PCOTH, a novel gene overexpressed in prostate cancers, promotes prostate cancer cell growth through phosphorylation of oncoprotein TAF-Ibeta/SET

Through genome-wide cDNA microarray analysis coupled with microdissection of prostate cancer cells, we identified a novel gene, prostate collagen triple helix (PCOTH), showing overexpression in prostate cancer cells and its precursor cells, prostatic intraepithelial neoplasia (PIN). Immunohistochemical analysis using polyclonal anti-PCOTH antibody confirmed elevated expression of PCOTH, a 100-amino-acid protein containing collagen triple-helix repeats, in prostate cancer cells and PINs. Knocking down PCOTH expression by small interfering RNA (siRNA) resulted in drastic attenuation of prostate cancer cell growth, and concordantly, LNCaP derivative cells that were designed to constitutively express exogenous PCOTH showed higher growth rate than LNCaP cells transfected with mock vector, suggesting the growth-promoting effect of PCOTH on prostate cancer cell. To investigate the biological mechanisms of this growth-promoting effect, we applied two-dimensional differential gel electrophoresis (2D-DIGE) to analyze the phospho-protein fractions in LNCaP cells transfected with PCOTH. We found that the phosphorylation level of oncoprotein TAF-Ibeta/SET was significantly elevated in LNCaP cells transfected with PCOTH than control LNCaP cells, and these findings were confirmed by Western blotting and in-gel kinase assay. Furthermore, knockdown of endogenous TAF-Ibeta expression by siRNA also attenuated viability of prostate cancer cells as well. These findings suggest that PCOTH is involved in growth and survival of prostate cancer cells thorough, in parts, the TAF-Ibeta pathway, and that this molecule should be a promising target for development of new therapeutic strategies for prostate cancers.     

前列腺癌特异抗原3对前列腺癌LNCaP细胞增殖的影响

背景与目的:前列腺癌抗原3(prostate cancer antigen 3,PCA3)作为一种长链非编码RNA已被证实在前列腺癌中特异性高表达,预示其可能在前列腺癌的发生、发展中发挥重要作用。本研究拟通过靶向干扰PCA3以初步观察其对前列腺癌细胞株LNCaP细胞增殖的影响。方法:使用脂质体介导小干扰RNA(siRNA)转染前列腺癌LNCaP细胞,荧光倒置显微镜观察转染效率,实时定量PCR检测siRNA对PCA3的抑制程度;CCK-8法和克隆形成实验检测转染前列腺癌LNCaP细胞后细胞的增殖能力。结果:荧光倒置显微镜观察转染后48 h转染效率为50%～70%;实时定量PCR检测结果发现,与PCA3 siRNA(si731)和PCA3 siRNA(si2060)比较,PCA3 siRNA(si164)对PCA3的抑制程度最大;细胞增殖实验显示,转染PCA3 siRNA(si164)组中LNCaP细胞增殖能力较阴性对照组明显下降(P<0.05)。结论:PCA3 siRNA(si164)在前列腺癌LNCaP细胞株中能靶向沉默PCA3的表达并抑制前列腺癌细胞增殖,表明PCA3通过调节细胞增殖促进前列腺癌发展,并可成为前列腺癌新的治疗靶点。     

Regulator of G-protein signaling 2 (RGS2) inhibits androgen-independent activation of androgen receptor in prostate cancer cells

Hormones acting through G protein-coupled receptors (GPCRs) can cause androgen-independent activation of androgen receptor (AR) in prostate cancer cells. Regulators of G-protein signaling (RGS) proteins, through their GTPase activating protein (GAP) activities, inhibit GPCR-mediated signaling by inactivating G proteins. Here, we identified RGS2 as a gene specifically downregulated in androgen-independent prostate cancer cells. Expression of RGS2, but not other RGS proteins, abolished androgen-independent AR activity in androgen-independent LNCaP cells and CWR22Rv1 cells. In LNCaP cells, RGS2 inhibited G(q)-coupled GPCR signaling. Expression of exogenous wild-type RGS2, but not its GAP-deficient mutant, significantly reduced AR activation by constitutively activated G(q)Q209L mutant whereas silencing endogenous RGS2 by siRNA enhanced G(q)Q209L-stimulated AR activity. RGS2 had no effect on RGS-insensitive G(q)Q209L/G188S-induced AR activation. Furthermore, extracellular signal-regulated kinase 1/2 (ERK1/2) was found to be involved in RGS2-mediated regulation of androgen-independent AR activity. In addition, RGS2 functioned as a growth suppressor for androgen-independent LNCaP cells whereas androgen-sensitive LNCaP cells with RGS2 silencing had a growth advantage under steroid-reduced conditions. Finally, RGS2 expression level was significantly decreased in human prostate tumor specimens. Taken together, our results suggest RGS2 as a novel regulator of AR signaling and its repression may be an important step during prostate tumorigenesis and progression.     

Enhanced combined tumor-specific oncolysis and suicide gene therapy for prostate cancer using M6 promoter

Enzyme pro-drug suicide gene therapy has been hindered by inefficient viral delivery and gene transduction. To further explore the potential of this approach, we have developed AdIU1, a prostate-restricted replicative adenovirus (PRRA) armed with the herpes simplex virus thymidine kinase (HSV-TK). In our previous Ad-OC-TK/ACV phase I clinical trial, we demonstrated safety and proof of principle with a tissue-specific promoter-based TK/pro-drug therapy using a replication-defective adenovirus for the treatment of prostate cancer metastases. In this study, we aimed to inhibit the growth of androgen-independent (AI), PSA/PSMA-positive prostate cancer cells by AdIU1. In vitro the viability of an AI- PSA/PSMA-expressing prostate cancer cell line, CWR22rv, was significantly inhibited by treatment with AdIU1 plus GCV (10 microg ml(-1)), compared with AdIU1 treatment alone and also cytotoxicity was observed following treatment with AdIU1 plus GCV only in PSA/PSMA-positive CWR22rv and C4-2 cells, but not in the PSA/PSMA-negative cell line, DU-145. In vivo assessment of AdIU1 plus GCV treatment revealed a stronger therapeutic effect against CWR22rv tumors in nude mice than treatment with AdIU1 alone, AdE4PSESE1a alone or in combination with GCV. Our results demonstrate the therapeutic potential of specific-oncolysis and suicide gene therapy for AI-PSA/PSMA-positive prostate cancer gene therapy.     

Metallothionein is up-regulated under hypoxia and promotes the survival of human prostate cancer cells

Tumor hypoxia is a common feature of several cancers, including prostate cancer, and is associated with tumor progression, acquisition of anti-apoptotic potential and therapeutic resistance. We explored hypoxia-inducible genes and examined the effect of knockdown of a target molecule with small interference RNA (siRNA) on the proliferation of human prostate cancer cells. Human prostate cancer cell lines (LNCaP and PC-3) were cultured in normoxia (21% O2) or hypoxia (0.5% O2). Hypoxia-inducible genes were identified by cDNA microarray analysis. Metallothionein (MT) expression was assessed by real-time RT-PCR, Western blot analysis and immunohistochemical staining. siRNA was transfected to knock down MT expression, and the cell cycle and apoptosis were evaluated by flow cytometry analysis. In cDNA microarray analysis, 22 genes (including MT) were up-regulated under hypoxia. MT-1X and MT-2A were up-regulated in real-time RT-PCR. In particular, MT-2A was increased 3-fold in LNCaP and 8-fold in PC-3. The siRNA-MT-2A treatment resulted in a 20% inhibition of cell growth and induced apoptosis in both LNCaP and PC-3. In human prostate tissue, intense staining of MT was observed in cancer cells and residual cancer cells after androgen ablation therapy, while normal tissue was only stained in patches. In conclusion, MT was up-regulated under hypoxia in prostate cancer cells and overexpressed in prostate cancer tissue and residual cancer cells after androgen ablation therapy. As down-regulation of MT by siRNA inhibited cell growth and induced cell death, MT may be a new molecular target for the treatment of human prostate cancer.     

Immunohistochemical detection of tyrosine phosphatase SHP‐1 predicts outcome after radical prostatectomy for localized prostate cancer

The protein tyrosine kinase (PTK) receptors and cytosolic signaling proteins as well as the protein tyrosine phosphatases (PTPs) have important roles in regulation of growth of the benign and malignant prostate gland. Here, we studied expression of the protein tyrosine phosphatase SHP‐1 in prostate cancer cell lines and in human prostatic tissues. SHP‐1 is expressed at a high level in LNCaP prostate cancer cells compared with PC3 cells. Silencing of SHP‐1 expression with siRNA in LNCaP cells led to an increased rate of proliferation, whereas overexpression of SHP‐1 by means of transient and stable transfection in PC3 cells led to a decrease in proliferation. Corresponding changes were observed in cyclin D1 expression. We further demonstrate that LNCaP and PC3 cells respond differently to IL‐6 stimulation. SHP‐1 overexpression in PC3 cells reversed IL‐6 stimulation of proliferation, whereas in SHP‐1‐silenced LNCaP cells, IL‐6 inhibition of proliferation was not affected. In addition, IL‐6 treatment led to higher levels of phosphorylated STAT3 in SHP‐1‐silenced LNCaP cells than in control cells. Next, SHP‐1 expression in human prostate cancer was analyzed by immunohistochemical staining of tissue microarrays comprising tumor specimens from 100 prostate cancer patients. We found an inverse correlation between the tumor level of SHP‐1 expression and time to biochemical recurrence and clinical progression among prostate cancer patients. In conclusion, our results suggest that a decreased level of SHP‐1 expression in prostate cancer cells is associated with a high proliferation rate and an increased risk of recurrence or clinical progression after radical prostatectomy for localized prostate cancer.     

Knockdown of STEAP1 inhibits cell growth and induces apoptosis in LNCaP prostate cancer cells counteracting the effect of androgens

Six transmembrane epithelial antigen of the prostate 1 (STEAP1) is overexpressed in numerous types of tumors, especially in prostate cancer. STEAP1 is located in the plasma membrane of epithelial cells and may play an important role in inter- and intracellular communication. Several studies suggest STEAP1 as a potential biomarker and an immunotherapeutic target for prostate cancer. However, the role of STEAP1 in cell proliferation and apoptosis remains unclear. Therefore, the role of STEAP1 in prostate cancer cells proliferation and apoptosis was determined by inducing STEAP1 gene knockdown in LNCaP cells. In addition, the effect of DHT on the proliferation of LNCaP cells knocked down for STEAP1 gene was evaluated. Our results demonstrated that silencing the STEAP1 gene reduces LNCaP cell viability and proliferation, while inducing apoptosis. In addition, we showed that the cellular and molecular effects of STEAP1 gene knockdown may be independent of DHT treatment, and blocking STEAP1 may reveal to be an appropriate strategy to activate apoptosis in cancer cells, as well as to prevent the proliferative and anti-apoptotic effects of DHT in prostate cancer.     

ARFGAP3, an androgen target gene, promotes prostate cancer cell proliferation and migration

ADP ribosylation factor GTPase-activating protein 3 (ARFGAP3) is a GTPase-activating protein that associates with the Golgi apparatus and regulates the vesicular trafficking pathway. In the present study, we examined the contribution of ARFGAP3 to prostate cancer cell biology. We showed that ARFGAP3 expression was induced by 100 nM of dihydrotestosterone (DHT) at both the mRNA and protein levels in androgen-sensitive LNCaP cells. We generated stable transfectants of LNCaP cells with FLAG-tagged ARFGAP3 or a control empty vector and showed that ARFGAP3 overexpression promoted cell proliferation and migration compared with control cells. We found that ARFGAP3 interacted with paxillin, a focal adhesion adaptor protein that is important for cell mobility and migration. Small interfering RNA (siRNA)-mediated knockdown of ARFGAP3 showed that ARFGAP3 siRNA markedly reduced LNCaP cell growth. Androgen receptor (AR)-dependent transactivation activity on prostate-specific antigen (PSA) enhancer was synergistically promoted by exogenous ARFGAP3 and paxillin expression, as shown by luciferase assay in LNCaP cells. Thus, our results suggest that ARFGAP3 is a novel androgen-regulated gene that can promote prostate cancer cell proliferation and migration in collaboration with paxillin.     

AKT3 promotes prostate cancer proliferation cells through regulation of Akt,B-Raf & TSC1/TSC2

The qRT-PCR analysis of 139 clinical samples and analysis of 150 on-line database clinical samples indicated that AKT3 mRNA expression level was elevated in primary prostate tumors. Immunohistochemical staining of 65 clinical samples revealed that AKT3 protein expression was higher in prostate tumors of stage I, II, III as compared to nearby normal tissues. Plasmid overexpression of AKT3 promoted cell proliferation of LNCaP, PC-3, DU-145, and CA-HPV-10 human prostate cancer (PCa) cells, while knockdown of AKT3 by siRNA reduced cell proliferation. Overexpression of AKT3 increased the protein expression of total AKT, phospho-AKT S473, phospho-AKT T308, B-Raf, c-Myc, Skp2, cyclin E, GSK3β, phospho-GSK3β S9, phospho-mTOR S2448, and phospho-p70S6K T421/S424, but decreased TSC1 (tuberous sclerosis 1) and TSC2 (tuberous Sclerosis Complex 2) proteins in PC-3 PCa cells. Overexpression of AKT3 also increased protein abundance of phospho-AKT S473, phospho-AKT T308, and B-Raf but decreased expression of TSC1 and TSC2 proteins in LNCaP, DU-145, and CA-HPV-10 PCa cells. Oncomine datasets analysis suggested that AKT3 mRNA level was positively correlated to BRAF. Knockdown of AKT3 in DU-145 cells with siRNA increased the sensitivity of DU-145 cells to B-Raf inhibitor treatment. Knockdown of TSC1 or TSC2 promoted the proliferation of PCa cells. Our observations implied that AKT3 may be a potential therapeutic target for PCa treatment.     

DcR2 (TRAIL-R4) siRNA and adenovirus delivery of TRAIL (Ad5hTRAIL) break down in vitro tumorigenic potential of prostate carcinoma cells

High levels of decoy receptor 2 (DcR2; TRAIL-R4) expression are correlated with TRAIL resistance in prostate cancer cells. In addition, upregulation of TRAIL death receptor (DR4 and DR5) expression, either by ionizing radiation or chemotherapy, can sensitize cancer cells to TRAIL. Considering more than half of human cancers are TRAIL resistant, modulation of surface TRAIL receptor expression appears to be an attractive treatment modality to counteract TRAIL resistance. In this study, three siRNA duplexes targeting DcR2 receptor were tested. Ad5hTRAIL infections were performed to overexpress human full-length TRAIL to induce cell death, and the in vitro tumorigenic potential of prostate cancer cells was assessed using colony-forming assays on soft agar. The DU145 and LNCaP prostate cancer cell lines, which express high levels of DcR2, were resistant to Ad5hTRAIL-induced death. Downregulation of surface DcR2 expression by siRNA sensitized these prostate cancer cell lines to Ad5hTRAIL. In addition, DcR2 siRNA-mediated knockdown of DcR2, followed by Ad5hTRAIL infection, dramatically reduced the in vitro tumorigenic potential of prostate cancer cells. Collectively, our results suggest the potential for combining receptor-specific siRNA with TRAIL in the treatment of certain cancers.     

Folate-linked nanoparticle-mediated suicide gene therapy in human prostate cancer and nasopharyngeal cancer with herpes simplex virus thymidine kinase

For targeted gene delivery to human prostate cancer LNCaP and PC-3 cells and nasopharyngeal cancer KB cells, we developed a folate-linked nanoparticle (NP-F), and evaluated the potential of NP-F-mediated suicide gene therapy in the cells and xenografts with herpes simplex virus thymidine kinase (HSV-tk) and connexin 43 (Cx43). An NP-F-plasmid DNA complex (NP-F nanoplex) showed high DNA transfection efficiency in KB, LNCaP and PC-3 cells. Cell growth inhibition in the presence of ganciclovir (GCV) was enhanced with HSV-tk and Cx43 genes in LNCaP cells. In suicide gene therapy, the tumor growths of KB and LNCaP xenografts were significantly inhibited when an NP-F nanoplex of the HSV-tk gene, and HSV-tk and Cx43 genes, respectively, was injected intratumorally and GCV was administered intraperitoneally. These findings suggested that the NP-F is a potential target vector in prostate and nasopharyngeal cancer for suicide gene therapy.     

Syndecan-1, a new target molecule involved in progression of androgen-independent prostate cancer

Heparan sulfate proteoglycan syndecan-1 (CD138) is well known to be associated with cell proliferation, adhesion and migration in various types of malignancies. In the present study, we focused on the role of syndecan-1 in human prostate cancer. Immunohistochemical analysis revealed either no or rare expression of syndecan-1 in normal secretory glands and prostate cancer cells at hormone naïve status, whereas the expression was significantly increased in viable cancer cells following neo-adjuvant hormonal therapy. Syndecan-1 expression was much higher in the androgen independent prostate cancer cell lines DU145 and PC3, rather than the androgen-dependent LNCaP, but the level in LNCaP was up-regulated in response to long-term culture under androgen deprivation. Silencing of syndecan-1 by siRNA transfection reduced endogenous production of reactive oxygen species through down-regulating NADPH oxidase 2 and induced apoptosis in DU145 and PC3 cells. Consistently, NADPH oxidase 2 knockdown induced apoptosis to a similar extent. Subcutaneous inoculation of PC3 cells in nude mice demonstrated the reduction of tumor size by localized injection of syndecan-1 siRNA in the presence of atelocollagen. Moreover, the mouse model and chorioallantoic membrane assay demonstrated significant inhibition of vascular endothelial growth factor and tumor angiogenesis by silencing of syndecan-1. In conclusion, syndecan-1 might participate in the process of androgen-dependent to -independent conversion, and be a new target molecule for hormone resistant prostate cancer therapy. ( Cancer Sci 2009; 100: 1248–1254)     

Systemic delivery of RafsiRNA using cationic cardiolipin liposomes silences Raf-1 expression and inhibits tumor growth in xenograft model of human prostate cancer

Raf-1, a protein serine-threonine kinase, plays a critical role in mitogen-activated protein kinase kinase (MKK/MEK)- mitogen-activated protein kinase (extracellular signal-regulated kinase) (MAPK/ERK) pathways. We show here that systemically delivered novel cationic cardiolipin liposomes (NeoPhectin-AT) containing a small interfering RNA (siRNA) against Raf-1 silence the expression of Raf-1 in tumor tissues and inhibit tumor growth in xenograft model of human prostate cancer. The knockdown of Raf-1 expression by siRNA is also associated with down-regulation of cyclin D1 expression in vivo.