LNCaP Sublines Research Articles

Androgen regulation of micro‐RNAs in prostate cancer

Androgens play a critical role in the growth of both androgen dependent and castration-resistant prostate cancer (CRPC). Only a few micro-RNAs (miRNAs) have been suggested to be androgen regulated. We aim to identify androgen regulated miRNAs. We utilized LNCaP derived model, we have established, and which overexpresses the androgen receptor (AR), the VCaP cell line, and 13 intact-castrated prostate cancer (PC) xenograft pairs, as well as clinical specimens of untreated (PC) and CRPC. The expression of miRNAs was analyzed by microarrays and quantitative RT-PCR (Q-RT-PCR). Transfection of pre-miR-141 and anti-miR-141 was also used. Seventeen miRNAs were > 1.5-fold up- or downregulated upon dihydrotestosterone (DHT) treatment in the cell lines, and 42 after castration in the AR-positive xenografts. Only four miRNAs (miR-10a, miR-141, miR-150*, and miR-1225-5p) showed similar androgen regulation in both cell lines and xenografts. Of those, miR-141 was found to be expressed more in PC and CRPC compared to benign prostate hyperplasia. Additionally, the overexpression of miR-141 enhanced growth of parental LNCaP cells while inhibition of miR-141 by anti-miR-141 suppressed the growth of the LNCaP subline overexpressing AR. Only a few miRNAs were found to be androgen-regulated in both cell lines and xenografts models. Of those, the expression of miR-141 was upregulated in cancer. The ectopic overexpression of miR-141 increased growth of LNCaP cell suggesting it may contribute to the progression of PC.

Androgen Receptor Regulation of Vitamin D Receptor in Response of Castration-Resistant Prostate Cancer Cells to 1 -Hydroxyvitamin D5: A Calcitriol Analog

Calcitriol (1,25(OH)(2)D3) is cytostatic for prostate cancer (CaP), but had limited therapeutic utility due to hypercalcemia-related toxicities, leading to the development of low-calcemic calcitriol analogs. We show that one analog, 1-α-Hydroxyvitamin-D5 (1α(OH)D5), induced apoptosis in castration-sensitive LNCaP prostate cancer cells, but unlike calcitriol, did not increase androgen receptor (AR) transcriptional activity. LNCaP-AI, a castrate-resistant (CRCaP) LNCaP subline, was resistant to 1α(OH)D5 in the presence of androgens; however, androgen withdrawal (AWD), although ineffective by itself, sensitized LNCaP-AI cells to 1α(OH)D5. Investigation of the mechanism revealed that the vitamin D receptor (VDR), which mediates the effects of 1α(OH)D5, is downregulated in LNCaP-AI cells compared to LNCaP in the presence of androgens, whereas AWD restored VDR expression. Since LNCaP-AI cells expressed higher AR compared to LNCaP and AWD decreased AR, this indicated an inverse relationship between VDR and AR. Further, AR stimulation (by increased androgen) suppressed VDR, while AR downregulation (by ARsiRNA) stimulated VDR levels and sensitized LNCaP-AI cells to 1α(OH)D5 similar to AWD. Another cell line, pRNS-1-1, although isolated from a normal prostate, had lost AR expression in culture and adapted to androgen-independent growth. These cells expressed the VDR and were sensitive to 1α(OH)D5, but restoration of AR expression suppressed VDR levels and induced resistance to 1α(OH)D5 treatment. Taken together, these results demonstrate negative regulation of VDR by AR in CRCaP cells. This effect is likely mediated by prohibitin (PHB), which was inhibited by AR transcriptional activity and stimulated VDR in CRCaP, but not castrate-sensitive cells. Therefore, in castration sensitive cells, although the AR negatively regulates PHB, this does not affect VDR expression, whereas in CRCaP cells, negative regulation of PHB by the AR results in concomitant negative regulation of the VDR by the AR. These data demonstrate a novel mechanism by which 1α(OH)D5 prolong the effectiveness of AWD in CaP cells.

Human Prostatic Acid Phosphatase, an Authentic Tyrosine Phosphatase, Dephosphorylates ErbB-2 and Regulates Prostate Cancer Cell Growth

Cellular prostatic acid phosphatase (cPAcP), an authentic tyrosine phosphatase, is proposed to function as a negative growth regulator of prostate cancer (PCa) cells in part through its dephosphorylation of ErbB-2. Nevertheless, the direct interaction between cPAcP and ErbB-2 has not been shown nor the specific dephosphorylation site of ErbB-2 by cPAcP. In this report, our data show that the phosphorylation level of ErbB-2 primarily at Tyr(1221/2) correlates with the growth rate of both LNCaP and MDA PCa2b human PCa cells. Further, cPAcP reciprocally co-immunoprecipitated with ErbB-2 in a non-permissive growth condition. Expression of wild type cPAcP, but not inactive mutant, by cDNA in cPAcP-null LNCaP C-81 cells results in decreased tyrosine phosphorylation of ErbB-2 including Tyr(1221/2). Concurrently, Tyr(317) phosphorylation of p52(Shc), proliferating cell nuclear antigen expression, and cell growth are decreased in these cells. Conversely, decreased cPAcP expression by short hairpin RNA in LNCaP C-33 cells was associated with elevated phosphorylation of ErbB-2 initially at Tyr(1221/2). Its downstream p52(Shc), ERK1/2, Akt, Src, STAT-3, and STAT-5 were activated, and cell proliferation, proliferating cell nuclear antigen, and cyclin D1 expression were increased. Stable subclones of C-33 cells by small interfering PAcP had elevated Tyr(1221/2) phosphorylation of ErbB-2 and exhibited androgen-independent growth and increased tumorigenicity in xenograft female animals. In summary, our data together indicate that in prostate epithelia, cPAcP interacts with and dephosphorylates ErbB-2 primarily at Tyr(1221/2) and hence blocks downstream signaling, leading to reduced cell growth. In PCa cells, decreased cPAcP expression is associated with androgen-independent cell proliferation and tumorigenicity as seen in advanced hormone-refractory prostate carcinomas.

Abstract 3184: TGF-β1 signaling inhibits proliferation but promotes motility with progression to castrate-resistant prostate cancer

Abstract Transforming growth factor-beta1 (TGF-B1) is a tumor suppressor in normal and early stage prostate cancer (PCa) cells, but exerts pro-tumorigenic effects during progression to metastatic disease, and correlates with poor prognosis. However, the mechanisms whereby TGF-B1 induces pro-metastatic action are uknown. Previously it was reported that the LNCaP cell line was insensitive to TGF-B-mediated growth suppression. Thus we sought to examine the TGF-B1-induced proliferative response of increasingly metastatic, lineage-related LNCaP sublines. Under reduced serum conditions we show that TGF-B1 inhibits cell proliferation profoundly and promotes apoptosis in castrate-insensitive LNCaP sublines, whereas inhibition of cell proliferation is marginal in LNCaP cells. Growth inhibitory effects and apoptosis are mediated by active TGF-B receptors, TBRII and TBRI, and the downstream Smad2/3 effectors. Knock down of TBRII with a dominant negative construct, as well as inhibition of kinase activity abrogrates these effects. Additionally, we show that TGF-B enhances cytoskeletal rearrangements and increases motility on selected extracellular matrices in metastatic LNCaP sublines. TGF-B signaling in castrate-resistant PCa cell lines is more robust than in hormonally sensitive clones and leads to selective activation of cell motilty that influences PCa cell metastatic behavior with potential consequences for bone colonization. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3184.

Abstract 3186: The role of fibroblast growth factor 9 for androgen-independent growth of prostate cancer cells

Abstract Introduction & Objectives: In prostate tissue, impairment of communication between epithelial and stromal cells through fibroblast growth factor 9 (FGF9) signaling pathway has been thought to cause failure of tissue homeostasis and development of malignant disease. Previous studies have reported that FGF9 expression increased in the prostate cancer xenografts derived from bone metastasis. The aim of our study is to investigate the role of FGF9 for progression of prostate cancer. Material & Methods: Immunohistochemical staining using anti-FGF9 antibody were performed for tissues derived from radical prostatectomy for 98 male patients. Cell viability and apoptosis of DU145, LNCaP and androgen-independent LNCaP subline (AI-LNCaP) were assessed with MTT assay and with Apopercentage apoptosis assay in the presence or absence of treatment with recombinant FGF9 or anti-FGF9 neutralizing antibody, respectively. Results: In immunohistochemical staining, FGF9 positive cells were detected in 12 samples. In cases with Gleason score 8 or higher, rates of cases with FGF9 positive cells were 32.1%, significantly higher than those in case with Gleason score 6 (3.3%, p=0.016) and 7 (5.0%, p=0.0079), respectively. 5-year biochemical relapse-free survival rate in cases with FGF9 positive cells were 31.3%, significantly lower than those in cases that FGF9 positive cells were not detectable (77.6%, p=0.0008). In condition with androgen-deprivated medium, cell viability of LNCaP was significantly lower than that of AI-LNCaP, and enhanced to the same level by treatment with FGF9. Furthermore, cell viability of AI-LNCaP and DU145 were significantly suppressed by treatment with anti-FGF9 neutralizing antibody. Significantly more apoptotic cells were detected in LNCaP than in AI-LNCaP by androgen deprivation, and decreased to same level as in AI-LNCaP by treatment with FGF9. Conclusions: These results indicate that FGF9 can contribute for progression of prostate cancer through stimulation for cell proliferation and anti-apoptotic effect. FGF9 may be candidates of novel therapeutic targets for hormone-refractory prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3186.

Abstract B153: Small molecule modulators of tumor-stromal cell interactions, new natural compounds from fungi

Abstract Tumor microenvironment is now recognized as one of attractive strategies for cancer therapy. We have been studying molecular targets for tumor-stromal cell interactions and searching for small molecules that modulate the interactions. We have previously reported that angiogenin and IGF-I play critical roles in the development of prostate cancer. Because small molecules that interfere with these targets could suppress tumor growth through tumor-stromal cell interactions, we have designed assay systems and conducted random screening to find active small molecules. Here we report new natural compounds that can modulate the interactions. Terrein glucoside, a new terrein, was isolated from Aspergillus sp. PF1381 as a specific inhibitor of angiogenin secretion. Angiogenin is one of angiogenic factors that confer high tumorigenicity in human prostate cancer LNCaP cells. Terrein glucoside inhibited angiogenin secretion from a tumorigenic LNCaP subline without effect on VEGF secretion and also inhibited tube formation of HUVEC. On the other hand, NBRI23477 A and B, new atpenins, were isolated from Penicillium atramentosum PF1420 by another assay system. Human prostate stromal cells (PrSC) increased the growth of human prostate cancer DU-145 cells in the coculture of both cells through IGF-I secreted from PrSC. NBRI23477 A and B inhibited the growth of DU-145 cells in the coculture with PrSC more strongly than that of DU-145 cells cultured alone suggesting that these compounds indirectly suppress the tumor growth through modulation of tumor-stromal cell interactions. We are now continuing these screening programs to find more potent compounds. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B153.

EB1089 inhibits the parathyroid hormone–related protein–enhanced bone metastasis and xenograft growth of human prostate cancer cells

Parathyroid hormone-related protein (PTHrP) plays a major role in prostate carcinoma progression and bone metastasis. Once prostate cancers become androgen-independent, treatment options become limited. Vitamin D analogues represent a potentially valuable class of agents in this clinical context. Using the prostate cancer cell line C4-2 as a model, we studied the effects of PTHrP and the noncalcemic vitamin D analogue EB1089 on markers of prostate cancer cell progression in vitro and in vivo. C4-2 is a second-generation androgen-independent LNCaP subline that metastasizes to the lymph nodes and bone when injected into nude mice and produces mixed lytic/blastic lesions, mimicking the in vivo situation. We report that PTHrP increases cell migration and invasion, and that a pathway via which EB1089 inhibits these processes is through down-regulation of PTHrP expression. PTHrP also increases anchorage-independent cell growth in vitro and xenograft growth in vivo; EB1089 reverses these effects. The in vivo PTHrP effects are accompanied by increased tumor cell proliferation and survival. Treatment with EB1089 reverses the proliferative but not the antiapoptotic effects of PTHrP. PTHrP also increases intratumor vessel density and vascular endothelial growth factor expression; EB1089 reverses these effects. Intracardially injected C4-2 cells produce predominantly osteoblastic lesions; PTHrP overexpression decreases the latency, increases the severity and alters the bone lesion profile to predominantly osteolytic. EB1089 largely reverses these PTHrP effects. A direct correlation between PTHrP immunoreactivity and increasing tumor grade is observed in human prostate cancer specimens. Thus, decreasing PTHrP production by treatment with vitamin D analogues may prove therapeutically beneficial for prostate cancer.

Improvement in predicting tumorigenic phenotype of androgen‐insensitive human LNCaP prostatic cancer cell subline in recombination with rat urogenital sinus mesenchyme

Hormone-refractory prostate cancer, a heterogeneous disease, has varying degrees of androgen sensitivity. To understand the physiological changes in the hormone-refractory state, the present study used a lineage-derived androgen receptor (AR)-positive, androgen-insensitive prostate cancer cell line and evaluated the tumorigenic phenotype, focusing on tumor-stromal interactions in vivo. First, tumorigenic differences of cancer cells alone were examined in an androgen-insensitive AR-positive LNCaP subline, AIDL, compared with those of the androgen-sensitive AR-positive parental LNCaP and the androgen-insensitive AR-negative PC-3 cells transplanted into subcutaneous, sub-renal and prostatic orthotopic graft sites. Next, cancer cells were recombined with rat urogenital sinus mesenchyme (rUGM) to simulate the tumor-stromal microenvironment. Tumors of AIDL and LNCaP without stromal components both formed well-defined globular tumors and contained large blood-filled areas, with no significant difference in tumor growth or histopathology regardless of the cell line's androgen sensitivity or graft site. In contrast, tumors of AIDL and LNCaP recombined with rUGM both showed reduction of blood-filled areas in the tumors and increased tumor growth compared with cancer cells alone. Tumors of AIDL + rUGM recombinants were approximately three times as large as those of LNCaP + rUGM recombinants, whereas tumors of AIDL and LNCaP without rUGM were not different in size. In addition to the tumor size, cell proliferation (Ki-67 labeling index) in tumors of AIDL + rUGM recombinants was significantly higher than that in tumors of LNCaP + rUGM recombinants. Immunoreactivities of AR, E-cadherin and beta-catenin were decreased in AIDL + rUGM recombinants relative to AIDL alone and LNCaP + rUGM recombinants. These results demonstrated that tumorigenic features of androgen-insensitive AR-positive prostate cancer cells could be significantly influenced by rUGM. Therefore, this in vivo recombination model with rUGM may be useful in developing new treatment strategies.

Suppression of androgen receptor signaling and prostate specific antigen expression by (−)-epigallocatechin-3-gallate in different progression stages of LNCaP prostate cancer cells

The green tea polyphenol, (−)-epigallocatechin-3-gallate (EGCG), inhibits the development and progression of prostate cancer in TRAMP mice and in men. We examined the effects of EGCG on LNCaP human prostate cancer sublines 104-S, 104-R1 and R1Ad representing different progression stages of prostate cancer. EGCG suppressed cell proliferation, prostate specific antigen (PSA) expression, and AR transcriptional activity in the different LNCaP sublines. Intraperitoneal administration of EGCG also suppressed the growth of relapsing R1Ad tumors and decreased tumor-derived serum PSA. Effects of EGCG on tumor PSA expression have the potential to affect accurate monitoring of patient tumor burden by serum PSA measurements.

Impairment of the DNA Repair and Growth Arrest Pathways by p53R2 Silencing Enhances DNA Damage–Induced Apoptosis in a p53-Dependent Manner in Prostate Cancer Cells

p53R2 is a p53-inducible ribonucleotide reductase that contributes to DNA repair by supplying deoxynucleotide triphosphate pools in response to DNA damage. In this study, we found that p53R2 was overexpressed in prostate tumor cell lines compared with immortalized prostatic epithelial cells and that the protein was induced upon DNA damage. We investigated the effects of p53R2 silencing on DNA damage in LNCaP cells (wild-type p53). Silencing p53R2 potentiated the apoptotic effects of ionizing radiation and doxorubicin treatment as shown by increased sub-G(1) content and decreased colony formation. This sensitizing effect was specific to DNA-damaging agents. Comet assay and gamma-H2AX phosphorylation status showed that the decreased p53R2 levels inhibited DNA repair. Silencing p53R2 also reduced the levels of p21(WAF1/CIP1) at the posttranscriptional level, suggesting links between the p53-dependent DNA repair and cell cycle arrest pathways. Using LNCaP sublines stably expressing dominant-negative mutant p53, we found that the sensitizing effect of p53R2 silencing is mediated by p53-dependent apoptosis pathways. In the LNCaP sublines (R273H, R248W, and G245S) that have defects in inducing p53-dependent apoptosis, p53R2 silencing did not potentiate DNA damage-induced apoptosis, whereas p53R2 silencing was effective in a LNCaP subline (P151S) which retains the ability to induce p53-dependent apoptosis. This study shows that p53R2 is a potential therapeutic target that could be used to enhance the effectiveness of ionizing radiation or DNA-damaging chemotherapy in a subset of patients with prostate cancer.

IMPAIRMENT OF THE DNA REPAIR AND GROWTH ARREST PATHWAYS BY p53R2 SILENCING ENHANCES DNA DAMAGE- INDUCED APOPTOSIS IN A p53-DEPENDENT MANNER IN PROSTATE CANCER CELLS

p53R2 is a p53-inducible ribonucleotide reductase that contributes to DNA repair by supplying deoxynucleotide triphosphate pools in response to DNA damage. In this study, we found that p53R2 was overexpressed in prostate tumor cell lines compared with immortalized prostatic epithelial cells and that the protein was induced upon DNA damage. We investigated the effects of p53R2 silencing on DNA damage in LNCaP cells (wild-type p53). Silencing p53R2 potentiated the apoptotic effects of ionizing radiation and doxorubicin treatment as shown by increased sub-G(1) content and decreased colony formation. This sensitizing effect was specific to DNA-damaging agents. Comet assay and gamma-H2AX phosphorylation status showed that the decreased p53R2 levels inhibited DNA repair. Silencing p53R2 also reduced the levels of p21(WAF1/CIP1) at the posttranscriptional level, suggesting links between the p53-dependent DNA repair and cell cycle arrest pathways. Using LNCaP sublines stably expressing dominant-negative mutant p53, we found that the sensitizing effect of p53R2 silencing is mediated by p53-dependent apoptosis pathways. In the LNCaP sublines (R273H, R248W, and G245S) that have defects in inducing p53-dependent apoptosis, p53R2 silencing did not potentiate DNA damage-induced apoptosis, whereas p53R2 silencing was effective in a LNCaP subline (P151S) which retains the ability to induce p53-dependent apoptosis. This study shows that p53R2 is a potential therapeutic target that could be used to enhance the effectiveness of ionizing radiation or DNA-damaging chemotherapy in a subset of patients with prostate cancer.

Androgen receptor mediates the expression of UDP‐glucuronosyltransferase 2 B15 and B17 genes

Enhanced androgen receptor (AR) activity by increased testosterone availability may play important roles in prostate cancer progressing to castration resistant state. Comparison of expression profiles in androgen dependent and independent prostate tumors demonstrated a marked increase of the expression of UDP-glucuronosyltransferase 2B15 (UGT2B15), an androgen catabolic enzyme. We investigated mechanisms controlling the differential expression of UGT2B15 and B17 in response to androgen treatments. Gene expression was determined by RT-PCR. The association of AR with UGT2B15/B17 genes was determined by Chromatin immuno-precipitation (CHIP). RNA interference was used to knock-down gene expression. UGT2B15 and B17 genes were not expressed in AR negative prostate cancer cell lines, PC3 and DU145, while they were expressed in AR positive cell lines, LNCaP, LNCaP-abl (an androgen independent LNCaP sub-line), and VCaP. The expression levels of UGT2B15/B17 were up-regulated in LNCaP-abl comparing to those in LNCaP. These results suggest the requirement of AR for the expression of UGT2B15/B17. Treatment with DHT down-regulated the expression of UGT2B15/B17 in LNCaP in a time and dose dependent manner and this down-regulation was competitively antagonized by flutamide and bicalutimide, suggesting a pathway mediated by AR. Further CHIP experiments demonstrated the direct interaction of AR with the promoter regions of UGT2B15/B17 genes. Knocking down AR expression in LNCaP significantly reduced the expression of UGT2B15/B17 and completely inhibited the DHT-induced down-regulation of UGT2B15/B17 genes. We demonstrated that UGT2B15 and B17 are primary androgen-regulated genes and AR is required for both their basal expression and their androgen-regulated expression.

Aberrant expression of extracellular signal-regulated kinase 5 in human prostate cancer

Abnormal intracellular signaling contributes to carcinogenesis and may represent novel therapeutic targets. mitogen/extracellular signal-regulated kinase kinase-5 (MEK5) overexpression is associated with aggressive prostate cancer. In this study, we examined the role of extracellular signal-regulated kinase (ERK5, an MAPK and specific substrate for MEK5) in prostate cancer. ERK5 immunoreactivity was significantly upregulated in high-grade prostate cancer when compared to benign prostatic hyperplasia (P<0.0001). Increased ERK5 cytoplasmic signals correlated closely with Gleason sum score (P<0.0001), bony metastases (P=0.0044) and locally advanced disease at diagnosis (P=0.0023), with a weak association with shorter disease-specific survival (P=0.036). A subgroup of patients showed strong nuclear ERK5 localization, which correlated with poor disease-specific survival and, on multivariant analysis, was an independent prognostic factor (P<0.0001). Analysis of ERK5 expression in matched tumor pairs (before and after hormone relapse, n=26) revealed ERK5 nuclear expression was significantly associated with hormone-insensitive disease (P=0.0078). Similarly, ERK5 protein expression was increased in an androgen-independent LNCaP subline. We obtained the following in vitro and in vivo evidence to support the above expression data: (1) cotransfection of ERK5wt and MEK5D constructs in PC3 cells results in predominant ERK5 nuclear localization, similar to that observed in aggressive clinical disease; (2) ERK5-overexpressing PC3 cells have enhanced proliferative, migrative and invasive capabilities in vitro (P<0.0001), and were dramatically more efficient in forming tumors, with a shorter mean time for tumors to reach a critical volume of 1000 mm(3), in vivo (P<0.0001); (3) the MEK1 inhibitor, PD184352, blocking ERK1/2 activation at low dose, did not suppress proliferation but did significantly decrease proliferation at a higher dose required to inhibit ERK5 activation. Taken together, our results establish the potential importance of ERK5 in aggressive prostate cancer.

Isolation and Characterization of LNCaP Sublines Differing in Hormone Sensitivity

Prostate cancer is a heterogeneous disease with varying degrees of androgen sensitivity. In this study, we performed a limiting dilution of human prostate LNCaP cells, and isolated two sublines, LNCaP-E9 and LNCaP-G4, with differential hormone-sensitivity. Two LNCaP sublines were obtained by the limiting dilution method. The growth of E9 cells was decreased in the presence of androgens, while that of androgen-treated G4 cells was biphasic. Although the androgen receptor expression level in E9 cells was similar to that seen in G4 cells, the expression of PSA mRNA and protein was significantly lower in the E9 cells. Moreover, the androgen-based stimulation of PSA mRNA expression was less sensitive in E9 cells than G4 cells. Intracellular zinc level did not differ between E9 and G4 cells, but ZnT3 mRNA expression was significantly higher in the E9 cells. When the cells were grafted at the subrenal capsule, the number of CD31-positive vessels with a lumen was approximately 2.5 times higher than that in G4 tumors. LNCaP-E9 cells show lower androgen sensitivity than LNCaP-G4 cells. E9 and G4 cells would be helpful for understanding the biology of hormone-refractory prostate cancer.

Met-Independent Hepatocyte Growth Factor-mediated regulation of cell adhesion in human prostate cancer cells

BackgroundProstate cancer cells communicate reciprocally with the stromal cells surrounding them, inside the prostate, and after metastasis, within the bone. Each tissue secretes factors for interpretation by the other. One stromally-derived factor, Hepatocyte Growth Factor (HGF), was found twenty years ago to regulate invasion and growth of carcinoma cells. Working with the LNCaP prostate cancer progression model, we found that these cells could respond to HGF stimulation, even in the absence of Met, the only known HGF receptor. The new HGF binding partner we find on the cell surface may help to clarify conflicts in the past literature about Met expression and HGF response in cancer cells.MethodsWe searched for Met or any HGF binding partner on the cells of the PC3 and LNCaP prostate cancer cell models, using HGF immobilized on agarose beads. By using mass spectrometry analyses and sequencing we have identified nucleolin protein as a novel HGF binding partner. Antibodies against nucleolin (or HGF) were able to ameliorate the stimulatory effects of HGF on met-negative prostate cancer cells. Western blots, RT-PCR, and immunohistochemistry were used to assess nucleolin levels during prostate cancer progression in both LNCaP and PC3 models.ResultsWe have identified HGF as a major signaling component of prostate stromal-conditioned media (SCM) and have implicated the protein nucleolin in HGF signal reception by the LNCaP model prostate cancer cells. Antibodies that silence either HGF (in SCM) or nucleolin (on the cell surfaces) eliminate the adhesion-stimulatory effects of the SCM. Likewise, addition of purified HGF to control media mimics the action of SCM. C4-2, an LNCaP lineage-derived, androgen-independent human prostate cancer cell line, responds to HGF in a concentration-dependent manner by increasing its adhesion and reducing its migration on laminin substratum. These HGF effects are not due to shifts in the expression levels of laminin-binding integrins, nor can they be linked to expression of the known HGF receptor Met, as neither LNCaP nor clonally-derived C4-2 sub-line contain any detectable Met protein. Even in the absence of Met, small GTPases are activated, linking HGF stimulation to membrane protrusion and integrin activation. Membrane-localized nucelolin levels increase during cancer progression, as modeled by both the PC3 and LNCaP prostate cancer progression cell lines.ConclusionWe propose that cell surface localized nucleolin protein may function in these cells as a novel HGF receptor. Membrane localized nucleolin binds heparin-bound growth factors (including HGF) and appears upregulated during prostate cancer progression. Antibodies against nucleolin are able to ameliorate the stimulatory effects of HGF on met-negative prostate cancer cells. HGF-nucleolin interactions could be partially responsible for the complexity of HGF responses and met expression reported in the literature.

Differential regulation of IGFBP-3 by the androgen receptor in the lineage-related androgen-dependent LNCaP and androgen-independent C4-2 prostate cancer models

Despite evidence implicating insulin-like growth factor binding protein-3 (IGFBP-3) as a growth inhibitor of prostate cancer (CaP), little is known about changes in its regulation and function during progression to androgen independence. The expression levels of IGFBP-3 were determined by cDNA microarray analysis and tissue microarrays (TMAs) after androgen ablations. LNCaP (LN-BP3) and C4-2 (C4-2-BP3) sublines were used to compare the apoptotic effects of IGFBP-3 in LNCaP (androgen-dependent) and C4-2 (androgen-independent) cells. After androgen deprivation, IGFBP-3 mRNA levels increased more in C4-2 compared to LNCaP cells. Androgens suppressed IGFBP-3 levels in a dose-dependent manner in LNCaP and C4-2 cell. IGFBP-3 expression was increased after NHT in human CaP tissues. Apoptotic rates increased in LN-BP3, but not C4-2-BP3 cells, following doxycycline-mediated IGFBP-3 induction. C4-2 cell survival in an androgen-depleted environment may be facilitated through differential resistance to the apoptotic effects elicited by IGFBP-3.

Expression of the inhibitors of apoptosis proteins in cisplatin-resistant prostate cancer cells

Acquired multi-drug resistance remains a major obstacle in the management of prostate cancer. The objective of this study was to examine whether chemoresistance could be due in part to the expression of the inhibitors of apoptosis proteins (IAPs). We established cisplatin-resistant LNCaP sublines. We examined the effects of cisplatin on cell growth and apoptosis in LNCaP cells and LNCaP sublines by 2-(4-lodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1) assay and Hoechst 33258 staining, and analyzed cross-resistance to adriamycin, 5-fluorouracil, taxol, taxotere, and etoposide. In addition, the expression of IAP-1, IAP-2, X-linked IAP (XIAP), neuronal apoptosis inhibitory protein, and survivin was investigated by immunoblot analysis in LNCaP sublines. Although the growth rates were reduced in a dose-dependent manner by cisplatin in LNCaP sublines, the anti-proliferative effects of cisplatin were significantly decreased in LNCaP sublines compared to LNCaP cells. Cisplatin-resistant sublines, LNCaP/C1, LNCaP/ C2, and LNCaP/C3 cells, were 6.3-, 9.1-, and 22.3-fold more resistant to cisplatin than LNCaP cells, respectively, and this resistance was paralleled with reduced induction of apoptosis. LNCaP/C3 cells showed cross-resistance to adriamycin, 5-fluorouracil, and etoposide whereas those cells exhibited no or only weak cross-resistance against taxol and taxotere. With the exception of survivin, all the IAPs were identified in LNCaP cells by immunoblot analysis. Interestingly, the expression of IAP-2, XIAP, and survivin gradually increased with the extent of cisplatin-resistance. Altered expression of IAP-2, XIAP, and survivin was involved in these phenotypes of cisplatin-resistant LNCaP sublines. IAPs may make an important contribution to the resistance to the apoptotic effect of cisplatin in prostate cancer.

Inhibition of BCL-2 by Stealth siRNA results in growth suppression of LNCaP cells

9626 Background: RNA interference is a powerful laboratory tool for gene silencing; however, the kinetics of unmodified small interfering RNA (siRNA) currently preclude most in vivo or clinical applications. Despite these limitations, targeted inhibition of key cancer survival genes would increase therapeutic options. We therefore tested Stealth siRNA (Invitrogen Corp), a novel modified siRNA with enhanced stability, directed against the Bcl-2 gene, a key anti-apoptotic regulator, in the prostate cell line LNCaP. Methods: Proof-of-principle experiments were conducted using an LNCaP sub-line constitutively expressing enhanced green fluorescent protein (eGFP). Fluorescent intensity was measured by flow cytometry and with a 96-well fluorescent plate reader controlling for cell number. Lipofectamine 2000 was used as a delivery vehicle. Bcl-2 silencing was measured by RT-PCR and Western blotting and cell growth was assessed by MTT assay and flow cytometry of DNA content. Non-silencing Stealth siRNA with matched GC content served as controls. Results: Stealth siRNA reduced fluorescent emission of the highly stable eGFP by 72% by day 5, with an increased duration of inhibition compared to conventional siRNA of the same sequence and concentration. Based on these findings, Stealth siRNA were developed against the BCL-2 gene. A single administration of anti-BCL-2 inhibitors (40nM) reduced BCL-2 mRNA to below the threshold of detection and greatly decreased protein levels. In a 96-well format, treatment resulted in substantial inhibition of LNCaP growth (>70%), compared to non-silencing controls. DNA content was measured on day 5 after transfection. At this time-point, minimal effect on cell cycling was observed, while apoptosis was modestly increased (8%). In androgen depleted media, where LNCaP growth is minimized, silencing of Bcl-2 resulted in a further reduction in cell number. Conclusions: In controlled comparisons measuring reduction in eGFP fluorescence, Stealth siRNA showed potent inhibition and an increased duration of response, likely stemming from its enhanced stability. Treatment with anti-Bcl-2 Stealth siRNA resulted in substantial growth inhibition of LNCaP cells, warranting further testing in in vivo models. No significant financial relationships to disclose.

Cisplatin inhibits the expression of X-linked inhibitor of apoptosis protein in human LNCaP cells

The purpose of the present study is to investigate the role of X-linked inhibitor of apoptosis protein (XIAP) in the regulation of apoptosis induced by cisplatin in human prostate cancer cell line (LNCaP). We examined the effects of cisplatin on cell growth and apoptosis in LNCaP by 2-(4-lodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1), flow cytometric analyses, and caspase-3 activity assay. In addition, to clarify the roles of the XIAP, we established clonal cell lines that overexpressed XIAP. The effects of cisplatin on the XIAP expression in the induction of apoptosis in LNCaP were examined by RT-PCR and immunoblot analyses. Although the growth rates were reduced in a dose- and time-dependent manner by cisplatin in LNCaP sublines, the anti-proliferative effects of cisplatin were significantly decreased in XIAP stably overexpressing cell lines. In addition, we found that cisplatin-induced apoptosis following activation of caspase-3, and that the overexpression of XIAP inhibited apoptosis by attenuating caspase-3 activity. Interestingly, treatment of LNCaP cells with 10 and 100 μM cisplatin for 48 h significantly decreased the expression of XIAP at both the protein and mRNA levels in a dose-dependent manner. Furthermore, 10-μM cisplatin treatment of LNCaP decreased XIAP mRNA and protein in a time-dependent manner. These results suggest that cisplatin induces apoptosis by the inhibition of XIAP expression, and that XIAP plays an important role in the regulation of cisplatin-induced apoptosis in LNCaP cells. The ability of cisplatin to down-regulate XIAP may be an important mechanism in chemosensitivity.

Changes in androgen receptor nongenotropic signaling correlate with transition of LNCaP cells to androgen independence.

A cure for prostate cancer (CaP) will be possible only after a complete understanding of the mechanisms causing this disease to progress from androgen dependence to androgen independence. To carry on a careful characterization of the phenotypes of CaP cell lines before and after acquisition of androgen independence, we used two human CaP LNCaP sublines: LNCaP(nan), which is androgen dependent (AD), and LNCaP-HP, which is androgen independent (AI). In AD LNCaP(nan) cells, dihydrotestosterone (DHT) stimulated in an androgen receptor (AR)-dependent way a phosphorylation signaling pathway involving steroid receptor coactivator (Src)-mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)-1/2-ERK-1/2-cAMP-response element binding-protein (CREB). Activation of this pathway was associated with increased [(3)H]thymidine incorporation and resistance to apoptosis. Use of dominant-negative forms of MEK-1/2 and CREB demonstrated in LNCaP(nan) cells that DHT induced [(3)H]thymidiine incorporation through a thus far unidentified molecule activated downstream of MEK-1/2, and antiapoptosis through phosphorylation of the transcription factor CREB. In contrast, in AI LNCaP-HP cells, the Src-MEK-1/2-ERK-1/2-CREB pathway was constitutively active. Because it was not further stimulated by addition of DHT, no increase of [(3)H]thymidine incorporation or apoptosis resistance was demonstrated in LNCaP-HP cells. Additional experiments showed that Src and the scaffold protein MNAR coimmunoprecipitated with AR, indicating a role for Src as an apical molecule in the Src-MEK-1/2-ERK-1/2-CREB pathway. Interestingly, differences between the two cell lines were that in LNCaP-HP cells presence of an AI phenotype and lack of response to DHT were associated with constitutive activation of the protein kinase Src and interaction among Src, AR, and MNAR. In contrast, in LNCaP(nan) cells, presence of an AD phenotype and ability to respond to DHT were associated with DHT-dependent activation of Src kinase activity and interaction among Src, AR, and MNAR. Intriguingly, in LNCaP(nan) cells, we found that transcription through the prototypical CREB-responsive promoter c-fos could be induced in a DHT-dependent way, and this action was inhibited by the AR antagonist Casodex and MEK-1 inhibitor PD98059. In contrast, transcription through the PSA P/E promoter, a prototypical AR-dependent promoter directly activated by agonist, was obliterated only by Casodex. Additional experiments with genital skin fibroblasts derived from patients with a variety of AR abnormalities indicated that nongenotropic AR signaling does not depend on an intact DNA-binding domain or on the ability of AR to translocate to the nucleus. The results suggest the following: (1) Constitutive activation of the Src-MEK-1/2-ERK-1/2-CREB pathway is associated with the AI phenotype observed in LNCaP-HP cells. (2) Activation of the Src-MEK-1/2-ERK-1/2-CREB pathway is DHT dependent in AD LNCaP(nan) cells. (3) DHT activation of this pathway is associated with induction of [(3)H]thymidine incorporation by a molecule activated downstream of MEK-1/2 and of antiapoptosis through activation of the transcription factor CREB in AD LNCaP(nan) cells. (4) AR regulates transcription either directly upon ligand binding and nuclear translocation or indirectly through kinase pathways leading to activation of downstream transcription factors. (5) Nuclear translocation and ability of the DNA-binding domain of AR to interact with DNA are not prerequisites for nongenotropic AR activity.