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.     

Calreticulin expression is associated with androgen regulation of the sensitivity to calcium ionophore-induced apoptosis in LNCaP prostate cancer cells

Calreticulin has been identified previously as one of the androgen-response genes in the prostate. The role of calreticulin in androgen action was studied using androgen-sensitive LNCaP and androgen-insensitive PC-3 human prostate cancer cell lines. Calreticulin appears to be a primary androgen-response gene in cultured LNCaP cells because androgen induction of calreticulin mRNA resists protein synthesis inhibition. Calreticulin is a high capacity intracellular Ca2+ binding protein, suggesting that calreticulin expression is likely to be associated with the intracellular Ca2+ buffering capacity that could regulate the sensitivity to cytotoxic intracellular Ca2+ overload. As expected, androgen protects androgen-sensitive LNCaP but not androgen-insensitive PC-3 cells from cytotoxic intracellular Ca2+ overload induced by Ca2+ ionophore A23187. To provide evidence for the role of calreticulin in reducing cytotoxic effect of Ca2+ influx in prostatic cells, we have shown that calreticulin antisense oligonucleotide down-regulates calreticulin protein level and significantly increases the sensitivity to A23187-induced apoptosis in both LNCaP and PC-3 cells. Furthermore, calreticulin antisense oligonucleotide reverses the androgen-induced resistance to A23187 in LNCaP cells. The above observations collectively suggest that calreticulin mediates androgen regulation of the sensitivity to Ca2+ ionophore-induced apoptosis in LNCaP cells.     

Castration-induced increases in insulin-like growth factor-binding protein 2 promotes proliferation of androgen-independent human prostate LNCaP tumors

Activation of alternative growth factor pathways after androgen withdrawal is one mechanism mediating androgen-independent (AI) progression in advanced prostate cancer. Insulin-like growth factor (IGF) I activation is modulated by a family of IGF binding proteins (IGFBPs). Although IGFBP-2 is one of the most commonly overexpressed genes in hormone refractory prostate cancer, the functional significance of changes in IGF-I signaling during AI progression remains poorly defined. In this article, we characterize changes in IGFBP-2 in the LNCaP tumor model after androgen withdrawal and evaluate its functional significance in AI progression using gain-of-function and loss-of-function analyses. IGFBP-2 mRNA and protein levels increase 2-3-fold after androgen withdrawal in LNCaP cells in vitro in LNCaP tumors during AI progression in vivo. Increased IGFBP-2 levels after castration were also identified using a human prostate tissue microarray of untreated and posthormone therapy-treated prostatectomy specimens. LNCaP cell transfectants that stably overexpressed IGFBP-2 progressed more rapidly after castration than control tumors. Antisense oligonucleotides (ASOs) targeting the translation initiation site of IGFBP-2 reduced IGFBP-2 mRNA and protein expression by >70% in a dose-dependent and sequence-specific manner. ASO-induced decreases in IGFBP-2-reduced LNCaP cell growth rates and increased apoptosis 3-fold. LNCaP tumor growth and serum prostate-specific antigen levels in mice treated with castration plus adjuvant IGFBP-2 ASOs were significantly reduced compared with mismatch control oligonucleotides. Increased IGFBP-2 levels after androgen ablation may represent an adaptive response that helps potentiate IGF-I-mediated survival and mitogenesis and promote androgen-independent tumor growth. Inhibiting IGFBP-2 expression using ASO technology may offer a treatment strategy to delay AI progression.     

Antiproliferative effects of AVN944, a novel inosine 5-monophosphate dehydrogenase inhibitor, in prostate cancer cells

Inosine 5-monophosphate dehydrogenase II, a key enzyme in the de novo synthesis of purine nucleotides, is expressed in prostate tumors and prostate cancer cells. AVN944 is a new, specific, noncompetitive IMPDH inhibitor. In this study, we investigated the effects of IMPDH inhibitor AVN944 on LNCaP, CWR22Rv1, DU145 and PC-3 human prostate cancer cells. AVN944 inhibited proliferation of these 4 prostate cancer cell lines and was associated with cell cycle G1 arrest of LNCaP cells and S-phase block of androgen-independent CWR22Rv1, DU145 and PC-3 cells. AVN944 induced caspase-dependentand caspase-independent cell death in LNCaP, CWR22Rv1, and DU145 cells. AVN944 induced expression of p53-target proteins Bok, Bax and Noxa in androgen-responsive cell lines and suppressed expression of survivin in prostate cancer cells regardless of their androgen sensitivity. AVN944 also induced differentiation of androgen-independent prostate cancer cells as indicated by morphological changes and increased expression of genes coding for prostasomal proteins, keratins and other proteins, including tumor suppressor genes MIG-6 and NDRG1. AVN944-differentiated androgen-independent DU145 and PC-3 cells are sensitized to TRAIL-induced apoptosis as demonstrated by induction of caspases and PARP cleavage. In summary, AVN944 inhibited the growth of human prostate cancer cells by inducing cell cycle arrest, cell death as well as differentiation. AVN944 is a novel, promising therapeutic agent that might be combined with other agents for treatment of human prostate cancer.     

Insulin-like growth factor binding protein-6 inhibits prostate cancer cell proliferation: implication for anticancer effect of diethylstilbestrol in hormone refractory prostate cancer

Diethylstilbestrol (DES) is a synthetic oestrogen, and its anticancer effects are exerted in androgen-dependent prostate cancer. The administration of DES decreases serum testosterone to castration levels. However, in androgen-independent prostate cancer patients, who are already orchiectomised, the administration of DES improves symptoms and decreases prostate-specific antigen (PSA). The mechanisms responsible for these direct inhibitory effects have been explained as biological actions not mediated by oestrogen receptors. We assessed the gene expression profiles of prostate cancer cells treated with DES, and investigated direct inhibitory effects of DES. DES inhibited the proliferation of LNCaP and PC-3 cells. cDNA microarray analysis showed that expression of many genes was downregulated by DES. However, insulin-like growth factor binding protein 6 (IGFBP-6) gene expression levels were upregulated in PC-3 cells. IGFBP-6 gene expression and protein levels significantly increased after DES treatment. Recombinant IGFBP-6 inhibited cell proliferation, and the inhibitory effect of DES was neutralised by anti-IGFBP-6 antibody. From the immunohistochemical analysis of IGFBP-6 using biopsy samples from androgen-independent prostate cancer, we found IGFBP-6 expression in androgen independent prostate cancer, and that DES treatment increased the IGFBP-6 staining intensity of the cancer cells in one sample. These findings suggested that DES induces IGFBP-6, which inhibits cell proliferation in an androgen-independent prostate cancer cell line, PC-3. IGFBP-6 therefore might be involved in the direct effects of DES in androgen-independent prostate cancer.     

Analysis of stromal-epithelial interactions in prostate cancer identifies PTPCAAX2 as a potential oncogene.

A PCR-based subtractive hybridisation technique was used to identify genes involved in stromal-epithelial interactions in prostate cancer. Eight genes were identified as being differentially expressed in benign prostatic fibroblast cells after stimulation with tumourigenic LNCaP conditioned media. One of these genes, protein tyrosine phosphatase CAAX2 (PTPCAAX2; also described as PTP4A and OV-1), has recently been shown to be oncogenic in hamster pancreatic epithelial cells. We show that PTPCAAX2 expression is up-regulated 4-fold in benign prostatic fibroblast cells 24 h after stimulation with LNCaP conditioned media and up-regulated 9-fold in prostatic tumour fibroblast cells. PTPCAAX2 overexpression was also detected in both androgen-dependent and androgen-independent prostate cancer cell lines and prostate tumour tissue, as determined by RT-PCR analysis and in situ hybridisation. These observations of PTPCAAX2 overexpression in prostate tumour cells and tissue suggest that PTPCAAX2 may potentially function as an oncogene in prostate cancer.     

Survivin mediates resistance to antiandrogen therapy in prostate cancer

Resistance to antiandrogen therapy in patients with metastatic prostate cancer poses a major challenge, which, if overcome, may lead to significant advances in the treatment of these patients. Hormone resistance of prostate cancer develops, in part, from upregulation of antiapoptotic genes after androgen deprivation. Given the accumulating evidence that Survivin, a new member of the inhibitor of apoptosis (IAP) family, is associated with both cancer progression and drug resistance, we hypothesized that Survivin plays a potentially important role in hormone therapy resistance, and that targeting of Survivin may enhance sensitivity to antiandrogen therapy in prostate cancer. Patterns of Survivin expression were assessed in three prostate cancer cell lines LNCaP, PC-3, and DU-145 using quantitative Western analysis. All three cell lines were found to strongly express Survivin. In LNCaP cells with intact androgen receptors (ARs), it was observed that androgen stimulation with 5alpha-dihydrotestosterone (DHT) increased Survivin expression. Conversely, treatment with Flutamide decreased Survivin expression in LNCaP cells. We next studied the functional effect of Survivin on sensitivity to Flutamide. LNCaP cells were infected with replication-deficient adenoviruses encoding either wild-type Survivin pAd-S(WT) or a phosphorylation-defective Survivin Thr34 --> Ala dominant-negative mutant pAd-S(T34A), and then treated with Flutamide. Cell viability and apoptosis were assessed in vitro and in vivo. It was determined that Survivin can mediate resistance to such antiandrogen therapies based on our assays. Direct androgen stimulation resulted in pan-cell cycle expression of Survivin, which was found to be mediated by AKT, as it was determined that exogenous insulin-like growth factor-1 (IGF-1), a known activator of AKT signaling, could increase Survivin expression and result in pan-cell cycle expression even in AR-negative prostate cancer cell lines PC-3 and DU-145. Given this alternative mechanism of Survivin expression and our findings that Survivin can mediate resistance to Flutamide treatment, we further investigated whether IGF-1-mediated activation of Survivin via AKT could mediate resistance to antiandrogen therapy. Both in vitro and in vivo, this was found to be the case, supporting a novel mechanism of resistance to antiandrogen therapy. Our study indicates that upregulation of Survivin via IGF-1 signaling confers resistance to Flutamide in prostate cancer cells. Targeted inhibition of Survivin appears to enhance the therapeutic effects of Flutamide in vitro and in vivo, revealing a novel strategy to enhance sensitivity to androgen ablation therapy.     

Guggulsterone-induced apoptosis in human prostate cancer cells is caused by reactive oxygen intermediate dependent activation of c-Jun NH2-terminal kinase

Guggulsterone, a constituent of Indian Ayurvedic medicinal plant Commiphora mukul, causes apoptosis in cancer cells but the sequence of events leading to cell death is poorly understood. We now show that guggulsterone-induced cell death in human prostate cancer cells is caused by reactive oxygen intermediate (ROI)-dependent activation of c-Jun NH(2)-terminal kinase (JNK). Exposure of PC-3 and LNCaP cells to apoptosis inducing concentrations of guggulsterone resulted in activation of JNK and p38 mitogen-activated protein kinase (p38 MAPK) in both cell lines and activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in LNCaP cells. The guggulsterone-induced apoptosis in PC-3/LNCaP cells was partially but statistically significantly attenuated by pharmacologic inhibition (SP600125) as well as genetic suppression of JNK activation. On the other hand, pharmacologic inhibition of p38 MAPK activation in PC-3 or LNCaP cells (SB202190) and ERK1/2 activation in LNCaP cells (PD98059) did not protect against guggulsterone-induced cell death. The guggulsterone treatment caused generation of ROI in prostate cancer cells but not in a normal prostate epithelial cell line (PrEC), which was also resistant to guggulsterone-mediated JNK activation. The guggulsterone-induced JNK activation as well as cell death in prostate cancer cells was significantly attenuated by overexpression of catalase and superoxide dismutase. In addition, guggulsterone treatment resulted in a decrease in protein level and promoter activity of androgen receptor in LNCaP cells. In conclusion, the present study reveals that the guggulsterone-induced cell death in human prostate cancer cells is regulated by ROI-dependent activation of JNK and guggulsterone inhibits promoter activity of androgen receptor.     

Role of SRC-1 in the promotion of prostate cancer cell growth and tumor progression

Prostate cancer is initially androgen dependent and there is evidence that androgen receptor continues to play a role in androgen-independent prostate cancer. Androgen receptor activity depends both on the level of androgens and on the level of coactivators that interact with androgen receptor. Our goal was to evaluate the role of the androgen receptor coactivator SRC-1 in prostate cancer progression. Using tissue arrays to measure SRC-1 protein levels, we found that increased SRC-1 expression in clinically localized, androgen-dependent cancer is associated with clinical and pathologic variables of increased tumor aggressiveness. Interestingly, there was variable expression of SRC-1 in normal prostate tissue which correlated with the staining intensity of the corresponding cancer tissue. To test the contribution of SRC-1, we examined its role in androgen-dependent LNCaP and androgen-independent C4-2 prostate cancer cell lines. Using small interfering RNA to reduce expression of androgen receptor, we found that androgen receptor was required both for cell growth and for basal expression of prostate-specific antigen in the androgen-independent C4-2 cell line. Thus, although the cells can grow in an androgen-depleted medium, they remained androgen receptor dependent. Reduction of SRC-1 expression significantly reduced growth and altered androgen receptor target gene regulation in both LNCaP and C4-2 cell lines whereas it had no effect on the growth of the androgen receptor-negative PC-3 and DU145 prostate cancer cell lines. Although the requirement for androgens and androgen receptor in the development of prostate cancer is well established, our study implicates enhanced androgen receptor activity through elevated expression of SRC-1 in the development of more aggressive disease in men with prostate cancer.     

Gene expression of angiogenic factors correlates with metastatic potential of prostate cancer cells

We hypothesize that expression of proangiogenic genes correlates with the metastatic potential of prostate cancer cells. LNCaP, DU-145, and PC-3 are prostate cancer cell lines with low, moderate, and high metastatic potential, respectively, as we demonstrated by their capacity to invade an extracellular matrix, an established tumor invasion assay. The constitutive gene expression of the proangiogenic factors, vascular endothelial growth factor, intercellular adhesion molecule-1, interleukin-8, and transforming growth factor-beta2, was significantly greater in the more metastatic DU-145 and PC-3 cells as compared with LNCaP cells. Matrix metalloproteinase (MMP)-9 is thought to contribute to the invasive phenotype of tumor cells. PC-3 cells showed increased expression of MMP-9 and membrane type 4-MMP as compared with LNCaP and DU-145. Tissue inhibitors of metalloproteinase 1 and 4 gene expression were elevated in DU-145 and PC-3 cells, but paradoxically, LNCaP cells had undetectable levels of these genes. We transfected and overexpressed MMP-9 in poorly metastatic LNCaP cells and measured their invasive activity. Transient expression of human MMP-9 in LNCaP cells produced a 3-5-fold increase in MMP-9 activity with a comparable increase in invasiveness. Antisense ablation of the expression of MMP-9 in DU-145 and PC-3 cells produced concomitant inhibition of the gene expression of the proangiogenic factors, vascular endothelial growth factor, and intercellular adhesion molecule-1 (ICAM-1). Treatment of DU-145 and PC-3 cells with a selective chemical inhibitor of MMP-9 proteinase activity also inhibited their invasive activity. These results support our hypothesis that metastatic potential of prostate cancer cells correlates with expression of proangiogenic factors.     

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)     

Heat shock protein 27 increases after androgen ablation and plays a cytoprotective role in hormone-refractory prostate cancer

Heat shock protein 27 (Hsp27) is a chaperone implicated as an independent predictor of clinical outcome in prostate cancer. Our aim was to characterize changes in Hsp27 after androgen withdrawal and during androgen-independent progression in prostate xenografts and human prostate cancer to assess the functional significance of these changes using antisense inhibition of Hsp27. A tissue microarray was used to measure changes in Hsp27 protein expression in 232 specimens from hormone naive and posthormone-treated cancers. Hsp27 expression was low or absent in untreated human prostate cancers but increased beginning 4 weeks after androgen-ablation to become uniformly highly expressed in androgen-independent tumors. Androgen-independent human prostate cancer PC-3 cells express higher levels of Hsp27 mRNA in vitro and in vivo, compared with androgen-sensitive LNCaP cells. Phosphorothioate Hsp27 antisense oligonucleotides (ASOs) and small interference RNA potently inhibit Hsp27 expression, with increased caspase-3 cleavage and PC3 cell apoptosis and 87% decreased PC3 cell growth. Hsp27 ASO and small interference RNA also enhanced paclitaxel chemosensitivity in vitro, whereas in vivo, systemic administration of Hsp27 ASO in athymic mice decreased PC-3 tumor progression and also significantly enhanced paclitaxel chemosensitivity. These findings suggest that increased levels of Hsp27 after androgen withdrawal provide a cytoprotective role during development of androgen independence and that ASO-induced silencing can enhance apoptosis and delay tumor progression.     

PC-1基因表达对前列腺癌细胞迁移能力的影响

背景与目的:PC-1基因在雄激素非依赖和高转移能力的前列腺癌C4-2细胞中高表达,在雄激素依赖及不转移的前列腺癌LNCaP细胞中低表达。本实验旨在研究PC-1对前列腺癌细胞迁移能力的影响。方法:构建PC-1稳定高表达的LNCaP细胞株和反义核酸调低内源性PC-1表达的C4-2细胞株。利用体外迁移系统检测PC-1表达对LNCaP和C4-2细胞迁移运动能力的影响。结果:体外迁移实验表明稳定转染提高PC-1的表达水平并未使LNCaP迁移细胞数增多(P>0.05),而反义核酸降低PC-1表达则使C4-2迁移细胞数降低(P<0.05)。PC-1蛋白水平升高不能提高LNCaP细胞迁移能力,但降低内源性PC-1表达则使C4-2细胞迁移能力明显降低。结论:PC-1可能在前列腺癌细胞侵袭过程中起一定作用。     

Androgen induction of urokinase gene expression in LNCaP cells is dependent on their interaction with the extracellular matrix

Urokinase-type plasminogen activator (uPA) plays a central role in tissue remodeling and cell invasion. In the present study, we examined the expression of uPA in the prostate cancer cell lines LNCaP, DU-145 and PC-3. In contrast to DU-145 and PC-3, the androgen-responsive cell line LNCaP does not express uPA. However, seeding LNCaP cells on fibronectin-coated plates stimulated a low level of uPA expression which was further induced upon exposure of the cells to dihydrotestosterone (DHT). Concomitant with the expression of uPA, an androgen-regulated expression of uPA receptor (uPAR) was induced. These results suggest that the interaction of LNCaP cells with the extracellular matrix plays a dominant role in the androgen control of uPA and uPAR gene expression.     

Calcium/calmodulin-dependent kinase II plays an important role in prostate cancer cell survival

It has recently been shown that the androgen receptor (AR) is the main factor that required for prostate cancer cells survival. We show that knocking down AR expression by siRNA induces PI3K-independent activation of Akt, which was mediated by calcium/calmodulin-dependent kinase II (CaMKII). We further show, for the first time, that prostate cancer cells express beta,gamma and delta CaMKII genes, and the expression of these genes is under the control of AR activity: active AR in the presence of androgens inhibits CaMKII gene expression whereas inhibition of AR activity results in elevated level of kinase activity and in enhanced expression of CaMKII-beta and -gamma genes. Overexpression of CaMKII genes results in resistance to apoptosis induced by KN-93, a CaMKII inhibitor, or wortmanninn, a PI3K/Akt inhibitor, in combination with doxorubicin, thapsigargin and TRAIL. Moreover, overexpression of CaMKII increases secretion of prostate specific antigen and promotes cell growth of LNCaP in steroid-free condition. Our data show that there is cross-talk between AR- and CaMKII-mediated pathways. The results of this study suggest that CaMKII is an important player in prostate cancer cells ability to escape apoptosis under androgen ablation and facilitate the progression of prostate cancer cells to an androgen independent state.     

The adrenal androgen androstenediol is present in prostate cancer tissue after androgen deprivation therapy and activates mutated androgen receptor

Despite an initial response to androgen deprivation therapy, prostate cancer (PCa) progresses eventually from an androgen-dependent to an androgen-independent phenotype. One of the mechanisms of relapse is antiandrogen withdrawal phenomenon caused by mutation of 877th amino acid of androgen receptor (AR). In the present study, we established a method to measure the concentration of androstenediol (adiol) in prostate tissue. We found that adiol maintains a high concentration in PCa tissue even after androgen deprivation therapy. Furthermore, adiol is a stronger activator of mutant AR in LNCaP PCa cells and induces more cell proliferation, prostate-specific antigen (PSA) mRNA expression, and PSA promoter than dihydrotestosterone (DHT). Because antiandrogen, bicalutamide, blocked adiol activity in LNCaP cells, it was suggested that adiol effect was mediated through AR. However, high concentration of bicalutamide was necessary to block completely adiol activity. These effects were specific to LNCaP cells because adiol had less effect in PC-3 PCa cells transfected with wild-type AR than DHT and had similar effect in PC-3 cells transfected with mutant AR. The mechanism that adiol activates mutant AR in LNCaP cells did not result from the increased affinity to mutant AR or from AR's association with coactivator ARA70. However, low concentration of adiol induced more AR nuclear translocation than DHT in LNCaP cells and not PC-3 cells transfected with AR. These results indicate that adiol may cause the progression of PCa even after hormone therapy.