Vikas Bhardwaj

CCAT2, a novel noncoding RNA mapping to 8q24, underlies metastatic progression and chromosomal instability in colon cancer

The functional roles of SNPs within the 8q24 gene desert in the cancer phenotype are not yet well understood. Here, we report that CCAT2, a novel long noncoding RNA transcript (lncRNA) encompassing the rs6983267 SNP, is highly overexpressed in microsatellite-stable colorectal cancer and promotes tumor growth, metastasis, and chromosomal instability. We demonstrate that MYC, miR-17-5p, and miR-20a are up-regulated by CCAT2 through TCF7L2-mediated transcriptional regulation. We further identify the physical interaction between CCAT2 and TCF7L2 resulting in an enhancement of WNT signaling activity. We show that CCAT2 is itself a WNT downstream target, which suggests the existence of a feedback loop. Finally, we demonstrate that the SNP status affects CCAT2 expression and the risk allele G produces more CCAT2 transcript. Our results support a new mechanism of MYC and WNT regulation by the novel lncRNA CCAT2 in colorectal cancer pathogenesis, and provide an alternative explanation of the SNP-conferred cancer risk.

Proteomic Profiling Identifies Dysregulated Pathways in Small Cell Lung Cancer and Novel Therapeutic Targets Including PARP1

UNLABELLED Small cell lung cancer (SCLC) is an aggressive malignancy distinct from non-small cell lung cancer (NSCLC) in its metastatic potential and treatment response. Using an integrative proteomic and transcriptomic analysis, we investigated molecular differences contributing to the distinct clinical behavior of SCLCs and NSCLCs. SCLCs showed lower levels of several receptor tyrosine kinases and decreased activation of phosphoinositide 3-kinase (PI3K) and Ras/mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) pathways but significantly increased levels of E2F1-regulated factors including enhancer of zeste homolog 2 (EZH2), thymidylate synthase, apoptosis mediators, and DNA repair proteins. In addition, PARP1, a DNA repair protein and E2F1 co-activator, was highly expressed at the mRNA and protein levels in SCLCs. SCLC growth was inhibited by PARP1 and EZH2 knockdown. Furthermore, SCLC was significantly more sensitive to PARP inhibitors than were NSCLCs, and PARP inhibition downregulated key components of the DNA repair machinery and enhanced the efficacy of chemotherapy. SIGNIFICANCE SCLC is a highly lethal cancer with a 5-year survival rate of less than 10%. To date, no molecularly targeted agents have prolonged survival in patients with SCLCs. As a step toward identifying new targets, we systematically profiled SCLCs with a focus on therapeutically relevant signaling pathways. Our data reveal fundamental differences in the patterns of pathway activation in SCLCs and NSCLCs and identify several potential therapeutic targets for SCLCs, including PARP1 and EZH2. On the basis of these results, clinical studies evaluating PARP and EZH2 inhibition, together with chemotherapy or other agents, warrant further investigation.

Therapeutic Delivery of miR-200c Enhances Radiosensitivity in Lung Cancer

The microRNA (miR)-200s and their negative regulator ZEB1 have been extensively studied in the context of the epithelial-mesenchymal transition. Loss of miR-200s has been shown to enhance cancer aggressiveness and metastasis, whereas replacement of miR-200 miRNAs has been shown to inhibit cell growth in several types of tumors, including lung cancer. Here, we reveal a novel function of miR-200c, a member of the miR-200 family, in regulating intracellular reactive oxygen species signaling and explore a potential application for its use in combination with therapies known to increase oxidative stress such as radiation. We found that miR-200c overexpression increased cellular radiosensitivity by direct regulation of the oxidative stress response genes PRDX2, GAPB/Nrf2, and SESN1 in ways that inhibits DNA double-strand breaks repair, increase levels of reactive oxygen species, and upregulate p21. We used a lung cancer xenograft model to further demonstrate the therapeutic potential of systemic delivery of miR-200c to enhance radiosensitivity in lung cancer. Our findings suggest that the antitumor effects of miR-200c result partially from its regulation of the oxidative stress response; they further suggest that miR-200c, in combination with radiation, could represent a therapeutic strategy in the future.

Modulation of c-Met signaling and cellular sensitivity to radiation: potential implications for therapy.

The c‐Met/hepatocyte growth factor receptor and its family members are known to promote cancer cell migration and invasion. Signaling within and beyond this pathway contributes to the systemic spread of metastases through induction of the epithelial‐mesenchymal transition, a process also implicated in mediating resistance to current anticancer therapies, including radiation. Induction of c‐Met has also been observed after irradiation, suggesting that c‐Met participates in radiation‐induced disease progression through the epithelial‐mesenchymal transition. Therefore, c‐Met inhibition is an attractive target for potentially mitigating radiation resistance. This article summarizes key findings regarding crosstalk between radiotherapy and c‐Met and discusses studies performed to date in which c‐Met inhibition was used as a strategy to increase cellular radiosensitivity. Cancer 2013.

C-Met Inhibitor MK-8003 Radiosensitizes c-Met–Expressing Non–Small-Cell Lung Cancer Cells With Radiation-Induced c-Met–Expression

Introduction: The radiation doses used to treat unresectable lung cancer are often limited by the proximity of normal tissues. Overexpression of c-Met, a receptor tyrosine kinase, occurs in about half of non–small-cell lung cancers (NSCLCs) and has been associated with resistance to radiation therapy and poor patient survival. We hypothesized that inhibiting c-Met would increase the sensitivity of NSCLC cells to radiation, enhancing the therapeutic ratio, which may potentially translate into improved local control. Methods: We tested the radiosensitivity of two high-c-Met–expressing NSCLC lines, EBC-1 and H1993, and two low-c-Met–expressing lines, A549 and H460, with and without the small-molecule c-Met inhibitor MK-8033. Proliferation and protein expression were measured with clonogenic survival assays and Western blotting, respectively. &ggr;-H2AX levels were evaluated by immunofluorescence staining. Results: MK-8033 radiosensitized the high-c-Met–expressing EBC-1 and H1993 cells but not the low-c-Met–expressing cell lines A549 and H460. However, irradiation of A549 and H460 cells increased the expression of c-Met protein at 30 minutes after the irradiation. Subsequent targeting of this up-regulated c-Met by using MK-8033 followed by a second radiation dose reduced the clonogenic survival of both A549 and H460 cells. MK-8033 reduced the levels of radiation-induced phosphorylated (activated) c-Met in A549 cells. Conclusions: These results suggest that inhibition of c-Met could be an effective strategy to radiosensitize NSCLC tumors with high basal c-Met expression or tumors that acquired resistance to radiation because of up-regulation of c-Met.

Failure of Pancreatic Cancer Chemotherapy: Consequences of Drug Resistance Mechanisms

Pancreatic cancer is one of the most lethal forms of cancer and it is estimated that there will be about 44,000 new cases in US in the year 2011. With 37,600 estimated deaths in 2011, pancreatic cancer is the fourth leading cause of cancer related deaths in US (American Cancer Society, 2011). In spite of numerous efforts, the 5-year survival rate for pancreatic cancer has not improved much for the last few decades. The suggested reasons for low survival among the pancreatic cancer patients include late disease diagnosis, highly invasive and metastatic nature, lack of effective therapies, and acquisition of resistant characteristics (American cancer Society 2007; Moore, et al., 2003; NIH 2007). Only two drugs – gemcitabine (GEM) and 5fluorouracil (5FU) – have been shown to improve the survival of patients consistently. 5FU was the first drug to be approved as adjuvant therapy for pancreatic cancer (Kalser and Ellenberg, 1985; Moertel, et al., 1981). Since then, GEM has been used as the first line chemotherapeutic drug for pancreatic cancer. However, GEM treatment does not always provide extended survival benefits. A study found that in post-operative patients, GEM treatment increased the survival by merely 6 months (Shore, et al., 2003). 5FU is also widely used as an adjuvant and neoadjuvant chemotherapeutic agent to treat pancreatic cancers (Ahlgren, 1996; Blaszkowsky, 1998; Snady, et al., 2000).

Abstract 1099: MiR-200c overexpression radiosensitizes lung cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL MiR-200 family are key regulators of the epithelial-to-mesenchymal transition (EMT) process and because this process is related to the development of tumor cell resistance and metastasis, these microRNAs represent potential biomarkers of cancer progression and therapy response. Nevertheless, their roles in resistance and sensitivity to radiotherapy are poorly understood. In the present work, we investigated the potential application of miR-200 family for overcoming radioresistence in non-small lung cancer cells (NSLC). First, we demonstrated that NSCLC cell lines that have gone through EMT lose the ability to express miR-200s. We established that miR-200s expression is lost in mesenchymal-like NSCLC cell lines by using real-time polymerase chain reaction analysis of two E-cadherin-positive cell lines H358 and H322 and two E-cadherin-negative cell lines A549 and H1299. We confirmed that restoring miR-200s in mesenchymal cells induces E-cadherin expression. E-cadherin expression was increased and ZEB1 and BIM expression were decreased after miR-200a overexpression in A549 cells. Moreover, we analyzed the effect of radiation treatment in an epithelial-like NSCLC cell line. H358 cells were treated with 4-Gys radiation doses over a 2-week period. Morphologic changes consistent with the development of mesenchymal-like characteristics were found and Western blotting demonstrated a 53% reduction in E-cadherin, suggesting that radiation drives epithelial cells into EMT. Next, we demonstrated that miR-200c replacement radiosensitizes mesenchymal-like cells. A549 cells with stable expression of miR-200c and cells transiently transfected with miR-200c mimics were exposed to ionizing radiation at 0, 2 and 4-Gys. Cell survival and DNA Double-strand breaks were monitored by clonogenic assay and the formation of γ-H2AX foci by immunofluorescence, respectively. We found that miR-200c treatment significantly sensitized A549 cells to radiation treatment. Moreover, miR-200c overexpression increased radiation-induced double-stranded DNA breaks relative to mock-transfected control cells. We also demonstrated that overexpression of miR-200c influenced two key DNA repair pathways, Rad-51 and Chk-1. To further verify the mechanism by which miR-200c promotes radiosensitization in lung cancer cells, we used reverse-phase protein arrays (RPPA) to identify EMT and DNA-repair proteins that are regulated by miR-200c. Comparison of several NSCLC lines that were or were not transfected with miR-200c revealed reductions in the expression of caveolin, AKT, phosphorylated AKT, phosphorylated SRC, and PI3K, BRCA1 and BRCA2, ATM, Ku80, XRCC1 and XIAP. Taken together, these results suggest that miR-200c can significantly impact the ability of lung cancer cells to respond to irradiation and may represent potential therapeutic target in lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1099. doi:1538-7445.AM2012-1099

Abstract 3454: Establishing role of KRAS mutation on NSCLC radio-sensitivity

Lung cancer is the leading cause of cancer related mortality in the US of which more than 75% cases are that of non-small cell lung carcinoma (NSCLC). Mutations in the proto-oncogene KRAS have been linked with poor prognosis for NSCLC patients. In this study, we aimed at analyzing the relationship between specific KRAS mutations and NSCLC cell radiosensitivity and protein expression patterns. We analyzed 22 NSCLC cell lines and stratified them according to their KRAS status. Our results show that NSCLC cells harbouring G12C mutations are more sensitive to radiation compared to cells with other KRAS status (SF2 = 0.35 ± 0.16 for G12C vs. 0.63 ± 0.17 for other KRAS mutants vs 0.54 ± 0.15 for wt KRAS). Our protein expression data suggests that G12C mutants have reduced expression of DNA-repair proteins such as ATM, Rad 50, Ku 80 and XRCC1 (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3454. doi:1538-7445.AM2012-3454