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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:

[email protected];

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

Research. on April 30, 2021. © 2017 American Association for Cancerclincancerres.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 December 15, 2017; DOI: 10.1158/1078-0432.CCR-17-2619

http://clincancerres.aacrjournals.org/

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.




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.




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.




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




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




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




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




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




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




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




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




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




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




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




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




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




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




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




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.




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.




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

SC

C-1

5+

PY

M

SC

C-1

5+

cD

DP

SC

C-1

5/P

YM

+P

YM

SC

C-1

5/P

YM

+cD

DP

Tum

or

weig

ht (g

)

Treatment

SCC-15 SCC-15/PYM

cD

DP

P

YM

P

BS

D

SC

C-2

5

SC

C-1

5

SC

C-9

CA

L27

SC

C-1

5/P

YM

HSP27

β-actin

CM

HS

P2

7 le

vel (n

g/m

l) E

ELIS

A I

n C

M

WB

in W

CL

- +

+

-

- -

- +

+

-

- -

**** ****

C

Tum

or

gro

wth

(m

m3)

***

***

****

***

****

PYM concentration (mg/L)

cDDP concentration (mg/L)

Cell

via

bili

ty

Cell

via

bili

ty




cDDP concentration (mg/L)


cDDP concentration (mg/L) cDDP concentration (mg/L)


Cell

via

bili

ty

Cell

via

bili

ty

Cell

via

bili

ty

Cell

via

bili

ty

Cell

via

bili

ty

Cell

via

bili

ty

A B C

Tum

or

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wth

(m

m3)

Tum

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wth

(m

m3)

Tum

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gro

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(m

m3)

Tum

or

weig

ht (g

)

Tum

or

weig

ht (g

)

Tum

or

weig

ht (g

)

Treatment Treatment Treatment

D E SCC-15/PYM SCC-15 SCC-25

Figure 2

***

***

***

***

****

*** ****

***


***

****

****

****




A Nuclear Lysates B

p65 His H3

Whole Lysates

HSP27

p-IκBα

IκBα

p65

Survivin

IL-6

β-actin

SC

C-2

5

SC

C-1

5

SC

C-9

CA

L27

SC

C-1

5/P

YM

SC

C-2

5

SC

C-1

5

SC

C-9

CA

L27

NF

-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

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pLE

X-H

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27

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on

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27

Nuclear Lysates

Whole Lysates

Mock

rhH

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27

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on

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SP

27

Mock

rhH

SP

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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+

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27

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C N

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Rela

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D

SCC-15 SCC-25 SCC-15 SCC-25

Figure 3

*** *** ***

*** ***

*** ***

*** ***

*** *** ***

*** ***

***

** **

SCC-15 SCC-25 **

** **

**

** **

*** *** ***

** ** **

** ** ** **

**

SC

C-1

5/P

YM

***

*** ***

***

***

***

***

***




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#

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-3#

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-4#

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+sh

-3#

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-3#

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+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

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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




β-actin

BAD

BAX

BIM

Cyto C

COX Ⅳ

Whole Lysates

Mitochondrial Fraction

Cytosolic Fraction

BAD

BAX

BIM

Cyto C

β-actin

Whole Lysates


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


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

+ + + - - -

- + - + - +

- - - - + +

- + - - - +

+ + + - - -

- + - + - +

- - - - + +

- + - - - +

+ + + - - - - - -

- - - + + + - - -

+ + + - - - - - -

- - - - - - + + +

- - - - - - + + +

+ + + - - - - -

- + - + - + + +

- - - - - + + +

- - - - - + + +

+ + + - - - - -

- + - + - + + +

- - - - - + + +

- - - - - + + +




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




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


HSP27

Cleaved Cas3 High 15 23

Low 33 13 0.0040


HSP27

Cleaved Cas3 High 14 26

Low 29 12 0.0018


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




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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HSP27 Confer Chemo Resistance in Squamous Cell Carcinoma ... · 12/15/2017 · old female Balb/C athymic nude . mice. All mice were housed and maintained und. er specific pathogen-free