Almost all epithelial tumours contain cancer stem-like cells, which possess a unique property of self-renewal and differentiation. In oral cancer, several biomarkers including cell surface molecules have been exploited for the identification of this highly tumorigenic population. Implicit is the role of CD44 in defining CSCs but CD24 is not well-explored. Here we show that CD44(high)CD24(low) cells isolated from the oral cancer cell lines, not only express stem cell related genes but also exhibit Epithelial-to-Mesenchymal transition (EMT) characteristics. This CD44(high)CD24(low) population gives rise to all other cell types upon differentiation. Typical Cancer Stem Cell (CSC) phenotypes like increased colony formation, sphere forming ability, migration and invasion were also confirmed in CD44(high)CD24(low) cells. Drug transporters were found to be over-expressed in CD44(high)CD24(low) sub-population thereby contributing to elevated chemo-resistance. To validate our findings in-vivo, we determined the relative expression of CD44 and CD24 in clinical samples of OSCC patients. CD44 expression was consistently high whereas CD24 showed significantly lower expression in tumour tissues. Further, the gene expression profile of the CSC and non-CSC population unravels the molecular pathways which may contribute to stemness. We conclude that CD44(high)CD24(low) represents cancer stem-like cells in Oral Squamous Cell Carcinoma.

Human NEIL2, one of five oxidized base-specific DNA glycosylases, is unique in preferentially repairing oxidative damage in transcribed genes. Here we show that depletion of NEIL2 causes a 6- to 7-fold increase in spontaneous mutation frequency in the HPRT gene of the V79 Chinese hamster lung cell line. This prompted us to screen for NEIL2 variants in lung cancer patients’ genomic DNA. We identified several polymorphic variants, among which R103Q and R257L were frequently observed in lung cancer patients. We then characterized these variants biochemically, and observed a modest decrease in DNA glycosylase activity relative to the wild type (WT) only with the R257L mutant protein. However, in reconstituted repair assays containing WT NEIL2 or its R257L and R103Q variants together with other DNA base excision repair (BER) proteins (PNKP, Polβ, Lig IIIα and XRCC1) or using NEIL2-FLAG immunocomplexes, an ~ 5-fold decrease in repair was observed with the R257L variant compared to WT or R103Q NEIL2, apparently due to the R257L mutant’s lower affinity for other repair proteins, particularly Polβ. Notably, increased endogenous DNA damage was observed in NEIL2 variant (R257L)-expressing cells relative to WT cells. Taken together, our results suggest that the decreased DNA repair capacity of the R257L variant can induce mutations that lead to lung cancer development.

The aim of this study is to understand the mechanism of EGFR overexpression in head and neck squamous cell carcinoma (HNSCC). For this reason, expression/mutation of EGFR were analyzed in 30 dysplastic head and neck lesions and 148 HNSCC samples of Indian patients along with 3 HNSCC cell lines. In addition, deletion/methylation/mutation/expression of SH3GL2 (associated with EGFR degradation) and CDC25A (associated with dephosphorylation of EGFR) were analyzed in the same set of samples. Our study revealed high frequency of EGFR overexpression (66–84%), low frequency of gene amplification (10–32.5%) and absence of functional mutation in the dysplastic lesions and HNSCC samples. No correlation was found between protein overexpression and mRNA expression/gene amplification status of EGFR. On the other hand, frequent alterations (deletion/methylation) of SH3GL2 (63–77%) and CDC25A (37–64%) were seen in the dysplastic and HNSCC samples. Two novel single nucleotide polymorphism (SNPs) were found in the promoter region of SH3GL2. Reduced expression of these genes showed concordance with their alterations. Overexpression of EGFR and p-EGFR were significantly associated with reduced expression and alterations of SH3GL2 and CDC25A respectively. In-vitro demethylation experiment by 5-aza-2′-deoxycytidine (5-aza-dC) showed upregulation of SH3GL2 and CDC25A and downregulation of EGFR expression in Hep2 cell line. Poor patient outcome was predicted in the cases with alterations of SH3GL2 and CDC25A in presence of human papilloma virus (HPV) infection. Also, low SH3GL2 and high EGFR expression was a predictor of poor patient survival. Thus, our data suggests that overexpression of EGFR due to its reduced degradation and dephosphorylation is needed for development of HNSCC.

Lens epithelium‐derived growth factor (LEDGF), a ubiquitously expressed nuclear protein, acts by interacting with DNA and protein and is involved in widely varying cellular functions. Despite its importance, the mechanism(s) that regulate naturally occurring LEDGF activity are unidentified. In the present study, we report that LEDGF is constitutively Sumoylated, and that the dynamical regulatory mechanism(s) (i.e. Sumoylation and deSumoylation) act as a molecular switch in modulating the DNA‐binding and transcriptional activity of LEDGF with the functional consequences. Using bioinformatics analysis coupled with in vitro and in vivo Sumoylation assays, we found that lysine (K) 364 of LEDGF was Sumoylated, repressing its transcriptional activity. Conversely, mutation of K364 to arginine (R) or deSumoylation by small ubiquitin‐like modifier (Sumo)‐specific protease‐1, a nuclear deSumoylase, enhanced the transactivation capacity of LEDGF and its cellular abundance. The enhancements were directly correlated with an increase in the DNA‐binding activity and small heat shock protein transcription of LEDGF, whereas the process was reversed in cells overexpressing Sumo1. Interestingly, cells expressing Sumoylation‐deficient pEGFP‐K364R protein showed increased cellular survival compared to wild‐type LEDGF protein. The findings provide insights into the regulation and regulatory functions of LEDGF in Sumoylation‐dependent transcriptional control that may be essential for modifying the physiology of cells to maintain cellular homeostasis. These studies also provide new evidence of the important role of post‐translational modification in controlling LEDGF function. Structured digital abstract  http://www.uniprot.org/uniprot/O75475 and http://www.uniprot.org/uniprot/P63165 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0403 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0416 (http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8384981)  http://www.uniprot.org/uniprot/O75475http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0914 with http://www.uniprot.org/uniprot/P63165 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0006 (View Interaction: http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8384827, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8384845, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8384873)