|Year : 2013 | Volume
| Issue : 4 | Page : 624-629
HSP70 induces TLR4 signaling in oral squamous cell carcinoma: An immunohistochemical study
Sindhu Nair1, Vijayalakshmi S Kotrashetti1, Ramakant Nayak1, Kishore Bhat2, Pradeep Somannavar1, Jagadish Hosmani1
1 Department of Oral Pathology, Maratha Mandal's NGH Institute of Dental Sciences, Belgaum, Karnataka, India
2 Department of Microbiology, Maratha Mandal's NGH Institute of Dental Sciences, Belgaum, Karnataka, India
|Date of Web Publication||11-Feb-2014|
Department of Oral Pathology, M.M.N.G.H institute of dental sciences and research centre, Belgaum - 590 010, Karnataka
Source of Support: None, Conflict of Interest: None
Objectives: Toll like receptors play an important role in innate and adaptive immune responses. Heat shock proteins play a significant role in cell proliferation, differentiation and oncogenesis. HSP70 acts as one of the ligands of TLR4 and binds to it in a CD14 dependent fashion to bring about proinflammatory cytokine production leading to an anti-tumor response. On the contrary, TLR4 has been implicated in carcinogenesis by secretion of anti-apoptotic proteins. Thus the aim of this study was to compare and correlate the association of HSP70 and TLR4 in various grades of oral squamous cell carcinoma.
Study Design: Twenty-seven cases of oral squamous cell carcinoma were considered. Ten cases each of well-differentiated (WDSCC) and moderately differentiated (MDSCC), 7 cases of poorly differentiated carcinoma (PDSCC) were considered. Sections were stained for HSP70 and TLR4 and were evaluated for staining degree and intensity.
Results and Conclusion: Positive expression of both HSP70 and TLR4 was found in all cases of WDSCC and MDSCC, whereas in PDSCC out of 7 cases only 6 showed positivity for TLR4 and 4 cases showed positivity for HSP70. Those cases that were positive for TLR4, also showed positivity for HSP70. HSP70 acts as a ligand and binds to TLR4 thus activating the My88 pathway resulting in production of proinflammatory cytokines, chemokines, growth factors etc., enhancing anti-cancer immunity in the early stages of disease. In later stages, TLRs expressed on cancer cells can produce anti-apoptotic proteins contributing to carcinogenesis and cancer cell proliferation.
Keywords: HSP70, oral squamous cell carcinoma, TLR4, toll like receptor
|How to cite this article:|
Nair S, Kotrashetti VS, Nayak R, Bhat K, Somannavar P, Hosmani J. HSP70 induces TLR4 signaling in oral squamous cell carcinoma: An immunohistochemical study. J Can Res Ther 2013;9:624-9
|How to cite this URL:|
Nair S, Kotrashetti VS, Nayak R, Bhat K, Somannavar P, Hosmani J. HSP70 induces TLR4 signaling in oral squamous cell carcinoma: An immunohistochemical study. J Can Res Ther [serial online] 2013 [cited 2020 May 25];9:624-9. Available from: http://www.cancerjournal.net/text.asp?2013/9/4/624/126460
| > Introduction|| |
Oral squamous cell carcinoma (OSCC) is the 6 th most common neoplasm occurring worldwide and has a high mortality rate.  Characteristic features include an aggressive growth pattern, local invasiveness, and spread to cervical lymph nodes. Patient outcome depends on the conventional prognostic factors used in clinical practice, but advances in the surgical and nonsurgical treatments have led to increased local tumor control in recent years. However, overall survival rates have not improved because of the prevalence of locoregional tumor recurrence and distant metastasis. Thus, molecular and histological markers must be identified to target tumors with a high likelihood of metastatic spread so as to improve the survival period of affected patients. 
Toll-like receptors (TLRs) are a family of conserved pattern recognition receptors. The activation of TLRs by microbial ligands triggers innate immune responses and primes antigen-specific adaptive immunity towards exogenous pathogens. There is a growing interest in the usage of TLR ligands as agonist for the prevention and treatment of infectious diseases, cancer, and autoimmune diseases.  Ligands of each TLR are different; while TLR2 recognizes bacterial lipoproteins, lipoteichoic acid and fungal zymosan, TLR3, TLR7, TLR8, and TLR9 recognize nucleic acids derived from virus or bacteria; lipopolysaccharide (LPS) from Gram negative bacteria cell wall is recognized by TLR4 and bacterial flagellin activates TLR5.  An increasing number of endogenous damage associated molecular pattern molecules (DAMPs) are being reported as candidate agonists of TLRs. Some of the DAMPs that serve as agonists of TLR4 include heat shock proteins (HSPs), high mobility group box 1 (HMGB1), and uric acid crystals.  HSPs are highly conserved proteins and following stressful stimuli their primary function is ascribed to intracellular molecular chaperones of aberrantly folded or mutated proteins, as well as in cytoprotection. Studies reveal that extracellular human HSP60 and HSP70 appear to activate the innate immunity by a CD14-dependent mechanism. HSP70 utilizes both TLR2 (receptor for Gram-positive bacteria) and TLR4 (receptor for Gram-negative bacteria) to transduce its proinflammatory signal in a CD14-dependent fashion. Thus, receptor complex of CD14 appears to be the principal receptor complex of LPS.  The net effect of TLR signaling activation by its respective ligands results in activation of transcriptional factors such as nuclear factor- κB (NF- κB) and activator protein 1 (AP-1) through myeloid differentiation factor 88 (MyD88) dependent and MyD88 independent pathway. This subsequently results in production of proinflammatory cytokines, chemokines, growth factors etc., thus suggestive of TLR-mediated signaling which enhances the anti-cancer immunity.  On the contrary, studies have also revealed that TLRs expressed on cancer cells can contribute to carcinogenesis and cancer cell proliferation. 
Numerous studies have validated the association between HSP70 and TLR4 in various pathologies. One study revealed that human HSP70 induced pelvic inflammation and thus regulated TLR4-mediated growth of endometriosis. In addition to the pelvic inflammation, endometriosis also produced a stress reaction and released endogenous HSPs in the pelvic environment as a result of tissue damage, tissue invasion and by inflammatory reaction itself.  In another study, HSP70-like protein 1 (HSP70L1) has also been suggested as a potent T helper cell (Th1) polarizing adjuvant that binds directly to TLR4 on the surface of dendritic cells thus activating mitogen activated protein kinase (MAPK) and NF-κB pathways which results in the activation of various chemokines contributing to anti-tumor response. 
On literature search, studies conducted on the expression of HSP70 and TLR4 in OSCC by immunohistochemistry were lacking and therefore the aim of our study was to correlate the association between HSP70 and TLR4 in various grades of OSCC and in normal oral mucosa.
| > Materials and Methods|| |
After obtaining the Institutional Review Board and Ethical Committee approval, a retrospective study was conducted. A total number of 32 cases were considered which included 5 cases of normal oral mucosa and 27 cases of OSCC [Table 1]. The normal oral mucosa was obtained from patients undergoing surgical removal of asymptomatic third molar for the purpose of orthodontic treatment. The study group included histologically diagnosed cases of oral squamous cell carcinoma which were graded based on WHO criteria. All the subjects of OSCC were associated with tobacco habit.
The OSCC group comprised of 10 cases each of well differentiated squamous cell carcinoma (WDSCC) and moderately differentiated squamous cell carcinoma (MDSCC) and 7 cases of poorly differentiated squamous cell carcinoma (PDSCC). The age of the patients in OSCC ranged from 39-75 years, with 20 males and 7 females.
Three sections each of 4 ΅m thickness were obtained and one section was stained with routine hematoxylin and eosin to confirm the diagnosis [Figure 1]a, [Figure 2]a, [Figure 3]a and [Figure 4]a, and other two sections were stained with immunohistochemistry (IHC). For IHC staining, sections were placed on 3-aminopropyl triethoxy silane (APES) (A3648Sigma) coated slides and staining protocol was performed by using supersensitive one step polymer HRP system (D-600N. Biogenex) with primary antibody being Mouse Monoclonal Anti-Human TLR4 (Imgenex) at a dilution of 1:200 and prediluted HSP70 (Clone BR-1-22, Biogenex).
|Figure 1: (a) Photomicrograph showing normal oral mucosa (H and E, ×10 magnification), (b) Photomicrograph showing positive nuclear and cytoplasmic staining for HSP70 in normal oral mucosa restricted to the basal cell layer (×10 magnification), (c) Photomicrograph showing positive cytoplasmic and membranous staining for TLR4 in normal oral mucosa in basal and suprabasal layers (×10 magnification)|
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|Figure 2: (a) Photomicrograph showing well differentiated squamous cell carcinoma (H and E, ×10 magnification), (b) Photomicrograph showing positive nuclear and cytoplasmic staining for HSP70 in WDSCC (×10 magnification), (c) Photomicrograph showing positive cytoplasmic and membranous staining for TLR4 in WDSCC (×10 magnification)|
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|Figure 3: (a) Photomicrograph showing moderately differentiated squamous cell carcinoma (H and E, ×10 magnification), (b) Photomicrograph showing positive nuclear and cytoplasmic staining for HSP70 in MDSCC (×10 magnification), (c) Photomicrograph showing positive cytoplasmic and membranous staining for TLR4 in MDSCC (×10 magnification)|
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|Figure 4: (a) Photomicrograph showing poorly differentiated squamous cell carcinoma (H and E, ×10 magnification), (b) Photomicrograph showing weak nuclear and cytoplasmic staining for HSP70 in PDSCC (×10 magnification), (c) Photomicrograph showing positive cytoplasmic and membranous staining for TLR4 in PDSCC (×10 magnification)|
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The IHC staining protocol was performed by using positive control as inflammatory oral mucous membrane for TLR4 and breast carcinoma for HSP70. Initially the slides were kept overnight in the incubator at 55°C for proper fixation of tissue to the slides, so that there will be limited chances of floating of tissues during antigen retrieval. Subsequently slides were deparaffinized and then dehydrated with graded alcohol and rinsed with distilled water. Antigen retrieval was standardized by using 2 different buffers by using citrate buffer in EZ- retrieval microwave at 96°C at 10 min for 2 cycles. After antigen retrieval, sections were allowed to cool till it reached room temperature. The sections were then thoroughly wiped with tissue paper, slides were rinsed with phosphate buffer saline (PBS) at pH 7.2-7.6 and excess surrounding the sections were wiped by blotting with tissue paper at every step of IHC. Endogenous peroxidase activity was blocked by incubating the slides with 3% H 2 O 2 for 10 min. Power block was used to make a thin casein layer so that all the epitopes were opened. Wash buffer was not used after this step. The slides were then incubated with primary antibody TLR4 (1:200 dilutions) with phosphate buffer and HSP70 for 45 min. Thereafter, slides were incubated with polymer HRP (horse radish peroxidase) secondary antibody for 30 min. To visualize the reaction, slides were finally incubated with DAB substrate chromogen for 10 min after which counterstaining was done with Harris hematoxylin for 5 seconds followed by blueing in running tap water. Thereafter, slides were dehydrated, dipped in xylene and mounted.
To evaluate the immunoexpression, slides were visualized at 10X magnification under binocular light microscope. The expression of both HSP70 and TLR4 was defined as light or dark brown granules. For HSP70 the expression was observed in cytoplasm and nucleus whereas for TLR4 it was observed in cytoplasm or on cell membrane. IHC staining was scored according to percentage of positive cells and staining degree as follows: 0, ≤10% positive; 1, 11% to 25% positive; 2, 26% to 50% positive; 3, 51% to 75% positive; 4, ≥76% positive. Based on intensity the scoring was done as follows - score 1 for light yellow, score 2 for yellow, and score 3 for brown. The two scores were multiplied and the final scores ranged from 0 to 12. A median score was then calculated. The tissue with scores equal to or more than median were defined as the high expression group and those with scores less than median were defined as the low expression group.
| > Results|| |
Expression of both HSP70 and TLR4 was positive in all cases of WDSCC and MDSCC, whereas in PDSCC out of 7 cases only 6 cases showed positivity for TLR4 and 4 cases showed positivity for HSP70 [Table 2] and [Table 3]. All cases of normal oral mucosa showed positivity for HSP70 and TLR4 with the expression of HSP70 being restricted to the basal cell layer and that of TLR4 in basal and suprabasal layers [Figure 1]a-c. High expression for TLR4 was observed in 10 cases (100%) of WDSCC, 6 cases (60%) of MDSCC and 4 cases of (57.14%) PDSCC [Table 3] and [Figure 2]c, [Figure 3]c and [Figure 4]c. Similarly, high expression was observed for HSP70 in 4 cases (40%) of WDSCC, 8 cases (80%) of MDSCC, but low intensity staining was observed in all cases of PDSCC [Figure 2]b, [Figure 3]b and [Figure 4]b. Those cases which showed positivity for HSP70 in WDSCC and MDSCC also showed positivity for TLR4.
|Table 2: Number of cases positive for HSP70 and TLR4 in normal oral mucosa and in OSCC|
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|Table 3: Expression of HSP70 and TLR4 in normal oral mucosa and in various grades of OSCC|
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| > Discussion|| |
Current evidence indicates that TLRs play a crucial role in the activation of innate immunity against invading pathogens, cytokine production, and development of adaptive immune responses.  While the protective role of TLR against pathogen infections have been widely discussed, studies have also suggested that TLR expressed on tumor cells contribute to tumor progression. The development of cancer has been associated with microbial infection, injury, inflammation, and tissue repair. The activation of TLRs in cancer cells and the ensuing signaling cascade with the cytokine/chemokine production may promote cancer cell survival, chemoresistance and therefore tumor progression.  Recent studies show that activated TLRs expressed on cancer cells can suppress the anti-tumor functions of infiltrating immune cells, and thus alter the inflammatory response in such a manner that promotes cancer progression. Positive TLR4 expression has been found in various carcinomas like colorectal cancer, gastric cancer, ovarian cancer, cervical cancer, lung cancer, prostate cancer, melanoma, brain cancer, breast cancer, hepatocellular carcinoma and laryngeal cancer. On the contrary, He et al., have demonstrated negative function of TLR4 expression in human lung cancer cell lines.  This proves that TLR4 action may be two-fold - stimulation of tumorigenesis or anti-tumor effect.
Researchers have found that MyD88 is required in a cell-autonomous fashion for RAS-MAPK signaling, leading to carcinogenesis. This indicates that MyD88 plays an important role not only in the pathway of TLR-mediated inflammation, but also in RAS-MAPK signaling, cell-cycle control, and cell transformation. NF- κB is one of the transcriptional factors for innate and adaptive immunity and plays a crucial role in inflammation against infectious molecules. Studies have found active NF- κB in a number of human malignancies thus recognizing that NF- κB signaling pathway acts as a link between chronic inflammation and tumor development. Recent studies have demonstrated that NF- κB plays an important role in TLRs-induced tumorigenesis when TLRs are activated by their respective ligands.  The ability of TLR to activate the adaptive immune system via its signaling pathway has led to attempts to harness this response against cancer cells through the use of exogenous administration of TLR ligands. More importantly, it has been shown that high doses of TLR agonists can lead to apoptosis and directly kill both tumor cells as well as ancillary cells of the tumor microenvironment, whereas low doses of TLR agonists promote cancer growth. 
In our study, we found high expression of TLR4 in WDSCC and MDSCC and poor expression in PDSCC [Table 3]. Similar results were obtained in separate immunohistochemical studies conducted by Szczepanski et al., on head and neck cancers and Sun et al., on oral cancer, in which a strong positive reaction for TLR4 was characteristic for well-differentiated or moderately differentiated tumors relative to moderate or weak staining intensity in poorly differentiated tumors. , Szczepanski et al., concluded that in vivo activation of the tumor-associated TLR4 by bacterial products in patients with well-differentiated tumors would be likely to induce abundant chronic inflammation, promote tumor growth, and protect tumor cells from apoptosis.  The above data confirms the role of TLR4 in tumorigenesis. Further literature survey showed that TLR4 in association with HSP70 induces proinflammatory signals thus activating the TLR pathway. TLR4-mediated growth of endometriosis by HSP70 has also been studied with positive results.  Considering this, we decided to study the role of HSP70 in the TLR4 signaling pathway in oral squamous cell carcinoma.
In our study, high expression of HSP70 was present in WDSCC and MDSCC, but poor expression in PDSCC [Table 3]. Expression of HSPs on the surface of tumor cells, instead of their normal intracellular location, suggests that they play a role in inducing an immune response against cancer. A tumor-specific transplantation antigen that appears to be an HSP has been identified by Ullrich et al., which is expressed on the cell surface and induces protective immunity.  Also, a differential pattern of expression of HSPs has been correlated with malignant transformation in experimental models.  More recently, the role of HSP70 in tumor progression has been investigated with studies suggesting that HSP70 promotes tumor cell growth by inhibiting apoptosis and/or stabilizing the lysosomal membranes. 
When the expression of HSP70 and TLR4 were analyzed, we found statistical correlation between the expression of HSP70 and TLR4 in oral squamous cell carcinoma (P < 0.005). Also, high expression of TLR4 and HSP70 was seen in WDSCC and MDSCC whereas poor expression was seen in PDSCC. Khan et al., studied the association between HSP70 and TLR4 and concluded that locally produced HSP70 might be responsible for TLR4-mediated induction of inflammatory reaction and directly promotes the growth of endometriosis. This growth promoting effect of combined LPS and HSP70 was significantly suppressed when the biological function of TLR4 was blocked with anti-TLR4 antibody. Thus, they came to the conclusion that growth of endometriosis may be regulated by endogenous HSP70 or LPS either alone or in combination after their binding with TLR4. 
Thus in agreement with Khan et al., we are of the opinion that HSP70 acts as a ligand and binds to TLR4 thus activating the My88 pathway resulting in the production of pro-inflammatory cytokines, chemokines, growth factors etc., and enhancing the anti-cancer immunity in the early stages of the disease. But, in the later stages, TLRs expressed on cancer cells can also upregulate the NF-κB cascade and produce anti-apoptotic proteins that contribute to carcinogenesis and cancer cell proliferation.
| > Conclusion|| |
As our results suggest that HSP70 acts as a ligand for TLR4, this association can be used as a therapeutic modality for cancer. Targeting agonists and antagonists of TLR4 can be used advantageously as a vaccine strategy to treat OSCC at a molecular level.
This was a pilot study done to establish a correlation between HSP70 and TLR4 in the various histological subtypes of oral squamous cell carcinoma by immunohistochemistry. Those cases which were positive for HSP70 were also positive for TLR4 thus pointing out that HSP70 serves as a TLR4 agonist. However, further studies are required to correlate this expression with the clinicopathological features and disease prognosis. Studies need to be done at a molecular level so as to confirm the association of TLR4 with HSP70 in tumorigenesis. This evidence can be used as a therapeutic approach for treating OSCC at its most basic level.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]