Search

In developed countries, prostate cancer (PCa) is one of the most commonly diagnosed cancers, identified by prostate-specific antigen (PSA) screening; PCa is also the third leading cause of cancer-related death among men. Most naïve PCa initially responds well to androgen-deprivation therapy (ADT). However, during ADT treatment, PCa cells acquire ADT treatment resistance and progress to a lethal pathology known as castration-resistant prostate cancer (CRPC). Cancer cells that have reached CRPC can cause distant metastasis, and effective treatments for patients with CRPC have not yet been established. Identification of the molecular pathogenesis underlying acquisition of androgen-independent and metastatic signaling pathways based on advanced genomic approaches is essential for further understanding of this disease.MicroRNAs (miRNAs) are endogenous small RNA (molecules 18-23 bases in length) that act as central players regulating the expression control of protein-coding and protein-noncoding RNAs. Interestingly, a single miRNA can directly regulate a vast number of RNAs in human cells. Therefore, aberrant expression of miRNAs can disrupt normal control of RNA expression in cancer cells. Furthermore, dysregulation of miRNAs is contributed to cancer cell malignancies, such as progression, metastasis, and treatment resistance. Analyses of RNA-sequencing of microRNA (miRNA) expression revealed that miR-99a-3p (passenger strand) is significantly downregulated in several types of cancers. In this study, we aimed to identify novel miR-99a-3p regulatory networks and therapeutic targets for CRPC. The expression levels of miR-99a-3p was significantly lower in PCa and CRPC tissues than in normal tissues (P = 0.0047 and P = 0.0001). Ectopic expression of miR-99a-3p significantly inhibited cancer cell proliferation, migration, and invasion in PCa cells (P < 0.0001). Non-SMC condensin I complex subunit G (NCAPG) was a direct target of miR-99a-3p in PCa cells. Overexpression of NCAPG was detected in CRPC clinical specimens and TCGA data showed that high NCAPG expression was significantly associated with shorter disease-free survival (P = 0.0009) and advanced clinical stage in PCa. Knockdown of NCAPG markedly inhibited cancer cell proliferation, migration, and invasion (P < 0.0001). Moreover, the proliferation, migration, and invasion abilities were recovered by cotransfection with NCAPG and miR-99a-3p. These results indicated that NCAPG affected the aggressiveness in PCa. The passenger strand miR-99a-3p acted as an antitumor miRNA in naïve PCa and CRPC. NCAPG was regulated by miR-99a-3p, and its overexpression was involved in CRPC pathogenesis. Involvement of passenger strand of miRNA in cancer pathogenesis is novel concept and identification of antitumor miRNA regulatory networks in CRPC might be provided novel prognostic markers and therapeutic targets for this disease. Citation Format: Takayuki Arai, Atsushi Okato, Yasutaka Yamada, Sho Sugawara, Akira Kurozumi, Satoko Kojima, Yukio Naya, Tomohiko Ichikawa, Naohiko Seki. Regulation of NCAPG by miR-99a-3p (passenger strand) inhibits cancer cell aggressiveness and is involved in CRPC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 415A.

Renal cell carcinoma (RCC) is the most common neoplasm of the kidney, and approximately 70-80% of RCCs are classified as clear cell-type (ccRCC). Approximately 350,000 new cases are diagnosed worldwide, with over 140,000 deaths, every year. Although surgical resection remains the only known curative treatment for localized RCC, a significant percentage of these patients develop relapses or metastatic disease, which are associated with a poor prognosis. Recently developed targeted molecular therapies and immunomodulatory agents are currently being widely used for patients with metastatic or recurrent RCC. However, those advanced treatments are restricted to RCC patients in advanced stages of disease and the 5-year survival rate of the patients is only 5-10%. MicroRNAs (miRNAs) belong to a class of noncoding RNA molecules that fine-tune the expression of protein coding/noncoding RNAs by repressing translation or cleaving RNA transcripts in a sequence-dependent manner. Aberrantly expressed miRNAs contribute to the initiation, development, and metastasis of several types of cancers, including RCC. Current advanced technologies can be used to identify dysregulated miRNAs in HNSCC clinical specimens. Identification of differentially expressed miRNAs is the first step to elucidating novel miRNA-mediated RNA networks in cancer cell. Analyses of miRNA expression signature by RNA sequencing revealed that miR-451a is significantly downregulated in RCC tissues. Here, we aimed to identify novel RNA networks for miR-451a regulation in RCC cells. Ectopic expression of miR-451a significantly inhibited cancer cell aggressiveness in RCC cells. The Cancer Genome Atlas (TCGA) showed that low expression of miR-451a was significantly associated with poor survival of patients with RCC (p=0.00305). Genome-wide gene expression analyses were applied to identify miR-451a target genes. A total of 16 genes were identified as putative targets of miR-451a in RCC cells. Among these targets, high expression of 8 genes (F2, CRELD2, MSC, LENG8, CLEC2D, SPC25, PMM2, BST2 and NSMAF) were significantly associated with poor prognosis of the patients with RCC by large numbers of cohort analyses. Downregulated miR-451a acted as an antitumor miRNA in RCC cells. Antitumor miR-451a regulatory network was contributed to RCC aggressiveness. The identification of antitumor-mediated oncogenic networks may lead to a better understanding of RCC pathogenesis. Citation Format: Yasutaka Yamada, Sho Sugawara, Takayuki Arai, Atsushi Okato, Satoko Kojima, Yukio Naya, Tomohiko Ichikawa, Naohiko Seki. Antitumor miR-451a regulatory network is associated with poor prognosis of renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 513.

Renal cell carcinoma (RCC) is a disease in which cells undergo oncogenic transformation in the kidney tubules. The five-year survival rate of advanced stage RCC is poor (5-10%) due to recurrence or distant metastasis. Recently, anti-angiogenic multi-tyrosine kinase inhibitors (TKIs) have been developed and have been used as first and second line treatments for RCC. However, these treatments extend progression-free survival only slightly, and relapse and metastasis eventually develop in most patients. The molecular mechanisms of RCC recurrence, metastasis and drug resistance are not yet fully understood. Therefore, analysis of the molecular mechanisms underlying RCC development and progression and studies of novel oncogenic pathways based on current genome-based approaches could significantly improve diagnosis, therapy, and prevention of the disease. miRNAs (miRNAs) are small noncoding RNAs that function to fine tune the expression of protein coding/noncoding RNAs by repressing translation or cleaving RNA transcripts in a sequence-depending manner. The unique characteristic function of miRNAs is to regulate RNA transcripts in human cells. Therefore, dysregulated expression of miRNAs can disrupt tightly regulated RNA networks in cancer cells. In this study, we constructed a miRNA expression signature to identify pathways activated by TKI treatment using autopsy specimens from patients with RCC. We have sequentially identified tumor-suppressive miRNA and its regulated RCC pathways based on the signature. The aim of this study was to investigate the functional significance of miR-10a-5p and to identify the molecular targets and pathways mediated by miR-10a-5p in RCC cells. The expression levels of miR-10a-5p were significantly reduced in RCC clinical specimens and RCC cell lines compared with non-cancerous kidney tissues (P < 0.001). TCGA data showed that the overall survival of low miR-10a-5p expression group was significantly shorter than that of high expression group (P = 0.00408). Restoration of miR-10a-5p significantly inhibited cancer cell migration and invasion in RCC cell lines (P < 0.0001). Spindle and kinetochore associated complex subunit 1 (SKA1) was identified as a direct target gene of miR-10a-5p by genome-wide gene expression analysis and in silico analysis. Overexpression of SKA1 was observed in RCC clinical specimens. Moreover, the overall survival of high SKA1 expression group was significantly shorter than that of low expression group by TCGA analysis (P = 1.44E-07). Tumor-suppressive miR-10a-5p was identified by using miRNA signature of patients with TKI failure. Overexpression of SKA1 might be involved in RCC aggressiveness, metastasis and drug resistance. Elucidation of tumor-suppressive miRNAs regulated molecular pathways and targets could provide new information on potential therapeutic strategies in the disease. Citation Format: Takayuki Arai, Atsushi Okato, Akira Kurozumi, Mayuko Kato, Yusuke Goto, Keiichi Koshizuka, Satoko Kojima, Yukio Naya, Tomohiko Ichikawa, Naohiko Seki. MicroRNA expression signature of patients with tyrosine kinase inhibitors failure: miR-10a-5p inhibits cancer cell aggressiveness in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2526. doi:10.1158/1538-7445.AM2017-2526

Introduction: Most prostate cancer (PCa) patients initially respond to androgen-deprivation therapy (ADT), but eventually acquire resistance to ADT and progress to castration-resistant prostate cancer (CRPC), resulting in metastasis causing PCa death. We have developed the microRNA (miRNA) expression signature of PCa using clinical specimens and determined the target genes of the tumor suppressive miRNAs, whose expressions are significantly decreased in PCa specimens. Based on the signature, our previous study revealed that miR-224 functions as a tumor suppressor, especially contributes to cancer cell metastasis directly targeting miR-224-TPD52 signaling. In human genome, miR-452 is located nearby tumor-suppressive miR-224, forming clustered miRNAs on chromosome Xq28 region. The aim of the study was to investigate the functional significance of miR-452 and to identify novel miR-452-mediated cancer pathways and responsible genes in PCa cells. Material and methods: Clinical prostate specimens were obtained from patients admitted to the Teikyo University Chiba Medical Center from 2008 to 2013. Ninety-two patients with elevated PSA levels underwent transrectal prostate needle biopsy, and three patients who died of CRPC underwent autopsies. PCa tissues (n=54), noncancerous prostate tissues (non-PCa, n=36), were used for analysis of expression levels of miRNAs. Functional studies of differentially expressed miRNAs were analyzed using PC3 and DU145 cells. The influence on CRPC-free survival of expression levels of miRNA was estimated using the Kaplan-Meier methods. In silico database and genome-wide gene expression analyses were performed to identify molecular targets regulated by the miRNAs. Results: (The miRNA expression signature of PCa specimens showed that the cluster miRNAs miR-224 was significantly downregulated, suggesting that this miRNA may act as tumor suppressor.) Clinical data using advanced PCa showed that low expression of miR-452 predicted a short duration of progression to CRPC. Restoration of miR-452 in PC3 and DU145 cells revealed significant inhibition of cancer cell migration and invasion. WW domain-containing E3 ubiquitin protein ligase-1 (WWP1) was confirmed as a direct target of miR-452 by in vitro transfection of miR-452 and Luciferase assay. Knockdown of the expression of WWP1 using siRNA showed inhibiting cell migration and invasion in PC3 and DU145 cells. Immunohistochemistry showed overexpressed WWP1 in advanced PCa. Conclusions: Loss of the tumor suppressive miR-452 enhanced migration and invasion in PCa cells. Regulation of the miR-452-WWP1 axis contributed to PCa cell migration and invasion, and elucidation of downstream signaling of this axis will provide new insights into the mechanisms of (progression to CRPC) metastasis of PCa. Citation Format: Satoko Kojima, Yusuke Goto, Akira Kurozumi, Mayuko Kato, Atsushi Okato, Takayuki Arai, Tomohiko Ichikawa, Yukio Naya, Naohiko Seki. miR-452 inhibits migration and invasion of prostate cancer cells by targeting E3 ubiquitin ligase-1 (WWP1) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2528. doi:10.1158/1538-7445.AM2017-2528

BACKGROUND: Prostate cancer (PCa) is the most frequently diagnosed cancer and the second leading cause of cancer death among men in developed countries. Disease progression and the development of hormone refractory disease remain major cause of cancer-related death.Understanding the molecular mechanisms of PCa progression and metastatic pathways using currently available genomic approaches would help to improve therapies for and prevention of the disease. Our recent study of microRNA (miRNA) expression signature of PCa has revealed that microRNA-218 (miR-218) was significantly downregulated in cancer tissues, suggesting that miR-218 may be a putative tumor-suppressive miRNA in PCa. The aim of this study was to investigate the functional significance of miR-218 in PCa and to identify novel miR-218-regulated cancer pathways and target genes involved in PCa oncogenesis and metastasis.METHODS: The expression levels of miR-218 were validated using real-time RT-PCR using PCa clinical specimens and cell lines (PC3 and DU145). Cell proliferation, migration and invasion assays were performed to investigate the functional significance of miR-218 and its target gene, LIM and SH3 protein 1 (LASP1) in PCa cell lines.Cells were transfected with mature miR-218 or si-LASP1 by reverse transfection methods. Genome-wide gene expression data and in silico analysis were used to identify the molecular pathways and putative targets regulated by miR-218.The luciferase reporter assay was applied to identify the actual binding site of miR-218 in target gene. The expression of LASP1 was investigated by immunohistochemistry.RESULTS: The expression levels of miR-218 were significantly downregulated in PCa tissues and PCa cell lines. Restoration of miR-218 in PCa cell lines revealed that miR-218 significantly inhibited cancer cell migration and invasion.Gene expression studies and luciferase reporter assays showed that LASP1 was directly regulated by miR-218. Silencing LASP1 resulted in significant inhibition of cell migration and invasion in PCa cells.The expression of LASP1 was upregulated in cancer tissues as demonstrated byimmunohistochemical staining.CONCLUSIONS: The miR-218 was frequently reduced in PCa clinical specimens and appeared to function as a tumor suppressor through regulation of oncogenic LASP1. The identification of novel molecularpathways and targets regulated by the tumor suppressive miRNAs may lead to a better understanding of PCa progression and metastasis, and the development of new therapeutic strategies to treat this disease. Citation Format: Rika Nishikawa, Yusuke Goto, Takashi Kinoshita, Shinichi Sakamoto, Takeshi Chiyomaru, Hideki Enokida, Satoko Kojima, Masayuki Nakagawa, Yukio Naya, Tomohiko Ichikawa, Naohiko Seki. Tumor suppressive microRNA-218 inhibits cancer cell migration and invasion via targeting LASP1 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 4371. doi:10.1158/1538-7445.AM2014-4371

(Background) Prostate cancer (PCa) is the most frequently diagnosed and second leading cause of cancer death among men in developing countries. Hormone-refractory PCa is currently difficult to treat and most clinical trials for advanced PCa have shown limited benefits, with disease progression and metastasis to bone or other sites. Thus, new prognostic markers and effective treatment strategies are urgently needed. Our expression signatures of human cancer including prostate cancer revealed that the expression of microRNA-1 (miR-1) and microRNA-133a (miR-133a) were significantly reduced in cancer cells. In human genome, miR-1 and miR-133a located same chromosomal regions called cluster. In this study, we investigated the functional significance of miR-1 and miR-133a and identified the novel molecular targets regulated by miR-1 and miR-133a commonly in PCa. (Methods) Cell proliferation, invasion and migration assays were performed by restoration of mature miR-1 and/or miR-133a in PCa cell lines (PC3 and DU145). Genome-wide gene expression analysis was performed to identify the molecular targets of miR-1 and miR-133a by microarray methods. A luciferase reporter assay was used to identify the actual binding site between miR-1 and miR-133a and its candidate target genes. Cell proliferation, invasion and migration assays were performed to investigate the targets genes in PCa cell lines. To investigate of expression of candidate target gene, immunohistochemistry was performed by using PCa tissue microarray. (Results) Restoration of miR-1 and/or miR-133a significantly inhibited cell proliferation, migration and invasion in cancer cells. Genome-wide molecular targets search and luciferase reporter assay showed that 6 genes: Transgelin2 (TAGLN2), WD repeat domain 78 (WDR78), chromosome 4 open reading frame 34 (C4orf34), purine nucleoside phosphorylase (PNP), LAG1 homologue, ceramide synthase 2 (LASS2) and syntaxin binding protein (STXBP4) had putative target sites of both miR-1 and miR-133a in their 3′UTR. Among them, PNP mRNA was highly expressed in the PCa clinical specimen compered with non-PCa tisses. The mRNA and protein expression levels of PNP were markedly downregulated in miR-1 and miR-133a -transfected PCa cells. A luciferase reporter assay suggested that miR-1 and miR-133a directly bind to specific sites on 3′UTR of PNP mRNA. Silencing of the target genes inhibited cell proliferation, migration, and invasion in PCa cells. Immunohistochemistry showed that PNP expression level was significantly higher in PCa than normal prostate tissues. (Conclusions) miR-1 and miR-133a function as tumor suppressor in PCa. PNP was directly regulated by tumor suppressive miR-1 and miR-133a commonly. PNP may function as oncogenes in PCa. Tumor suppressive miR-1 and miR-133a mediates novel molecular targets provide new insights of molecular mechanisms of PCa oncogenesis. 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 3166. doi:1538-7445.AM2012-3166