Suppressed Prostate Cancer Cell Proliferation Research Articles

Knockdown of LncRNA GHET1 suppresses prostate cancer cell proliferation by inhibiting HIF-1α/Notch-1 signaling pathway via KLF2.

Prostate cancer (PC) is one of the most common cancers in male groups worldwide. Long noncoding RNAs (LncRNAs) are reported to be dysregulated in a variety of cancers, including PC. This study aimed to explore the role of LncRNA GHET1 in the pathogenesis of PC. RT-qPCR was carried out to examine the relative expression level of GHET1 in PC patients. In vitro, GHET1 siRNA (si-GHET1) was used to investigate the biological role of GHET1 in PC cell lines. Cell proliferation was detected by CCK-8 and colony formation assay, while cell cycle and cell apoptosis were analyzed using flow cytometry. Moreover, western blot was carried out to measure the protein expression levels of KLF2 and HIF-1α/Notch-1 signal pathway. We found that GHET1 showed higher expression in PC tissues and had a negative correlation with KLF2 expression. Knockdown of GHET1 significantly suppressed the cell proliferation, induced cell cycle arrest at G0/G1 phase and promoted cell apoptosis. Additionally, si-GHET1 transfection induced KLF2 upregulation and HIF-1α/Notch-1 signal pathway suppression, which could be rescued by si-KLF2 transfection. These results suggest the key role of GHET1 in PC progression. Moreover, GHET1 might be explored to be a potential target for clinical treatment of PC. © 2019 BioFactors, 45(3):364-373, 2019.

Long noncoding RNA PCA3 regulates prostate cancer through sponging miR-218-5p and modulating high mobility group box 1.

The purpose of this study was to investigate the mechanism of long noncoding RNA (lncRNA) prostate cancer antigen 3 (PCA3) in prostate cancer (PCa) via regulating the miR-218-5p/high mobility group box 1 (HMGB1) axis. The Cancer Genome Atlas database was used to divide differentially expressed lncRNAs, microRNAs, and messenger RNA (mRNAs). The mRNA expressions of lncRNA PCA3, miR-218-5p, and HMGB1 were determined by reverse transcription polymerase chain reaction. Cell propagation was evaluated using the Cell Counting Kit-8 assay and the apoptotic rate was examined by flow cytometry. Cell migration and invasion were observed through the wound healing assay and transwell assay. Target relationships among PCA3, miR-218-5p, and HMGB1 were validated via dual-luciferase reporter gene assay. A nude mouse model in vivo was designed to evaluate the effect of PCA3 on prostate tumor growth. PCA3 and HMGB1 were high-expressed in PCa, whereas miR-218-5p was low-expressed. PCA3 knockdown or miR-218-5p overexpression suppressed PCa cell proliferation, migration, and invasion, but promoted apoptosis. Besides, targeted relationships and interactions on the expression between miR-218-5p and PCA3 or HMGB1 were elucidated. PCA3 weakened cell viability and mobility whereas induced apoptosis through binding with miR-218-5p. Meanwhile, miR-218-5p also inhibited PCa tumorigenesis via downregulation of HMGB1. Knockdown of PCA3 impeded tumor growth by downregulating its downstream gene HMGB1. lncRNA PCA3 facilitated PCa progression through sponging miR-218-5p and regulating HMGB1.

Abstract A070: Targeting N-myristoyltransferase 1 inhibits prostate cancer progression

Abstract N-myristoyltransferase (NMT) facilitates co/post-translational myristoylation of several oncogenic proteins, regulating their functions in tumor progression. N-myristoyltransferase (NMT), which has been shown to be upregulated in a variety of cancers, is overexpressed in androgen-independent prostate cancer cells. We demonstrate that genetically, ablation of NMT1 inhibited proliferation of prostate cancer cells, tumor growth, and suppressed myristoylation profile of prostate cancer cells. Screening a panel of myristoyl-CoA analogs against purified human NMT1 protein leads to identifying the B13 as an inhibitor for NMT1. B13 significantly suppressed prostate cancer cell proliferation, migration, and invasion by cell cycle arrest, and inhibited the growth of prostate xenograft tumors with minimal pathologic effect on major organs in vivo. Structure activity relationship based optimization of B13 led to LCL204, which showed better inhibitory properties towards NMT1. We demonstrate that targeting protein myristoylation is a potential therapeutic approach to inhibit prostate tumor progression. Citation Format: Omar Alsaidan, Sungjin Kim, Octavia Goodwin, Qianjin Li, Aiping Bai, Alicja Bielawska, George Zheng, Iryna Lebedyeva, Thomas Albers, James S. Norris, Scott D. Pegan, Houjian Cai. Targeting N-myristoyltransferase 1 inhibits prostate cancer progression [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A070.

Toll-Like Receptor 4 (TLR4)/Cyclooxygenase-2 (COX-2) Regulates Prostate Cancer Cell Proliferation, Migration, and Invasion by NF-κB Activation.

BackgroundToll-like receptor 4 (TLR4)-mediated signaling has been implicated in invasion, metastasis, and survival of various cancers. Activation of TLR4 can promote cyclooxygenase-2 (COX-2) and nuclear factor-κB (NF-κB). However, little is known about the effects of TLR4/COX-2 in prostate cancer (PCa).Material/MethodsIn our study, TLR4 and COX-2 expressions were detected by quantitative real-time reverse transcription PCR (qRT-PCR) in PCa tissues (n=34). Cell proliferation was measured by Cell Counting Kit-8 (CCK-8) and carboxyfluorescein succinimidyl ester (CFSE) assays. The migration and invasion abilities were detected by wound healing and Transwell assays. qRT-PCR and western blot assays were performed to detect TLR4, COX-2, matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of matrix metalloproteinases (TIMP)-1, epithelial-cadherin (E-cadherin), vimentin, NF-κB (p65), and p-p65 expressions.ResultsThe results revealed that TLR4 and COX-2 were upregulated in PCa tissues; Silencing of TLR4 or COX-2 inhibited PCa cell proliferation, migration, and invasion, and TLR4 siRNAs combined with COX-2 siRNAs synergistically suppressed PCa cell proliferation, migration, and invasion. Silencing of TLR4 or COX-2 also downregulated MMP-2, MMP-9, and E-cadherin expressions, and upregulated TIMP-1 and vimentin expressions. In addition, silencing of TLR4 or COX-2 inhibited p65 phosphorylation and had a synergistic effect.ConclusionsWe demonstrated that TLR4/COX-2 inhibits PCa cell proliferation, migration, and invasion by regulating NF-κB.

ASC-J9® suppresses prostate cancer cell invasion via altering the sumoylation-phosphorylation of STAT3

The androgen-deprivation therapy (ADT) to either reduce the androgen biosynthesis (for example, Abiraterone) or to prevent binding of androgen to the androgen receptor (AR), for example using Casodex or Enzalutamide, which may result in .decrease of the prostate cancer (PCa) cell growth, yet may also increase the PCa cell invasion. In contrast, the recently identified AR degradation enhancer ASC-J9® may function via degrading the AR protein to simultaneously suppress the PCa cell proliferation and invasion. The details of this unique mechanism, however, remain unclear. Here we found that ASC-J9® could suppress PCa cell invasion via inducing the sumoylation of STAT3, thereby inhibiting the STAT3 phosphorylation that led to suppress the EMT-SNAIL2 signals in both PCa DU145 and PC3 AR-negative cells. Mutation of lysine-679 on the sumoylation site of the STAT3 effectively blocked the ASC-J9®-suppressed PCa cell invasion in both in vitro cell lines and in vivo mouse models. These results suggest that in addition to degrading AR to suppress PCa cell proliferation, ASC-J9® can also function through an AR-independent mechanism via modulating the STAT3 sumoylation to alter the phospho-STAT3 status to suppress the PCa cell invasion. These dual functions of ASC-J9® to suppress PCa proliferation and invasion (via altering STAT3 sumoylation) may help us to develop a better anti-AR compound that may overcome the current antiandrogens' unwanted side-effect of increasing the metastasis to better suppress the castration-resistant PCa progression.

Long non-coding RNA CASC2 regulates Sprouty2 via functioning as a competing endogenous RNA for miR-183 to modulate the sensitivity of prostate cancer cells to docetaxel

Prostate cancer (PC) is the most common cancer in men; however, limited effect is obtained due to the therapy resistance. CASC2 acts as a tumor suppressor in human malignancies serving as a ceRNA for miRNAs; Sprouty2 (SPRY2), a key antagonist of RTK signaling, also serves as a tumor suppressor. Herein, CASC2 and SPRY2 expression was down-regulated in PC tissues and cell lines; the overexpression of CASC2 and SPRY2 could suppress PC cell proliferation, promote PC cell apoptosis, and enhance the sensitivity of PC cells to docetaxel. CASC2 positively regulated SPRY2 expression and inhibited downstream extracellular regulated protein kinases (ERK) signaling activation through SPRY2. By using online tools, miR-183 might be a direct target of CASC2, and might simultaneously bind to the 3′UTR of SPRY2. The direct binding between CASC2, miR-183 and SPRY2 was then validated; miR-183 inhibition enhanced the cytotoxicity of docetaxel on PC cells, which could be partially attenuated by SPRY2 knockdown. In summary, CASC2 competes with SPRY2 for miR-183 binding to rescue the expression of SPRY2 in PC cells, thus enhancing the sensitivity of PC cells to docetaxel through SPRY2 downstream ERK signaling pathway; CASC2 and SPRY2 might be novel adjuvants for docetaxel-based chemotherapy for PC.

MicroRNA181c inhibits prostate cancer cell growth and invasion by targeting multiple ERK signaling pathway components.

The ERK signaling pathway is frequently deregulated in tumorigenesis, mostly by classical mechanisms such as gene mutation of its components (eg, RAS and RAF). However, whether and how multiple key components of ERK pathway are regulated by microRNAs are not clear. We firstly predicted post-transcriptional regulation of multiple key components of the ERK signaling pathway by miR181c through bioinformatics analysis, and then confirmed the post-transcriptional regulation by dual luciferase reporter gene assays and Western blot analysis. The biological effects of miR181c on prostate cancer cell proliferation, apoptosis, migration, and invasion were measured by CCK-8 assay, flow cytometry, wound scratch assay, transwell cell migration, and invasion assays. miR181c post-transcriptionally regulated multiple key members of the ERK signaling pathway, including extracellular signal-regulated kinase 2 (ERK2), ribosomal S6 kinase 2 (RSK2), serum response factor (SRF), and FBJ murine osteosarcoma viral oncogene homolog (c-Fos). Ectopic expression of miR181c mimics effectively suppressed prostate cancer cell proliferation, migration, and invasion, but promoted cell apoptosis. Furthermore, miR181c treatment combined with the multi-kinase inhibitor sorafenib significantly enhanced these anti-tumor effects. Downregulation of miR181c results in deregulated ERK signaling and promotes prostate cancer cell growth and metastasis.

Suppression of microRNA-454 impedes the proliferation and invasion of prostate cancer cells by promoting N-myc downstream-regulated gene 2 and inhibiting WNT/β-catenin signaling

MicroRNA-454 (miR-454) is emerging as critical regulator in tumorigenesis; it may function as an oncogene or a tumor suppressor. However, the role of miR-454 in prostate cancer remains unknown. In this study, we aimed to investigate the function and molecular mechanisms of miR-454 in prostate cancer. We found that miR-454 was highly expressed in prostate cancer tissues and cell lines (*p<0.05), as detected by real-time quantitative polymerase chain reaction (RT-qPCR). Cell counting kit-8 assay, colony formation assay and cell invasion assay showed that the inhibition of miR-454 significantly suppressed prostate cancer cell proliferation and invasion (*p<0.05), whereas the overexpression of miR-454 markedly promoted prostate cancer cell proliferation and invasion (*p<0.05). Bioinformatics analysis showed that N-myc downstream-regulated gene 2 (NDRG2), a well-known tumor suppressor, was identified as a potential target gene of miR-454. Dual-luciferase reporter assay showed that miR-454 directly targeted the 3′-untranslated region of NDRG2. RT-qPCR and western blot showed that miR-454 overexpression significantly decreased NDRG2 expression (*p<0.05), whereas miR-454 inhibition markedly promoted NDRG2 expression (*p<0.05). Spearman’s correlation analysis showed that miR-454 expression was inversely correlated with NDRG2 expression in prostate cancer tissues (r=−0.8932; p<0.0001). Moreover, miR-454 inhibition significantly suppressed the protein expression of β-catenin (*p<0.05) and blocked the activation of WNT signaling (*p<0.05). In addition, small interfering RNA mediated NDRG2 knockdown significantly reversed the antitumor effect of miR-454 inhibition on prostate cancer cell proliferation and invasion (*p<0.05). Taken together, these results reveal an oncogenic role of miR-454, which promotes prostate cancer cell proliferation and invasion by downregulation of NDRG2. These results also suggest miR-454 as a potential therapeutic target for the treatment of prostate cancer.

Combined targeting of Arf1 and Ras potentiates anticancer activity for prostate cancer therapeutics

BackgroundAlthough major improvements have been made in surgical management, chemotherapeutic, and radiotherapeutic of prostate cancer, many prostate cancers remain refractory to treatment with standard agents. Therefore, the identification of new molecular targets in cancer progression and development of novel therapeutic strategies to target them are very necessary for achieving better survival for patients with prostate cancer. Activation of small GTPases such as Ras and Arf1 is a critical component of the signaling pathways for most of the receptors shown to be upregulated in advanced prostate cancer.MethodsThe drug effects on cell proliferation were measured by CellTiter 96® AQueous One Solution Cell Proliferation Assay. The drug effects on cell migration and invasion were determined by Radius™ 24-well and Matrigel-coated Boyden chambers. The drug effects on apoptosis were assessed by FITC Annexin V Apoptosis Detection Kit with 7-AAD and Western blot with antibodies against cleaved PARP and Caspase 3. A NOD/SCID mouse model generated by subcutaneous injection was used to assess the in vivo drug efficacy in tumor growth. ERK activation and tumor cell proliferation in xenografts were examined by immunohistochemistry.ResultsWe show that Exo2, a small-molecule inhibitor that reduces Arf1 activation, effectively suppresses prostate cancer cell proliferation by blocking ERK1/2 activation. Exo2 also has other effects, inhibiting migration and invasion of PCa cells and inducing apoptosis. The Ras inhibitor salirasib augments Exo2-induced cytotoxicity in prostate cancer cells partially by enhancing the suppression of ERK1/2 phosphorylation. In a xenograft mouse model of prostate cancer, Exo2 reduces prostate tumor burden and inhibits ERK1/2 activation at a dose of 20 mg/kg. Synergistic treatment of salirasib and Exo2 exhibits a superior inhibitory effect on prostate tumor growth compared with either drug alone, which may be attributed to the more efficient inhibition of ERK1/2 phosphorylation.ConclusionThis study suggests that simultaneous blockade of Arf1 and Ras activation in prostate cancer cells is a potential targeted therapeutic strategy for preventing prostate cancer development.

An Ω-3 fatty acid desaturase-expressing gene attenuates prostate cancer proliferation by cell cycle regulation.

Previous studies have reported that Ω-6 and Ω-3 fatty acids have opposing effects on cancer development. Consuming high levels of long-chain Ω-3 polyunsaturated fatty acids (PUFAs) has been shown to reduce prostate cancer risk and increase chemotherapy sensitivity. The sdd17 gene encodes an Ω-3 fatty acid desaturase, which converts arachidonic acid into eicosapentaenoic acid (EPA). However, little is known regarding the function of the sdd17 gene in tumor cells in vitro. In the present study, prostate cancer cells were infected with the msdd17 gene, which allowed the endogenous production of Ω-3 PUFAs. The cells that expressed the msdd17 gene had high levels of long-chain Ω-3 PUFAs compared with the control cells. Expression of the msdd17 gene significantly inhibited prostate cancer cell proliferation. EPA exposure and msdd17 gene transfection each induced G2 cell cycle arrest and reduced E2F transcription factor 1 expression in prostate cancer cells. These results suggest that msdd17 gene transfection suppressed prostate cancer cell proliferation and induced G2 cell cycle arrest.

The biological functions and mechanism of miR-212 in prostate cancer proliferation, migration and invasion via targeting Engrailed-2

Accumulating evidence indicates that Engrailed-2 (EN-2), which is a homeobox-containing transcription factor, act as a candidate oncogene in prostate cancer (PCa). Even though there are some treatments targeting EN-2, however, it is limited because the mechanism of EN-2 upregulation in PCa cells is still unknown. In this study, we investigate the role of miR-212 on EN-2 expression and explored the mechanism of prostate cancer survival and metastasis. The relative expression levels of miR-212 and EN-2 in PCa samples and adjacent normal tissues as well as in PCa cell lines were detected by using quantitative real-time PCR. CCK-8, TUNEL and Transwell assays were used to analyze cell proliferation, apoptosis and invasion, respectively. EN-2 was identified as a direct target of miR-212 via luciferase reporter and western blot assays. Results showed that the expression level of miR-212 was downregulated in both PCa samples and PCa cell lines when compared with prostate epithelial cells and the adjacent no tumor tissues. Moreover, we found that overexpression of miR-212 suppressed PCa cell proliferation and invasion, promoted PCa cell apoptosis. EN-2 was identified as a direct target gene of miR-212 by using luciferase reporter and western blot assays. Also, the expression of EN-2 and miR-212 in the PCa cells had an opposite correlation. The critical role of miR-212 in inhibiting prostate tumor growth was verified in xenograft models of prostate cancer. These findings highlighted the role of miR-212 in PCa progression. More importantly, we speculate that EN-2 is a direct target gene of miR-212.

Knockdown of prostaglandin reductase 1 (PTGR1) suppresses prostate cancer cell proliferation by inducing cell cycle arrest and apoptosis.

Chemoresistance is a serious problem for the treatment of androgen-independent prostate cancer (PC). The underlying molecular mechanisms by which androgen-independent PC cells acquire the capacity to proliferate remain largely unclear. The aim of this study was to investigate the biological role of prostaglandin reductase 1 (PTGR1) in prostate cancer. Data from the Oncomine database showed that PTGR1 is commonly upregulated in PC tissue in comparison to corresponding normal controls. Two PTGR1-specific short hairpin RNA (shRNA) sequences were used to block the expression of PTGR1 via a lentivirus-mediated system in the androgen-independent PC cell lines DU145 and PC 3. Functional analysis revealed that knockdown of PTGR1 significantly inhibited proliferation and colony formation by PC cells. The inhibition of cell proliferation was related to arrest of the cell cycle in the G0/G1 phase and increased apoptosis in response to PTGR1 knockdown as indicated by flow cytometry. PTGR1 silencing was found to mechanically enhance the expression of p21, caspase 3, and cleaved PARP and to decrease the level of cyclin D1. In conclusion, PTGR1 plays an essential role in PC cells and may be a potential therapeutic target for PC.

MicroRNA-613 represses prostate cancer cell proliferation and invasion through targeting Frizzled7

A growing number of studies have indicated that microRNAs (miRNAs) are critical regulators of carcinogenesis and cancer progression and may serve as potential therapeutic tools for cancer therapy. Frizzled7 (Fzd7), the most important receptor of the Wnt signaling pathway, is extensively involved in cancer development and progression. However, the role of Fzd7 in prostate cancer remains unclear. In this study, we aimed to explore the expression of Fzd7 in prostate cancer and test whether modulating Fzd7 expression by miR-613 would have an impact on prostate cancer cell proliferation and invasion. We found that Fzd7 was highly expressed in prostate cancer cell lines. Through bioinformatics analysis, Fzd7 was predicted as a target gene of miR-613, which was validated by dual-luciferase reporter assays, real-time quantitative polymerase chain reaction and Western blot analysis. By gain of function experiments, we showed that overexpression of miR-613 significantly suppressed prostate cancer cell proliferation and invasion. Furthermore, miR-613 overexpression markedly downregulated the Wnt signaling pathway. Through a rescue experiment, we showed that overexpression of Fzd7 could abrogate the inhibitory effect of miR-613 on cell proliferation and invasion as well as Wnt signaling. Additionally, these results were further strengthened by data showing that miR-613 was significantly downregulated in prostate cancer tissues, exhibiting an inverse correlation with Fzd7 expression. In conclusion, our study suggests that miR-613 functions as a tumor suppressor, partially through targeting Fzd7, and is a potential therapeutic target for prostate cancer.

Up-Regulation of LAT1 during Antiandrogen Therapy Contributes to Progression in Prostate Cancer Cells

Cancer cells require massive amounts of amino acids for survival. LAT1 (L-type amino acid transporter 1) transports essential amino acids, including leucine, which trigger the downstream mTOR (mammalian target of rapamycin) pathway. We examined the association between androgen receptor and LAT1, and the association between LAT1 expression and the acquisition of castration resistance. Western blot and real-time polymerase chain reaction were performed to study protein and mRNA expression. siRNA was used to knock down target genes. A total of 92 prostate biopsy specimens of patients who underwent androgen deprivation therapy were used for immunohistochemical analyses. Cox hazard proportional models and the Kaplan-Meier method were used for statistical analyses. LAT1 was highly expressed in hormone resistant prostate cancer cell lines. Knockdown of LAT1 in LNCaP and C4-2 cells significantly suppressed cell proliferation, migration and invasion. Androgen receptor siRNA or androgen receptor blocking through bicalutamide (10 μM) or MDV3100 (10 μM) significantly increased LAT1 expression (p <0.01). Treatment with dihydrotestosterone (0.1 to 10 nM) reduced LAT1 expression in a dose dependent manner (p<0.01). Bicalutamide/MDV3100 plus siLAT1 synergistically suppressed prostate cancer cell proliferation compared to single inhibition by androgen receptor or LAT1 (p<0.01). High LAT1 expression correlated with significantly shorter prostate specific antigen recurrence-free survival in patients receiving androgen deprivation therapy (p <0.0001). LAT1 expression was an independentpredictor of castration resistance on multivariate analysis (HR 3.56, p=0.0133). The current data may indicate a novel mechanism to acquire castration resistance through activation of the amino acid transporter LAT1.

MiR-503 suppresses tumor cell proliferation and metastasis by directly targeting RNF31 in prostate cancer

Microarray data analyses were performed to search for metastasis-associated oncogenes in prostate cancer (PCa). RNF31 mRNA expressions in tumor tissues and benign prostate tissues were evaluated. The RNF31 protein expression levels were also analyzed by western blot and immunohistochemistry. Luciferase reporter assays were used to identify miRNAs that can regulate RNF31. The effect of RNF31 on PCa progression was studied in vitro and in vivo. We found that RNF31 was significantly increased in PCa and its expression level was highly correlated with seminal vesicle invasion, clinical stage, prostate specific antigen (PSA) level, Gleason score, and BCR. Silence of RNF31 suppressed PCa cell proliferation and metastasis in vitro and in vivo. miR-503 can directly regulate RNF31. Enforced expression of miR-503 inhibited the expression of RNF31 significantly and the restoration of RNF31 expression reversed the inhibitory effects of miR-503 on PCa cell proliferation and metastasis. These findings collectively indicated an oncogene role of RNF31 in PCa progression which can be regulated by miR-503, suggesting that RNF31 could serve as a potential prognostic biomarker and therapeutic target for PCa.

MiR-93/miR-106b/miR-375-CIC-CRABP1: a novel regulatory axis in prostate cancer progression.

Capicua (CIC) has been implicated in pathogenesis of spinocerebellar ataxia type-1 (SCA1) neurodegenerative disease and some types of cancer; however, the role of CIC in prostate cancer remains unknown. Here we show that CIC suppresses prostate cancer progression. CIC expression was markedly decreased in human prostatic carcinoma. CIC overexpression suppressed prostate cancer cell proliferation, invasion, and migration, whereas CIC RNAi exerted opposite effects. We found that knock-down of CIC derepresses expression of ETV5 and CRABP1 in LNCaP and PC-3 cells, respectively, thereby promoting cell proliferation and invasion. We also discovered that miR-93, miR-106b, and miR-375, which are known to be frequently overexpressed in prostate cancer patients, cooperatively down-regulate CIC levels to promote cancer progression. Altogether, we suggest miR-93/miR-106b/miR-375-CIC-CRABP1 as a novel key regulatory axis in prostate cancer progression.

MicroRNA-302a Suppresses Tumor Cell Proliferation by Inhibiting AKT in Prostate Cancer.

Micro (mi) RNAs are important regulators involved in various physical and pathological processes, including cancer. The miRNA-302 family has been documented as playing a critical role in carcinogenesis. In this study, we investigated the role of miRNA-302a in prostate cancer (PCa). MiRNA-302a expression was detected in 44 PCa tissues and 10 normal prostate tissues, and their clinicopathological significance was analyzed. Cell proliferation and cell cycle analysis were performed on PCa cells that stably expressed miRNA-302a. The target gene of miRNA-302a and the downstream pathway were further investigated. Compared with normal prostate tissues, miRNA-302a expression was downregulated in PCa tissues, and was even lower in PCa tissues with a Gleason score ≥8. Overexpression of miRNA-302a induced G1/S cell cycle arrest in PCa cells, and suppressed PCa cell proliferation both in vitro and in vivo. Furthermore, miRNA-302a inhibits AKT expression by directly binding to its 3΄ untranslated region, resulting in subsequent alterations of the AKT-GSK3β-cyclin D1 and AKT-p27Kip1 pathway. These results reveal miRNA-302a as a tumor suppressor in PCa, suggesting that miRNA-302a may be used as a potential target for therapeutic intervention in PCa.

MP37-07 UP REGULATION OF AMINOACID TRANSPORTER LAT1 DURING ANDROGEN DEPRIVATION THERAPY CONTRIBUTES TO ACQUISITION OF CASTRATION RESISTANCE IN PROSTATE CANCER CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research I1 Apr 2015MP37-07 UP REGULATION OF AMINOACID TRANSPORTER LAT1 DURING ANDROGEN DEPRIVATION THERAPY CONTRIBUTES TO ACQUISITION OF CASTRATION RESISTANCE IN PROSTATE CANCER CELLS Min Hui Xu, Shinichi Sakamoto, Shuhei Kamda, Mayuko Kato, Akira Kurozumi, Rika Nishikawa, Yusuke Goto, Miki Fuse, Yoshikatu Kanai, Naohiko Seki, and Tomohiko Ichikawa Min Hui XuMin Hui Xu More articles by this author , Shinichi SakamotoShinichi Sakamoto More articles by this author , Shuhei KamdaShuhei Kamda More articles by this author , Mayuko KatoMayuko Kato More articles by this author , Akira KurozumiAkira Kurozumi More articles by this author , Rika NishikawaRika Nishikawa More articles by this author , Yusuke GotoYusuke Goto More articles by this author , Miki FuseMiki Fuse More articles by this author , Yoshikatu KanaiYoshikatu Kanai More articles by this author , Naohiko SekiNaohiko Seki More articles by this author , and Tomohiko IchikawaTomohiko Ichikawa More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1270AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES L-type amino acid transporter 1(LAT1) transports essential amino acids, including leucine, which trigger the downstream m-TOR pathway. In a previous analysis, we identified up-regulation of LAT1 in androgen receptor (AR)-negative cells. We hypothesized that expression of LAT1 may be regulated by AR. This study examined 1) the association between AR and LAT1 expression, and 2) contribution of LAT1 in acquisition of castration resistance. METHODS Western blotting and real-time PCR were used to study protein and mRNA expressions. SiRNA was used to knockdown target genes. A total of 155 radical prostatectomy and prostate biopsy samples was used for immunohistochemical analyses. Cox hazard proportional models and the Kaplan-Meier method were used for statistical analysis. RESULTS LAT1 expresion was low in LNCaP cells but high in C4-2 and PC-3 cells. Knockdown of LAT1 in C4-2 cells significantly suppressed cell proliferation, migration, and invasion. Knockdown of LAT1 inhibited m-TOR related phosphorylation of p-70S6K and 4EBP-1. SiRNA of AR or blocking of AR through MDV3100(10µM) significantly increased LAT1 expression (P<0.01), while treatment with DHT (10nM) reduced LAT1 expression (P<0.05). MDV3100 plus SiLAT1 synergistically suppressed prostate cancer cell proliferation compared to single inhibition of AR or LAT1 (P<0.001). On immunohistochemical analysis, LAT1 expression was significantly higher in prostate cancer (PC) than in normal gland (P<0.001). LAT1 expression correlated with pathological stage (P=0.038) and Gleason Score (P=0.04). On Kaplan-Meier analysis, the high LAT1 group showed significantly shorter time to castration-resistant PC (CRPC) than the low LAT1 group after initiation of androgen deprivation therapy (ADT) (P=0.0002). When assessing the risk of castration resistance, lymph node metastasis (HR 4.29, P=0.0.072), bone metastasis (HR 6.63, P=0.0363), and LAT1 expression (HR 10.1, P=0.0274) were independent predictors on multivariate analysis. CONCLUSIONS During ADT, blocking of AR signaling up-regulated LAT1 expression and activated the downstream m-TOR pathway, contributing to CRPC. Dual inhibition of AR and LAT1 may be therapeutically useful in CRPC patients. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e440 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Min Hui Xu More articles by this author Shinichi Sakamoto More articles by this author Shuhei Kamda More articles by this author Mayuko Kato More articles by this author Akira Kurozumi More articles by this author Rika Nishikawa More articles by this author Yusuke Goto More articles by this author Miki Fuse More articles by this author Yoshikatu Kanai More articles by this author Naohiko Seki More articles by this author Tomohiko Ichikawa More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Monoamine oxidase A inhibitor-near-infrared dye conjugate reduces prostate tumor growth.

Development of anti-cancer agents with high tumor-targeting specificity and efficacy is critical for modern multidisciplinary cancer research. Monoamine oxidase A (MAOA), a mitochondria-bound enzyme, degrades monoamine neurotransmitters and dietary monoamines. Recent evidence suggests a correlation between increased MAOA expression and prostate cancer (PCa) progression with poor outcomes for patients. MAOA induces epithelial-mesenchymal transition (EMT) and augments hypoxic effects by producing excess reactive oxygen species. Thus, development of MAOA inhibitors which selectively target tumors becomes an important goal in cancer pharmacology. Here we describe the design, synthesis, and in vitro and in vivo evaluation of NMI, a conjugate that combines a near-infrared dye for tumor targeting with the moiety derived from the MAOA inhibitor clorgyline. NMI inhibits MAOA with low micromolar IC50, suppresses PCa cell proliferation and colony formation, and reduces migration and invasion. In mouse PCa xenografts, NMI targets tumors with no detectable accumulation in normal tissues, providing effective reduction of the tumor burden. Analysis of tumor specimens shows reduction in Ki-67(+) and CD31(+) cells, suggesting a decrease of cell proliferation and angiogenesis and an increase in M30(+) cells, indicating increased apoptosis. Gene expression profiles of tumors treated with NMI demonstrate reduced expression of oncogenes FOS, JUN, NFKB, and MYC and cell cycle regulators CCND1, CCNE1, and CDK4/6, along with increases in the levels of tumor suppressor gene TP53, cell cycle inhibitors CDKN1A and CDKN2A, and MAOA-downstream genes that promote EMT, tumor hypoxia, cancer cell migration, and invasion. These data suggest that NMI exerts its effect through tumor-targeted delivery of a MAOA-inactivating group, making NMI a valuable anti-tumor agent.

MicroRNA-224 and its target CAMKK2 synergistically influence tumor progression and patient prognosis in prostate cancer.

We previously demonstrated that microRNA (miR)-224 expression was significantly reduced in human prostate cancer (PCa) tissues and predicted unfavorable prognosis in patients. However, the underlying mechanisms of miR-224 have not been fully elucidated. In this study, calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) was identified as a target gene of miR-224. Then, we found that enforced expression of miR-224 could suppress PCa cell proliferation and cell cycle by regulating the expression of CAMKK2 in vitro. In addition, the expression levels of miR-224 in PCa tissues were negatively correlated with those of CAMKK2 mRNA significantly (Spearman's correlation: r = -0.66, P = 0.004). Moreover, combined low miR-224 expression and high CAMKK2 expression (miR-224-low/CAMKK2-high) was closely correlated with advanced clinical stage (P = 0.028). Furthermore, PCa patients with miR-224-low/CAMKK2-high expression more frequently had shorter overall survival than those in groups with other expression patterns of two molecules. In conclusion, our data offer the convincing evidence that miR-224 and its target gene CAMKK2 may synergistically contribute to the malignant progression of PCa. Combined detection of miR-224 and CAMKK2 expressions represents an efficient predictor of patient prognosis and may be a novel marker which can provide additional prognostic information in PCa.