Progression to androgen-independent LNCaP human prostate tumors: cellular and molecular alterations

Lethal phenotypes of human prostate cancer are characterized by progression to androgen-independence and metastasis. For want of a clinically relevant animal model, mechanisms behind this progression remain unclear. Our study used an in vivo model of androgen-sensitive LNCaP human prostate cancer cell xenografts in male SCID mice to study the cellular and molecular biology of tumor progression. Primary tumors were established orthotopically, and the mice were then surgically castrated to withdraw androgens. Five generations of androgen-independent tumors were developed using castrated host mice. Tumor samples were used to determine expressions of cellular and molecular markers. Androgen-independent tumors had increased proliferation and decreased apoptosis compared to androgen-sensitive tumors, outcomes associated with elevated expression of p53, p21/waf1, bcl-2, bax and the bcl-2/bax ratio. Blood vessel growth in androgen-independent tumor was associated with increased expression of vascular endothelial growth factor. Overexpression of androgen receptor mRNA and reduced expression of androgen receptor protein in androgen-independent tumors suggest that the androgen receptor signaling pathway may play an important role in the progression of human prostate cancer to androgen-independence. The in vivo orthotopic LNCaP tumor model described in our study mimics the clinical course of human prostate cancer progression. As such, it can be used as a model for defining the molecular mechanisms of prostate cancer progression to androgen-independence and for evaluating the effect of preventive or therapeutic regimens for androgen-independent human prostate cancer.     

Androgen causes growth suppression and reversion of androgen-independent prostate cancer xenografts to an androgen-stimulated phenotype in athymic mice

Most prostate cancer patients develop androgen-independent recurrent prostate tumors a few years after androgen ablation therapy. No therapy, however, has been shown to substantially extend survival in these patients. Previously, we reported that androgen suppresses the growth of androgen-independent LNCaP prostate tumor cells both in vitro and in vivo. In cell culture, androgen receptor (AR)-rich androgen-independent LNCaP 104-R1 cells adapt to growth suppression by androgen and then their growth is androgen stimulated. Because maintaining androgen dependency of prostate tumor cells should prolong the usefulness of androgen ablation therapy, we determined if androgen-independent prostate tumors would revert to an androgen-stimulated phenotype in vivo upon androgen treatment. Growth of the LNCaP 104-R1 tumors was suppressed by androgen, but tumors then adapted to suppression by androgen and growth became androgen stimulated. Tumor AR and prostate-specific antigen mRNA and protein were initially high in 104-R1 tumors but decreased during adaptation. Subsequent removal of androgen decreased the serum prostate-specific antigen level further and stopped the growth of the adapted tumors. Because androgen caused growth suppression and then reversion of androgen-independent tumors to an androgen-stimulated phenotype and because the growth of androgen-stimulated tumors could be restrained by androgen ablation, these results suggest a novel therapy for AR-positive androgen-independent prostate cancer.     

Antiproliferative effect of LXR agonists T0901317 and 22(R)-hydroxycholesterol on multiple human cancer cell lines

Previously, we and other groups reported that liver X receptor (LXR) agonists T0901317, 22(R)-hydroxycholesterol, and 24(S)-hydroxycholesterol suppressed the proliferation of prostate and breast cancer cells. In this study, we report that T0901317 and 22(R)-hydroxycholesterol treatment inhibited the proliferation of different progression stages of LNCaP human prostate cancer cells, as well as different commonly used human cancer cell lines. Cancer cell lines with higher LXRα mRNA expression were more sensitive to 22(R)-hydroxycholesterol-induced inhibition. T0901317 treatment decreased the percentage of the cell population in S-phase and caused G(1) cell cycle arrest. Overexpression of S-phase kinase-associated protein 2 (Skp2) partially blocked the suppressive effect of T0901317 treatment. Modulating LXR signaling is therefore a potential adjuvant therapy for advanced prostate cancer and other types of cancer.     

Combination antiangiogenic and androgen deprivation therapy for prostate cancer: a promising therapeutic approach.

PURPOSE: Androgen ablation therapy leads to mild regression or stabilization of prostate cancer, followed by progression to the fatal androgen-independent state. Whereas androgen ablation diminishes tumor angiogenesis by suppressing vascular endothelial growth factor (VEGF) production, androgen-independent disease is marked by androgen-independent VEGF expression. We examined combined androgen ablation and inhibition of VEGF signaling in an androgen-sensitive human prostate cancer xenograft model (LNCaP) that is known to develop androgen-independent growth after androgen ablation. EXPERIMENTAL DESIGN: N-(4-Bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine (ZD6474) is an orally active inhibitor of VEGF receptor tyrosine kinase activity, with additional activity against epidermal growth factor receptor tyrosine kinase. ZD6474 (50 mg/kg/d, per os) was administered to groups of castrated and noncastrated athymic mice bearing established (4-616 mm3) LNCaP xenografts. To evaluate the extent of tumor regrowth after ZD6474, treatment was stopped after 40 days of continuous dosing, and subsequent tumor growth was monitored. Prostate-specific antigen expression was assessed to determine the effect of ZD6474 on androgen-regulated genes. RESULTS: In comparison with orchiectomy, ZD6474 treatment produced greater tumor growth inhibition (P < 0.001), inducing complete cytostasis for the duration of dosing. An analysis of serum prostate-specific antigen concentration and tumor weight indicated that ZD6474 did not have a direct effect on androgen-related gene expression. Combination therapy (castration plus ZD6474) produced a comparable therapeutic effect to treatment with ZD6474 alone (in noncastrated mice), for the duration of ZD6474 administration. However, when ZD6474 treatment was discontinued, the rate of tumor regrowth was significantly less in the combination group. Tumors from mice receiving combined treatment were also found to be more necrotic than tumors from mice receiving either androgen ablation or ZD6474 alone. CONCLUSIONS: These data indicate that inhibition of VEGF signaling produces a highly significant inhibition of tumor growth in a human androgen-dependent prostate tumor model, which far exceeds that produced by androgen ablation alone. However, when ZD6474 treatment is removed, concurrent androgen ablation produces a greater inhibition of tumor regrowth than is observed in mice without androgen ablation. Increased necrosis observed in tumors from orchiectomized mice receiving ZD6474 also suggests benefit from combining anti-androgen and anti-VEGF signaling approaches.     

NE-10 neuroendocrine cancer promotes the LNCaP xenograft growth in castrated mice

Increases in neuroendocrine (NE) cells and their secretory products are closely correlated with tumor progression and androgen-independent prostate cancer. However, the mechanisms by which NE cells influence prostate cancer growth and progression, especially after androgen ablation therapy, are poorly understood. To investigate the role of NE cells on prostate cancer growth, LNCaP xenograft tumors were implanted into nude mice. After the LNCaP tumors were established, the NE mouse prostate allograft (NE-10) was implanted on the opposite flank of these nude mice to test whether NE tumor-derived systemic factors can influence LNCaP growth. Mice bearing LNCaP tumors with or without NE allografts were castrated 2 weeks after NE tumor inoculation, and changes in LNCaP tumor growth rate and gene expression were investigated. After castration, LNCaP tumor growth decreased in mice bearing LNCaP tumors alone, and this was accompanied by a loss of nuclear androgen receptor (AR) localization. In contrast, in castrated mice bearing both LNCaP and NE-10 tumors, LNCaP tumors continued to grow, had increased levels of nuclear AR, and secreted prostate-specific antigen. Therefore, in the absence of testicular androgens, NE secretions were sufficient to maintain LNCaP cell growth and androgen-regulated gene expression in vivo. Furthermore, in vitro experiments showed that NE secretions combined with low levels of androgens activated the AR, an effect that was blocked by the antiandrogen bicalutamide. Because an increase in AR level has been reported to be sufficient to account for hormone refractory prostate cancers, the NE cell population ability to increase AR level/activity can be another mechanism that allows prostate cancer to escape androgen ablation therapy.     

PC-1/PrLZ contributes to malignant progression in prostate cancer

PC-1/PrLZ gene overexpression has been identified to be associated with prostate cancer progression. Previous studies have revealed that PC-1 possesses transforming activity and confers malignant phenotypes to mouse NIH3T3 cells. However, the functional relevance of PC-1 expression changes during prostate cancer development and progression remains to be evaluated. In this study, gain-of-function and loss-of-function analyses in LNCaP and C4-2 cells, respectively, were implemented. Experimental data showed that PC-1 expression was in positive correlation with prostate cancer cell growth and anchor-independent colony formation in vitro, as well as tumorigenicity in athymic BALB/c mice. Moreover, PC-1 expression was also found to promote androgen-independent progression and androgen antagonist Casodex resistance in prostate cancer cells. These results indicate that PC-1 contributes to androgen-independent progression and malignant phenotypes in prostate cancer cells. Furthermore, molecular evidence revealed that PC-1 expression stimulated Akt/protein kinase B signaling pathway, which has been implicated to play important roles in promoting androgen refractory progression in prostate cancer. Increased PC-1 levels in C4-2 cells may represent an adaptive response in prostate cancer, mediating androgen-independent growth and malignant progression. Inhibiting PC-1 expression may represent a novel therapeutic strategy to delay prostate cancer progression.     

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.     

PI3K-Akt signaling is involved in the regulation of p21(WAF/CIP) expression and androgen-independent growth in prostate cancer cells

The purpose of this study is to investigate the role of PI3K-Akt signaling in prostate cancer cell growth and androgen receptor (AR)-mediated gene expression. Androgen-dependent LNCaP cells and their androgen-independent counterpart, LNCaP-AI cells, were used. We found that PI3K-Akt signaling is elevated in LNCaP-AI cells compared to that in LNCaP cells and is involved in androgen-independent growth. More importantly, PI3K-Akt signaling enhances AR activity and is involved in the induction of AR target genes, such as p21(WAF/CIP), a gene with anti-apoptosis activity and associated with androgen-independent growth in human prostate cancer. A receptor tyrosine kinase inhibitor also inhibits the PI3K-Akt signaling and compromises AR activity and cell growth. These findings suggest that the PI3K-Akt cell growth survival pathway and its downstream-regulated gene, p21(WAF/CIP), are targets for developing novel therapies against prostate cancer, especially those androgen-independent diseases.     

Deregulation of the Rho GTPase, Rac1, suppresses cyclin-dependent kinase inhibitor p21(CIP1) levels in androgen-independent human prostate cancer cells

Abnormally suppressed levels of cyclin-dependent kinase inhibitors (CKIs) are associated with aggressive androgen-independent prostate cancer and contribute to uncontrolled proliferation. The androgen-independent human prostate cancer cell lines, LNCaP-104R1, ALVA31 and PC-3, express low levels of the CKI, p21(CIP1), compared to the less-malignant, androgen-dependent LNCaP cells. We investigated the mechanism underlying this suppression by examining the role of Rho GTPases, signaling proteins that play important roles in cell cycle progression, at least in part through regulation of CKIs. Inhibition of Rac1 induced p21 expression in androgen-independent lines but had no effect on the higher p21 levels characteristic of LNCaP cells. This induction of p21 was functionally significant as evidenced by inhibition of cyclin-dependent kinase 2 activity and decreased cell proliferation. Conversely, overexpression of constitutively active Rac1 suppressed the higher p21 levels seen in LNCaP cells. Thus, Rac1 activity is both necessary and sufficient for suppression of p21 in prostate cancer cells. Furthermore, Rac1 activity was significantly higher in all three androgen-independent cell lines compared to LNCaP cells. Thus in three models of aggressive human prostate cancer, hyperactivity of Rac1 corresponds to suppressed levels of p21. These results are unique in describing a role for Rac1 in p21 regulation and may implicate the Rac1 signaling pathway as a potential therapeutic target for controlling prostate cancer cell growth following progression to androgen independence.     

Inhibition of p53 function diminishes androgen receptor-mediated signaling in prostate cancer cell lines

Current therapy for advanced prostate cancer is mainly based on androgen deprivation, although most patients relapse to androgen-insensitive disease. Several mechanisms contributing to androgen-independent growth including alterations in the structure or expression of the androgen receptor (AR) and its cofactors have been identified. Recent evidence suggests that p53 is involved in androgen signaling. The analysis of the effect of p53 on androgen signaling was performed in 22Rv1 and LNCaP prostate cancer cells that express both p53 and AR. The overexpression of p53 diminished the androgenic response in both cell lines in a reporter gene assay. Conversely, the inhibition of p53 by three different p53 inhibitors, Pifithrin-1alpha (PFT-1alpha), an inhibitor of p53-dependent transactivation; MDM2, a regulator of p53 expression; and a dominant-negative N-terminally truncated p53 gene also reduced transactivation of androgen-dependent reporter genes. The inactivation of p53 by PFT-1alpha decreased AR-protein expression in both 22Rv1 and LNCaP cells. Our findings confirm that the overexpression of wild-type p53 decreases androgen function, whereas p53 expression at physiological levels stabilizes AR signaling. Thus, our findings suggest that there is a balance of AR and p53 expression during the androgen-dependent growth of prostate cancer, which is obliterated during further progression of the disease.     

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.     

Interleukin-4 enhances prostate-specific antigen expression by activation of the androgen receptor and Akt pathway

Androgen receptor (AR) plays an important role in the development and progression of prostate cancer upon the action of androgen through the binding of the androgen-responsive elements (AREs) on the target genes. Abnormal activation of the AR by nonandrogen has been implicated in the progression of androgen-independent prostate cancer. The levels of interleukin-4 (IL-4) are significantly elevated in sera of patients with hormone refractory prostate cancer. The potential role of IL-4 on the activation of AR was investigated in prostate cancer cells. IL-4 enhances AR-mediated prostate-specific antigen (PSA) expression and ARE-containing gene activity through activation of the AR in the androgen ablation condition in human prostate cancer cells. The AR can also be sensitized by IL-4 and activated by significantly lower levels of androgen (10 pM of R1881) in prostate cancer cells. IL-4 enhances nuclear translocation of AR and increases binding of the AR to the ARE in LNCaP prostate cancer cells. Blocking of the Akt pathway by an Akt-specific inhibitor LY294002 abrogates IL-4-induced PSA expression and AR signaling. These results demonstrate that IL-4 enhances PSA expression through activation of the AR and Akt signaling pathways in LNCaP prostate cancer cells. Understanding IL-4-induced signaling leading to abnormal activation of AR will provide insights into the molecular mechanisms of androgen-independent progression of prostate cancer cells.     

Androgen suppresses proliferation of castration-resistant LNCaP 104-R2 prostate cancer cells through androgen receptor, Skp2, and c-Myc

Androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, this therapy is associated with several undesired side-effects, including increased risk of cardiovascular diseases. To study if termination of long-term androgen ablation and restoration of testosterone levels could suppress the growth of relapsed hormone-refractory prostate tumors, we implanted testosterone pellets in castrated nude mice carrying androgen receptor (AR)-positive LNCaP 104-R2 cells, which relapsed from androgen-dependent LNCaP 104-S cells after long-term androgen deprivation. 104-R2 tumor xenografts regressed after testosterone pellets were implanted. Of 33 tumors, 24 adapted to elevation of testosterone level and relapsed as androgen-insensitive tumors. Relapsed tumors (R2Ad) expressed less AR and prostate-specific antigen. We then studied the molecular mechanism underlying the androgenic regulation of prostate cancer cell proliferation. Androgen suppresses proliferation of 104-R2 by inducing G(1) cell cycle arrest through reduction of S-phase kinase-associated protein 2 (Skp2) and c-Myc, and induction of p27(Kip1). 104-R2 cells adapted to androgen treatment and the adapted cells, R2Ad, were androgen-insensitive cells with a slower growth rate and low protein level of AR, high levels of c-Myc and Skp2, and low levels of p27(Kip1). Nuclear AR and prostate-specific antigen expression is present in 104-R2 cells but not R2Ad cells when androgen is absent. Overexpression of AR in R2Ad cells regenerated an androgen-repressed phenotype; knockdown of AR in 104-R2 cells generated an androgen-insensitive phenotype. Overexpression of Skp2 and c-Myc in 104-R2 cells blocked the growth inhibition caused by androgens. We concluded that androgens cause growth inhibition in LNCaP 104-R2 prostate cancer cells through AR, Skp2, and c-Myc.     

Mitochondrial DNA determines androgen dependence in prostate cancer cell lines

Prostate cancer progresses from an androgen-dependent to androgen-independent stage after androgen ablation therapy. Mitochondrial DNA plays a role in cell death and metastatic competence. Further, heteroplasmic large-deletion mitochondrial DNA is very common in prostate cancer. To investigate the role of mitochondrial DNA in androgen dependence of prostate cancers, we tested the changes of normal and deleted mitochondrial DNA in accordance with the progression of prostate cancer. We demonstrated that the androgen-independent cell line C4-2, established by inoculation of the androgen-dependent LNCaP cell line into castrated mice, has a greatly reduced amount of normal mitochondrial DNA and an accumulation of large-deletion DNA. Strikingly, the depletion of mitochondrial DNA from androgen-dependent LNCaP resulted in a loss of androgen dependence. Reconstitution of normal mitochondrial DNA to the mitochondrial DNA-depleted clone restored androgen dependence. These results indicate that mitochondrial DNA determines androgen dependence of prostate cancer cell lines. Further, mitochondrial DNA-deficient cells formed tumors in castrated athymic mice, whereas LNCaP did not. The accumulation of large deletion and depletion of mitochondrial DNA may thus play a role in the development of androgen independence, leading to progression of prostate cancers.     

Interleukin-8 is a molecular determinant of androgen independence and progression in prostate cancer

The proinflammatory chemokine interleukin-8 (IL-8) is undetectable in androgen-responsive prostate cancer cells (e.g., LNCaP and LAPC-4), but it is highly expressed in androgen-independent metastatic cells, such as PC-3. In this report, we show IL-8 functions in androgen independence, chemoresistance, tumor growth, and angiogenesis. We stably transfected LNCaP and LAPC-4 cells with IL-8 cDNA and selected IL-8-secreting (IL8-S) transfectants. The IL8-S transfectants that secreted IL-8 at levels similar to that secreted by PC-3 cells (100-170 ng/10(6) cells) were characterized. Continuous or transient exposure of LNCaP and LAPC-4 cells to IL-8 reduced their dependence on androgen for growth and decreased sensitivity (>3.5x) to an antiandrogen. IL-8-induced cell proliferation was mediated through CXCR1 and was independent of androgen receptor (AR). Quantitative PCR, immunoblotting, and transfection studies showed that IL8-S cells or IL-8-treated LAPC-4 cells exhibit a 2- to 3-fold reduction in PSA and AR levels, when compared with vector transfectants. IL8-S cells expressed 2- to 3-fold higher levels of phospho-EGFR, src, Akt, and nuclear factor kappaB (NF-kappaB) and showed increased survival when treated with docetaxel. This increase was blocked by NF-kappaB and src inhibitors, but not by an Akt inhibitor. IL8-S transfectants displayed a 3- to 5-fold increased motility, invasion, matrix metalloproteinase-9 and vascular endothelial growth factor production. LNCaP IL8-S cells grew rapidly as tumors, with increased microvessel density and abnormal tumor vasculature when compared with the tumors derived from their vector-transfected counterparts. Therefore, IL-8 is a molecular determinant of androgen-independent prostate cancer growth and progression.     

Inhibition of interleukin-6 with CNTO328, an anti-interleukin-6 monoclonal antibody, inhibits conversion of androgen-dependent prostate cancer to an androgen-independent phenotype in orchiectomized mice

Initially, prostate cancer is androgen dependent. However, most cases progress to an androgen-independent state through unknown mechanisms. Interleukin-6 (IL-6) has been associated with prostate cancer progression including activation of the androgen receptor (AR). To determine if IL-6 plays a role in the conversion of prostate cancer from androgen dependent to androgen independent, we established androgen-dependent LuCaP 35 human prostate cancer xenografts in nude mice, castrated the mice, and blocked IL-6 activity using a neutralizing antibody (CNT0328) for a period of 18 weeks. IL-6 inhibition increased survival of mice and inhibited tumor growth, as reflected by decreased tumor volume and prostate-specific antigen levels, compared with that in mice receiving isotype control antibody. To test the effect of IL-6 inhibition on the conversion from androgen dependent to androgen independent, tumor cells from the treated mice were assessed for their androgen dependence both in vitro and by implanting them into sham-operated or orchiectomized mice. Tumor cells derived from the isotype-treated animals converted to androgen-independent state, whereas tumor cells from the anti-IL-6 antibody-treated mice were still androgen dependent in vitro and in vivo. Although there was no difference in AR levels between the androgen-independent and androgen-dependent tumors, IL-6 inhibition promoted both apoptosis and inhibited cell proliferation in tumors and blocked the orchiectomy-induced expression of histone acetylases, p300 and CBP, which are AR cofactors. These data show that IL-6 contributes to the development of androgen independence in prostate cancer and suggest that it mediates this effect, in part, through modulation of p300 and CBP.