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.

Multifaceted role of keratins in epithelial cell differentiation and transformation.

Keratins, the epithelial-predominant members of the intermediate filament superfamily, are expressed in a pairwise, tissuespecific and differentiation-dependent manner. There are 28 type I and 26 type II keratins, which share a common structure comprising a central coiled coil α-helical rod domain flanked by two nonhelical head and tail domains. These domains harbor sites for major posttranslational modifications like phosphorylation and glycosylation, which govern keratin function and dynamics. Apart from providing structural support, keratins regulate various signaling machinery involved in cell growth, motility, apoptosis etc. However, tissue-specific functions of keratins in relation to cell proliferation and differentiation are still emerging. Altered keratin expression pattern during and after malignant transformation is reported to modulate different signaling pathways involved in tumor progression in a context-dependent fashion. The current review focuses on the literature related to the role of keratins in the regulation of cell proliferation, differentiation and transformation in different types of epithelia.

Keratin 5/14­mediated cell differentiation and transformation are regulated by TAp63 and Notch­1 in oral squamous cell carcinoma­derived cells.

Keratins 5/14 (K5/14) are intermediate filament proteins expressed in the basal layer of stratified epithelial cells and are known targets of p63. Previous research in our laboratory showed that upon K5/14 downregulation in oral squamous cell carcinoma (OSCC)­derived cells, there was an increase in intracellular Notch­1 levels and differentiation markers such as involucrin, keratin 1 and a decrease in tumorigenic potential in vivo. However, the molecules involved in the K14 regulated cell differentiation and transformation are not known to date. In order to understand the possible role of TAp63, we downregulated TAp63 in a K14­knockdown background. We observed that there was a decrease in the expression of Notch­1. Expression levels of differentiation markers such as involucrin, K1, loricrin and filaggrin were also decreased. Furthermore, TAp63 downregulation led to an increase in invasion, migration and in vivo tumorigenic potential of these cells. We observed a decrease in ß­catenin signaling in K14­downregulated cells. Notably, when TAp63 was downregulated in K14­knockdown cells, there was increase in non­phospho ß­catenin levels. Hence, this study indicates that TAp63 plays an important role in K14­downregulated cells possibly by regulating the Notch­1 expression. K14 regulates the expression of TAp63 which in turn regulates expression of Notch­1. The present study is a step forward in our quest to understand the functional significance of molecules that regulate the process of differentiation and tumorigenesis in stratified epithelial cells.

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).

Quantitative phosphoproteomic analysis reveals system-wide signaling pathways regulated by site-specific phosphorylation of Keratin-8 in skin squamous cell carcinoma derived cell line.

Keratin 8/18, a simple epithelia specific keratin pair, is often aberrantly expressed in squamous cell carcinomas (SCC) where its expression is correlated with increased invasion and poor prognosis. Majority of Keratin 8 (K8) functions are governed by its phosphorylation at Serine73 (head-domain) and Serine431 (tail-domain) residues. Although, deregulation of K8 phosphorylation is associated with progression of different carcinomas, its role in skin-SCC and the underlying mechanism is obscure. In this direction, we performed tandem mass tag-based quantitative phosphoproteomics by expressing K8 wild type, phosphodead, and phosphomimetic mutants in K8-deficient A431 cells. Further analysis of our phosphoproteomics data showed a significant proportion of total phosphoproteome associated with migratory, proliferative, and invasive potential of these cells to be differentially phosphorylated. Differential phosphorylation of CDK1T14,Y15 , EIF4EBP1T46,T50 , EIF4BS422 , AKT1S1T246,S247 , CTTN1T401,S405,Y421 , and CAP1S307/309 in K8-S73A/D mutant and CTTN1T401,S405,Y421 , BUB1BS1043 , and CARHSP1S30,S32 in K8-S431A/D mutants as well as some anonymous phosphosites including MYCS176 , ZYXS344 , and PNNS692 could be potential candidates associated with K8 phosphorylation mediated tumorigenicity. Biochemical validation followed by phenotypic analysis further confirmed our quantitative phosphoproteomics data. In conclusion, our study provides the first global picture of K8 site-specific phosphorylation function in neoplastic progression of A431 cells and suggests various potential starting points for further mechanistic studies.

Identification of morphological and biochemical changes in keratin-8/18 knock-down cells using Raman spectroscopy.

Accurate understanding of cellular processes and responses to stimuli is of paramount importance in biomedical research and diagnosis. Raman spectroscopy (RS), a label-free and nondestructive spectroscopic method has the potential to serve as a novel 'theranostics' tool. Both fiber-optic and micro-Raman studies have demonstrated efficacy in diagnostics and therapeutic response monitoring. In the present study, we have evaluated the potential of micro-Raman spectroscopic maps in identifying changes induced by loss of K8/18 proteins in a tongue cancer cell line. Furthermore, we also evaluated the efficacy of less expensive and commercially available fiber probes to identify K8/18 wild and knock-down cell pellets, in view of the utility of cell pellet-based studies. The findings suggest that major differences in the cellular morphology and biochemical composition can be objectively identified and can be utilized for classification using both micro-Raman and fiber-probe-based RS. These findings highlight the potential of fiber-optic probe-based RS in noninvasive cellular phenotyping for diagnosis and therapeutic response monitoring, especially in low-resource settings.

Functional Implications of O-GlcNAcylation-dependent Phosphorylation at a Proximal Site on Keratin 18.

Keratins 8/18 (K8/18) are phosphoglycoproteins and form the major intermediate filament network of simple epithelia. The three O-GlcNAcylation (Ser(29), Ser(30), and Ser(48)) and two phosphorylation (Ser(33) and Ser(52)) serine sites on K18 are well characterized. Both of these modifications have been reported to increase K18 solubility and regulate its filament organization. In this report, we investigated the site-specific interplay between these two modifications in regulating the functional properties of K18, like solubility, stability, and filament organization. An immortalized hepatocyte cell line (HHL-17) stably expressing site-specific single, double, and triple O-GlcNAc and phosphomutants of K18 were used to identify the site(s) critical for regulating these functions. Keratin 18 mutants where O-GlcNAcylation at Ser(30) was abolished (K18-S30A) exhibited reduced phosphorylation induced solubility, increased stability, defective filament architecture, and slower migration. Interestingly, K18-S30A mutants also showed loss of phosphorylation at Ser(33), a modification known to regulate the solubility of K18. Further to this, the K18 phosphomutant (K18-S33A) mimicked K18-S30A in its stability, filament organization, and cell migration. These results indicate that O-GlcNAcylation at Ser(30) promotes phosphorylation at Ser(33) to regulate the functional properties of K18 and also impact cellular processes like migration. O-GlcNAcylation and phosphorylation on the same or adjacent sites on most proteins antagonize each other in regulating protein functions. Here we report a novel, positive interplay between O-GlcNAcylation and phosphorylation at adjacent sites on K18 to regulate its fundamental properties.

Differential Ratios of Omega Fatty Acids (AA/EPA+DHA) Modulate Growth, Lipid Peroxidation and Expression of Tumor Regulatory MARBPs in Breast Cancer Cell Lines MCF7 and MDA-MB-231.

Omega 3 (n3) and Omega 6 (n6) polyunsaturated fatty acids (PUFAs) have been reported to exhibit opposing roles in cancer progression. Our objective was to determine whether different ratios of n6/n3 (AA/EPA+DHA) FAs could modulate the cell viability, lipid peroxidation, total cellular fatty acid composition and expression of tumor regulatory Matrix Attachment Region binding proteins (MARBPs) in breast cancer cell lines and in non-cancerous, MCF10A cells. Low ratios of n6/n3 (1:2.5, 1:4, 1:5, 1:10) FA decreased the viability and growth of MDA-MB-231 and MCF7 significantly compared to the non-cancerous cells (MCF10A). Contrarily, higher n6/n3 FA (2.5:1, 4:1, 5:1, 10:1) decreased the survival of both the cancerous and non-cancerous cell types. Lower ratios of n6/n3 selectively induced LPO in the breast cancer cells whereas the higher ratios induced in both cancerous and non-cancerous cell types. Interestingly, compared to higher n6/n3 FA ratios, lower ratios increased the expression of tumor suppressor MARBP, SMAR1 and decreased the expression of tumor activator Cux/CDP in both breast cancer and non-cancerous, MCF10A cells. Low n6/n3 FAs significantly increased SMAR1 expression which resulted into activation of p21WAF1/CIP1 in MDA-MB-231 and MCF7, the increase being ratio dependent in MDA-MB-231. These results suggest that increased intake of n3 fatty acids in our diet could help both in the prevention as well as management of breast cancer.

Versatile hemidesmosomal linker proteins: structure and function.

Hemidesmosomes are anchoring junctions which connect basal epidermal cells to the extracellular matrix. In complex epithelia like skin, hemidesmosomes are composed of transmembrane proteins like α6ß4 integrin, BP180, CD151 and cytoplasmic proteins like BPAG1e and plectin. BPAG1e and plectin are plakin family cytolinker proteins which anchor intermediate filament proteins i.e. keratins to the hemidesmosomal transmembrane proteins. Mutations in BPAG1e and plectin lead to severe skin blistering disorders. Recent reports indicate that these hemidesmosomal linker proteins play a role in various cellular processes like cell motility and cytoskeleton dynamics apart from their known anchoring function. In this review, we will discuss their role in structural and signaling functions.

14-3-3γ-Mediated transport of plakoglobin to the cell border is required for the initiation of desmosome assembly in vitro and in vivo.

The regulation of cell-cell adhesion is important for the processes of tissue formation and morphogenesis. Here, we report that loss of 14-3-3γ leads to a decrease in cell-cell adhesion and a defect in the transport of plakoglobin and other desmosomal proteins to the cell border in HCT116 cells and cells of the mouse testis. 14-3-3γ binds to plakoglobin in a PKCµ-dependent fashion, resulting in microtubule-dependent transport of plakoglobin to cell borders. Transport of plakoglobin to the border is dependent on the KIF5B-KLC1 complex. Knockdown of KIF5B in HCT116 cells, or in the mouse testis, results in a phenotype similar to that observed upon 14-3-3γ knockdown. Our results suggest that loss of 14-3-3γ leads to decreased desmosome formation and a decrease in cell-cell adhesion in vitro, and in the mouse testis in vivo, leading to defects in testis organization and spermatogenesis.

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.

Alterations in keratins and associated proteins during 4- Nitroquinoline-1-oxide induced rat oral carcinogenesis.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is the sixth largest group of malignancies globally and the single largest group of malignancies in the Indian subcontinent. Despite the advances in treatment and therapeutic modalities the five year survival rate of OSCC has not changed in the last few decades, and remains less than 40%. Several studies have focused on defining molecular markers that can either detect cancer at an early stage or can predict patient's outcome. However, such markers are still undefined. Keratins (K) are epithelia predominant intermediate filament proteins which are expressed in a differentiation dependent and site specific manner. Keratins are being used as biomarkers in different epithelial disorders including cancer. They are associated with desmoplakin and α6ß4 integrin which are components of desmosomes and hemidesmosomes respectively. MATERIALS AND METHODS: 4-Nitroquinoline 1-Oxide (4NQO) was used as a carcinogen for the development of various stages of oral carcinogenesis in rat lingual mucosa. Two-Dimentional gel electrophoresis was performed for the separation of Keratins followed by western blotting for their specific identification. Western blotting and RT PCR was carried out for desmoplakin and α6ß4 integrin respectively to understand their levels. Immunohistochemical analysis was carried out to further study the localization of desmoplakin and α6 integrin. RESULTS: In this study we have analysed the alterations in Keratins and associated proteins during sequential stages of 4NQO induced rat oral carcinogenesis. Our results showed that the alterations primarily begin after the dysplastic changes in the lingual epithelium like the elevation of Keratins 5/6a, ectopic expression of Keratin 8, increase in suprabasal expression of α6 integrin and increase in desmoplakin levels. Most of these alterations persisted till the development of SCC except desmoplakin, the levels of which were downregulated in papillomatous lesions and SCC. Many of these alterations have also been documented in human oral carcinogensis. CONCLUSION: Thus, 4NQO model of rat lingual carcinogenesis reproduces majority of the changes that are seen in human oral carcinogenesis and it can be exploited for the development of biomarkers.

Polymeric black tea polyphenols modulate the localization and activity of 12-O-tetradecanoylphorbol-13-acetate-mediated kinases in mouse skin: mechanisms of their anti-tumor-promoting action.

Polymeric black tea polyphenols (PBPs) have been shown to possess anti-tumor-promoting effects in two-stage skin carcinogenesis. However, their mechanisms of action are not fully elucidated. In this study, mechanisms of PBP-mediated antipromoting effects were investigated in a mouse model employing the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Compared to controls, a single topical application of TPA to mouse skin increased the translocation of protein kinase C (PKC) from cytosol to membrane. Pretreatment with PBPs 1-3 decreased TPA-induced translocation of PKC isozymes (α, ß, η, γ, ε) from cytosol to membrane, whereas PBPs 4 and 5 were less effective. The levels of PKCs δ and ζ in cytosol/membrane were similar in all the treatment groups. Complementary confocal microscopic evaluation showed a decrease in TPA-induced PKCα fluorescence in PBP-3-pretreated membranes, whereas pretreatment with PBP-5 did not show a similar decrease. Based on the experiments with specific enzyme inhibitors and phosphospecific antibodies, both PBP-3 and PBP-5 were observed to decrease TPA-induced level and/or activity of phosphatidylinositol 3-kinase (PI3K) and AKT1 (pS473). An additional ability of PBP-3 to inhibit site-specific phosphorylation of PKCα at all three positions responsible for its activation [PKCα (pT497), PKC PAN (ßII pS660), PKCα/ßII (pT638/641)] and AKT1 at the Thr308 position, along with a decrease in TPA-induced PDK1 protein level, correlated with the inhibition of translocation of PKC, which may impart relatively stronger chemoprotective activity to PBP-3 than to PBP-5. Altogether, PBP-mediated decrease in TPA-induced PKC phosphorylation correlated well with decreased TPA-induced NF-κB phosphorylation and downstream target proteins associated with proliferation, apoptosis, and inflammation in mouse skin. Results suggest that the antipromoting effects of PBPs are due to modulation of TPA-induced PI3K-mediated signal transduction.

Plakophilin3 loss leads to an increase in PRL3 levels promoting K8 dephosphorylation, which is required for transformation and metastasis.

The desmosome anchors keratin filaments in epithelial cells leading to the formation of a tissue wide IF network. Loss of the desmosomal plaque protein plakophilin3 (PKP3) in HCT116 cells, leads to an increase in neoplastic progression and metastasis, which was accompanied by an increase in K8 levels. The increase in levels was due to an increase in the protein levels of the Phosphatase of Regenerating Liver 3 (PRL3), which results in a decrease in phosphorylation on K8. The increase in PRL3 and K8 protein levels could be reversed by introduction of an shRNA resistant PKP3 cDNA. Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation. Further, the K8 PKP3 double knockdown clones showed a decrease in colony formation in soft agar and decreased tumorigenesis and metastasis in nude mice. These results suggest that a stabilisation of K8 filaments leading to an increase in migration and transformation may be one mechanism by which PKP3 loss leads to tumor progression and metastasis.

Fascin overexpression promotes neoplastic progression in oral squamous cell carcinoma.

BACKGROUND: Fascin 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. METHODS: To 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. RESULTS: Fascin 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. CONCLUSION: In 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.

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.

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

Keratins are cytoplasmic intermediate filament proteins preferentially expressed by epithelial tissues in a site-specific and differentiation-dependent manner. The complex network of keratin filaments in stratified epithelia is tightly regulated during squamous cell differentiation. Keratin 14 (K14) is expressed in mitotically active basal layer cells, along with its partner keratin 5 (K5), and their expression is down-regulated as cells differentiate. Apart from the cytoprotective functions of K14, very little is known about K14 regulatory functions, since the K14 knockout mice show postnatal lethality. In this study, K14 expression was inhibited using RNA interference in cell lines derived from stratified epithelia to study the K14 functions in epithelial homeostasis. The K14 knockdown clones demonstrated substantial decreases in the levels of the K14 partner K5. These cells showed reduction in cell proliferation and delay in cell cycle progression, along with decreased phosphorylated Akt levels. K14 knockdown cells also exhibited enhanced levels of activated Notch1, involucrin, and K1. In addition, K14 knockdown AW13516 cells showed significant reduction in tumorigenicity. Our results suggest that K5 and K14 may have a role in maintenance of cell proliferation potential in the basal layer of stratified epithelia, modulating phosphatidylinositol 3-kinase/Akt-mediated cell proliferation and/or Notch1-dependent cell differentiation.

Loss of keratins 8 and 18 leads to alterations in α6ß4-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 α6ß4 integrin levels and α6ß4-integrin-dependent signalling events, which have been reported to play an important role in neoplastic progression in epithelial tissues. Therefore, modulation of α6ß4 integrin signalling might be one of the mechanisms by which K8 and K18 promote malignant transformation and/or progression in OSCCs.

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.

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.