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MiR-145 is downregulated in various cancers including prostate cancer. However, the underlying mechanisms of miR-145 downregulation are not fully understood. Here, we reported that miR-145 was silenced through DNA hypermethylation and p53 mutation status in laser capture microdissected (LCM) prostate cancer and matched adjacent normal tissues. In 22 of 27 (81%) prostate tissues, miR-145 was significantly downregulated in the cancer compared with the normal tissues. Further studies on miR-145 downregulation mechanism showed that miR-145 is methylated at the promoter region in both prostate cancer tissues and 50 different types of cancer cell lines. In seven cancer cell lines with miR-145 hypermethylation, 5-aza-2'-deoxycytidine treatment dramatically induced miR-145 expression. Interestingly, we also found a significant correlation between miR-145 expression and the status of p53 gene in both LCM prostate tissues and 47 cancer cell lines. In 29 cell lines with mutant p53, miR-145 levels were downregulated in 28 lines (97%), whereas in 18 cell lines with wild-type p53 (WT p53), miR-145 levels were downregulated in only 6 lines (33%, P < 0.001). Electrophoretic mobility shift assay showed that p53 binds to the p53 response element upstream of miR-145, but the binding was inhibited by hypermethylation. To further confirm that p53 binding to miR-145 could regulate miR-145 expression, we transfected WT p53 and MUT p53 into PC-3 cells and found that miR-145 is upregulated by WT p53 but not with MUTp53. The apoptotic cells are increased after WT p53 transfection. In summary, this is the first report documenting that downregulation of miR-145 is through DNA methylation and p53 mutation pathways in prostate cancer.

The purpose of this study was to optimize the conditions for polymerase chain reaction (PCR). The most common problem with conventional PCR is the presence of nonspecific products and primer-dimers formation, which could be due to several factors such as annealing temperature, primer concentration, and Taq DNA polymerase activity during setup of the PCR. Recently, a neutralizing monoclonal antibody (TaqStart) that blocks Taq DNA Polymerase activity has been developed. In the present study, we determined the effects of Taq DNA polymerase monoclonal antibody (TaqStart antibody) and other parameters, such as annealing temperature and oligonucleotide primer concentrations, on the specificity of PCR products. The results of these experiments suggest that TaqStart antibody inhibits nonspecific products and primer-dimers formation by blocking Taq DNA polymerase activity until the reaction components are heated in the thermal cycler (hot start). Other factors such as annealing temperature, oligonucleotide primer concentration, and magnesium concentration are equally important for specificity of PCR products.

ARHI is an imprinted tumor suppressor gene and is downregulated in various malignancies. However, ARHI expression, function, and mechanisms of action in prostate cancer have not been reported. Here, we report that ARHI mRNA and protein levels were downregulated in prostate cancer tissues compared with adjacent normal tissues. Overexpression of ARHI inhibited cell proliferation, colony formation, invasion, and induced apoptosis. Further studies on a new mechanism of ARHI downregulation showed a significant inverse relationship between ARHI and miR-221 and 222, which were upregulated in prostate cancer cell lines. Transfection of miR-221 and 222 inhibitors into PC-3 cells caused a significant induction of ARHI expression. A direct interaction of miR-221 or 222 with a target site on the 3′UTR of ARHI was confirmed by a dual luciferase pMIR-REPORT assay. Finally, we also found that genistein upregulates ARHI by downregulating miR-221 and 222 in PC-3 cells. In conclusion, ARHI is a tumor suppressor gene downregulated in prostate cancer, and overexpression of ARHI can inhibit cell proliferation, colony formation, and invasion. This study demonstrates for the first time that prostate cancer cells have decreased level of ARHI which could be caused by direct targeting of 3′UTR of ARHI by miR221/222. Genistein, a potential nontoxic chemopreventive agent, restores expression of ARHI and may be an important dietary therapeutic agent for treating prostate cancer. Cancer Prev Res; 4(1); 76–86. ©2010 AACR. Cancer Prev Res; 4(1); 76–86. ©2010 AACR.

Cytochrome P450 17 (CYP17) encodes cytochrome P450c17α, an enzyme with 17α-hydroxylase and 17, 20-lyase activities involved in estradiol biosynthesis. Here we examine the role of CYP17 gene in endometrial carcinogenesis. Immunohistochemistry staining of endometrial carcinoma and corresponding uninvolved tissues showed that CYP17 is upregulated in endometrial cancers (15 of 24, 62.5%). To understand the functional significance of this upregulation, we silenced CYP17 gene by introduction of siRNA into endometrial cancer cell line KLE followed by functional studies. Further, to understand the molecular basis of the role of CYP17, we profiled the expression of key pathway-specific genes and identified several components of the apoptosis and invasion pathways that are potentially regulated by CYP17. Silencing of CYP17 caused decreased cell proliferation and induced apoptosis. Significantly, CYP17 depletion leads to downregulation of anti-apoptotic genes B cell lymphoma 2 (Bcl-2) and telomerase reverse transcriptase (TERT), indicating induced apoptosis. Also, attenuation of CYP17 decreased the cellular invasion ability and regulated expression of several invasion pathway components such as melanoma cell adhesion molecule (MCAM), matrix metallopeptidase 2 and 9 (MMP-2 and MMP-9), and tissue inhibitor of metalloproteinase 3 (TIMP3). In conclusion, this is the first report documenting that upregulation of CYP17 in endometrial cancers play a crucial role in endometrial carcinogenesis by targeting multiple components of apoptosis and invasion pathways. Further studies are required to understand the detailed mechanisms underlying CYP17-mediated regulation of these components.

Background: MicroRNAs (miRNAs) are small noncoding RNAs that have important roles in numerous cellular processes. Recent studies have shown aberrant expression of miRNAs in prostate cancer tissues and cell lines. On the basis of miRNA microarray data, we found that miR-145 is significantly downregulated in prostate cancer. Methods and results: We investigated the expression and functional significance of miR-145 in prostate cancer. The expression of miR-145 was low in all the prostate cell lines tested (PC3, LNCaP and DU145) compared with the normal cell line, PWR-1E, and in cancerous regions of human prostate tissue when compared with the matched adjacent normal. Overexpression of miR-145 in PC3-transfected cells resulted in increased apoptosis and an increase in cells in the G2/M phase, as detected by flow cytometry. Investigation of the mechanisms of inactivation of miR-145 through epigenetic pathways revealed significant DNA methylation of the miR-145 promoter region in prostate cancer cell lines. Microarray analyses of miR-145-overexpressing PC3 cells showed upregulation of the pro-apoptotic gene TNFSF10, which was confirmed by real-time PCR and western analysis. Conclusion: One of the genes significantly upregulated by miR-145 overexpression is the proapoptotic gene TNFSF10. Therefore, modulation of miR-145 may be an important therapeutic approach for the management of prostate cancer.

The functional significance of Wnt antagonist DKK1 has not been investigated in renal cell carcinoma (RCC). Therefore, we hypothesized that DKK1 may be a tumor suppressor gene and is epigenetically silenced, thus decreased DKK1 may cause progression of RCC. To assess the function of DKK1, we established stable DKK1 transfected cells and monitored them regarding cell viability, colony formation, apoptosis, cell cycle, and invasive capability. RCC cell lines had decreased levels of DKK1, which were increased after treatment with 5-Aza-2'-deoxycytidine and trichostatin A. In chromatin immunoprecipitation assay, the level of dimethyl H3K9 and trimethyl H3K27 was decreased after 5-Aza-2'-deoxycytidine/trichostatin A treatment in RCC cell lines. Increased methylation was also associated with higher pathological stages in primary RCC tissues. T-cell factor/lymphoid enhancer factor activity and nuclear beta-catenin expression were not changed in DKK1 transfectants. Also the expression of cyclinD1 and c-Myc was not changed in DKK1 transfectants. These results suggest that DKK1 may not be involved in the beta-catenin dependent pathway. We also evaluated the expression of various related genes. Cleaved caspase3, p53, p21 and puma expression were significantly upregulated in the DKK1 transfected cells. The population of apoptotic cells was increased in stable DKK1 cells and tumor growth suppression was also observed in nude mice with DKK1 transfected cells. In conclusion, this is the first report to show that DKK1 expression is epigenetically silenced in kidney cancer and its reexpression induces apoptosis and cell cycle arrest in RCC.

BACKGROUND: MicroRNAs (miRNAs) are small noncoding RNAs that regulate the expression of approximately 60% of all human genes. They play important roles in numerous cellular processes, including development, proliferation, and apoptosis. Currently, it is believed that miRNAs elicit their effect by silencing the expression of target genes. In this study, the authors demonstrated that miRNA-205 (miR-205) induced the expression the interleukin (IL) tumor suppressor genes IL24 and IL32 by targeting specific sites in their promoters. METHODS: The methods used in this study included transfection of small RNAs; quantitative real-time polymerase chain reaction; in situ hybridization; fluorescence-labeled in situ hybridization; cell cycle, apoptosis, cell viability, migratory, clonability, and invasion assays; immunoblotting; and luciferase reporter, nuclear run-on, and chromatin immunoprecipitation assays. RESULTS: The results revealed that miR-205 was 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. Micro-RNA-205 induced the expression of tumor suppressor genes IL24 and IL32 at both the messenger RNA and protein levels. The induction of in vitro transcription and enrichment of markers for transcriptionally active promoters in the IL24 and IL32 genes was observed in response to miR-205. CONCLUSIONS: In this study, a new function for miR-205 was identified that specifically activated tumor suppressor genes by targeting specific sites in their promoters. These results corroborate a newly identified function that miRNAs have in regulating gene expression at the transcriptional level. The specific activation of tumor suppressor genes (eg, IL24, IL32) or other dysregulated genes by miRNA may contribute to a novel therapeutic approach for the treatment of prostate cancer. Cancer 2010. © 2010 American Cancer Society.

The study was aimed to explore the value of minimal residual disease (MRD) for risk stratification in childhood precursor-B-acute lymphoblastic leukemia. MRD was monitored at two time points (TP1, after induction and TP2, before consolidation therapy) by quantitative detection of monoclonal immunoglobulin heavy chain gene rearrangements. This study stratified 105 patients into three MRD risk groups: standard-risk, MRD −4 at both TP1 and TP2; high-risk, TP1 ≥ 10 −2 or TP2 ≥ 10 −3 ; and others were classified as intermediate-risk. We incorporated this MRD risk information to refine risk stratification among these patients and developed a new classification system that predicted the treatment outcomes more successfully than did the traditional risk classification criteria.

A subset of colorectal cancers with CpG island methylator phenotype-high (CIMP-H) is frequently associated with MSI and BRAF V600E mutation. Since limited data are available on different histological types of colorectal polyps, we compared the pattern and the frequency of promoter methylation, CIMP-H, MSI, KRAS and BRAF V600E mutations and the relationship among these molecular parameters and the clinicopathologic characteristics in 110 serrated polyps (48 hyperplastic polyps, 32 sessile serrated adenomas and 30 serrated adenomas) and 32 tubular adenomas using 7 commonly used tumor-associated gene loci. No significant difference in the frequency of overall methylation frequency (86% vs. 100%) and CIMP-H (39% vs. 28%) between serrated polyps and tubular adenomas was observed, but proximally located serrated polyps showed more frequent methylation at 5 of 7 loci examined, and were more likely to be CIMP-H (62% vs. 22%). MGMT methylation was more common in tubular adenomas while MLH1 and HIC1 were more frequently methylated in serrated polyps. BRAF mutation was frequently present in all types of serrated polyps (80%), but was absent in tubular adenomas and was not associated with CIMP or MSI status. These results show comparable frequencies of promoter methylation of tumor-associated genes and CIMP-H, but distinct differences in gene-specific or colonic site-specific methylation profiles occur in serrated polyps and tubular adenomas. BRAF mutation occurs independently of CIMP and MSI in all types of serrated polyps and may serve as a marker of serrated pathway of colorectal carcinogenesis.

Objective To determine the value of histology in identifying Lynch syndrome among those patients with early onset of colorectal cancer (CRC). Methods Demographic, clinical and cancer history data from patients diagnosed with CRC before 60 years of age, and treated at our institution between 1997 and 2005, were collected from medical records and direct interview. Their tumors were assessed to identify histological features suggestive of high frequency microsatellite instability (MSI-H): tumor infiltrating lymphocytes, Crohn’s like inflammatory reaction, mucinous, signet ring cells, medullary growth pattern and then, tested for microsatellite instability (MSI) and MLH1/ MSH2 protein expression. Results Sixty-five patients were included in the study. The mean age at diagnosis was 48 ± 9.9 years. Overall, 28 (43%) patients, including 13 of 35 diagnosed between ages 50 and 60, had tumor demonstrating one or more histological features suggestive of MSI-H. These patients were younger (45 vs. 50 years, P = 0.02) and more commonly had family history of Lynch syndrome-related cancers (36 vs. 19%), though the latter feature did not reach statistical significance (P = 0.07). Eleven of 25 tumors with MSI-H histology, but only 1 of 29 tumors without special histological features were found to be MSI-H (P < 0.0001). Histology had a positive predictive value of 44% and a negative predictive value of 97% for identifying MSI-H tumors. Conclusions Limiting MSI analysis only to those tumors with suggestive histology would have reduced the need for testing by nearly 60% of all tumors from patients that met the revised Bethesda guidelines.

Mismatch repair (MMR) enzymes have been shown to be deficient in prostate cancer. MMR can influence the regulation of tumor development in various cancers but their role on prostate cancer has not been investigated. The aim of the present study was to determine the functional effects of the mutL-homolog 1 (MLH1) gene on growth of prostate cancer cells. The DU145 prostate cancer cell line has been previously established as MLH1-deficient and thus, this cell line was utilized to determine effects of MLH1 by gene expression. Lack of MLH1 expression was confirmed by Western blotting in DU145 cells whereas levels were high in normal prostatic PWR-1E and RWPE-1 cells. Stably expressing MLH1 in DU145 resulted in decreased cell proliferation, migration and invasion properties. Lack of cell growth using athymic nude mice model also indicated a tumor suppressive effect by MLH1. Interestingly, MLH1 caused an increase in apoptosis along with phosphorylated c-Abl, and treatment with MLH1 siRNAs countered this effect. Furthermore, inhibition of c-Abl with imatinib (STI571) also abrogated the effect on apoptosis caused by MLH1. These results demonstrate MLH1 protects against prostate cancer development by inducing c-Abl-mediated apoptosis. Citation Format: Shinichiro Fukuhara, Inik Chang, Yozo Mitsui, Soichiro Yamamura, Takeshi Chiyomaru, Shahana Majid, Sharanjot Saini, Hiroshi Hirata, Guoren Deng, Ankurpreet Gill, Darryn K. Wong, Hiroaki Shiina, Norio Nonomura, Rajvir Dahiya, Yuichiro Tanaka. Effects of MLH1 on prostate cancer cell function. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1238. doi:10.1158/1538-7445.AM2015-1238

Background The most frequent alteration in the prostate oncogenome is the loss of chromosome (chr) 8p21 that has been traditionally associated with the loss of homeodomain protein, NKX3.1, that plays important role in prostate carcinogenesis. Genomic deletions of this region increase significantly with tumor grade and are associated with poor prognosis in prostate cancer (PCa) suggesting the critical involvement of this region in PCa progression. Recent genomic studies suggest that this region harbors alternative tumor suppressor genes apart from NKX3.1. However, the identity of these tumor suppressors has largely remained elusive. Here we propose a novel, paradigm shifting hypothesis that this frequently deleted locus is associated with a microRNA (miRNA) gene- miR-3622a- that is lost in prostate cancer and play an important mechanistic role in PCa progression and metastasis by regulating Epithelial-mesenchymal transition (EMT). Methods miRNA expression profiling was performed in microdissected PCa tissues and matched adjacent normal regions by real-time PCR. To understand the functional significance of miR-3622a in PCa, we performed knockdown studies using non-transformed prostate epithelial cell line and overexpression studies employing PCa cell lines. We also examined the role of miR-3622a in vivo in an orthotopic mouse model of PCa. Further, we explored the regulatory mechanisms underlying the loss of this miRNA in prostate cancer by performing methylation analysis and copy number analysis on PCa tissues and cell lines. Results Expression analyses in a cohort of clinical specimens showed that miR-3622a expression is widely attenuated in prostate cancer and is significantly correlated with poor survival outcome and tumor progression. Functional studies showed that this miRNA inhibits PCa EMT, progression and metastasis by repression of TFs of SNAIL and ZEB family. In vivo studies demonstrated that miR-3622a overexpression reduces tumor progression significantly. Analyses of the regulatory mechanisms underlying low miR-3622a expression in clinical tissues and prostate cell lines suggested that apart from genomic deletions of this locus, epigenetic events such as DNA methylation regulate its expression coordinately. Conclusions Here we provide the first direct evidence that a miRNA gene located in a frequented deleted region of prostate cancer inhibits prostate cancer EMT, progression and metastasis. We propose that frequent loss of miR-3622a at chr8p21 region by genetic and epigenetic mechanisms leads to induction of EMT that in turn, promote PCa progression and metastasis. This study has potentially significant implications in prostate cancer field as it supports a novel concept that seeks to connect a long standing observation of frequent loss of a chromosomal region with a novel miRNA in prostate cancer. Citation Format: Shinichiro Fukuhara, Nathan Bucay, Shahana Majid, Varahram Shahryari, Laura Z. Tabatabai, Kirsten Greene, Soichiro Yamamura, Yozo Mitsui, Yuichiro Tanaka, Guoren Deng, Rajvir Dahiya, Sharanjot Saini. MicroRNA located in frequently deleted locus 8p21 regulates prostate cancer EMT, progression and metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3134. doi:10.1158/1538-7445.AM2015-3134

Introduction and Objective Bone morphogenetic protein-2 (BMP-2) has been shown to function as a tumor suppressor with several different cancers, while its expression level is down-regulated by CpG hypermethylation. We hypothesized that impaired regulation of BMP-2 through epigenetic pathways is associated with the pathogenesis of renal cell carcinoma (RCC). Methods To test our hypothesis, CpG methylation of the BMP-2 gene was analyzed using 2 RCC cell lines, and 96 matched RCC and normal renal tissues. We also performed functional analysis using BMP-2 restored RCC cells. Results BMP-2 mRNA expression was significantly enhanced in the RCC cells by 5-aza-2′-deoxycitidie treatment. The prevalence of BMP-2 promoter methylation was significantly higher, while BMP-2 mRNA expression was significantly lower in RCC samples as compared to normal kidney samples. Furthermore, a significant correlation was found between BMP-2 promoter methylation and mRNA transcription in the tumors. There was also a significant association of BMP-2 mRNA expression with tumor stage and lymph node involvement, while lower BMP-2 mRNA expression was significantly associated with poor overall survival after a radical nephrectomy. In RCC cells, BMP-2 restoration significantly inhibited cell proliferation, migration, invasion, and colony formation. In addition, BMP-2 overexpression effectively induced p21WAF1/CIP1 and p27KIP1 expression, and cellular apoptosis in RCC cells. Conclusion This is the first study to show inactivation of the BMP-2 gene via epigenetic pathways in RCC. Aberrant BMP-2 methylation and the resultant loss of BMP-2 expression may be useful as a molecular marker for designing improved diagnostic and therapeutic strategies for RCC. Citation Format: Yozo Mitsui, Hiroshi Hirata, Naoko Arichi, Miho Hiraki, Hiroaki Yasumono, Inik Chang, Shinichiro Fukuhara, Soichiro Yamamura, Varahram Shahryari, Guoren Deng, Sharanjot Saini, Shahana Majid, Yuichiro Tanaka, Rajvir Dahiya, Hiroaki Shiina. Dysregulation of bone morphogenetic protein 2 serves as a candidate molecular marker in human renal cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3823. doi:10.1158/1538-7445.AM2015-3823

Silibinin is the major active constituent of silymarin, an extract of the milk thistle seeds, containing a mixture of flavonolignans such as silibinin and isosilibinin. Silibinin has been shown to have significant anti-cancer effects in a variety of cancer types, including bladder, kidney, prostate, skin, breast, lung and colon cancer. Silibinin (10 μM) significantly suppressed proliferation and invasion of T24 and UM-UC-3 bladder cancer cells. Western blot analysis showed that silibinin significantly reduces expression of vimentin and slug in these bladder cells. Vimentin is overexpressed in various cancers and is known to promote tumor growth, invasion, and poor prognosis. Slug, a transcriptional repressor which mediates epithelial to mesenchymal transition (EMT), has been implicated in tumor metastasis. These results indicate that silibinin inhibits proliferation and invasion by suppressing vimentin and slug in bladder cancer cells. We studed the anti-cancer effects of silibinin in bladder cancer cells using RNA-Seq, a transcriptome profiling technique that uses deep-sequencing to quantitatively investigate the global behavior of transcriptomes. We will discuss the RNA-Seq profiling of the anti-cancer effects of silibinin in bladder cancer cells, including the vimentin and slug networks. Citation Format: Soichiro Yamamura, Yozo Mitsui, Nathan Bucay, Sharanjot Saini, Shahana Majid, Guoren Deng, Varahram Shahryary, Rajvir Dahiya, Yuichiro Tanaka. RNA-Seq profiling of silibinin effects on bladder cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 902. doi:10.1158/1538-7445.AM2015-902

Regional DNA hypermethylation and global DNA hypomethylation are 2 epigenetic alterations associated with colorectal cancers. However, their correlation with microsatellite instability (MSI) and chromosomal instability (CIN) in colorectal cancer, and their relationship with chromatin conformation and histone modification are not clear. In this study, we analyzed regional and global methylation in 16 cell lines and 64 primary colorectal cancers. We found that MSI and CIN are 2 alternative events in most cell lines and tumors. Furthermore, regional hypermethylation and global hypomethylation are also alternative events in most cases. We also observed a strong correlation between MSI and regional hypermethylation and between CIN and global hypomethylation. We further analyzed chromatin conformation and histone acetylation in cell lines with CIN or MSI. CIN cancers had open chromatin conformation and enriched histone acetylation in repetitive as well as in gene-specific regions. MSI cancers, on the other hand, had closed chromatin conformation and low levels of histone acetylation. After a MSI cell line was treated with 5-aza-2'-deoxycytidine or trichostatin A, the closed chromatin conformation became open, and histone acetylation was enriched. These observations support our hypothesis that in colorectal cancer, regional hypermethylation and global hypomethylation are associated with altered chromatin conformation and histone acetylation, which might have a causal correlation with MSI and CIN, respectively.

Genomic studies suggest that deletions at chromosome (chr) 5q region (particularly chr5q14-q23) are frequent in prostate cancer, implicating this region in prostate carcinogenesis. However, the genes within this region are largely unknown. Here we report for the first time, the widespread attenuation of miR-3607 - a microRNA gene located at chr5q14 region- in prostate cancer. Expression analyses of miR-3607 in a clinical cohort of prostate cancer (PCa) specimens showed that miR-3607 is significantly attenuated and low miR-3607 partment of Urology, UCSF School of Mediacinehip and guidance. his guidance. expression is correlated with tumor progression and poor survival outcome in prostate cancer. Our analyses suggest that miR-3607 expression may be a clinically significant parameter with an associated diagnostic potential. We examined the functional significance of miR-3607 in prostate cancer cell lines and found that miR-3607 overexpression led to significantly decreased proliferation, apoptosis induction and decreased invasiveness. Further, our results suggest that miR-3607 directly represses oncogenic SRC family kinases, LYN and SRC, in prostate cancer. In view of our results, we propose that miR-3607 plays a tumor suppressive role in prostate cancer by regulating SRC kinases that in turn regulates prostate carcinogenesis. To our knowledge this is the first report that: (i) identifies a novel role for miR-3607 located in a frequently deleted region of prostate cancer and (ii) defines novel miRNA mediated regulation of SRC kinases in prostate cancer. Since SRC kinases play a central role in prostate cancer progression and metastasis and are attractive targets, this study has potential implications in the design of better therapeutic modalities for prostate cancer management. Citation Format: Sharanjot Saini, Sumit Arora, Shahana Majid, Varahram Shahryari, Laura Z. Tabatabai, Soichiro Yamamura, Yuichiro Tanaka, Rajvir Dahiya, Guoren Deng. Regulation of SRC kinases by microRNA-3607 located in a frequently deleted locus in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5211. doi:10.1158/1538-7445.AM2014-5211

Catechol-compounds such as 4-hydroxy-estrogen have been shown to play a role in the renal carcinogenesis process. To counter the effects of the highly reactive and mutagenic catechol-estrogens, the catechol-O-methyltransferase (COMT) gene detoxifies these compounds by forming methoxy compounds, which have been shown to be biologically inactive. Proper COMT function is thus vital for cells and a mechanism by which activity levels can be affected is through polymorphisms. The Val(158)Met polymorphism that correlates with renal cancer has been shown to reduce COMT enzymatic activity, but also of importance are those variants in the promoter region which can lead to alterations in gene expression levels. The aim of this study was to determine if polymorphisms of the COMT promoter can influence their transcription levels. To examine this, plasmids containing wildtype regions of the COMT promoter were obtained and site-directed mutagenesis used to create polymorphic variants. Constructs were then transfected into the Caki-1 renal cell line to measure their effects on gene expression by using a luciferase reporter assay. A total of 8 polymorphic sites covering the distal and proximal promoter region were evaluated. Results indicate that the variant at polymorphic sites rs2020917 (C to T), rs13306278 (C to T), and rs6269 (A to G) caused a significant reduction (P Citation Format: Inik Chang, Shinichiro Fukuhara, Darryn K. Wong, Ankurpreet Gill, Varahram Shahryari, Soichiro Yamamura, Shahana Majid, Sharanjot Saini, Hiroshi Hirata, Koji Ueno, Guoren Deng, Rajvir Dahiya, Yuichiro Tanaka. Activity of catechol-O-methyltransferase gene promoter polymorphisms in renal cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3406. doi:10.1158/1538-7445.AM2014-3406

HOTAIR is a long non-coding RNA (lncRNA) which interacts with the Polycomb Repressive Complex and suppresses its target genes. HOTAIR has also been demonstrated to promote malignancy. MicroRNA-141 (miR-141) has been reported to play a role in the epithelial to mesenchymal transition (EMT) process and the expression of miR-141 is inversely correlated with tumorigenicity and invasiveness in several human cancers. We found that HOTAIR expression is inversely correlated to miR-141 expression in renal carcinoma cells. HOTAIR promotes malignancy including proliferation and invasion whereas miR-141 suppresses malignancy in renal carcinoma cells. miR-141 binds to HOTAIR in a sequence specific manner, and suppresses HOTAIR expression and functions including proliferation and invasion. Both HOTAIR and miR-141 were associated with the immunoprecipitated Argaunaute2 (Ago2) complex and the Ago2 complex cleaved HOTAIR in the presence of miR-141. These results demonstrate that HOTAIR is suppressed by miR-141 in an Ago2 dependent manner. Citation Format: Takeshi Chiyomaru, Shinichiro Fukuhara1, Sharanjot Saini, Shahana Majid, Guoren Deng, Varahram Shahryary, Inik Chang, Yuichiro Tanaka, Hideki Enokida, Masayuki Nakagawa, Rajvir Dahiya, Soichiro Yamamura. Long noncoding RNA HOTAIR is targeted and regulated by microRNA-141 in renal carcinoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4357. doi:10.1158/1538-7445.AM2014-4357

Purpose: The relationship between global hypomethylation, chromosomal instability (CIN), and microsatellite instability (MSI) remains unclear in colorectal cancer. The aim of this study was to investigate the relationship between global methylation status, loss of heterozygosity (LOH), and MSI in sporadic colorectal cancer. Experimental Design: We determined global methylation levels in 80 sporadic colorectal cancers, 51 adjacent normal tissues, and 20 normal tissues using the long interspersed nucleotide elements–combined bisulfite restriction analysis method. We also analyzed 80 colorectal cancers for MSI status and LOH at chromosomes 5q21, 8p12-22, 17p13, and 18q21. Results: We identified 14 cases of MSI (17.5%) and 58 cases of LOH (72.5%). LOH was observed more frequently in microsatellite stable (MSS) cancers than in MSI cancers at all loci. Colorectal cancers showed significantly lower global methylation levels than did normal tissues (41.0 ± 9.7% versus 54.3 ± 6.5%; P < 0.001). MSS cancers showed significantly lower global methylation levels when compared with MSI cancers (39.5 ± 9.4% versus 48.2 ± 8.2%; P = 0.003). Tumors with global hypomethylation (with ≤40% of methylation levels) had a significantly increased number of chromosomal loci with LOH than did tumors without global hypomethylation (1.9 versus 0.9; P < 0.001); 11 tumors (13.9%) lacked both MSI and LOH. This subgroup had significantly higher global methylation levels (46.8 ± 8.7%) than did MSS cancers with LOH (38.0 ± 9.0%; P = 0.006). Conclusions: These data showed a significant association between global hypomethylation and chromosomal instability in sporadic colorectal cancer. This suggests that global hypomethylation plays an important role in inducing genomic instability in colorectal carcinogenesis.

We compared the frequency of CpG island methylation phenotype (CIMP), inactivation of APC, p53 and DCC genes and K-ras and BRAF mutations in 39 mucinous carcinomas (MC) and 34 non-mucinous carcinomas (NMC) of the colorectum with different microsatellite instability (MSI) status. The higher incidence of MSI (36% vs. 18%) was observed in MC compared with NMC. APC inactivation and K-ras mutations occurred more frequently in NMC (APC, 88%, p

As an important regulator in Wnt-signaling pathway, the APC gene is involved in apoptosis and cell cycle arrest. The loss of APC function is observed in most familial adenomatous polyposis-associated and sporadic colorectal cancer. APC gene is frequently inactivated by DNA mutations. However, hypermethylation in APC gene promoter was also observed in different cancers. In this study, by analyzing the methylation status of APC promoter in 22 colorectal cancer cell lines with different APC expression levels, we identified Regions A and B in the promoter, where the methylation of CpG sites was invariably correlated with the loss of gene expression. By nuclease accessibility assay, we also observed a correlation between the closed chromatin conformation in APC promoter and loss of gene expression. When the nonexpressing cell lines were treated with a DNA methyltransferase inhibitor, 5-Aza-2′-Deoxycytidine, the APC expression in these cells was induced, CpG sites were demethylated, and closed chromatin conformation was opened. However, when these cell lines were treated with a histone deacetylase inhibitor, Trichostatin A, no significant changes in APC expression, methylation status, and chromatin conformation were observed. Using transient transfection assay, a CCAAT box located in Region B was identified, which was involved in up-regulation of APC expression. Methylation of CpG sites around the CCAAT box resulted in a significant inhibition in the gene expression. The specific binding of a transcription factor CCAAT-binding factor (CBF) to the CCAAT box was determined by electrophoretic mobility shift analysis. The binding was inhibited after CpG sites close to the CCAAT box were methylated, indicating that DNA methylation can silence gene expression through interfering with the binding of transcription factors to the promoter. The biological function of CBF in APC gene regulation was further indicated by the decrease of luciferase activities in cells cotransfected with a plasmid carrying APC promoter/luciferase gene and a plasmid expressing dominant negative CBF mutant. In summary, methylation of CpG sites around CCAAT box in APC promoter inhibits the gene expression by changing the chromatin conformation and interfering with the binding of transcription factor CBF to CCAAT box.

Anomalous expression of certain types of mucins occurs in pancreatic tumors, but little is known about the causes. MUC2 and MUC5AC are not expressed in normal pancreas. In the present study an immunohistochemical screening showed that MUC2 antigen was expressed in 6% of invasive tumors. Of the pancreatic cell lines, 2.4% expressed MUC2 message and antigen. In contrast, MUC5AC antigen was expressed in 86-100% of invasive adenocarcinoma (depending on the antibody). MUC5AC message and antigen were expressed in 66.7% of the cell lines tested. A polymerase chain reaction based assay was used to determine if methylation of CpG sites immediately 5' of the transcription initiation sites of the MUC2 and MUC5AC genes could be related to mucin expression in the cell lines. Digestibility by the methylation sensitive restriction enzyme HpaII correlated with the presence or absence of mRNA in 100% and 77.8% of the cell lines for MUC2 and MUC5AC, respectively. Treatment of a cell line that did not express MUC2 or MUC5AC gene products with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine resulted in an increase in MUC2 message but no change in MUC5AC message. In summary, the expression of MUC2 gene products correlated well with methylation of the proximal region of the promoter whereas expression of MUC5AC may involve additional regions or other mechanisms.

Microsatellite instability is present in over 80% of the hereditary non-polyposis colorectal carcinoma and about 15–20% of the sporadic cancer. Microsatellite instability is caused by the inactivation of the mismatch repair genes, such as primarily hMLH1, hMSH2. To study the mechanisms of the inactivation of mismatch repair genes in colorectal cancers, especially the region-specific methylation of hMLH1 promoter and its correlation with gene expression, we analysed microsatellite instability, expression and methylation of hMLH1 and loss of heterozygosity at hMLH1 locus in these samples. Microsatellite instability was present in 17 of 71 primary tumours of colorectal cancer, including 14 of 39 (36%) mucinous cancer and three of 32 (9%) non-mucinous cancer. Loss of hMLH1 and hMSH2 expression was detected in nine and three of 16 microsatellite instability tumours respectively. Methylation at CpG sites in a proximal region of hMLH1 promoter was detected in seven of nine tumours that showed no hMLH1 expression, while no methylation was present in normal mucosa and tumours which express hMLH1. However, methylation in the distal region was observed in all tissues including normal mucosa and hMLH1 expressing tumours. This observation indicates that methylation of hMLH1 promoter plays an important role in microsatellite instability with a region-specific manner in colorectal cancer. Loss of heterozygosity at hMLH1 locus was present in four of 17 cell lines and 16 of 54 tumours with normal hMLH1 status, while loss of heterozygosity was absent in all nine cell lines and nine tumours with abnormal hMLH1 status (mutation or loss of expression), showing loss of heterozygosity is not frequently involved in the inactivation of hMLH1 gene in sporadic colorectal cancer. British Journal of Cancer (2002) 86, 574–579. DOI: 10.1038/sj/bjc/6600148 www.bjcancer.com © 2002 Cancer Research UK

The Abelson (c-Abl) proto-oncogene encodes a highly conserved nonreceptor protein tyrosine kinase that plays a role in cell proliferation, differentiation, apoptosis and cell adhesion. c-Abl represents a specific anti-cancer target in prostate cancer as aberrant activity of this kinase has been implicated in the stimulation of prostate cancer growth and progression. However, the mechanism of regulation of c-Abl is not known. Here we report that Abl kinases are regulated by a novel microRNA, miR-4723, in prostate cancer. Expression profiling of miR-4723 expression in a cohort of prostate cancer clinical specimens showed that miR-4723 expression is widely attenuated in prostate cancer. Low miR-4723 expression was significantly correlated with poor survival outcome and our analyses suggest that miR-4723 has significant potential as a disease biomarker for diagnosis and prognosis in prostate cancer. To evaluate the functional significance of decreased miR-4723 expression in prostate cancer, miR-4723 was overexpressed in prostate cancer cell lines followed by functional assays. miR-4723 overexpression led to significant decreases in cell growth, clonability, invasion and migration. Importantly, miR-4723 expression led to dramatic induction of apoptosis in prostate cancer cell lines suggesting that miR-4723 is a pro-apoptotic miRNA regulating prostate carcinogenesis. Analysis of putative miR-4723 targets showed that miR-4723 targets integrin alpha 3 and Methyl CpG binding protein in addition to Abl1 and Abl2 kinases. Further, we found that the expression of Abl kinase is inversely correlated with miR-4723 expression in prostate cancer clinical specimens. Also, Abl1 knockdown partially phenocopies miR-4723 reexpression in prostate cancer cells suggesting that Abl is a functionally relevant target of miR-4723 in prostate cancer. In conclusion, we have identified a novel microRNA that mediates regulation of Abl kinases in prostate cancer. This study suggests that miR-4723 may be an attractive target for therapeutic intervention in prostate cancer.

A new method of measuring gene copy number in small samples of DNA was used to measure amplification of the erbB-2 gene and a reference gene in breast cancers. The method, termed 'competitively differential polymerase chain reaction' (CD-PCR), combines the advantages of two other techniques for measuring amplification by PCR, namely differential PCR (D-PCR) and competitive PCR (C-PCR). The CD-PCR methodology was evaluated for sensitivity and specificity by comparing amplification measured by CD-PCR with that obtained by fluorescence in situ hybridization (FISH), C-PCR, and Southern blotting analysis. CD-PCR analysis proved to be an accurate predictor of amplification. CD-PCR also overcomes the problems involved in variation of PCR efficiencies and DNA concentrations in tumor samples, and the problems caused by the plateau effect in PCR.

Recent studies have shown that chromosome 8p21-22 is the main site of frequent loss of heterozygosity (LOH) in breast cancers. However, the detailed molecular analysis of chromosome 8 so far in breast cancer has been variable. Most of the literature pertaining to LOH in breast cancer is mainly on short arm of chromosome 8. In the present study, we have examined LOH on both short and long arm of chromosome 8 using fifteen different polymorphic DNA markers in microdissected samples of normal breast epithelium and carcinoma from the same patients. For this purpose, DNA was extracted from the microdissected normal and tumor cells of 66 breast cancers, amplified by PCR and analyzed for LOH on chromosome 8 using fifteen different polymorphic DNA markers (D8S264, D8S298, D8S535, D8S255, D8S1098, D8S589, D8S567, D8S591, D8S285, D8S1102, D8S1763, D8S260, D8S530, D8S1772, and D8S1844). Expression of estrogen receptor, progesterone receptors, and p53 antigens was determined by immunohistochemistry using specific monoclonal antibodies. The results of this study suggest that LOH on chromosome 8 was identified in 40 of 66 cases (61%) with at least one marker. Three distinct regions of loss detected were: i) at 8p12, at loci between D8S535 and D8S255; ii) at 8p11, on loci D8S567, D8S591, D8S285, and D8S1102; iii) at 8q11-12, on loci D8S1763, D8S260 and D8S530. We found 45% (30 out of 66 informative cases) of the tumors showed LOH at 8p12; 52% (34 out of 66 informative cases) had LOH at 8p11; and 39% (26 out of 66 informative cases) had LOH at 8q11-12. Deletion at 8q11-12 was significantly correlated with the grade of the breast cancer specimens. Moderate to poorly differentiated specimens had higher incidence of LOH at 8q11-12 as compared to well differentiated specimens. Deletion at 8p12 and 8p11 was significantly higher in clinical stages III and IV of breast cancer tissues as compared to stage I and II cases. Tissues with lymph node involvement showed higher incidence of LOH at 8p12 as compared to the tissues with no lymph node involvement. There was no correlation of LOH at these loci with either the age of the patients, tumor size, BrdU labeling index, expression of estrogen receptor, progesterone receptor, and p53 in breast cancer specimens. These experiments, for the first time, report multiple sites of LOH on chromosome 8 in human breast cancer, and these deletions have differential correlation with clinical parameters of breast cancer samples.

MicroRNAs (miRNAs) are highly conserved, single stranded, non-coding RNAs of approximately 21 - 24 nucleotides that regulate gene expression by posttranscriptional silencing of specific target RNAs, by repressing translation or cleaving RNA transcripts by binding to the 3’ untranslated region (3’UTR) of the target messenger RNAs. miRNAs regulate diverse cellular processes such as cell-cycle progression, proliferation, apoptosis and development. miRNAs also have been shown to function as oncogenes or tumor suppressor genes. MicroRNA profiling has shown that miR-720 was up-regulated in cancer tissues, however the function of miR-720 in cancer has not been studied. Here we studied the functional effects of miR-720 on prostate cancer cells. We found that miR-720 was up-regulated in human prostate cancer tissues compared to adjacent normal tissues. We also found that miR-720 was up-regulated in prostate PC-3 cancer cell line compared with normal prostate cell line, RWPE1 cells. Transfection of miR-720 into prostate cancer cells, PC-3 and DU145 cells, decreased apoptosis, promoted cell proliferation and cell invasion. Transfection of anti-miR-720 into PC-3 cells increased apoptosis, inhibited cell proliferation and cell invasion. These results indicated that miR-720 has oncogenic functions in prostate cancer cells. In-silico analysis suggests that miR-720 targets genes of DR1, a down-regulator of global transdcription, and SWI5, a component of a complex required for double-strand break repair, which have tumor suppressive functions. Luciferase assays indicated that miR-720 binds to the 3’ UTR of these genes in prostate cancer cells, suggesting that these genes are targets of miR-720. Our results suggest that miR-720 promotes malignancy by targeting DR1 and SWI5 in prostate cancer cells. Funding: National Institutes of Health through Grant Number RO1CA138642, RO1CA130860 and RO1CA160079 and VA Merit Review and VA Program Project. Citation Format: Soichiro Yamamura, Takeshi Chiyomaru, Shinichiro Fukuhara, Sharanjot Saini, Shahana Majid, Guoren Deng, Vary Shahryary, Sumit Arora, Inik Chang, Yuichiro Tanaka, Peter Carroll, Rajvir Dahiya. MicroRNA-720 promotes cell proliferation and invasion in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5340. doi:10.1158/1538-7445.AM2013-5340

Prostate cancer is one of the most prevalent cancers in males and ranks the second most common cause of cancer related deaths. 2-methoxyestradiol (2-ME), an endogenous estrogen metabolite, is a promising anticancer agent for various types of cancers. Although 2-ME has been shown to activate Jun N-terminal kinase (JNK) and mitochondrial dependent apoptotic signaling pathways, the underlying mechanisms including downstream effectors remain unclear. Here, we report that the human Bcl-2 homology 3 (BH3)-only protein harakiri (Hrk) is a critical effector of 2-ME-induced-JNK dependent apoptosis in prostate cancer cells. Hrk mRNA and protein are preferentially up-regulated by 2-ME, and Hrk induction is dependent on JNK activation of c-Jun. Hrk knockdown prevents 2-ME-mediated apoptosis by attenuating the decrease in mitochondrial membrane potential, subsequent cytochrome c (cyt c) release and caspase activation. Conversely, Hrk over-expression increases caspase-dependent prostate cancer cell apoptosis. Involvement of the pro-apoptotic protein Bak in this process suggested the possible interaction between Hrk and Bak. Thus, Hrk activation by 2-ME or its over-expression displaced Bak from the complex with anti-apoptotic protein Bcl-xL, while deletion of the Hrk BH3 domain abolished its interaction with Bcl-xL, reducing the pro-apoptotic function of Hrk. Finally, Hrk is also involved in the 2-ME-mediated reduction of X-linked inhibitor of apoptosis (XIAP) through Bak activation in prostate cancer cells. Together, our findings suggest that induction of the BH3-only protein Hrk is a critical step in 2-ME activation of the JNK-induced apoptotic pathway targeting mitochondria by liberating pro-apoptotic protein Bak. Citation Format: Inik Chang, Shahana Majid, Sharanjot Saini, Mohd S. Zaman, Soichiro Yamamura, Takeshi Chiyomaru, Varahram Shahryari, Shinichiro Fukuhara, Guoren Deng, Rajvir Dahiya, Yuichiro Tanaka. Hrk mediates 2-methoxyestradiol-induced mitochondrial apoptotic signaling in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4375. doi:10.1158/1538-7445.AM2013-4375

Introduction: Genistein has been shown to inhibit various cancers but its mechanism of action is still unknown. In this study, we tested the hypothesis that genistein can inhibit prostate cancer (PCa) through modulation of microRNA (miRNAs) using various in vitro models. We also tested the functional significance of miRNAs in PCa cell lines and clinical significance of miRNAs in different stages and grades of PCa. Methods: Subconfluent cells (60-70% confluent) were treated with genistein (25 μM). Genistein administered everyday along with a change of medium, and the cells were grown for 96 h. The miRNA microarray analysis was carried out and analyzed by Phalanx Biotech using the human v3 miRNA OneArray platform that is designed to contain 100% of miRBase Sequence Database Release 17.0. The expression levels of miR-574-3p were analyzed by TaqMan quantitative real-time PCR and normalized to RNU48. The 48 clinical specimens were obtained from patients in San Francisco Veterans Affairs Medical Center from 1998 to 2004. For gain-of-function assay, cell proliferation (MTS assay), migration (wound healing assay), invasion (matrigel invasion assay) and apoptosis (flow cytometry analysis) were evaluated in human PCa cell lines, PC3 and DU145. Predicted target genes and their target miRNA binding site seed regions were investigated using TargetScan. To identify the biological processes or pathways potentially regulated by miR-574-3p, we performed GeneCodis analysis. To identify networks among the miRNAs and their target genes, we analyzed and characterized those genes in KEGG pathway categories. Results: Using miRNA microarray analysis and real-time RT-PCR we observed that miR-574-3p was significantly up-regulated in PCa cells treated with genistein compared with vehicle control. The expression of miR-574-3p was significantly lower in PCa cell lines and clinical PCa tissues compared with normal prostate cells (RWPE-1) and adjacent normal tissues. Low expression level of miR-574-3p was correlated with advanced tumor stage and higher Gleason score in PCa specimens. Re-expression of miR-574-3p in PCa cells significantly inhibited cell proliferation, migration and invasion. miR-574-3p restoration induced apoptosis through reducing Bcl-xL and activating caspase-9 and caspase-3. Using GeneCodis software analysis, several pathways affected by miR-574-3p were identified, such as ‘Pathways in cancer’, ‘Jak-STAT signaling pathway’, and ‘Wnt signaling pathway’. Luciferase reporter assays demonstrated that miR-574-3p directly binds to the 3’ UTR of several target genes (such as RAC1, EGFR and EP300) that are components of ‘Pathways in cancer’. Conclusions: Our results show that genistein up-regulates tumor suppressor miR-574-3p expression targeting several cell signaling pathways. These findings enhance understanding of how genistein regulates with miRNA in PCa. Citation Format: Takeshi Chiyomaru, Soichiro Yamamura, Shinichiro Fukuhara, Hideo Hidaka, Shahana Majid, Sharanjot Saini, Sumit Arora, Guoren Deng, Varahram Shahryari, Inik Chang, Yuichiro Tanaka, Z. Laura Tabatabai, Hideki Enokida, Naohiko Seki, Masayuki Nakagawa, Rajvir Dahiya. Genistein inhibits prostate cancer through activation of tumor suppressor microRNA-574-3p. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4161. doi:10.1158/1538-7445.AM2013-4161

A challenging aspect of prostate cancer (PCa) is the high rates of recurrence associated with the disease. About 40% of men with localized prostate cancer suffer from relapse after initial therapy. In these cases, androgen ablation therapy is used to reduce tumor burden/circulating PSA to low or undetectable limits. However, most patients suffer from disease recurrence, as monitored by rising PSA and tumor progression to hormone refractory/castration resistant prostate cancer that is essentially untreatable. Tumor recurrence has been attributed to the existence of tumor-initiating cells (TIC) within the bulk of the tumor; CD44 is an important marker for these cells. In this study, we demonstrate that miR-708 is a key negative regulator of CD44+ subpopulation of PCa cells. miR-708 was underexpressed in CD44+ cells from PCa xenografts. Reconstitution of miR-708 in PCa cell lines or CD44+ PCa cells led to decreased tumorigenicity in vitro. Intratumoral delivery of synthetic miR-708 oligonucleotides triggered regression of established tumors in a murine xenograft model of human prostate cancer. Conversely, miR-708 silencing in a purified CD44- population of PCa cells promoted tumor growth. Functional studies validated CD44 to be a direct target of miR-708. Also, we identified Androgen Receptor (AR) and the serine/threonine kinase AKT2, a member of the PI3K/AKT signaling pathway as functional targets of miR-708. Clinically, low miR-708 expression was associated significantly with poor survival outcome, tumor progression and recurrence in prostate cancer patients. In view of these results we propose that reduced miR-708 expression leads to prostate cancer initiation, progression and development by regulating expression of CD44, AR, AKT2, which in turn regulate proliferative, invasive and migratory abilities of prostate cancer cells. Therefore, miR-708 may represent a novel therapeutic target for prostate cancer. Citation Format: Sharanjot Saini, Sumit Arora, Shahana Majid, Varahram Shahryari, Soichiro Yamamura, Inik Chang, Takeshi Chiyomaru, Shinichiro Fukuhara, Guoren Deng, Yuichiro Tanaka, Rajvir Dahiya. CD44+ prostate cancer cells are regulated by microRNA-708: A novel role of microRNA with potential prognostic and diagnostic significance in prostate cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5322. doi:10.1158/1538-7445.AM2013-5322

Prostate cancer is one of the most common malignancies in men and ranks the second most common cause of cancer-related deaths. Mismatch repair (MMR) enzymes act as proofreading complexes that maintain the genomic integrity and MMR-deficient cells show an increased mutation rate. MMR is essential to the survival of the cell and health of the individual. Recent studies have shown that MMR influence the regulation of tumor development in various cancers, such as colon, ovarian, and others, but their impact on prostate cancer growth has not been investigated thus far. One important gene in the MMR complex is MutL homolog 1 (MLH1). The aim of the present study was to investigate the effect of MLH1 on growth properties of prostate cancer. MLH1 levels were characterized in patients with prostate cancer. Immunohistochemical analysis was done on tissue microarrays (TMAs) by using a monoclonal antibody for MLH1. TMAs consisted of cores sampled from the center of donor tissue blocks from 36 prostate cancer cases of various Gleason's grades. Staining intensity was measured to estimate the expression level of MLH1 proteins in each core and compared. As MLH1 levels differed between grades of cancer, we then evaluated the functional effects of MLH1 on prostate cancer cells. Expression of MLH1 was initially measured in normal and cancerous prostate cell lines by real-time PCR and western blotting. Cancerous cells displaying the lowest levels of MLH1 as compared to normal cells was then transfected with MLH1 to determine gene effects on cell proliferation by utilizing the MTS assay, and apoptosis by flow cytometry. Immunohistochemical analysis showed patients with high Gleason's score to have reduced expression of MLH1 compared to those with low grade prostate cancer. To determine the role of MLH1 on prostate cancer cell growth, gene transfection studies were then performed. Of the various prostate cell lines, DU145 cells had dramatically reduced expression levels of MLH1 as measured by real-time PCR with no protein detected by western blotting, whereas levels were high in normal prostate cell lines. Interestingly, over-expressing the MLH1 gene in DU145 cells decreased cell proliferation and increased apoptosis compared to vector control. These results demonstrate MLH1 to protect against prostate cancer progression and to have a functional role by affecting apoptosis of prostate cancer cells. Citation Format: Shinichiro Fukuhara, Inik Chang, Takeshi Chiyomaru, Shahana Majid, Sharanjot Saini, Soichiro Yamamura, Sumit Arora, Varahram Shahryari, Koji Ueno, Hiroshi Hirata, Guoren Deng, Rajvir Dahiya, Yuichiro Tanaka. DNA mismatch repair protein MLH1 induces apoptosis in human prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 625. doi:10.1158/1538-7445.AM2013-625

CpG island hypermethylation is one of the most common causes of the loss of tumor-suppressor microRNAs (miRNA) in cancers. Here we show that miR-34b is silenced in human prostate cancer (PCa) and the mechanism is through CpG hypermethylation. We provide evidence that miR-34b directly targets methyltransferases (DNMT) and deacetylases (HDACs). We show that miR-34b expression could predict overall survival and recurrence free survival such that patients with high miR-34b levels had longer survival. Further we elucidate the tumor suppressor role of miR-34b using both in vitro and in vivo models. Functionally miR-34b inhibits cell proliferation, colony formation, migration/invasion and triggers G0/G1 cell cycle arrest and apoptosis in cancer cells. The mechanism is partly through the down regulation of AKT pathway as miR-34b directly targets AKT and causes depletion of its downstream genes. Our results show that over-expression of miR-34b caused a decline in the mesenchymal markers Vimentin, ZO1, N-cadherin and Snail whereas there was an increase in E-cadherin expression. Finally we demonstrated the antitumor effect of miR-34b in vivo. MiR-34b caused a dramatic decrease in tumor growth in nude mice compared to cont-miR. These findings offer new insight into the role of miR-34b in the inhibition of prostate cancer through demethylation, active chromatin modification and AKT pathways and may provide a rationale for the development of new strategies targeting epigenetic regulation of miRNAs for the treatment of prostate cancer. Citation Format: Shahana Majid, Altaf A. Dar, Sharanjot Saini, Varahram Shahryari, Sumit Arora, Inik Chang, Soichiro Yamamura, Yuichiro Tanaka, Takeshi Chiyomaru, Shinichiro Fukuhara, Guoren Deng, Peter R. Carroll, Rajvir Dahiya. Pleiotropic tumor suppressor role of microRNA-34b in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3069. doi:10.1158/1538-7445.AM2013-3069

The Abelson (c-Abl) proto-oncogene encodes a highly conserved nonreceptor protein tyrosine kinase that has been implicated in many cellular processes including regulation of cell growth and survival, oxidative stress and DNA-damage responses, actin dynamics and cell migration. Aberrant activity of this tyrosine kinase has been implicated in the stimulation of cancer growth and progression. Abl tyrosine kinase represents a target for specific anti-cancer therapy in prostate cancer and Abl inhibitors are being tested clinically for the treatment of prostate cancer bone metastasis. Here we report that Abl kinases are regulated by a novel microRNA- miR-4723-5p in prostate cancer. Expression profiling of miR-4723-5p expression in a cohort of prostate cancer clinical specimens showed that miR-4723-5p expression is widely attenuated in prostate cancer. Low miR-4723-5p expression was significantly correlated with poor survival outcome and tumor progression and recurrence in prostate cancer. Further, our analyses suggest that miR-4723-5p has significant potential as a disease biomarker for diagnosis and prognosis in prostate cancer. To evaluate the functional significance of decreased miR-4723-5p expression in prostate cancer, miR-4723-5p was reintroduced in prostate cancer cell lines followed by functional assays. The over expression of miR-4723-5p led to significant decreases in cell growth, clonability, invasion and migration. Importantly, miR-4723-5p reexpression led to dramatic induction of apoptosis in prostate cancer cell lines suggesting that miR-4723-5p is a pro-apoptotic microRNA that regulates prostate carcinogenesis. Analysis of putative miR-4723-5p targets showed that miR-4723-5p targets Integrin alpha 3 (ITGA3) and Methyl CpG binding protein (MeCP2) in addition to Abl1 and Abl2 kinases. In conclusion, we have identified a novel microRNA mediated regulation of Abl tyrosine kinases in prostate cancer. This study suggests that miR-4723-5p may be an attractive target for therapeutic intervention in prostate cancer. Citation Format: Sumit Arora, Sharanjot Saini, Shahana Majid, Varahram Shahryari, Soichiro Yamamura, Takeshi Chiyomaru, Shinichiro Fukuhara, Inik Chang, Guoren Deng, Yuichiro Tanaka, Rajvir Dahiya. MicroRNA 4723-5p is novel tumor suppressor microRNA in prostate cancer that directly regulates Abelson family of nonreceptor protein tyrosine kinases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3068. doi:10.1158/1538-7445.AM2013-3068

Loss of heterozygosity (LOH) was detected in morphologically normal lobules adjacent to breast cancers. The most frequent aberration was at chromosome 3p22-25; of ten cases with this LOH in the carcinoma, six displayed the same LOH in adjacent normal lobules. This suggests that in a subset of sporadic breast cancers, a tumor suppresser gene at 3p22-25 may be important in initiation or early progression of tumorigenesis. Among sixteen breast cancers with LOH at 17p13.1 and five breast cancers with LOH at 11p15.5, one case each displayed the same LOH in adjacent normal lobules. Thus the molecular heterogeneity that characterizes invasive breast cancers may occur at the earliest detectable stages of progression.

Mutations in the p53 tumour-suppressor gene are among the most common genetic alterations in human cancers. In the present study we analysed the mutations in the p53 tumor-suppressor gene in 25 primary and 20 metastatic human prostate cancer specimens. DNA extracted from the paraffin-embedded sections was amplified by hot-start polymerase chain reaction, and p53 gene mutations in the conserved mid-region (exons 4-9) were examined using single-strand conformation polymorphism (SSCP) analysis and immunohistochemistry. In the present study, we used a novel hot-start PCR-SSCP technique using DNA Taq polymerase antibody, which eliminates primer-dimers and non-specific products. Because of this new technique, the results of PCR-SSCP showed very high resolution. Polymerase chain reaction products were sequenced directly for point mutations for the p53 gene. Mutations were found in 2 out of 25 primary prostate cancers (8%) and 4 out of 20 metastatic cancers (20%). Mutations were observed exclusively in exon 7 and not in exons 4, 5, 6, 8 or 9. Nuclear accumulation of p53 protein, determined by immunohistochemistry, correlated with the degree of metastasis in prostatic cancer. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7

In male kidney, 4-hydroxy-estradiol has been shown to play a role in the carcinogenesis process. This metabolite is formed from estradiol and due to the hydroxylation activity of the cytochrome P450 1B1 (CYP1B1) enzyme. Prior reports show polymorphisms of CYP1B1 to result in higher catalytic activity and thus, we hypothesize that single nucleotide polymorphisms of the CYP1B1 gene are involved in the malignant transformation of the renal cell. To test this hypothesis, the genetic distributions of four missense CYP1B1 polymorphisms were analyzed in 383 normal healthy subjects and 384 sporadic renal cell carcinoma (RCC) cases by sequence-specific PCR and sequencing. All subjects were men and from a Caucasian population. The polymorphic sites evaluated were at codons 48 (ArgαGly, rs10012), 119 (AlaαSer, rs1056827), 432 (LeuαVal, rs1056836), and 453 (AsnαSer, rs1800440). Results of these experiments demonstrate a trend for the codon 119 Ser/Ser genotype to be a risk for RCC (χ2, P=0.09). Odds ratio for the Ser/Ser genotype was calculated as 1.85 with 95% confidence interval of 1.07-3.20 as compared to the Ala/Ala genotype. The Leu allele on codon 432 was also found to approach significance as a risk for RCC as compared to Val (χ2, P=0.09). Codons 119 and 432 were observed to be linked (R2=0.25) and haplotype analyses show Ala and Val, representing non-risk alleles at these sites, respectively, to be significantly reduced in RCC cases (P=0.06). No association was found however, when analyzing polymorphic sites with clinical stage of RCC. These results demonstrate missense polymorphisms of CYP1B1 to be associated with renal carcinogenesis and are of importance in understanding their role in the pathogenesis of RCC. 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 2640. doi:1538-7445.AM2012-2640

Introduction and Objective: Genistein has been shown to suppress the growth of several cancer cell lines in vitro and in vivo, reducing the expression of several oncogenic microRNAs. However, the effects of genistein on the regulation of oncogenic miR-151 have not been reported. In this study, we investigated whether genistein could alter the expression of oncogenic miR-151 and its target genes that are involved in the progression and metastasis of prostate cancer. Methods: The expression levels of miR-151 were analyzed by TaqMan quantitative real-time PCR and normalized to RNU48. Nineteen clinical specimens were obtained from patients at the San Francisco Veterans Affairs Medical Center from 2000 to 2006. For gain-and loss-of-function assay, cell proliferation (MTS assay), migration (wound healing assay), and invasion (matrigel invasion assay) were evaluated in human prostate cancer cell lines, PC3 and DU145. Predicted target genes and their target microRNA binding site seed regions were investigated using TargetScan and microRNA.org. Genistein was administered every day along with a change of medium, and the cells were grown for four days. Results: Real-time RT-PCR showed that the expression of miR-151 was higher in PC3 and DU145 cells compared with RWPE-1 cells. Treatment of PC3 and DU145 cells with 25 μM genistein down-regulated the expression of miR-151 compared with vehicle control. Kaplan-Meier curves and log-rank tests revealed that high expression levels of miR-151 had a significant adverse effect on survival rate. Inhibition of miR-151 in prostate cancer cells by genistein significantly inhibited cell migration and invasion. By in-cilico analysis, several genes (N4BP1, CASZ1, IL1RAPL1, LGI1, SOX17 and TOM1L1) that are suggested to have tumor suppressive function in several types of cancers were detected as candidate target genes of miR-151. Conclusions: We demonstrated that genistein down-regulated miR-151 that functions as an oncogenic miRNA in prostate cancer. This study suggests that genistein mediated suppression of oncogenic miRNAs can be an important dietary therapeutic strategy for the treatment of prostate cancer. Source of Funding: Grants R01CA160079-01, R01CA138642, VA Research Enhancement Award Program (REAP), and VA Merit Review grants 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 2299. doi:1538-7445.AM2012-2299

MicroRNAs have been implicated in the etiology, progression and metastasis of various malignancies. We identify miR-708, a little studied member of a set of microRNAs that have been implicated in stress control, as an important tumor suppressor in prostate cancer. In this study we identify that miR-708 expression is significantly attenuated in prostate cancer cell lines and human prostate clinical specimens. To evaluate the functional significance of decreased miR-708 expression, miR-708 was reintroduced in prostate cancer cell lines followed by functional assays. Restoration of miR-708 expression in prostate cancer cell lines decreased cell growth, clonability, invasion, migration and elicited a dramatic increase in apoptosis. Moreover, intratumoral delivery of miR-708 was sufficient to trigger in vivo regression of established tumors in a murine xenograft model of human prostate cancer. Further, analysis of putative miR-708 targets showed that miR-708 targets the serine/threonine kinase Akt2/PKBβ, a member of the PI3K/Akt signaling pathway that plays a key role in cellular survival. Phenocopy experiments in prostate cancer cell lines suggest that Akt2 is a functionally relevant target of miR-708 in prostate cancer. In conclusion, our data suggests that miR-708 is a tumor suppressor microRNA that inhibits prostate cancer growth both in vitro and in vivo through targeting Akt2 pathways. 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 134. doi:1538-7445.AM2012-134

Introduction and Objective: MicroRNAs are small noncoding RNAs that regulate the expression of >90% of all human genes, either inhibiting target mRNA translation or inducing its degradation. These genes are abnormally expressed in human malignancies, making their biological importance increasingly apparent. The main objective of this study was to investigate the role of miRNA-23b in prostate carcinogenesis and assess whether miR-23b has any prognostic implication in prostate cancer (PC). Methods: The methods employed in this study include quantitative-real time PCR; methylation specific quantitative-real time PCR; western blot; in-situ hybridization; fluorescence-activated cell sorting assays for cell cycle distribution and apoptosis; assays for cell viability, clonability, migration and invasiveness of prostate cancer cells. Luciferase reporter and mutational assays and in-vivo studies in nude mice were also performed. Results: The expression of miR-23b was significantly suppressed or silenced in human PC tissue samples and cell lines when compared with normal tissues and a non-malignant cell line. The mechanism of miR-23b silencing in PC was found to be due to hypermethylation that was reversed by 5Aza-deoxycitidine (5Aza) treatments. Lower expression of miR-23b also correlated with higher pathological stage and higher Gleason grade making it a useful prognostic biomarker in prostate cancer. Ectopic expression of miR-23b induced G0/G1 cell cycle arrest and apoptosis, suppressed cell proliferation, colony formation, migration, and invasion in prostate cancer cells. miR-23b exerted these functional effects by directly targeting the proto-oncogene Src kinase. Over-expression of miR-23b significantly suppressed the luciferase activity of reporter plasmid containing the 3′UTR sequences complementary to Src kinase, which was abolished by mutations in these 3′UTR regions. Genistein treatment also induced cell cycle arrest and apoptosis, and suppressed Src kinase expression at the protein level in prostate cancer cells. Genistein and miR-23b reduced the Src-regulated downstream ERK/AKT pathway molecules and also inhibited Src kinase and tumor cell growth in vivo. Conclusion: This is the first study demonstrating that i) miRNA-23b is silenced by CpG hypermethylation in PC; ii) miR-23b acts as a tumor suppressor and is a prognostic biomarker in PC; iii) genistein, a nontoxic chemopreventive agent induces the expression of miR-23b and iv) both genistein and miR-23b inhibited oncogenic Src kinase in-vitro and in-vivo. The use of RNAi is currently being implemented as a gene-specific approach for molecular medicine. By the same principle, it is possible that reexpression of silenced miR-23b by nontoxic chemopreventive agents may cause down-regulation of target oncogenes thereby leading to the development of novel therapeutic approaches for the treatment of prostate 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 2303. doi:1538-7445.AM2012-2303

Long-term exposure to estrogen and its metabolites may play an important role in renal cell carcinogenesis. Catechol-O-methyltransferase (COMT) participates in the estrogen metabolism pathway by neutralizing toxic substances. Although reduced COMT activity has been suggested to be a risk factor for estrogen-associated cancers, no studies have investigated the biological significance of COMT in the pathogenesis of human renal cell cancers (RCC). Using quantitative PCR, Western blotting and immunohistochemical staining, we initially found that COMT levels are significantly decreased in human RCC tissues and cells suggesting a suppressive role of COMT in tumor development. However, transient over-expression of COMT has no effect on the proliferation, apoptosis and cell cycle progression of RCC cell lines. In contrast, when cells over-expressing COMT are treated with its substrate 4-hydroxyestradiol (4-OHE2), growth is inhibited by apoptotic cell death. We also found that COMT over-expression combined with 4-OHE2 induces up-regulation of growth arrest- and DNA damage-inducible protein ≤ (GADD45α). We further show that down-regulation of GADD45α by a siRNA-mediated approach inhibits cell death, indicating the essential role of GADD45α in the underlying mechanism of COMT action in response to 4-OHE2. Finally, 4-methoxyestradiol (4-ME) fully reproduces the anti-proliferative function of COMT with 4-OHE2 by promoting GADD45α induction. Together, our study shows that COMT in the presence of 4-OHE2 prevents RCC cell proliferation by enhancing apoptosis, and that GADD45α plays a critical role in the COMT-mediated inhibition of RCC. These findings will provide new direction for the biomarker detection and corresponding treatment in the prevention of human RCC. 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 1860. doi:1538-7445.AM2012-1860

MicroRNAs (miRNAs) constitute an evolutionarily conserved class of small non-coding RNAs that primarily regulate gene expression at the post-transcriptional level via sequence-specific interaction with the 3’-untranslated regions of cognate mRNA targets. Recent evidence indicates that dysregulated miRNA expression leads to tumorigenesis and miRNAs have been implicated to function as either tumor suppressors or oncogenes. The main objective of the present study was to identify miRNA genes that regulate renal cell carcinomas (RCC). Towards this, we performed microRNA expression profiling in normal immortalized renal cell line (HK2) and primary renal cancer cell lines (A498 and Caki2) and identified that miR-708 expression is significantly attenuated in renal cancer cell lines. This attenuation was validated by RT-PCR and in situ hybridization analysis of miR-708 in RCC tissue specimens. Further, trichostatin A, a pharmacological inhibitor of histone deacetylases, strongly increased miR-708 levels in cell lines from renal cancer suggesting that epigenetic silencing may lead to miR-708 downregulation in RCC. To evaluate the functional significance of decreased miR-708 expression, miR-708 was reintroduced in renal cancer cell lines followed by functional assays. Overexpression of miR-708 in the A498 cell line leads to significant decreases in cell growth, clonability, invasiveness and migratory properties. Importantly, miR-708 reexpression leads to dramatic induction of apoptosis in A498 cells suggesting that restoration of miR-708 expression in A498 cells suppressed tumorigenecity in vitro by primarily affecting the apoptotic program. In vivo, intratumoral delivery of miR-708 led to a regression of tumor growth in established renal tumor xenografts. Further, analysis of putative miR-708 targets showed that miR-708 targets survivin/BIRC5, a small inhibitor of apoptosis (IAP) protein that plays a pivotal role in renal cancer progression. In conclusion, our data suggests a crucial tumor suppressive role for miR-708 in RCC. 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 3981. doi:10.1158/1538-7445.AM2011-3981

Junction plakoglobin (JUP) is a member of the catenin family that forms complexes with cadherins and these molecules play a role in cell adhesion and motility. Prior studies have demonstrated genetic alterations of the JUP gene to be a risk for diseases. In prostate, expression of JUP has been shown to be down-regulated in the carcinogenesis process and mutations of this gene may play a role in this process. In this study, we hypothesize that polymorphic variants of the JUP gene can be a risk factor for prostate cancer. To test this hypothesis, the genetic distributions of five JUP polymorphisms were analyzed in 131 normal healthy subjects, 132 benign prostatic hyperplasia (BPH), and 177 sporadic prostate cancer samples by sequence-specific PCR and sequencing. The polymorphic sites evaluated were codon 697 (Met to Leu, rs1126821) along with four locations in the promoter region at the following base pairs prior to the ATG start site: -3633 (C to G, rs12450138), -5971 (A to C, 11650969), -9598 (T to C, rs4479305), and -14959 (G to A, rs12937988). Results of these experiments demonstrate that the variants at the -5971 (P 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 2773. doi:10.1158/1538-7445.AM2011-2773

MicroRNA-21 is up-regulated in a variety of cancers, such as breast, colorectal, lung, head and neck, etc. However, the regulation of miR-21 in kidney cancer has not yet been studied systematically. We measured miR-21 levels in 40 pairs of kidney cancers and their normal matched tissues by real-time PCR. In 36 cases (90%), miR-21 was increased (ratios of miR-21 levels in tumors to normal tissues T/N were greater than 1.5). The expression level of miR-21 was correlated with 5 year survival and the stage of the patients. For patients with low miR-21 expression, all survived 5 years after the operation, while in case of high miR-21 expression, only 59% survived. In groups of patients having low expression (T/N 10), the percent of early stage (Stage I) decreased: 100%, 76% and 43%, respectively. To study the biological effect of miR-21 up-regulation, a kidney cancer cell line A498 was transfected with miR-21 anti-miRNA inhibitor. The miR-21 level was reduced by more than 99%, as compared to the negative control. Flow cytometry analysis of the cell cycle showed a significant increase in G0/G1 phase (9.1%), with a concomitant reduction in the G2/M phase (6.8%), indicating that the forced reduction of miR-21 expression in A498 cells could inhibit the cell growth and cause G0/G1 arrest. Apoptosis assay using flow cytometry showed a significant increase in the number of total apoptotic cells in the miR-21 anti-miRNA inhibitor transfected cells as compared to the negative control. MiR-21 gene was mapped to chromosome 17q23, and increased copy numbers of this section, which includes oncogenes like erb-B2/HER-2, RPS6KB1, etc., were frequently present in cancers. We compared the miR-21 expression levels with the amplification of this section in 30 cancer cell lines, including 5 kidney cancer, 6 breast cancer, 15 colorectal cancer, 3 prostate cancer, and 1 bladder cancer. We observed a significant correlation between the amplification of 17q and high miR-21 expression. In conclusion, the up-regulated miR-21 in kidney cancer could be used as a tumor marker for diagnosis. The inhibition of miR-21 and/or its adjacent oncogenic genes may prove to be useful in controlling cancers with up-regulated miR-21. 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 4934. doi:10.1158/1538-7445.AM2011-4934

Introduction and Objective: 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. We investigated the role of miR-205 in inhibition of Src-mediated oncogenic pathways in renal cancer. Methods: The methods employed in this study include quantitative-real time PCR; western blot; fluorescence-activated cell sorting assays for cell cycle distribution and apoptosis; assays for cell viabiltity, clonability, migration and invasiveness of prostate cancer cells. Luciferase reporter and mutational assays; generation of miR-205 stable cells and in-vivo study in nude mice was also performed. Results: The 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 proto-oncogenic Src family of kinases indicating a therapeutic potential of miR-205 in the treatment of renal cancer. Conclusion: This study demonstrates that miRNA-205 inhibits proto-oncogenic Src family of kinases indicating a therapeutic potential of miR-205 in the treatment of renal cancer. 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 2936. doi:10.1158/1538-7445.AM2011-2936

MicroRNA-34a (miR-34a), a potent p53 mediator, has been reported to function as a tumor suppressor. The proto-oncogene c-Myc regulates cell proliferation and transformation both transcriptionally and non-transcriptionally and is frequently deregulated in human cancers. Rho GTPases are small G proteins that regulate various cellular processes, including cytoskeletal dynamics, migration, cell proliferation, apoptosis, and transcription. RhoA has been shown to control cancer metastasis and progression and cross-talk between RhoA and c-Myc has been reported. We studied the functional effects of miR-34a on c-Myc and RhoA in PC-3 prostate cancer cells. Transfection of miR-34a into PC-3 cells strongly inhibited in vitro cell proliferation, in vivo xenograft growth and cell invasion and promoted apoptosis. miR-34a downregulated expression of c-Myc by targeting its 3’ UTR as shown by luciferase reporter assays. miR-34a was also found to repress RhoA expression. Overexpression of c-Myc reversed miR-34a suppression of RhoA expression and invasion, suggesting that miR-34a inhibits invasion by suppressing RhoA through c-Myc. We also found that miR-34a downregulated guanine nucleotide exchange factors (GEFs) which convert inactive GTPases to an active form. Our results demonstrate that miR-34a suppresses malignancy by targeting c-Myc in PC-3 prostate cancer cells. 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 3968. doi:10.1158/1538-7445.AM2011-3968

The North American region has the highest rates of renal cancers in the entire world. MicroRNAs are small noncoding RNAs that play important roles in numerous cellular processes including development, proliferation, and apoptosis. miR-23b is part of a cluster of miRNAs which includes among others miR-24-1, miR-27b and miR-30a. Recent studies have demonstrated that miR-23b plays different roles in different organs of the body. This led us to speculate its role in urological cancers in general and renal cancer in particular. In the current study we observe the role of miR-23b in renal cancer. The expression of miR-23b was checked in 30 pairs of renal cancer and normal tissues. The samples included three different types of renal cancer tissues, clear cell, papillary and tubular. 55% of clear cell carcinoma and all tubular samples showed high expression of miR-23b as compared to the normal. Interestingly, papillary carcinoma showed a contrasting result in which miR-23b levels were found to be down regulated in almost 90% of the samples. The expression in renal cancer cell lines, A498, ACHN, Caki-1 and Caki-2 was found to be significantly higher as compared to the normal cell line HK-2. The chemo preventive agent Genistein (25µM for 96 hours) was able to reduce the expression of miR-23b in A498 cells by more than 50%. Inhibition of miR-23b expression by the anti-miR-23b antagonist in A498 cells resulted in a significant increase in apoptosis, as detected through flow cytometry analysis. In contrast over expression of mir-23b in A498 cells resulted in a decrease in apoptosis. No significant effect on cell cycle was observed in either over expressing or inhibiting miR-23b in A498 cells. Cell viability assays also did not show any major effect due to the inhibition of expression of miR-23b. Moreover, the expression of miR-23b was correlated with 5 year survival rate of patients. For patients with low levels of miR-23b, 85% of patients survived after 5 years of operation, whereas in case of high expression of miR-23b only 50% survived. Our data suggests that miR-23b acts as an oncogenic mi-RNA in renal cancer. 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 128. doi:10.1158/1538-7445.AM2011-128

Exposure to carcinogenic catechol estrogens plays a role in the development of kidney cancer. Catechol-O-methyltransferase (COMT) neutralizes the genotoxic effects of these compounds by methoxylation. Although reduced activity of COMT has been suggested as a risk factor for estrogen-associated cancers, its critical role in kidney cancer and underlying mechanisms of renal cell carcinogenesis are largely unknown. To address the role and function of COMT in renal cell carcinomas (RCC), we initially determined levels of COMT in normal kidney and clear cell RCC. The expression of COMT mRNA was down-regulated in clear cell RCC tissues as determined by quantitative PCR. Low expression of COMT protein in clear cell RCC tissues was also confirmed by immunohistochemical counting of staining score (2.5 vs. 0.5 in normal kidney and clear cell RCC, respectively, p 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 1343. doi:10.1158/1538-7445.AM2011-1343