Neuropilin-1 is upregulated in the adaptive response of prostate tumors to androgen-targeted therapies and is prognostic of metastatic progression and patient mortality.

Recent evidence has implicated the transmembrane co-receptor neuropilin-1 (NRP1) in cancer progression. Primarily known as a regulator of neuronal guidance and angiogenesis, NRP1 is also expressed in multiple human malignancies, where it promotes tumor angiogenesis. However, non-angiogenic roles of NRP1 in tumor progression remain poorly characterized. In this study, we define NRP1 as an androgen-repressed gene whose expression is elevated during the adaptation of prostate tumors to androgen-targeted therapies (ATTs), and subsequent progression to metastatic castration-resistant prostate cancer (mCRPC). Using short hairpin RNA (shRNA)-mediated suppression of NRP1, we demonstrate that NRP1 regulates the mesenchymal phenotype of mCRPC cell models and the invasive and metastatic dissemination of tumor cells in vivo. In patients, immunohistochemical staining of tissue microarrays and mRNA expression analyses revealed a positive association between NRP1 expression and increasing Gleason grade, pathological T score, positive lymph node status and primary therapy failure. Furthermore, multivariate analysis of several large clinical prostate cancer (PCa) cohorts identified NRP1 expression at radical prostatectomy as an independent prognostic biomarker of biochemical recurrence after radiation therapy, metastasis and cancer-specific mortality. This study identifies NRP1 for the first time as a novel androgen-suppressed gene upregulated during the adaptive response of prostate tumors to ATTs and a prognostic biomarker of clinical metastasis and lethal PCa.

Prognostic value of Ki67 in localized prostate carcinoma: a multi-institutional study of >1000 prostatectomies.

BACKGROUND: Expanding interest in and use of active surveillance for early state prostate cancer (PC) has increased need for prognostic biomarkers. Using a multi-institutional tissue microarray resource including over 1000 radical prostatectomy samples, we sought to correlate Ki67 expression captured by an automated image analysis system with clinicopathological features and validate its utility as a clinical grade test in predicting cancer-specific outcomes. METHODS: After immunostaining, the Ki67 proliferation index (PI) of tumor areas of each core (three cancer cores/case) was analyzed using a nuclear quantification algorithm (Aperio). We assessed whether Ki67 PI was associated with clinicopathological factors and recurrence-free survival (RFS) including biochemical recurrence, metastasis or PC death (7-year median follow-up). RESULTS: In 1004 PCs (∼4000 tissue cores) Ki67 PI showed significantly higher inter-tumor (0.68) than intra-tumor variation (0.39). Ki67 PI was associated with stage (P<0.0001), seminal vesicle invasion (SVI, P=0.02), extracapsular extension (ECE, P<0.0001) and Gleason score (GS, P<0.0001). Ki67 PI as a continuous variable significantly correlated with recurrence-free, overall and disease-specific survival by multivariable Cox proportional hazard model (hazards ratio (HR)=1.04-1.1, P=0.02-0.0008). High Ki67 score (defined as ⩾5%) was significantly associated with worse RFS (HR=1.47, P=0.0007) and worse overall survival (HR=2.03, P=0.03). CONCLUSIONS: In localized PC treated by radical prostatectomy, higher Ki67 PI assessed using a clinical grade automated algorithm is strongly associated with a higher GS, stage, SVI and ECE and greater probability of recurrence.

Regulation of c-Myc expression by the histone demethylase JMJD1A is essential for prostate cancer cell growth and survival.

The histone demethylase JMJD1A, which controls gene expression by epigenetic regulation of H3K9 methylation marks, functions in diverse activities, including spermatogenesis, metabolism and stem cell self-renewal and differentiation. Here, we found that JMJD1A knockdown in prostate cancer cells antagonizes their proliferation and survival. Profiling array analyses revealed that JMJD1A-dependent genes function in cellular growth, proliferation and survival, and implicated that the c-Myc transcriptional network is deregulated following JMJD1A inhibition. Biochemical analyses confirmed that JMJD1A enhances c-Myc transcriptional activity by upregulating c-Myc expression levels. Mechanistically, JMJD1A activity promoted recruitment of androgen receptor (AR) to the c-Myc gene enhancer and induced H3K9 demethylation, increasing AR-dependent transcription of c-Myc mRNA. In parallel, we found that JMJD1A regulated c-Myc stability, likely by inhibiting HUWE1, an E3 ubiquitin ligase known to target degradation of several substrates including c-Myc. JMJD1A (wild type or mutant lacking histone demethylase activity) bound to HUWE1, attenuated HUWE1-dependent ubiquitination and subsequent degradation of c-Myc, increasing c-Myc protein levels. Furthermore, c-Myc knockdown in prostate cancer cells phenocopied effects of JMJD1A knockdown, and c-Myc re-expression in JMJD1A-knockdown cells partially rescued prostate cancer cell growth in vitro and in vivo. c-Myc protein levels were positively correlated with those of JMJD1A in a subset of human prostate cancer specimens. Collectively, our findings identify a critical role for JMJD1A in regulating proliferation and survival of prostate cancer cells by controlling c-Myc expression at transcriptional and post-translational levels.

DAB2IP loss confers the resistance of prostate cancer to androgen deprivation therapy through activating STAT3 and inhibiting apoptosis.

Loss of DAB2IP, a novel tumor suppressor gene, is associated with the high risk of aggressive prostate cancer (PCa). Previously, we reported that DAB2IP modulated androgen receptor activation in the development of castration-resistant PCa; however, its direct action on the failure of androgen deprivation therapy (ADT) remains largely unknown. In this study, we showed that DAB2IP knockdown could significantly enhance in vitro growth and colony formation of PCa cells following ADT as well as tumorigenicity in pre-castrated nude mice. In addition, DAB2IP loss stabilized mitochondrial transmembrane potential, prevented release of cytochrome c, Omi/HtrA2 and Smac from the mitochondria to the cytoplasm and inhibited intrinsic apoptosis induced by ADT. Mechanistically, DAB2IP could interact with the signal transducer and activator of transcription 3 (STAT3) via its unique PR domain and suppress STAT3 phosphorylation and transactivation, leading to the inhibition of survivin expression in PCa cells. Moreover, the luminal epithelia in DAB2IP(-/-) mice with more activated STAT3 and survivin expression were resistant to castration-induced apoptosis. Consistently, DAB2IP expression inversely correlated with STAT3 phosphorylation and survivin expression in PCa patients. Together, our data indicate that DAB2IP loss reprograms intracellular signal transduction and anti-apoptotic gene expression, which potentiates PCa cell survival from ADT-induced cell death.

Lyn tyrosine kinase regulates androgen receptor expression and activity in castrate-resistant prostate cancer.

Castrate-resistant prostate cancer (CRPC) progression is a complex process by which prostate cells acquire the ability to survive and proliferate in the absence or under very low levels of androgens. Most CRPC tumors continue to express the androgen receptor (AR) as well as androgen-responsive genes owing to reactivation of AR. Protein tyrosine kinases have been implicated in supporting AR activation under castrate conditions. Here we report that Lyn tyrosine kinase expression is upregulated in CRPC human specimens compared with hormone naive or normal tissue. Lyn overexpression enhanced AR transcriptional activity both in vitro and in vivo and accelerated CRPC. Reciprocally, specific targeting of Lyn resulted in a decrease of AR transcriptional activity in vitro and in vivo and prolonged time to castration. Mechanistically, we found that targeting Lyn kinase induces AR dissociation from the molecular chaperone Hsp90, leading to its ubiquitination and proteasomal degradation. This work indicates a novel mechanism of regulation of AR stability and transcriptional activity by Lyn and justifies further investigation of the Lyn tyrosine kinase as a therapeutic target for the treatment of CRPC.Oncogenesis (2014) 3, e115; doi:10.1038/oncsis.2014.30; published online 18 August 2014.

The role of DAB2IP in androgen receptor activation during prostate cancer progression.

Altered androgen-receptor (AR) expression and/or constitutively active AR are commonly associated with prostate cancer (PCa) progression. Targeting AR remains a focal point for designing new strategy of PCa therapy. Here, we have shown that DAB2IP, a novel tumor suppressor in PCa, can inhibit AR-mediated cell growth and gene activation in PCa cells via distinct mechanisms. DAB2IP inhibits the genomic pathway by preventing AR nuclear translocation or phosphorylation and suppresses the non-genomic pathway via its unique functional domain to inactivate c-Src. Also, DAB2IP is capable of suppressing AR activation in an androgen-independent manner. In addition, DAB2IP can inhibit several AR splice variants showing constitutive activity in PCa cells. In DAB2IP(-/-) mice, the prostate gland exhibits hyperplastic epithelia, in which AR becomes more active. Consistently, DAB2IP expression inversely correlates with AR activation status particularly in recurrent or metastatic PCa patients. Taken together, DAB2IP is a unique intrinsic AR modulator in normal cells, and likely can be further developed into a therapeutic agent for PCa.

Fibroblast growth factor and ornithine decarboxylase 5'UTRs enable preferential expression in human prostate cancer cells and in prostate tumors of PTEN(-/-) transgenic mice.

In this study, we have taken advantage of over-expression of eukaryotic translation initiation factor 4E (eIF4E) in prostate cancer cells to design a viral-based targeting system of prostate cancer. Three different lengths of 5'-untranslated regions (5'UTRs) derived from either fibroblast growth factor-2 (FU-FGF2-GW) or ornithine decarboxylase (FU-ODC149-GW and FU-ODC274-GW) were inserted upstream of enhanced green fluorescent protein (GFP) gene in a lentiviral backbone. Both nonmalignant control (PNT1B and BPH-1) and neoplastic (LNCaP, C4-2, DU145 and PC-3) prostate cell lines were transfected with each plasmid or virus alone, or in the presence of siRNA against eIF4E, and their expression was monitored via GFP protein levels. Two 5'UTRs (FU-FGF2-GW and FU-ODC-GW) were selected as being most sensitive to eIF4E status. Lentiviruses containing these sequences were injected directly into the prostates of PTEN(-/-) (tumor-bearing) and control mice. Immunofluorescence data and western blot analyses determined that a lentivirus containing a 5'UTR derived from FGF-2 is the best candidate for directing selective gene expression in the prostate tumors of PTEN(-/-) mice in vivo. This study demonstrates that judicious selection of a complex 5'UTR can enhance selective targeting of viral-based gene therapies for prostate cancer.

Heat shock protein 27 confers resistance to androgen ablation and chemotherapy in prostate cancer cells through eIF4E.

One strategy to improve therapies in advanced prostate cancer (PC) involves targeting genes that are activated by androgen withdrawal to delay the emergence of the androgen-independent (AI) phenotype. Heat shock protein 27 (Hsp27) expression becomes highly upregulated in PC cells after androgen withdrawal or chemotherapy, in which it functions as a cytoprotective chaperone to confer broad-spectrum treatment resistance. The purpose of this study is to elucidate anti-apoptotic pathways regulated by Hsp27 that are activated during PC progression. Using two-hybrid experiment, we found that Hsp27 was having a major role in the protein translational initiation process. Furthermore, using complementary DNA (cDNA) microarray analysis, 4E binding protein 1 was identified as being proportionately and highly regulated by Hsp27. These data led us to analyze the protein synthesis initiation pathway, which is a prerequisite for cell growth and proliferation. Using northern and western blot analysis, we found that Hsp27 downregulation decreased eukaryotic translation initiation factor 4E (eIF4E) expression at the protein, but not mRNA, level. The cytoprotection afforded by Hsp27 overexpression was attenuated by eIF4E knockdown using specific eIF4E short interfering RNA (siRNA). Co-immunoprecipitation and co-immunofluorescence confirmed that Hsp27 colocalizes and interacts directly with eIF4E. Hsp27-eIF4E interaction decreases eIF4E ubiquitination and proteasomal degradation. By chaperoning eIF4E, Hsp27 seems to protect the protein synthesis initiation process to enhance cell survival during cell stress induced by castration or chemotherapy. Forced overexpression of eIF4E induces resistance to androgen-withdrawal and paclitaxel treatment in the prostate LNCaP cells in vitro. These findings identify Hsp27 as a modulator of eIF4E and establish a potential mechanism for the eIF4E-regulated apoptosis after androgen ablation and chemotherapy. Targeting Hsp27-eIF4E interaction may serve as a therapeutic target in advanced PC.

Lentiviruses with trastuzumab bound to their envelopes can target and kill prostate cancer cells.

In this study, we took advantage of the overexpression of human epidermal growth factor receptor 2 (HER-2) in prostate cancers to design lentiviruses with modified envelope proteins that bind antibodies to specific cell-surface antigens. When bound to trastuzumab (Herceptin, Genentech, CA), lentiviruses were able to selectively infect androgen-sensitive LNCaP and castration-resistant C4-2 human prostate cancer cell lines, both of which express high levels of HER-2. To test for a therapeutic effect, we engineered our antibody-binding lentiviruses to express thymidine kinase, which can convert the non-toxic pro-drug ganciclovir (GCV) into a cytotoxic form. LNCaP and C4-2 cells infected by these viruses were sensitive to GCV killing. In vivo, C4-2 xenograft tumors treated either intratumorally or i.v. with trastuzumab-bound lentivirus expressed luciferase, although the latter route was less tumor specific. When a prostate-specific promoter for governing luciferase expression was combined with trastuzumab-mediated delivery, there was a further enrichment in targeting viral gene expression in prostate tumors. In conclusion, we found that although prostate cancers that express high levels of HER-2 are resistant to the killing effects of trastuzumab, they can be targeted for selective gene expression and destruction by viruses with envelope proteins engineered to bind this antibody.

Targeting and killing of prostate cancer cells using lentiviral constructs containing a sequence recognized by translation factor eIF4E and a prostate-specific promoter.

To develop a gene therapy that would selectively kill prostate cancer cells while sparing normal cells, we have constructed lentiviral vectors that contain a therapeutic gene with a short DNA sequence in the 5'-untranslated region (UTR) that is recognized by the translation initiation factor, eIF4E, which is often overexpressed in malignant cells. Infection of cancer (LNCaP, PC-3M, DU145, and MCF-7 cells) and noncancer cell lines (BPH-1, 267-B1, Plat-E, and Huvec-c cells) with lentivirus having a CMV-promoter and EGFP reporter resulted in high levels of EGFP expression in all cells, whereas, inclusion of the eIF4E UTR recognition sequence restricted high expression to cancer cells and Plat-E cells, which also express substantial levels of eIF4E. Infection of the cells with lentiviral vectors having this UTR in front of the HSV thymidine kinase suicide gene resulted in differential sensitivity to the killing effects of ganciclovir, with at least 100-fold more drug required to kill noncancer cells than cancer cells. Furthermore, in experiments where the CMV promoter was replaced by the prostate-specific ARR(2)PB promoter, the killing effects of ganciclovir were restricted to prostate cancer cells and not seen in nonprostate cancer cells. Our results indicate that combined translational regulation, by incorporation of an eIF4E-UTR recognition sequence into a therapeutic gene, together with transcriptional regulation with a prostate-specific promoter, may provide a means to selectively destroy prostate cancer cells while sparing normal prostate cells.

Integrin-linked kinase, a promising cancer therapeutic target: biochemical and biological properties.

Integrin-linked kinase (ILK) is an ankyrin repeat-containing Ser/Thr kinase that interacts with the cytoplasmic domains of beta(1) and beta(3) integrins. ILK is widely expressed in tissues throughout the body, and, as might be expected, appears to mediate a diversity of functions relating to its role in coupling integrins and growth factor receptors to downstream signaling pathways. Through its downstream targets protein kinase B/Akt and glycogen synthase kinase-3beta, ILK appears to be involved in several oncogenesis-related events, including suppression of apoptosis and promotion of cell survival, as well as cell migration and invasion. Over-expression of ILK in epithelial cells results in anchorage-independent cell growth with increased cell cycle progression. Inoculation of nude mice with ILK over-expressing cells leads to tumor formation. Furthermore, increased ILK expression and activity have been correlated with malignancy in several human tumor types, including breast, prostate, brain, and colon carcinomas. Based on these findings, ILK represents an excellent therapeutic target for the prevention of tumor progression. Here, we provide an overview of the physical and biochemical properties of ILK, and present data describing the impact of small-molecule ILK inhibitors on several ILK-mediated cellular functions.