Shanaya Patel

Current Drugs and Drug Targets in Non-Small Cell Lung Cancer: Limitations and Opportunities

Lung cancer is a serious health problem and leading cause of death worldwide due to its high incidence and mortality. More than 80% of lung cancers feature a non-small cell histology. Over few decades, systemic chemotherapy and surgery are the only treatment options in this type of tumor but due to their limited efficacy and overall poor survival of patients, there is an urge to develop newer therapeutic strategies which circumvent the problems. Enhanced knowledge of translational science and molecular biology have revealed that lung tumors carry diverse driver gene mutations and adopt different intracellular pathways leading to carcinogenesis. Hence, the development of targeted agents against molecular subgroups harboring critical mutations is an attractive approach for therapeutic treatment. Targeted therapies are clearly more preferred nowadays over systemic therapies because they target tumor specific molecules resulting with enhanced activity and reduced toxicity to normal tissues. Thus, this review encompasses comprehensive updates on targeted therapies for the driver mutations in non-small cell lung cancer (NSCLC) and the potential challenges of acquired drug resistance faced in the field of targeted therapy along with the imminent newer treatment modalities against lung cancer.

Role of miRNA dynamics and cytokine profile in governing CD44v6/Nanog/PTEN axis in oral cancer: modulating the master regulators

Late diagnosis, low therapeutic response, and metastasis are accountable for poor 5-year survival rate of OSCC. These failures are attributed to the existence of “cancer stem cell (CSC)” subpopulation. Hence, it is necessary to identify and understand the mechanism of CSCs in tumor development, metastasis, and chemotherapeutic response. Propelling evidences suggest that microRNA (miRNA)-mediated regulation and cytokines of tumor microenvironment have the ability to modulate CSC signalling pathway; however, their exact mechanism needs to be elucidated. Thus, in this study, we characterized CSC markers and highlighted the miRNA dynamics and cytokine profile regulating these CSCs in a pathway-dependent manner. Our results demonstrated CD44+ subpopulation as tumor-initiating cells with self-renewal capability, tumorigenic growth potential and intrinsic chemoresistance. These tumors exhibited increased expression of CSC markers (CD44v3, CD44v6, Nanog, and Bmi1) and significantly reduced expression of PTEN and ATM in OSCC patients. Pathway analysis of these CSC markers demonstrated a prospective pathway regulated by miRNA and cytokine network. On analyzing these modulators, we observed decreased expression of miRNA542-3p, miRNA34a and miRNA9, and significant upregulation of miRNA21, thus forming an unexplored axis. Cytokine profiling revealed significantly increased levels of IL-6 and IL-8 compared to normals and demonstrated their strong association with CD44v6. Collectively, this study indicates that miR5423p and miR34a targets the CD44v6-Nanog-PTEN axis, thus playing a vital role in regulating the CSC properties. Furthermore, we speculate an impinging role of cytokines IL-6 and IL-8 in regulating this CSC-mediated pathway which can have prognostic and therapeutic implications.

Circulating tumor stem like cells in oral squamous cell carcinoma: An unresolved paradox

OBJECTIVE Circulating tumor cells (CTCs) are increasingly gaining importance due to their immense potential in enhancing diagnosis, prognosis and response to therapy in solid malignancies. Therefore, we aimed to comprehend the molecular diversity and critical role of this disseminated tumor population in OSCC. METHODOLOGY CD44+ subpopulation was isolated using immuno-magnetic cell separation and their purity was validated using flow cytometry. Characterisation of self renewal potential and resistance to chemotherapy was assessed using tumor sphere forming and cytotoxicity assay. Gene expression profile of pertinent CSC (CD44s, CD44v3, CD44v6) and stemness markers (Bmi1 and Nanog) was carried out in CD44+ cells using Real Time PCR. Predominantly expressed markers and their association with clinico-pathological conditions were substantiated in 30 OSCC patients. RESULT Flow cytometry analysis depicted a predominant population of CD44+CD24-CD45- cells suggesting that circulating tumor cells had a subpopulation of CSC like cells in the circulation. These cells demonstrated increased sphere forming capability and intrinsic chemo-resistance compared to non-CSC, thus indicating the CSC features of self-renewal and chemo-resistance. Additionally, CD44+ cells showed significantly increased expression levels of CD44v6 and Nanog compared to CD44- cells. Clinically, expression pattern of CD44v6 and Nanog correlated with different anatomical subsites, loco-regional aggressiveness of the disease and recurrence, thus opening newer avenues that can be explored for better prognostic and therapeutic implications. CONCLUSION This study explored the inevitable role of CD44v6 and Nanog as circulating stem like cell markers in assessment of loco-regional aggressiveness, detection of relapse and therapeutic response and resistance.

Insights into the structural perturbations of spliced variants of CD44: a modeling and simulation approach

Transient interactions between cancer stem cells and components of the tumor microenvironment initiate various signaling pathways crucial for carcinogenesis. Predominant hyaluronan (HA) receptor, CD44 is structurally and functionally one of the most variable cell surface receptors having the potential to generate a diverse repertory of CD44 isoforms by alternative splicing of variant exons and post-translational modifications. A structurally distinctive variant of CD44, CD44v10, has an inevitable role in malignant progression, invasion, and metastasis. This can be attributed to the binding of HA with CD44v10, which demonstrates a completely different behavioral pattern as compared to the other spliced variants of CD44 molecule. Absence of a comprehensively predicted crystal structure of human CD44s and CD44v10 is an impediment in understanding the resultant structural alterations caused by the binding of HA. Thus, in this study, we aim to predict the CD44s and CD44v10 structures to their closest native confirmation and study the HA binding-induced structural perturbations using homology modeling, molecular docking, and MD simulation approach. The results depicted that modeled 3D structures of CD44s and CD44v10 isoforms were found to be stable throughout MD simulations; however, a substantial decrease was observed in the binding affinity of HA with CD44v10 (−5.355 kcal/mol) as compared to CD44s. Furthermore, loss and gain of several H-bonds and hydrophobic interactions in CD44v10–HA complex during the simulation process not only elucidated the reason for decreased binding affinity for HA but also prompted toward the plausible role of HA-induced structural perturbations in occurrence and progression of carcinogenesis.

Uncovering the potential of CD44v/SYNE1/miR34a axis in salivary fluids of oral cancer patients

OBJECTIVES Late-stage diagnosis is one of the major confounders for poor prognosis of patients with oral cancer owing to lack of a biomarker to diagnose this disease at an early stage. Moreover, till date, invasive biopsies are the only option to assess disease occurrence and progression in this malignancy. Thus, this study aims to identify and assess potential salivary markers in OSCC patients in order to open newer avenues in the field of non-invasive biopsies. METHODOLOGY Bioinformatic-based analysis was performed to identify potential biomarkers that could be assessed in OSCC patients. The expression patterns of CD44v and its genetic and epigenetic modulators were assessed in saliva of OSCC patients, leukoplakia, and controls using real-time and methylation-specific PCR. Statistical analysis was conducted to understand the significance of these markers in terms of their clinical relevance. RESULTS CD44v/SYNE1/miR34a axis was identified using bioinformatic analysis, and the expression profile of these markers was assessed in saliva of OSCC patients. CD44v6 and CD44v10 demonstrated a significantly increased expression, whereas SYNE1 and miR34a depicted a significantly decreased expression in OSCC patients. Statistical analysis suggested a probable role of CD44v6, SYNE1, and miR34a in early stages of the malignancy, whereas a strong association was observed between CD44v6, CD44v10, and miR34a expression with locoregional aggressiveness and histopathological conditions. CONCLUSION Collectively, these findings suggested a plausible role of CD44v/SYNE1/miR34a axis as non-invasive salivary biomarkers to diagnose this disease at an early stage and predict the early onset of metastasis.

Significant Role of Segmental Duplications and SIDD Sites in Chromosomal Translocations of Hematological Malignancies: A Multi-parametric Bioinformatic Analysis

Recurrent non-random chromosomal translocations are hallmark characteristics of leukemogenesis, and however, molecular mechanisms underlying these rearrangements are less explored. The fundamental question is, why and how chromosomes break and reunite so precisely in the genome. Meticulous understanding of mechanism leading to chromosomal rearrangement can be achieved by characterizing breakpoints. To address this hypothesis, a novel multi-parametric computational approach for characterization of major leukemic translocations within and around breakpoint region was performed. To best of our knowledge, this bioinformatic analysis is unique in finding the presence of segmental duplications (SDs) flanking breakpoints of all major leukemic translocation. Breakpoint islands (BpIs) were analyzed for stress-induced duplex destabilization (SIDD) sites along with other complex genomic architecture and physicochemical properties. Our study distinctly emphasizes on the probable correlative role of SDs, SIDD sites and various genomic features in the occurrence of breakpoints. Further, it also highlights potential features which may be playing a crucial role in causing double-strand breaks, leading to translocation.

Ocimum basilicum miRNOME revisited: A cross kingdom approach

O. basilicum is medicinally important herb having inevitable role in human health. However, the mechanism of action is largely unknown. Present study aims to understand the mechanism of regulation of key human target genes that could plausibly modulated by O. basilicum miRNAs in cross kingdom manner using computational and system biology approach. O. basilicum miRNA sequences were retrieved and their corresponding human target genes were identified using psRNA target and interaction analysis of hub nodes. Six O. basilicum derived miRNAs were found to modulate 26 human target genes which were associated `with PI3K-AKTand MAPK signaling pathways with PTPN11, EIF2S2, NOS1, IRS1 and USO1 as top 5 Hub nodes. O. basilicum miRNAs not only regulate key human target genes having a significance in various diseases but also paves the path for future studies that might explore potential of miRNA mediated cross-kingdom regulation, prevention and treatment of various human diseases including cancer.

Unravelling the link between embryogenesis and cancer metastasis

PURPOSE Cancer as opposed to embryonic development is characterized by dysregulated, uncontrolled and clonal growth of cells. Inspite of that they share certain commonality in gene expression patterns and a number of cellular & molecular features. Consequently, in the present study we aimed to evaluate the role of a definite set of genes in fetal liver, primary liver cancers and metastatic liver tissue. METHODS The relative expression of fourteen candidate genes obtained by data mining and manual curation of published data (CXCL12, CXCR4, CK7, CDH1, CTNNB1, CLDN4, VEGFA, HIF1A, MMP9, p53, OPN, CDKN2A, TGFBR2, MUC16, β-actin) were performed on 62 tissues (32 liver metastasis tissues and 30 primary Liver cancer tissues), Fetal liver tissues (below and above 20weeks of gestation) and 2 sets of control samples by real-time quantitative reverse transcription PCR (qRT-PCR). RESULTS Results showed significant down-regulation of MMP9 and TP53 in Fetal liver above 20weeks of gestation whereas it was up-regulated in fetal liver below 20weeks of gestation, primary liver cancers and liver metastasis. Contradictory to that OPN and CDKN2A were significantly up-regulated in primary liver cancer, liver metastasis; down-regulated in fetal liver above 20weeks of gestation but were not expressed during early embryo development (below 20weeks of gestation). Moreover, MMP9 and TP53 demonstrated a strong correlation with MUC16 whereas CDKN2A and OPN showed correlation with CXCL12/CXCR4 signifying that MUC16, CXCL12/CXCR4 might be involved in the complex process of cancer metastasis. CONCLUSION MMP9, OPN, TP53 and CDKN2A were the identified markers that were expressed in a similar pattern in early embryonic development and cancer development & invasion suggesting that these genes are activated during embryogenesis and might be re-expressed in cancer metastasis. Moreover, these genes govern a pathway that might be activated during cancer metastasis. Thus, targeting these molecules may provide better treatment for metastatic liver cancers.

Abstract 3357: Role of miRNA dynamics in governing cancer stem cell properties via CD44v6/Nanog/ PTEN axis in head and neck cancer: Modulating the master regulators

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Background: Head and Neck Squamous cell carcinoma (HNSCC) is a major cause of morbidity and mortality worldwide. Late diagnosis, metastasis and therapeutic resistance are major confounders for the poor 5 year survival rate and are attributed to the existence of Cancer Stem Cells (CSCs). Several evidences have demonstrated the key role of nicotine in Tobacco related Cancers; however, the plausibility of nicotine modulating CSC markers still needs to be elucidated. Propelling evidences strongly suggest that HNSCC is a genetic disease, regulated by epigenetic mechanisms. Thus, understanding the role of miRNAs in regulation of CSCs and its contributions to improvise the prognostic and therapeutic implications is of great importance. Methodology: Orosphere was generated from primary habituated and non-habituated HNSCC tumors using CD44+ immunomagnetic cell separation and ex-vivo expansion. Characterization of these in-vitro models to evaluate spheroid formation ability and expression profile of CSC markers (CD44v3, CD44v6, BMI1 and Nanog) was carried out using quantitative Real Time PCR. Further, miRNA profiling of the upstream regulators (miR5423p, miR34a and miR21) was conducted in order to understand the miRNA mediated pathway that regulate these CSC features in tumor development, metastasis and response to chemotherapy. Correlation analysis of these miRNA profile along with the expression levels of its CSC target gene, were analyzed in specimens from patients with HNSCC. Result: Orosphere assay facilitated the propagation of CD44+ cells that retained CSC phenotype and self-renewal property. In-vitro model of habituated tumor showed an increased spheroid forming efficiency as compared to non-habituated models. In addition, these models showed higher expressions of CSC markers (CD44v3, CD44v6, Nanog & BMI1) and a significantly reduced expression of PTEN and ATM as compared to the healthy individual; however the habituated and non-habituated were found to have a trivial difference in their behavioral patterns. Furthermore, a significantly increased expression of upstream miRNA was observed which eventually leads to the modulation CD44 interaction with Nanog/PTEN, thus forming an unexplored axis which can have prognostic and therapeutic implications. Clinically, altered miRNA expression not only positively correlated with CSC expression patterns but also associated with increased progression of the disease. Conclusion: Collectively, this study indicates that miR5423p and miR34a targets the HA-induced CD44v6 -Nanog-PTEN axis playing a vital role in regulating the CSC properties comprising of self renewal, clonal proliferation and resistance to chemotherapy in Head and Neck Cancers. Furthermore, our results depict an undisputable role of these miRNA mediated pathways in the development of tobacco related carcinogenesis Citation Format: Shanaya Patel, Dr. Rakesh Rawal. Role of miRNA dynamics in governing cancer stem cell properties via CD44v6/Nanog/ PTEN axis in head and neck cancer: Modulating the master regulators. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3357.

Tobacco habituated and non-habituated subjects exhibit different mutational spectrums in head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common non-skin cancer in the world. Tobacco chewing is implicated with most of the cases of HNSCC but this type of cancer is increasing in non-tobacco chewers as well. This study was instigated to provide comprehensive variant and gene-level data in HNSCC subjects of the Indian population and fill the gap in the literature on comparative assessment of gene mutations in cancer subjects with a habit of tobacco and those without any habit using targeted amplicon sequencing. We performed targeted Amplicon sequencing of 409 tumor suppressor genes and oncogenes, frequently mutated across many cancer types, including head and neck. DNA from primary tumor tissues and matched blood was analyzed for HNSCC patients with a habit of tobacco and those without any habit. PDE4DIP, SYNE1, and NOTCH1 emerged as the highly mutated genes in HNSCC. A total of 39 candidate causal variants in 22 unique cancer driver genes were identified in non-habitual (WoH) and habitual (WH) subjects. Comparison of genes from both the subjects, showed seven unique cancer driver genes (KIT, ATM, RNF213, GATA2, DST, RET, CYP2C19) in WoH, while WH showed five (IL7R, PKHD1, MLL3, PTPRD, MAPK8) and 10 genes (SETD2, ATR, CDKN2A, NCOA4, TP53, SYNE1, KAT6B, THBS1, PTPRT, and FGFR3) were common to both subjects. In addition to this NOTCH1, NOTCH2, and NOTCH4 gene were found to be mutated only in habitual subjects. These findings strongly support a causal role for tobacco, acting via PI3K and MAPK pathway inhibition and stimulation of various genes leading to oncogenic transformations in case of tobacco chewers. In case of non-tobacco chewers it appears that mutations in the pathway affecting the squamous epithelial lineage and DNA repair genes lead to HNSCC. Somatic mutation in CYP2C19 gene in the non-habitual subjects suggests that this gene may have a tobacco independent role in development and progression of HNSCC. In addition to sharing high mutation rate, NOTCH gene family was found to be mutated only in habitual sample. Further, presence of mutated genes not earlier reported to be involved in HNSCC, suggest that the Indian sub-continent may have different sets of genes, as compared to other parts of the world, involved in the development and progression of HNSCC.