Milind M. Vaidya

Novel function of keratins 5 and 14 in proliferation and differentiation of stratified epithelial cells

Keratin expression in stratified epithelia is tightly regulated during squamous cell differentiation. Keratins 5 and 14 are expressed in mitotically active basal layer cells, but their function is not well defined. Reported here is the possible role of K14 in regulation of cell proliferation/differentiation in stratified epithelial cells.

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.

O-GlcNAcylation Determines the Solubility, Filament Organization, and Stability of Keratins 8 and 18

Keratins 8 and 18 (K8/18) are intermediate filament proteins expressed specifically in simple epithelial tissues. Dynamic equilibrium of these phosphoglycoproteins in the soluble and filament pool is an important determinant of their cellular functions, and it is known to be regulated by site-specific phosphorylation. However, little is known about the role of dynamic O-GlcNAcylation on this keratin pair. Here, by comparing immortalized (Chang) and transformed hepatocyte (HepG2) cell lines, we have demonstrated that O-GlcNAcylation of K8/18 exhibits a positive correlation with their solubility (Nonidet P-40 extractability). Heat stress, which increases K8/18 solubility, resulted in a simultaneous increase in O-GlcNAc on these proteins. Conversely, increasing O-GlcNAc levels were associated with a concurrent increase in their solubility. This was also associated with a notable decrease in total cellular levels of K8/18. Unaltered levels of transcripts and the reduced half-life of K8 and K18 indicated their decreased stability on increasing O-GlcNAcylation. On the contrary, the K18 glycosylation mutant (K18 S29A/S30A/S48A) was notably more stable than the wild type K18 in Chang cells. The K18-O-GlcNAc mutant accumulated as aggregates upon stable expression, which possibly altered endogenous filament architecture. These results strongly indicate the involvement of O-GlcNAc on K8/18 in regulating their solubility and stability, which may have a bearing on the functions of these keratins.

Loss of keratins 8 and 18 leads to alterations in α6β4-integrin-mediated signalling and decreased neoplastic progression in an oral-tumour-derived cell line.

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 and invasion in squamous cell carcinomas (SCCs). To understand the molecular basis by which K8 promotes neoplastic progression in oral SCC (OSCC), K8 expression was inhibited in AW13516 cells. The K8-knockdown clones showed a significant reduction in tumorigenic potential, which was accompanied by a reduction in cell motility, cell invasion, decreased fascin levels, alterations in the organization of the actin cytoskeleton and changes in cell shape. Furthermore, K8 knockdown led to a decrease in α6β4 integrin levels and α6β4-integrin-dependent signalling events, which have been reported to play an important role in neoplastic progression in epithelial tissues. Therefore, modulation of α6β4 integrin signalling might be one of the mechanisms by which K8 and K18 promote malignant transformation and/or progression in OSCCs.

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.

Fascin overexpression promotes neoplastic progression in oral squamous cell carcinoma

BackgroundFascin is a globular actin cross-linking protein, which plays a major role in forming parallel actin bundles in cell protrusions and is found to be associated with tumor cell invasion and metastasis in various type of cancers including oral squamous cell carcinoma (OSCC). Previously, we have demonstrated that fascin regulates actin polymerization and thereby promotes cell motility in K8-depleted OSCC cells. In the present study we have investigated the role of fascin in tumor progression of OSCC.MethodsTo understand the role of fascin in OSCC development and/or progression, fascin was overexpressed along with vector control in OSCC derived cells AW13516. The phenotype was studied using wound healing, Boyden chamber, cell adhesion, Hanging drop, soft agar and tumorigenicity assays. Further, fascin expression was examined in human OSCC samples (N = 131) using immunohistochemistry and level of its expression was correlated with clinico-pathological parameters of the patients.ResultsFascin overexpression in OSCC derived cells led to significant increase in cell migration, cell invasion and MMP-2 activity. In addition these cells demonstrated increased levels of phosphorylated AKT, ERK1/2 and JNK1/2. Our in vitro results were consistent with correlative studies of fascin expression with the clinico-pathological parameters of the OSCC patients. Fascin expression in OSCC showed statistically significant correlation with increased tumor stage (P = 0.041), increased lymph node metastasis (P = 0.001), less differentiation (P = 0.005), increased recurrence (P = 0.038) and shorter survival (P = 0.004) of the patients.ConclusionIn conclusion, our results indicate that fascin promotes tumor progression and activates AKT and MAPK pathways in OSCC-derived cells. Further, our correlative studies of fascin expression in OSCC with clinico-pathological parameters of the patients indicate that fascin may prove to be useful in prognostication and treatment of OSCC.

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.

Keratins: Markers of cell differentiation or regulators of cell differentiation?

Keratins are epithelia-specifi c intermediate fi lament proteins, which are expressed in a tissue-specifi c manner (Moll et al 1982). Around 50 keratin genes have been discovered across the species. Keratins have been classifi ed according to their molecular weights and isoelectric points. They have been further subdivided into two subtypes, type I, which are acidic and have low molecular weights, and type II, which are basic or neutral and have high molecular weights. Keratins are obligatory heteropolymers, and the expression of at least one member of each subfamily is essential for proper fi lament formation (Coulombe and Omary 2002). The transcription factors regulating keratin gene expression include members of the AP1 and SP1 families (for keratin 16; Wang and Chang 2003), C/EBP and AP-2 (for keratin 10; Maytin et al 1999), and AP1 and ETS family members (keratin 8/18; Hendrix et al 1996). However, these transcription factors do not govern their tissue-specifi c expression. Keratins, like all other intermediate fi laments, form are highly insoluble structures and require conditions such as 8–9.5 M urea or 1% sodium dodecyl sulphate with boiling for their dissolution. Because of their highly insoluble nature, they were thought to be static structures having only structural functions. It is now clear that keratin fi laments are highly dynamic in nature and post-translational modifi cations such as phosphorylation contribute to their dynamic nature (Omary et al 2006). Their role in the structural integrity of cells was emphasized by the appearance of keratinopathies, which have been shown to be the direct result of single-point mutations in keratin genes (Coulombe and Omary 2002). Some of the severe skin disorders which result from point mutations include epidermolysis bullosa simplex (mutations in keratin 5/14 genes), epidermolytic hyperkeratosis (mutations in keratin 1/10 genes) and epidermolytic palmoplantar keratoderma (mutations in the keratin 9 gene). The point mutations in these keratin genes result in the formation of abnormal keratin fi laments or protein aggregates, and lead to malfunctioning. In these diseases, the skin becomes scaly and even the slightest insult to the skin results in boils and erythema. Similar phenotypes have been observed in respective transgenic mice (Magin et al 2000). Mutations in the K8/18 genes have also been associated with diseases such as acute pancreatitis and liver cirrhosis (Omary et al 2004). The effects of mutation in a particular keratin are more deleterious due to protein aggregation rather than the absence of that keratin, probably because it would also result in defective interactions with membrane proteins, affecting their function of imparting structural resilience. Similar effects have been observed as a result of mutations in keratin-associated proteins such as plectin and desmoplakin (Omary et al 2004). Well-established structural functions alone cannot explain the diversity and dynamic nature of keratin fi laments. Several questions pertaining to the multiplicity of keratins and their probable tissue function remain unanswered. These unresolved issues include: