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Introduction
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April 2012 - present
July 2005 - January 2012
July 2005 - November 2011
Publications
Publications (85)
Amplification of the CCNE1 locus on chromosome 19q12 is prevalent in multiple tumour types, particularly in high-grade serous ovarian cancer, uterine tumours and gastro-oesophageal cancers, where high cyclin E levels are associated with genome instability, whole-genome doubling and resistance to cytotoxic and targeted therapies1–4. To uncover thera...
Ataxia telangiectasia and Rad3-related (ATR) kinase protects genome integrity during DNA replication. RP-3500 is a novel, orally bioavailable clinical-stage ATR kinase inhibitor (NCT04497116). RP-3500 is highly potent with IC50 values of 1.0 and 0.33 nM in biochemical and cell-based assays, respectively. RP-3500 is highly selective for ATR with 30-...
Background: Ataxia telangiectasia and Rad3-related (ATR) protein kinase is a key mediator of cellular DNA damage repair (DDR) and is activated in response to DNA replication stress. ATR is attractive as a drug target in tumors with loss-of-function alterations in complimentary DDR pathways, including ataxia-telangiectasia mutated (ATM) and BRCA. RE...
Keratin 8/18, the predominant keratin pair of simple epithelia, is known to be aberrantly expressed in several squamous cell carcinomas, 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 post‐translational mod...
Amplification of the gene encoding cyclin E (CCNE1) is an oncogenic driver in several malignancies and is associated with chemoresistance and poor prognosis. To uncover therapeutic targets for CCNE1-amplified tumors, we undertook genome-scale CRISPR/Cas9-based synthetic lethality screens in cellular models of CCNE1 amplification. Here, we report th...
Histone methyltransferase KMT2D harbors frequent loss-of-function somatic point mutations in several tumor types, including melanoma. Here, we identify KMT2D as a potent tumor suppressor in melanoma through an in vivo epigenome-focused pooled RNAi screen and confirm the finding by using a genetically engineered mouse model (GEMM) based on condition...
Histone methyltransferase KMT2D harbors frequent loss-of-function somatic point mutations in several tumor types, including melanoma. Here, we identify KMT2D as a potent tumor suppressor in melanoma through an in vivo epigenome-focused pooled RNAi screen and confirm the finding by using a genetically engineered mouse model (GEMM) based on condition...
Epigenetic modifiers frequently harbor loss-of-function mutations in lung cancer, but their tumor-suppressive roles are poorly characterized. Histone methyltransferase KMT2D (a COMPASS-like enzyme, also called MLL4) is among the most highly inactivated epigenetic modifiers in lung cancer. Here, we show that lung-specific loss of Kmt2d promotes lung...
Epigenetic modifiers often harbor loss-of-function mutations in lung cancer, but their tumor-suppressive roles are poorly characterized. Here we show that lung-specific loss of the gene encoding the histone methyltransferase MLL4 (alias KMT2D; a COMPASS-like enzyme), which is ranked the most highly inactivated epigenetic modifier in lung cancer, st...
Epigenetic modifiers have emerged as important regulators of tumor progression. We identified histone methyltransferase KMT2D as a potent tumor-suppressor through an in vivo epigenome-focused pooled RNAi screen in melanoma. KMT2D harbors frequent somatic point mutations in multiple tumor types. How these events contribute to tumorigenesis and wheth...
Lung adenocarcinoma (LUAD) is a major form of lung cancer, which is the leading cause of cancer death. Histone methylation reader proteins mediate the effect of histone methylation, a hallmark of epigenetic and transcriptional regulation of gene expression. However, their roles in LUAD are poorly understood. Here our bioinformatic screening and ana...
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 Notch‑1 levels and differentiation m...
Vimentin is an intermediate filament protein, predominantly expressed in cells of mesenchy-mal origin, although its aberrant expression is seen in many carcinomas during epithelial mes-enchymal transition. In cancer, vimentin expression is associated with the transition from a more differentiated epithelial phenotype to a dedifferentiated state. In...
Graph showing fold change in the protein level of vimentin and K8 upon vimentin downregulation.
Graph shows quantitation of western blots using densitometry. Fold-change in vimentin and K8 protein level in vimentin knockdown clones is shown relative to that of its vector control clone. Error bars denote ± SEM. from three independent experiments.
(T...
Graph showing fold change in the protein level of K14, K5, K17 and K18 upon vimentin downregulation.
Graph shows quantitation of western blots using densitometry. Fold-change in K14, K5, K17 and K18 protein level in vimentin knockdown clones is shown relative to that of its vector control clone. Error bars denote ±S.E.M. from three independent expe...
Proliferation-related markers like PCNA and Ki67 remained unchanged upon vimentin downregulation.
(A) Representative immunofluorescence images (Bar: 10μm) of PCNA (green) staining in vimentin knockdown and vector control clones. (B) Representative immunofluorescence images (Bar: 10μm) of Ki67 (red) staining in vimentin knockdown and vector control...
Oct-4 mRNA levels decreased significantly upon vimentin downregulation.
QRT-PCR analysis of Oct-4 in vimentin knockdown and its vector control clones.
(TIF)
Vimentin downregulation led to a significant increase in the protein level of differentiation specific marker involucrin.
(A) Western blot analysis shows protein level of involucrin from whole cell lysates of vimentin knockdown and its vector control clones. (B) Graph shows quantitation of western blot using densitometry. Fold-change in involucrin...
Tumors formed in mice from vimentin knockdown cells showed an increase in the expression of involucrin.
Representative images (Bar: 200μm) of IHC staining for expression of involucrin and PCNA in tumor tissues of mice, injected with either vimentin knockdown or vector control clones. The negative control images represent tissue sections incubated w...
Graphs showing fold change in the protein level of vimentin, K5, K14 and involucrin across different cell lines.
Graphs show quantitation of western blots using densitometry. (A and B) Fold-change in vimentin, K5, K14, K17 and involucrin protein level in vimentin overexpressing clones of A431vim and HaCatvim is shown relative to its respective vect...
K5 re-expression alone in vimentin knockdown background failed to rescue the protein level of differentiation specific marker involucrin.
(A) Immunofluorescence (Bar: 10 μm) images of K5 overexpressing (K5) and its vector control clone (K5vc) using antibodies against K5 and K14. (B) Western blot analysis shows protein level of K5, K14 and involucri...
Tumors formed in mice from K5/K14 overexpressing cells showed a decrease in the expression of involucrin.
Representative images (Bar: 200μm) of IHC staining for expression of involucrin and PCNA in tumor tissues of mice, injected with either K5/K14 overexpressing or its vector control clones. The negative control images represent tissue sections in...
Oct-4 mRNA levels did not change significantly upon ΔNp63α re-expression.
qRT-PCR analysis of Oct-4 in flag-ΔNp63α overexpressing and its vector control clones.
(TIF)
Notch1 (independently or through NF-κB) may regulate the expression of ΔNp63.
(A) Western blot analysis shows the protein levels of notch intracellular domain (NICD) in vimentin knockdown and its vector control cells. (B) The confocal images (Bar: 20μm) show the distribution of NF-κB (p65) (red) in the cytoplasmic vs. nuclear compartment in vimenti...
Confocal images showing filament organization of K5 and K14, after their re-expression in vimentin knockdown background.
(A and B) Confocal microscopy analysis (Bar: 10μm) shows levels and filament networks of K5 and K14 respectively in K5/K14 (K5 and K14 overexpressing) as compared to its vector control vcvc clones (empty vectors of K5 and K14 tog...
Graphs showing fold change in the protein level of ΔNp63 and its related molecules.
Graphs show quantitation of western blots using densitometry. (A) Fold-change in ΔNp63, p21 and p27 protein level in vimentin knockdown clones is shown relative to that of its vector control clone. (B) Fold-change in flag- tagged ΔNp63α protein level in flag-ΔNp63α...
Graphs showing fold change in the protein level of K5, K14 and vimentin upon K5/K14 re-expression in vimentin knockdown background.
Graphs show quantitation of western blots using densitometry. (A and B) Fold-change in K5 and K14 protein level in K5/K14 overexpressing clone is shown relative to that of its vector control clone. (C) Fold-change in v...
K5/K14 re-expression in vimentin knockdown background led to a significant decrease in the protein level of differentiation specific marker involucrin.
(A) Western blot analysis shows protein level of involucrin from whole cell lysates of K5/K14 overexpressing as compared to its vector control clones. (B) Graph shows quantitation of western blot us...
Oct-4 mRNA levels increased significantly upon K5/K14 re-expression in vimentin knockdown background.
qRT-PCR analysis of Oct-4 in K5/K14 overexpressing and its vector control clones.
(TIF)
K14 re-expression alone in vimentin knockdown background failed to rescue the protein level of differentiation specific marker involucrin.
(A) Immunofluorescence (Bar: 10 μm) images shows K14 overexpression in overexpressing (K14) and its vector control clone (K14vc). K5 levels remained unchanged between the clones. (B) Western blot analysis shows...
Confocal images showing filament organization of K5 and K14, upon re-expression of ΔNp63α in vimentin knockdown cells.
(A and B) Confocal microscopy analysis (Bar: 10μm) shows levels and filament networks of K5 and K14 respectively in ΔNp63α (flag tagged ΔNp63α overexpressing) as compared to vbb (vector control clone).
(TIF)
Reagents used in this study.
The table shows a list of reagents along with their particulars.
(TIF)
Correlations of high vimentin-K5-K14-low K1 expression with clinicopathological parameters of the oral cancer patients (n = 100).
(TIF)
Graphs showing fold change in the protein level of NICD, nuclear NF-κB and transcript levels of their respective target genes.
(A) Fold-change in NICD protein level in vimentin knockdown clone is shown relative to that of its vector control clone. Also, fold-change in NICD protein level in flag-ΔNp63α overexpressing clone is shown relative to that...
Antibodies used in this study.
The table shows a list of antibodies along with their particulars.
(TIF)
Primers used in this study.
The table shows a list of primer sequences used for RT-PCR and qRT-PCR analysis.
(TIF)
Immunohistochemistry analysis on human oral tumor tissues showed positive correlation between K5/K14 and vimentin expression.
(A) The upper panel show images (Bar: 200μm) of IHC staining for K1, K5, K14 and vimentin expression in cut margin tissues while lower panel show images (Bar: 200μm) of IHC staining for K1, K5, K14 and vimentin expression in...
List of cell lines used in this study.
The table shows a list of cell lines along with their particulars.
(TIF)
Correlations of vimentin-K14 expression with clinicopathological parameters of the oral cancer patients (n = 100).
(TIF)
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 diagnos...
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 t...
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...
Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA
The molecular etiology of non-small cell lung cancer (NSCLC) is heterogeneous and mostly dominated by alterations in kinase signaling pathways (i.e. KRAS, EGFR, EML4-ALK, PI3K, MEK1). Epigenetic modifiers, including histone methyltransferase and demethylases, have emerged as imp...
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 (PG) and other desmosomal proteins to the cell border in HCT116 cells and in the mouse testis. 14-3-3γ binds to PG in a...
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...
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 caus...
To investigate the clinical significance of vimentin expression at early and late events of tobacco/areca nut-associated oral tumorigenesis.
Immunohistochemistry (IHC) was carried out on paraffin-embedded tissues of oral mucosa normal (n = 10), inflammatory lesions (n = 19), leukoplakia (n = 52), submucous fibrosis (n = 71) and tumours/cut margins...
OBJECTIVE: To investigate the clinical significance of vi-mentin expression at early and late events of tobacco/ areca nut–associated oral tumorigenesis. MATERIALS AND METHODS: Immunohistochemistry (IHC) was carried out on paraffin-embedded tissues of oral mucosa normal (n = 10), inflammatory lesions (n = 19), leukoplakia (n = 52), submucous fibros...
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 c...
Analysis of differentiation status of K8 up−/down-regulated clones by lipid droplets staining using Nile red. Representative confocal images of lipid droplets staining (A) MDA MB 435 K8 over-expressed (K8C1) and vector control (K8Vc) clones. Histogram showing the mean intensity of MDA MB 435 K8 over-expressed (K8C1 and C2) and vector control (K8Vc)...
High salt Keratin extraction. The keratin profile of MDA MB 468 K8 down-regulated (ShC1 and C2) and vector control (Vc) clones after high-salt extraction. The arrow indicates position of K8 band on the gel at molecular weight (M.W. ∼52 kDa). The numbers indicated on the left hand side indicates the gel pieces taken for the mass spectrometry analysi...
Analysis of soft agar colony forming potential in MCF10A, MDAMB 468 and MDA MB 435 cell lines. Representative phase contrast images (10X) of colonies formed in soft agar per plate by (A) MCF10A, MDA MB 468 and MDA MB 435. (B) Histogram showing number of colonies formed in soft agar. (C) Histogram showing volume of colonies formed in soft agar. Size...
Details of the bands with keratin identity.
(DOCX)
Real time PCR analysis of K8 and K18 expression in K8 up-regulated MDA MB 435 clones. Real time PCR analysis of genes encoding (A) K8 gene in MDAMB 435 K8 over-expressed (K8C1, C2) and vector control (K8Vc) clones and (B) K18 gene in K8 over-expressed MDA MB 435 clones (K8C1 and C2) as compared to vector control clone (K8Vc) using GAPDH as internal...
Analysis of K8, K18 and vimentin in MCF10A, MDA MB 468 and MDA MB 435 cell lines. Representative confocal images of K8, K18 and vimentin filaments formed in (A) MCF10A, MDA MB 468 and MDA MB 435 cells using mAb to K8, K18 and vimentin respectively. (B) Western blot analysis of K8 and K18 in MCF10A, MDA MB 468 and MDA MB 435 cells using mAb to K8 an...
MCF10A K8/18 down-regulation. (A) Histogram showing % protein expression (± S.E.) for three independent experiments of K8 in MCF10A K8 down-regulated (MShC1, C2 and C3) and vector control (MVc) clones.(B) Histogram showing % protein expression (± S.E.) for three independent experiments of K18 in MCF10A K8 down-regulated (MShC1, C2 and C3) and vecto...
Analysis of K8 expression by flow cytometry in K8 up-regulated clones of MDA MB 435, K8 down-regulated clones of MDA MB 468 and MCF10A. Histograms showing mean fluorescence intensity of K8 (± S.E.) for three independent experiments (lower panel); in (A) K8 up-regulated MDA MB 435 clones (K8C1 and C2) as compared to parental MDA MB 435 and vector co...
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 i...
PRL3 localization in PKP3 knockdown cells. A. PRL3 localization in PKP3 knockdown cells. The vector control (pTU6) or PKP3 knockdown clones (S9 and S10) were stained with antibodies to PRL3 and imaged by confocal microscopy. Note that PRL3 levels increase in the PKP3 knockdown clones with an increase in perinuclear localization and a slight increas...
K8 and K18 filament formation in PKP3 knockdown clones. A. Localization of phosphorylated K8 in PKP3 knockdown clones. The vector control (pTU6) or PKP3 knockdown clones (S9 and S10) were stained with phospho-specific antibodies to PRL3 (S73 and S431) and imaged by confocal microscopy. Note that the intensity of the signal for pS73 increases due to...
Characterization of K8 and PKP3 double knockdown clones. A. Generation of PKP3 and K8 double knockdown clones. Protein extracts from the S10 derived K8 (8.21, 8.24 and 8.28) knockdown clones or the vector alone (S10P3) or the vector control (pTU6) were resolved on SDSPAGE gels followed by Western blotting with antibodies to PKP3 and β-actin. B. The...
K8 and K18 organization in PKP3 knockdown cells. A. K18 forms a complex with PKP3 in HCT116 cells. EBC extracts prepared from HCT116 cells were incubated with a control antibody (IgG) or antibodies to K18 (K18). The reactions were resolved on SDS-PAGE gels and Western blots performed with antibodies to PKP3 and K18. (WCE = Whole cell extract. IP =...
Post-translational modifications on K8 and K18 upon PKP3 knockdown. A. Migration of K18 in two-dimensional gel electrophoresis. Protein extracts from the vector control and PKP3 knockdown clones were resolved in two dimensional gels followed by Western blotting with antibodies to K18. Note that the levels of K18 are higher in the PKP3 knockdown clo...
Transformation induced upon keratin loss in the PKP3 knockdown clones. A. The S10 derived K18 (18.22 and 18.23) knockdown clones or the vector alone (S10P3) were plated in soft agar and colony formation determined after 2–3 weeks. The number of colonies formed by the clones per 20 low power fields (10X) was counted in triplicate in each experiment...
Keratins are one of most widely used markers for oral cancers. Keratin 8 and 18 are expressed in simple epithelia and perform both mechanical and regulatory functions. Their expression are not seen in normal oral tissues but are often expressed in oral squamous cell carcinoma. Aberrant expression of keratins 8 and 18 is most common change in human...
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 a...
Table S1. Clinico-pathological parameters of the OSCC patients.
Figure S1. (A) Cell morphology (shape) of fascin-overexpressed and vector control clone analyzed by phase contrast microscopy. Scale bar: 100 μm. (B) Western blot analysis of fascin-overexpressed (AW-Fascin-1 and AW-Fascin-2) and vector control clones (AW-GFP-Cont) with antibody to vimentin. β-actin was used as a loading control. (C) Representative...
Table S3. Correlations of fascin in combination with K8 and β4-integrin expression with clinico-pathological parameters of the OSCC patients.
Figure S4. Representative images of IHC staining with antibodies against fascin on paraffin embedded sections o f primary tumor and lymph node metastasized tumor of human OSCC tissues. Sections were counter stained with eosin (Magnification: 200×).
Table S2. The Fascin overexpressed clones show a decrease in cell-cell adhesion. Cell adhesion was measured by the hanging drop assay as described. 2 × 104 cells of the indicated clones were resuspended in 35 μl of complete medium on the lid of a 24 well dish. 16 h later the cells were fixed and the number and area of aggregates in fifteen fields w...
Figure S2. Representative images of haematoxylin and eosin along with immunohistochemical staining with antibodies against fascin, K8 and β4-integrin on paraffin embedded sections of human in oral tumors (A) and non malignant tissues (B). Sections were counter stained with eosin (Magnification: 200×).
Figure 3. (A) Representative images of immunofluorescence staining with antibodies against β4-integrin and K1 of paraffin embedded sections of non malignant oral tissues. Sections were counter stained with DAPI. Scale bar: 50 μm. (B) Representative images of immunofluoroscence staining with antibodies against fascin, β4-integrin and K14 of paraffin...
Figure R1. (A and B)Western blot analysis of fascin-overexpressed (AW-Fascin-1 and AW-Fascin-2) and vector control clones (AW-GFP-Cont) with antibodies to α6-integrin, β4-integrin and pFAK. β-actin and FAK were used as loading control respectively. Densitometric analysis for the quantification of level of α6-integrin, β4-integrin and pFAK obtained...
IHC analysis of K8 expression in malignant and non-malignant human oral tissues. Representative images of H&E and IHC staining using antibody against K8 on paraffin embedded sections of human oral tumors and non-malignant tissues. Note that non-malignant oral tissues showed negative staining while malignant tissues showed positive staining of K8.
(...
IHC staining of phosphorylated K8 in human OSCC. Representative images of H&E staining along with positive IHC staining with antibodies specific to phosphorylated Ser73 and Ser431 of K8 on paraffin embedded sections of human OSCC.
(TIF)
Clinico-pathological parameters of OSCC patients (
n
= 52).
(DOC)
