Gopalan Natarajan

Knockdown of RNA binding protein musashi-1 leads to tumor regression in vivo.

BACKGROUND & AIMS In the gut, tumorigenesis is thought to arise from the stem cell population located near the base of intestinal and colonic crypts. The RNA binding protein musashi-1 (Msi-1) is a putative intestinal and progenitor/stem cell marker. Msi-1 expression is increased during rat brain development and in APC(min/+) mice tumors. This study examined a potential role of Msi-1 in tumorigenesis. METHODS Msi-1 small interfering RNA (siRNA) was administered as a liposomal preparation to HCT116 colon adenocarcinoma xenografts in athymic nude mice and tumor volume was measured. Cell proliferation was assessed by hexosaminidase and 3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyltetrazolium bromide MTT assays. siRNA-transfected cells were subjected to 12 Gy gamma-irradiation. Apoptosis was assessed by immunoreactive activated caspase-3 and mitosis was assessed by phosphorylated histone H3 staining. The tumor xenografts were stained similarly for phosphorylated histone H3, activated caspase-3, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, Notch-1, and p21(WAF1). Furthermore, siRNA-transfected cells were subjected to cell-cycle analysis and Western blot analyses for Notch-1 and p21(WAF1). RESULTS Knockdown of Msi-1 resulted in tumor growth arrest in xenografts, reduced cancer cell proliferation, and increased apoptosis alone and in combination with radiation injury. siRNA-mediated reduction of Msi-1 lead to mitotic catastrophe in tumor cells. Moreover, there was inhibition of Notch-1 and up-regulation of p21(WAF1) after knockdown of Msi-1. CONCLUSIONS Our results show the involvement of Msi-1 in cancer cell proliferation, inhibition of apoptosis, and mitotic catastrophe, suggesting an important potential mechanism for its role in tumorigenesis.

Translation regulatory factor RBM3 is a proto-oncogene that prevents mitotic catastrophe

RNA-binding proteins play a key role in post-transcriptional regulation of mRNA stability and translation. We have identified that RBM3, a translation regulatory protein, is significantly upregulated in human tumors, including a stage-dependent increase in colorectal tumors. Forced RBM3 overexpression in NIH3T3 mouse fibroblasts and SW480 human colon epithelial cells increases cell proliferation and development of compact multicellular spheroids in soft agar suggesting the ability to induce anchorage-independent growth. In contrast, downregulating RBM3 in HCT116 colon cancer cells with specific siRNA decreases cell growth in culture, which was partially overcome when treated with prostaglandin E2, a product of cyclooxygenase (COX)-2 enzyme activity. Knockdown also resulted in the growth arrest of tumor xenografts. We have also identified that RBM3 knockdown increases caspase-mediated apoptosis coupled with nuclear cyclin B1, and phosphorylated Cdc25c, Chk1 and Chk2 kinases, implying that under conditions of RBM3 downregulation, cells undergo mitotic catastrophe. RBM3 enhances COX-2, IL-8 and VEGF mRNA stability and translation. Conversely, RBM3 knockdown results in loss in the translation of these transcripts. These data demonstrate that the RNA stabilizing and translation regulatory protein RBM3 is a novel proto-oncogene that induces transformation when overexpressed and is essential for cells to progress through mitosis.

Wnt inhibitory factor 1 induces apoptosis and inhibits cervical cancer growth, invasion and angiogenesis in vivo.

Aberrant activation of Wingless-type (Wnt)/β-catenin signaling is widespread in human cervical cancer. However, the underlying mechanisms of Wnt activation and the therapeutic potential of Wnt inhibition remain largely unknown. Here, we demonstrate that the Wnt inhibitory factor 1 (WIF1), a secreted Wnt antagonist, is downregulated in all human primary cervical tumors and cell lines analyzed. Our data reveal that WIF1 downregulation occurs due to promoter hypermethylation and is an early event in cervical oncogenesis. WIF1 re-expression upon 5-aza-2′-deoxycytidine treatment or WIF1 gene transfer induces significant apoptosis and G2/M arrest, and inhibits cervical cancer cell proliferation in vitro. Consistent with this, treatment of established mice tumor xenografts with peritumoral WIF1 gene transfer results in a significant inhibition of cancer growth and invasion. WIF1 treatment causes a significant decrease in intracellular WNT1 and TCF-4 proteins revealing novel Wnt-regulatory mechanisms. Thus, WIF1 causes a major cellular re-distribution of β-catenin and a significant inhibition of the Wnt/β-catenin pathway in tumor cells, as documented by a remarkable reversion in the expression of Wnt/β-catenin transcriptional target genes (E-cadherin, c-Myc, cyclin D1, CD44 and VEGF). Consequently, multiple critical events in tumor progression and metastasis such as cell proliferation, angiogenesis and invasion were inhibited by WIF1. In addition, WIF1 modulated the expression of specific anti-apoptotic and apoptotic proteins, thereby inducing significant apoptosis in vivo. Our findings demonstrate for the first time that WIF1 downregulation by epigenetic gene silencing is an important mechanism of Wnt activation in cervical oncogenesis. Of major clinical relevance, we show that peritumoral WIF1 gene transfer reduces not only cancer growth but also invasion in well-established tumors. Therefore, our data provide novel mechanistic insights into the role of WIF1 in cervical cancer progression, and the important preclinical validation of WIF1 as a potent drug target in cervical cancer treatment.

CUGBP2 downregulation by prostaglandin E2 protects colon cancer cells from radiation-induced mitotic catastrophe

Prostaglandin E(2) (PGE(2)) is a potent inhibitor of ionizing radiation (IR)-induced cell death. Exposure of colon cancer cells to IR leads to increased CUGBP2 expression. Therefore, we tested the hypothesis that PGE(2) radioprotects colon cancer cells by inhibiting CUGBP2 expression. Exposure of HCT-116 cells to gamma-IR (0-12 Gy) resulted in a dose-dependent reduction in cell growth and an increase in the G(2)-M phase of the cell cycle. Western blot analyses demonstrated increased levels of activated caspase 9 and caspase 3. In addition, whereas Bax expression is increased, that of Bcl-2 and Bcl-x(L) was reduced. Further analyses demonstrated increased activation of Chk1 and Chk2 kinases, coupled with higher levels of nuclear cyclin B1 and Cdc2. Pretreatment with PGE(2) suppressed the activation of caspase 3 and caspase 7 and inhibited Bax expression. In addition, PGE(2) treatment restored growth and colony formation to control levels. IR significantly upregulated the expression of CUGBP2 in the cells, which was suppressed when cells were pretreated with PGE(2). Ectopic overexpression of CUGBP2 also induced apoptosis. Furthermore, it reversed the PGE(2)-mediated protection from IR-induced mitotic catastrophe. Furthermore, there was an increase in nuclear localization of cyclin B1 and Cdc2 coupled with increased phosphorylation of p53, Chk1, Chk2, and Cdc25c proteins. Cell cycle analysis also demonstrated increased G(2)-M transition. In contrast, siRNA-mediated suppression of CUGBP2 expression restored normal cell cycle progression and decreased IR-induced apoptosis. Taken together, these data demonstrate that PGE(2) protects colon cancer cells from IR-induced mitotic catastrophe in part through suppression of CUGBP2 expression.

Abstract C21: Wnt inhibitory factor 1 is silenced by promoter hypermethylation in cervical cancer and its restoration suppresses tumor growth

Background and Purpose: Cervical cancer is the leading cause of morbidity and mortality among women worldwide. It is also the second most common cause of cancer deaths in women. Recent studies suggest that aberrant activation of the Wingless-type (Wnt) pathway plays an important role in cervical cancer. However, the mechanisms and implications of Wnt activation in human cervical cancer are yet to be determined. We hypothesized that the Wnt inhibitory factor 1 (WIF1), a secreted Wnt antagonist, might be silenced in human cervical cancer. Therefore, we characterized the methylation status of WIF1 gene, its mRNA and protein expression in human cervical cancer samples. We also determined the effects of WIF1 treatment on tumor growth in a xenograft mouse model. Methods: To study WIF1 promoter methylation, genomic DNA was isolated from human normal and cancerous cervical samples, processed for bisulfite modification using EZ DNA methylation kit and WIF1 methylation-specific PCR was performed. WIF1 mRNA and protein expression in human cervical normal epithelium and tumor samples were assessed by real-time RTPCR and immunohistochemistry, respectively. To determine the tumor suppressive effects of WIF1, tumor xenograft studies were performed by injecting HeLa cells subcutaneously into the flanks of nude mice. Palpable tumors were treated for 7 weeks with peritumoral injection of pCI-blast-WIF1 expression vector that expresses the full WIF1 protein. Results: Our study demonstrates that WIF1 promoter hypermethylation is a frequent mechanism leading to WIF1 down-regulation as observed by decreased WIF1 mRNA and protein expression in cervical cancer compared with normal tissue. Furthermore, treatment of human cervical cancer in a xenograft mouse model by WIF1 gene transfer significantly inhibited the tumor growth by decreasing the expression of TCF-4, β-catenin, c-myc, cyclin D1 and CD44. WIF1 elicited its tumor suppressive effects by acting at multiple levels in Wnt/β-catenin pathway, in addition to modulating the expression of antiapoptotic (Bcl-2) and apoptotic proteins (p53, p21 and caspase-3), leading to a significant reduction in tumor cell proliferation and induction of massive apoptosis. Of major clinical relevance, while vector treated tumors were highly infiltrative, WIF1 treated tumors presented mainly with pushing borders, a characteristic feature of benign tumors. In addition, WIF1 treatment decreased the expression of angiogenic factors, VEGF and CD31, and significantly reduced the tumor vascular irrigation. Conclusions: Our findings for the first time demonstrate that WIF1 is silenced by promoter hypermethylation and identify WIF1 down-regulation as an important mechanism of Wnt activation in cervical cancer. Remarkably, our in vivo study emphasizes the anti-invasive, anti-angiogenic and tumor suppressive effects of WIF1 and therefore its potential therapeutic value in the treatment of cervical cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C21.

Abstract 2347: Inhibition of metastasis: A novel role for RNA binding protein CUGBP2

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: Cancer metastasis is the key determinant of a patients death. Interestingly, cells have an effective system to inhibit the ability to metastasize, termed metastasis suppression, and loss of this regulatory mechanism has been associated with cancer progression. In the present study, we have extended our previous observation that RNA binding protein CUGBP2 is not only downregulated in various cancers, but also that there is a clear-cut correlation with CUGBP2 expression and the metastatic state of a cancer cell. Methods: Human colon epithelial cell line SW620, SW480 and mouse fibroblast cell line NIH3T3 were cultured under recommended conditions. Migration assay was performed using a 24-well transwell plate, and fetal bovine serum was used as a chemoattractant. We have developed a metastatic animal model where NIH-3T3 cells overexpressing RBM3 induce tumors in nude mice and contain malignant DCAMKL-1 (a bonafide stem cell marker) positive cells. When these cells were sorted and injected into the flanks of athymic nude mice, tumors were observed after 4 weeks not only in the flanks, but also in the lungs and liver. Total RNA and protein were isolated for Real Time RT-PCR and western blot analyses, respectively. Tumors were formalin fixed and used for immunohistochemistry. Results: Lower levels of CUGBP2 were present in highly metastatic SW620 when compared to the poorly metastatic SW480 cells. Furthermore, CUGBP2 levels were significantly decreased in lung and liver metastasis when compared to the primary tumors in the flanks of the nude mice. Similarly, CUGBP2 levels were reduced in SW620 cells that had migrated through the collagen in the in vitro cell invasion assay. Forced overexpression of CUGBP2 in SW620 cells however, significantly reduced cell migration through the collagen. Moreover, CUGBP2 overexpression reduced cell migration in the in vitro scratch plate/migration assay. Conversely, siRNA mediated knockdown of CUGBP2 in SW480 cells increased the migration of cells in the scratch plate assay. In previous studies, we have demonstrated that CUGBP2 inhibit the translation of mRNAs that contain AU-rich sequences in the 3′ untranslated region of transcripts such as cyclooxygenase-2 (COX-2). Immunoprecipitation coupled with RT-PCR assay demonstrated the binding of CUGBP2 protein with MMP-9 and COX-2 mRNAs. To confirm that the CUGBP2 is playing a role in regulating MMP-9 expression, we also determined the effect of CUGBP2 downregulation in the cells. CUGBP2 was downregulated with the specific siRNA in HCT-116 cells. There was significant increase in MMP-9 and COX-2 protein levels in the cells with reduced CUGBP2, which was not affected when cells were also transfected with the scrambled siRNA. Conclusion: Taken together, these data suggest that RNA binding protein CUGBP2 is a critical player in modulating tumor metastasis. 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 2347.