HSP27-mediated Extracellular and Intracellular Signaling Pathways Synergistically
Confer Chemo-Resistance in Squamous Cell Carcinoma of Tongue
Guopei Zheng*1, Zhijie Zhang*1, Hao Liu1, Yan Xiong2, Liyun Luo1, Xiaoting Jia1,
Cong Peng1, Qiong Zhang1, Nan Li1, Yixue Gu1, Minying Lu1, Ying Song1, Hao Pan3,
Jinbao Liu4, Wanqing Liu5, Zhimin He1
1Affiliated Cancer Hospital & Institute of Guangzhou Medical University; Guangzhou Key Laboratory
of "Translational Medicine on Malignant Tumor Treatment”, Hengzhigang Road 78#, Guangzhou
510095, Guangdong, China.
2Guangzhou Institute of Snake Venom Research, School of Pharmaceutical Sciences; 4Protein
Modification and Degradation Lab, Department of Pathophysiology, Guangzhou Medical University,
Guangzhou, Guangdong 510182, China
3Department of Oral & Maxillofacial Surgery, Xiangya Stomatological Hospital & School of
Stomatology, Central South University, Changsha 410078, Hunan, China
5Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health
Sciences; Department of Pharmacology, School of Medicine; Barbara Ann Karmanos Cancer Institute,
Wayne State University, Detroit, MI 48201, USA
* Equal Contributors
Corresponding Authors: Wanqing Liu, PhD., Department of Pharmaceutical Sciences,
Eugene Applebaum College of Pharmacy and Health Sciences; Department of
Pharmacology, School of Medicine; Barbara Ann Karmanos Cancer Institute, Wayne State
University, Wayne State University, Detroit, MI 48201, USA. Tel: 1-313-577-3375; E-mail:
Zhimin He MD., PhD., Affiliated Cancer Hospital & Institute of Guangzhou Medical
University; Guangzhou Key Laboratory of "Translational Medicine on Malignant Tumor
Treatment”, Hengzhigang Road 78#, Guangzhou 510095, Guangdong, China. Tel:
86-020-83492353; E-mail: [email protected].
Running title: Extra-and intra-cellular HSP27 in chemo-resistance
Keywords: tongue cancer, chemo-resistance, HSP27, NF-κB, apoptosis
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Translational Relevance
As an important type of head and neck cancer, squamous cell carcinoma of tongue (SCCT)
has generally poor response to chemotherapy with unclear mechanisms. In this study,
HSP27 was identified to play a critical role in the chemo-resistance of SCCT cells. Via in
vitro, in vivo as well as human studies, it was found that extracellular HSP27 can
maintain SCCT cell survival via interacting with TLR5 to activate NF-κB signaling. On the
other hand, intracellular HSP27 binds to BAX and BIM to inhibit the mitochondrial
apoptotic pathway. In SCCT patients, HSP27 protein level in both serum and SCCT tissues
represents a potential biomarker to predict the response to chemotherapy. This study
suggests that HSP27 can be a target for effective therapeutic intervention to enhance
the efficacy of chemotherapy against SCCTs.
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ABSTRACT
Purpose: Squamous cell carcinoma of tongue (SCCT) is the most common type of the
oral cavity carcinoma. Chemo-resistance in SCCT is common and the underlying
mechanism remains largely unknown. We aim to identify key molecules and signaling
pathways mediating chemo-resistance in SCCT.
Experimental Design: Using a proteomic approach we identify that the HSP27 was a
potential mediator for chemo-resistance in SCCT cells. To further validate this role of
HSP27, we performed various mechanistic studies using in vitro and in vivo models as
well as serum and tissue samples of SCCT patients.
Results: The HSP27 protein level was significantly increased in the multidrug-resistant
SCCT cells and cell culture medium. Both HSP27 knockdown and anti-HSP27 antibody
treatment reversed chemo-resistance. Inversely, both HSP27 overexpression and
recombinant human HSP27 protein treatment enhanced chemo-resistance. Moreover,
chemotherapy significantly induced HSP27 protein expression in both SCCT cells and
their culture medium, so is that in tumor tissues and serum of SCCT patients. HSP27
overexpression predicts a poor outcome of SCCT patients receiving chemotherapy.
Mechanically, extracellular HSP27 binds to TLR5 and then activates NF-κB signaling to
maintain SCCT cells survival. TLR5 knockdown or restored IκBα protein level disrupts
extracellular HSP27-induced NF-κB transactivation and chemo-resistance. Moreover,
Intracellular HSP27 binds to BAX and BIM to repress their translocation to
mitochondrion and subsequent cytochrome C release upon chemotherapy, resulting into
inhibition of the mitochondrial apoptotic pathway.
Conclusions: HSP27 plays pivotal role in chemo-resistance of SCCT cells via a synergistic
extracellular and intracellular signaling. HSP27 may represent a potential biomarker and
therapeutic target for precision SCCT treatment.
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INTRODUCTION
Oral cavity carcinoma (OCC) is a major cause of morbidity and mortality in patients with
head and neck cancer (1). Despite the progress of treatment options, the outcomes
remain poor in patients with advanced oral cavity carcinoma (2). Squamous cell
carcinoma of tongue (SCCT) is the most common type of the oral cavity carcinoma. In
the United States, it has been estimated approximately 12,060 new cases and 2030
deaths from SCCT in 2011 (3). The majority of patients with SCCT are treated with
surgery, radiotherapy, and chemotherapy. In China, Pingyangmycin (PYM)- and/or
Cisplatin (cDDP)-based chemotherapy demonstrates favorable outcomes, but often
chemo-resistance develops later on and results in failure of this therapy. Both basic
researches and clinical studies demonstrate that chemo-insensitivity of SCCTs are
correlated with more aggressive cancer behavior and a worse clinical outcome (4). Thus,
there is an urgent need to fully understand the molecular mechanisms for
chemo-resistance of SCCT cells, and to identify new therapeutic targets to improve the
efficacy of chemotherapy.
Several mechanisms that mediate chemo-resistance in cancer have been reported,
including activation of antiapoptotic cellular defense, increased DNA repair, and
induction of drug-detoxifying mechanisms (5). However, there are limited reports
regarding the mechanisms for tongue cancer chemo-resistance (6-8). The precise
mechanisms of chemo-resistance in SCCT remain elusive. In this study, we compared the
protein expression profiles between established multidrug-resistant (MDR) SCCT cell line
SCC-15/PYM and its parental cell line SCC-15, and identified that increased HSP27 level
contributes to chemo-resistance and predicts poor clinical outcome. Moreover, we
demonstrated that both extracellular and intracellular HSP27 synergistically confers
chemo-resistance via enhancing TLR5/NF-κB signaling and interacting with BAX and BIM
to inhibit the mitochondrial apoptotic pathway, respectively.
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MATERIALS AND METHODS
Ethical statement
This study was reviewed and approved by the Ethnics Committees of the Central South
University and Affiliated Xiangya Stomatological Hospital (Changsha, Hunan, China), and
Guangzhou Medical University and Affiliated Cancer Hospital (Guangzhou, Guangdong,
China). The study was conducted in accordance with the Declaration of Helsinki.
Cell culture, transfection and tissue samples
The human SCCT cell lines SCC-15, SCC-25, SCC-9 and CAL-27 were obtained
commercially from American Type Culture Collection at the beginning of this study. The
cell lines were last authenticated by short tandem repeat DNA fingerprinting on May 28,
2014. Cells were cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, USA)
supplemented with 10% fetal bovine serum (Gibco), and incubated at 37°C in a
humidified incubator containing 5% CO2. The stable MDR-SCCT cell line SCC-15/PYM was
established in our lab. To maintain the chemo-resistance phenotype, SCC-15/PYM cells
were cultured in the medium with 0.5mg/L PYM. For establishing stable transfectants
with knockdown or overexpression, cell lines were transfected with psi-LVRU6GP vectors
with HSP27 shRNAs (target sequence for sh-1#: 5'-CCTCAAACGGGTCATTGCCATTAAT-3',
sh-2#: 5'-CATTGCCATTAATAGAGACCTCAAA-3', sh-3#:
5'-GCGTGTCCCTGGATGTCAACCACTT-3', sh-4#:5'-TTCCGCGACTGGTACCCGCAT-3' or
pLEX-MCS vectors with HSP27 overexpressing constructs, and stable clones were
selected using puromycin. In some experiments, related cells were cultured in medium
with 10μg/ml anti-HSP27 antibody (Enzo Life Sciences, Farmingdale, NY, USA ) or 1μg/ml
recombinant human protein (rhHSP27) (R&D Systems, Minneapolis, MN, USA) excepting
special mention. Primary tongue cancer tissue samples were obtained from 84 patients
at the Affiliated Caner Hospital of Guangzhou Medical University, and 81 patients at the
Xiangya Stomatological Hospital & School of Stomatology, Central South University. All
samples were collected with informed consent from patients and all related procedures
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were performed with the approval of the internal review and ethics boards of the two
hospitals.
Comparative proteomic analysis
To compare different protein expression profiles between the drug-resistant
SCC-15/PYM cells and SCC-15 cells, we first did two-dimensional gel electrophoresis
analysis of total lysates prepared from these two cell lines. Three well-reproducible 2-DE
gels were performed. Following coomassie blue staining, the gels were subjected to
scan with MagicScan software on Imagescanner, and PDQuest analysis software was
used for spot intensity calibration. Protein spots were subjected to MALDI-TOF-MS
analysis and ESI-Q-MS analysis to identify differential proteins. Protein spots that had
consistent differences (>2 fold) between the two cell lines in triplicate experiments were
chosen as differential protein spots.
Caspase-3 activity assay
Caspase-3 activity was measured by caspase-3 activity kit (Beyotime, Shanghai, China)
according to the manufacturer’s instruction. To evaluate the activity of caspase-3, cell
lysates were prepared after related treatments. Assays were performed on 96-well
plates by incubating 10 μL of cell lysate per sample in 80 μL of reaction buffer and 10 μL
of caspase 3 substrate (Ac-DEVD-pNA, 2 mm). After incubation at 37 °C for 4 h, the
absorbance at 405 nm was recorded for each well on the BioTek Synergy 2, and the
relative caspase-3 activity was calculated.
Xenograft model in athymic mice
Xenograft tumors were generated by subcutaneous injection of related cell lines at 1x106
cells in 200 μl, respectively, into the armpit of 4-6 week-old female Balb/C athymic nude
mice. All mice were housed and maintained under specific pathogen-free conditions at
27˚C with 12:12 h light:dark cycle and fed with sterilized food and water, and all animal
experiments were approved by the Experimental Animal Ethics Committee of
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Guangzhou medical University and performed in accordance with institutional guidelines.
15 days later, the mice were grouped randomly and injected intraperitoneally with PYM
(30mg/kg) or Cisplatin (cis-diamminedichloridoplatinum, cDDP) (3mg/kg). The treatment
was administered every 3 days for ten cycles. Tumor growth was examined every 3 days
during the animal experiment. The mice were sacrificed with 120 μl 10% hydral
(Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) at the experimental endpoint
and the tumors were harvested and weighed.
MTS assay, Hoechst staining, Western blot, ELISA assay, Immunohistochemistry, NF-κB
activity assay, Real-time PCR, Co-immunoprecipitation and Immunofluorescence
The methods used are described in the Supplemental Experimental Procedures.
Statistical Analysis
Statistical analyses were performed using SPSS version 16.0 (SPSS, Chicago, IL) and
GraphPad Prism 6 (La Jolla, CA). Data are presented as mean ± SD. The difference
between two groups for statistical significance was analyzed using Student’s t test. To
compare multiple groups, One-way ANOVA analysis was used. Pearson correlation
analysis was performed to determine the correlation between two variables. Pearson’s
Chi-square test was used to analyze the clinical variables. Survival curves were plotted
using the Kaplan-Meier method and compared using the log-rank test. A P value < 0.05
was considered statistically significant.
RESULTS
Construction of the chemo-resistant tongue cancer cell line
In order to investigate the mechanisms for chemo-resistance in tongue cancer, we
established a multidrug-resistant human SCCT cell line SCC-15/PYM derived from SCC-15
by pingyangmycin (PYM) induction. Here, we firstly confirmed the chemo-resistant
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characteristics of SCC-15/PYM cells. After treatment with different concentrations of
PYM or cDDP, the dose-response curves for cells were plotted and IC50 values were
calculated. SCC-15/PYM cells were significantly more resistant to chemotherapy induced
cytotoxicity as compared to the parental cells (Fig.1A and Fig.S1A). Because of the wide
use of PYM and cDDP in chemotherapy for SCCT, we selected PYM and cDDP for our
further experiments. Consistently, SCC-15/PYM cells possessed enhanced anti-apoptotic
ability in response to chemotherapy measured by Hoechest staining (Fig.S1B). Moreover,
chemotherapy significantly induced caspase3 activation in SCC-15 cells, but caspase3
activation was attenuated in SCC-15/PYM cells upon chemotherapy (Fig.1B).
We further confirmed chemo-resistance of SCC-15/PYM cells in vivo using a xenograft
mouse model. We separately injected the SCC-15 and SCC-15/PYM cells subcutaneously
into the armpit of athymic mice. After 15 days, mice were treated with PBS (Control),
PYM or cDDP every 3 days for ten cycles. At the experimental end point, the mice were
euthanized. The tumors were excised and weighted. We found that the tumors formed
by SCC-15 and SCC-15/PYM cells grew at a similar rate. Chemotherapy persistently
inhibited the growth of tumors formed by SCC-15 cells, but tumors formed by
SCC-15/PYM cells showed only a short growth delay and then started re-grow (Fig.1C
and Fig.S1C). Consistently, chemotherapy induced more caspase3 activation (cleaved
caspase3) in tumors formed by SCC-15 cells compared to tumors formed by SCC-15/PYM
cells in vivo (Fig.1D). Together with the aforementioned in vitro study, these data verified
the stable chemo-resistant characteristics of SCC-15/PYM cells.
HSP27 protein level is increased in chemo-resistant tongue cancer cells
In order to identify key molecules involved into the chemo-resistance, we compared the
protein profiles between SCC-15/PYM and SCC-15 cell lines using the 2D gel proteomic
approach (Fig.S1D and Table S1). Among the differentially expressed proteins, HSP27,
Alpha-enolase (Enolase-1) and Lamin-A/C (LAMN) were selected to be further validated
(Fig.S1E). We found that protein level of HSP27 is significantly higher in SCC-15/PYM cell
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line, compared to other SCCT cell lines (Fig.1E). Because HSP27 is a secreted protein that
is suitable to serve as a biomarker, we selected HSP27 to explore its roles and
mechanisms for chemo-resistance. We further determined the protein level of HSP27 in
cell culture medium (CM) of SCCT cell lines. The ELISA result confirmed the significantly
higher HSP27 protein level in CM of SCC-15/PYM cell line, as compared to the CM of the
SCC-15 cell line (Fig.1E). As a negative control, β-actin protein is non-detectable in the
CM of SCCT cell lines, indicating that the HSP27 protein detected in the cell culture
medium was extracellular protein that was secreted from the cells (Fig. S1F). Taken
together, these results verified the significant changes in HSP27 protein express in both
its intracellular and extracellular levels in chemo-resistant SCCT cells.
HSP27 enhances chemo-resistance in tongue cancer cells
Given the increased HSP27 in chemo-resistant SCCT cells, we then investigated whether
the up-regulation of both extracellular and intracellular HSP27 plays a causal effect on
the chemo-resistance. We found that HSP27 knockdown with shRNA significantly
enhanced the sensitivity of SCC-15/PYM cells to PYM and cDDP in vitro (Fig.2A and
Fig.S2A). Inversely, the chemo-sensitivity of SCC-15 cells was dramatically decreased
after HSP27 was overexpressed (Fig.2B and Fig.S2B). This effect of HSP27 on
chemo-resistance was also validated in another SCCT cell line SCC-25. Similarly, HSP27
overexpression (Fig.S2C) also enhanced chemo-resistance in SCC-25 cells (Fig.2B).
We further validated the effect of HSP27 on chemo-sensitivity in the xenograft tumor
models by subcutaneously injecting athymic mice with SCC-15/PYM cells with HSP27
knockdown, SCC-15, SCC-25 cells with HSP27 overexpression or their related control cells.
After 15 days, these mice were treated with PBS (Control), PYM or cDDP every 3 days for
ten cycles. We found that HSP27 overexpression did not significantly influence tumor
growth, but markedly affected the chemo-sensitivity in vivo. The tumors formed by
control cells showed a short initial response to chemotherapy prior to re-growing, but
chemotherapy led to sustained growth inhibition of tumors formed by SCC-15/PYM cells
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with HSP27 knowdown (Fig.2D). The volume and weight of the tumors formed by
SCC-15/PYM cells with HSP27 knockdown were decreased to significantly greater extent
than that of control xenografts, in response to PYM and cDDP treatment (Fig.2D and
Fig.S2C). On the other hand, the tumor volume and weight of xenografts formed by
SCC-15 and SCC-25 cells with HSP27 overexpression showed an inverse effect upon
chemotherapy (Fig.2E and Fig.S2D).
We further sought to assess the role of extracellular HSP27 in the cells with altered
HSP27 expression. We observed that HSP27 knockdown also reduced HSP27 protein
level in CM of SCC-15/PYM cells (Fig.S2A), while HSP27 overexpression increased HSP27
protein level in CM of SCC-15 and SCC-25 cell lines (Fig.S2B). To investigate whether
extracellular HSP27 was also involved in chemo-resistance, anti-HSP27 antibody was
used to block extra-cellular HSP27 function while recombinant human protein (rhHSP27)
was used to stimulate SCCT cells. We found that anti-HSP27 antibody treatment also
sensitized SCC-15/PYM cells to chemotherapy (Fig.2A), but rhHSP27 treatment enhanced
chemo-resistance of SCC-15 (Fig.2B) and SCC-25 (Fig.2C) cell lines. In summary, these
lines of evidence strongly suggest that both extracellular and intracellular HSP27
mediates chemo-resistance in SCCT cells.
HSP27 activates NF-κB signaling
In order to elucidate the detailed molecular mechanism mediating the chemo-resistance
effect of HSP27, we examined NF-κB transactivation in a series of SCCT cells lines using
NF-κB reporter assay. We found that NF-κB transactivation activity in SCC-15/PYM cells
was significantly higher than in the other SCCT cells lines (Fig.3A). The expression of
NF-κB target genes such as survivin and IL-6 were also significantly up-regulated in
SCC-15/PYM cells (Fig.3A). Moreover, compared to other SCCT cell lines, p65 nuclear
translocation and p-IκBα level were also increased in SCC-15/PYM cells, but the total
IκBα level was decreased (Fig.3A). These data indicated that NF-κB signaling was
markedly activated in SCC-15/PYM cells. As expected, HSP27 knockdown significantly
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repressed NF-κB transactivation, as reflected by decreased expression of NF-κB target
genes survivin and IL-6, decreased p65 nuclear translocation, decreased p-IκBα level and
increased total IκBα level (Fig.3B). On the other hand, treatment with anti-HSP27
antibody in the culture medium showed the similar effect of HSP27 knockdown on NF-κB
signaling in SCC-15/PYM cells (Fig.3B). However, overexpression of HSP27 enhanced
NF-κB signaling activation in SCC-15 and SCC-25 cell lines. Treating the cells with
rhHSP27 exerted the similar effect of HSP27 overexpression (Fig.3C).
In order to examine whether HSP27 regulates NF-κB signaling via modulating IκBα level,
we used a dominant negative model where a mutant gene encoding a
non-phosphorylatable IκBα, which was cloned into the pBabe plasmid (pBabe-IκBα),
resulting into the constitutive suppression of NF-κB signaling despite the presence of its
activators (9). We found that pBabe-IκBα transfection or treatment with NF-κB inhibitor
Bay11-7082 prevented IκBα degradation and impaired rhHSP27 induced NF-κB signaling
activation (Fig.3D). Together, these data indicated that the effect of HSP27 in
chemo-resistance is mediated by NF-κB signaling activation in SCCT cells, and both
extracellular and intracellular HSP27 are involved in this process.
HSP27 interacts with TLR5 to activate NF-κB signaling
We sought to further explore the key mediator linking between HSP27 and the
activation of NF-κB signaling. Given the high correlation between HSP27 function and
the expression of Toll-like receptors (TLRs), and that TLR5 has been reported to be highly
expressed in tongue cancer (10), we examined TLR5 protein level in SCCT cell lines, and
found that TLR5 is highly expressed in SCCT cells (Fig.S3A). To validate whether TLR5 was
involved in extracellular HSP27 mediated chemo-resistance, we knocked down TLR5
expression in SCCT cell lines with shRNA (Fig.S3B). We then assessed whether
extracellular HSP27 mediated chemo-resistance via TLR5. TLR5 knockdown sensitized
SCC-15/PYM cells to chemotherapy (Fig.S3C). TLR5 knockdown impaired the
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chemo-resistance induced by rhHSP27 in SCC-15 and SCC-25 cells (Fig.S3D). Levels of
NF-κB transactivation, p65 nuclear translocation, p-IκBα, and expression of survivin and
IL-6 were also significantly decreased after TLR5 knockdown in SCC-15/PYM cells, but the
total IκBα level increased (Fig.4A and Fig.S3E).
Moreover, the efficiency of rhHSP27 treatment on NF-κB signaling activation was
attenuated in SCC-15 cell after TLR5 knockdown (Fig.4B and Fig.S3F). We also confirmed
the role of TLR5 in another SCCT cell line SCC-25 in response to rhHSP27 treatment
(Fig.4C and Fig.S3G). Furthermore, Co-IP assay demonstrated that extracellular HSP27
could physically interact with TLR5 after rhHSP27 incubation in SCC-15 and SCC-25 cell
lines (Fig.4D). In addition, immunofluorescent staining also confirmed the co-localization
of the two proteins on the cell membrane of SCC-15 cells after rhHSP27 incubation
(Fig.4E). These results indicated that extracellular HSP27 activates NF-κB signal via a
TLR5 dependent manner to mediate chemo-resistance in SCCT cells.
Intracellular HSP27 inhibits BAX and BIM function to repress apoptotic signal
As shown in Fig. 2, the chemo-sensitizing effect of HSP27 knockdown was greater than
anti-HSP27 antibody treatment in SCC-15/PYM cells (Fig. 2A). The chemo-resistant effect
of HSP27 overexpression also was greater than that of rhHSP27 treatment in SCC-15 and
SCC-25 cell lines (Fig.2B and C), suggesting that intracellular HSP27 may also play a role
in chemo-resistance. To further explore the mechanism underlying this effect, we further
determined the activation of apoptotic executant caspase3 in SCCT cells resulted from
intracellular and extracellular interference of HSP27 function. We found that in
SCC-15/PYM cells, anti-HSP27 antibody treatment enhanced PYM or cDDP induced
Caspase3 activation and cleaved Caspase3 level, but HSP27 knockdown demonstrated
stronger enhancement on caspase3 activation upon chemotherapy (Fig.S4A). Similarly,
in SCC-15 and SCC-25 cell lines, rhHSP27 treatment attenuated PYM- or cDDP-induced
Caspase3 activation and cleaved Caspase3 level, but the efficiency resulted from
overexpressed HSP27 was greater than rhHSP27 treatment (Fig.S4B). These findings
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implied that HSP27 mediates chemo-resistance via both intra- and extracellular
mechanisms rather than extracellular-only mechanisms.
In order to investigate the role of HSP27 in apoptosis, we detected changes of
pro-apoptotic proteins levels upon chemotherapy. We found that,regardless of HSP27
protein level, chemotherapy induced protein expression of BCL-2 family members BAX,
BAD and BIM in SCCT cell lines SCC-15 and SCC-25 (Fig.5A), even in SCC-15/PYM cells
(Fig.5B). However, HSP27 overexpression repressed BAX and BIM mitochondria
translocation, and cytochrome C (Cyto C) release from mitochondria, and subsequently
repressed the increase of cytosolic Cyto C level upon chemotherapy in SCC-15 and
SCC-25 cell lines (Fig.5A). Inversely, in SCC-15/PYM cells, HSP27 knockdown enhanced
the mitochondria translocation of BAX and BIM, and the release of Cyto C from
mitochondria, and subsequently enhanced the increase of cytosolic Cyto C level (Fig.5B).
To assess whether intracellular HSP27 mediates the translocation of pro-apoptotic
proteins, we detected HSP27 protein level in mitochondria fraction, but there was no
detectable HSP27 protein in the mitochondrial fraction. Moreover, using Co-IP assay, we
found HSP27 could physically interact with BAX and BIM, and the interacting level was
increased when HSP27 was overexpressed in response to chemotherapy (Fig.5C).
Additionally, immunofluorescent staining also confirmed the co-localization HSP27 and
BAX and BIM in SCC-15/PYM cells upon cDDP treatment (Fig.S4C). These results suggest
that intracellular HSP27 interacts with BAX and BIM to sequester them in the cytoplasm
from mitochondria translocation upon chemotherapy.
Chemotherapy induces HSP27 expression
In order to explore the role of HSP27 in chemotherapy, we examined the expression
change of HSP27 protein in a series of SCCT cell lines in response to chemotherapy. We
found that PYM or cDDP treatment induced increased levels of HSP27 protein in SCCT
cells and cell culture medium (Fig.6A). In the xenograft tumors formed by SCC-15 and
SCC-25 cell lines and their HSP27 overexpressed cell lines (Fig.2E and Fig.S2D), PYM and
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CDDP treatment induced increased expression of HSP27 protein in xenograft tumors and
mice serum (Fig.6B). Notably, chemotherapy did not induce significant HSP27 protein
expression in tumors formed by HSP27 overexpressed SCCT cells, but resulted in
significant HSP27 protein increase in mice serum. Additionally, we found that
chemotherapy induced increased expression of HSP27 protein in the mice normal liver
tissues collected from both mice group harboring tumors formed by
HSP27-overexpressing and control cells, but not in the mice normal lung and kidney
tissues (Fig.S5). These data suggests that chemotherapy maybe also induce HSP27
secretion from other tissues rather than only from tumors.
In order to examine whether chemotherapy alters HSP27 expression, we determined
HSP27 protein levels in the serum from 20 healthy controls, and 23 tongue cancer
patients pre-chemotherapy and post-chemotherapy respectively. These patients
underwent treatment with PYM and/or cDDP based chemotherapy. We found that
HSP27 protein level was higher in the serum from tongue cancer patients as compared
to that of healthy controls, and that chemotherapy significantly induced expression of
HSP27 protein in the serum of 15 out of the 23 (65.22%) tongue cancer patients, who
showed poor response to chemotherapy (Fig.6C). Moreover, we detected HSP27 protein
level in the 23 paired SCCT tissues collected before and after PYM and/or cDDP based
chemotherapy. HSP27 protein level in the 15 tissues from patients who had HSP27
protein increased in serum after treatment, were dramatically increased after
chemotherapy (Fig.6C). These results demonstrated that chemotherapy could induce
HSP27 expression in tongue cancer cells as well as in SCCT patients.
HSP27 expression is correlates with poor clinical outcome in tongue cancer patients
To further explore the clinical significance of HSP27 in tongue cancer treatment, we
evaluated HSP27 status in two independent cohorts of human SCCT tissues collected
from SCCT patients received PYM and/or cDDP based chemotherapy. In the cohort 1
tissues, 84 SCCT tissues were obtained at Affiliated Cancer Hospital of Guangzhou
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Medical University (Guangzhou, Guangdong Province, China). In the cohort 2 tissues, 81
SCCT tissues were obtained at Xiangya Stomatological Hospital & School of Stomatology,
Central South University (Changsha, Hunan Province, China). The SCCT patients from
both cohort 1 and cohort 2 had no distant metastasis. Using immunohistochemistry
staining, we found that HSP27 was highly expressed in 57.14% (48/84) and 53.09%
(43/81) SCCT tissue samples of cohort 1 and cohort 2, respectively. High HSP27 levels
were closely associated with tumor stage and recurrence of patients with SCCT (Table S2
and S3). TLR5 protein was usually highly expressed in SCCT tissues (77.38% for cohort 1
and 75.31% for cohort 2). Tissue samples with high HSP27 protein level possessed a high
nuclear p65 protein level, but a low cleaved caspase3 protein level (Fig.S6). HSP27
protein level was positively correlated with p65 nuclear translocation, but negatively
correlated with caspase3 activation (Fig.7A). Importantly, SCCT patients with high HSP27
protein level in their tumor samples had significantly shorter overall survival times
(Fig.7B). On the basis of our analysis, we conclude that high HSP27 protein levels predict
worse treatment outcomes.
DISCUSSION
Our study reveals that HSP27 is a key modulator that is at least in part, responsible for
the chemo-resistance of tongue cancer. Our findings provide several insights into the
detailed mechanisms for the role of HSP27 in chemo-resistance of SCCT: (1)
Chemotherapy induced HSP27 protein level increase in SCCT cells and extracellular
environment to (2) enhance chemo-resistance. (3) Extracellular HSP27 interacts with
TLR5 to activate NF-κB signaling, while (4) intracellular HSP27 sequesters BAX and BIM
from mitochondria translocation to inhibit apoptotic signaling upon chemotherapy
(Fig.7D).
Our study for the first time revealed a novel function of HSP27 in cancers. Heat shock
proteins (HSPs) include several different protein families, which can be induced in cells
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by many physiological and pathophysiological stresses (11). HSPs are expressed in either
a constitutive or inductive pattern in different cell types and subcellular compartments
(12). Cancer cells usually possess high expression of chaperones to meet the high
metabolic requirements and the abundant signal transduction, subsequently to maintain
cancer cells survival. HSPs mediate the oncogenic signal transduction by folding and
stabilizing oncoproteins. Thus, treatment strategies based on HSPs inhibition are
recognized as an important approach to deplete oncoproteins and attack oncogenic
signal pathways required for cancer development and progression (13). HSP27 is an
important small HSP and is induced in response to varieties of cellular stresses, such as
exposure to mitogens, growth factors, hormones, inflammatory cytokines, and
anticancer treatment (14). It has been reported that HSP27 was elevated in some human
cancer types, such as breast cancer and prostate cancer and aberrant HSP27 expression
correlates with tumor growth, metastasis, as well as therapy resistance (15,16). In
consistent with these previous observations, we found that the protein expression of
HSP27 was significantly up-regulated in MDR-SCCT cell line and its culture medium (CM).
Both HSP27 knockdown and HSP27 neutralizing antibody treatment sensitized
MDR-SCCT cells to chemotherapy. Also, both ectopic HSP27 overexpression and
recombinant human HSP27 protein treatment enhanced chemo-resistance in SCCT cell
lines. This observation further highlighted that HSP27 is a key player in both maintaining
cancer cell function and more importantly, facilitating cancer cells to escape the stress
from chemotherapy.
Our study also delineated the detailed mechanism underlying the HSP27 signaling
involved in both extra- and intracellular function. The extracellular HSP27 level in CM
from MDR-SCCT cell line was higher than from other SCCT cell lines. Interestingly,
chemotherapy induced the increase of HSP27 protein levels in CM from SCCT cell lines in
vitro and in the serum of mouse model bearing tongue cancer. Notably, chemotherapy
did not induce significant HSP27 protein increase in tumors formed by HSP27
overexpressed SCCT cells, but resulted in significant HSP27 protein increase in mice
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serum, suggesting that chemotherapy may induce HSP27 secretion from both SCCT cells
and other tissues. As expected, the serum HSP27 levels are indeed elevated after
chemotherapy in SCCT patients. The elevated serum HSP27 level also has been reported
in breast cancer patients (17). These lines of evidence highlighted the potential role of
HSP27 in the microenvironment of SCCT and possibly other cancers as well. This role in
the microenvironment may be associated with both endocrine and paracrine
mechanisms. It is worthwhile noting that this elevated HSP27 in both tumor
microenvironment and entire circulatory system may globally affect cancer recurrence,
metastasis and drug resistance in general. This notion is supported by previously studies
where extracellular HSP27 affects the biological behaviors of both cancer cells and
stromal cell, and then contributes to cancer progression, e. g. extracellular HSP27 in
cancer microenvironment promotes VEGF secretion in endothelial cells depending on
TLR3 and then promotes angiogenesis (18). Extracellular HSP27 also exhibited biological
effects on monocytes, resulting in the secretion of IL-10 and TNFα (19), blocking the
differentiation of monocytes to normal dendritic cells (20), and driving the
differentiation of monocytes to tumor-associated macrophages (TAMs) to promote
cancer progression (21). Whether the role of HSP27 we have discovered in this study
would be further extended to these cancer biological and physiological processes in
SCCT patients and further contributes to the chemo-resistance and other cancer
function remains to be further investigated.
Our study also for the first time identified the major signaling pathways downstream of
HSP27 in the chemo-resistance SCCT cells. Extracellular HSP27 executes roles intensely
through toll-like receptors (TLRs) activated NF-κB signaling (18,22). We demonstrated
that tongue cancer cells and tissues possess high TLR5 protein levels, and that
extracellular HSP27 triggered NF-κB signaling via binding to TLR5. Moreover, we found
that chemotherapy induced expression of pro-apoptotic proteins BAX, BAD and BIM
which are major BH-3 domain containing BLC-2 family members. We demonstrated that
HSP27 interacts with BAX and BIM to attenuate their translocation to mitochondria and
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subsequently blocks cytochrome C release. Our observation is consistent with previous
findings where HSP27 is able to prevent cells from apoptosis by interacting with
cytochrome C (24) or by interfering the apoptotic signaling upstream of the
mitochondrial cytochrome C release (25,26). In addition, it was reported that HSP27 as a
subunit of AUF1 protein complexes, can also act as a RNA-binding protein to mediate
mRNA decay (27). Enhanced HSP27 expression in response to oxidative stress binds to
the 3'-UTR of BIM mRNA to repress its translation, and subsequently prevent neuronal
death in cerebellar granule neurons (28). Whether this could be also a potential
mechanism in tongue cancer as well as other cancer types should be further investigated
in future studies.
Lastly, our study has important translational implications. Our findings indicates that
HSP27 and its associated key molecules can be critical targets for developing new
therapeutics for tongue cancer. In particular, we showed that HSP27 is induced among
SCCT patients by chemotherapy, which indicates that increased HSP27 may also be a
useful biomarker for selecting patients toward developing precision medication for
tongue cancer treatment. In addition, it is of importance to keep in mind that HSP27
targeting strategies should control both intracellular and extracellular function of HSP27.
Our study thus warranted continued investigation to these translational directions.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
No potential conflicts of interest were disclosed.
AUTHORS' CONTRIBUTIONS
Conception and design: Guopei Zheng, Yan Xiong, Wanqing Liu, Zhimin He
Development of methodology: Guopei Zheng, Zhijie Zhang, Liyun Luo, Xiaoting Jia, Hao
Pan
Acquisition of data (provided animals, acquired and managed patients, provided
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facilities, etc.): Guopei Zheng, Qiong Zhang, Nan Li, Minying Lu, Ying Song
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational
analysis): Guopei Zheng, Zhijie Zhang, Yixue Gu, Hao Liu
Writing, review, and/or revision of the manuscript: Guopei Zheng, Wanqing Liu, Hao Liu,
Zhimin He
Administrative, technical, or material support (i.e., reporting or organizing data,
constructing databases): Guopei Zheng, Liyun Luo, Cong Peng, Wanqing Liu
Study supervision: Jinbao Liu, Zhimin He
ACKNOWLEDGMENTS
We thank Key Laboratory of Cancer Proteomics of Chinese Ministry of Health (Xiangya
Hospital, Central South University, Changsha, China) for the technical support of
proteomics.
GRANT SUPPORT
This study was supported by grants from the National Natural Science Foundation of
China: No. 81402196 (G Zheng), No.81672616 (G Zheng), No.81272450 (Z He) and No.
81401989 (N Li), supported by grants from Guangdong Natural Science Funds for
Distinguished Young Scholars No.2016A030306003 (G Zheng), supported by by
Guangzhou key medical discipline construction project fund, supported by grants from
Science and Technology Program of Guangzhou, China (201710010100) (G Zheng),
Guangzhou Municipal University Scientific Research project (1201610027) (G Zheng).
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Figure Legends
Figure 1. HSP27 protein level increase in chemo-resistant SCCT cell line and its culture medium.
(A) Dose-response curves of SCC-15 and SCC-15/PYM cells to PYM or cDDP. Each point
represents the mean of three independent experiments. (B) Caspase3 activation was
determined by detecting cleaved caspase3 level using western blot and performing caspase3
activation assay after PYM (80mg/L) or cDDP (5mg/L) treatment for 24 hours. β-actin was used
as a loading control for western blot. Student’s t-test, Mean±s.d. (n=3), **** p < 0.0001. (C) The
growth and chemo-sensitivity to PYM (30mg/kg) or cDDP (3mg/kg) in vivo of tumors formed by
SCC-15 and SCC-15/PYM were monitored, tumor volume was periodically measured for each
mouse and tumor growth curves were plotted. The wet weight was recorded. Student’s t-test,
Mean±s.d. (n=3/group), *** p < 0.001, **** p < 0.0001. (D) Representative immunohistochemical
staining for cleaved caspase3 in tissues from xenograft mouse model (scale bar, 50μm). (E)
HSP27 protein levels in cell culture medium (CM) detected by ELISA (upper) and in SCCT cell
lines detected by western blot in the whole cell lysates (WCL) (lower). One-way ANOVA and
Dunnett’s multiple comparison test, Mean±s.d. (n = 3 biological replicates with n = 3 technical
replicates each), **** p < 0.0001.
Figure 2. HSP27 enhances chemo-resistance in SCCT cells. (A-C) The effects of HSP27 expression
interference and function interference (10μg/ml HSP27 antibody; 1μg/ml rhHSP27) on
sensitivity of SCCT cell lines to PYM or cDDP were detected by MTS assay. Each point represents
the mean of three independent experiments. (D and E) Subcutaneous xenograft assays of
HSP27-knockdown and control SCC-15/PYM cells (D), of HSP27 overexpression and control
SCC-15 cells (E, left) and HSP27 overexpression and control SCC-25 cells (E, wright) in nude mice
with PYM (30mg/kg) or cDDP (3mg/kg) treatment. Tumor growth and chemo-sensitivity in vivo
were monitored, tumor volume was periodically measured for each mouse and tumor growth
curves were plotted. The tumor wet weights were recorded. Student’s t-test, Mean±s.d.
(n=3/group), *** p < 0.001, **** p < 0.0001.
Figure 3. Extracellular HSP27 triggers NF-κB signaling activation. (A, B and D) NF-κB transactivity
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was detected by NF-κB activation reporter assay, relative survivin and IL-6 expression was
detected by qRT-PCR, and proteins levels in nuclear lysates and whole cell lysates were detected
by western blot. GAPDH was used as an internal control for qRT-PCR, Histone H3 was used as a
loading control for nuclear lysates and β-actin was used as a loading control for the whole cell
lysates. One-way ANOVA and Dunnett’s multiple comparison test (A), vs Mock or sh-con,
Student’s t-test (B), vs Mock or pLEX-con, Student’s t-test (D), Mean±s.d. (n=3), ** p < 0.01, *** p
<0.001. (C) NF-κB transactivation was detected by NF-κB activation reporter assay and relative
survivin and IL-6 expression was detected by qRT-PCR in SCC-15 and SCC-25 cell lines after
treatment of rhHSP27 and/or pBabe-IκBα, and treatment of rhHSP27 and/or Bay11-7082
(2μg/ml), GAPDH was used as an internal control for qRT-PCR. Student’s t-test, Mean±s.d., vs
mock or rhHSP27, ** p < 0.01, *** p<0.001.
Figure 4. Extracellular HSP27 activates NF-κB signaling depending on TLR5. (A-C) NF-κB
transactivation was detected by NF-κB activation reporter assay, and proteins levels in nuclear
lysates and whole cell lysates were detected by western blot. β-actin was used as a loading
control for western blot. vs sh-con (A), vs rhHSP27 (A and B), Student’s t-test, Mean±s.d. (n=3),
*** p < 0.001. (D) The interaction between HSP27 and TLR5 in SCC-15 and SCC-25 cells after
rhHSP27 (5μg/ml) incubation was determined by CoIP assay. (E) Representative
immunofluorescent staining images showing the colocation of HSP27 and TLR5 on the cell
membrane of SCC-15 cells after rhHSP27 (5μg/ml) incubation in SCC-15 cells. Nuclei was
counterstained with DAPI.
Figure 5. Intracellular HSP27 inhibits mitochondrial apoptotic pathway via interacting with BAX
and BIM function to repress apoptotic signal. (A-B) Related proteins levels in different cellular
fraction were detected by western blot upon varieties of treatments. The loading control for
whole cell lysates and cytosolic fraction was β-actin, and for mitochondrial fraction was COX Ⅳ.
(C) The interaction between HSP27 and BAX and BIM in response to varieties of treatments was
determined by CoIP assay.
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Figure 6. Chemotherapy induces HSP27 expression in SCCT. (A) HSP27 protein levels in cell
culture medium and intra-cell were determined by ELISA (upper) and western blot (lower),
respectively, after cells were treated with PYM (80mg/L) or cDDP (5mg/L). vs no-treatment,
Student’s t-test, Mean±s.d. (n=3), * p < 0.05, ** p < 0.01, *** p < 0.001. (B) HSP27 protein levels in
xenograft tumors and serums from mice bearing tumors as shown in Figure 2E and Figure S2E
were examined using ELISA (left) and immunohistochemistry (wright) respectively. vs
PBS-treatment, Student’s t-test, Mean±s.d. (n=3), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p <
0.0001. (C) HSP27 protein levels in serums from tongue cancer patients were examined using
ELISA (upper). HSP27 protein levels in tissues were valuated using immunohistochemistry
staining (lower). Student’s t-test, Mean±s.d., ** p < 0.01, **** p < 0.0001.
Figure 7. HSP27 level correlates with poor clinical outcome in tongue cancer patients. (A)
Correlation between HSP27 and nuclear p65 and cleaved caspase3 protein levels in human
tongue cancers. Statistical significance was determined by a χ2 test. (B) Kaplan-Meier curves
showing the overall survival of patients with high or low protein level of HSP27 in their tongue
cancers. Statistical significance was determined by a log-rank test. (C) The working model of
regulation of chemo-resistance by HSP27.
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A B
Figure 1
Caspase
3 a
ctivity
β-actin
Caspase3
Uncleaved
Cleaved
SCC-15 SCC-15/PYM
PYM
cDDP
SC
C-1
5
SC
C-1
5/P
YM
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C-1
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PY
M
SC
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YM
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)
Treatment
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YM
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- +
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Tum
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***
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***
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PYM concentration (mg/L)
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Cell
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ty
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cDDP concentration (mg/L)
PYM concentration (mg/L)
cDDP concentration (mg/L) cDDP concentration (mg/L)
PYM concentration (mg/L)
Cell
via
bili
ty
Cell
via
bili
ty
Cell
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ty
Cell
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A B C
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Tum
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Treatment Treatment Treatment
D E SCC-15/PYM SCC-15 SCC-25
Figure 2
***
***
***
***
****
*** ****
***
PYM concentration (mg/L)
***
****
****
****
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A Nuclear Lysates B
p65 His H3
Whole Lysates
HSP27
p-IκBα
IκBα
p65
Survivin
IL-6
β-actin
SC
C-2
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SC
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-κB
activity
Rela
tive e
xpre
ssio
n
p65
His H3
HSP27
p-IκBα
IκBα
p65
Survivin
IL-6
β-actin
Mock
an
ti-H
SP
27
sh
-Con
sh
-1#
sh
-2#
Mock
an
ti-H
SP
27
sh
-Con
sh
-1#
sh
-2#
Nuclear Lysates
Whole Lysates
NF
-κB
activity
Rela
tive e
xpre
ssio
n
Mock
rhH
SP
27
pLE
X-C
on
pLE
X-H
SP
27
Mock
rhH
SP
27
pLE
X-C
on
pLE
X-H
SP
27
Nuclear Lysates
Whole Lysates
Mock
rhH
SP
27
pLE
X-C
on
pLE
X-H
SP
27
Mock
rhH
SP
27
pLE
X-C
on
pLE
X-H
SP
27
p65
His H3
Hsp27
p-IκBα
IκBα
p65
Survivin
IL-6
β-actin
NF
-κB
activity
Rela
tive e
xpre
ssio
n
Mock
rhH
SP
27
Bay11
-70
82
Bay11
-708
2+
rhH
SP
27
pB
ab
e-IκBα
pB
ab
e-IκBα+rhHSP27
Mock
rhH
SP
27
Bay11
-70
82
Bay11
-708
2+
rhH
SP
27
pB
ab
e-IκBα
pB
ab
e-IκBα+rhHSP27
C N
F-κ
B a
ctivity
Rela
tive e
xpre
ssio
n
D
SCC-15 SCC-25 SCC-15 SCC-25
Figure 3
*** *** ***
*** ***
*** ***
*** ***
*** *** ***
*** ***
***
** **
SCC-15 SCC-25 **
** **
**
** **
*** *** ***
** ** **
** ** ** **
**
SC
C-1
5/P
YM
***
*** ***
***
***
***
***
***
Research. on April 30, 2021. © 2017 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
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NF
-κB
activity
A
Nuclear Lysates
p65
His H3
β-actin
p-IκBα
IκBα
p65
NF
-κB
activity
Whole Lysates
sh
-Con
sh
-3#
sh
-4#
NF
-κB
activity
Nuclear Lysates Nuclear Lysates
Whole Lysates Whole Lysates
Mock
rhH
SP
27
rhH
SP
27
+sh
-3#
sh
-3#
sh
-4#
rhH
SP
27
+sh
-4#
Mock
rhH
SP
27
rhH
SP
27
+sh
-3#
sh
-3#
sh
-4#
rhH
SP
27
+sh
-4#
p65 His H3
β-actin
p-IκBα
IκBα
p65
p65 His H3
β-actin
p-IκBα
IκBα
p65
B C
IP: HS
P27
IgG
HS
P27
IgG
IP: HS
P27
IgG
HS
P27
IgG
IP: TLR
5
IgG
TLR
5
IgG
IP: TLR
5
IgG
TLR
5
IgG
HSP27
TLR5
HSP27
TLR5
HSP27
TLR5
HSP27
TLR5
D DAPI TLR5
Merged HSP27
SCC-15 SCC-25
E
Figure 4
Mock rhHsp27 Mock rhHsp27
SCC-15 SCC-25
*** *** *** *** *** ***
SCC-15/PYM
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β-actin
BAD
BAX
BIM
Cyto C
COX Ⅳ
Whole Lysates
Mitochondrial Fraction
Cytosolic Fraction
BAD
BAX
BIM
Cyto C
β-actin
Whole Lysates
Mitochondrial Fraction
Cytosolic Fraction
β-actin
BAD
BAX
BIM
Cyto C
COX Ⅳ
BAD
BAX
BIM
Cyto C
β-actin
cDDP
pLEX-HSP27
pLEX-Con
PYM
cDDP
pLEX-HSP27
pLEX-Con
PYM
SCC-15 SCC-25 A
B
β-actin
BAD BAX
BIM
Cyto C
COX Ⅳ
BAD
BAX
BIM
Cyto C
β-actin
sh-Con
cDDP
sh-1#
sh-2#
PYM
Whole Lysates
Mitochondrial Fraction
Cytosolic Fraction
cDDP
pLEX-HSP27
pLEX-Con
PYM
IP: HSP27 IgG
BAX
BIM
cDDP
pLEX-HSP27
pLEX-Con
PYM
BAX
BIM
IP: HSP27 IgG
SCC-15
SCC-25
C SCC-15/PYM
Figure 5
+ + + - - -
- + - + - +
- - - - + +
- + - - - +
+ + + - - -
- + - + - +
- - - - + +
- + - - - +
+ + + - - - - - -
- - - + + + - - -
+ + + - - - - - -
- - - - - - + + +
- - - - - - + + +
+ + + - - - - -
- + - + - + + +
- - - - - + + +
- - - - - + + +
+ + + - - - - -
- + - + - + + +
- - - - - + + +
- - - - - + + +
Research. on April 30, 2021. © 2017 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
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SC
C-2
5
SC
C-1
5
SC
C-9
CA
L27
SC
C-1
5/P
YM
PB
S
PY
M
cD
DP
PB
S
PY
M
cD
DP
PB
S
PY
M
cD
DP
PB
S
PY
M
cD
DP
pLEX-Con pLEX-HSP27
pLEX-Con pLEX-HSP27
pLEX-Con pLEX-HSP27 pLEX-Con pLEX-HSP27
PB
S
PY
M
cD
DP
SCC-15 SCC-25
N=20 N=23 N=23
Pre-chemo Post-chemo
CM
HS
P27 le
vel (n
g/m
L)
Seru
m H
SP
27 le
vel (n
g/m
L)
Seru
m H
SP
27 le
vel (n
g/m
L)
Seru
m H
SP
27 le
vel (n
g/m
L)
β-actin
HSP27
A
B
C
Figure 6
WB
in W
CL
E
LIS
A I
n C
M
** ** ***
** *** *** ***
***
* *
*
**
* *
*** ****
SCC-15
** **
**** ****
SCC-25
Research. on April 30, 2021. © 2017 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
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Nuclear p65 High 38 16
Low 10 20 0.0013
Expression High Low p value
HSP27
Nuclear p65 High 36 20
Low 7 18 0.0036
Expression High Low p value
HSP27
Cleaved Cas3 High 15 23
Low 33 13 0.0040
Expression High Low p value
HSP27
Cleaved Cas3 High 14 26
Low 29 12 0.0018
Expression High Low p value
HSP27
Cohort 1 Cohort 2
NF-κB
…Survivin
IL-6…
IκBα
Apoptosis
Chemo-resistance
HSP27
HSP27
NF-κB
TLR
5
Bax
Bim
Figure 7
A
C Cohort 1
n=48 n=36 p=0.0081
Cohort 2
n=43 n=38 p=0.0024
Overa
ll S
urv
ival (%
)
Month
Overa
ll S
urv
ival (%
)
Month
B
Research. on April 30, 2021. © 2017 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
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Published OnlineFirst December 15, 2017.Clin Cancer Res Guopei Zheng, Zhijie Zhang, Hao Liu, et al. Squamous Cell Carcinoma of TonguePathways Synergistically Confer Chemo-Resistance in HSP27-mediated Extracellular and Intracellular Signaling
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