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Prostate cancer is one of the most prevalent cancers in males and ranks as 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 c-Jun-NH2-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/mitochondria–dependent apoptosis in prostate cancer cells. Hrk mRNA and protein are preferentially upregulated by 2-ME, and Hrk induction is dependent on the 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. Involvement of the proapoptotic protein Bak in this process suggested the possible interaction between Hrk and Bak. Thus, Hrk activation by 2-ME or its overexpression displaced Bak from the complex with antiapoptotic protein Bcl-xL, whereas deletion of the Hrk BH3 domain abolished its interaction with Bcl-xL, reducing the proapoptotic function of Hrk. Finally, Hrk is also involved in the 2-ME–mediated reduction of X-linked inhibitor of apoptosis 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 proapoptotic protein Bak. Mol Cancer Ther; 12(6); 1049–59. ©2013 AACR.

PDF file - 321K, Figure S1. Induction of BH3-only protein family members by 2-ME in prostate cancer cells. Figure S2. Hrk knockdown prevents 2-ME-mediated cell death in time-dependent manner. Figure S3. Mitochondrial localization of Hrk. Figure S4. No change in total cell number after 2-ME treatment. Figure S5. Bax is not involved in 2-ME-mediated apoptotic cell death. Figure S6. Endogenous Bak interacts with Bcl-xL but not with Bcl-2 in prostate cancer cells. Figure S7. Hrk requires the BH3 domain for pro-apoptotic function.

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PDF file - 76K, Supplemental Table 1. Clinicopathologic characteristics of prostate cancer patients. Supplemental Table 2. Complimentary sequences in 3'UTRs of genes for miR-34b and sequences of primers and probes.

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PDF file - 325K, Supplemental Figure 1: Downregulation of miR-34b expression, its potential diagnostic and prognostic significance and methylation status in PCa. A) Box plot representation of mir-34b expression in a cohort of unmatched tissue samples. B) Box plot representation of ISH results of tissue samples. (Ave- Average; T/N- Tumor/Normal). C) Chi-square test showing correlation of clinicopathological characteristics with miR-34b expression. D) CpG islands within the 1.0 kb region upstream of the miR-34b gene. Arrow indicates the start of the miR-34b gene. Locations for primers and probes are also shown on the miR-34b gene. F- Forward, R-Reverse, M/U Methylation and Unmethylation probes, CF1, CF2, CR1, CR2- ChiP Forward1-2, ChIP Reverse 1-2. E) Correlation of miR-34b expression with average percent methylation indicating cases with low miR-34b expression have higher methylation and vice versa.

PDF file - 141K, miR-23b directly targets Akt in prostate cancer

PDF file - 65K, Clinicopathological characteristics of patients and their correlation with miR-23b

PDF file - 967K, Depletion of Src kinase by an additional siRNA sequence

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Supplementary Figure 4 from MicroRNA-205 Inhibits Src-Mediated Oncogenic Pathways in Renal Cancer

Supplementary Figure Legends 1-4 from MicroRNA-205 Inhibits Src-Mediated Oncogenic Pathways in Renal Cancer

PDF file - 567K, miR-708 induces morphological changes indicative of increased apoptosis in PCa cell lines

Tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) is cytotoxic to a wide variety of transformed cells, but not to most normal cells, implying potential therapeutic value against advanced cancer. However, signal transduction in TRAIL-mediated apoptosis is not clearly understood compared with other TNF family members. Specifically, it is not yet understood how TRAIL controls nuclear factor κB (NF-κB) activation and overcomes its antiapoptotic effect. We explored the regulation of NF-κB activity by TRAIL and its role in apoptosis. TRAIL combined with IκBα-“superrepressor” induced potent apoptosis of SK-Hep1 hepatoma cells at low concentrations of TRAIL that do not independently induce apoptosis. Apoptosis by high concentrations of TRAIL was not affected by IκBα-superrepressor. Although TRAIL alone did not induce NF-κB activity, TRAIL combined with z-VAD significantly increased NF-κB activation. Analysis of the NF-κB activation pathway indicated that TRAIL unexpectedly induced cleavage of p65 at Asp97, which was blocked by z-VAD, accounting for all of these findings. p65 expression abrogated apoptosis and increased NF-κB activity in TRAIL-treated cells. Cleavage-resistant p65D97A further increased NF-κB activity in TRAIL-treated cells, whereas the COOH-terminal p65 fragment acted as a dominant-negative inhibitor. XIAP levels were increased by TRAIL in combination with z-VAD, whereas XIAP levels were decreased by TRAIL alone. Cleavage of p65 was also detected after FRO thyroid cancer cells were treated with TRAIL. These results suggest that TRAIL induces NF-κB activation, but simultaneously abrogates NF-κB activation by cleaving p65, and thereby inhibits the induction of antiapoptotic proteins such as XIAP, which contributes to the strong apoptotic activity of TRAIL compared with other TNF family members.

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Supplementary Figures 1-2 from Caspase-Mediated p65 Cleavage Promotes TRAIL-Induced Apoptosis

PDF file - 145K, AKT2 expression in CD44-ve and CD44+ve subpopulations of PC3 xenografts

Supplementary Figure 3 from MicroRNA-205 Inhibits Src-Mediated Oncogenic Pathways in Renal Cancer

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Supplementary Figure Legends from Caspase-Mediated p65 Cleavage Promotes TRAIL-Induced Apoptosis

Supplementary Figure 2 from MicroRNA-205 Inhibits Src-Mediated Oncogenic Pathways in Renal Cancer

PDF file - 73K, Clinicopathologic characteristics of prostate cancer patients

PDF file - 91K, Correlation of miR-708 expression with serum PSA levels

Supplementary Figure 1 from MicroRNA-205 Inhibits Src-Mediated Oncogenic Pathways in Renal Cancer

Supplementary Table 1 from MicroRNA-205 Inhibits Src-Mediated Oncogenic Pathways in Renal Cancer

PDF file - 402K, In situ hybridization analysis of miR-708 expression in PCa tissues

Cytochrome P450 (CYP) 1A1 is a phase I enzyme that can activate various compounds into reactive forms and thus, may contribute to carcinogenesis. In this study, we investigated the expression, methylation status, and functional role of CYP1A1 on prostate cancer cells. Increased expression of CYP1A1 was observed in all cancer lines (PC-3, LNCaP, and DU145) compared to BPH-1 (P < 0.05); and was enhanced further by 5-aza-2′-deoxycytidine treatment (P < 0.01). Methylation-specific PCR (MSP) and sequencing of bisulfite-modified DNA of the xenobiotic response element (XRE) enhancer site XRE-1383 indicated promoter methylation as a regulator of CYP1A1 expression. In tissue, microarrays showed higher immunostaining of CYP1A1 in prostate cancer than normal and benign prostatic hyperplasia (BPH; P < 0.001), and methylation analyses in clinical specimens revealed significantly lower methylation levels in cancer compared to BPH at all enhancer sites analyzed (XRE-1383, XRE-983, XRE-895; P < 0.01). Interestingly, smoking affected the XRE-1383 site where the methylation level was much lower in cancer tissues from smokers than non-smokers (P < 0.05). CYP1A1 levels are thus increased in prostate cancer and to determine the functional effect of CYP1A1 on cells, we depleted the gene in LNCaP and DU145 by siRNA. We observe that CYP1A1 knockdown decreased cell proliferation (P < 0.05) and increased apoptosis (P < 0.01) in both cell lines. We analyzed genes affected by CYP1A1 silencing and found that apoptosis-related BCL2 was significantly down-regulated. This study supports an oncogenic role for CYP1A1 in prostate cancer via promoter hypomethylation that is influenced by tobacco smoking, indicating CYP1A1 to be a promising target for prostate cancer treatment.

Cytochrome P450 1B1 (CYP1B1) is recognized as a universal tumor biomarker and a feasible therapeutic target due to its specific overexpression in cancer tissues. Despite its up-regulation in prostate cancer (PCa), biological significance and clinicopathological features of CYP1B1 are still elusive. Here, we show that overexpression or hyperactivation of CYP1B1 stimulated proliferative, migratory and invasive potential of non-tumorigenic PCa cells. Attenuation of CYP1B1 with its specific small hairpin (sh) RNAs greatly reduced proliferation through apoptotic cell death and impaired migration and invasion in PCa cells. Intratumoral injection of CYP1B1 shRNA attenuated growth of pre-existing tumors. The antitumor effect of CYP1B1 shRNA was also observed in prostate tumor xenograft mouse models. Among the genes altered by CYP1B1 knockdown, reduction of caspase-1 (CASP1) activity attenuated the antitumor effect of CYP1B1 inhibition. Indeed, CYP1B1 regulates CASP1 expression or activity. Finally, CYP1B1 expression was increased in higher grades of PCa and overall survival was significantly reduced in patients with high levels of CYP1B1 protein. CYP1B1 expression was reversely associated with CASP1 expression in clinical tissue samples. Together, our results demonstrate that CYP1B1 regulates PCa tumorigenesis by inhibiting CASP1 activation. Thus, the CYP1B1-CASP1 axis may be useful as a potential biomarker and a therapeutic target for PCa.

Cytochrome P450 1B1 (CYP1B1) converts xenobiotics to carcinogens and how lifestyle choices may interact with CYP1B1 polymorphisms and affect prostate cancer risk was assessed. Blood genomic DNA from a Caucasian population was analysed at polymorphic sites of the 5′ untranslated region of CYP1B1 using TaqMan genotyping assays. Overall, drinker status and minor alleles at rs2551188, rs2567206 and rs10175368 were associated with prostate cancer. Linkage was observed between rs2551188, rs2567206, rs2567207 and rs10175368, and the G‐C‐T‐G haplotype (major allele at respective sites) was decreased in cancer. Interestingly when classified by lifestyle factors, no associations of genotypes were found for non‐smokers and non‐drinkers, whereas on the contrary, minor type at rs2567206 and rs10175368 increased and major G‐C‐T‐G decreased risk for cancer among smokers and drinkers. Interestingly, rs2551188, rs2567206 and rs10175368 minor genotypes correlated with increased tissue CYP1B1 as determined by immunohistochemistry. Further, rs10175368 enhanced luciferase activity and mobility shift show stronger binding of nuclear factor for the minor allele. These results demonstrate smoking and alcohol consumption to modify the risks of CYP1B1 polymorphisms for prostate cancer which may be through rs10175368, and this is of importance in understanding their role in the pathogenesis and as a biomarker for this disease.

// Shinichiro Fukuhara 1, 2, 3, * , Inik Chang 1, 4, * , Yozo Mitsui 1, 2, 5 , Takeshi Chiyomaru 1, 2, 6 , Soichiro Yamamura 1, 2 , Shahana Majid 1, 2 , Sharanjot Saini 1, 2 , Guoren Deng 1, 2 , Ankurpreet Gill 1 , Darryn K. Wong 1 , Hiroaki Shiina 5 , Norio Nonomura 3 , Yun-Fai C. Lau 7, 8 , Rajvir Dahiya 1, 2 , Yuichiro Tanaka 1, 2 1 Department of Surgery/Urology, Veterans Affairs Medical Center, San Francisco, California 94121, United States of America 2 Department of Urology, University of California, San Francisco, California 94121, United States of America 3 Department of Urology, Osaka University Graduate School of Medicine, Suita 565-0871, Japan 4 Department of Oral Biology, Yonsei University College of Dentistry, Seoul 120-752, South Korea 5 Department of Urology, Shimane University Faculty of Medicine, Izumo 693-8501, Japan 6 Department of Urology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima 890-8544, Japan 7 Department of Medicine, Veterans Affairs Medical Center, San Francisco, California 94121, United States of America 8 Institute for Human Genetics, University of California, San Francisco, California 94121, United States of America * These authors have contributed equally to this work Correspondence to: Yuichiro Tanaka, e-mail: yuichiro.tanaka@ucsf.edu Keywords: PMS2, apoptosis, prostate cancer Received: January 02, 2015 Accepted: April 24, 2015 Published: May 06, 2015 ABSTRACT DNA mismatch repair (MMR) enzymes act as proofreading complexes that maintains genomic integrity and MMR-deficient cells show an increased mutation rate. MMR has also been shown to influence cell signaling and the regulation of tumor development. MMR consists of various genes and includes post-meiotic segregation (PMS) 2 which is a vital component of mutL-alpha. In prostate, the functional role of this gene has never been reported and in this study, our aim was to investigate the effect of PMS2 on growth properties of prostate cancer (PCa) cells. Previous studies have shown PMS2 to be deficient in DU145 cells and this lack of expression was confirmed by Western blotting whereas normal prostatic PWR-1E and RWPE-1 cells expressed this gene. PMS2 effects on various growth properties of DU145 were then determined by creating stable gene transfectants. Interestingly, PMS2 caused decreased cell proliferation, migration, invasion, and in vivo growth; and increased apoptosis as compared to vector control. We further analyzed genes affected by PMS2 expression and observe the apoptosis-related TMS1 gene to be significantly upregulated whereas anti-apoptotic BCL2A1 was downregulated. These results demonstrate a functional role for PMS2 to protect against PCa progression by enhancing apoptosis of PCa cells.

Objective The oral cavity is often exposed to not only diverse external pathogens but also dramatic temperature changes. In this study, we investigated the effect of thermal stress on PDL cells with a focus on the inflammatory responses and bone homeostasis. Design The PDL cells were isolated from healthy premolar extracted for orthodontic reasons, and examined using intracellular calcium concentration ([Ca2+]i) measurement and reverse transcription-polymerase chain reaction for pro-inflammatory cytokines and bone remodelling mediators. Results We detected the expression of thermosensitive transient receptor potential (TRP) channels, such as TRPV1, TRPV2, TRPV3, TRPM8, and TRPA1. Functional activation of the channels by thermal stress and their specific agonists increased [Ca2+]i and interleukin (IL)-6 and IL-8 mRNA expression. A selective Ca2+ chelator, BAPTA-AM, prevented TRP channel agonists-mediated IL-6 and IL-8 induction. Unlike pro-inflammatory cytokines, the expression of bone remodelling mediators, including receptor activator of nuclear factor-kappa B ligand and osteoprotegerin, was not altered by treatment with TRP channel agonists. Conclusions The activation of thermosensitive TRP channels induced IL-6 and IL-8 expression by increasing [Ca2+]i in human PDL cells. Therefore, thermal stress may play a critical role in the inflammatory responses of PDL cells.

Despite high protein expression and enzymatic activity of cytochrome P450 1B1 (CYP1B1) in renal cell cancer (RCC), its functional significance has not been elucidated. Here we explored the functional role and regulatory mechanism of CYP1B1 in RCC. Reduction of CYP1B1 levels fail to prevent in vitro tumorigenicity such as proliferation, apoptosis, and cell cycle progression of RCC cells. Moreover, the expression levels are not associated with tumor type, stage, Fuhrman grade and 5-year survival probability after surgery. Instead, alteration of CYP1B1 expression regulates the chemosensitivity of RCC cells to docetaxel suggesting its critical contribution to the chemoresistance. Additionally, miR-200c, which is significantly down-regulated in RCC regulates CYP1B1 expression and activity. An inverse association was also observed between the expression levels of miR-200c and CYP1B1 protein in RCC tissues. Finally, alteration of miR-200c levels affects the chemosensitivity of RCC cells. Restoration of docetaxel resistance by exogenous expression of CYP1B1 in miR-200c-over-expressing cells indicates that CYP1B1 is a functional target of miR-200c. These results suggest that CYP1B1 up-regulation mediated by low miR-200c is one of the mechanisms underlying resistance of RCC cells to docetaxel. Therefore, expression of CYP1B1 and miR-200c in RCC may be useful as a prediction for docetaxel response.

Mismatch repair (MMR) enzymes have been shown to be deficient in prostate cancer (PCa). MMR can influence the regulation of tumor development in various cancers but their role on PCa 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 PCa cells. The DU145 cell line has been established as MLH1-deficient and thus, this cell line was utilized to determine effects of MLH1 by gene expression. Lack of MLH1 protein expression was confirmed by Western blotting in DU145 cells whereas levels were high in normal PWR-1E and RWPE-1 prostatic cells. MLH1-expressing stable transfectant DU145 cells were then created to characterize the effects this MMR gene has on various growth properties. Expression of MLH1 resulted in decreased cell proliferation, migration and invasion properties. Lack of cell growth in vivo 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 STI571 also abrogated the effect on apoptosis caused by MLH1. These results demonstrate MLH1 protects against PCa development by inducing c-Abl-mediated apoptosis.

Homer proteins are scaffold molecules with a domain structure consisting of an N-terminal Ena/VASP homology 1 protein-binding domain and a C-terminal leucine zipper/coiled-coil domain. The Ena/VASP homology 1 domain recognizes proline-rich motifs and binds multiple Ca2+-signaling proteins, including G protein-coupled receptors, inositol 1,4,5-triphosphate receptors, ryanodine receptors, and transient receptor potential channels. However, their role in Ca2+ signaling in nonexcitable cells is not well understood. In this study, we investigated the role of Homer2 on Ca2+ signaling in parotid gland acinar cells using Homer2-deficient (Homer2−/−) mice. Homer2 is localized at the apical pole in acinar cells. Deletion of Homer2 did not affect inositol 1,4,5-triphosphate receptor localization or channel activity and did not affect the expression and activity of sarco/endoplasmic reticulum Ca2+-ATPase pumps. In contrast, Homer2 deletion markedly increased expression of plasma membrane Ca2+-ATPase (PMCA) pumps, in particular PMCA4, at the apical pole. Accordingly, Homer2 deficiency increased Ca2+ extrusion by acinar cells. These findings were supported by co-immunoprecipitation of Homer2 and PMCA in wild-type parotid cells and transfected human embryonic kidney 293 (HEK293) cells. We identified a Homer-binding PPXXF-like motif in the N terminus of PMCA that is required for interaction with Homer2. Mutation of the PPXXF-like motif did not affect the interaction of PMCA with Homer1 but inhibited its interaction with Homer2 and increased Ca2+ clearance by PMCA. These findings reveal an important regulation of PMCA by Homer2 that has a central role on PMCA-mediated Ca2+ signaling in parotid acinar cells.

The cytochrome P450 1B1 (CYP1B1) enzyme activates xenobiotics to reactive forms as well as convert estradiol to 4-hydroxy-estradiol that has been shown to play a role in the carcinogenesis process of the kidney in male but not female animals. Prior reports show polymorphic variants of CYP1B1 to alter catalytic activity, and thus, we hypothesize that polymorphisms of the CYP1B1 gene are involved in the malignant transformation of the renal cell in men. The genetic distributions of five CYP1B1 polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism in 480 normal healthy subjects and 403 sporadic renal cell carcinoma cases. All subjects were Caucasian men. The sites evaluated were codons 48 (C → G, Arg → Gly, rs10012), 119 (G → T, Ala → Ser, rs1056827), 432 (C → G, Leu → Val, rs1056836), 449 (C → T, Asp, rs1056837), and 453 (A → G, Asn → Ser, rs1800440). A trend was demonstrated for the 432 Val/Val (χ2, P = 0.06) and 449 T/T (χ2, P = 0.1) genotypes to play a protective role against renal cancer. Odds ratio (95 % confidence interval) for Val/Val compared to Leu/Leu at codon 432 was 0.65 (0.44-0.95) and T/T compared to C/C at codon 449 was 0.67 (0.45-0.99). Codons 432 and 449 were observed to be linked (D = 0.24), and haplotype involving 432 Val and 449 T was significantly reduced in cancer cases (P = 0.04). No association was found, however, when analyzing polymorphic sites with clinical stage of cancer. These results demonstrate polymorphisms of CYP1B1 to be associated with renal carcinogenesis and are of importance in understanding their role in the pathogenesis of renal cell carcinoma.

Prostate cancer is one of the most prevalent cancers in males and ranks as 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 c-Jun-NH2-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/mitochondria–dependent apoptosis in prostate cancer cells. Hrk mRNA and protein are preferentially upregulated by 2-ME, and Hrk induction is dependent on the 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. Involvement of the proapoptotic protein Bak in this process suggested the possible interaction between Hrk and Bak. Thus, Hrk activation by 2-ME or its overexpression displaced Bak from the complex with antiapoptotic protein Bcl-xL, whereas deletion of the Hrk BH3 domain abolished its interaction with Bcl-xL, reducing the proapoptotic function of Hrk. Finally, Hrk is also involved in the 2-ME–mediated reduction of X-linked inhibitor of apoptosis 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 proapoptotic protein Bak. Mol Cancer Ther; 12(6); 1049–59. ©2013 AACR.