Hunain Alam

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.

KDM2A promotes lung tumorigenesis by epigenetically enhancing ERK1/2 signaling

Epigenetic dysregulation has emerged as a major contributor to tumorigenesis. Histone methylation is a well-established mechanism of epigenetic regulation that is dynamically modulated by histone methyltransferases and demethylases. The pathogenic role of histone methylation modifiers in non-small cell lung cancer (NSCLC), which is the leading cause of cancer deaths worldwide, remains largely unknown. Here, we found that the histone H3 lysine 36 (H3K36) demethylase KDM2A (also called FBXL11 and JHDM1A) is frequently overexpressed in NSCLC tumors and cell lines. KDM2A and its catalytic activity were required for in vitro proliferation and invasion of KDM2A-overexpressing NSCLC cells. KDM2A overexpression in NSCLC cells with low KDM2A levels increased cell proliferation and invasiveness. KDM2A knockdown abrogated tumor growth and invasive abilities of NSCLC cells in mouse xenograft models. We identified dual-specificity phosphatase 3 (DUSP3) as a key KDM2A target gene and found that DUSP3 dephosphorylates ERK1/2 in NSCLC cells. KDM2A activated ERK1/2 through epigenetic repression of DUSP3 expression via demethylation of dimethylated H3K36 at the DUSP3 locus. High KDM2A levels correlated with poor prognosis in NSCLC patients. These findings uncover an unexpected role for a histone methylation modifier in activating ERK1/2 in lung tumorigenesis and metastasis, suggesting that KDM2A may be a promising therapeutic target in NSCLC.

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.

Histone methylation modifiers in cellular signaling pathways

Histone methyltransferases and demethylases epigenetically regulate gene expression by modifying histone methylation status in numerous cellular processes, including cell differentiation and proliferation. These modifiers also control methylation levels of various non-histone proteins, such as effector proteins that play critical roles in cellular signaling networks. Dysregulated histone methylation modifiers alter expression of oncogenes and tumor suppressor genes and change methylation states of effector proteins, frequently resulting in aberrant cellular signaling cascades and cellular transformation. In this review, we summarize the role of histone methylation modifiers in regulating the following signaling pathways: NF-κB, RAS/RAF/MEK/MAPK, PI3K/Akt, Wnt/β-catenin, p53, and ERα.

Transcriptional Repression of Histone Deacetylase 3 by the Histone Demethylase KDM2A Is Coupled to Tumorigenicity of Lung Cancer Cells

Background: Overexpression of the epigenetic repressor KDM2A promotes lung tumorigenesis. Results: Transcriptional inhibition of HDAC3 expression by KDM2A releases cell cycle and proinvasive genes from HDAC3-mediated repression and positively regulates cell proliferation and invasiveness. Conclusion: KDM2A-mediated repression of HDAC3 is linked to KDM2A-promoted tumorigenicity. Significance: Our findings provide a novel epigenetic insight into how KDM2A promotes lung tumorigenesis and have implications for therapeutic intervention. Dysregulated expression of histone methyltransferases and demethylases is an emerging epigenetic mechanism underlying cancer development and metastasis. We recently showed that the histone H3 lysine 36 (H3K36) demethylase KDM2A (also called FBXL11 and JHDM1A) is necessary for tumorigenic and metastatic capabilities of KDM2A-overexpressing non-small cell lung cancer (NSCLC) cells. Here, we report that KDM2A transcriptionally represses the histone deacetylase 3 (HDAC3) gene by removing methyl groups from dimethylated H3K36 at the HDAC3 promoter in KDM2A-overexpressing NSCLC cells. KDM2A depletion reduced expression levels of cell cycle-associated genes (e.g. CDK6) and cell invasion-related genes (e.g. NANOS1); these levels were rescued by ectopic expression of KDM2A but not its catalytic mutant. These genes were occupied and down-regulated by HDAC3. HDAC3 knockdown significantly recovered the proliferation and invasiveness of KDM2A-depleted NSCLC cells as well as the levels of CDK6 and NANOS1 expression in these cells. Similar to their previously reported functions in other cell types, CDK6 and NANOS1 were required for the proliferation and invasion, respectively, of KDM2A-overexpressing NSCLC cells. In a mouse xenograft model, HDAC3 depletion substantially restored the tumorigenic ability of KDM2A knockdown cells. These findings reveal a novel cancer-epigenetic pathway in which the antagonistic effect of KDM2A on HDAC3 expression releases cell cycle-associated genes and cell invasion-related genes from HDAC3 repression and indicate the importance of this pathway for tumorigenicity and invasiveness of KDM2A-overexpressing NSCLC cells.

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.

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.

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

ABSTRACT The regulation of cell–cell adhesion is important for the processes of tissue formation and morphogenesis. Here, we report that loss of 14-3-3&ggr; 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. 14-3-3&ggr; binds to plakoglobin in a PKC&mgr;-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 14-3-3&ggr; knockdown. Our results suggest that loss of 14-3-3&ggr; 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.