Topology of NGEP, a prostate-specific cell:cell junction protein widely expressed in many cancers of different grade level

New gene expressed in prostate (NGEP) is a prostate-specific polytopic membrane protein found at high concentrations at cell:cell contact regions. To determine if NGEP is a useful target for antibody-based therapy of prostate cancer, we performed an immunohistochemical analysis of 126 human prostate carcinoma samples using polyclonal anti-NGEP sera and found that 91% of the cancers express NGEP protein. To elucidate the topology of NGEP and guide the development of monoclonal antibodies (mAb) reacting with the extracellular regions of NGEP, a hemagglutinin epitope tag was inserted at several positions within the NGEP sequence. The tagged proteins were expressed in 293T cells and locations of the tags were determined by immunofluorescence in intact or permeabilized cells. The results indicate that NGEP contains eight transmembrane domains with both the NH(2) and COOH termini of NGEP located inside the cell. We produced mAb to three regions that are predicted to be intracellular based on the epitope tag data (amino acids 1-352, 441-501, and 868-933), and as predicted, the mAb only detected the protein in permeabilized cells. NGEP is a glycoprotein with predicted glycosylation sites at N809 and N824. When these residues were converted to glutamine, glycosylation was abolished, confirming that the residues are extracellular. Our findings on the expression and the orientation of the NGEP protein serve as an important framework for the development of mAb targeting the extracellular regions of NGEP that could be used for prostate cancer immunotherapy.     

Identification and characterization of prostein, a novel prostate-specific protein

In this report, we describe the application of a systematic, genome-based approach to identify prostein, a novel prostate-specific protein expressed in normal and malignant prostate tissues. Characterization of the prostein gene shows that prostein cDNA encodes a 553-amino acid protein. The protein is predicted to be a type IIIa plasma membrane protein with a cleavable signal peptide and 11 transmembrane-spanning regions. The prostein gene is located on chromosome 1 at the WI-9641 locus between q32 and q42. Prostein mRNA is shown to be uniquely expressed in normal and cancerous prostate tissues using Northern blot, eDNA microarray, and real-time PCR analyses. Furthermore, prostein mRNA expression does not appear to be prostate tumor grade related and is restricted exclusively to prostate cell lines. Immunohistochemical staining using a mouse monoclonal antibody generated against prostein demonstrates that this protein is specifically detected in prostate tissues both at the plasma membrane and in the cytoplasm. Prostein expression is androgen responsive because treatment of LNCaP cells with androgen up-regulates prostein message and protein expression levels. These results validate prostein as a prostate-specific marker with potential utility in the diagnosis and treatment of 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.     

Expression of c-erb B-2/neu proto-oncogene in human prostatic cancer tissues and cell lines.

Both amplification and overexpression of c-erb B-2/neu have been associated with the progression and possible prognosis of a number of human cancers. In this study, we demonstrated that c-erb B-2/neu may also play an important role in human prostate cancer. Our conclusion is based on the following observations: (1) A monoclonal antibody raised against a peptide sequence from the C-terminal domain of the human c-erb B-2/neu gene product reacted positively with 68.7% (11 of 16) of the human prostatic cancer tissue extracts analyzed by western blot procedure. These results were supported by the immunohistochemical staining of the prostatic cancer specimens; 80% (12 of 15) showed positive staining, primarily around the plasma membranes of the prostatic cancer cells. c-erb B-2/neu oncoprotein was not detectable in normal prostate tissues (five examined by immunohistochemical staining and three by western blotting) or in human benign prostatic hyperplasia (two examined by immunohistochemical staining and six by western blotting) and was expressed less abundantly with lower intensity in "normal" human prostate tissues adjacent to cancerous prostate tissue (5 of 12 examined by immunohistochemical staining). We observed no evidence of c-erb B-2/neu gene amplification in 10 fresh human prostatic cancer specimens examined by Southern blotting and in the cultured human prostatic cancer cell lines PC-3, DU-145, and LNCaP. (2) The c-erb B-2/neu protein was detected in both androgen-receptor-positive (LNCaP) and -negative (PC-3 and DU-145) human prostate cancer cell lines. Positive immunostaining of c-erb B-2/neu protein was found to be associated predominantly with the plasma membranes of PC-3 cells, but was also found to be widespread in the cytoplasmic region of the LNCaP cells and in the perinuclear region of the DU-145 cells. (3) Like prostate-specific antigen (PSA) expression, c-erb B-2/neu mRNA expression was also positively regulated by androgen in androgen-receptor-positive LNCaP cells in vitro and LNCaP tumors in vivo. When LNCaP tumors were grown in castrated male hosts, levels of c-erb B-2/neu and PSA mRNA expression decreased initially, but rebounded at 3 wk to levels comparable to those expressed by tumors maintained in intact adult male hosts.     

PSMA specific single chain antibody-mediated targeted knockdown of Notch1 inhibits human prostate cancer cell proliferation and tumor growth

The down-regulation of Notch1 by small interfering RNA (siRNA) can significantly inhibit human prostate cancer cell growth. The delivery of siRNA into specific cells is a key requirement for its clinical application. Recent reports have indicated that antibody-mediated siRNA delivery is an effective approach for targeted knockdown of specific genes in appropriate cells. Prostate-specific membrane antigen (PSMA) is regarded as an ideal target for the delivery of therapeutic agents to prostate cancer cells. The purpose of the present study was to evaluate whether siRNA can be efficiently delivered into PSMA-positive prostate cancer cells using two fusion proteins, s-tP and sFH-tP. These fusion proteins are composed of an anti-PSMA single chain antibody (scFv, abbreviated as an “s”) and a truncated protamine (tP); and in sFH-tP a furin cleavage site and an HA2 fragment sequence (FH) were inserted between the scFv and tP domains. Our results showed that siRNA can be specifically delivered into PSMA-positive LNCaP cells by these two fusion proteins, with the sFH-tP fusion protein being more effective. Efficient knockdown of Notch1 by siNotch1 delivered by either fusion protein was observed in PSMA-positive LNCaP cells and in LNCaP xenografted nude mice. Further experiments confirmed that the fusion protein-delivered siNotch1 could efficiently inhibit PSMA-positive LNCaP cell proliferation and promote apoptosis both in vitro and in vivo. Our data describe a promising strategy for the targeted delivery of siRNA to PSMA-positive prostate cancer cells using anti-PSMA scFv fusion proteins.     

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.     

Overexpression of tissue factor pathway inhibitor-2 (TFPI-2), decreases the invasiveness of prostate cancer cells in vitro

Human tissue factor pathway inhibitor-2 (TFPI-2) is a 32-kDa serine protease inhibitor that inhibits plasmin, trypsin, chymotrypsin, cathepsin G, and plasma kallikrein but not urokinase and tissue-type plasminogen activators or thrombin. After discovering that TFPI-2 expression is down-regulated or lost during tumor progression, we investigated the role of TFPI-2 in the invasiveness of the prostate cancer cell line (LNCaP). We stably transfected LNCaP cells with a 0.7-kb vector expressing TFPI-2 in the sense orientation and measured the expression of TFPI-2 protein and mRNA by these cells by western and northern blotting. Neither TFPI-2 protein nor mRNA was expressed by parental LNCaP cells or vector-transfected controls, but levels of both protein and mRNA were significantly increased in the sense-TFPI-2 clones. The sense clones were less invasive than the control cells in Matrigel invasion and spheroid migration assays. This is the first demonstration that upregulation of TFPI-2 plays a significant role in the invasive behavior of human prostate cancer cells.     

Calcitriol-induced prostate-derived factor: autocrine control of prostate cancer cell growth

Calcitriol (1alpha,25-dihydroxycholecalciferol) seems to play an important role in the complex control of prostate cell growth. It inhibits proliferation and induces differentiation and apoptosis in prostate cancer cells. However, the molecular mechanisms of the antiproliferative activity of calcitriol are not completely understood. The expression of prostate-derived factor (PDF), a member of the transforming growth factor-beta (TGF-beta) superfamily, has been shown to be associated with proapoptotic and antimitotic activities. We show that calcitriol induces PDF expression in LNCaP human prostate cancer cells in a concentration- and time-dependent manner. In LNCaP cells, the suppression of cell growth by calcitriol is accompanied by stimulation of PDF mRNA and protein synthesis. Human recombinant PDF inhibits LNCaP cell growth. We do not detect any effect of PDF-specific antibody on the basal growth of LNCaP cells, but this antibody partially reverses the suppression of LNCaP cell growth by calcitriol, suggesting that the effect of calcitriol on cell growth is at least partially mediated by PDF. In PC-3 cells, which are less responsive to the growth-inhibitory effect of calcitriol, it has no effect on PDF expression. We do not detect an effect of recombinant PDF on SMAD phosphorylation in LNCaP cells, but ERK1/2 kinases are transiently phosphorylated in response to PDF, which suggests that in LNCaP cells PDF may exert its action through pathway alternative to the classical TGF-beta signaling pathway. The present study describes the regulation of PDF, the proapoptotic protein of the TGF-beta superfamily, by calcitriol in androgen-responsive prostate cancer cells. This is a new link between calcitriol and growth factors of TGF-beta superfamily in the control of prostate cell growth.     

Characterization of a zinc-finger protein and its association with apoptosis in prostate cancer cells

BACKGROUND: The transition from androgen-dependent to androgen-independent prostate cancer is not fully understood but appears to involve multiple genetic changes. We have identified a gene, GC79, that is more highly expressed in androgen-dependent LNCaP-FGC human prostate cancer cells than in androgen-independent LNCaP-LNO human prostate cancer cells. Physiologic levels (0.1 nM:) of androgens repress expression of GC79 messenger RNA (mRNA) in LNCaP-FGC cells. To determine the role of GC79, we cloned its complementary DNA (cDNA) and functionally characterized its product. METHODS: The differentially expressed GC79 gene was cloned from human prostate cDNA libraries, sequenced, and transfected into mammalian cells to study its function. Expression of GC79 was analyzed in various adult and fetal human tissues and in prostate glands of castrated rats. The association of GC79 expression and apoptosis was investigated in COS-1 and LNCaP cells transfected with GC79 cDNA. All statistical tests are two-sided. RESULTS: Sequence analysis indicates that GC79 encodes a large, complex, multitype zinc-finger protein, containing nine C(2)H(2)-type zinc-finger domains, a cysteine-rich region, and a GATA C(4)-type zinc-finger domain. Castration-induced androgen withdrawal increased the expression of GC79 mRNA in the regressing rat ventral prostate, suggesting that the expression of GC79 mRNA is associated with the process of apoptotic cell death in the rat ventral prostate. Transfection and induction of GC79 cDNA in both COS-1 and LNCaP prostate cancer cells led to an apoptotic index that was eightfold higher (P:<.001, two-sided Student's t test) than that observed in uninduced transfected cells. CONCLUSIONS: We have cloned an androgen-repressible gene, GC79, that is potentially involved in apoptosis. This finding may have implications for the development of androgen-independent prostate cancer and, ultimately, for the treatment of prostate cancer.     

Inhibition of tumorigenic potential and prostate-specific antigen expression in LNCaP human prostate cancer cell line by 13-cis-retinoic acid

Prostate-specific antigen (PSA) is a member of the kallikrein family and has been an important biological marker for prostate cancer. The mechanisms regulating PSA expression in prostatic cancer cells are unclear. The present study was designed to elucidate the role of 13-cis-retinoic acid (RA) in regulation of PSA and the tumorigenic potential of the human prostate cancer cell line LNCaP. The growth regulation of LNCaP cells was examined by DNA synthesis and doubling time. The tumorigenic potential of prostate cancer cells was analyzed by soft agar colony-forming assay, in vitro invasion assay, type IV collagenase assay and binding to extracellular matrix assay. The nuclear receptors for retinoic acid (RARα, -β, -γ and RXRα, -β,-γ) as well as PSA mRNA were determined by Northern blot using specific oligonucleotide probes. Our results suggest that 13-cis-RA significantly inhibits PSA secretion and expression both at the mRNA and protein levels compared with untreated cells. Electron microscopic studies suggest that after 13-cis-RA treatment, cells become more differentiated as they contain lumina, lined by plasma membrane and microvilli. Prostate cancer cell growth and tumorigenic potential after 13-cis-RA treatment was significantly decreased compared with controls. Nude mice tumorigenicity studies showed that 13-cis-RA-treated cells produced significantly smaller tumors compared with untreated cell tumors. There was also a significant increase in the expression of RXRa mRNA after 13-cis-RA treatment compared with untreated cells.     

T-cell receptor gamma chain alternate reading frame protein (TARP) expression in prostate cancer cells leads to an increased growth rate and induction of caveolins and amphiregulin.

Previously, we showed that prostate and prostate cancer cells express a truncated T-cell receptor gamma chain mRNA that uses an alternative reading frame to produce a novel nuclear T-cell receptor gamma chain alternate reading frame protein (TARP). TARP is expressed in the androgen-sensitive LNCaP prostate cancer cell line but not in the androgen-independent PC3 prostate cancer cell line, indicating that TARP may play a role in prostate cancer progression. To elucidate the function of TARP, we generated a stable PC3 cell line that expresses TARP in a constitutive manner. Expression of TARP in PC3 cells resulted in a more rapid growth rate with a 5-h decrease in doubling time. cDNA microarray analysis of 6538 genes revealed that caveolin 1, caveolin 2, amphiregulin, and melanoma growth stimulatory activity alpha were significantly up-regulated, whereas IL-1beta was significantly down-regulated in PC3 cells expressing TARP. We also demonstrated that TARP expression is up-regulated by testosterone in LNCaP cells that express a functional androgen receptor. These results suggest that TARP has a role in regulating growth and gene expression in prostate cancer cells.     

The emergence of protocadherin-PC expression during the acquisition of apoptosis-resistance by prostate cancer cells

In order to identify gene products associated with the development of acquired therapeutic resistance by prostate cancer cells, we created two novel apoptosis-resistant prostate cancer cell lines, LNCaP-TR (phorbol-ester [TPA]-Resistant) and LNCaP-SSR (Serum Starvation-Resistant) by repeated transient exposure of cultured human LNCaP cells to apoptotic stimuli followed by expansion of surviving cell populations. These cell lines were found to be cross-resistant to the alternative selective agent and also hormone-resistant when xenografted into castrated male immunodeficient mice. RNA from the LNCaP-TR line was comparatively screened using a subtractive hybridization-PCR procedure. This allowed us to identify a 249 bp cDNA fragment that hybridized to a 4.8 kb mRNA preferentially expressed by the apoptosis-resistant cells. Using RACE procedures, we cloned and sequenced the complete 4.8 kb cDNA. It is an unusual member of the protocadherin gene family containing two large overlapping open reading frames encoding homologous polypeptides, one having a signal sequence and the other lacking a signal sequence and we refer to it as protocadherin-PC. LNCaP cells directly transformed with protocadherin-PC cDNA were comparatively resistant to phorbol-ester induced apoptosis. Antibody recognition studies demonstrating the cytoplasmic nature of the protcadherin-PC translation product and its propensity to bind beta-catenin suggest that it might influence the apoptotic sensitivity of prostate cancer cells through a unique mechanism.     

Increased prostate-specific membrane antigen expression in LNCaP cells following treatment with bispecific antisense oligonucleotides directed against bcl-2 and EGFR

Antisense oligonucleotides have been employed against in vivo and in vitro prostate cancer models. While most oligos consist of a single mRNA binding site, targeting a single gene product or others sharing sequence homology, our laboratory has developed bispecific oligos directed toward even unrelated proteins. This study evaluates the inhibition of in vitro propagating LNCaP cells employing mono- and bispecific oligos directed against bcl-2 [the second bispecific binding site was directed against the epidermal growth factor receptor (EGFR)]. Employing RT-PCR, the expression of non-targeted proteins encoded by mRNA for prostate-specific membrane antigen (PSMA) and prostate-specific antigen (PSA) were subsequently evaluated. When LNCaP prostate tumor cells were incubated with bispecific oligos (directed against bcl-2 and EGFR) and compared to lipofectin-containing controls significant growth inhibition resulted. In subsequent experiments, the levels of mRNA encoding PSMA were unexpectedly found to be elevated following treatment with the bispecific oligos but not with the monospecific directed solely against bcl-2. No differences were detected in mRNA levels encoding PSA following treatment with either mono- or bispecific oligos. Previously, we suggested that cell growth inhibition produced by some bispecifics could be attributed to complementary double-stranded regions formed by intra-strand base pairs. Double-stranded nucleic acids are known inducers of interferon, which promote expression of cell surface HLA type antigens. If induced, perhaps this cytokine also enhances PSMA expression, making prostate tumor cells a more recognizable target for cytotoxic T cells.     

The therapeutic and preventive effect of RRR-alpha-vitamin E succinate on prostate cancer via induction of insulin-like growth factor binding protein-3.

PURPOSE: Insulin-like growth factor binding protein-3 (IGFBP-3) is a well-known antiproliferative and proapoptotic molecule in prostate cancer, suggesting that targeting IGFBP-3 might produce clinical benefits. In prostate cancer cells, RRR-alpha-vitamin E succinate (VES) inhibits cell proliferation and induces apoptosis, yet the mechanisms remain to be elucidated. We hypothesize that the protective effects of VES in prostate cancer are mediated by IGFBP-3 up-regulation. Using prostate cancer models, the involvement of IGFBP-3 in the anticancer effect of VES was investigated. EXPERIMENTAL DESIGN: IGFBP-3 mRNA and protein were determined by real-time PCR and Western blotting in prostate cancer cells, xenografted tumors of nude mice, and prostate tumors of transgenic adenocarcinoma mouse prostate (TRAMP) mice. The serum levels of IGFBP-3 were assessed by ELISA. The importance of IGFBP-3 in VES-mediated antitumor effects was confirmed by small interfering RNA knockdown strategy. RESULTS: We found that VES induced IGFBP-3 mRNA and protein levels in human prostate cancer cell lines. Knockdown of IGFBP-3 by small interfering RNA attenuated VES-induced IGFBP-3 expression and VES-mediated antiproliferative and proapoptotic functions. Furthermore, administration of VES resulted in a significant therapeutic effect on LNCaP and PC3 xenografts and a preventive effect on tumorigenic progression in the TRAMP model without overt toxicity. Notably, the therapeutic and preventive efficacy of VES correlated with increased accumulation of IGFBP-3 in mouse serum as well as in the xenograft tumors and TRAMP prostate samples. Consequently, reduced proliferation and induced apoptosis were witnessed. CONCLUSIONS: VES mediates its therapeutic and preventive effects against prostate cancer at least partially through up-regulating IGFBP-3, which inhibits cell proliferation and promotes cell apoptosis.