Role of Exosomes in Prostate Cancer Metastasis.

Prostate cancer remains a life-threatening disease among men worldwide. The majority of PCa-related mortality results from metastatic disease that is characterized by metastasis of prostate tumor cells to various distant organs, such as lung, liver, and bone. Bone metastasis is most common in prostate cancer with osteoblastic and osteolytic lesions. The precise mechanisms underlying PCa metastasis are still being delineated. Intercellular communication is a key feature underlying prostate cancer progression and metastasis. There exists local signaling between prostate cancer cells and cells within the primary tumor microenvironment (TME), in addition to long range signaling wherein tumor cells communicate with sites of future metastases to promote the formation of pre-metastatic niches (PMN) to augment the growth of disseminated tumor cells upon metastasis. Over the last decade, exosomes/ extracellular vesicles have been demonstrated to be involved in such signaling. Exosomes are nanosized extracellular vesicles (EVs), between 30 and 150 nm in thickness, that originate and are released from cells after multivesicular bodies (MVB) fuse with the plasma membrane. These vesicles consist of lipid bilayer membrane enclosing a cargo of biomolecules, including proteins, lipids, RNA, and DNA. Exosomes mediate intercellular communication by transferring their cargo to recipient cells to modulate target cellular functions. In this review, we discuss the contribution of exosomes/extracellular vesicles in prostate cancer progression, in pre-metastatic niche establishment, and in organ-specific metastases. In addition, we briefly discuss the clinical significance of exosomes as biomarkers and therapeutic agents.

Role of the PI3K/Akt pathway in cadmium induced malignant transformation of normal prostate epithelial cells.

This study investigated the role of the PI3K/Akt pathway in cadmium (Cd) induced malignant transformation of normal prostate epithelial (PWR1E and RWPE1) cells. Both PWR1E and RWPE1 cells were exposed to 10 µM Cd for one year and designated as Cd-PWR1E and Cd-RWPE1. Cd-RWPE1 cells robustly formed tumors in athymic nude mice. Functionally, Cd-exposure induced tumorigenic attributes indicated by increased wound healing, migration and invasion capabilities in both cell lines. RT2-array analysis revealed many oncogenes including P110α, Akt, mTOR, NFKB1 and RAF were induced whereas tumor suppressor (TS) genes were attenuated in Cd-RWPE1. This was validated by individual quantitative-real-time-PCR at transcriptional and by immunoblot at translational levels. These results were consistent in Cd-PWR1E vs parental PWR1E cells. Gene Set Enrichment Analysis revealed that five prostate cancer (PCa) related pathways were enriched in Cd-exposed cells compared to their normal controls. These pathways include the KEGG- Pathways in cancer, Prostate Cancer Pathway, ERBB, Apoptosis and MAPK pathways. We selected up- and down-regulated genes randomly from the PI3K/Akt pathway array and profiled these in the TCGA/GDC prostate-adenocarcinoma (PRAD) patient cohort. An upregulation of oncogenes and downregulation of TS genes was observed in PCa compared to their normal controls. Taken together, our study reveals that the PI3K/Akt signaling is one of the main molecular pathways involved in Cd-driven transformation of normal prostate epithelial cells to malignant form. Understanding the molecular mechanisms involved in the Cd-driven malignant transformation of normal prostate cells will provide a significant insight to develop better therapeutic strategies for Cd-induced prostate cancer.

Role of a novel race-related tumor suppressor microRNA located in frequently deleted chromosomal locus 8p21 in prostate cancer progression.

The prostate cancer (PCa) genome is characterized by deletions of chromosome 8p21-22 region that increase significantly with tumor grade and are associated with poor prognosis. We proposed and validated a novel, paradigm-shifting hypothesis that this region is associated with a set of microRNA genes-miR-3622, miR-3622b, miR-383-that are lost in PCa and play important mechanistic roles in PCa progression and metastasis. Extending our hypothesis, in this study, we evaluated the role of a microRNA gene located in chromosome 8p-miR-4288-by employing clinical samples and cell lines. Our data suggests that (i) miR-4288 is widely downregulated in primary prostate tumors and cell lines; (ii) miR-4288 expression is lost in metastatic castration-resistant PCa; (ii) miR-4288 downregulation is race-related PCa alteration that is prevalent in Caucasian patients and not in African Americans; (iii) in Caucasians, miR-4288 was found to be associated with increasing tumor grade and high serum prostate-specific antigen, suggesting that miR-4288 downregulation/loss may be associated with tumor progression specifically in Caucasians; (iv) miR-4288 possess significant potential as a molecular biomarker to predict aggressiveness/metastasis; and (v) miR-4288 is anti-proliferative, is anti-invasive and inhibits epithelial-to-mesenchymal transition; and (vi) miR-4288 directly represses expression of metastasis/invasion-associated genes MMP16 and ROCK1. Thus, the present study demonstrates a tumor suppressor role for a novel miRNA located with a frequently lost region in PCa, strengthening our hypothesis that this locus is causally related to PCa disease progression via loss of microRNA genes. Our study suggests that miR-4288 may be a novel biomarker and therapeutic target, particularly in Caucasians.

Influence of lifestyle choices on risks of CYP1B1 polymorphisms for prostate cancer.

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.

MicroRNAs as Regulators of Prostate Cancer Metastasis.

Prostate cancer causes significant morbidity in men and metastatic disease is a major cause of cancer related deaths. Prostate metastasis is controlled by various cellular intrinsic and extrinsic factors, which are often under the regulatory control of various metastasis-associated genes. Given the dynamic nature of metastatic cancer cells, the various factors controlling this process are themselves regulated by microRNAs which are small non-coding RNAs. Significant research work has shown differential microRNA expression in primary and metastatic prostate cancer suggesting their importance in prostate pathogenesis. We will review the roles of different microRNAs in controlling the various steps in prostate metastasis.

Long non-coding RNA HOTAIR is targeted and regulated by miR-141 in human cancer cells.

HOTAIR is a long non-coding RNA that 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 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 human cancer 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 Ago2 (Argonaute2) 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.

Cytochrome P450 1B1 polymorphisms and risk of renal cell carcinoma in men.

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.

A novel exosome based therapeutic intervention against neuroendocrine prostate cancer.

Neuroendocrine prostate cancer (NEPC) is a highly lethal variant of castration-resistant prostate cancer (CRPC) with poor survival rates. Current treatment options for NEPC are limited to highly toxic platinum drugs highlighting the urgent need for new therapies. This study aimed to develop a novel therapeutic approach using engineered exosomes against NEPC. Exosomes were modified to target CEACAM5, an NEPC surface antigen, by attaching CEACAM5 antibodies to HEK293T exosomes. These exosomes were loaded with drugs inhibiting EZH2 and the androgen receptor (AR) as recent research shows a persistent role of AR in NEPC wherein it plays a concerted role with EZH2 in driving neuronal gene programs. In vitro experiments with NEPC cell lines demonstrated that CEACAM5-targeted exosomes were specifically taken up by NEPC cells, leading to reduced cellular viability and decreased expression of neuronal markers. Further in vivo tests using a NEPC patient-derived xenograft model (LuCaP145.1) showed significant tumor regression in mice treated with engineered exosomes compared to control mice receiving IgG-labeled exosomes. These results suggest that CEACAM5-engineered exosomes hold promise as a targeted therapy for NEPC. Importantly, our exosome engineering strategy is versatile and can be adapted to target various surface antigens in prostate cancer and other diseases.

Role of transcription factors and chromatin modifiers in driving lineage reprogramming in treatment-induced neuroendocrine prostate cancer.

Therapy-induced neuroendocrine prostate cancer (NEPC) is a highly lethal variant of prostate cancer that is increasing in incidence with the increased use of next-generation of androgen receptor (AR) pathway inhibitors. It arises via a reversible trans-differentiation process, referred to as neuroendocrine differentiation (NED), wherein prostate cancer cells show decreased expression of AR and increased expression of neuroendocrine (NE) lineage markers including enolase 2 (ENO2), chromogranin A (CHGA) and synaptophysin (SYP). NEPC is associated with poor survival rates as these tumors are aggressive and often metastasize to soft tissues such as liver, lung and central nervous system despite low serum PSA levels relative to disease burden. It has been recognized that therapy-induced NED involves a series of genetic and epigenetic alterations that act in a highly concerted manner in orchestrating lineage switching. In the recent years, we have seen a spurt in research in this area that has implicated a host of transcription factors and epigenetic modifiers that play a role in driving this lineage switching. In this article, we review the role of important transcription factors and chromatin modifiers that are instrumental in lineage reprogramming of prostate adenocarcinomas to NEPC under the selective pressure of various AR-targeted therapies. With an increased understanding of the temporal and spatial interplay of transcription factors and chromatin modifiers and their associated gene expression programs in NEPC, better therapeutic strategies are being tested for targeting NEPC effectively.

Therapeutic potential of pomegranate juice-derived nanovesicles in nude mouse benign prostatic hyperplasia (BPH) xenograft model.

Benign prostatic hyperplasia (BPH) and associated lower urinary tract symptoms affect a large percentage of the male population and places a substantial burden on the world health system. Current therapies include 5-alpha reductase inhibitors and alpha-blockers that are only partially effective and pose a huge economic burden, emphasizing the urgent need for effective, economical therapies. We isolated nanovesicles from pomegranate juice (Punica Granatum) (referred to as 'POM-NVs') and report to our knowledge for the first time, that these vesicles possess therapeutic potential against BPH. Following extensive characterization of POM-NVs, we tested their therapeutic potential in vitro using BPH1 cell line and identified a potential anti-proliferative and pro-apoptotic effect. We further tested these vesicles using a clinically relevant xenograft mouse BPH model derived from human BPH tissues. Remarkably, POM-NVs could reverse the BPH phenotype conferred by TGF-ß mediated signaling and induced epithelial-to-mesenchymal (EMT) reversal, leading to the restoration of prostate epithelial states in vivo and in vitro. Furthermore, these vesicles attenuated bone morphogenic protein 5 (BMP5) signaling, a cardinal alteration that is instrumental in driving BPH. Considering the large incidences of BPH and its associated economic burdens, our study has important implications and can potentially improve the clinical management of BPH.

miR-410 Is a Key Regulator of Epithelial-to-Mesenchymal Transition with Biphasic Role in Prostate Cancer.

The molecular basis of prostate cancer (PCa) progression from the primary disease to metastatic castration-resistant prostate cancer (CRPC) followed by therapy-induced neuroendocrine prostate cancer is not fully understood. In this study, we elucidate the role of miR-410, a little-studied microRNA located on chromosome 14q32.31 within the DLK1-DIO3 cluster, in PCa. miR-410 expression analyses in primary and metastatic PCa tissues and cell lines show that its levels are decreased in initial stages and increased in advanced PCa. Functional studies were performed in a series of PCa cell lines. In LNCaP cells, miR-410 overexpression led to decreases in cellular viability, proliferation, invasiveness, and migration. On the other hand, miR-410 overexpression in PC3 and C42B cells led to increased viability, proliferation, and invasiveness. Our data suggest that miR-410 represses epithelial-to-mesenchymal transition (EMT) in LNCaP cells by directly repressing SNAIL. However, it promotes EMT and upregulates PI3K/Akt signaling in PC3 and C42B cells. In vivo studies with PC3 xenografts support an oncogenic role of miR-410. These data suggest that miR-410 acts as a tumor suppressor in the initial stages of PCa and play an oncogenic role in advanced PCa. Our findings have important implications in understanding the molecular basis of PCa progression with potential translational implications.

MicroRNA-34a suppresses malignant transformation by targeting c-Myc transcriptional complexes in human renal cell carcinoma.

We investigated the functional effects of microRNA-34a (miR-34a) on c-Myc transcriptional complexes in renal cell carcinoma. miR-34a down-regulated expression of multiple oncogenes including c-Myc by targeting its 3' untranslated region, which was revealed by luciferase reporter assays. miR-34a was also found to repress RhoA expression by suppressing the c-Myc-Skp2-Miz1 transcriptional complex that activates RhoA. Overexpression of c-Myc reversed miR-34a suppression of RhoA expression and inhibition of cell invasion, suggesting that miR-34a inhibits invasion by suppressing RhoA through c-Myc. miR-34a was also found to repress the c-Myc-P-TEFb transcription elongation complex, indicating one of the mechanisms by which miR-34a has profound effects on cellular functions. Our results demonstrate that miR-34a suppresses assembly and function of the c-Myc complex that activates or elongates transcription, indicating a novel role of miR-34a in the regulation of transcription by c-Myc.

Catechol-O-methyltransferase-mediated metabolism of 4-hydroxyestradiol inhibits the growth of human renal cancer cells through the apoptotic pathway.

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 (RCCs). We initially found that COMT levels are significantly decreased in human RCC tissues and cells suggesting it plays a suppressive role in tumor development. However, transient overexpression of COMT has no functional effect on RCC cell lines. In contrast, when cells overexpressing COMT are treated with its substrate 4-hydroxyestradiol (4-OHE(2)), growth is inhibited by apoptotic cell death. We also found that COMT overexpression combined with 4-OHE(2) induces upregulation of growth arrest- and DNA damage-inducible protein α (GADD45α). We further show that downregulation of GADD45α by a small interfering RNA-mediated approach inhibits cell death, indicating the essential role of GADD45α in the underlying mechanism of COMT action in response to 4-OHE(2). Finally, 4-methoxyestradiol fully reproduces the antiproliferative function of COMT with 4-OHE(2) by promoting GADD45α induction. Together, these findings show that COMT in the presence of 4-OHE(2) prevents RCC cell proliferation by enhancing apoptosis and that GADD45α plays a critical role in the COMT-mediated inhibition of RCC.

Role of Swi6/HP1 self-association-mediated recruitment of Clr4/Suv39 in establishment and maintenance of heterochromatin in fission yeast.

Swi6/HP1, an evolutionarily conserved protein, is critical for heterochromatin assembly in fission yeast and higher eukaryotes. In fission yeast, histone deacetylation by histone deacetylases is thought to be followed by H3-Lys-9 methylation by the histone methyltransferase Clr4/Suv39H1. H3-Lys-9-Me2 interacts with the chromodomain of Swi6/HP1. Swi6/HP1 is thought to act downstream of Clr4/Suv39, and further self-association of Swi6/HP1 is assumed to stabilize the heterochromatin structure. Here, we show that the self-association-defective mutant of Swi6 does not interact with Clr4. It not only fails to localize to heterochromatin loci but also interferes with heterochromatic localization of H3-Lys-9-Me2 (and thereby Clr4) and the endogenous Swi6 in a dominant negative manner. Thus, self-association of Swi6/HP1 helps in binding to and recruitment of Clr4 and thereby in establishment and maintenance of heterochromatin by a concerted rather than a sequential mechanism.

Wnt signaling pathways in urological cancers: past decades and still growing.

The Wnt signaling pathway is involved in a wide range of embryonic patterning events and maintenance of homeostasis in adult tissues. The pathological role of the Wnt pathway has emerged from studies showing a high frequency of specific human cancers associated with mutations that constitutively activate the transcriptional response of these pathways. Constitutive activation of the Wnt signaling pathway is a common feature of solid tumors and contributes to tumor development, progression and metastasis in various cancers. In this review, the Wnt pathway will be covered from the perspective of urological cancers with emphasis placed on the recent published literature. Regulation of the Wnt signaling pathway by microRNAs (miRNA), small RNA sequences that modify gene expression profiles will also be discussed. An improved understanding of the basic genetics and biology of Wnt signaling pathway will provide insights into the development of novel chemopreventive and therapeutic strategies for urological cancers.

Role of MicroRNAs in Neuroendocrine Prostate Cancer.

Therapy-induced neuroendocrine prostate cancer (t-NEPC/NEPC) is an aggressive variant of prostate cancer (PCa) that frequently emerges in castration-resistant prostate cancer (CRPC) under the selective pressure of androgen receptor (AR)-targeted therapies. This variant is extremely aggressive, metastasizes to visceral organs, tissues, and bones despite low serum PSA, and is associated with poor survival rates. It arises via a reversible trans-differentiation process, referred to as 'neuroendocrine differentiation' (NED), wherein PCa cells undergo a lineage switch and exhibit neuroendocrine features, characterized by the expression of neuronal markers such as enolase 2 (ENO2), chromogranin A (CHGA), and synaptophysin (SYP). The molecular and cellular mechanisms underlying NED in PCa are complex and not clearly understood, which contributes to a lack of effective molecular biomarkers for diagnosis and therapy of this variant. NEPC is thought to derive from prostate adenocarcinomas by clonal evolution. A characteristic set of genetic alterations, such as dual loss of retinoblastoma (RB1) and tumor protein (TP53) tumor suppressor genes and amplifications of Aurora kinase A (AURKA), NMYC, and EZH2, has been reported to drive NEPC. Recent evidence suggests that microRNAs (miRNAs) are important epigenetic players in driving NED in advanced PCa. In this review, we highlight the role of miRNAs in NEPC. These studies emphasize the diverse role that miRNAs play as oncogenes and tumor suppressors in driving NEPC. These studies have unveiled the important role of cellular processes such as the EMT and cancer stemness in determining NED in PCa. Furthermore, miRNAs are involved in intercellular communication between tumor cells and stromal cells via extracellular vesicles/exosomes that contribute to lineage switching. Recent studies support the promising potential of miRNAs as novel diagnostic biomarkers and therapeutic targets for NEPC.

Secreted frizzled-related protein-5 is epigenetically downregulated and functions as a tumor suppressor in kidney cancer.

Secreted frizzled-related protein-5 (sFRP-5) has been identified as 1 of the secreted antagonists that bind Wnt protein. However, the functional significance of sFRP-5 in renal cell cancer (RCC) has not been reported. We hypothesized that sFRP-5 may be epigenetically downregulated through DNA methylation and histone modification and function as a tumor suppressor gene in RCC. Using tissue microarray and real-time RT-PCR, we found that sFRP-5 was significantly downregulated in kidney cancer tissues and cell lines, respectively. DNA bisulfite sequencing of the sFRP-5 promoter region in RCC cell lines showed it to be densely methylated, whereas there was few promoter methylation in normal kidney. The sFRP-5 expression was restored and the acetylation of H3 and H4 histones associated with the sFRP-5 promoter region were significantly increased after treatment with demethylation agent (5-Aza-dc) and histone deacetylase inhibitor (TSA). When RCC cells were transfected with the sFRP-5 gene, significant inhibition of anchorage independent colony formation and cell invasion were observed compared to controls. The sFRP-5 transfection also significantly induced apoptosis in RCC cells. In conclusion, this is the first report documenting that the sFRP-5 is downregulated by promoter methylation and histone acetylation and functions as a tumor suppressor gene by inducing apoptosis in RCC cells.

Wnt antagonist DKK1 acts as a tumor suppressor gene that induces apoptosis and inhibits proliferation in human renal cell carcinoma.

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.

Cytochrome P450 17 (CYP17) is involved in endometrial cancinogenesis through apoptosis and invasion pathways.

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.

An RNAi-independent role of AP1-like stress response factor Pap1 in centromere and mating-type silencing in Schizosaccaromyces pombe.

Gene silencing in S. pombe occurs by heterochromatin formation at the centromere (cen), mating-type (mat) and telomere loci. It is mediated by silencing factors including Swi6, Clr1-4, Rhp6 and Pola. RNAi pathway also plays a role in establishment of silencing at the mat and cen loci. Recently, the stress response factors, Atf1 and Pcr1were shown to play an RNAi-independent role in silencing at the mat3 locus through a cis-acting Atf1-binding site located within the repression element REIII and recruitment of the silencing factors Clr3 and Clr6. Another cis-acting site, named repression element REII abutting the mat2 locus, also establishes heterochromatin structure through Clr5 and histone deacetylases but independently of H3-Lys9-methylation and RNAi. Here, we report the occurrence of binding sites for another oxidative response factor, the pombe AP1- like factor Pap1, at the mating-type, centromere and telomere loci. By genetic studies we show that these sites play a role in silencing at the outer repeats of centromeres as well as mating-type locus and this effect is mediated through Pap1 binding site and interaction with and recruitment of the HP1/Swi6. Importantly, pap1Δ cells display a silencing defect even in absence of the oxidative stress. Such a role of Pap1 in heterochromatin formation may be evolutionarily conserved.