Sharada Sawant

Implications of cytokeratin 8/18 filament formation in stratified epithelial cells: Induction of transformed phenotype

The cytokeratin (CK) pair 8 and 18 is normally expressed in all simple epithelia. This pair is not normally seen in stratified epithelial cells. Squamous cell carcinomas derived from stratified epithelia show anomalous expression of this CK pair. It is not known whether CKs 8 and 18 in any way contribute to the malignant phenotype of these cells. We used an immortalised, nontransformed human foetal buccal mucosa (FBM) cell line that expresses significantly higher amounts of CK18 compared to CK8. FBM cells were transfected with the full‐length CK8 gene to study the role of CKs 8 and 18 in malignant transformation. Clones with higher expression of CK8 compared to untransfected FBM cells were studied for changes in their phenotypic characteristics. Immunofluorescence studies using antibodies to CKs 8 and 18 revealed well‐decorated filaments throughout the cytoplasm in CK8 gene–transfected cells vs. untransfected FBM cells. Transmission images showed that FBM cells were isolated while transfected cells were in groups of well‐spread cells with cellular projections. Transfected cells were independent of growth supplement requirements and showed anchorage‐independent growth in soft agar assay and significantly reduced doubling time compared to nontransfected FBM cells. DNA flow‐cytometric studies revealed increased DNA content and prolonged S phase in transfected clones vs. FBM cells. Injection of cells s.c. obtained from soft agar colonies developed from 2 of the clones resulted in tumour formation at the site of injection. In both cases, lung metastasis was also seen. Thus, in conclusion, it appears that increased expression of CK8 in some way changes the phenotypic characteristics of stratified epithelial cells, resulting in malignant transformation.

Understanding the role of keratins 8 and 18 in neoplastic potential of breast cancer derived cell lines.

Background Breast cancer is a complex disease which cannot be defined merely by clinical parameters like lymph node involvement and histological grade, or by routinely used biomarkers like estrogen receptor (ER), progesterone receptor (PGR) and epidermal growth factor receptor 2 (HER2) in diagnosis and prognosis. Breast cancer originates from the epithelial cells. Keratins (K) are cytoplasmic intermediate filament proteins of epithelial cells and changes in the expression pattern of keratins have been seen during malignant transformation in the breast. Expression of the K8/18 pair is seen in the luminal cells of the breast epithelium, and its role in prognostication of breast cancer is not well understood. Methodology/Principal Findings In this study, we have modulated K8 expression to understand the role of the K8/18 pair in three different breast epithelium derived cell lines: non-transformed MCF10A, transformed but poorly invasive MDA MB 468 and highly invasive MDA MB 435. The up-regulation of K8 in the invasive MDA MB 435 cell line resulted in a significant decrease in proliferation, motility, in-vitro invasion, tumor volume and lung metastasis. The down-regulation of K8 in MDA MB 468 resulted in a significant increase in transformation potential, motility and invasion in-vitro, while MCF10A did not show any changes in cell transformation assays. Conclusions/Significance These results indicate the role of K8/18 in modulating invasion in breast cancer -its presence correlating with less invasive phenotype and absence correlating with highly invasive, dedifferentiated phenotype. These data may have important implications for prognostication of breast cancer.

Cytokeratin fragments in the serum: Their utility for the management of oral cancer

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy and is a major cause of cancer morbidity and mortality worldwide. Oral cancer is the most predominant malignancy in the Indian subcontinent due to the widespread habits of chewing tobacco and related products. Patients with oral tumours have a high risk of early locoregional relapse. Early detection of disease progression remains a challenging task mainly due to the lack of adequate early prognostic markers. CEA, SCC Ag, CA-125, serum cytokeratin (CK) fragments, Cyfra 21-1 (CK 19), TPS (CK 18), TPA (CK 8, 18, and 19) etc. are being used as serum markers for the prediction of prognosis of various malignancies. This review presents the available literature on serum CK markers in different malignancies evaluates their utility in the management of oral cancer, and identifies the lacunae which need to be addressed to develop sensitive and specific assays for early detection of recurrence, prognosis, and treatment monitoring.

Cytokeratin expression in precancerous lesions of the human oral cavity

Cytokeratin (CK) expression was studied in buccal mucosa (BM) from 20 leucoplakia and 7 submucous fibrosis patients using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting and two-dimensional gel electrophoresis with iso-electric focussing (IEF) as the first dimension. Normal BM expresses CK 4, 5, 13, 14 and perhaps 19. Of 20 leucoplakia samples analysed, CK 5 was not detected in 17 samples, while CK 14 was not found in 13 samples. CK 1 and CK 8 were aberrantly expressed in six and seven samples, respectively. CK expression in contralaterally collected uninvolved tissues from 3 patients showed a normal pattern in two samples. Non-expression of CK 5 was observed in five of seven submucous fibrosis samples, while CK 14 was not detected in only two samples. CK 8 was aberrantly expressed in three samples. All the leucoplakia patients were chronic tobacco chewers. Thus, non-expression of CK 5 may be an early event occurring in tobacco-associated pathological changes in the BM.

Loss of keratin 8 phosphorylation leads to increased tumor progression and correlates with clinico-pathological parameters of OSCC patients

Background Keratins are cytoplasmic intermediate filament proteins expressed in tissue specific and differentiation dependent manner. Keratins 8 and 18 (K8 and K18) are predominantly expressed in simple epithelial tissues and perform both mechanical and regulatory functions. Aberrant expression of K8 and K18 is associated with neoplastic progression, invasion and poor prognosis in human oral squamous cell carcinomas (OSCCs). K8 and K18 undergo several post-translational modifications including phosphorylation, which are known to regulate their functions in various cellular processes. Although, K8 and K18 phosphorylation is known to regulate cell cycle, cell growth and apoptosis, its significance in cell migration and/or neoplastic progression is largely unknown. In the present study we have investigated the role of K8 phosphorylation in cell migration and/or neoplastic progression in OSCC. Methodology and Principal Findings To understand the role of K8 phosphorylation in neoplastic progression of OSCC, shRNA-resistant K8 phospho-mutants of Ser73 and Ser431 were overexpressed in K8-knockdown human AW13516 cells (derived from SCC of tongue; generated previously). Wound healing assays and tumor growth in NOD-SCID mice were performed to analyze the cell motility and tumorigenicity respectively in overexpressed clones. The overexpressed K8 phospho-mutants clones showed significant increase in cell migration and tumorigenicity as compared with K8 wild type clones. Furthermore, loss of K8 Ser73 and Ser431 phosphorylation was also observed in human OSCC tissues analyzed by immunohistochemistry, where their dephosphorylation significantly correlated with size, lymph node metastasis and stage of the tumor. Conclusion and Significance Our results provide first evidence of a potential role of K8 phosphorylation in cell migration and/or tumorigenicity in OSCC. Moreover, correlation studies of K8 dephosphorylation with clinico-pathological parameters of OSCC patients also suggest its possible use in prognostication of human OSCC.

Prognostic value of tissue polypeptide antigen in oral squamous cell carcinoma

Human oral cancer has a high risk of locoregional relapse which is difficult to diagnose early due to lack of prognostic markers. We and others have shown aberrant expression of cytokeratin (CK) 8 and 18 in human oral cancer. Aberrant supra-basal expression of CK19 has been shown earlier. In this study, our aim was to develop a non-invasive test for prognostication of human oral cancer. Immunoradiometric assay (IRMA) was used to measure the circulating levels of TPA in the sera of 80 oral cancer patients before surgery and seven days after surgery. Elevated serum TPA levels were noted in the post surgery samples of 28 out of the 50 patients for whom clinical follow-up was available, as compared to their pre-surgery samples. TPA levels in the pre-surgery serum samples of patients were significantly higher than levels in the sera of healthy controls (p=0.001). Elevated levels in patients correlated significantly with stage (p=0.02), development of recurrence (p<0.006), and impacted survival (p<0.033). IHC analysis showed statistically significant correlation between expression of CK8, 18 and 19 in surrounding uninvolved tissues of the tumour and post surgery elevated serum TPA levels (p<0.001). This suggests the possibility that CK fragments are released from the surrounding uninvolved tissues into the sera of patients after surgical removal of the tumour. Thus, our study indicates that TPA can be a useful tumour marker for the prediction of recurrence and poor prognosis in human oral cancer.

Prognostic role of Oct4, CD44 and c-Myc in radio–chemo-resistant oral cancer patients and their tumourigenic potential in immunodeficient mice

ObjectiveIn the present study, we have investigated the prognostic value of known stem cell-associated molecules such as Oct4, CD44 and c-Myc in patients with oral SCC who had received post-surgery radio- and/or chemotherapy.Materials and methodsImmunohistochemistry was performed to analyse the expression of Oct4, CD44 and c-Myc in 87 tumour tissues, and the expression profile obtained was correlated with clinicopathological parameters of the patients with oral cancer. Tumourigenic potential of these molecules was also evaluated by in vivo studies.ResultsOur results showed significant correlation of Oct4 (OS, p = 0.003; DFS, p = 0.001) and c-Myc (OS, p = 0.01; DFS, p = 0.03) with overall survival and disease-free survival independently. Furthermore, all the three markers in combinations of two markers each, i.e. Oct4 + CD44 (OS, p = 0.003; DFS, p = 0.001), Oct4 + c-Myc (OS, p = 0.0001; DFS, p = 0.0001), CD44 + c-Myc (OS, p = 0.008; DFS, p = 0.02) and in combinations of three markers each, i.e. Oct4 + CD44 + c-Myc (OS, p = 0.0001; DFS, p = 0.0001) also significantly correlated with overall survival and disease-free survival. Univariate and multivariate analyses further established the independent prognostic value of Oct4. Oct4-, CD44- and c-Myc-enriched populations independently induced sarcomatoid carcinomas whereas primary keratinocytes developed poorly differentiated carcinomas in immunodeficient mice.ConclusionsOct4 and c-Myc independently as well as in combination with CD44 might be useful for the prediction of local recurrence and poor survival of patients with oral cancer which is the novel finding of this study.Clinical relevanceOct4, c-Myc and CD44 can be used to predict local recurrence and the outcome of treatment in oral cancer patients. In addition, these molecules may find use as molecular targets for effective therapy.

Demonstration of cytokeratin-5 non-expression in tobacco related oral carcinogenesis—use of reverse transcriptase polymerase chain reaction as a sensitive assay

Cytokeratins (CK) are the epithelia specific intermediate filament proteins. We have shown consistent non-expression of CK-5 protein in human oral pre-cancer and cancer, in earlier studies. To investigate whether non-expression of CK-5 protein is the result of transcriptional or translational block and to evaluate the possibility if CK-5 non-expression can be used as a marker for early diagnosis of tobacco related oral cancer, RT-PCR using CK-5 specific primers was conducted. Out of 36 precancerous lesions and 29 squamous cell carcinomas (SCC) of buccal mucosa (BM) samples studied, 11 and 13 samples respectively of precancer and SCC did not show CK-5 product in RT-PCR. Down regulation of CK-5 mRNA expression was also observed in some samples. Thus, in conclusion, our results have shown that CK-5 non-expression is the result of transcriptional block. We proposed CK-5 non-expression as a potential marker for the early diagnosis of tobacco related oral cancer.

Vimentin regulates differentiation switch via modulation of keratin 14 levels and their expression together correlates with poor prognosis in oral cancer patients

Vimentin is an intermediate filament protein, predominantly expressed in cells of mesenchymal origin, although its aberrant expression is seen in many carcinomas during epithelial mesenchymal transition. In cancer, vimentin expression is associated with the transition from a more differentiated epithelial phenotype to a dedifferentiated state. In view of the perceived role of keratins (Ks) as regulators of differentiation in epithelia, it was important to understand whether vimentin modulates differentiation through the reprogramming of keratins, in transformed cells. To address this, vimentin was stably downregulated in oral cancer derived cells. Further, global keratin profiling was performed after high salt keratin extraction. K5/K14 pair was found to be significantly downregulated, both at protein and mRNA levels upon vimentin downregulation. The previous study from our laboratory has shown a role of the K5/K14 pair in proliferation and differentiation of squamous epithelial cells. Vimentin depleted cells showed an increase in the differentiation state, marked by an increase in the levels of differentiation specific markers K1, involucrin, filaggrin and loricrin while its proliferation status remained unchanged. Rescue experiments with the K5/K14 pair overexpressed in vimentin knockdown background resulted in decreased differentiation state. ΔNp63 emerged as one of the indirect targets of vimentin, through which it modulates the expression levels of K5/K14. Further, immunohistochemistry showed a significant correlation between high vimentin-K14 expression and recurrence/poor survival in oral cancer patients. Thus, in conclusion, vimentin regulates the differentiation switch via modulation of K5/K14 expression. Moreover, vimentin-K14 together may prove to be the novel markers for the prognostication of human oral cancer.

Vimentin-mediated regulation of cell motility through modulation of beta4 integrin protein levels in oral tumor derived cells

Vimentin expression correlates well with migratory and invasive potential of the carcinoma cells. The molecular mechanism by which vimentin regulates cell motility is not yet clear. Here, we addressed this issue by depleting vimentin in oral squamous cell carcinoma derived cell line. Vimentin knockdown cells showed enhanced adhesion and spreading to laminin-5. However, we found that they were less invasive as compared to the vector control cells. In addition, signaling associated with adhesion behavior of the cell was increased in vimentin knockdown clones. These findings suggest that the normal function of β4 integrin as mechanical adhesive device is enhanced upon vimentin downregulation. As a proof of principle, the compromised invasive potential of vimentin depleted cells could be rescued upon blocking with β4 integrin adhesion-blocking (ASC-8) antibody or downregulation of β4 integrin in vimentin knockdown background. Interestingly, plectin which associates with α6β4 integrin in the hemidesmosomes, was also found to be upregulated in vimentin knockdown clones. Furthermore, experiments on lysosome and proteasome inhibition revealed that perhaps vimentin regulates the turnover of β4 integrin and plectin. Moreover, an inverse association was observed between vimentin expression and β4 integrin in oral squamous cell carcinoma (OSCC). Collectively, our results show a novel role of vimentin in modulating cell motility by destabilizing β4 integrin-mediated adhesive interactions. Further, vimentin-β4 integrin together may prove to be useful markers for prognostication of human oral cancer.