A summary sheet of the results of the decision counseling session
will be provided to the patient and to his clinical team to further guide discussions about
treatment preference. A member of the research team will follow-up with each patient to
document the patient’s decision about treatment, as well as a medical chart audit will verify
treatment status. We expect that the proportion of patients who choose and initiate AS will be
higher than that is observed historically in the JMDC.
Specific aims of the study are to:
1. Assess feasibility of implementing the decision counseling protocol in practice
2. Determine the proportion of patients who choose AS and AT
3. Among those patients who initially choose AS, determine the proportion that actually initiate
AS
4. Identify patient factors that are associated with choosing AS.
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 29
Principal Investigator
Ronald E. Myers, PhD
Director, Division of Population Science, Department of Medical Oncology
Thomas Jefferson University
125 S. 9th Street
Philadelphia, PA 19107
Other Participating Researchers
Amy Leader, DrPH, MPH; Anett Petrich, MSN, RN; Anna Quinn, MPH; James Cocroft, MA –
employed by Thomas Jefferson University
Expected Research Outcomes and Benefits
Men diagnosed with early-stage, low risk prostate cancer face complex decisions about
treatment. This decision may provoke anxiety and is likely to affect clinical outcomes. It is
important to facilitate well-informed, shared decision making about these options in clinical care.
However, the literature suggests that men with early-stage, low-risk prostate cancer often make
decisions about treatment with a limited understanding of available options. Furthermore, little
research has been reported in the literature on the use of decision support interventions in this
patient population. It is expected that patients who participate in the pilot study will be more
knowledgeable about AS and AT, feel informed about treatment decision making, and report a
high level of satisfaction with the decisions that they make about their care. Furthermore,
clinicians will be able to engage patients in shared decision making about treatment and clinical
trials participation effectively and efficiently.
The proposed pilot will lay the foundation for a larger randomized, control trial aimed at
developing and testing the impact of decision counseling, a novel approach to facilitating
informed, shared decision making in cancer care.
Summary of Research Completed
Development of Protocol and Study Materials
Members of the research team met weekly to review potential materials, discuss study design
and flow, and prepare for study implementation. Materials, including study surveys and
educational charts and pamphlets, were pre-tested with three patients in the Jefferson
Genitourinary Multidisciplinary Clinic (JGMC), the location of patient recruitment. Pre-testing
involved assessing the amount of time each study component required for implementation (so as
not to disrupt the patient flow of the JGMC), as well as assessing readability and patient
comprehension of the study materials. From the pre-testing exercise, we altered or included
additional language in the educational materials, at the request of the patients. We also reduced
the number of items on certain surveys, to reduce the time burden to the patient.
Next, the research team finalized the study protocol. According to the study protocol, the nurse
educator receives a list of all the patients who have scheduled appointments at the JGMC,
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 30
including the patient’s clinical information and diagnosis. If one or more patients on the list
meet the study’s eligibility criteria, the nurse educator attends the tumor board meeting, where
the patient cases are reviewed by the clinical team in preparation for the morning’s scheduled
appointments. At this meeting, the clinical team makes a final determination about the patient’s
tumor stage and study eligibility. The research assistant approaches each eligible patient as he
waits for his clinic appointment and assesses his interest in participating in the study. For
patients who are interested, the research assistant obtains informed consent.
Following consent, the research assistant administers a baseline survey. The survey assesses
knowledge about treatment options, health perceptions, and decisional conflict related to the
decision about treatment, as well as background and demographic characteristics of the
patient. After the patient completes the survey, a nurse educator reviews an informational page
about treatment decisions with the patient. Then, the nurse uses an online Decision Counseling
Program©
(DCP) protocol to identify decision factors (i.e. reasons) that are likely to influence the
patient to choose AS or AT. During the DCP session, the patient states how important each
factor is (factor weighting). From this information, a decision preference score that ranges from
0.000 to 1.000 is calculated. A higher decision preference score indicates a preference for AS,
while a lower score indicates a preference for AT. The nurse validates the score with the patient
and assesses that he understands and agrees with the results. A copy of the summary report is
given to the patient and the clinicians for use in shared decision making about treatment during
the clinical encounter.
Three to five days after the participant visit, the nurse educator calls him by phone and
administers a brief endpoint survey to assess the patient’s treatment decision, level of decisional
conflict, perception of decision counseling, and whether he needs further information from the
clinical team.
Thirty days after the initial visit, the research assistant contacts each study participant by
telephone to determine his treatment status. Additionally, the research assistant elicits reasons
for the decision and assesses patient satisfaction with their decision.
Six months after the visit, the research assistant conducts a medical chart audit to determine the
patient’s actual treatment decision.
All study data are entered into a Microsoft Excel spreadsheet and are analyzed using SAS
(v.9.1). For decisions about treatment, the proportion of patients who choose AS, along with an
exact binomial 95% confidence interval will be computed. We will test this proportion against
the JGMC’s historical rate via an exact binomial test. Furthermore, we will determine the
likelihood that patients actually undergo the type of treatment their preference report from the
DCP indicates that they preferred.
Study Recruitment and Enrollment
We approached 26 potentially eligible patients, as determined by the clinical team at the JGMC.
Of those approached, 23 consented and enrolled in the study. Only 3 patients declined
enrollment. Of the 23 who enrolled in the study, 16 have completed a 30-day survey and 5 have
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 31
been reviewed by medical chart audit. Figure 1 outlines the flow of patients in the study.
Study Results
Of the 5 participants who have completed the study, 3 were Caucasian and 2 were African
American. Three participants were between the ages of 50 and 59, while 2 were 60 years or
older. Four participants had more than a high school diploma, and all 5 were married or living
with a partner. From the decision counseling session, we determined that participants identified
a total of 12 decision factors; 6 factors were in favor of AS (pro factors), while 6 were in favor of
AT (con factors). At the end of decision counseling, 4 participants weighed AS and AT equally,
while 1 preferred AS. From the baseline survey to the 30-day endpoint survey, treatment-based
knowledge for the 5 participants had increased by 18% from baseline, and decisional conflict
decreased by 47% from baseline. At the 90-day chart audit, 4 s participants had chosen AS and 1
participant had chosen AT.
Figure 1. Decision Counseling and Active Surveillance Study: CONSORT Flow Diagram
Approached:
26
Consented & enrolled:
23
Declined: 3
Endpoint Survey
Complete:
16
22
Endpoint Survey Pending: 5
Lost to follow-up: 2
Endpoint Chart Audit:
5
Endpoint Survey Pending: 11
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 32
Research Project 6: Project Title and Purpose
Cell Fate Factor Protein DACH1 Modification in Signaling and Cancer – The DACH1 protein
has features of a tumor suppressor, however very little is known of its modification by
phosphorylation or acetylation, which may in turn govern signaling by this protein. DACH1 was
cloned as a Drosophila homologue (Dac) that blocks Epidermal Growth Factor Receptor (EGFR)
signaling. Hyperactive growth factor signaling is a common feature of terminal cancer.
Identifying new mechanisms to attenuate hyperactive growth signaling in cancer may provide
important new therapeutic options. Identifying the functional modifications of DACH1 may
allow us to target the DACH1 protein and its activity in tumors.
Anticipated Duration of Project
1/1/2013 – 12/31/2016
Project Overview
The aims of these studies are to define at a higher level of resolution of the mechanism by which
DACH1 governs cancer onset and progression. Hyperactive growth factor receptor signaling
remains active in many tumors that resist current therapies. The Retinal Determination Gene
Network (RDGN), consisting of the Dachshund (dac), eyes absent (eya), eyeless, and sine oculis
(so) genes, regulates cell fate determination in metazoans. The Drosophila dac gene was cloned
as a dominant inhibitor of the hyperactive EGFR (Elipse). Genetic deletion of Dach1 in the
mouse results in perinatal lethality, therefore, we will use conditional Dach1 knockout tri-
transgenic systems.
In previous studies, we showed DACH1 inhibits breast cellular proliferation and metastasis. The
current studies will determine the mechanisms by examining post-translational modification of
DACH1. We hypothesize that post-translational modification of DACH1 is a key signaling event
contributing to tumorigenesis and metastasis. We will determine the post-translational
modifications of DACH1 and assess their role in tumor growth and metastasis. Functional
analyses of DACH1 post-translational modification may lead to the identification of new cancer
targets.
Specific Aims:
Aim 1. Determine the mechanisms by which DACH1-phosphorylation determines lung and
breast tumor cellular proliferation.
Aim 2. Determine the mechanisms by which DACH1-acetylation determines lung and breast
tumor cellular proliferation.
Aim 3. Determine the role of DACH1 post-translational modification in chromatin occupancy
and gene expression.
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 33
Principal Investigator
Richard G. Pestell, MD, PhD
Professor and Chairman
Department of Cancer Biology
Thomas Jefferson University
125 S. 9th Street
Philadelphia, PA 19107
Other Participating Researchers
Chenguang Wang, PhD; Xuanmao Jiao, PhD – employed by Thomas Jefferson University
Expected Research Outcomes and Benefits
Resistance to Non-Small Cell Lung Cancer (NSCLC) and breast cancer to current therapies kills
a substantial number of patients each year in the United States. New molecular targets governing
tumor onset and progression are needed urgently. DACH1 represents a novel tumor suppressor
pathway. Reduced DACH1 expression correlates with poor prognosis in NSCLC and human
breast cancer. Reintroduction of DACH1 into human mammary epithelial cells transformed with
distinct oncogenes (Ras, ErbB2, c-Myc) reverts the transformed phenotype in 3D culture.
Reversion of the tumor phenotype and the associated genotype led to the DACH1 gene being
referred to as a “commandeering” tumor suppressor. DACH1 is structurally distinct from the
current known tumor suppressors. We will identify key functional modifications of DACH1
required for inhibition of NSCLC and breast tumor growth. As DACH1 is a cell fate
determination factor, understanding the mechanisms by which DACH1 inhibits tumorigenesis
may have important implications for the role of cell fate in lung and breast tumorigenesis.
Summary of Research Completed
Substantial progress has been made with this project. We have shown that DACH1 is
phosphorylated, identified the individual phosphorylation sites and shown that these residues
regulate nuclear translocation. We have also shown that the phosphorylation sites determine the
ability of DACH1 to inhibit epithelial-mesenchymal transition (EMT) via translational
suppression of Snail.
DACH1 binds YB-1 in breast cancer cells. DACH1 has been implicated in the regulation of stem
cells of several tissue types, including muscle, neural and breast. Aproteomic analysis was
conducted in order to identify candidate DACH1-binding partners that may govern this function.
DACH1 protein complexes were prepared from HEK293T cells transfected with the FLAG-
DACH1 expression vector, and DACH1-associated proteins were resolved on a 6 – 10% percent
hydrochloride gel and silver stained. Analysis of the proteins recovered from the gel through in-
gel trypsin digestion, and in sequential ms/ms identified a ~50 kDa protein, identical to YB-1.
HEK293T cells expressing FLAG-tagged DACH1 and Myc-tagged YB-1, demonstrated the
association of YB-1 with DACH1 in immunoprecipitation-Western blotting. MDA-MB-231 cells
were used to examine the binding of endogenous YB-1 to DACH1. The MDA-MB-231 cells
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 34
were stably transduced with an expression vector encoding DACH1 and immunoprecipitation,
conducted with a FLAG antibody directed to the amino-terminus of DACH1, co-precipitated
endogenous YB-1.
The cold-shock domain of YB-1 binds to the carboxyl terminus of DACH1. The YB-1 protein
consists of three key domains; a C-terminal domain (CTD), which contributes to DNA/RNA
binding, a highly conserved cold shock domain (CSD) which forms an antiparallel β barrel to
facilitate binding to nucleic acids as a chaperone protein, and an non-conserved variable N-
terminal domain characterized by four alternating clusters of basic and acidic amino acids, which
is both alanine and proline rich and is thought to have been involved in transactivation. Nuclear
cytoplasmic shuffling of YB-1 is controlled by both a distinct nuclear localization signal and
cytoplasmic retention signals in the CTD. HEK293T cells were transfected with a series of
FLAG-tagged YB-1 mutant expression vectors and HA-tagged DACH1 (Fig. 1A). The FLAG-IP
precipitated YB-1 fragment. Western blotting for DACH1 using the HA antibody demonstrated
the association of DACH1 with each YB-1 fragment but a failure to bind the CSD alone (Fig.
1B). Immunoprecipitation of DACH1 with the HA antibody demonstrated DACH1 binding to
the YB-1 C-terminus (Fig. 1C). In GST pulldown 6-His DACH1 bound GST-expressed YB-1.
The DACH1 DBD did not bind YB-1 (Fig. 1D). Thus, the C-terminus of YB-1 was sufficient for
binding DACH1 and the YB-1 CSD domain was incapable of binding DACH1 (Fig. 1A, B).
DACH1 exists in three isoforms (DACH1a, b, c), and each of the DACH1 isoforms were capable
of binding YB-1 with similar affinity (Fig. 1E,F). DACH1 mutant expression vectors were co-
expressed with YB-1 WT in order to define the domains of DACH1 governing association with
YB-1 (Fig. 1G). Deletion of the DNA-binding domain (ΔDBD) of DACH1 did not affect binding
to YB-1, however deletion of the C-terminus (DACH1 ΔC) abolished YB-1 binding and the C-
terminus was sufficient for YB-1 binding (Fig. 1H).
Phosphorylation determines DACH1 nuclear-cytoplasmic translocation. In order to determine
whether DACH1 is phosphorylated, cellular extracts from HEK293T cells were examined before
and after treatment with calf intestinal phosphatase (CIP). The treatment of cells with CIP
reduced the mobility of DACH1. Mass spectrometry was used to identify the phosphorylation
sites and point mutation of the residues with highest Mascot Scores was conducted. The
electrophoretic mobility of the DACH1 protein expressing a mutation of S439 was similar to the
CIP treated cells expressing DACH1 wt. By IHC, the DACH1 protein was located primarily in
the nucleus. The S439A S441 and S529 mutants were cytoplasmic. These findings suggest
DACH1 phosphorylation at S439 determines the cytoplasmic vs. nuclear location.
DACH1 inhibits and YB-1 induces breast cancer cellular EMT. As YB-1 located in the
cytoplasmic pool regulates translational control to induce EMT and DACH1 expression reduced
cytoplasmic YB-1, we examined the possibility that DACH1 expression may reduce YB-1-
dependent EMT. The MBA-MB-231 breast cancer cell line stably transduced with a
ponasterone-inducible DACH1 expression vector demonstrated the expression of FLAG-tagged
DACH1 upon the addition of Ponasterone A, which reduced the abundance of Ezrin, SNAIL and
ZEB-1 (Fig. 2A). Vinculin and GDI, two loading controls for protein abundance were
unchanged. Consistent with a role for YB-1 to induce EMT, shRNA to YB-1, reduced the
abundance of SNAIL and ZEB-1 (Fig. 2A). Immunohistochemical staining demonstrated a
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 35
reduction in ZEB-1 upon expression of DACH1 (Fig. 2B). Similarly, shRNA to YB-1 reduced
ZEB-1 abundance (Fig. 2B). The abundance of SNAIL was reduced by either DACH1
expression (Fig. 2C) or shRNA to YB-1, (Fig. 2C). The reduction in SNAIL abundance by YB-1
shRNA in MDA-MB-231 cells is consistent with prior findings in MCF10A-Ras breast cancer
Cells. E-cadherin abundance is reduced upon EMT induction and DACH1 expression or YB-1
shRNA induced E-cadherin immunostaining (Fig. 2D). Together these studies suggest DACH1
inhibits YB-1 induced EMT .
DACH1 expression regulates nuclear cytoplasmic distribution of YB-1 and thereby YB-1-
mediated cap-independent translation of Snail. The finding that DACH1 expression of YB-1
shRNA induced EMT, without affecting expression of genes governing EMT, led us to consider
alternate mechanisms. Cytoplasmic YB-1 is known to activate cap-independent translation of
mRNA encoding Snail and other factors implicated in activators of mesenchymal genes. As
cytoplasmic, not nuclear YB-1, governs EMT, we considered the possibility that DACH1
expression may regulate the relative abundance of cytoplasmic vs. nuclear pools of YB-1.
To examine the effects of DACH1 expression, on YB-1 sub-cellular localization, IHC for
DACH1 and YB-1 was conducted on postasterone-inducible DACH1 expressing breast cancer
cell lines. Expression of DACH1 in SUM159 cells was readily detectable upon Ponasterone A
treatment in a nuclear compartment, detected by the FLAG antibody to the N-terminal tag of
DACH1. YB-1 was predominately cytoplasmic in location. Upon expression of DACH1, YB-1
became predominantly nuclear (Fig. 3A). Similar findings were made in MDA-MB-231 cells in
which expression of DACH1 was associated with increased nuclear abundance of YB-1, detected
either by immunohistochemistry (Fig. 3B) or by Western blot of nuclear vs. cytoplasmic
fractions (Fig. 3C). Although, relatively little is known of the factors associated with nuclear-
cytoplasmic shuttling of YB-1, the stimuli known to induce cytoplasmic to nuclear shuttling of
YB-1 include DNA damaging agents, wherein YB-1 binds to damaged DNA. HEK293T cells
were treated with DNA damaging agents (cisplatinum 2 mmol or paclitaxel, 2 nmol). The
addition of DNA damaging agents induced the nuclear localization of YB-1 which co-associated
with nuclear DACH1.
We had shown the DACH1 expression or YB-1 shRNA reduced SNAIL abundance. The role of
SNAIL1 as an inducer of EMT is well established. The Snail1 mRNA harbors specific motifs in
its 5’UTR that are conserved and are responsible for cap-independent translation by YB-1. YB-1
has been shown to induce cap-independent translation of genes enhancing EMT including Snail
in MCF10AT cells. (The MCF10AT subclone is derived by transformation of the immortalized
benign MCF10A human mammary epithelial cells with oncogenic Ha-Ras and subsequent
passaging through mouse xenotransplants). We examined the role of DACH1 in YB-1-dependent
regulation of Snail, and deployed a Snail1 5’UTR linked to the luciferase reporter gene.
Conditions of starvation, or starvation combined with rapamycin, restricts cap-dependent
translation. Consistent with previous observations that YB-1 induces cap-dependent translation
of Snail1 5’UTR-Luc activity, we also show that YB-1 induced Snail 5’UTR-Luc activity.
Ectopic expression of DACH1 repressed YB-1 activation (Fig. 3D). The induction of Snail
5’UTR-Luc activity by YB-1 requires the cytoplasmic pool. DACH1 expression reduces the
cytoplasmic YB-1. The phosphorylation point mutants of DACH1 are localized to the cytoplasm
and fail to undergo nuclear translocation. Consistent with a model in which DACH1 inhibits YB-
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 36
1 cytoplasmic EMT function through inducing nuclear translocation, each of the DACH1
phosphorylation point mutants failed to repress YB-1-mediated induction of Snail 5’UTR-Luc
activity, indeed the mutants conveyed a dominant positive effect on Snail1 5’UTR-Luc activity .
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 37
Figure 1. DACH1 binding requires the YB-1 Cold Shock Domain (CSD). A) Schematic representation of
DACH1, YB-1 and YB-1 mutant expression vectors. B) IP-Western blot was conducted of HEK293T cells transfected with expression vectors encoding either N-terminal 3x FLAG-tagged YB-1 or HA-tagged DACH1 expression vectors. In (B) the IP was conducted with an antibody to the FLAG-tag of YB-1 and in (C) the IP was directed to the Ha tag of DACH1. DACH1 binding to YB-1 was derived from N=3 separate experiments. (D) DACH1-YB-1 binding characterized in pulldown using 6-His-tagged DACH1 or DACH1 DBD region and GST-YB-1. (E-G) Schematic representation of YB-1 and DACH1 mutant expression vectors. (F-H) IP-Western blot of Myc-tagged YB-1 and associated DACH1 expression vectors transfected into HEK293T cells. Antibodies were directed for IP to the 3x FLAG tag of DACH1 and Western blot to the Myc epitope of YB-1. Antibodies are directed to the proteins as indicated. Data are representative of N=3 separate experiments. The DACH1 isoforms are indicated as DACH1a, b and c.
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 38
Figure 2. DACH1 inhibits YB-1 induced EMT. A) MDA-MB-231 cells stably transduced with a Ponasterone A-inducible FLAG-tagged DACH1 expression vector were sequentially transduced with shRNA to YB-1. Pon A treatment (2 μM) was used to induce DACH1. Western blot was conducted with the antibodies as indicated. (B-D) Immunohistochemistry for markers of mesenchymal cells include, (B) ZEB1, (C) SNAIL, (D) E-cadherin.
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 39
Figure 3. DACH1 promotes nuclear translocation of YB-1, inhibiting YB-1 dependent translation. Confocal
microscopic images of SUM159 (CA) and MDA-MB-231 cells (B) with ectopic expression of DACH1 using antibodies as indicated. (C) Western blot analysis of cytoplasmic and nuclear protein as indicated. (D) MCF10A-RAS cell transfected with YB-1, DACH1 and SNAIL-5’UTR Luc reporter were starved and treated with rapamycin (uM). Luciferase activity was measured after 24 hours. The data are mean ±SEM.
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 40
Research Project 7: Project Title and Purpose
Molecular Aberrations in Adenocarcinoma and Neuroendocrine Prostate Cancer – We
hypothesize that Adenocarcinoma (AdCa) and Neuroendocrine (NE) Prostate Cancer carry
different molecular aberrations that may explain the aggressive phenotype of NE Prostate Cancer
(NEPC). Using two mouse models of prostate cancer, we plan to delineate the signaling
aberrations by which neuroendocrine prostate cancer (NEPC) differ from AdCa. In both in vivo
mouse models, we will profile all samples for aberrant expression and activities of signaling
molecules and identify the molecules which contribute to an aggressive cancer cell phenotype.
This study presents an unmet clinical need for NEPC patients with advanced disease.
Anticipated Duration of Project
1/1/2013 – 12/31/2016
Project Overview
We hypothesize that Adenocarcinoma (AdCa) and Neuroendocrine (NE) Prostate Cancer carry
different molecular aberrations that may explain the aggressive phenotype of NE Prostate Cancer
(NEPC). This study presents an unmet clinical need for NEPC patients with advanced disease.
The main goal of this proposal is to delineate the molecular aberrations that occur in AdCa and
NEPC. The specific aims of this proposal are as follows:
1) To delineate the signaling aberrations by which NEPC differ from AdCa. We will use
TRAMP and castrated PTEN conditional knock-out mouse models, which are known to develop
both AdCa and NEPC. Using gene expression analysis, AdCa and NEPC tumors will be isolated
by laser capture microdissection and analyzed. We will profile all samples in both in vivo mouse
models for aberrant expression of signaling molecules. We will analyze this signature in
TRAMP and castrated PTEN conditional knock-out mice; 20 mice will be used/group and
routine staining for synathophysin and chromogranin will be used to define neuroendocrine
differentiation. All techniques to be used, including qRT-PCR, are routinely performed in our
laboratories and have been previously described by our group.
2) To validate the findings in Aim 1 using primary and metastatic samples from NEPC tumors.
We will profile all samples in both in vivo mouse models for aberrant activities of signaling
molecules. Several measures of signaling aberrations will be performed using immunoblot
analysis to assess the differences in kinase activities, DNA-repair proteins, apoptotic response
and proliferation rates. Upon transfection of the identified aberrant signaling molecules,
anchorage-independent growth assay, proliferation, androgen receptor (AR) localization and
activity, mitochondrial isolation and import, DNA damage response and apoptosis will be
measured to confirm that the aberrant signaling contribute to an aggressive cancer cell
phenotype. The study will make use of a murine tissue bank available in our laboratory from
mice at various stages of AdCa and NEPC (primary tumors as well as metastatic tumors).
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 41
Principal Investigator
Lucia R. Languino, PhD
Professor
Thomas Jefferson University
125 S. 9th Street
Philadelphia, PA 19107
Other Participating Researchers
Sheikh Aejaz Sayeed, PhD – employed by Thomas Jefferson University
Expected Research Outcomes and Benefits
This study will provide new insights for therapeutic intervention in patients with aggressive
neuroendocrine prostate cancer. Understanding signaling aberrations occurring in AdCa and in
NEPC will help to characterize the pathways that contribute to the aggressive phenotype found in
NEPC and develop strategies to successfully treat NEPC. We will make use of a murine tissue
bank available in our laboratory from mice at various stages of AdCa and NEPC (primary tumors
as well as metastatic tumors). Protein aberrations will be correlated with the degree of
neuroendocrine differentiation, other pathologic features and survival. This study presents an
unmet clinical need for NEPC patients with advanced disease. The fundamental knowledge
gained from these studies will clearly be beneficial and applicable to populations with
historically poor cancer prognoses.
Summary of Research Completed
We have started our investigation on the basis of our initial hypothesis that Adenocarcinoma
(AdCa) and Neuroendocrine (NE) prostate cancer carry different molecular aberrations that may
explain the aggressive phenotype of NE Prostate Cancer (NEPC). During the current reporting
period, we have focused predominantly on characterizing the available prostate mouse tissue
specimens isolated from TRAMP and castrated PTEN conditional knock-out mice (Aim 1). As
described in the first specific aim of this proposal, these mice are known to develop both AdCa
and NEPC. Prostate tissue specimens from ten TRAMP mice have been characterized. Indeed,
histological examination of these specimens shows expression of both types of cancer.
Additional analysis has been performed to demonstrate that the murine tissues express
synathophysin and chromogranin A, which are known to define NE differentiation. We have
optimized the conditions to detect, by immunohistochemistry, synathophysin and chromogranin
A, and show that all our specimens express both markers of NE cancer. A representative
example of this immunohistochemical staining is shown in Figure 1.
In parallel, we have optimized the conditions to analyze integrin expression and have stained all
ten specimens for av6 integrin. Using previously published protocols, we show that av6 integrin
is expressed in AdCa but not in NE cancer. This result will allow us to differentiate the two
types of cancer; however, if confirmed, this indicates that therapeutic approaches that target
integrins will inhibit only one type of cancer but not NE. We are in the process of characterizing
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 42
additional specimens to reach the number requested to complete the first task planned in Aim 1.
We have also obtained NCI-H660 cells and N-Myc- LNCaP cells known to be NE cells, to
develop an in vitro system to characterize molecular aberrations in NE cancer. A preliminary
analysis shows that surface expression of cell adhesion receptors in these cells is different. We
are currently completing this characterization.
METHOD optimized in our laboratory to stain for Synaptophysin and Chromogranin A by
Immunohistochemistry.
DAY 1:
1. Place slides in glass holders, tissue facing out. Bake at 60o C for 2 hrs.
2. Wash with xylene to deparaffinize the slides: 3 x 10 mins each
During the 3rd
wash pour 10mM sodium citrate in the plastic coplin jars and heat to 95oC for
about 15 minutes (using DDH20 in the bottom of the steamer)
Cover steamer with lid and place a thermometer in the lid of the steamer with the tip of the
thermometer in the sodium citrate
Prepare a stock of 1M Sodium citrate (pH 6.0): 294g of sodium citrate (dehydrate granular
powder; J.T. Baker – Catalog # 3646-01) in 1 liter of ddH2O, bring to pH 6.0 using 1 M citric
acid (Make citric acid using monohydrate granular citric acid powder (J.T. Baker – Catalog #
0110-01). NOTE: Make a fresh 10mM solution of sodium citrate at pH 6.0 for each experiment.
3. Rehydrate the slides. Discard alcohol in sink.
a. 95% ethanol- 2 x 2 mins
b. 70% ethanol- 1 x 2 mins
c. 50% ethanol- 1 x 2 mins
d. DH2O- 1 x 2 mins
4. Briefly dip slides in warm water (< 30 seconds).
5. Place slides in coplin jars filled with hot sodium citrate. (Use tweezers for protection
from the heat). Make sure that the temperature of the sodium citrate does not rise above
100oC; (carefully open the lid of the steamer to decrease temp).
a. Place pipet tips between the edges of the coplin jar and the outermost slides.
b. Wait until the temperature of the sodium citrate returns to 95oC to start the timer;
boil for 23 mins.
c. Afterwards, take out the coplin jars individually, and allow the slides to cool for
about 20 minutes (until the sodium citrate is cool enough to touch).
d. Place into glass containers, filled with RT DH20 for 3-4 mins.
e. After the slides have reached RT, submerge them in TBS for (3-4 mins)
Prepare 100 ml of 3% of H202 and submerge the slides in it for 5 minutes. Wear gloves to discard
H202
6. Wash slides with tap water, place on shaker for 5 mins. Wash DDH20 for 2 mins, then
with TBS for 2 mins.
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 43
7. Dry slides. Wipe. Block for 20 mins.
8. Prepare antibody (Ab) solutions into numbered eppendorf tubes: Rabbit Antibody to
Synaptophysin (Invitrogen, 18-0130); Rabbit Ab to Chromogranin A (Invitrogen,
180094). Store everything on ice. Dilute in TBS solution (TBS solution=TBS +
0.2%BSA + 0.02%Na3)
9. After blocking, wipe the back dry, and use a kimwipe to get around the tissue. Incubate
with the antibodies, using the humidity tray. Put on shelf in cold room overnight.
DAY 2:
1. Put slides in glass container. Wash with TBS 3 x 3 min each
2. Prepare secondary Ab in TBS. Incubate with secondary for 30 mins at room temperature
in the humidity tray.
a) If primary Ab was raised in rabbits, use 3 g/ml biotinylated Goat anti-Rabbit IgG
(Vector Laboratories; 1:500)
b) If primary Ab was raised in mice, use 3 g/ml biotinylated Horse anti-Mouse IgG
(Vector Laboratories; 1:500)
c) If primary Ab was mouse monoclonal IgM, use 3.3 g/ml biotinylated goat anti-mouse
IgM (Vector Laboratories; 1:150)
3. Wash with TBS 3 x 3 min each
4. Meanwhile, prepare strept-avidin peroxidase (SAP)-(0.5 units/ml; Boehringer) (1:1000 in
TBS)
a. wipe slides. Incubate for 30 mins at room temperature in the humidity tray
5. Wash with TBS-3x3 mins.
6. Incubate with di-aminobenzidine for 5 mins at room temperature, shaking, and wrapped
in aluminum foil. DAB—0.5mg/ml in TBS+0.012% H2O2
i.e. for 100mL DAB:
Dissolve 0.05ug DAB in 10mL TBS. (solution A)
Add 12uL H2O2 to 90mL H2O (solution B).
Mix solution A and solution B right before incubating the slides.
7. Wash under running tap water for 5 mins
8. Rinse in DH2O for 2 mins
9. Stain with hematoxylin for 1 min and then run under tap water for a few minutes
Dehydrate slides:
a. 50% ethanol-2 mins
b. 70% ethanol-2 mins
c. 95% ethanol-2 x 2 mins
d. xylene-3 x 2 mins
10. Wipe the slides, and mount using permount: 20 µl per slide
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 44
Fig 1. Neuroendocrine markers expressed in mouse prostate tumors.
Mouse #5224, TRAMP, age 28.6 weeks. A: Rabbit Antibody to Synaptophysin (Invitrogen, 18-
0130); B: Rabbit Antibody to Chromogranin A (Invitrogen, 180094).
Research Project 8: Project Title and Purpose
Anoikis-Resistance in Metastatic Breast Cancer Cells and the Role of the Focal Adhesion Kinase
(FAK1) – Many breast cancer patients die after the removal of primary tumors because of
metastasis. A very aggressive type called inflammatory breast cancer (IBC) is responsible for a
disproportionate number of deaths due to its propensity to rapidly metastasize. IBC primarily
affects younger women under the age of 50 at diagnosis, and is difficult to detect as it does not
present as a lump, but rather occurs as tumor emboli. Anchorage-independent growth (anoikis-
resistance) is a crucial step in metastases. The focal adhesion kinase (FAK1) is an important
regulator of anoikis-resistance. In this project, we propose to study the mechanism responsible
of anoikis-resistance in IBC to determine if FAK1 inhibition suppresses the metastatic potential
of these cells.
Anticipated Duration of Project
1/1/2013 – 12/31/2015
Project Overview
The goal of this proposal is to understand the gene expression and protein changes involved in
the mechanism of epithelial-mesenchymal transition (EMT) and anoikis-resistance that drives
cancer cells to invade and metastasize. We hypothesize that the understanding of the molecular
mechanisms driving EMT and anoikis-resistance in inflammatory breast cancer (IBC) patients
will provide novel tools for blocking the metastatic process.
A B
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 45
Specific Aims.
1) Characterize the process of EMT and anoikis-resistance in metastatic IBC cells. We propose
to study the expression of proteins related to EMT and anoikis resistance in cancer cells from
patients with IBC.
2) Determine if CEP-37440 suppresses breast tumor growth and metastasis in animal models.
We are going to determinate if FAK1 inhibition enhances anoikis of IBC cells and suppresses
their metastasis in vivo.
3) Determine if FAK1 is responsible for anoikis-resistance of circulating tumor cells (CTC).
Cancer cells in peripheral blood, known as CTCs, play an important role in tumor dissemination
and the characterization of these cells is fundamental to develop therapeutic targets against
metastatic lesions. CTCs have also be used in preclinical models to evaluate the various phases
of the metastatic process and the phenotypic properties of tumorigenic cells.
Principal Investigator
Massimo Cristofanilli, MD
Professor of Medical Oncology
Deputy Director of Translational Research, Kimmel Cancer Center
Thomas Jefferson University
125 S. 9th Street
Philadelphia, PA 19107
Other Participating Researchers
Sandra Fernandez, PhD; R. Katherine Alpaugh, PhD; Zhaomei Mu, MD – employed by Thomas
Jefferson University
Expected Research Outcomes and Benefits
Metastasis is the main cause of death of women with breast cancer. Epithelial-mesenchymal
transition (EMT) and anoikis-resistance, are key processes for metastasis and they share common
regulators, such as Twist, Snail, E-cadherin, and N-cadherin. Therefore, understanding the
pathways, as well as specific molecules, that critically regulate EMT and anoikis-resistance
could lead to the development of new therapeutic strategies that decrease metastatic potential of
breast cancer cells. Women with triple negative IBC often develop metastasis in the lungs, brain,
and, in some cases, the bones.
FC-IBC02, a new IBC triple negative cell line, and breast xenograft model recently developed
previously by the PI, will be used in this study. Moreover, we are going to generate SUM149 and
FC-IBC-02 cell lines that stably express the inducible FAK1 shRNA lentiviral vector, and to
study the effect of FAK1 downregulation on both primary tumor growth and metastasis
formation. The formation of distant metastasis in lungs, lymph nodes and other target organs will
be monitored in vivo. Our studies will determine if FAK1 inhibition could inhibit the metastatic
process of IBC. We propose to downregulate FAK1 using FAK1 shRNA although, we expect to
be able to test in the near future a commercially available small molecule FAK1 inhibitor. IBC is
a very aggressive form of breast cancer, and currently there is no adequate therapy to help these
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 46
patients. If these studies are successful, the inhibition of FAK1 may be incorporated into future
adjuvant therapies to prevent local and distant metastasis in patients with breast cancers from
basal subtypes.
Summary of Research Completed
During this initial reporting period, we finalized the Animal Protocol necessary to conduct the
in-vivo studies and we obtained final IRB approval. Moreover, we were able to secure a contract
with Teva Pharmaceuticals that will provide additional funds (Direct plus indirect cost $150K)
for the conduct of this research project.
We also recently published a peer-reviewed original article describing the preclinical model used
in this project, FC-IBC02 cell line and xenograft model: Inflammatory breast cancer (IBC): clues
for targeted therapies. Fernandez et al., Breast Cancer Res Treat. 2013 Jul; 140 (1):23-33.
Research Project 9: Project Title and Purpose
Long Non-coding RNAs Regulate Protein-coding Transcripts in Colon Cancer with the Help of a
Specific Family of MicroRNAs – Decades of research have been invested towards understanding
genetic defects in colon cancer. Until recently, the lack of available tools and of critical insights
resulted in the community having paid only limited attention to the mechanisms of post-
transcriptional regulation of the colon cancer cell. The work we propose will explore unexpected
facets of microRNA-mediated regulation of colon cancer-related genes by long non-coding
RNAs. Our goal is two-fold: a) provide evidence for a novel paradigm of microRNA targeting
in colon cancer cells; and b) demonstrate that non-coding RNAs can regulate important protein-
coding genes through interactions that obviate direct molecular contact (“decoying”).
Anticipated Duration of Project
1/1/2013 – 12/31/2015
Project Overview
Colorectal cancer (CRC) is the third most frequently diagnosed cancer in the United States with
~150,000 new cases and ~50,000 deaths expected in 2012. The objective of this project is to
show that long non-coding RNAs (lncRNAs) use endogenous microRNAs (miRNAs) to regulate
protein-coding messenger RNAs (mRNAs) that are expressed in CRC; also, that many of the
interactions between these miRNAs and their targets transcend what has been known as the
“standard model” of targeting, and instead comprise heteroduplexes with one or more guanine-
uracil (G:U) wobbles and mismatches in the miRNA’s “seed” region (“expanded model”).
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 47
Specific Aims:
Aim 1. Show that lncRNAs can alter the expression levels of mRNAs in colon cancer cells. For
two of the three lncRNAs that we have cloned (currently unpublished), we will show that
modulation of their expression can alter the expression of mRNAs in colon cancer cells. Using
commercial expression microarrays, we will identify those mRNAs that are affected following
the over-expression and silencing respectively of each of the two lncRNAs.
Aim 2. Show that the mRNAs of genes expressed in CRC are affected by a specific miR family,
and that many of the underlying interactions are not anticipated by the standard miRNA targeting
model. Using the results of Aim 1 and the rna22 target prediction tool, we will predict targets for
the specific miR-family, putting emphasis on heteroduplexes that are not expected by the
“standard” miRNA targeting model. These mRNAs may have known CRC relevance. Using
reporter assays with pre-miRNAs/scrambled-control, we will examine the validity of these novel
predictions.
Aim 3. Show that the expression of the two lncRNAs of interest is affected by a specific miR-
family of miRNAs. Using at least two miRNA target prediction tools that can process ncRNA
sequences, we will predict target sites in the two of three cloned lncRNAs as well as the
corresponding heteroduplexes for members of the specific miR- family of miRNAs. We will
again use reporter assays to demonstrate the validity of the predicted heteroduplexes.
Principal Investigator
Isidore Rigoutsos, PhD
Professor and Director, Computational Medicine Center
Thomas Jefferson University
125 S. 9th Street
Philadelphia, PA 19107
Other Participating Researchers
Yi Jing, MD, PhD; Phillipe Loher, MBA; Peter Clark, PhD – employed by Thomas Jefferson
University
Expected Research Outcomes and Benefits
This project will help advance our understanding of important regulatory interactions in CRC
biology. We expect to reveal facets of post-transcriptional regulation that involve currently
unsuspected players and novel interactions, and which could not be elucidated by classical
approaches. Preliminary work by us and others in different contexts has demonstrated that
lncRNAs can play important regulatory roles by modulating the expression of mRNAs through
competition for binding to endogenous miRNAs – such transcripts are known as ‘competing
endogenous RNAs’ (ceRNAs) and impact one another in the absence of direct molecular contact.
The two lncRNAs on which we focus are up-regulated in colon cancer cell lines compared to
normal samples and are predicted to be targeted extensively by multiple members of the specific
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 48
miR-family of miRNAs. In both cancer and normal samples, the specific miR-family members
are present and abundantly expressed. Moreover, our analysis of public CLIP-seq data, i.e. next
generation sequencing data of RNAs obtained from UV-crosslinking followed by
immunoprecipitation with an Argonaute (AGO) antibody, revealed many miRNA:mRNA
heteroduplexes involving these miR-family members: importantly for this project, the majority
of these heteroduplexes can be discerned only under the “expanded” miRNA targeting model.
Upon completion, we will have demonstrated the existence of lncRNAs that can regulate
mRNAs in the CRC context. Also, we will have established the importance of this miR-family
for CRC biology. Lastly, we will have shed light on what is now a lurking regulatory landscape
in CRC biology and involves both known (miRNAs and mRNAs) and novel (lncRNAs) players
that participate in unexpected interactions with one another.
Summary of Research Completed
MiRNAs are known to regulate the expression levels of many proteins by directly targeting the
corresponding mRNAs. More recently, miRNAs were also shown to regulate the expression
levels of long non-coding RNA transcripts, generically referred to as lncRNAs. We have
initiated experiments to test our hypothesis that in the CRC context changes in the expression
levels of specific lncRNAs can modulate the expression of specific protein-coding mRNAs in the
absence of direct molecular interactions. If present, these ‘decoying-driven’ interactions would
involve lncRNAs and mRNAs that are directly and simultaneously targeted by endogenous
miRNAs and be the result of competition for binding to these endogenous miRNAs.
Aim 1. Show that lncRNAs can alter the expression levels of messenger RNAs in colon cancer
cells. This aim is focused on determining whether the two lncRNAs of interest can modulate the
expression of mRNAs in CRC.
First we investigated whether we could extend our preliminary findings from colon cell lines to
actual metastatic colorectal cancer (mCRC) samples. To this end we profiled de-identified
samples from patients undergoing operations for metastatic colorectal cancer. The samples were
a kind gift by Dr. Jen Jen Yeh from the University of North Carolina at Chapel Hill (UNC) and
released by the Tissue Procurement Core Facility under a UNC IRB-approved protocol; the
samples were harvested after engraftment into athymic (nu/nu) mice. As can be seen from
Figure 1, pyk-reg-10 was present in 13 and pyk-reg-83 was present in 16 of the 21 tested
samples.
Further, we sought to answer the question of tissue specificity for the two lncRNAs of interest. In
particular, we sought to determine whether the two lncRNAs of interest are present in cell types
other than colon. To this end, we profiled them in five pancreas cell lines: HPNE, CaPan-2,
MiaPaCa, PL-5, and PL-45 and found both to be present in all five cell lines (Figure 2).
Additionally, we profiled the two lncRNAs in five breast and two prostate cell lines: pyk-reg-83
was found present in all seven cell lines whereas pyk-reg-10 was present in six of the seven (data
not shown).
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 49
In specific Aim 1, we proposed to design one or more siRNAs for the purpose of silencing pyk-
reg-10 and pyk-reg-83. Of these two lncRNAs, pyk-reg-83 is a) present in all of the tested cell
lines, b) present in the majority of the tested mCRC samples, and, c) consistently more abundant
than pyk-reg-10. We thus began our investigations by focusing on pyk-reg-83 and designed two
siRNAs against it. We have just completed testing the two siRNAs separately as well as in
combination using luciferase constructs and found them to be very effective (Figure 3). Next, we
will be testing the siRNA efficiency against the full length pyk-reg-83.
The specific family of miRNAs on which we focus is endogenous in both colon and pancreas
cells (normal and cancer) and so are the two lncRNAs of interest (see above). This co-
occurrence would suggest that any direct interactions between the miRNA family and pyk-reg-
10/pyk-reg-83 likely persist in both cell contexts. Given that we have just began the project, we
wanted to leverage miRNA-targeting information, which we have generated through a parallel
study on pancreatic cancer, in the context of Aims 2 and 3. The findings might help us discard
any non-promising candidates (target sites of interest, mRNAs of interest) and focus on
promising ones. To this end, we carried out these first experiments (Figure 3) with the two
pancreas cell lines HPNE and MiaPaCa. We are currently gearing up to assay the two siRNAs
we designed in colon cell lines.
Aim 2. Show that the mRNAs of genes expressed in CRC are affected by a specific miRNA
family and that many of the underlying interactions are not anticipated by the standard miRNA
targeting model. This aim is focused on demonstrating that the endogenous miRNA family on
which we have focused can regulate the abundance of mRNAs through interactions that are not
anticipated by the “standard” model of miRNA targeting.
We began with first addressing step 2 of Aim 2 as it could proceed independently. Therein, we
proposed to design and implement the next generation of the MuTaMe algorithm that we first
described in (Tay et al. Cell 2011). The original implementation that was used in this publication
was done in shell-script language, which does not render itself to speed and to the processing of
the larger datasets that will be used in this project. In light of this, during this reporting period,
we focused on re-implementing the originally described MuTaMe algorithm in the C
programming language. We have just completed the implementation and are currently in the last
stages of testing with specific test cases to ensure the accuracy of results. As intended, this
version of MuTaMe is significantly faster than the original and can handle much bigger datasets
of miRNA target predictions with great ease.
Aim 3. Show that the expression of the two lncRNAs of interest is affected by a specific miR-
family of miRNAs. This aim is focused on demonstrating that the endogenous family of
miRNAs on which we have focused can regulate the abundance of the pyk-reg-10 and pyk-reg-
83, just like they do with mRNAs. This is an interesting novel aspect of regulatory control by
miRNAs in that, among other things, the standard model does not anticipate miRNA targeting
beyond the confines of protein-coding mRNAs.
In the context of this aim, we first examined whether transfection with pre-miRNAs for several
of the miRNAs of the family of interest has any impact on the expression level of pyk-reg-83.
This would suggest the possibility of direct targeting by these miRNAs, a prerequisite for them
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 50
to control endogenous mRNAs through decoying. A first set of transfection experiments with
five of the miRNA family members (herein denoted as miR-a, -b, -c, -d and –e) at a
concentration of 25 nM indicated that overexpression of all five of them can induce a very
notable reduction to the level of pyk-reg-83 in HPNE cells (Figure 4). We are gearing up to
repeat the experiments and establish the level of statistical significance for these findings.
In parallel, through computational analysis of the sequence of pyk-reg-83, we have identified
two sequence segments, one near pyk-reg-83’s 5´ and the other near its 3´ end. According to our
analysis, each of these two segments is likely targeted by multiple members of the miRNA
family of interest and at nearby locations. We are in the process of constructing Renilla
luciferase assays in order to test whether the two segments are indeed targeted directly by these
miRNAs.
Figure 1. Investigating the presence of pyk-reg-10 and pyk-reg-83 in 21 metastatic
colorectal cancer samples.
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 51
Figure 2. Investigating the presence of pyk-
reg-10 and pyk-reg-83 in five pancreas cell
lines.
Figure 3. Impact of the two designed siRNAs on
the abundance of pyk-reg-83 in two different cell
lines. (* indicates a p-value that was ≤ 0.01)
Figure 4. Impact of transfection with pre-miRNAs for five
members of the miRNA family of interest on the abundance of
pyk-reg-83 (n=1).
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 52
Research Project 10: Project Title and Purpose
Mechanisms of Presynaptic Dysfunction of Dopaminergic Neurotransmission in Human LRRK2
(R1441G) Transgenic Mouse Model – The major goal of this project is to understand the role of
synaptic mitochondria in dopamine (DA) release and DAergic axon degeneration in LRRK2
transgenic mouse. We hypothesize that mutant LRRK2 impairs mitochondrial function at the
pre-synaptic terminals, which in turn diminishes local ATP synthesis or calcium buffering
required for normal DA release. We will utilize a combination of fast cyclic voltammetry
recording and two-photon imaging in living brain slices, and mouse genetics to uncover
mechanisms underlying DAergic transmission deficits in Parkinson’s (PD). These findings will
likely provide new insights into pathogenesis of PD and open new avenues for therapeutic
intervention.
Anticipated Duration of Project
1/1/2013 – 12/31/2016
Project Overview
Emerging evidence suggests that synaptic dysfunction of dopamine (DA) neurons is an early
event in the pathogenesis of Parkinson disease (PD) occurring prior to the onset of symptoms.
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent causes of
familial and sporadic PD, demonstrating an unprecedented significant role in PD pathogenesis.
Recently, a transgenic mouse model with over-expression of human LRRK2-R1441G has been
shown to recapitulate robust motor behavioral, neurochemical and pathological features of PD,
and we have found age-dependent deficits in DA release in the striatum in this model. Both
genetic and environmental causes of PD have highlighted the importance of mitochondrial
dysfunction in the pathogenesis of PD.
Essentially, synaptic mitochondria play a critical role in the function and organization of synaptic
vesicle pools and in neurotransmitter release. However, behavior of synaptic mitochondria in
mutant LRRK2 associated-PD has not been well studied. Therefore, it is important to understand
the role of synaptic mitochondria in DA release and DAergic axon degeneration in LRRK2
transgenic mice. We hypothesize that mutant LRRK2 impairs mitochondrial function at the pre-
synaptic terminals, which in turn diminishes local ATP synthesis or calcium buffering required
for normal DA release. We will utilize a combination of fast cyclic voltammetry recording and
two-photon imaging in living brain slices, and mouse genetics to uncover mechanisms
underlying DAergic transmission deficits in PD. These findings will likely provide new insights
into pathogenesis of PD and open new avenues for therapeutic intervention.
Specific Aims:
Aim 1. To study the defects in synaptic vesicle release of DAergic neurons and its association
with mitochondria.
Aim 2. To analyze synaptic mitochondrial function and its correlation with the synaptic
dysfunction in hLRRK2-R1441G transgenic mice.
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 53
Aim 3. To evaluate basal oxidation level using mito-roGFP measurement to see whether there is
a high basal oxidation in the hLRRK2-R1441G mice and whether this is age dependent.
Aim 4. To evaluate the mitochondrial distribution and trafficking within the axons of the
nigrostriatal projecting DAergic neurons.
Principal Investigator
Hui Zhang, PhD
Assistant Professor
Thomas Jefferson University
123 S. 9th Street
Philadelphia, PA 19107
Other Participating Researchers
None
Expected Research Outcomes and Benefits
LRRK2 mutations are the most common genetic cause of Parkinson’s disease. In this study, we
plan to determine whether LRRK2 mutation will cause dysfunction of presynaptic mitochondria,
which in turn diminishes local adenosine triphosphate (ATP) synthesis or calcium buffering
required for normal dopamine release. We will utilize a combination of fast cyclic voltammetry
recording and two-photon imaging in living brain slices as well mouse genetics to address these
questions. After the completion of this project, we will know whether there are presynaptic
mitochondria defects in the LRRK2-R1441G transgenic mice and whether this is one of the
underlying mechanisms leading to synaptic transmission deficits that we have observed. These
findings will likely provide new insights into pathogenesis of PD and help to develop early
therapies for preventing and/or delaying onset of symptoms.
Summary of Research Completed
Milestone(s) for 1/1/2013-6/30/2013:
Postdoc recruitment; finish 50% of Aim 1A; mouse crossing and establish the mouse line (pTH-
GFP/LRRK2) for Aim 2.
We have successfully finished the postdoc recruitment and the postdocs will start to work on the
project on July 01, 2013.
Aim 1. To study the defects in synaptic vesicle release of DAergic neuron and its association
with mitochondria.
We have completed 50% of Aim 1A, confirming the preliminary results as planned.
We use a paired pulse stimulation to characterize evoked DA release and recycling/refilling by
cyclic voltammetry (CV) and amperometry in acute corticostriatal slices. The paired pulse ratio
reflects the recycling /refilling of synaptic vesicles from a reserve pool to the releasable pool. We
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 54
found that mitochondria inhibitor, rotenone (1 µM, 1 hr) slows down the recovery of DA release
stimulated by paired stimuli at 5, 10 and 20 sec interval, mimicking the decreased vesicle
recycling/refilling in hLRRK2-R1441G TG mice shown in the preliminary results. Mitochondria
membrane potential depolarizing reagent carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 3
µM, 10 min) has similar effects. These results indicate that mitochondria dysfunction affects the
recycling /refilling of synaptic vesicles and the slower recovery of DA release in the mutant
could stem from mitochondria dysfunction.
We also examined the vulnerability of DA release to CCCP in 3 pairs of 5 month-old of
transgenic R1441G-LRRK2 and their WT littermates. Bath application of CCCP (3 µM, 20 min)
causes a more rapid decrease in evoked DA release and mass release of DA indicating the
“dying” of the DA terminals in the living brain slices. The onset of mass release of DA as
indicated by the shift up of the baseline is 600 ± 50 sec for WT and 386 ± 70 sec for TG
respectively (N=3, n=6, p<0.05).
Aim 2. To analyze synaptic mitochondrial function and its correlation with the synaptic
dysfunction in LRRK2-R1441G transgenic mice.
In order to use 2PLSM: TMRM mitochondrial imaging in living brain slices to examine how
mitochondrial function (membrane potential) is altered in the dopaminergic terminals in
hLRRK2-R1441G mice as specified in Aim 2A, we haves started to cross pTH-GFP mice with
LRRK2-R1441G mice to generate mice that have specific GFP labeling in the DA terminals. It is
now in the 3rd generation. We expect to have the 5th generation, which will be used for
experiments in Aim 2A, around December 2013.
We have started to measure the mitochondrial respiratory function, i.e., ATP level in the WT
and TG ahead of time. Preliminary result shows that there is a 30±1% decrease of ATP in the
striatal slices from LRRK2-R1441G mice compared to their WT littermates (2 pairs of mice at 5
month old).
General Methods
1. Slice preparation: Mice are decapitated without anesthesia. Coronal brain slices are cut on a
vibratome at a thickness of 300 µm and then separated at the midline to obtain two usable
hemislices. Slices are allowed to recover for 1.5 hr in a holding chamber in oxygenated artificial
cerebrospinal fluid (aSCF) at room temperature, and then placed in a recording chamber, and
super- fused (1.5ml/min) with aCSF (in mM: NaCl 125, KCl 2.5, NaHCO3 26, CaCl2 2.4,
MgSO4 1.3, KH2PO4 0.3, glucose 10) at 36 ºC.
2. Stimulation and recording: Striatal slices are electrically stimulated by an Iso-Flex stimulus
isolator triggered by a Master-8 pulse generator (A.M.P.I., Jerusalem, Israel) using a bipolar
stimulating electrode placed at ~100 µm distance from the recording electrode. Disk carbon fiber
electrodes of 5 µm diameter with a freshly cut surface are placed into the striatum about 50 µm
into the slice. For CV, a triangular voltage wave (-400 to +1000 mV at 300 V/s versus Ag/AgCl)
is applied to the electrode every 100 ms. Current is recorded with an Axopatch 200B amplifier
(Axon Instrument, Foster City, CA), with a low-pass Bessel Filter setting at 10 kHz, digitized at
20 kHz (ITC-18 board, Instrutech Corporation, Great Neck, New York). Triangular wave
_____________________________________________________________________________________________
Pennsylvania Department of Health – 2012-2013 Annual C.U.R.E. Report
Thomas Jefferson University - 2012 Formula Grant – Page 55
generation and data acquisition are controlled by a PC computer running a house-written IGOR
program. Background-subtracted cyclic voltammograms serve for electrode calibration and to
identify the released substance.
3. ATP assay was perfomed following the manual of commercil kit (K354-100, Biovision).
Briefly, one brain slice containing both stiatum and cortex (300μm, about 20mg) was
homogenized in 200μl ATP assay buffer, after centrifugation ice cold at 15,000g for 2 min, the
supernatant was deproteinized by perchloric acid method (K808-200, Biovision). Take 25μl of
the deproteinized supernatant from each sample to a 96-well plate which already contains 25μl
ATP assay buffer in the wells. Add 50μl ATP reaction mix (contains 2μl ATP probe, 2μl ATP
converter, and 2μl Developer mix in ATP assay buffer) per well and measure O.D. at 570nm
after 30 min incubation at room temperature protecting from light. Each sample was measured in
triplicate. The ATP levels were calculated based on the ATP standard curve done at the same
time, and then normalized to μg protein of the original lysates.
4. Data Analysis and Statistics: The two-tailed Student’s t-test is used for paired data and one-
way ANOVA followed by Student-Newmann-Keuls’s test is used for group’s data (GB Stat
software, Silverspring, MD). The critical level of significance is p<0.05 (*). Means will be
reported ±SEM. N: mouse number; n: experiment number.