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Dynamic and Nuclear expression of Pdgfrα and Igf1r in Alveolar Rhabdomyosarcoma

Running Title: Nuclear Receptor Tyrosine Kinases in Rhabdomyosarcoma

M. Imran Aslam1,2†#, Simone Hettmer3,4#, Jinu Abraham2, Dorian LaTocha1, Anuradha Soundararajan5,

Elaine T. Huang2, Martin W. Goros6, Joel E. Michalek4, Shuyu Wang7,

Atiya Mansoor8, Brian J. Druker1,9, Amy J. Wagers3*, Jeffrey W. Tyner1*, Charles Keller2*

1Knight Cancer Institute, Division of Hematology and Medical Oncology, Oregon Health & Science University, Portland, OR 97239 USA

2Pediatric Cancer Biology Program, Papé Family Pediatric Research Institute, Department of Pediatrics, Oregon Health & Science University, Portland, OR 97239 USA

5Greehey Children’s Cancer Research Institute and 6Department of Epidemiology & Biostatistics,

University of Texas Health Science Center, San Antonio, TX 78229 USA

3Howard Hughes Medical Institute, Department of Stem Cell and Regenerative Biology, Harvard

University, Harvard Stem Cell Institute, and Joslin Diabetes Center, 7 Divinity Ave., Cambridge, MA

02138 USA 4Department of Pediatric Oncology, Dana Farber Cancer Institute and Division of Pediatric Hematology/ Oncology, Children’s Hospital, 7Harvard University, Boston, MA 02115 USA

8Department of Pathology, Oregon Health & Science University, Portland, OR 97239 USA

9Howard Hughes Medical Institute, Portland, OR 97239 USA

†Howard Hughes Medical Institute Medical Research Fellows Program, 4000 Jones Bridge Rd., Chevy Chase, MD 20815 USA # contributed equally *Correspondence: Charles Keller, 3181 SW Sam Jackson Park Rd, MC-L321, Portland, OR 97239. Phone: (503) 494-1210, Fax (503) 418-5044, [email protected], and Jeffrey W. Tyner, 3181 SW Sam Jackson Park Rd., MC-L592, Portland, OR 97239. Phone: (503) 346-0603; Fax: (503) 494-3688, [email protected]; Amy J.Wagers, 7 Divinity Avenue, Cambridge, MA 02215, Phone: (617)309-2590, Fax: (617)309-2593, [email protected]

Key words: Pdgfrα, Igf1r, rhabdomyosarcoma, nucleus, receptor tyrosine kinase

Conflict of Interest Statement: The authors have no conflicts to declare related to these studies.

on April 10, 2020. © 2013 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2013; DOI: 10.1158/1541-7786.MCR-12-0598

http://mcr.aacrjournals.org/

1

Abstract

Since the advent of tyrosine kinase inhibitors as targeted therapies in cancer, several receptor tyrosine

kinases (RTKs) have been identified that are operationally important for disease progression.

Rhabdomyosarcoma (RMS) is an example of a malignancy in need of new treatment options, and a

better understanding of the heterogeneity of RTK expression could advance this goal. We investigated

populations of alveolar RMS (aRMS) tumor cells derived from a transgenic mouse model expressing

two such RTKs, Pdgfrα and Igf1r, by fluorescent activated cell sorting. Sorted subpopulations that were

positive or negative for Pdgfrα and Igf1r dynamically altered their RTK cell surface expression profile

as early as the first cell division. Interestingly, too, a difference in total Pdgfrα expression and nuclear

Igf1r expression was conserved in populations. Nuclear Igf1r expression was greater than cytoplasmic

Igf1r in cells with initially high cell surface Igf1r. Cells with high nuclear expression of Igf1r established

tumors most efficiently in vivo. RNAi-mediated silencing of Igf1r in the subpopulation of cells initially

harboring higher cell surface and total Igf1r resulted in a comparatively greater reduction in anchorage-

independent colony formation than sorted with initially lower cell surface and total Igf1r expression. In

accordance with our findings in murine aRMS, human aRMS were also found to express nuclear Igf1r.




2

Introduction

Rhabdomyosarcoma is the most frequent soft tissue sarcoma of childhood. Of the common subtypes

for this aggressive muscle cancer, patients with alveolar rhabdomyosarcoma (aRMS) have the most

dismal outcome despite maximal therapeutic intervention (1-3). We have developed and validated a

genetically-engineered, conditional mouse model of aRMS (4, 5) and therein identified the receptor

tyrosine kinase (RTK) targets Pdgfrα and Igf1r as potential therapeutic targets (6, 7). In agreement with

our mouse model studies, careful work by other groups has ascribed biological and clinical significance

to the expression of these and other RTKs for aRMS (8, 9).

In the current era of biomarker-stratified and personalized cancer therapy clinical trials, great

importance is being ascribed to biomarker expression from the solitary biopsy sample taken from each

patient at a single time point (10). However, intra- and inter-tumor heterogeneity for individual patients

is increasingly recognized (11). Several models developed to interpret the heterogeneity of individual

cells that compose a tumor have been proposed, with strong evidence that solid tumors consist of subsets

of cells distinct at the genetic and phenotypic level (11-15). However, the concept of isolating unique

populations of cells by expression of surface membrane proteins lends itself towards controversy

because cells reverting to a state reflecting the heterogeneity of the original tumor has been described as

well (16). Thus, the ability to profile cell populations using cell surface markers, particularly RTKs

expressed by only a subpopulation of tumor cells, might be informative if RTK expression is stable.

Otherwise, understanding mechanisms that dynamically modulate subcellular localization of RTKs is

necessary (17-23). In this regard, the mechanism of intracellular (nuclear) trafficking of RTKs has been

described with most detail for the EGFR (18, 20, 21). Other RTKs, such as FGFR, Igf1r, and ErbB4

have also been shown to localize to the cell nucleus in cancer (17, 19, 22).




3

Since Pdgfrα and Igf1r are reported to act in concert for resistance to Igf1r small molecule inhibition

in rhabdomyosarcoma (24), we sought to understand if tumor cell subpopulations can be subdivided into

distinct RTK profiles and if isolated cells behave as phenotypically static or dynamic populations.

Furthermore, we aimed to determine whether differential subcellular localization of these RTKs had a

biologically significant role in tumor establishment.




4

Materials and Methods

Cell Culture

Mouse tumor cell lines were established and cultured previously described (7). Cells were grown in

DMEM supplemented with 10% fetal bovine serum and penicillin (100 U/ml)/streptomycin (100μg/ml)

(Invitrogen, Carlsbad, CA) in 5% CO2 in air at 37°C.

Flow Cytometry

Murine aRMS cells were grown to ~50% confluency then trypsinized, washed with PBS and stained

with primary APC-conjugated antibody to Pdgfrα (1:40, eBiosciences, San Diego, CA) in FACS buffer

(2% FBS in PBS) on ice for 30 minutes, washed in FACS buffer, then stained with Igf1r (1:100,

ab32823, Abcam, Cambridge, MA) primary antibody for 30 minutes on ice. Cells were again washed in

FACS buffer, then stained with anti-chicken secondary antibody conjugated to Cy3.5 (1:80, ab97146,

Abcam) for 30 minutes on ice. Cells were washed in FACS buffer and resuspended in buffer and

analyzed by a BD FACSAria III Cell Sorter (BD Biosciences, San Jose, CA). As a control, cells were

stained with isotype antibody conjugated to APC at the same concentration as Pdgfrα antibody (#17-

1401-81, eBiosciences) and subsequently with secondary antibody conjugated to Cy3.5 only, at the

aforementioned concentration. Stained cells were gated according to control and sorted by quadrant for

Pdgfrα and Igf1r expression. Once cells were sorted, re-analysis by FACS was done to assess purity of

sorted populations before cells were recounted and allowed to recover in culture before further

experiments were performed. Analysis of .fcs files was done with FlowJo (Tree Star, Inc., PC version

7.6, Ashland, OR).

Immunoblotting and Immunoprecipitation

Cell lysates were prepared using Cell Lysis Buffer (#9803, Cell Signaling Technology, Danvers,

MA) supplemented with the appropriate protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis,




5

MI) and standard immunoblotting procedures were subsequently performed and proteins visualized with

chemiluminescent signal using Super Signal Western Pico or Dura Substrate (Pierce Biotechnology,

Rockford, IL). Mouse tumors were washed in PBS then lysed in RIPA buffer supplemented with

protease and phosphatase inhibitors. Lysates were homogenized and centrifuged at maximum speed for

10 minutes at 4°C and supernatant used for western blotting. Antibodies used for immunoblotting were

Pdgfrα (1:1000, #3164, Cell Signaling Technologies), Igf1rβ (1:1000, sc-713, Santa Cruz

Biotechnology, Santa Cruz, CA), p-Igf1rβ recognizing phospho-Tyr 1161 (1:1000, sc-101703, Santa

Cruz Biotechnology), phospho and total p42/44 MAP K from Cell Signaling Technology (used at

1:1000), β-actin (1:10,000, Sigma), α-tubulin (1:10,000, Sigma), and Sp1 (1:1000, Millipore, Billerica,

MA) and pan-Tyr antibody (1:10,000, 4G-10, Upstate Biotechnology). Immunoprecipitation was

performed by standard protocol using Protein A beads for rabbit IgG (Invitrogen).

Cytosolic and Nuclear fractionation

The nuclear fraction of cells was isolated by the following protocol: cells were lifted from a 100 mm

culture dish and washed with PBS once. Cell pellet was resuspended in 600 µl Buffer 1 (25 mM

HEPES, pH 7.9, 5 mM KCl, 0.5 mM MgCl2, 1 mM DTT, 100 mM PMSF in H2O) then 600 µl of Buffer

2 added (Buffer 1 + 1% Nonidet-P40), then centrifuged at 2600 rpm for 5 minutes. The supernatant

(cytoplasmic fraction) was transferred to a new tube. 100 µl of Buffer 3 (1:1 mix of Buffer 1 and 2) was

added to the cell pellet and gently mixed, centrifuged at 2600 rpm and supernatant added to cytoplasmic

fraction. Cell pellet was then washed 2-3x with Buffer 1 and supernatant discarded for purity of

subsequent soluble nuclear protein extraction. 500 µl of Buffer 4 (25 mM HEPES, pH 7.9, 360 mM

NaCl, 10% Dextrose, 0.05% Nonidet-P40) was added to cell pellet and tube rotated for 1 hour at 4°C.

Tube was centrifuged at 13200 rpm for 10 minutes and supernatant (soluble nuclear fraction) transferred

to a new tube. At this point both the cytoplasmic and nuclear fractions were clarified again by




6

centrifuged at 13200 rpm and transferring supernatant to a new tube. Protein was quantified using BCA

Protein Assay Reagent (Pierce) and immunoblotting performed as mentioned above.

RNA-interference mediated silencing of Igf1r and anchorage-independent colony formation assay

For RNAi studies, siRNA targeted to Igf1r (#SI01074017) and non-specific siRNA (#1027280) were

purchased from Qiagen (Valencia, CA). Experiments with siRNA were performed at 100 nM

concentration out using Lipofectamine 2000 in Opti-MEM Reduced Serum Media (Invitrogen). Cells

were transfected with non-specific siRNA or siRNA targeting Igf1r and on day 2, trypsinized and in

each well of a 6 well plate, 6,000 cells were suspended in 1.5 mL of 0.7% agarose (at 37°C) in DMEM

with 10% fetal calf serum. The cells in agarose were plated atop a 1.4% agarose layer and were allowed

to grow for 2 weeks before visualizing the colonies by light microscopy.

Immunohistochemistry

Human aRMS tissue microarrays were obtained from the COG Biorepository (TMA #3000-30-

P6897). Histology was performed using standard protocol. Briefly, formalin-fixed paraffin embedded

sections (FFPE) were dewaxed and dehydrated in xylene and graded alcohol concentrations. Heat

induced epitope retrieval (HIER) was utilized with sodium citrate buffer (pH 6.0) for 20 minutes, slides

blocked with 2% normal goat serum (NGS) and for endogenous biotin using an avidin/biotin blocking

kit (Vector Labs, Burlingame, CA). Primary antibody for Igf1r (Santa Cruz) was used at 1:50 overnight

in 2% NGS. VECTASTAINTM Elite ABC kit (Rabbit IgG) and ImmPACT DAB Peroxidase Substrate

kit was utilized according to manufacturer’s protocol to visualize staining (Vector Lab, Burlingame,

CA). Slides were counterstained with hematoxylin for 5 minutes, rinsed, dehydrated and mounted with

xylene based mounting medium.

Immunocytochemistry




7

Confocal microscopy was performed as follows: cells were allowed to recover post-sort in culture for

2-3 days then replated in 8 chamber slides (BD Biosciences) at low confluency and allowed to incubate

for 2-3 days. Cells were then fixed in 4% paraformaldehyde (PFA) in PBS at room temperature for 20

minutes. Slides were carefully washed with PBS 3x and incubated at room temperature in 5% normal

goat serum (Invitrogen) and 0.01% Triton-X in PBS for 1 hour to inhibit nonspecific binding of

antibodies. Cells were washed again and primary antibody diluted in 5% normal goat serum in PBS was

added overnight at 4°C. Alexafluor488-conjugated or Alexafluor594-conjugated anti-rabbit IgG

antibody (Invitrogen) at 1:200 was added and cells were incubated for 1 hour at room temperature.

Slides were mounted using the VECTASHIELDTM Mounting Medium with DAPI (Vector Laboratories)

and visualized with a Zeiss LSM 700 confocal microscope. Pdgfrα antibody was used at 1:100 (#3164,

Cell Signaling Technologies), Igf1rβ at 1:50 (sc-713, Santa Cruz Biotechnology). Images were taken at

40x and 63x oil immersion for further magnification.

In vivo studies

All animals were treated humanely and the experiments were conducted in accordance with the

Institutional Animal Care and Use Committee approved protocols. SCID/hairless/outbred (Crl:SHO-

PrkdcscidHrhr) mice purchased from Charles River at 8 weeks of age were injected with 1 x 105 U23674

cells into the gastrocnemius muscle that had been pre-injured 24 hours prior with 0.85 µg/mouse

cardiotoxin (from Naja mossambica, Sigma, St. Louis, MI). Tumor volumes (cm3) were measured 3-

dimensionally with electronic calipers and calculated from formula (π/6) x length x width x height,

assuming tumors to be spheroid. Once tumor volume reached 2 cm3, mice were euthanized and the

tumor was harvested at necropsy.




8

Results

Murine aRMS express Pdgfrα and Igf1r by FACS and can be isolated by cell sorting

The murine alveolar rhabdomyosarcoma (aRMS) primary cell culture, U23674, was derived from a

left upper extremity primary tumor that developed in a 46 day old mouse from our genetically-

engineered, conditional mouse model of aRMS harboring the Pax3:Fkhr oncogene and homozygous p53

deletion (4, 5). This lesion was histologically diagnosed as an aRMS tumor and utilized for these studies

at low passage number (p<10). Initially, U23674 cells were stained for Pdgfrα (APC) and Igf1r (Cy3.5)

and analyzed by fluorescent activated cell sorting (FACS), indicating ~35% of cells express both Pdgfrα

and Igf1r, but ~15% of cells express either receptor exclusively (Figure 1A). However, tumor cells

displayed a continuum of expression of Pdgfrα and Igf1r rather than neatly distinct populations

expressing either RTK individually or together. Similar results were seen in 2 other murine aRMS cell

lines analyzed by flow cytometry for cell surface expression of Pdgfrα and Igf1r (Supplemental Figure

S1).

Next, after U23674 cells were stained for Pdgfrα and Igf1r and cells were sorted by expression of

each receptor into 4 populations. Gates were determined using control cells stained with isotype control

antibody in addition to a Cy3.5 conjugated secondary antibody as a control. Cells positive or negative

for Pdgfrα or Igf1R by the assigned gates were designated as Pdgfrαhi/Igf1rhi or Pdgfrαlo/Igf1rlo,

respectively (Figure 1B). For instance, cells sorted for high Igf1r expression, but negative for Pdgfrα

were denoted as PdgfrαloIgf1rhi. Immediately following sorting, cells were re-analyzed by flow

cytometry to assess purity of the sort (Figure 1B). Figure 1C shows distinct morphology of cells grown

in culture for 7 days post-sort. Igf1rhi expression, but not Pdgfrαhi expression, was associated with

spindle morphology.

Pdgfrα and Igf1r cell surface expression is dynamic




9

Once U23674 cells were sorted by Pdgfrα and Igf1r expression, cells were allowed to recover post-

sort in culture and reanalyzed by flow cytometry for surface expression of both RTKs at serial time

points. FACS analysis revealed that cell surface expression of both Pdgfrα and Igf1r varied dramatically

as cells were grown in culture post-sort. Two such time points are shown in Figure 2A at 2 days and 14

days post-sort, compared to day 0 (day 7 was similar to day 14, Supplemental Figure S2). These results

illustrated an initial dramatic change in expression of Pdgfrα and Igf1r post-sort within a 48-hour time

period, tending towards a pre-sort profile. With the observation that cell surface expression profile

shifted considerably within a short period of time, we sought to understand if altered receptor expression

was a function of many or few rounds of replication. Thus, after a purity check was performed for sorted

populations post-sort, cells were recounted and plated at a known cell number. Exactly 48 hours after the

initial sort, cells were re-counted and FACS analysis for Pdgfrα and Igf1r expression was done. The

number of cells comprising each of the four populations was calculated from the FACS profile of the

initial sort and 48 hour post sort (Figure 2A). Doubling times were on the order of 30-42 hours

(Supplemental Table S1); therefore, dynamic alterations of Pdgfrα and/or Igf1r cell surface expression

appear to start after 0-1 cell divisions.

Total expression of Pdgfrα and Igf1r by immunoblot in sorted cells differ from cell surface

expression profile by FACS

To determine if total expression of either Pdgfrα or Igf1r in the 4 sorted cell populations reflected the

surface expression profile of each RTK by FACS, we performed immunoblot on cells grown in culture

for 7 days post-sort. Surprisingly, total cellular Pdgfrα expression appeared to vary dramatically among

the sorted populations (Figure 3A). Higher Pdgfrα expression at day 7 correlated with the populations

which were sorted for high Pdgfrα expression by FACS initially. Conversely, total Igf1r expression by

immunoblot did not show a dramatic difference in expression among sorted populations (Figure 3A). In

terms of activation, phosphorylation of Pdgfrα was elevated in the PdgfrαhiIgf1rlo and PdgfrαhiIgf1rhi




10

populations (Figure 3A). Despite lack of differential expression for total Igf1r, Igf1r phosphorylation

was increased in the PdgfrαhiIgf1rhi populations as well as (modestly) in the PdgfrαloIgf1rhi population.

Because we observed activated receptor levels varied across sorted populations, we looked at activation

of common cell survival and proliferation pathways downstream of both Pdgfrα and Igf1r such as Akt

and Erk 1/2, respectively. Although we did not see a difference in Akt phosphorylation levels between

the sorted cells (data not shown), Erk1/2 phosphorylation levels were remarkably elevated in the

PdgfrαhiIgf1rhi population and conversely decreased in the PdgfrαloIgf1rlo population; p-Erk 1/2 levels

appeared to correlate with levels of phosphorylated Pdgfrα and Igf1r receptor.

Thus far, our results indicated that Pdgfrα and Igf1r in tumor cells are expressed in a dynamic manner

with respect to surface expression profile by FACS, as well as total protein expression by immunoblot.

However, the dramatic difference in total Pdgfrα expression (Figure 3A), for example, did not reflect the

percentage of cells positive for Pdgfrα by FACS analysis at the same time point (day 7; Figure 2A).

Therefore, to determine if the amount of Pdgfrα or Igf1r expressed on the surface membrane of the

sorted cells was reflective of total expression of each receptor, we looked at mean fluorescence intensity

(MFI) of cells stained for Pdgfrα and Igf1r at day 7. Figure 3B shows MFI of both RTKs in each of the

four sorted populations. The MFI values indicated cells sorted initially for Pdgfrα or Igf1r displayed a

higher cell surface expression of each receptor 1 week post-sort. Taken together, these results

demonstrated that surface expression did not completely correlate with total expression level of either

receptor, indicating, among other possibilities, differential subcellular localization of these RTKs.

Total Pdgfrα and Igf1r expression in vivo reflects in vitro behavior of tumor cells and Igf1rhi sorted

cells establish tumors in vivo earlier than Igf1rlo cells

To determine if tumor cells retained an expression profile of Pdgfrα and Igf1r in vivo reflective of in

vitro behavior (Figure 3A), we utilized an orthotopic allograft in vivo model of aRMS and implanted




11

cells immediately post-sort into the right gastrocnemius muscle of immunocompromised mice.

Immunoblot of tumors formed in mice showed that Pdgfrα and Igf1r expression of subpopulations

(Figure 4A) mirrored the expression of both receptors in vitro (Figure 3A). Leg measurements taken

daily to monitor tumor growth revealed the PdgfrαloIgf1rhi and PdgfrαhiIgf1rhi sorted cells more quickly

established tumors in mice compared to PdgfrαhiIgf1rlo and PdgfrαloIgf1rlo cells (Figure 4B, 4C). The

control group of mice implanted with unsorted U23674 murine aRMS cells established tumors with the

same latency as PdgfrαloIgf1rlo sorted cells (Figure 4D).

To confirm the results that Igf1rhi sorted cells establish tumors in vivo earlier than Igf1rlo cells, we

sorted the independent murine aRMS primary cell culture U21459 into Igf1rhi and Igf1rlo cells and

implanted the cells immediately post-sort in recipient NOD.SCID host mice. Compared to Igf1rlo cells,

Igf1rhi cells sorted from U21459 engrafted with decreased latency and led to shorter survival

(Supplemental Figure S3A-C).

Pdgfrα and Igf1r demonstrate nuclear localization in Pdgfrαhi and Igf1rhi sorted cells and nuclear

Igf1r is associated with increased anchorage-independent colony formation ability

Based on the observation that cells sorted for either Pdgfrα or Igf1r exhibited an apparent

discrepancy between cell surface and total expression of each receptor, we hypothesized that differential

cellular localization of Pdgfrα and Igf1r was the etiology for these disparate results. We first performed

confocal microscopy on sorted cells looking at Pdgfrα and Igf1r localization. Interestingly, we observed

that in PdgfrαhiIgf1rhi and PdgfrαhiIgf1rlo cells, Pdgfrα was predominantly localized to the nucleus, while

chiefly cytosolic or membranous in PdgfrαloIgf1rhi and PdgfrαloIgf1rlo cells (Figure 5, Supplemental

Figure S3). Similarly, in PdgfrαloIgf1rhi and PdgfrαhiIgf1rhi cells, Igf1r appeared to be principally

localized to the nucleus, while more abundant in the cytosolic compartment/cell membrane of

PdgfrαhiIgf1rlo and PdgfrαloIgf1rlo cells (Figure 5, Supplemental Figure S4). In the biologically-




12

independent U21459 cell culture, immunocytochemistry also revealed that Igf1r was commonly

localized to the nucleus in Igf1rhi cells and Igf1rlo cells, respectively (Supplemental Figure S3D). These

results subsequently led us to question whether the ability of Igf1rhi cells to establish tumors earlier in

vivo compared to Igf1rlo cells was due to higher nuclear expression of Igf1r in these cells. Focusing our

efforts on Igf1r because of the orthotopic allograft results, we next confirmed whether Igf1r was indeed

expressed at higher levels in the nucleus in Igf1rhi cells, compared to Igf1rlo cells, by separating cytosolic

and nuclear proteins of all 4 sorted populations 7 days following isolation of cells by FACS. We

subsequently immunoblotted for Igf1r, using Sp1 and α-tubulin as confirmation of enrichment of nuclear

and cytosolic cell fractions, respectively (Figure 6A). These results confirmed a higher nuclear

expression of Igf1r in Igf1rhi cells compared to Igf1rlo cells.

To confirm our hypothesis that nuclear expression of Igf1r contributed to the ability of tumor cells to

establish more aggressive tumors in vivo, we used RNA-interference (RNAi) to silence Igf1r in

PdgfrαhiIgf1rhi and PdgfrαloIgf1rlo cells. We then assessed the effect of Igf1r knockdown on these cells’

ability to form colonies in an anchorage-independent colony formation assay (Figure 6B). Knockdown

efficiencies of Igf1r in both sorted cells are shown (Figure 6B). Colony formation ability of

PdgfrαhiIgf1rhi was decreased ~45% compared to control (non-specific siRNA) when Igf1r was silenced

by RNAi, whereas PdgfrαloIgf1rlo cells demonstrate only a ~27% reduction in colony formation ability

with Igf1r knockdown. Similarly, studies with the nuclear Igf1r-expressing murine aRMS primary cell

culture U21459 showed that 3 independent siRNA for Igf1r suppressed colony formation in soft agar

(Supplemental Figure S3E). Taken together, these results indicate that nuclear expression of Igf1r

confers an increased tumorigenic phenotype to murine aRMS cells.

Normal human tissue expresses cytosolic Igf1r whereas human aRMS has high frequency of

nuclear Igf1r by immunohistochemistry




13

To determine whether nuclear Igf1r occurs in human aRMS, we performed immunohistochemistry

for Igf1r on a human aRMS tumor microarray with 31 sections of 19 unique cases. We observed that

16/19 (~84%) specimens had nuclear expression of Igf1r (>10% nuclei of section positive). Tumor

sections demonstrated either cytosolic (Figure 7A), or predominantly nuclear staining of Igf1r (Figure

7B). Normal human tissue (pancreatic and skeletal muscle) was used as positive controls for Igf1r, and

showed only cytosolic staining of Igf1r; no nuclear Igf1r was seen in these sections (data not shown).

Details of sample scores are given in Supplemental Table 2.




14

Discussion

In this report, we describe that the receptor tyrosine kinases (RTK) Pdgfrα and Igf1r exhibit a

dynamic cell surface expression profile in murine aRMS cells. While we were able to isolate relatively

pure populations of cells expressing high or low Pdgfrα and/or Igf1r, all sorted populations rapidly and

dynamically alter their cell surface expression profile before the second cell division. Kept isolated in

culture, sorted populations eventually equilibrated their cell surface expression profile towards a pre-sort

profile. In the context of previous studies illustrating the phenotypically dynamic state of cancer cells,

including stem and non stem-cancer cells (16, 25), our study is a key next step into understanding the

complexity and potential pitfalls of assigning therapy based on surface expression profiles or other static

biomarkers. Previously, several studies have utilized cell surface markers (e.g., CD133, CD44, CD24,

etc) as stable markers for a specific cell population (12, 15, 26). RTKs, which are known to undergo

ligand-dependent subcellular trafficking and recycling (18, 27, 28), may however be particularly

undesirable as cell surface biomarkers. Nevertheless, our results then pointed us to differences in Pdgfrα

and Igf1r subcellular localization that paralleled membrane expression. Igf1r nuclear localization, in

particular, exhibited an important functional effect in contributing towards a more aggressive tumor

phenotype in vivo (summarized in Supplemental Figure S5).

Nuclear localization of RTKs or their substrates is an interesting phenomenon which has been

described in select early reports (19-22, 29). Igf1r had previously been shown to be a poor prognostic

indicator in clear cell renal cell carcinoma (30), and a new study was able to further show that among

renal tumors expressing Igf1r, intense or widespread nuclear expression by immunohistochemistry

portends a decrease in survival probability (17). Interestingly, nuclear localization of the Igf1r

holoreceptor was dependent upon tyrosine phosphorylation of putative residues on the Igf1rβ subunit of

the receptor (31). In our sorted populations, the Igf1rhi cells which displayed a higher nuclear expression

of Igf1r also had increased phosphor-Igf1rβ. A typical biological response when an RTK is stimulated




15

by its respective ligand is receptor internalization and subsequent degradation (31). However,

phosphorylation of Igf1rβ is one of two necessary post-translation modifications of the receptor required

for nuclear trafficking. Sumoylation of K1025, K1100, and K1120 residues in the kinase domain of

Igf1rβ are also critical to nuclear import of the holoreceptor (32). Further investigation has shown that

nuclear Igf1r originates from surface Igf1r only (33). Of note, we used an antibody to sort cells based on

surface Igf1r expression (the α subunit of Igf1r) and a different antibody to detect Igf1rβ at the nuclear

fraction by immunoblot, but given the covalent assembly of α and β subunits in the mature receptor we

do not expect this to have altered our results. Another intriguing aspect of our studies was that nuclear

Pdgfrα did not appear to have an adverse functional role with respect to tumor establishment in vivo.

While we were able to identify a difference in receptor localization among the sorted populations of

murine aRMS, we did not observe a differential sensitivity of these cells to small molecule inhibitors

which target Pdgfrα or Igf1r (data not shown). An interesting question these results raise is if small

molecule inhibitors designed to inhibit kinase activity of RTKs are able to inhibit the function of these

RTKs when localized to the nucleus. Understanding if kinase activity contributes to the role these

receptors play in the nucleus would be one step towards answering this question. Presumably, small

molecule inhibitors would have an advantage over neutralizing antibodies in this respect. Prior studies

have already shown that certain RTKs, such as EGFR, interact with DNA-binding proteins to directly

regulate the transcription of putative gene targets (34). The exact role of Igf1r in the nucleus of aRMS

will be an active area of further investigation, particularly given the potential translation from mouse to

human and the expression of nuclear IGF1R in clinical formalin-fixed paraffin embedded (FFPE)

samples. The mechanism by which nuclear Igf1r contributes to the establishment of new (metastatic)

tumors will be a key subject of further study and perhaps an operative step towards a temporal

biomarker-driven and personalized medicine approach in cancer therapy with regards to Igf1r inhibition.




16

Acknowledgments

MIA is a Howard Hughes Medical Institute Medical Research Fellow. This work was supported by

5R01CA133229-05 and -06 to CK. These studies were supported in part from a gift from the Ethan

Jostad Foundation for Childhood Cancer. Pilot studies leading to this work were supported by an

innovation award from Alex’s Lemonade Stand. BJD is an investigator of the Howard Hughes Medical

Institute. JWT is supported by grants from the William Lawrence and Blanche Hughes Foundation,

Leukemia & Lymphoma Society, and the National Cancer Institute. AJW is an Early Career Scientist of

the Howard Hughes Medical Institute.

Conflict of Interest Statement:

The authors have no conflicts to declare related to these studies.




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Figure Legends

Figure 1. Murine aRMS isolated by Pdgfrα and Igf1r expression. (A) U23674 (murine aRMS) cell expression profile by FACS of Pdgfrα (APC) and Igf1r (Cy3.5) gated with respect to isotype and secondary only control. (B) Purity of isolated cells by FACS shown. Isolated populations according to cells positive and negative for each receptor designated as hi and lo, respectively. (C) Phase contrast microscopy of sorted populations demonstrating differential morphology of cells grown in culture 7 days post-sort. Scale bar: 200 µm.

Figure 2. Murine aRMS demonstrate dynamic expression of Pdgfrα and Igf1r by FACS in a temporal manner. (A) FACS profile of Pdgfrα and Igf1r expression in U23674 cells at day of sort (Day 0), and 2 and 14 days post sort. Day 7 was similar to Day 14.

Figure 3. Functional Pdgfrα and Igf1r expression post-sort. (A) Immunoblot of sorted populations grown in culture for 1 week post-sort, demonstrating phosphorylated as well as total Pdgfrα, Igf1r and Erk 1/2. (B) Representative data showing mean fluorescence intensity (MFI) of U23674 sorted cells 7 days post sort indicating surface expression of Pdgfrα and Igf1r.

Figure 4. Cells sorted for Igf1r establish more aggressive tumors in vivo. (A) Tumors established from murine aRMS cells sorted for Pdgfrα and Igf1r show a relative dynamic and static expression of each receptor, respectively, reflective of cells in vitro. (B) PdgfrαhiIgf1rlo sorted cells demonstrate a significantly slower growth rate in an in vivo orthotopic allograft mouse model of aRMS compared to PdgfrαloIgf1rhi sorted cells. (C) PdgfrαhiIgf1rhi sorted cells grow at a significantly faster growth rate in an in vivo orthotopic allograft mouse model of aRMS compared to PdgfrαloIgf1rlo sorted cells. (D) Tumor establishment or growth rate of unsorted U23674 cells in vivo do not differ from PdgfrαloIgf1rlo sorted cells.

Figure 5. Nuclear localization of Pdgfrα and Igf1r correlates with cells sorted by FACS for surface expression of the respective receptor. (A) Representative confocal images of U23674 sorted populations showing localization of Pdgfrα and Igf1r (40x) with inset box enlarging image of a single cell to appreciate localization. Scale bar: 100 µm. Larger magnifications of insets are given in Supplemental Figure S3.

Figure 6. Igf1r is present in soluble nuclear fraction of sorted cells and is associated with anchorage-independent colony formation ability. (A) Cytosolic and nuclear fractions of sorted U23674 cells show cells sorted by Igf1rhi express higher levels of nuclear Igf1r. (B) Cells with higher nuclear Igf1r, show a significantly further decrease in colony formation ability compared to control (non-specific siRNA) when Igf1r is silenced by siRNA. * indicated p <0.05 compared to non-specific siRNA by student’s t-test.

Figure 7. IGF1R localization in human aRMS. (A) Human aRMS sections with chiefly cytosolic IGF1R expression. (B) Images of aRMS sections demonstrating both cytosolic (left) and nuclear (right) IGF1R expression. Scale bar: 100 μM.




Published OnlineFirst August 8, 2013.Mol Cancer Res M. Imran Alsam, Simone Hettmer, Jinu Abraham, et al. Rhabdomyosarcoma

and Igf1r in AlveolarαDynamic and Nuclear expression of Pdgfr

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