Karunagaran Devarajan

Lung cancer causes high mortality without a declining trend and non small cell lung cancer represents 85% of all pulmonary carcinomas. MicroRNAs (miRNAs) serve as fine regulators of proliferation, migration, invasion/metastasis and angiogenesis of normal and cancer cells. Using TargetScan6.2, we predicted that the ubiquitin ligase, β-TRCP2, could be a target for two of the constituent miRNAs of the miR-106b-25 cluster (miR-106b and miR-93). We generated a stable clone of miR-106b-25 cluster (CL) or the empty vector (EV) in H1299 (non small cell lung cancer) cells. The expression of β-TRCP2 mRNA was significantly lower in CL than that in EV cells. Transient expression of miR-93 but not antimiR-93 decreased the expression of β-TRCP2 mRNA in H1299 cells. β-TRCP2-3'UTR reporter assay revealed that its activity in CL cells was only 60% of that in EV cells. Snail protein expression was higher in CL than that in EV cells and H1299 cells exhibited an increase in the expression of Snail upon transient transfection with miR-93. miR-106b-25 cluster-induced migration of CL measured by scratch assay was more than that in EV cells and no significant difference in migration was observed between antimiR-93-transfected H1299 cells and the corresponding control-oligo-transfected cells. miR-106b-25 cluster-induced migration of CL cells was again confirmed in a Boyden chamber assay without the matrigel. CL cells were more invasive than EV cells when assessed using Boyden chambers with matrigel but there were no significant changes in the cell viabilities between EV and CL cells. Colony formation assay revealed that the CL cells formed more number of colonies than EV cells but they were smaller in size than those formed by EV cells. The supernatant from CL cells was more effective than that from EV cells in inducing tube formation in endothelial cells. Taken together, our data indicate that miR-106b-25 cluster may play an important role in the metastasis of human non-small cell lung cancer cells by directly suppressing the β-TRCP2 gene expression with a consequent increase in the expression of Snail. © 2013 Elsevier Inc.

microRNA-145 (miR-145) has been shown to act as a tumor suppressor in colorectal cancer but its role in the regulation of epithelial-mesenchymal transition (EMT) is unclear. Ectopic expression of miR-145 suppressed the proliferation, migration and invasion in SW480 but surprisingly enhanced these traits in its metastatic counterpart, SW620 cells, while, antimiR-145 reversed the effects of miR-145 in both of these human colorectal cancer cells. In SW480 and SW620 cells, SMAD-interacting protein 1 (SIP1), was identified as a target of miR-145, and its expression was suppressed both at mRNA and protein levels, and siRNA-SIP1 mimicked the effects of miR-145. Further, re-introduction of SIP1 alone or its co-expression with miR-145, rescued SW480 and SW620 cells from the effects of miR-145, indicating that the distinct functions of miR-145 might be mediated, in part, through SIP1. Since Wnt signaling plays an essential role in EMT in CRC progression, the effects of miR-145 on the expression of Wnt signaling intermediates and EMT markers were studied. Re-expression of miR-145 was found to downregulate the expression of CTNNB1, TCF4, CCND1, VIM, and SNAI, but, upregulated CDH1 expression in SW480 cells. On the other hand, miR-145 exhibited an oncogenic potential in SW620 cells by actuating Wnt signaling and the expression of EMT-relevant markers. These results strongly hint that the paradoxical functions of miR-145 in the regulation of proliferation, migration and invasion might be mediated through downregulation of SIP1, and differential tuning of Wnt signaling and EMT-mediators.

Human papilloma virus is associated with cervical and other tumors, and several cellular conditions also play an important role in carcinogenesis. Human papilloma virus (HPV)-infected cells exhibit activation of NF-κB and STAT3 (mediators of inflammation), but little is known about their regulation by HPV. This study attempts to understand the role of HPV16 E2, an important early protein of HPV16, in the regulation of NF-κB and STAT3 by reporter assays, quantitative reverse transcriptase-polymerase chain reaction, and immunoblotting. We demonstrate that E2 enhances NF-κB activation induced by TNF-α, a proinflammatory cytokine, in both non-tumor- and tumor-derived epithelial cell lines besides potentiating STAT3 transcriptional activity induced by TNF-α in HEK293 cells. E2 increases the expression of RelA and its transcriptional activation, and retention of E2 was observed in the nucleus with significant interaction with RelA (immunoprecipitation) upon TNF-α treatment. Transfection with shRNA-RelA or pretreatment with a STAT3 inhibitor had a negative effect on the ability of E2 to enhance TNF-α-induced NF-κB activation. Experiments with co-expression of a mutant of STAT3 with E2 also suggested that the activation of STAT3 is indispensible for TNF-α-induced NF-κB activation. Inhibition of STAT3 activation enhanced E2-induced apoptosis, whereas parallel activation of NF-κB and STAT3 by the combined action of E2 and TNF-α increased the expression of their common targets, cyclin-D1, c-Myc, survivin, and Bcl-2, leading to a decrease in E2-induced apoptosis (viability and cell cycle). Our results reveal novel mechanisms by which E2 may regulate NF-κB and STAT3 activation in the presence of TNF-α with implications on the survival of HPV-infected cells.

Breast cancer cells evade cell death by overexpressing SLC7A11, which functions by transporting cystine into cells in exchange for intracellular glutamate facilitating glutathione synthesis and reducing reactive oxygen species (ROS)–mediated stress. Using an in silico approach, we predicted an miRNA (miR-5096) that can target and downregulate SLC7A11. We demonstrated SLC7A11 as a target of miR-5096 by 3′UTR luciferase assay and further validated it by identifying reduced mRNA and protein levels of SLC7A11 upon miR-5096 overexpression. miR-5096–induced ferroptotic cell death in human breast cancer cells was confirmed by concurrently increased ROS, OH−, lipid ROS, and iron accumulation levels and decreased GSH and mitochondrial membrane potential (MitoTracker™ Orange) with mitochondrial shrinkage and partial cristae loss (observed by TEM). miR-5096 inhibited colony formation, transwell migration, and breast cancer cell invasion, whereas antimiR-5096 promoted these tumorigenic properties. Ectopic expression of SLC7A11 partly reversed miR-5096-mediated effects on cell survival, ROS, lipid peroxides, iron accumulation, GSH, hydroxyl radicals, mitochondrial membrane potential, and colony formation. miR-5096 modulated the expression of epithelial-mesenchymal transition markers in vitro and inhibited the metastatic potential of MDA-MB-231 cells in a tumor xenograft model of zebrafish larvae. Our results demonstrate that miR-5096 is a tumor-suppressive miRNA in breast cancer cells, and this paper discusses its therapeutic implications.

Curcumin, the active ingredient of turmeric (Curcuma longa) used in culinary and medical practices in Asia, has immense potential for being used in cancer chemotherapy because of its control over the cell growth regulatory mechanisms. The present chapter throws light on the role of curcumin in modulating the various phases of the cell cycle and its apoptosis-inducing effects. This is followed by a discussion on the implications of these effects of curcumin for its use as a chemotherapeutic agent in cancer. © 2007 Springer.

Development of colon adenocarcinoma (COAD) metastasis involves several mediators including fluid shear stress (FSS), intracellular ROS levels, and non-coding RNAs. In our present study, we identified and investigated the role of regulatory non-coding RNA molecules specifically involved in COAD metastasis and their association with FSS and ROS. Interactions between the mRNAs associated with FSS and ROS, the corresponding microRNAs (miRNAs), long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) in COAD metastasis were used to generate the mRNA-miRNA-lncRNA-circRNA network. Experimental validation of the identified RNA hubs using quantitative real-time PCR demonstrated a direct effect of the FSS on their expression levels in cancer cells. FSS resulted in the downregulation of HMGA1 and RAN, as well as the upregulation of HSP90AA1, PMAIP1 and BIRC5. Application of shear stress also led to downregulation of hsa-miR-26b-5p and hsa-miR-34a-5p levels in HCT116 cells. Further, functional enrichment and survival analysis of the significant miRNAs, as well as the OncoPrint and the survival analyses of the selected mRNAs were performed. Subsequently, their functional role was also corroborated with existing literature. Ten significant miRNA hubs were identified, out of which hsa-miR-17-5p and hsa-miR-20a-5p were found to interact with lncRNA (CCAT2) while hsa-miR-335 was found to interact with four circRNAs. Fifteen significant miRNAs were identified in 10 different modules suggesting their importance in FSS and ROS-mediated COAD metastasis. Finally, 10 miRNAs and 3 mRNAs associated with FSS and/or ROS were identified as significant overall survival markers; 33 mRNAs were also identified as metastasis-free survival markers whereas 15 mRNAs showed > 10% gene alterations in TCGA-COAD data and may serve as promising therapeutic biomarkers in the COAD metastasis.

An oncogenic transformation has been associated with different factors such as unhealthy lifestyle and diet, exposure to harmful environmental conditions and infectious agents. These extrinsic and intrinsic carcinogens can be detrimental to cellular, genetic, and epigenetic functions of the cell, leading to abnormal cell growth and division. However, a significant hallmark of cancer cell metabolism is elevated intracellular reactive oxygen species (ROS) level. The increased ROS levels are known to promote cell proliferation, cell-to-cell adhesion, and motility, as well as angiogenesis in tumors. On the other hand, many cancer therapeutics exert their cytotoxic effects on cancer cells by mediating excess intracellular ROS generation, thus implicating a dual role of ROS in tumorigenesis and cancer cell death. Constraint-based modeling (CBM) and genome-scale metabolic models (GSMMs) have been widely used to study diverse biological systems at the metabolic level as well as to understand human diseases better. For instance, reprogrammed metabolism is central to sustaining growth and proliferation in transformed cells and hence needs to be modeled. For this purpose, a constraint-based reconstruction and analysis (COBRA) modeling strategy has been used to analyze cancer-specific metabolism and phenotypes, using context-specific metabolic models. Context-specific GSMMs have also proven advantageous in uncovering potential therapeutic targets. However, unlike numerous experimental studies that have already established the relevance of ROS in cancer, a CBM approach has not considered the role of ROS generation in understanding complex cancer metabolism thus far. This work discusses the significance of CBM in studying the scope of ROS in cancer and in devising novel cancer therapeutics.

The onset of colorectal cancer (CRC) is often attributed to gut bacterial dysbiosis, and thus gut microbiota are highly relevant in devising treatment strategies. Certain gut microbes, like Enterococcus spp., exhibit remarkable anti-neoplastic and probiotic properties, which can aid in silver nanoparticle (AgNPs) induced reactive oxygen species (ROS)-based CRC treatment. However, the effects of AgNPs on gut microbial metabolism have not been reported thus far. In this study, a detailed systems-level understanding of ROS metabolism in Enterococcus durans (E. durans), a representative gut microbe, was gained using constraint-based modeling, wherein, the critical association between ROS and folate metabolism was established. Experimental studies involving low AgNP concentration treatment of E. durans cultures confirmed these modeling predictions (an increased extracellular folate concentration by 52%, at the 9th h of microbial growth, was observed). Besides, the computational studies established various metabolic pathways involving amino acids, energy metabolites, nucleotides, and SCFAs as the key players in elevating folate levels on ROS exposure. The anti-cancer potential of E. durans was also studied through MTT analysis of HCT 116 cells treated with microbial culture (AgNP treated) supernatant. A decrease in cell viability by 19% implicated the role of microbial metabolites (primarily folate) in causing cell death. The genome-scale modeling approach was then extended to extensively model CRC metabolism, as well as CRC–E. durans interactions in the context of CRC treatment, using tissue-specific metabolic models of CRC and healthy colon. These findings on further validation can facilitate the development of robust and effective cancer therapy.

Re-expression of E2 in human papillomavirus (HPV) transformed tumour cells can induce apoptosis; however, some evidences also attribute an important role to E2 in sustaining tumorigenesis. In the present paper, we studied the effects of tumour necrosis factor (TNF)-α -mediated NF-κ B (nuclear factor kappa-light-chain-enhancer of activated B-cells) activation on E2-induced senescence in HPV16-integrated SiHa cells. The results show that E2 inhibits endogenous E6 gene expression and sensitizes SiHa cells to (TNF)-α -induced NF-κ B activation. Under this condition there was an increase in the expression of senescent proteins p53, p21, p27 and p16 and senescence-associated (SA)-β- galactosidase activity indicating that (TNF)-α augments E2-mediated senescence. Re-expression of E2 expression with (TNF)-α treatment resulted in an increase in the expression of anti-apoptotic Bcl2 (B-cell lymphoma 2) protein and other pro-survival genes like cyclin D1 (cyc D1), survivin and hTERT (human telomerase reverse transcriptase). Concomitantly, E2 + (TNF)-α combination increased the survival of SiHa cells by positive changes in viability, proliferation and colony formation. E2-induced apoptotic tendency shifted towards senescence in presence of (TNF)-α by arresting the cells at both G0/G1 and G2/M phases, thus enhancing cell survival. Another observation in the present study is the significant up-regulation of key senescence messaging factors regulated by NF-κ B namely interleukin (IL)-6, IL-8, high-mobility group protein A (HMGA)1 and B (HMGB)1 in E2-transfected cells treated with (TNF)-α . Our data provide a mechanistic basis and a new insight for the role of (TNF)-α and E2 in linking cellular senescence, tumorigenesis and HPV re-infection.

Background: Epithelial cell adhesion molecule (EpCAM), a type I transmembrane protein of the epithelial tissues and known cell adhesion molecule, has been demonstrated to have critical role in carcinogenesis. In breast cancer, EpCAM expression has been associated with poor prognosis. The expression pattern of EpCAM across molecular subtypes of breast carcinoma has been studied in patients reporting to a South Indian multispecialty tertiary care hospital. The prognostic significance of EpCAM expression pattern and probable response to therapy has also been addressed. Materials and Methods: EpCAM expression was assessed by immunohistochemical studies on 200 breast carcinoma tissue samples of different molecular subtypes, including luminal A, luminal B, Her2Neu, and triple-negative breast cancer (TNBC). The expression was scored using the standard scoring system. A correlation was drawn with detailed clinicopathologic annotation and available outcomes data to analyze the influence of EpCAM on prognosis. Results: EpCAM expression varied significantly in the different intrinsic subtypes of breast carcinoma. Differential expression was also established with different grades of breast carcinoma with varying levels of differentiation. We observed strong EpCAM expression in TNBC among other subtypes. Conclusion: The differential expression of EpCAM among intrinsic subtypes of breast cancer and the correlation of EpCAM expression with high-grade breast carcinoma shown in the study have important implications in understanding the role of EpCAM and might form the basis for developing targeted therapies in breast cancer in the future.