Role of heat shock proteins in oral squamous cell carcinoma : A systematic review

Background. Environmental and patho-physiologic stresses stimulate synthesis of heat shock proteins (HSPs) which enable the cell to survive and recover from stressful conditions, by as yet incompletely understood mechanisms. Heat shock proteins show an increased expression in a wide range of human cancers and have been associated with tumor cell proliferation, differentiation, invasion, metastasis, death, and recognition by the immune system. Yet the role of heat shock proteins in oral cancer is ambiguous. The objective of this review was to systematically assess the data available on the role of HSP expression in oral cancer with special reference to its role in diagnosis, prognosis and treatment. Methods and Results. A systematic review of studies that investigated the HSP expression in oral squamous cell carcinoma using Scopus, Medline, Embase and Google scholar databases from their inceptions to 2013, without language restrictions was conducted. We selected 24 studies from which data extraction and validations were performed. Conclusion. The literature search revealed differential expression of HSPs during oral tumorigenesis with implications for the specific role of HSPs in the pathogenesis of oral cancer. HSP expression has been regarded as an independent prognostic factor for oral squamous cell carcinoma patients and HSPs are being explored as potent vehicles for delivery of preventive and treatment vaccines in cancer and other diseases.


INTRODUCTION
Heat shock proteins are a group of highly conserved proteins which first came to fame as gene products whose expression is induced by heat and other stresses 1,2 .More recently the focus of research has shifted to understanding the roles of HSPs as molecular chaperones [3][4][5] .They are now known to play diverse roles, even in unstressed cells, in successful folding, assembly, intracellular localization, secretion, regulation, and degradation of other proteins 6 .Failure of these activities is thought to underlie numerous serious human diseases 7 .Heat shock proteins appear to play dual and contradictory roles in cancer pathogenesis.On the one hand, they endow tumor cells with stress resistance and also promote growth and survival of tumor cells by engaging misfolded or aggregated proteins involved in cell proliferation.However they can also promote tumor immunity by stimulating the innate immunity mechanisms and enhancing cross presentation of tumor antigens to lymphocytes 8 .
In this article we have systematically reviewed the data available on the role of heat shock proteins (HSPs) expression in oral squamous cell carcinoma (OSCC) with special emphasis to their role in diagnosis, prognosis and treatment of the disease.

MATERIALS AND METHODS
The scheme used in the literature review was to identify all reports of investigations that had been undertaken to assess the role of Heat Shock Proteins in Oral Squamous Cell Carcinoma.A structured search was done of diverse subject-unique databases.The literature searches were done using short-string Boolean-based methodologies.Different Boolean search phrases were applied.These are denoted within < > brackets.The search phrase "operators" are presented in capital letters (e.g., < heat shock proteins AND cancer>).Various search terms were incorporated in the searches (e.g., "oral cancer", "oral squamous cancer", "heat shock proteins","chaperones" and "molecular chaperones").All searches were free of bias and were unrestricted (i.e., excluded limits); thus, the explorations included all (a) fields (e.g., author and title), (b) languages, (c) dates, (d) subjects (e.g., humans and animals), and (e) database subject subsets (e.g., cancer and heat shock protein).The literature search, done using PubMed, Pub med central, Scopus, Medline, Embase and Google scholar databases identified articles published from 1995 to 2013.
The literature searches were extended by using the "Related Articles" link to articles recovered with PubMed.Thereafter, a search was done of the literature references cited in these articles to identify additional writings that reported the results of research defining the role of heat shock proteins in oral squamous cell carcinoma.At this juncture, all articles were assembled and arranged in reverse chronological order.The articles were read, and off topic articles were excluded.By way of example, rejected articles included those that reported research on oral cancer other than heat shock proteins.Likewise, reports of investigations of heat shock proteins that were not associated with oral squamous cell carcinoma (e.g.oesophageal cancer) or had a more diverse group (e.g. head and neck cancer) were excluded.

RESULTS
A selection of 24 research articles was included for systematic review.Of these 17 research articles had used the immunohistochemical expression of HSPs in oral squamous cell carcinoma biopsy tissue, with 11 studies evaluating the concomitant HSP expression in dysplastic tissue and normal oral epithelium.The studies were read and on the basis of the role of heat shock proteins they were categorized under the following subheads: Implications in  1), Implications in prognosis (Table 2) and Therapeutics implications (Table 3).It was noted that most studies in Table 1 and 2 were retrospective studies using immunostaining techniques.Whereas the studies on therapeutic implications were done on oral squamous cell carcinoma cell lines using flowcytometry or other methods such as RNA transfection.

DISCUSSION
HSPs are now also more generally known as stress proteins because they are induced in response to a wide variety of physiological and environmental insults 9 .The HSPs are categorized as molecular chaperones, proteins, which have in common the property of modifying the structures and interactions of other proteins 10 .Mammalian HSPs have been classified into 5 families according to their molecular weight: HSP110, HSP90, HSP70, HSP60 and the family of small HSPs (ref. 9).Among the different HSPs, the ATP-dependent chaperone families HSP70 and HSP90 are the most studied by their involvement in cancer 9 .In our review we found that amongst all the HSPs the role of HSP70 in oral cancer had been explored the most, followed by HSP 27, HSP 90 and HSP 60.
HSP expression is tailored for induction by the stress response, and the proximal signal for HSP induction is apparently the accumulation of denatured proteins 11 .The molecular mechanisms responsible for over expression of heat-shock proteins in cancer cells are yet to be understood but may be tumor specific 12 .Suggested hypothesis is that the physiopathological features of the tumor microenvironment (low glucose, pH, and oxygen) tend toward HSP induction 13 .Another proposed mechanism states that oncoproteins may appear during carcinogenesis (e.g., mutated p53), and these mutated conformationally altered proteins may elicit an HSP response.Kaur J et al. 14 (1996) proposed that p53-HSP70 complex formation may be one of the mechanisms of stabilisation of p53 protein resulting in its increased levels in potentially malignant and malignant oral lesions and may be implicated in oral carcinogenesis.

DIAGNOSTIC IMPLICATIONS OF HSP IN ORAL CANCER
HSP expression was analyzed in relation to the histopathological characteristics of the tumor tissues (e.g.grade of differentiation), with the expression of other molecules (e.g.mutated p53), and with patient parameters like sex and age.In addition, HSP expression has been evaluated in the spectrum of normal, premalignancy to malignancy.Table 1 summarizes the work done in context with the diagnostic implications of the HSPs in cancer.
The following inferences were derived by analyzing the available data.(a) HSP expression levels can help indicate the presence of abnormal changes during the process of carcinogenesis.For example, HSP70 is intensely expressed in OSCC, shows increased expression in premalignant lesions and is not expressed in normal oral mucosa 23 .(b) HSP expression correlates with the degree of differentiation in oral cancer.Most studies show an increased HSP70 expression in poorly differentiated oral squamous cell carcinoma.Most studies on HSP 27 in oral squamous cell carcinoma revealed a strong association with decreased expression of HSP27 being linked to poor tumor differentiation 24,25 although some studies showed no correlation between HSP27 expression and histological grade 20 .
HSP70 has been involved not only with poor tumor differentiation but also with higher clinical stage 15 and advanced age 26 .
(c) HSPs are co-expressed in cancer tissues.In addition, certain HSPs can be significantly associated with other molecules.For example, co-immunoprecipitation of p53 and HSP70 in potentially malignant lesions (dysplasia) and oral squamous cell carcinomas (SCCs).This co-expression is suggestive of a physical association, resulting in p53-HSP70 complex formation 14 .
(d) HSPs are not particularly useful in diagnostic immunopathology since they are expressed in a wide range of malignant cells and tissues 13 .

PROGNOSTIC IMPLICATIONS OF HSP IN ORAL CANCER
Heat shock proteins have been shown to be necessary for survival and proliferation of oral tumor cells 18 and their association with oral carcinogenesis and differentiation of tumor cells is also well established.Therefore, it was logical to study the prognostic implications of HSPs in order to establish their role as markers which can be used to predict treatment outcomes and survival rates of the patient.
On evaluating the available literature (Table 2) we found that most studies associated poor prognosis of oral cancer patients with decreased expression of HSPs (ref. 21,22,24).Although some authors correlated HSP expression inversely with survival time 16,21 .A few other studies demonstrated to prognostic correlation with HSP expression 17 .These variable results may be accountable for the fact that Heat shock proteins till date are not in the list of useful prognostic markers in oral cancer.

THERAPEUTIC IMPLICATIONS
Intracellular HSPs have a protective role in allowing cells to survive potentially lethal conditions largely attributable to their anti-apoptotic properties.HSP 27, HSP70 and HSP90 can directly interact with different proteins of the tightly regulated programmed cell death machinery and thereby block the apoptotic process at distinct key points 9 .In cancer cells, the remarkably increased HSP In contrast to intracellular HSPs, extracellularly located or membrane-bound HSPs mediate immunological functions.They can elicit an immune response providing a link between innate and adaptive immune systems 9 .
In context to oral cancer therapeutics it was found that most research was done on OSCC cell lines and focussed on the extracellular HSPs exploiting their carrier function for immunogenic peptides.The following inferences were drawn from the studies (Table 3

CONCLUSION
Our analysis of heat shock proteins in oral cancer indicated an immense role for HSP in many aspects of tumor progression and response to therapy.Although at the diagnostic level HSPs did not seem very relevant, they were found to be helpful biomarkers for carcinogenesis in oral cancer tissues and point towards their degree of differentiation and aggressiveness.
The established anti-apoptotic function of HSPs 27, 70 and 90 explains their increased expression in cancer cells.Yet the surface and extracellular HSPs constitute an important arm of adaptive and innate immune responses against cancer cells.This dual role of HSPs depending on their intracellular or extracellular location may be a big challenge in cancer therapy.
The comprehension of the role of HSPs in oral squamous cell carcinomas is still in an early stage, and little tangible information is available on how HSP affects the molecular events involved with tumor proliferation and invasiveness.More research will be necessary in order to correctly construe the role of HSPs in oral cancer and targeting HSPs in oral cancer therapy.

Table 1 .
Implications in diagnosis of oral cancer.
26Oral dysplasia and cancer p53-HSP70 complex formation may be one of the mechanisms of stabilisation of p53 protein resulting in its increased levels in potentially malignant and malignant oral lesions and may be implicated in oral carcinogenesis.26OSCC, OS Increase in HSP70 expression from OSMF to OSCC Inc HSP exp >50yrs in OSCC pts No association with gender , habits and duration of habits diagnosis (Table

Table 2 .
Implications in prognosis of oral cancer.

Table 3 .
Implications in therapuetics of oral cancer.
). HSPs are vital to survival and proliferation of oral tumor cells.Studies by yonekura et al. showed HSPs 27 over expression in oral squamous cell carcinoma cell lines and resistance to apoptotic stimuli conferring a poorer prognosis.IFN-gamma downregulates HSP 27 expression and therefore promotes cells to a proapoptotic state and / aborted apoptosis.The authors suggest combining INF gamma with other anticancer drugs and propose that HSP 27 may be a useful marker of sensitivity to anticancer drugs in OSCC.Studies carried out by Atre et al. demonstrated a novel strategy adapted by oral tumor cells to escape immune recognition.They explained the paradoxical role of HSPs in immune recognition by activated γδT lymphocytes of tumor cells and also subsequent apoptosis induced cell death of these γδT lymphocytes.Kim et al suggested that HSP 27 might contribute to regulating photodynamic therapy induced apoptosis in the photodynamic therapy resistant oral tumor cells.They recommended targeting HSP 27 as an adjunctive therapy to photodynamic therapy.