Harekrushna Panda

Endometrial miR-200c is altered during transformation into cancerous states and targets the expression of ZEBs, VEGFA, FLT1, IKKβ, KLF9, and FBLN5.

A number of microRNAs (miRNAs), including miR-200 family, are aberrantly expressed in endometriosis and endometrial cancer. Here we assessed the expression and functional aspects of miR-200c in endometrial tissues (N = 52) from normal endometrial biopsies (N = 15), endometrial tissues including those exposed to hormonal therapies (N = 20), and grade I-III endometrial cancer (N = 17). miR-200c expression was elevated in normal endometrial biopsies from mid- and late-luteal phase, and in endometrial tumors as compared to endometrial tissues from peri- and postmenopausal period (P < .05) and its pattern of temporal expression displayed an inverse relationship with the expression of ZEBs. The expression of E-cadherin (CDH1) varied, but expressed at low levels, specifically in endometrial tissues and endometrial tumors. The endometrial expression of ZEBs and CDH1 in patients who were exposed to Depo-Provera and gonadotropin-releasing hormone agonist GnRHa displayed a trend toward lower expression as compared to proliferative phase; however, treatment of Ishikawa cells with 17β-estradiol, progesterone, and medroxy progesterone acetate had modest effects on the expression of miR-200c and ZEBs without affecting CDH1 expression. Gain of function of miR-200c in Ishikawa cells repressed ZEBs, as well as VEGFA, FLT1, IKKβ, and KLF9 expression at transcriptional and translational levels through direct interaction with their respective 3′untranslated regions and increased the rate of their proliferation. These results indicated that endometrial miR-200c expression undergoes dynamic changes during transition from normal into cancerous states; possibly influenced by hormonal milieu and by targeting the expression of specific genes with key regulatory functions in cellular transformation, inflammation, and angiogenesis may influence these events during normal and disease progression.

miR-200c is aberrantly expressed in leiomyomas in an ethnic-dependent manner and targets ZEBs, VEGFA, TIMP2, and FBLN5.

MicroRNA-200c (miR-200c) through repression of specific target genes has been associated with cellular transition, tumorigenesis, and tissue fibrosis. We explored the expression and functional aspects of miR-200c in genesis of leiomyomas (LYO), benign uterine tumors with fibrotic characteristic. Using LYO and matched myometrium (MYO; n=76) from untreated and from patients exposed to hormonal therapies (GNRH agonist (GNRHa), Depo-Provera, and oral contraceptives), we found that miR-200c was expressed at significantly lower levels (P<0.05) in LYO as compared with MYO. These levels were lower in LYO from African Americans as compared with Caucasians, patients experiencing abnormal uterine bleeding and those exposed to GNRHa therapy. Gain-of-function of miR-200c in isolated leiomyoma smooth muscle cells (LSMCs), myometrial smooth muscle cells (MSMCs), and leiomyosarcoma cell line (SKLM-S1) repressed ZEB1/ZEB2 mRNAs and proteins, with concurrent increase in E-cadherin (CDH1) and reduction in vimentin expression, phenotypic alteration, and inhibition of MSMC and LSMC proliferations. We further validated TIMP2, FBLN5, and VEGFA as direct targets of miR-200c through interaction with their respective 3′ UTRs, and other genes as determined by microarray analysis. At tissue levels, LYO expressed lower levels of TIMP2 and FBLN5 mRNAs but increased protein expressions, which to some extent altered due to hormonal exposure. Given the regulatory functions of ZEBs, VEGFA, FBLN5, and TIMP2 on cellular activities that promote cellular transition, angiogenesis, and matrix remodeling, we concluded that altered expression of miR-200c may have a significant impact on the outcome of LYO growth, maintenance of their mesenchymal and fibrotic characteristics, and possibly their associated symptoms.

A novel inhibitor of DNA polymerase β enhances the ability of Temozolomide to impair the growth of colon cancer cells

The recent emerging concept to sensitize cancer cells to DNA-alkylating drugs is by inhibiting various proteins in the base excision repair (BER) pathway. In the present study, we used structure-based molecular docking of DNA polymerase β (Pol-β) and identified a potent small molecular weight inhibitor, NSC-666715. We determined the specificity of this small molecular weight inhibitor for Pol-β by using in vitro activities of APE1, Fen1, DNA ligase I, and Pol-β–directed single-nucleotide and long-patch BER. The binding specificity of NSC-666715 with Pol-β was also determined by using fluorescence anisotropy. The effect of NSC-666715 on the cytotoxicity of the DNA-alkylating drug temozolomide (TMZ) to colon cancer cells was determined by in vitro clonogenic and in vivo xenograft assays. The reduction in tumor growth was higher in the combination treatment relative to untreated or monotherapy treatment. NSC-666715 showed a high specificity for blocking Pol-β activity. It blocked Pol-β–directed single-nucleotide and long-patch BER without affecting the activity of APE1, Fen1, and DNA ligase I. Fluorescence anisotropy data suggested that NSC-666715 directly and specifically interacts with Pol-β and interferes with binding to damaged DNA. NSC-666715 drastically induces the sensitivity of TMZ to colon cancer cells both in in vitro and in vivo assays. The results further suggest that the disruption of BER by NSC-666715 negates its contribution to drug resistance and bypasses other resistance factors, such as mismatch repair defects. Our findings provide the “proof-of-concept” for the development of highly specific and thus safer structure-based inhibitors for the prevention of tumor progression and/or treatment of colorectal cancer. (Mol Cancer Res 2009;7(12):1973–83)

NSC666715 and Its Analogs Inhibit Strand-Displacement Activity of DNA Polymerase β and Potentiate Temozolomide-Induced DNA Damage, Senescence and Apoptosis in Colorectal Cancer Cells.

Recently approved chemotherapeutic agents to treat colorectal cancer (CRC) have made some impact; however, there is an urgent need for newer targeted agents and strategies to circumvent CRC growth and metastasis. CRC frequently exhibits natural resistance to chemotherapy and those who do respond initially later acquire drug resistance. A mechanism to potentially sensitize CRC cells is by blocking the DNA polymerase β (Pol-β) activity. Temozolomide (TMZ), an alkylating agent, and other DNA-interacting agents exert DNA damage primarily repaired by a Pol-β-directed base excision repair (BER) pathway. In previous studies, we used structure-based molecular docking of Pol-β and identified a potent small molecule inhibitor (NSC666715). In the present study, we have determined the mechanism by which NSC666715 and its analogs block Fen1-induced strand-displacement activity of Pol-β-directed LP-BER, cause apurinic/apyrimidinic (AP) site accumulation and induce S-phase cell cycle arrest. Induction of S-phase cell cycle arrest leads to senescence and apoptosis of CRC cells through the p53/p21 pathway. Our initial findings also show a 10-fold reduction of the IC50 of TMZ when combined with NSC666715. These results provide a guide for the development of a target-defined strategy for CRC chemotherapy that will be based on the mechanisms of action of NSC666715 and TMZ. This combination strategy can be used as a framework to further reduce the TMZ dosages and resistance in CRC patients.

Abstract 4201: Role of adenomatous poliposis coli (APC) in cigarette smoke condensate-induced accumulation of apurinic/apyrimidinic lesions and enhanced transformation of normal breast epithelial cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Active and passive smoking are major risk factors for cancer and have been implicated in the etiology of breast cancer. The mechanisms through which smoking promotes breast cancer are poorly understood. Our previous studies have shown that cigarette smoke condensate (CSC), a surrogate for cigarette smoke, induces transformation of normal human breast epithelial (MCF10A) cells in culture. We have described the involvement of a multifunctional protein, adenomatous polyposis coli (APC), in the CSC-induced transformation of MCF10A cells through the interaction with DNA polymerase β and 5’-flap endonuclease 1 (Fen1), and thus, the blockade of the BER pathway. In the present study, we have examined the underlying mechanisms involved in CSC and Benzo[α]pyrene (B[α]P)-induced transformation of MCF10A cells in which APC was overexpressed or knocked down. Our results suggest a positive correlation between the increased levels of APC expression with the decreased BER activity and concomitant accumulation of apurinic/apyrimidinic (AP)-site lesions. If not repaired on time, the cytotoxic and mutagenic AP-site lesions ultimately lead to enhanced transformation of MCF10A cells. These studies suggest a possible mechanism by which active and/or passive cigarette smoking might be linked with breast carcinogenesis. This work was supported by Flight attendant Medical Research Institute, Miami, FL to SN. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4201. doi:10.1158/1538-7445.AM2011-4201

Exposure to Environmental Mutagens: APC and Colorectal Carcinogenesis

Environmental mutagens are global health hazards and a major risk factor for colon cancer development. Environmental mutagens include a variety of genotoxic carcinogenic compounds including polycyclic aromatic hydrocarbons, heterocyclic amines, nitrosamines, and aromatic amines. Their sources are pollution, diet, alcohol and cigarette smoke. These mutagens come in contact with large bowel through blood circulation or direct ingestion. Once these mutagens are in contact with DNA of the gut epithelial cells, they form DNA-adducts. If the damage exceeds the repair capacity of the epithelial cells, then the persistence of mutations can lead to the transformation of these cells. Mutations in adenomatous polyposis coli (APC) gene is considered to be one of the earliest events in the genesis of colorectal cancer, and its role has been well documented in a broad spectrum of functions ranging from cell adhesion to cell migration, regulation of Wnt/β-catenin-signaling pathway, cell cycle control, apoptosis, and chromosomal segregation at mitosis. The contribution of environmental mutagens on the APC gene mutations is very scanty in the literature. In the past several years, we have initiated studies to examine the interaction of APC with pathways induced by environmental mutagens. We showed that APC can be transcriptionally upregulated after DNA damage subsequent to the exposure of environmental mutagens including cigarette smoking. In addition, we recently discovered a novel role of APC in base excision repair (BER) pathway. APC interacts with DNA polymerase β and Fen-1 and blocks Pol-β-directed BER. Thus, APC-mediated block of BER in response to environmental mutagens, especially the DNA-alkylating agents, can be detrimental to the cell.

Abstract 692: Antitumor activity of novel structure-based chemotherapeutic agent for the intervention of colorectal cancer

Colorectal cancer is the second most common cause of cancer-related death in both men and women in the Western hemisphere. The success of treatment of colon cancer patients depends on matching the most effective therapeutic regimen with the characteristics of the individual patient. The primary challenge in achieving this goal is the heterogeneity of the disease. In the past 10 years, the overall survival of colon cancer patients has significantly improved with adjuvant trials of drugs; the recurrence rate over 5 years is still high. Thus, there is clearly an urgent need for the development of new chemotherapeutic drugs and strategies. The recent emerging concept to sensitize cancer cells to DNA-alkylating drugs is by inhibiting various proteins in the base excision repair (BER) pathway. We used structure-based molecular docking of DNA polymerase β (Pol-β) and identified a potent small molecular weight inhibitor (SMI), NSC-666715. We determined the specificity of this SMI for Pol-β by using in vitro activities of APE1, Fen1, DNA ligase I, and Pol-β-directed single nucleotide (SN)- and long-patch (LP)-BER. The binding specificity of NSC-666715 with Pol-β was also determined by using fluorescence anisotropy. The effect of NSC-666715 on the cytotoxicity of the DNA-alkylating drug, Temozolomide (TMZ), to colon cancer cells was determined by in vitro clonogenic and in vivo xenograft assays. The reduction in tumor growth was higher in the combination treatment relative to untreated or monotherapy treatment. NSC-666715 showed a high specificity for blocking Pol-β activity. It blocked Pol-β-directed SN- and LP-BER without affecting the activity of APE1, Fen1 and DNA ligase I. Fluorescence anisotropy data suggested that NSC-666715 directly and specifically interacts with Pol-β and interferes with binding to damaged DNA. NSC-666715 drastically induces the sensitivity of TMZ to colon cancer cells both in vitro and in vivo assays. The results further suggest that the disruption of BER by NSC-666715 negates its contribution to drug-resistance and bypasses other resistance factors, such as mismatch repair defects. Our findings provide the “proof-of-concept” for the development of highly specific and thus safer structure-based inhibitors for the prevention of tumor progression and/or treatment of colorectal cancer. Funded by NCI-NIH grants CA-097031 and CA-100247 to S.N. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 692.