HSP105 expression in oral squamous cell carcinoma: correlation with clinicopathological features and outcomes

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(1)Article. HSP105 expression in oral squamous cell carcinoma: correlation with clinicopathological features and outcomes ARVANITIDOU, Souzana, et al.. Abstract Heat shock proteins (HSPs) are released in response to stress situations, such as heat, inflammation, infection. They are also involved in the tumor cell proliferation and prevention of apoptosis. Heat shock protein 105 (Hsp105/110) is a high-molecular-weight protein which has been reported in many cancer types but few studies have been carried out on oral squamous cell carcinoma (OSCC). In the current study we have focused on HSP105 expression on OSCC and evaluated their correlation with tumor clinicopathological parameters and patients' survival.. Reference ARVANITIDOU, Souzana, et al. HSP105 expression in oral squamous cell carcinoma: correlation with clinicopathological features and outcomes. Journal of Oral Pathology and Medicine, 2020. PMID : 32128880 DOI : 10.1111/jop.13007. Available at: http://archive-ouverte.unige.ch/unige:132462 Disclaimer: layout of this document may differ from the published version..

(2) Accepted Article. DR NICOLAS DULGUEROV (Orcid ID : 0000-0001-8676-3698) PROFESSOR TOMMASO LOMBARDI (Orcid ID : 0000-0001-9347-3736). Article type. : Original Article. HSP105 expression in oral squamous cell carcinoma: correlation with clinicopathological features and outcomes.. Souzana Arvanitidou1, Carla P. Martinelli-Kläy1, Samson Jacky1, Johannes A. Lobrinus 2, Nicolas Dulguerov3, Tommaso Lombardi1. 1- Laboratory of Oral & Maxillofacial Pathology, Oral Medicine and Oral and Maxillofacial Pathology Unit, Division of Oral Maxillofacial Surgery, Department of Surgery, Geneva University Hospitals, University of Geneva, Switzerland.. 2- Division of Clinical Pathology, Geneva University Hospitals, University of Geneva, Switzerland. 3- Department of Oto-Rhino-Laryngology, Head and Neck Surgery, Geneva University Hospitals, University of Geneva, Switzerland.. Running head: HSP105 in OSCC This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/JOP.13007 This article is protected by copyright. All rights reserved.

(3) Accepted Article. Reprint requests and correspondence to: Prof. T. Lombardi Laboratory of Oral & Maxillofacial Pathology, Oral Medicine and Oral and Maxillofacial Pathology Unit Rue Michel-Servet 1 1211 Genève 4 Switzerland.. e-mail: tommaso.lombardi@hcuge.ch Phone number: +41 22 379 4034. Abstract. Background. Heat shock proteins (HSPs) are released in response to stress situations, such as heat, inflammation, infection.. They are also involved in the tumor cell proliferation and prevention of apoptosis. Heat shock protein 105 (Hsp105/110) is a high-molecular-weight protein which has been reported in many cancer types but few studies have been carried out on oral squamous cell carcinoma (OSCC). In the current study we have focused on HSP105 expression on OSCC and evaluated their correlation with tumor clinicopathological parameters and patients’ survival. Methods: A retrospective study included 70 patients with OSCC of which 50 patients (71.4%) were male and 20 (28.6%) were female. The patient´s information, including age, location, TNM stage, histological grade, regional metastasis, recurrence and survival, were collected. Immunohistochemical staining for HSP105 was performed. The healthy oral mucosa (n=10) was used as a control. The staining intensity and percentage of. This article is protected by copyright. All rights reserved.

(4) Accepted Article. stained cells were semi-quantitatively evaluated and HSP105 expression was correlated with tumor clinicopathological features and patient survival. Results: Statistical analysis for HSP105 showed that there was no significant correlation with tumor clinicopathological features. However, HSP105 overexpression was associated with a decrease in the duration of patients’ survival (p= 0.042). Conclusion: This result suggests that the increased expression of the HSP105 in the OSCC could be a prognostic factor for malignancy. Key words: heat shock protein 105, oral squamous cell carcinoma, oral cancer, immunohistochemistry.. Introduction. Heat shock proteins (HSPs) participate in the maintenance of protein homeostasis in physiological state and under stressful conditions. It functions as molecular chaperons that bind to proteins denoted as “client proteins” and, together, form multi molecular complexes. The failure of these activities is suggested to underlie human diseases like cancer. It has been shown that the HSP chaperone machinery is associated with various client proteins which are involved in tumour cell growth and survival, collectively termed as oncoproteins. The chaperoning of these client proteins allows tumour cells to tolerate the mutations of multiple signalling pathways that, otherwise, would be lethal, thus, playing an important role in the control of the cell cycle, growth and apoptosis.1-3 HSPs have been classified into 6 major families according to their molecular size: the large HSPs of 105-110kDa, the HSP of 90kDa, the HSP of 70kDa, the HSP of 60kDa, the HSP of 40kDa and the small HSPs of 18 to 30kDa. Among other HSPs, HSP 105 plays an important role in the oncogenesis.1-3. Hsp105 is a high-molecular-weight protein belonging to a subgroup of the HSP70 family, HSP105/110. It has only been studied for the past few years and its cellular interactions are not yet fully recognized. Among other functions, HSP105 have an anti-apoptotic function and be involved in tumorigenesis as its overexpression is essential for cancer cells to survive.2,4 HSP 105 can suppress cell apoptosis by preventing the release of cytochrome c from the mitochondria5 or, it can contribute to the stabilization of p53 proteins in the testicular germ cells.6 The upregulation of HSP105 expression has been reported in many cancer types7-9 but few studies have been carried out on OSCC.. This article is protected by copyright. All rights reserved.

(5) Accepted Article. The aim of this study was to analyze the HSP105 expression in the OSCC and to determine its relation with tumor clinicopathological parameters and patient survival.. Materials and methods This retrospective study included initially 103 (74 male and 29 female) patients with oral squamous cell carcinoma who underwent surgical tumour resection between 1993 and 2003 in the Department of Oto-RhinoLaryngology and Cervico-Facial Surgery of the Geneva Medical University Hospital, Geneva, Switzerland. From the initial pool of 103 patients, 33 patients were excluded for inadequate clinical information. Neither chemotherapy nor radiotherapy was administered prior to surgery and additionally the absence of distant (haematogenous) metastases was confirmed by PET scan. Approval for tissue collection and patient clinical information for medical research purposes was granted by the board of the Geneva University Hospital Central Ethical Committee (protocol number 07-149R). The biopsies and/or excisions of oral squamous cell carcinomas were obtained from the archives of the Department of Pathology and Immunology, Geneva Medical University Hospital, Geneva, Switzerland. Healthy oral mucosa samples (n=10) were used as control and comprehended 1 case of the vestibular mucosa, 1 of the lower lip, 6 of the buccal mucosa and 2 of the palatine mucosa. Seventy patients were included in this study. The tumour staging was assessed by using the UICC (Union for International Cancer Control) TNM staging system of malignant tumours10 and further classified as stage I, II, III, IV. The OSCC cases were evaluated histologically from tissues stained with haematoxylin/eosin and graded according to the system proposed by World Health Organisation (WHO). The data are summarized in Table 1. Immuohistochemical staining for HSP105. Four-micrometer-thick sections were deparanized in xylene, rehydrated in graded alcohol and rinsed with distilled water. The epitope retrieval was performed by pressurized boiling (Pascal Pressurized Heating Chamber - DakoCytomation, Glostrup, Denmark) using EDTA buffer (pH 7, 3 minutes). The sections were then rinsed in Tris/Tween solution for 10 min. The endogenous peroxidase activity was blocked after treatment with Dako REAL™ Peroxidase-Blocking Solution for 5 minutes. This was followed by incubation with the anti-HSP105 polyclonal primary antibody (code: sc-6241, Santa-Cruz Biotechnology Inc., Santa Cruz, California, concentration: 200μg/ml) diluted at 1:200, for 60 min, at room temperature. After several washes with Tris/Tween buffer solution for 5 minutes, the sections were incubated with an anti-Rabbit immunoglobulin (IgG) EnVision™ + Dual Link System-HRP (Dako Laboratories), for 30 min. The sections. This article is protected by copyright. All rights reserved.

(6) Accepted Article. were then rinsed gently with Tris/ Tween buffer solution for 5 minutes. The staining was developed with 3,3’diaminobenzidine-hydrogen peroxidase (DAB+) chromogen for 10 minutes, rinsed and counterstained with haematoxylin for 2 minutes (Dako REAL ™ Hematoxylin). Afterwards, the slides were rinsed with alcohol and in xylene solutions. The slides were then mounted and examined by light microscopy (Zeiss, Axiostar plus) in 100x and 400x magnification. Negative controls sections in each run of staining were prepared using normal mouse immunoglobulin (isotype: IgG1, concentration: 100μg/ml, Dako) diluted at 1:15000.. Evaluation of HSP105 expression HSP105 expression was characterized by semi-quantitative evaluation of both intensity and proportion of stained cells in each slice separately. The intensity and the extent of immunostaining were evaluated by two different oral pathologists (CPM, TL). The immunohistochemical labeling was inhomogeneous with staining varying from negative to strongly positive in the same sample. Therefore, the intensity of the positive tumor cells was evaluated with the help of a 5-grade scale as follows: 0 (-), 1 (-) to (+), 2(-) to (++), 3 (-) to (+++), 4 (+) to (++), 5 (+) to (+++). The extent of the positive tumour cells was scored using a 5-grade scale as follows: (0) no staining, (1) 1 to 10%, (2) 11 to 25%, (3) 26% to 50%, (4) 51% to 70% (5) more than 70% positively stained tumor cells. For the interpretation of the results due to the fact that the previous mentioned 5-grade scales were overdetailed for the number of the available samples, the extent of expression was classified as negative to weak when the score was (0), (1) and (2) thus 0% to 25%, as moderate when (3) 26% to 50% and as strong when the score was (4) and (5) 51% to 100%. Overall, when the stained cells accounted for > 25% of the total in the samples examined, the expression of HSP105 was evaluated as moderate to strongly positive. If the frequency was lower than < 25% the expression was considered as weak to negative. Similarly, the intensity of immunoreactivity was classified as negative to weak when the score was (0) and (1) thus (-) and (+), as moderate when (2) thus (-) up to (++) and strong when (3), (4) and (5) thus up to (+++). Overall, when staining intensity of tissue samples was + and between ++ and +++ the expression of HSP105 was characterized as weak and moderate to strongly positive respectively. In addition, the percentage of the subcellular localization of HSP105 was evaluated as cytoplasmic, nuclear or both. Statistical analysis The statistical significance of the data was analyzed by the chi-square test. The survival advantage was calculated by the Kaplan-Meier method and statistical significance was analyzed by log rank test. Multivariate analysis was performed based on Cox proportional hazard regression model to identify the independent prognostic factors. The Spearman rank correlation test was used to assess the relationship among HSP105. This article is protected by copyright. All rights reserved.

(7) Accepted Article. extent and intensity of expression. Statistical analysis was performed using SPSS software for Windows. (Version.17.0, SPSS Inc., Chicago, IL USA). Statistical significance was set at p<0.05 throughout the study. Results. In healthy oral mucosa epithelia, a weak cytoplasmic immunoreactivity for HSP105 was occasionally observed (Fig 1A). In OSCC both cytoplasm and nucleus staining were found in 75.7% of tumors (53 out of 70 cases) while in 5.7% (4 out of 70 cases) there was only cytoplasmic staining. Thirteen cases (18.6%) were totally negative for HSP 105. Representative tissue immunostaining for HSP105 in OSCC samples are illustrated in Fig 1 (B and C). The intensity of HSP105 expression was moderate and strong in 64.3% (45 of 70 cases), whereas 35.7% (25 of 70 cases) showed negative and weak expression. The staining extent of HSP105 in OSCC samples was moderate and strong in 68.6 % (48 of 70 cases), whereas 31.4% (22 of 70 cases) showed negative and weak expression. To verify the possible role of HSP105 as a biomarker of prognosis in the malignant expression, correlations of the HSPs immunostaining with patients’ characteristics and tumor clinicopathological features (tumor stage, histological grade, lymph node invasion, tumor location, and recurrence) were achieved and are presented in Table 2. A moderate to strong expression of HSP105 appeared in 11 cases of stage I (61.1%), 4 cases of stage II (50%), 10 cases of stage III (71.4%) and 23 cases of stage IV (76.7%). No statistically significant correlation was found between HSP105 expression and advanced tumor stages (p=0.17). Similarly, a moderate to strong expression of HSP105 was detected in 18 (62.1%) well differentiated, in 26 (74.3%) moderate and 4 (66.7%) poor differentiated OSCC samples (p=0.47). Our results revealed no statistical significance between HSP105 expression and advanced histological grades. Collectively, no statistically significant correlation between HSP105 expression pattern and tumor clinicopathological parameters examined or patient’s characteristics was revealed. Univariate analysis showed that HSP105 extent (p=0.028) and intensity (p=0.050) of expression was found significantly associated with patients’ survival. More particularly, mortality among patients with no observed presence of the protein (that is, percentage 0%) is notably lower (15.4%) than mortality of patients with higher HSP105 percentage 37.5%, 75%, 43.8% and 53.6% for (1-10%), (26-50%), (51-70%) and (>70%) respectively (Table 3). In multivariate analysis using the Cox hazards regression model, HSP105 extent and intensity of expression was identified to be among the strongest independent prognostic factors together with the positive lymph node involvement for OSCC patients (p=0.031). As shown in Table 4 that depicts the significant variables, the relative hazard for high HSP105 expression (2.93) is slightly lower than that of a positive lymph node status (3.46). The Kaplan Meyer analysis of HSP105 protein expressions (negative to weak versus moderate to strong) was performed and the. This article is protected by copyright. All rights reserved.

(8) Accepted Article. cumulative survival curves for OSCC patients are shown in Fig 2. A lower survival rate was found in patients with moderate to strong HSP105 expression compared with other patients (p= 0.042). Discussion. HSPs play a role in carcinogenesis as they participate in numerous pathways that help cancer cells keep a certain level of functional integrity in tumor proliferation and prevention of apoptosis.1-4 Therefore, in the. current study we have focused on HSP105 and evaluated its expression patterns in OSCC. In healthy oral mucosa epithelium, the expression of HSP105 was considered weak. Previous reports in the literature observed a slight immunoreactivity in normal skin epidermis11,12 or a weak HSP105 staining in normal colorectal mucosa13 which is consistent with the findings of this study. On the contrary, no staining was found in normal esophageal7,14 and colorectal mucosa.7 These variations regarding HSP105 staining may be the result of different case selection and of different immunohistochemical techniques used. In OSCC, a moderate to strong HSP105 overexpression was identified in 68.5% of the cases examined in this study. The fact that oral carcinoma displays a clearly stronger HSP105 expression than the healthy oral epithelium may be indicative of its role in carcinogenesis and represent a tumor selective therapeutic target. A study has successfully demonstrated a high HSP105 expression in skin SCC that accounted for a total of 60% of the samples investigated.11 In line with the study, an upregulation of HSP105 expression in squamous cell carcinoma of the lung has been identified.15 Also, Monhtasham16 reports progressive increase of HSP105 expression in advanced squamous cell carcinoma samples and postulates the idea of an association of this marker with the lesion stage and worse prognosis. Serum analysis of esophageal squamous cell carcinoma patients showed greater expression of HSP 105 (39.1%) when compared to healthy individuals which had only 5% of positivity.14 In a previous study, Kai et al7 found 100% of HSP105 immunoreactivity in esophageal cancer. However, the same study reported no expression in squamous cell carcinoma of the uterine cervix. In addition, the HSP 105 overexpression was observed during colorectal cancer progression.9 HSP105 immunoreactivity was mainly located in both the cytoplasmic and the nuclear compartment of the cancerous cells. This result is in consistent with the previous reports in skin11 and in esophageal cancer14 and. may be indicative of a translocation that underlines its functional activity.6 It also seems that there are two forms of HSP105, 105α, and 105β. HSP105α is constitutively expressed in the cell cytoplasm while HSP105β is localized in the nucleus and is induced strictly by heat. According to Saito et al,17 HSP105b enhanced the expression of HSP70 in mammalian cells, which suggested that both, together, play an important role in the protection of cells against various stresses. However, the exact role of these two distinct forms of HSP105 has not yet been carefully studied.. This article is protected by copyright. All rights reserved.

(9) Accepted Article. With regard to the correlation between HSP105 expression and clinicopathological parameters, a statistically significant relationship was found between its expression and patient mortality which was also confirmed by the univariate analysis. However, no other association was noted with important tumor clinicopathological characteristics. It can be suggested that this difference may be explained by the probability that the biological effects of HSP105 expression in oral cancer on its clinical behavior might be modulated by the different therapeutic modalities in the course of disease progression. The data of this study are in conformity with the ones presented in SCC of the tongue16 where there was no relationship between histological grade of the lesion and HSP105 expression. Differently, the HSP105 positivity in colorectal cancer was correlated to advanced clinical stages and positive lymph node suggesting a role of the HSP105 in metastatic progression of the tumor.13. When the correlation between HSP105 expression and survival was analyzed, this study showed a statistically significant positive correlation. Moreover, multivariate analysis confirmed that HSP105 expression is an independent risk factor predicting the probability of survival and highlighting its possible role in oral squamous carcinoma patients. This could suggest that its upregulated expression is a prerequisite for the increased growth of tumor cells and may represent a possible biological marker for disease progression. According to Yu et al.,18 HSP110 expression was correlated to upregulation of β-catenin and transcription of Wnt target genes in colorectal and breast cancers. It is known that, Wnt signaling plays a crucial role in the cell physiology regulation (proliferation, differentiation, survival, and self-renewal of stem cells). Abnormal activation of the levels of Wnt ligands or altered activities of the pathway components, can contribute to diverse diseases, including cancer.19 At our knowledge, the current study performed on OSCC is the first that implies a prognostic relevance between survival period of oral cancer patients and HSP105 expression. Similar results are presented in squamous cell carcinoma of the lung15 in which a relationship between low expression of HSP105 and longer patient survival is confirmed and also in lung adenocarcinoma,20 where high HSP105 expression score is an unfavorable factor. However, Kawai et al.,21showed that a high HSP105 score was significantly associated with a favorable prognosis in patients with urinary bladder cancer treated with radical cystectomy. In line with our study, the poor prognosis was also associated with HSP 105 overexpression in myeloblastic syndromes,8 in colorectal carcinomas22 and in gastric cancer.23 Different findings suggest that HSP105 may function differently in bladder cancer or elsewhere, compared to oral squamous cell carcinoma and squamous cell carcinoma of the lung. This study proved a statistically significant positive correlation between the expression of HSP105 and patient overall survival and suggested that its upregulation may be a predictive factor for oral carcinoma patients and. This article is protected by copyright. All rights reserved.

(10) Accepted Article. indicative of a poor prognosis. Moreover, multivariate analysis confirmed that HSP105 expression is an independent risk factor predicting the probability of survival and highlighting its possible role in oral squamous carcinoma patients. This novel result for the HSP105 could contribute to the suggestion that the upregulation of its expression is a perquisite for increased growth of tumor cells and may represent a possible biological marker for disease progression. Taking also into consideration the accumulative evidence that confirms an involvement of HSP105 in the prevention of apoptosis by binding to the tumor suppressor gene p53,24 we can speculate that overexpression of HSP105 is responsible for the increased survival of malignant cells. However, there is limited information concerning the exact mechanism as it has not yet been determined. HSP105 function and its immunohistochemical expression profile deserve a more rigorous future study in order to help understand its role in oral carcinoma which may lead to new therapeutic approaches in cancer treatment via the development of HSP105 inhibitors. Conclusion. HSP105 overexpression represented an independent predictive prognostic marker, highlighting its potential role in disease progression. In spite of the limited sample size, the finding of this study indicates that augmentation in HSP105 expression is correlated with poor prognosis. However, further larger scale studies are needed to establish the usefulness of HSP 105 overexpression in oral carcinomas as a prognostic tool in patients’ survival. Acknowledgements: The authors greatly thank technicians Eliane Dubois and Claire Herrmann, for their valuable contribution. Conflict of interest statement: The authors have no conflict of interests.. References. 1. Bablani Popli D, Sircar K, Chowdhry A, Rani V. Role of heat shock proteins in oral squamous cell carcinoma: A systematic review. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2015; 159:366371.. This article is protected by copyright. All rights reserved.

(11) Accepted Article. 2. Zuo D, Subjeck J, Wang XY. Unfolding the Role of Large Heat Shock Proteins. New Insights and Therapeutic Implications. Front Immunol. 2016; 7:75. 3. Calderwood SK. Molecular cochaperones: tumor growth and cancer treatment. Scientifica. (Cairo) 2013; 2013:217513.. 4. Hosaka S, Nakatsura T, Tsukamoto H, Hatayama T, Baba H, Nishimura Y. Synthetic small interfering RNA targeting heat shock protein 105 induces apoptosis of various cancer cells both in vitro and in vivo. Cancer Sci. 2006; 97:623-632. 5. Yamagishi N, Ishihara K, Saito Y, Hatayama T. Hsp105 family proteins suppress taurosporine-induced apoptosis by inhibiting the translocation of Bax to mitochondria in HeLa cells. Exp Cell Res. 2006; 312:32153223.. 6. Kumagai J,Fukuda J, Kodama H, Murata M, Kawamura K, Itoh H, Tanaka T. Germ cell-specific heat shock protein 105 binds to p53 in a temperature-sensitive manner in rat testis. Eur J Biochem. 2000; 267:3073-3078. 7. Kai M, Nakatsura T, Egami H, Senju S, Nishimura Y, Ogawa M. Heat shock protein 105 is overexpressed in a variety of human tumors. Oncol Rep. 2003; 10:1777-1782. 8. Duval A, Olaru D, Campos L, Flandrin P, Nadal N, Guyotat D. Expression and prognostic significance of heat-shock proteins in myelodysplastic syndromes. Haematologica. 2006; 91:713-714. 9. Slaby O, Sobkova K, Svoboda M, Garajova I, Fabian P, Hrstka R, Nenutil R, Sachlova M, Kocakova, I, Michalek J, Smerdova T, Knoflickova D, Vyzula R. Significant overexpression of Hsp110 gene during colorectal cancer progression. Oncol Rep. 2009; 21:1235-1241. 10. TNM Classification of malignant tumours Sobin LH, Wittekind C. 5th edition New York, Wiley Liss 1997. 11. Muchemwa FC, Nakatsura T, Ihn H, Kageshita T. Heat shock protein 105 is overexpressed in squamous cell carcinoma and extramammary Paget disease but not in basal cell carcinoma. Br J Dermatol. 2006; 155:582-585.. 12. Park HS, Park CH, Choi BR, Lim MS, Heo SH, Kim CH, Kang SG, Whang KU, Cho MK. Expression of heat shock protein 105 and 70 in malignant melanoma and benign melanocytic nevi. J Cutan Pathol. 2009; 36:511-516.. 13. Hwang TS, Han HS, Choi HK, Lee YJ, Kim YJ, Han MY, Park Y. M. Differential, stage-dependent expression of Hsp70, Hsp110 and Bcl-2 in colorectal cancer. J Gastroenterol Hepatol 2003; 18:690-700.. This article is protected by copyright. All rights reserved.

(12) Accepted Article. 14. Gao H, Zheng Z, Mao Y, Wang W, Qiao Y, Zhou L, Liu F, He H, Zhao X. Identification of tumor antigens that elicit a humoral immune response in the sera of Chinese esophageal squamous cell carcinoma patients by modified serological proteome analysis. Cancer Lett. 2014; 344:54-61.. 15. Ullmann R, Morbini P, Halbwedl I, Bongiovanni M, Gogg-Kammerer M, Papotti M, Gabor S, Renner H, Popper HH. Protein expression profiles in adenocarcinomas and squamous cell carcinomas of the lung generated using tissue microarrays. J Pathol. 2004; 203:798-807. 16. Mohtasham N, Babakoohi S, Montaser L,Memar B, Salehinejad J, Rahpeyma A, Khageh-Ahmady S, Marouzi P, Firooz A, Pazoki-Toroudi H, Anvari K. The expression of heat shock proteins 27 and 105 in squamous cell carcinoma of the tongue and relationship with clinicopathological index. Med Oral Patol Oral Cir Bucal. 2011; 6:730-735. 17. Saito Y, Yamagishi N, Hatayama T. Nuclear localization mechanism of Hsp105beta and its possible function in mammalian cells. J Biochem. 2009; 45:185-191. 18. Yu N, Kakunda M, Pham V, Lill JR, Du P, Wongchenko M, Yan Y, Firestein R, Huang X.. HSP105 recruits protein phosphatase 2A to dephosphorylate β-catenin. Mol Cell Biol. 2015; 35:1390-1400. 19. MacDonald BT, Tamai K, He X. Wnt/β-catenin signaling: components, mechanisms, and diseases. Dev Cell. 2009; 17:9-26. 20. Oda T, Morii E, Inoue M, Ikeda J, Aozasa K, Okumura M. Prognostic significance of heat shock protein 105 in lung adenocarcinoma. Mol Med Reports 2009; 2: 603-607. 21. Kawai T, Enomoto Y, Morikawa T, Matsushita H, Kume H, Fukayama M, Yamaguchi H, Kakimi K, Homma Y. High expression of heat shock protein 105 predicts a favorable prognosis for patients with urinary bladder cancer treated with radical cystectomy. Mol Clin Oncol. 2014; 2:38-42.. 22. Oh HJ, Kim JH, Lee TH, Park HE, Bae JM, Lee HS, Kang GH. Dominant high expression of wild-type HSP110 defines a poor prognostic subgroup of colorectal carcinomas with microsatellite instability: a wholesection immunohistochemical analysis. APMIS. 2017; 125:1076-1083. 23. Kimura A, Ogata K, Altan B, Yokobori T, Ide M, Mochiki E, Toyomasu Y, Kogure N, Yanoma T, Suzuki M, Bai T, Oyama T, Kuwano H. Nuclear heat shock protein 110 expression is associated with poor prognosis and chemotherapy resistance in gastric cancer. Oncotarget. 2016; 7:18415-18423.. This article is protected by copyright. All rights reserved.

(13) Accepted Article. 24. Kampinga H, Hagerman HJ, Vos MJ, Kubota H, Tanguay RM, Bruford EA, Cheetham ME, Chen B, Hightower LE. Guidelines for the nomenclature of the human heat shock proteins. Cell Stress Chaperones 2009; 14: 105-11.. Legends. Figure 1. HSP105 expression: positive slightly cytoplasm in the healthy oral squamous epithelium (A; immunoperoxidase, 100x) and, moderate to strong expression (B; immunoperoxidase, 200x) in both nuclear and cytoplasmic departments in OSCC (C; immunoperoxidase, 400x). Figure 2. Cumulative survival curves for OSCC patients according to HSP105 expression: A significant association between HSP105 status and lower survival curves is observed (p=0.042). Table 1. List of the distribution of patients’ clinicopathological parameters. Table 2. Expression status of HSP105 and correlations with clinicopathological features.. This article is protected by copyright. All rights reserved.

(14) Accepted Article. Table 3. Univariate analysis in relation to survival. Table 4. Cox multivariate analysis between survival and other clinicopathological variables indicating independent factors.. This article is protected by copyright. All rights reserved.

(15) Accepted Article. Summary of patient population and tumour characteristics. Sex. Male. 50 (71.4). Female. 20 (28.6). Age (median,mean,range). 60y, 60.9y, 32-83y. TNM Stage I. 18 (25.7). II. 8 (11.4). III. 14 (20.0). IV(IVA, IVB). 30 (42.9). Histological grade Well differentiated (G1). 29 (41.4). Moderately differentiated (G2). 35 (50.0). Poorly differentiated (G3). 6 (8.6). Node invasion Negative. 39 (55.7). Positive. 31 (44.3). Tumour sublocation Mouth floor. 28 (40.0). Tongue. 29 (41.4). Other. 24 (34.3). Table 1.. This article is protected by copyright. All rights reserved.

(16) Accepted Article. Staining extent Number. Staining intensity. (%). Moderate/Strong. Male. 50 (71.4). 30 (60.0). Female. 20 (28.6). 15 (75.0). 16 (80.0). 18 (25.7). 10 (55.6). 11 (61.1). II. 8 (11.4). 4 (50.0). III. 14 (20.0). 9 (64.3). 10 (71.4). IV. 30 (42.9). 22 (73.3). 23 (76.7). G1. 29 (41.4). 18 (62.1). G2. 35 (50.0). 23 (65.7). G3. 6 (8.6). 4 (66.7). Negative. 39 (55.7). 24 (61.5). Positive. 31 (44.3). 21 (67.7). Variables. Moderate/ P Value. Strong. P Value. 32 (64). 0.19. Gender 0.24. TMN, Stage I. 0.16. 4 (50.0). 0.17. Histological Grade 18 (62.1) 0.76. 26 (74.3). 0.47. 4 (66.7). Lymphatic Invasion 0.59. 25 (64.1). 0.47. 23 (74.2). Location Mouth Floor. 42 (60). 18 (64.3). 1. 18 (64.3). 0.53. Tongue. 29 (41.4). 16 (55.2). 0.18. 18 (62.1). 0.32. Other. 24 (34.3). 16 (66.7). 0.76. 17 (70.8). 0.77. 42 (60.0). 29 (69.0). 0.31. 31 (73.8). 0.25. Relapse during Follow up. Table 2. This article is protected by copyright. All rights reserved.

(17) Parameters Gender Male Female Age 32-50 51-60 61-70 71-83 TNM, Stage I II III IV Histological Grade G1 G2 G3 Neck lymph node metastasis Negative Positive Location Mouth Floor Tongue Other Relapse during Follow up Yes No HSP-105 intensity None 0 to + 0 to ++ 0 to +++ + to ++ + to +++ HSP-105 percentage 0% 1-10% 11-25% 26-50% 51-70% >70%. Table 3. Number (%). Deceased. RR. (95% CI). P Value. 50 (71.4) 20 (28.6). 20 (40.0) 10 (50.0). 1 1.35. (0.63-2.90). 0.44. 12 (17.1) 25 (35.7) 21 (30.0) 12 (17.1). 3 (25.0) 10 (40.0) 9 (42.9) 8 (66.7). 1 2.01 2.34 3.1. 0.55-7.34 0.63-8.74 0.82-10.72. 0.051. 18 (25.7) 8 (11.4) 4 (20.0) 30 (42.9). 3 (16.7) 1 (12.5) 8 (57.1) 18 (60.0). 1 0.63 3.5 4.82. 0.06-6.04 0.93-13.24 1.41-16.50. 29 (41.4) 35 (50.0) 6 (8.6). 10 (34.5) 19 (54.3) 1 (16.7). 1 1.96 0.47. 0.91-4.22 0.06-3.71. 0.74. 39 (55.7) 31 (44.3). 11 (28.2) 19 (61.3). 1 2.93. 1.38-6.22. 0.005. 42 (60) 29 (41.4) 24 (34.3). 18 (42.9) 11 (37.9) 11 (45.8). 42 (60) 28 (40.0). 26 (61.9) 4 (14.3). 1 5.46. 13 (18.6) 12 (17.1) 6 (8.6) 36 (51.4) 0 (0) 3 (4.3). 2 (15.4) 5 (41.7) 4 (66.7) 17 (47.2) 2 (66.7). 1 4 8.35 3.83. 13 (18.6) 8 (11.4) 1 (1.4) 4 (5.7) 16 (22.9) 28 (40.0). 2 (15.4) 3 (37.5) 0 (0) 3 (75.0) 7 (43.8) 15 (53.6). 1 3.33. 0.001. 1 0.79 0.72. 5.35. 5.6 4.04 4.45. 1.90-15.69. <0.001. (0.77 – 20.85) (1.51 – 46.07) (0.88 – 16.58) (0.75 – 38.14). 0.05. (0.55 – 20.09) 0 (0.93 – 33.64) (0.84 – 19.49) (1.02 – 19.48). 0.028.

(18) Accepted Article. 95% CI. RR. p value. 1.13-2.39. 1.64. 0.010. tongue or mouth floor. 0.18-0.99. 0.42. 0.046. Nodal status positive vs negative. 1.57-7.62. 3.46. 0.002. HSP105 26-100% versus 0-25%. 1.10-7.78. 2.93. 0.031. Age (for 10 years) Primary location other than. Table 4. This article is protected by copyright. All rights reserved.

(19) Accepted Article. jop_13007_f1.tif. This article is protected by copyright. All rights reserved.

(20) Accepted Article. jop_13007_f2.tif. This article is protected by copyright. All rights reserved.

(21)