Standard therapeutic approaches of cytotoxics and radiation in cancer are not only highly toxic, but also of limited efficacy in treatment of a significant number of cancer patients. The molecular analysis of the cancer genomes have shown a remarkable complexity and pointed to key genomic and epigenomic alterations in cancer. These discoveries are paving the way for targeted therapy approaches. However, although there are a large number of potential targets, only a few can regulate key cellular functions and intersect multiple signaling networks. The Aurora kinase family members (A, B, and C) are a collection of highly related and conserved serine-threonine kinases that fulfill these criteria, being key regulators of mitosis and multiple signaling pathways. Alterations in Aurora kinase signaling are associated with mitotic errors and have been closely linked to chromosomal aneuploidy in cancer cells. Several studies have shown amplification and/or overexpression of Aurora kinase A and B in hematologic malignancies and solid tumors. Over the past several years, Aurora kinases have become attractive targets. Several ongoing clinical trials and bench-based research are assessing the unique therapeutic potential of Aurora-based targeted therapy.Mol Cancer Ther; 9(2); 268-78. ©2010 AACR.

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Background MicroRNAs (miRNAs) are small noncoding RNAs that regulate the expression of approximately 30% of all human genes. They play important roles in numerous cellular processes including development, proliferation, and apoptosis. It is currently believed that miRNAs elicit their effect by silencing the expression of target genes. Here we show that microRNA-205 (miR-205) induces the expression of IL24 and IL32 tumor suppressor genes by targeting specific sites in their promoters. Methods Methods used in this study include transfection of small RNAs, quantitative-real-time-PCR, in-situ hybridization, fluorescence labeled in-situ hybridization, cell cycle, apoptosis, cell viability, migratory, clonability and invasion assays, immunoblotting, luciferase reporter, nuclear run-on and chromatin immunoprecipitation assays. Results Our results revealed that miR-205 is silenced in prostate cancer. Its re-expression induced apoptosis and cell cycle arrest. It also impaired cell growth, migration, clonability and invasiveness of prostate cancer cells. MicroRNA-205 induced tumor suppressor genes IL24 and IL32 at both mRNA and protein levels. Induction of in-vitro transcription and enrichment of markers for transcriptionally active promoters in IL24 and IL32 genes was observed in response to miR-205. Conclusion In this study we identify a new function for miR-205 to specifically activate tumor suppressor genes by targeting specific sites in their promoters. These results corroborate a new function that miRNAs have in regulating gene expression at the transcriptional level. The specific activation of tumor suppressor genes (e.g., IL24, IL32) or other dysregulated genes by microRNAs may contribute to the novel therapeutic approach in the treatment of prostate cancer.

BTG3/ANA/APRO4 has been reported to be a tumor suppressor gene in some malignancies. It constitutes important negative regulatory mechanism for Src-mediated signaling, a negative regulator of the cell cycle and inhibits transcription factor E2F1. We report that BTG3 is downregulated in renal cancer and that the mechanism of inactivation is through promoter hypermethylation. Quantitative real-time polymerase chain reaction (PCR) showed that BTG3 was downregulated in cancer tissues and cells. Genistein and 5-aza-2'-deoxycytidine (5Aza-C) induced BTG3 messenger RNA (mRNA) expression in A498, ACHN and HEK-293 renal cell carcinoma (RCC) cell lines. Bisulfite-modified PCR and DNA sequencing results showed complete methylation of BTG3 promoter in tumor samples and cancer cell lines. Genistein and 5Aza-C treatment significantly decreased promoter methylation, reactivating BTG3 expression. Chromatin immunoprecipitation assay revealed that genistein and 5Aza-C increased levels of acetylated histones 3, 4, 2H3K4, 3H3K4 and RNA polymerase II at the BTG3 promoter indicative of active histone modifications. Enzymatic assays showed genistein and 5Aza-C decreased DNA Methyltransferase, methyl-CpG-binding domain 2 activity and increased HAT activity. Cell cycle and 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide cell proliferation assays showed that genistein has antiproliferative effect on cancer cell growth through induction of cell cycle arrest. This is the first report to show that BTG3 is epigenetically silenced in RCC and can be reactivated by genistein-induced promoter demethylation and active histone modification. Genistein had similar effects to that of 5Aza-C, which is a potent demethylating agent with high toxicity and instability. Genistein being a natural, non-toxic, dietary isoflavone is effective in retarding the growth of RCC cells, making it a promising candidate for epigenetic therapy in renal carcinoma.

Aurora kinase A (AURKA) is located at 20q13, a region that is frequently amplified in gastric cancer. In this study, we have investigated the role of AURKA in regulating GSK-3β and β-catenin/TCF complex in gastric cancer cells. Our results demonstrate a significant increase in the phosphorylation of GSK-3β at Ser 9 following the over-expression of AURKA in AGS cells. The immunoprecipitation with antibodies specific for AURKA and GSK-3β indicated that the two proteins coexist in the same protein complex. The recombinant human AURKA protein phosphorylated the GSK-3β protein at Ser 9 in a concentration dependent manner, in vitro. The phosphorylation of β-catenin (Ser33/37/Thr41) by GSK-3β is known to target β-catenin towards degradation. In line with our findings, the increase in phospho-GSK-3β level was accompanied by a significant decrease in β-catenin phosphorylation (Ser33/37/Thr41) and accumulation of β-catenin protein. The knockdown of AURKA reversed the phosphorylation of GSK-3β and the β-catenin protein levels. The immunofluorescence analysis demonstrated co-localization of AURKA and GSK-3β proteins and a significant increase in the nuclear β-catenin levels in cells overexpressing AURKA. The β-catenin/TCF transcription activity was measured using the pTopFlash and its mutant pFopFlash luciferase reporter vectors. Indeed, AURKA over-expression led to a significant increase in the pTopFlash reporter activity, whereas kinase dead AURKA mutant (D274A) had no effect. Consistent with these findings, we detected a significant mRNA up-regulation of several direct targets of the β-catenin/TCF transcription complex (cyclin D1, c-MYC, c-MYC binding protein, CLDN1, FGF18, and VEGF), and a two-fold increase in the proliferation rate in AURKA over-expressing cells. We conclude that the AURKA/GSK-3β interaction plays an important role in regulating β-catenin, underscoring a novel oncogenic potential for AURKA in gastric tumorigenesis.

MicroRNAs (miRNAs) have great potential as biomarkers and therapeutic agents owing to their ability to control multiple genes and potential to influence cellular behavior. Here we identified that miR-23b is a methylation-silenced tumor suppressor in prostate cancer (PCa). We demonstrated that miR-23b expression is controlled by promoter methylation and has great promise as a diagnostic and prognostic biomarker in PCa. High levels of miR-23b expression are positively correlated with higher overall and recurrence-free survival in PCa patients. Further we elucidated the tumor suppressor role of miR-23b using in vitro and in vivo models. We demonstrated that proto-oncogene Src kinase and Akt are direct targets of miR-23b. Increased expression of miR-23b inhibited proliferation, colony formation, migration/invasion and triggered G0/G1 cell cycle arrest and apoptosis in PCa. Over-expression of miR-23b inhibited epithelial to mesenchymal transition (EMT) causing a decline in mesenchymal markers Vimentin and Snail and increasing the epithelial marker, E-cadherin. Depletion of Src by RNA interference conferred similar functional effects as that of miR-23b reconstitution. miR-23b expression caused a dramatic decrease in tumor growth in nude mice and attenuated Src expression in excised tumors compared to a control miR. These findings suggest that miR-23b is a methylation-silenced tumor suppressor that may be useful biomarker in PCa. Loss of miR-23b may confer proliferative advantage and promote PCa migration and invasion and re-expression of miR-23b may contribute to the epigenetic therapy for PCa.

The Src family of protein kinases (SFKs) plays key roles in regulating fundamental cellular processes, including cell growth, differentiation, cell shape, migration and survival, and specialized cell signals in various malignancies. The pleotropic functions of SFKs in cancer make them promising targets for intervention. Here we sought to investigate the role of miR-205 in inhibition of Src-mediated oncogenic pathways in renal cancer. We report that expression of miR-205 was significantly suppressed in renal cancer cell lines and tumors when compared with normal tissues and a non-malignant cell line, and is correlated inversely with the expression of SFKs. miR-205 significantly suppressed the luciferase activity of reporter plasmids containing the 3’UTR sequences complementary to either Src, Lyn or Yes, which was abolished by mutations in these 3’UTR regions. Over-expression of miR-205 in A498 cells reduced Src, Lyn and Yes expression both at mRNA and protein levels. Proliferation of renal cancer cells was suppressed by miR-205, mediated by the phosphoSrc-regulated ERK1/2 pathway. Cell motility factor- FAK and STAT3 activation was also inhibited by miR-205. Transient as well as stable over-expression of miR-205 in A498 cells resulted in induction of G0/G1 cell cycle arrest and apoptosis as indicated by decreased levels of cyclin D1 and cMyc, suppressed cell proliferation, colony formation, migration, and invasion in renal cancer cells. miR-205 also inhibited tumor cell growth in vivo. This is the first study demonstrating that miRNA-205 inhibits protooncogenic Src family of kinases indicating a therapeutic potential of miR-205 in the treatment of renal cancer.

Background BTG3/ANA/APRO4 is a candidate tumor suppressor gene in some malignancies. We report here that BTG3 is transcriptionally down-regulated in prostate cancer and the mechanism of inactivation is through promoter hypermethylation. Methods Prostate cancer and normal cell lines were treated with different doses of genistein and 5-aza-2’-deoxycytidine (5Aza-C). BTG3 mRNA expression was determined by quantitative real-time PCR in tissues and cell lines. BS-PCR, cloning and sequencing were used to examine promoter methylation in tumor samples and cell lines. Enzyme activity/inhibition assays were done to check the effect of genistein and 5Aza-C on DNA methyltransferases. ChIP assay was performed to analyze chromatin modifications caused by genistein treatment. Results BTG3 mRNA expression was down-regulated in cancer tissues and cells. Genistein and 5Aza-C induced BTG3 mRNA expression in all PC cell lines. Complete methylation of BTG3 promoter in tumor samples and cancer cell lines was observed. Genistein and 5Aza-C treatment significantly decreased promoter methylation, reactivating BTG3 expression. Genistein and 5Aza-C increased levels of acetylated histones 3, 4, 2H3K4, 3H3K4 and Pol II, decreased DNMTase, MBD2 activity and increased HAT activity. Conclusion This is the first report to show that BTG3 is silenced in prostate cancer and can be reactivated by genistein induced promoter demethylation and active histone modification. Genistein showed similar effects to that of 5Aza-C, which is currently undergoing phase II clinical trials as a treatment for prostate cancer. Since genistein is a natural, non-toxic, dietary isoflavone, these results indicate that genistein is a novel, advantageous therapeutic agent for treating prostate cancer.