| Abstract|| |
Background: It is well established that chronic exposure to tobacco induces head and neck cancers but the exact etiopathogenesis is not known. Though studies have shown expression of TIMP1, EPS8 and AXL in cancers, their role in tobacco-induced cancers is not known. We aimed this study to evaluate the role of these molecules in oral and oropharyngeal squamous cell cancers (SCC). Materials and Methods: In this single institutional study, 31 patients of oral and oropharyngeal SCC with history of chewing tobacco were included. Smokers were excluded from the study. After informed consent biopsies were taken from affected and contralateral normal mucosa. Paraffin blocks were made and tissue microarray (TMA) were constructed using these blocks. Immunohistochemistry (IHC) for TIMP1, EPS8, AXL kinase was carried out on these tissue microarrays. The intensity of staining was scored from 0 to 3+, related to expression of each of the three molecules. Results: The expression of TIMP1, EPS8 and AXL kinase was significantly more in the cancerous mucosa versus non-cancerous mucosa (P = 0.000 in all three) in oral and oropharyngeal SCC exposed to chewing tobacco. Conclusion: Immunohistochemical expression of these molecular markers in oral and oropharyngeal SCC correlated with their molecular based studies. Significant IHC expression of TIMP1, EPS8 and AXL establishes their role in the pathogenesis of oral and oropharyngeal squamous cell carcinomas. Novel targeted therapies may be researched that can detect and target these molecules at an earlier stage of pathogenesis of these tumors.
Keywords: AXL, chewing tobacco, EPS8, immunohistochemistry, oral cancer, TIMP1, tissue microarray
|How to cite this article:|
Agarwal A, Garg C, Ganesh M S, Reddy S. Molecular mechanisms of tobacco induced oral and oropharyngeal cancer: Results of a tissue microarray and immunohistochemistry-based study from a tertiary cancer center in India. Indian J Pathol Microbiol 2020;63:7-12
|How to cite this URL:|
Agarwal A, Garg C, Ganesh M S, Reddy S. Molecular mechanisms of tobacco induced oral and oropharyngeal cancer: Results of a tissue microarray and immunohistochemistry-based study from a tertiary cancer center in India. Indian J Pathol Microbiol [serial online] 2020 [cited 2020 Mar 28];63:7-12. Available from: http://www.ijpmonline.org/text.asp?2020/63/1/7/277425
| Introduction|| |
The overall 5-year survival rate for people worldwide with oral or oropharyngeal cancer is 64%. If the cancer is diagnosed at an early stage, the 5-year survival rate is 83%. Survival rates for oral and oropharyngeal cancer vary widely depending on the original location, cancer due to HPV, and the extent of the disease.
In India, the exact incidence is not known but as per NCRP (National Cancer Registration Program) data, it is more than western countries with highest incidence in Bhopal.
An estimated 49,670 adults (35,720 men and 13,950 women) in the United States were diagnosed with oral and oropharyngeal cancer in the year 2015-2016. Rates of these cancers are more than twice as high in men as in women. Cancer of the oral cavity is the ninth most common cancer among men. The average age of diagnosis is 62 years.
The use of smokeless tobacco (SLT, tobacco consumed without combustion) has become prevalent all over the world. SLT is placed inside the oral cavity in contact with the mucus membranes where the nicotine is absorbed to provide the desired effect. SLT has been used in many forms in different parts of the world. Use of oral snuff (wet or moist snuff) is more common in the west and the middle east. Betel quid chewing, in a variety of forms and various ingredients is widespread in Asia, where it is a custom and cultural habit, especially in the Indian subcontinent. Betel quid (also referred to as pan or paan) usually contains betel-leaf (leaf of Piper betel vine), areca nut, slaked lime, and tobacco. In India, other ingredients are often added namely spices such as cardamom, cloves, aniseed. Some of the common forms of these mixtures are khaini (tobacco and lime), zarda (boiled tobacco), gadakhu (tobacco and molasses), and mawa (tobacco, lime and areca) consumed in different parts of India.
Considerable research has been focused in the recent past on the carcinogenic, mutagenic, and genotoxic potential of betel quid ingredients, especially tobacco and areca nut. In vitro studies on oral mucosal fibroblasts using DNA damage, cytotoxicity, and cell proliferation assays have shown that some essential betel quid ingredients are genotoxic, cytotoxic, and also stimulate cell proliferation. It has been shown that reactive oxygen species (ROS), methylating agents, and reactive metabolic intermediates from betel quid induce various kinds of DNA damage.
Studies have been done at molecular level to know the expression of different molecules in oral cancers. Though studies have shown the role of TIMP1, EPS8 and AXL in oral and oropharyngeal cancers, the role of these proteins in tobacco-induced cancers is not reported among Indian population where the prevalence of tobacco use is high. In an in vitro study, mass spectrometry-based analysis revealed over expression of Tissue Inhibitor of Metalloproteinases1 (TIMP1), Epidermal Growth Factor Receptor Pathway Substrate 8 (EPS8) and AXL Receptor tyrosine kinase (AXL) at 2.7-, 2.0- and 1.6-fold, respectively, in response to chewing tobacco in the cell lines of oral mucosal cells that were chronically treated with tobacco over a period of 6 months.
We decided to conduct an in vivo study to evaluate the role of these proteins in cases of oral and oropharyngeal squamous cell cancer (OSCC) exposed as well as not exposed to tobacco by looking into the expression of these molecules, thereby trying out to figure out the links in the etiopathogenesis of oral cancer.
| Materials and Methods|| |
The sources of data for the study were patients presenting with oral and OSCC at Department of Surgical Oncology, Vydehi Institute of Medical Sciences, Bangalore, India. Duration of study was between January 2016 and January 2018. Sample size was taken as 31 as per the Hospital based Cancer Registry.
All patients presenting for the first time to the institute irrespective of age, sex and stage of disease with oral and OSCC and with history of use of chewing tobacco.
- Smokers (patients who were both tobacco chewers as well as smokers were also excluded from the study)
- Prior chemotherapy or radiation.
Sampling and tissue processing
Informed consent was taken followed by biopsies from affected mucosa and from contralateral normal looking mucosa of the same patient serving as control. Paraffin blocks were made and tissue microarray (TMA) were constructed using these blocks; 2 mm cores from each paraffin block were embedded into a recipient paraffin block. IHC was carried out on these TMAs. The primary antibodies were used in the following dilutions: TIMP-1 (1:500), EPS8 (1:250) and AXL (1:100).
The expression and cellular location of the molecular markers was examined in affected and normal oral mucosa by IHC. All slides were scored independently by two pathologists without knowledge of the clinical impression to remove any bias. Occasional disagreements were discussed to reach a consensus.
Immunohistochemistry (IHC) interpretation
For TIMP-1 [Figure 1], staining was assessed on cell membrane and cytoplasm of the tumor cells. Staining for TIMP-1 was measured as the percentage of positively stained tumor cells using light microscopy. Intensity of staining was classified as negative, weak, moderate and strong., Four categories were assigned: score 0, negative; score 1, less than 10%, weak; score 2, more than 10% and less than 50%, moderate; score 3, more than 50% positive staining, strong.
|Figure 1: (a) Positive Control; (b) TIMP-1 Negative, score 0. (TIMP-1 IHC × 200); (c) TIMP-1 expression with weak cytoplasmic staining, score1+. (TIMP-1 IHC × 100); (d) TIMP-1 expression with 10%-50% moderate to strong cytoplasmic and membranous positive staining, score2+. (TIMP-1 IHC × 200); (e and f) TIMP-1 expression with >50% strong cytoplasmic and membranous positive staining, score3+. (TIMP-1 IHC × 100)|
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[Figure 2], the staining was assessed in tumor cell cytoplasm and ranged from negative (0), focal and weak (1+), moderate (2+), strong and diffuse (3+).
|Figure 2: (a) EPS8 Negative, score 0 (EPS 8 IHC × 100); (b) EPS8 expression with weak cytoplasmic staining, score 1+. (EPS8 IHC × 100); (c) EPS8 expression with moderate cytoplasmic staining, score 2+. (EPS8 IHC × 100); (d) EPS8 expression with strong and diffuse cytoplasmic positive staining, score3+. (EPS8 IHC × 400)|
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For AXL kinase
[Figure 3], staining was scored as 0, 1+, 2+, and 3+ in the tumor cell cytoplasm., Score 0, no reactivity in any tumor cell; Score 1+, tumor cells cluster with a faint reactivity irrespective of percentage of tumor cells stained; Score 2+, tumor cells cluster with a moderate reactivity irrespective of percentage of tumor cells stained; Score 3+, tumor cells cluster with a diffuse and strong reactivity.
|Figure 3: (a) AXL Negative, score 0. (AXL IHC × 400); (b) AXL expression in tumor cell cluster with faint cytoplasmic staining, score 1+. (AXL IHC × 400); (c) AXL expression with moderate cytoplasmic staining, score 2+. (AXL IHC × 100); (d) AXL expression with strong and diffuse cytoplasmic staining, score 3+. (AXL IHC × 400)|
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The quantitative data comparison was done using Chi-square test. P value ≤ 0.05 was considered statistically significant.
| Results|| |
Out of 31 patients, 11 (35.5%) patients were male and 19 (61.3%) patients were female with M:F ratio of 1:1.73. The patients selected for the study aged from 28 to 85 years. Of the 31 cases, cancer of oral cavity was seen in 26 patients (83.8%) while oropharyngeal cancers in 5 patients (16.1%).
Out of 31 patients, G1 (well differentiated) cases were 12 (38.7%); G2 (moderately differentiated) 18 (58%) and G3 (poorly differentiated) 1 (3.2%).
For Tissue Inhibitor of Metalloproteinases1 (TIMP1) IHC staining, 20 out of 31 patients (64.3%) with squamous cell carcinoma had 3+ staining in affected mucosa, while 19 out of 31 biopsies (61.3%) from normal control mucosa were negative for TIMP1 (P-value 0.000) [Table 1].
In Epidermal Growth Factor Receptor Pathway Substrate 8 (EPS8), it was found that the normal mucosa did not stain in 16 out of 31 cases (51.6%) while the cancerous mucosa showed 2+ staining in 18 out of 31 patients (58.1%) and 3+ strong in 12 out of 31 patients (38.7%) (P value = 0.000) [Table 2].
Normal mucosa in AXL Receptor tyrosine kinase (AXL) in 16 out of 31 cases (51.6%) did not show any staining while the samples from squamous cell carcinoma showed 2+ staining in 18 out of 31 patients (58.1%) and 3+ staining in 13 out of 31 patients (41.9%) (P value = 0.000) [Table 3].
| Discussion|| |
Smokeless or chewing tobacco is a known risk factor in the etiopathogenesis of oral cancer. Although the tumor inducing property of chewing tobacco was proven in the year 1964, the molecular events that lead to tumor growth and progression due to its consumption are not well known.
The molecular mechanisms and pathobiology of oral cancer resulting from chewing tobacco differs significantly from that of cigarette smoking because of difference in their composition. Chewing tobacco contains several compounds such as nicotine tobacco specific N-nitrosamines and polycyclic aromatic hydrocarbons which are known to be carcinogenic. As mentioned earlier tobacco demonstrates its carcinogenic effect through long-term (chronic) exposure and not of short duration. Studies have been done at molecular levels to know the expression of different molecules in oral cancers.
Tissue inhibitors of metalloproteinases (TIMP) are glycoproteins that belong to TIMP gene family and are inhibitors of matrix metalloproteinases (MMPs). MMPs are group of peptidases which are involved in the degradation of the extracellular matrix. In addition to its inhibitory role against most of the known MMPs, TIMPs family of proteins also regulates cell proliferation in a wide range of cell types and has an anti-apoptotic function.
Studies have shown that TIMPs can promote cancer progression through MMP-dependent and -independent mechanisms though the underlying mechanisms are not well understood.
EPS8 is a novel tumor-associated antigen shown to regulate cellular proliferation and metastasis., It is a signaling adapter that controls various cellular protrusions by regulating actin cytoskeleton dynamics and architecture. Depending on its association with other signal transducers, can regulate different processes. Together with SOS1 and ABI1, forms a trimeric complex that participates in transduction of signals from Ras to Rac by activating the Rac-specific guanine nucleotide exchange factor (GEF) activity.
AXL is an inhibitor of the innate immune response. The function of activated AXL in normal tissues includes the efficient clearance of apoptotic material and the dampening of TLR-dependent inflammatory responses and natural killer cell activity. AXL is a putative driver of diverse cellular processes that are critical for the development, growth, and spread of tumors, including proliferation, invasiveness and migration, epithelial-to-mesenchymal transition, stemness, angiogenesis, and immune modulation.
Though studies have shown the role of TIMP1, EPS8 and AXL in oral and oropharyngeal cancers, role of these proteins in tobacco-induced cancers is not well reported especially in Indian population with a high prevalence of tobacco use.
In a study done by Nanjappa et al., an in vitro cell line model was developed by treating the normal oral keratinocytes, OKF6/TERT1 chronically with chewing tobacco (referred to as OKF6/TERT1-tobacco). They found a 2.7-fold over expression of TIMP1 in the cell line treated with tobacco. In a study by Ragini D et al., tissue analysis showed 2.4- and 1.3-fold increase in TIMP-1 and TIMP-2 mRNA expression levels in comparison to histologically adjacent normal mucosa respectively. Another that included 68 oral squamous cell carcinoma by Vincent et al. expression of TIMP-1 by IHC was detected in 45 cases (66.2%). In all of these TIMP-1 was expressed in tumoral tissue, and in 19 of them also in the surrounding stroma.
In the present study, all of the cases of squamous cell cancer mucosa showed expression of TIMP 1 staining. When the tobacco exposed cancer tissue were compared with the normal mucosal tissue of the same patient a statistically significant over expression was observed in the oral squamous cell cancer tissue (P value = 0.000).
Nanjappa et al. also demonstrated a 2.0-fold increase in the expression of EPS8 in their cell line (as OKF6/TERT1-tobacco) when treated with tobacco. In a study by Yap et al., EPS 8 expression was enhanced by >5-fold in the OSCC cell lines relative to normal keratinocytes. Eps8 expression by IHC was detected in 19 of 59 (32%) of the tumors examined. The staining was cytoplasmic and ranged from weak and focal to strong and diffuse. In a study by Chu et al., Eps8 expression was identified in 186 of the 205 cases of OSCC (91%) and the aberrance occurred primarily in the cytoplasm of OSCC cells.
In our study the expression was strong (3+) in the tissues from cancerous mucosa (mostly in the cytoplasm) of 12 patients (38.7%). Correlating this staining pattern with that of the normal mucosa, it was found to be statistically significant (P = 0.000).
As far as AXL kinase is concerned, Nangappa et al. demonstrated a 1.6-fold increase in the expression of AXL kinase in cancerous mucosa with respect to normal mucosa. In a study by Lee C et al., the immunoreactivity of AXL was found low in normal epithelium, and a progressively increased positive percentage was noted, from normal/hyperplastic epithelium (10.9%) to dysplasia (30.8%) to cancer tissue (54.5%). AXL expression correlated with lymph node status (P = 0.001) and clinical stage (P = 0.014) of OSCC. Patients with high expression of AXL showed poor prognosis compared with those with low AXL expression patients (P < 0.001).
In our study, 18 out of 31 patients (58.1%) samples from cancer mucosa showed 2+ staining and 13 out of 31 patients (41.9%) had 3+ staining (P value = 0.000).
| Conclusion|| |
Immunohistochemical expression of these molecular markers in oral and OSCC correlated with their molecular-based studies. Over expression of TIMP1, EPS8 and AXL kinase signifies their role in the pathogenesis of oral and OSCC. Novel targeted therapies can be researched that can detect and target these molecules at an earlier stage of pathogenesis of these tumors. Future directions can also be in assessment margins during surgery in cases of microscopic negativity as well in deciding the role of adjuvant radiation.
| Limitations of the Study|| |
Further studies with a normal individual oral mucosa (non-oral cancer) as compared with the normal mucosa of diseased individual will substantiate the involvement of these molecules in the etiopathogenesis of oral squamous cell cancer. If these results are repeated in a larger population then future directions can include evaluation of local recurrences by the expression of these molecules in suspected areas on follow-up.
Institute of Bioinformatics, Bangalore, India for their support in conducting Tissue Microarray Analysis of the samples.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
NCRP - Three year report of Population Based Cancer Registries 2012-2014, National Cancer Registry Programme (Indian Council of Medical Research), Bangalore, 2016.
Sinha DN, Suliankatchi RA, Gupta PC, Thamarangsi T, Agarwal N, Parascandola M, et al
. Global burden of all-cause and cause-specific mortality due to smokeless tobacco use: Systematic review and meta-analysis. Tob Control 2018;27:35-42.
Kumar M, Nanavati R, Modi TG, Dobariya C. Oral cancer: Etiology and risk factors: A review. J Can Res Ther 2016;12:458-63.
] [Full text]
Nanjappa V, Renuse S, Sathe G, Raja R, Syed N, Radhakrishnan A, et al
. Chronic exposure to chewing tobacco selects for overexpression of stearoyl-CoA desaturase in normal oral keratinocytes. Cancer Biol Ther 2015;16:1593-603.
Yoshizaki T, Maruyama Y, Sato H, Furukawa M. Expression of tissue inhibitor of matrix metalloproteinase-2 correlates with activation of matrix metalloproteinase-2 and predicts poor prognosis in tongue squamous cell carcinoma. Int J Cancer 2001;95:44-50.
de Vicente JC, Fresno MF, Villalain L, Vega JA, Arranz JS. Immunoexpression and prognostic significance of TIMP-1 and-2 in oral squamous cell carcinoma. Oral Oncol 2005;41:568-79.
Benn TM, Jenei V, Lopes VV, Moutasim KA, Paterson IC, Robinson CM, et al
. Upregulation of Eps8 in oral squamous cell carcinoma promotes cell migration and invasion through integrin-dependent Rac1 activation. Oncogene2009;28:2524-34.
Hsieh M-S, Yang P-W, Wong L-F, Lee J-M. The AXL receptor tyrosine kinase is associated with adverse prognosis and distant metastasis in esophageal squamous cell carcinoma. Oncotarget 2016;7:36956-70.
Brand TM, Iida M, Stein AP, Corrigan KL, Braverman CM, Coan JP, et al
. AXL is a logical molecular target in head and neck squamous cell carcinoma. Clin Cancer Res 2015;21:2601-12.
Singh R, Haridas N, Patel J, Shah F, Shukla S, Shah P, et al
. Matrix metalloproteinases and their inhibitors: Correlation with invasion and metastasis in oral cancer. Indian J Clin Biochem 2010;25:250-9.
Leemans CR, Braakhuis BJ, Brakenhoff RH. The molecular biology of head and neck cancer. Nat Rev Cancer 2011;11:9-22.
Boffetta P, Aagnes B, Weiderpass E, Andersen A. Smokeless tobacco use and risk of cancer of the pancreas and other organs. Int J Cancer 2005;114:992-5.
Boffetta P, Hecht S, Gray N, Gupta P, Straif K. Smokeless tobacco and cancer. Lancet Oncol 2008;9:667-75.
EPS8-Epidermal growth factor receptor kinase substrate 8 - Homo sapiens (Human) - EPS8 gene & protein [Internet]. Uniprot.org. 2017. Available from: http://www.uniprot.org/uniprot/Q12929
. [Last accessed on 2017 Dec 10].
Yap L, Jenei V, Robinson C, Moutasim K, Benn T, Threadgold S, et al
. Upregulation of Eps8 in oral squamous cell carcinoma promotes cell migration and invasion through integrin-dependent Rac1 activation. Oncogene 2009;28:2524-34.
Chu PY, Liou JH, Lin YM, Chen CJ, Chen MK, Lin SH, et al
. Expression of Eps8 correlates with poor survival in oral squamous cell carcinoma. Asia Pac J Clin Oncol 2012;8:77-81.
Lee C, Yen C, Liu S, Chen C, Chiang C, Shiah S, et al
. Axl is a prognostic marker in oral squamous cell carcinoma. Ann Surg Oncol 2011;19:500-8.
Department of Pathology, Rohilkhand Medical College and Hospital, Bareilly, Uttar Pradesh - 243 001
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]