Generation of a tissue-specific transgenic model for K8 phosphomutants: A tool to investigate the role of K8 phosphorylation during skin carcinogenesis in vivo.

Keratin 8/18, the predominant keratin pair of simple epithelia, is known to be aberrantly expressed in several squamous cell carcinomas (SCCs), where its expression is often correlated with increased invasion, neoplastic progression, and poor prognosis. The majority of keratin 8/18 structural and regulatory functions are governed by posttranslational modifications, particularly phosphorylation. Apart from filament reorganization, cellular processes including cell cycle, cell growth, cellular stress, and apoptosis are known to be orchestrated by K8 phosphorylation at specific residues in the head and tail domains. Even though deregulation of K8 phosphorylation at two significant sites (Serine73 /Serine431 ) has been implicated in neoplastic progression of SCCs by various in vitro studies, including ours, it is reported to be highly context-dependent. Therefore, to delineate the precise role of Kereatin 8 phosphorylation in cancer initiation and progression, we have developed the tissue-specific transgenic mouse model expressing Keratin 8 wild type and phosphodead mutants under Keratin 14 promoter. Subjecting these mice to 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate-mediated skin carcinogenesis revealed that Keratin 8 phosphorylation may lead to an early onset of tumors compared to Keratin 8 wild-type expressing mice. Conclusively, the transgenic mouse model developed in the present study ascertained a positive impact of Keratin 8 phosphorylation on the neoplastic transformation of skin-squamous cells.

Role of Electron Microscopy in Early Detection of Altered Epithelium During Experimental Oral Carcinogenesis.

Early detection of altered epithelium can help in controlling the further progression by timely intervention. Alterations in cellular adhesion are one of the hallmarks of cancer progression, which can be detected at the intracellular level using high-resolution electron microscopy. This study aimed to evaluate the role of electron microscopy in the establishment of ultrastructural markers for early detection of altered epithelium using tissues from 4-Nitroquinoline-1-Oxide (4NQO) induced rat tongue carcinogenesis. Our previous study using light microscopy displayed no histopathological alterations in 4NQO treated tissues until 40 days of treatment, while dysplasia, papilloma and carcinoma were detected at 80/120, 160 and 200 days, respectively. However, electron microscopy detected alterations such as detachment of desmosomes from cell membranes and their clustering in the cytoplasm, increased tonofilaments, keratohyaline granules and thickened corneum in 40 days treated corresponding tissues. These alterations are apparent with hyperkeratosis/hyperplasia but remained undetected using light microscopy. Further, in dysplasia, papilloma and carcinoma, gradual and significant loss of desmosomes, leading to the significant widening of intercellular spaces, was observed using iTEM software. These parameters may serve as indicators for progression of oral cancer. Our results highlight the importance of electron microscopy in the early detection of subcellular changes in the altered epithelium.

Alterations in desmosomal adhesion at protein and ultrastructure levels during the sequential progressive grades of human oral tumorigenesis.

With the aim of developing early diagnostic/prognostic markers for oral cancer, desmosomal adhesion in sequentially progressive grades of tissues from oral normal/disorders (normal, hyperplastic, dysplastic, non-metastatic/metastatic tumours, and metastatic nodes) was investigated at protein and ultrastructural levels using immunohistochemistry and transmission electron microscopy, respectively. The expression of desmosomal proteins was higher in hyperplastic tissues than in normal tissues but was significantly decreased in subsequent progressive stages of the disease. Altered expression of desmosomal proteins was significantly correlated with local recurrence and disease-free survival. Ultrastructural analysis in the corresponding tissues revealed cytoplasmic clustering of desmosomes in hyperplasia; in more advanced disease stages, a significantly lower number of desmosomes and widened intercellular spaces were observed. Altered protein expression resulting in structural changes was confirmed by knocking down desmoplakin expression in non-transformed cells, which failed to form normal desmosome structures and induced a cell-transformation phenotype. Our data suggest that alterations in desmosomal assembly initiate at an early hyperplastic grade and, with more advanced disease stages, the severity of the alterations gradually becomes higher. Alterations in desmosomal adhesion can be useful for early detection of high-risk premalignant lesions, as well as for identification of invasive characteristics of primary non-metastatic tumours. Early detection will help to control further progression of disease by timely intervention.

Prognostic significance of elevated serum CD44 levels in patients with oral squamous cell carcinoma.

BACKGROUND: Availability of reliable methods distinguishing high-risk recurrent tumours from regressive tumours prior to surgery could help in better management of the disease. This study was aimed to estimate pre-surgical serum CD44 concentration and assess the possibility of using it as a non-invasive prognostic tool in oral cancer. METHODS: ELISA was performed on pre-surgical serum samples from 64 primary oral cancer patients and 16 healthy individuals to estimate soluble CD44 levels. Immunohistochemistry was performed on parallel 64 solid tumours and 10 recurrent tumours. All patients clinically followed up for median period of 19.2 months and obtained prognostic information correlated with CD44 concentration in serum as well as in tumours. RESULTS: Serum CD44 concentration was found significantly high in patients as compared to healthy individuals (P < .001) and also in patients whose disease locally recurred as compared to those did not recur (P = 0026). High serum CD44 concentration inversely affected on patients survival (P = .032). CD44v6 staining intensity was detected significantly high in recurrent tumours as compared to primary tumours (P < .001), and it also correlated with poor survival (P < .001). Furthermore, in combination, patients with increased CD44 concentration in serum and CD44v6 expression in tumours significantly correlated with local recurrence (P < .001) and poor survival (P < .001). CONCLUSION: Our data suggest that the ELISA-based estimation of pre-surgical serum CD44 concentration could be a non-invasive reliable method distinguishing high-risk recurrent tumours which can further assist in post-surgery treatment planning.

Depletion of keratin 8/18 modulates oncogenic potential by governing multiple signaling pathways.

Keratin 8/18, the predominant keratin pair of simple epithelia, is often aberrantly expressed in various squamous cell carcinomas (SCCs) including skin SCC. Its aberrant expression is correlated with increased invasiveness and poor prognosis of the same, although the underlying mechanism is still unclear. A previous report from our laboratory has shown K8-mediated regulation of α6ß4 integrin signaling and thereby tumorigenic potential of oral SCC-derived cells. Another study on transgenic mouse model has shown that during skin carcinogenesis, K8 favors conversion of papillomas toward malignancy. In order to understand the role of K8 and allied mechanism in skin SCC, K8 was stably knocked down in a skin epidermoid carcinoma-derived A431 cells. K8 downregulation significantly reduced the tumorigenic potential of these cells. In agreement with our phenotypic data, differential quantitative proteomics followed by IPA analysis showed altered expression of many proteins associated with biological functions including 'Cancer', 'Cellular movement', 'Cell death and survival', and 'Cellular morphology'. Some of these proteins were TMS1, MARCKSL1, RanBP1, 14-3-3γ, Rho-GDI2, etc. Furthermore, to our surprise, there was a significant reduction in K17 protein stability upon loss of K8, probably due to its caspase-mediated degradation. This was supported by altered TMS1-NF-κB signaling, leading to increased apoptotic sensitivity of A431 cells which in turn affected 'Cell death and survival'. Moreover, MARCKSL1-Paxillin1-Rac axis was found to be deregulated bestowing a possible mechanism behind altered 'Cellular movement' pathway. Altogether our study unravels a much broader regulatory role of K8, governing multiple signaling pathways and consequently regulating oncogenic potential of skin SCC-derived cells. DATABASE: Proteome Xchange Consortium via PRIDE database (dataset identifier PXD007206).