CWR22 Xenograft Research Articles

Abstract 4541: Binding and transcriptional activity of the androgen receptor isoforms in 22Rv1 prostate cancer cells

Abstract Prostate carcinoma (CaP) is a heterogeneous multifocal disease where gene expression and regulation is altered as the disease progresses but the androgen receptor (AR) continues to be a pivotal regulatory molecule. The 22Rv1 castrate resistant cell line, derived from the androgen dependent CWR22 xenograft model, continues to express the AR and remains androgen responsive. However, in additional to the full length AR, these cells also express several low molecular weight isoforms. These isoforms are the predominant (95%) forms present in the nucleus in castrate androgen levels. siRNA knockdown of all AR isoforms in castrate levels of androgen decreases cell growth indicating that AR is required for proliferation. Analysis of ChIP-promoter array data identified 128 binding sites (FDR<=0.05) in Rv1 cells proliferating in androgen-depleted media. A closer examination of these 128 binding sites revealed that 20% of the sites showed exactly the same start and end position in the absence or presence of DHT. The remainder (80%) were within ∼35-1000bp upstream of the start position or downstream of the end position. Only 6% of the binding sites contained the palendromic consensus AR-response element and 48% contained the AR half-site motif. The 128 binding sites were associated with 118 genes. To define the role of AR binding in regulating transcription, the expression of 20 of the 118 genes was analyzed following knockdown of AR in castrate levels of androgen. One third of these genes are transactivated, while 1/3 are repressed, arguing that AR activating and repressing complexes co-exist in these cells. Notably the expression of CDKN1B (p27) RNA and protein is highly activated following AR knockdown, suggesting that AR-dependent repression of this cell cycle inhibitor may be important for proliferation of 22Rv1 cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4541. doi:10.1158/1538-7445.AM2011-4541

Longitudinal Magnetic Resonance Imaging-Based Assessment of Vascular Changes and Radiation Response in Androgen-Sensitive Prostate Carcinoma Xenografts under Androgen-Exposed and Androgen-Deprived Conditions

Prostate cancer (PCa) patients receive androgen-deprivation therapy (ADT) to reduce tumor burden. However, complete eradication of PCa is unusual, and recurrent disease is evident within approximately 2 years in high-risk patients. Clinical evidence suggests that combining ADT with radiotherapy improves local control and disease-free survival in these patients compared with radiotherapy alone. We investigated whether vascularization of androgen-sensitive PCa xenografts changed after ADT and whether such therapy affected radiation response. CWR22 xenografts received combinations of ADT by castration (CWR22-cas) and 15 Gy of single-dose irradiation. At a shortest tumor diameter of 8 mm, vascularization was visualized by dynamic contrast-enhanced magnetic resonance imaging before radiation and 1 and 9 days after radiation. Voxel-wise quantitative modeling of contrast enhancement curves extracted the hemodynamic parameter Ktrans, reflecting a combination of permeability, density, and blood flow. Tumor volumes and prostate-specific antigen (PSA) were monitored during the experiment. The results showed that Ktrans of CWR22-cas tumors 36±4 days after ADT was 47.1% higher than Ktrans of CWR22 tumors (P = .01). CWR22-cas tumors showed no significant changes in Ktrans after radiation, whereas Ktrans of CWR22 tumors at day 1 decreased compared with pretreatment values (P = .04) before a continuous increase from day 1 to day 9 followed (P = .01). Total PSA in blood correlated positively to tumor volume (r = 0.59, P < .01). In conclusion, androgen-exposed xenografts demonstrated radiation-induced reductions in vascularization and tumor volumes, whereas androgen-deprived xenografts showed increased vascularization and growth inhibition, but no significant additive effect of radiation.

Abstract 602: Silencing of the androgen receptor (AR) with a novel mRNA antisense oligonucleotide causes antitumor effects in xenograft models of prostate cancer

Abstract Background: The AR is a ligand-activated transcriptional factor that plays an important role in prostate cancer. While androgen-deprivation therapies typically inhibit tumor growth, tumor recurrence due to the castration resistant form of the disease frequently occurs and leads to mortality. Nevertheless, castration-resistant tumors are still dependent on a functional (AR). Therefore, inhibition of AR expression, rather than systemic androgen deprivation, may provide a novel strategy for treatment of advanced prostate cancer. We have developed a novel locked nucleic acid (LNA)-based antisense oligonucleotide (ON), EZN-4176, which down-modulated AR and inhibited tumor growth. Methods: The target mRNA down-modulation, growth inhibition, luciferase activity, protein inhibition, and reduction of prostate specific antigen (PSA; an AR downstream target and biomarker of prostate cancer) were evaluated by qRT-PCR, MTS, SteadyGlo, western blot analysis, and ELISA assay, respectively, in LNCaP cancer cell lines. A mismatched LNA antisense oligo (EZN-4176MM) and an AR negative cell line (PC-3 prostate cancer) served as controls. Additionally, the effect of EZN-4176 on DHT-induced AR transcriptional activity was evaluated in LNCaP cells containing an AR-responsive luciferase reporter. In vivo, therapeutic efficacy of EZN-4176 was evaluated in the AR-positive CWR22 (androgen dependent). Results: In vitro, in the absence of any transfection reagent, EZN-4176 but not the mismatched control (EZN-4176MM) inhibited the growth of DHT-induced LNCaP cells. The growth reduction correlated with AR down-modulation and its down-stream target gene expression and AR-responsive luciferase activity. In vivo, EZN-4176, prepared in the absence of a delivery vehicle, demonstrated dose-dependent inhibition of mRNA of AR, PSA, and TMPRSS2 as well as protein level of AR in androgen-sensitive CWR22 tumor xenografts, resulting in significant tumor growth inhibition (TGI) (∼55%) (similar to high-dose Casodex® [bicalutamide] treatment). Cy5.5-labeled EZN-4176 was shown to localize in subcutaneous CWR22 or LNCaP tumor xenografts using xenogen imaging system. In castration-resistant tumor cells, EZN-4176 potently and specifically inhibited the growth of C4-2b cells as well as down-regulated AR-luciferase reporter systems. More importantly, AR and PSA mRNA were inhibited in both castration-resistant C4-2b and 22RV1 tumor xenografts. Conclusions: Our data suggest that AR mRNA down modulation by EZN-4176 inhibits prostate tumor growth and provides a novel strategy to treat advanced 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 602.

Abstract 4900: MAGE-A11 expression in castration-recurrent prostate cancer: association with a global epigenetic defect

Abstract Despite the initial clinical response to androgen deprivation therapy, most prostate cancer patients subsequently develop castration-recurrent disease. Elucidation of the mechanisms driving this process is expected to lead to improved strategies to prevent and treat advanced prostate cancer. Epigenetic alterations, including DNA hypomethylation, play a key role in human cancer development. DNA hypomethylation is thought to promote oncogenesis in part by driving oncogene expression, but its potential role in castration-recurrent prostate cancer is unknown. Melanoma Antigen A-11 (MAGE-A11) is a cancer germline (CG) antigen that stabilizes the androgen receptor (AR), facilitates AR association with co-activators, and promotes AR transcriptional activity; thus it appears to play an oncogenic role in castration-recurrent disease. We have recently reported that MAGE-A11 is over-expressed in prostate cancer cell lines, prostate cancer xenografts, and human prostate cancer tissues; in each setting MAGE-A11 overexpression is associated with DNA hypomethylation at the MAGE-A11 promoter CpG island (Karpf et al., Molecular Cancer Research 7; 523-535, 2009). Critically, MAGE-A11 expression and promoter hypomethylation correlate with castration-recurrent cancer growth, both in the CWR-22 xenograft model and in primary human prostate cancer tissues. In the current study we examined whether MAGE-A11 expression and hypomethylation in prostate cancer is an isolated molecular event, or alternatively whether it coincides with a global DNA hypomethylation defect. To test this, we analyzed the global DNA methylation status of prostate cancer cell lines, CWR-22 cancer xenografts, and primary prostate cancer, in which MAGE-A11 expression and methylation was also characterized. We assessed global DNA methylation by measuring total genomic 5-methyl-2′-deoxycytidine (5mdC), LINE-1 repetitive DNA element methylation, and the expression and methylation of several other CG antigen genes, including MAGE-A1, NY-ESO-1, XAGE-1, and BORIS/CTCFL. Taken together, our data provide evidence for a global DNA hypomethylation phenotype in castration-recurrent prostate cancers that express MAGE-A11. Ongoing studies are using HpaII tiny fragment enrichment by ligation-mediated PCR (HELP assay) to define the epigenome of castration-recurrent prostate cancer displaying MAGE-A11 expression. In summary, our data provide evidence that global DNA hypomethylation contributes to castration-recurrent prostate cancer, at least in part by promoting MAGE-A11 expression. 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 4900.

ERK Regulates Calpain 2-induced Androgen Receptor Proteolysis in CWR22 Relapsed Prostate Tumor Cell Lines

Androgen ablation therapy is effective in treating androgen-dependent prostate tumors; however, tumors that can proliferate in castrate levels of androgen eventually arise. We previously reported that in CWR22Rv1 (Rv1) cells, the protease calpain 2 can cleave the androgen receptor (AR) into a constitutively active approximately 80,000 low molecular weight (LMW) form. In this study, we further dissect the mechanisms that produce the AR LMW forms using Rv1 cells and the related CWR22-R1 (R1) cells. The 39-amino acid insertional mutation in the Rv1-AR (E3DM-AR) sensitizes this AR to calpain 2 proteolysis. R1 cells encode the same AR molecule as the parental CWR22 xenograft. Using calpain 2 small interfering RNA and calpeptin, we find that calpain 2 plays a role in the generation of the LMW-AR in R1 cells. Furthermore, LMW-AR expression is regulated by the activation of calpain 2 by ERK 1 and 2. Inhibition of ERK phosphorylation or small interfering RNA-mediated decrease of ERK expression reduces LMW-AR levels in R1 cells. Conversely, activation of the MAPK pathway results in increased ERK phosphorylation and increased levels of LMW-AR. Finally, analyses of human tumor samples found that LMW-AR levels are higher in tumors that have an increased calpain/calpastatin ratio and/or increased levels of phospho-ERK (pERK). This suggests that a higher calpain/calpastatin ratio collaborates with activated ERK to promote the generation of the LMW-AR.

Abstract C144: A locked nucleic acid antisense oligonucleotode against androgen receptor, down-modulates target mRNA and causes antitumor effects in xenograft models of prostate cancer

Abstract Background: Androgen-deprivation therapies remain the first line treatment for prostate cancer. Despite an excellent initial response, most of these patients will succumb to the castration resistant form of the disease. Interestingly, even in the presence of castration levels of circulating androgens, these tumors are still dependent on a functional androgen receptor (AR). Therefore, inhibition of AR expression, rather than systemic androgen deprivation, may provide a novel strategy for treatment of advanced prostate cancer. We have developed a novel locked nucleic acid (LNA)-based antisense oligonucleotide (ASO), EZN-4176, that silence AR and is associated with tumor growth inhibition. Methods: Target mRNA and protein knockdown, growth inhibition, and apoptosis induction effects of EZN-4176 were evaluated by qRT-PCR, western blot analysis, ELISA, MTS assay, and caspase3/7 activity assay, respectively, in AR-positive cancer cell lines (LNCaP, 22RV1) or AR-negative cell line (PC3) in the presence or absence of lipofection (transfection reagent). In vivo, therapeutic efficacy of EZN-4176 was evaluated in AR-positive CWR22 (androgen dependent) and 22Rv1 (castration refractory) tumor models. Results: EZN-4176 resulted in potent down-modulation of AR mRNA or protein (IC50 &amp;lt;5 nM) when transfected in LNCaP or 22Rv1 cells. Consequently, cell growth was inhibited (IC50 &amp;lt; 10 nM), and caspase 3/7 activity was significantly increased. Additionally, the PSA mRNA and protein levels were significantly decreased after EZN-4176 treatment (IC50 &amp;lt; 5 nM). The biological effects of EZN-4176 were specific as the scrambled LNA-ASO did not down regulate target mRNA, nor were inhibitory effects on growth observed with EZN-4176 in an AR-negative cell line (PC3). In non-transfected cells, EZN-4176 but not the scrambled LNA-ASO down-regulated AR and inhibited the growth of DHT-induced LNCaP cells. In vivo, dose-dependent reduction of AR mRNA (up to 80%) in the liver was observed following intravenous administration of the EZN-4176. Additionally, EZN-4176 treatment dose-dependently inhibited AR (up to 40%), PSA (up to 85%) and TMPRSS2 (up to 50%), mRNA in, 22Rv1 or CWR22 tumor xenografts. In CWR22 tumor xenografts, EZN-4176 treatment also resulted in significant tumor growth inhibition (TGI) (∼55%) (similar to high-dose Casodex® [bicalutamide] treatment). TGI and mRNA knockdown effects in xenografts were specific as scrambled LNA-ASO resulted in no to insignificant response and EZN-4176 had no TGI in PC3 xenograft model. Cy5.5-labeled EZN-4176 was shown to localize in subcutaneous CWR22 or LnCaP tumor xenografts using xenogen imaging system. Conclusions: Our data suggest that EZN-4176 is a potent LNA-based ASO of AR that causes target down-modulation and TGI effects in vivo and hence may provide a novel strategy to treat advanced prostate cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C144.

The relationship among tumor architecture, pharmacokinetics, pharmacodynamics, and efficacy of bortezomib in mouse xenograft models

Understanding a compound's preclinical pharmacokinetic, pharmacodynamic, and efficacy relationship can greatly facilitate its clinical development. Bortezomib is a first-in-class proteasome inhibitor whose pharmacokinetic/pharmacodynamic parameters are poorly understood in terms of their relationship with efficacy. Here we characterized the bortezomib pharmacokinetic/pharmacodynamic/efficacy relationship in the CWR22 and H460 xenograft models. These studies allowed us to specifically address the question of whether the lack of broad bortezomib activity in solid tumor xenografts was due to insufficient tumor penetration. In vivo studies showed that bortezomib treatment resulted in tumor growth inhibition in CWR22 xenografts, but not in H460 xenografts. Using 20S proteasome inhibition as a pharmacodynamic marker and analyzing bortezomib tumor exposures, we show that efficacy was achieved only when suitable drug exposures drove proteasome inhibition that was sustained over time. This suggested that both the magnitude and duration of proteasome inhibition were important drivers of efficacy. Using dynamic contrast-enhanced magnetic resonance imaging and high-resolution computed tomographic imaging of vascular casts, we characterized the vasculature of CWR22 and H460 xenograft tumors and identified prominent differences in vessel perfusion, permeability, and architecture that ultimately resulted in variations in bortezomib tumor exposure. Comparing and contrasting the differences between a bortezomib-responsive and a bortezomib-resistant model with these techniques allowed us to establish a relationship among tumor perfusion, drug exposure, pharmacodynamic response and efficacy, and provided an explanation for why some solid tumor models do not respond to bortezomib treatment.

Continuous and intermittent dosing of lonafarnib potentiates the therapeutic efficacy of docetaxel on preclinical human prostate cancer models

Lonafarnib is a potent, selective farnesyltransferase inhibitor (FTI) undergoing clinical studies for the treatment of solid tumors and hematological malignancies. Preclinically, a number of FTIs, including lonafarnib, interact with taxanes to inhibit cancer cell growth in an additive/synergistic manner. These observations provided rationale for investigating the effects of combining lonafarnib and docetaxel on preclinical prostate cancer models. To date, docetaxel is the only chemotherapeutic agent in clinical use for hormone-refractory prostate cancer. In vitro experiments with 22Rv1, LNCaP, DU-145, PC3 and PC3-M prostate cancer cell lines showed significantly enhanced inhibition of cell proliferation and apoptosis when lonafarnib was added to docetaxel. In human tumor xenograft models, continuous coadministration of lonafarnib with docetaxel caused marked tumor regressions (24-47%) in tumors from all of the cell types as well as parental CWR22 xenografts. Intermittent dosing of lonafarnib (5 days on then 5 days off) coadministered with docetaxel produced similar regressions in hormone-refractory 22Rv1 tumors. 22Rv1 tumors progressing on docetaxel treatment also responded to treatment with intermittent lonafarnib (5 days on then 5 days off). Moreover, animals did not exhibit any signs of toxicity during coadministration of lonafarnib and docetaxel. In conclusion, coadministration of continuous and intermittent lonafarnib enhanced the antitumor activity of docetaxel in a panel of prostate cancer models. An intermittent dosing schedule of lonafarnib coadministered with docetaxel may allow enhanced efficacy to that of continuous dosing by improving the tolerability of higher doses of lonafarnib.

Increased Expression of Androgen Receptor Coregulator MAGE-11 in Prostate Cancer by DNA Hypomethylation and Cyclic AMP

Melanoma antigen gene protein-A11 (MAGE-11) of the MAGE family of cancer germ-line antigens increases androgen receptor (AR) transcriptional activity through its interaction with the AR NH(2)-terminal FXXLF motif. The present study investigated the regulatory mechanisms that control MAGE-11 expression during androgen deprivation therapy and prostate cancer progression. Studies include the CWR22 xenograft model of human prostate cancer, clinical specimens of benign and malignant prostate, and prostate cancer cell lines. MAGE-11 mRNA levels increased 100- to 1,500-fold during androgen deprivation therapy and prostate cancer progression, with highest levels in the castration-recurrent CWR22 xenograft and clinical specimens of castration-recurrent prostate cancer. Pyrosequencing of genomic DNA from prostate cancer specimens and cell lines indicated the increase in MAGE-11 resulted from DNA hypomethylation of a CpG island in the 5' promoter of the MAGE-11 gene. Sodium bisulfite sequencing of genomic DNA from benign and malignant prostate tumors and prostate cancer cell lines revealed DNA hypomethylation at individual CpG sites at the transcription start site were most critical for MAGE-11 expression. Cyclic AMP (cAMP) also increased MAGE-11 expression and AR transcriptional activity in prostate cancer cell lines. However, cAMP did not alter DNA methylation of the promoter and its effects were inhibited by extensive DNA methylation in the MAGE-11 promoter region. Increased expression of the AR coregulator MAGE-11 through promoter DNA hypomethylation and cAMP provides a novel mechanism for increased AR signaling in castration-recurrent prostate cancer.

Commentary on Splicing of a novel androgen receptor exon generates a constitutively active androgen receptor that mediates prostate cancer therapy resistance

The standard systemic treatment for prostate cancer (PCa) is androgen ablation, which causes tumor regression by inhibiting activity of the androgen receptor (AR). Invariably, PCa recurs with a fatal androgen-refractory phenotype. Importantly, the growth of androgen-refractory PCa remains dependent on the AR through various mechanisms of aberrant AR activation. Here, we studied the 22Rv1 PCa cell line, which was derived from a CWR22 xenograft that relapsed during androgen ablation. Three AR isoforms are expressed in 22Rv1 cells: a full-length version with duplicated exon 3 and two truncated versions lacking the COOH terminal domain (CTD). We found that CTD-truncated AR isoforms are encoded by mRNAs that have a novel exon 2b at their 3′ end. Functionally, these AR isoforms are constitutively active and promote the expression of endogenous AR-dependent genes, as well as the proliferation of 22Rv1 cells in a ligand-independent manner. AR mRNAs containing exon 2b and their protein products are expressed in commonly studied PCa cell lines. Moreover, exon 2b-derived species are enriched in xenograft-based models of therapy-resistant PCa. Together, our data describe a simple and effective mechanism by which PCa cells can synthesize a constitutively active AR and thus circumvent androgen ablation.

An Oncogenic Role for the Phosphorylated h-Subunit of Human Translation Initiation Factor eIF3

Dysregulation of protein synthesis has been implicated in oncogenesis through a mechanism whereby "weak" mRNAs encoding proteins involved in cell proliferation are strongly translated when the protein synthesis apparatus is activated. Previous work has determined that many cancer cells contain high levels of eIF3h, a protein subunit of translation initiation factor eIF3, and overexpression of eIF3h malignantly transforms immortal NIH-3T3 cells. This is a general feature of eIF3h, as high levels also affect translation, proliferation, and a number of malignant phenotypes of CHO-K1 and HeLa cells and, most significantly, of a primary prostate cell line. Furthermore, overexpressed eIF3h inhibits Myc-dependent induction of apoptosis of primary prostate cells. eIF3h appears to function through translation, as the initial appearance of overexpressed eIF3h in rapidly induced NIH-3T3 cells correlates tightly with the stimulation of protein synthesis and the generation of malignant phenotypes. This oncogenic potential of eIF3h is enhanced by phosphorylation at Ser(183). Finally, reduction of eIF3h levels in breast and prostate cancer cell lines by short interfering RNA methods reduces their rates of proliferation and anchorage-independent growth in soft agar. The results provide compelling evidence that high eIF3h levels directly stimulate protein synthesis, resulting in the establishment and maintenance of the malignant state in cells.

Dietary n-3 Polyunsaturated Fatty Acids Enhance Hormone Ablation Therapy in Androgen-Dependent Prostate Cancer

Hormone ablation therapy typically causes regression of prostate cancer and represents an important means of treating this disease, particularly after metastasis. However, hormone therapy inevitably loses its effectiveness as tumors become androgen-independent, and this conversion often leads to death because of subsequent poor responses to other forms of treatment. Because environmental factors, such as diet, have been strongly linked to prostate cancer, we examined the affects of dietary polyunsaturated fatty acids (PUFAs; at 1.5 wt%) on growth of androgen-dependent (CWR22) and androgen-independent (CWR22R) human prostatic cancer xenografts, the acute response of CWR22 tumors to ablation therapy, and their progression to androgen independence. Significant diet-induced changes in tumor n-3 or n-6 PUFA content had no affect on CWR22 or CWR22R tumors growing with or without androgen support, respectively. However, dietary changes that increased tumor eicosapentaenoic acid and linoleic acid content enhanced responses to ablation therapy, measured by cancer cell apoptosis and mitosis. In addition, relapse to androgen-independent growth (measured by renewed increases in tumor volume and serum prostate-specific antigen after ablation) positively correlated with tumor arachidonic acid content. There was no correlation between expression of 15-lipoxygenase isozymes or their products and tumor growth or responses to ablation. In conclusion, dietary n-3 PUFA may enhance the response of prostate cancer to ablation therapy and retard progression to androgen-independent growth by altering tumor PUFA content.

Splicing of a Novel Androgen Receptor Exon Generates a Constitutively Active Androgen Receptor that Mediates Prostate Cancer Therapy Resistance

The standard systemic treatment for prostate cancer (PCa) is androgen ablation, which causes tumor regression by inhibiting activity of the androgen receptor (AR). Invariably, PCa recurs with a fatal androgen-refractory phenotype. Importantly, the growth of androgen-refractory PCa remains dependent on the AR through various mechanisms of aberrant AR activation. Here, we studied the 22Rv1 PCa cell line, which was derived from a CWR22 xenograft that relapsed during androgen ablation. Three AR isoforms are expressed in 22Rv1 cells: a full-length version with duplicated exon 3 and two truncated versions lacking the COOH terminal domain (CTD). We found that CTD-truncated AR isoforms are encoded by mRNAs that have a novel exon 2b at their 3' end. Functionally, these AR isoforms are constitutively active and promote the expression of endogenous AR-dependent genes, as well as the proliferation of 22Rv1 cells in a ligand-independent manner. AR mRNAs containing exon 2b and their protein products are expressed in commonly studied PCa cell lines. Moreover, exon 2b-derived species are enriched in xenograft-based models of therapy-resistant PCa. Together, our data describe a simple and effective mechanism by which PCa cells can synthesize a constitutively active AR and thus circumvent androgen ablation.

Human PARM‐1 is a novel mucin‐like, androgen‐regulated gene exhibiting proliferative effects in prostate cancer cells

In this paper we characterize hPARM-1, the human ortholog of rat PARM-1 (prostatic androgen-repressed message-1) and demonstrate its role in prostate cancer. Immunofluorescence microscopy and ultrastructural analysis revealed the localization of hPARM-1 to Golgi, plasma membrane and the early endocytic pathway but not in lysosomes. Biochemical and deglycosylation studies showed hPARM-1 as a highly glycosylated, mucin-like type I transmembrane protein. Analysis of expression of hPARM-1 in various human tissues revealed its presence in most human tissues with especially high expression in heart, kidney and placenta. Androgen controls the expression of the gene as a marked 7-fold increase is seen in the androgen-dependent prostate cancer cell line, LNCaP on androgen stimulation. This is further supported by its decrease in expression in CWR22 xenograft upon castration. Moreover, ectopic expression of hPARM-1 in PC3 prostate cancer cells increased colony formation, suggesting a probable role in cell proliferation. These results suggest that hPARM-1 may have a role in normal biology of the prostate cell and in prostate cancer.

Bevacizumab plus 5-fluorouracil induce growth suppression in the CWR-22 and CWR-22R prostate cancer xenografts

Prostate cancer is the most common malignancy in men. Although patients with metastatic prostate cancer can benefit from androgen ablation, most of them will die of prostate cancer progression to an androgen-refractory state. In the present study, the effects of docetaxel, bevacizumab, 5-fluorouracil (5-FU), bevacizumab plus docetaxel, and bevacizumab plus 5-FU on the growth of human CWR-22 (androgen-dependent) and CWR-22R (androgen-independent) prostate carcinoma xenografts were investigated. We report that i.p. administration of 10 mg/kg docetaxel at 1-week interval, 5 mg/kg/ bevacizumab once every 2 weeks, or 12.5 mg/kg 5-FU, bevacizumab/docetaxel, or bevacizumab/5-FU weekly to severe combined immunodeficient mice bearing prostate cancer xenografts (12 mice per treatment group) for 21 days resulted in 22.5 +/- 8%, 23 +/- 7%, 31 +/- 8%, 22 +/- 6%, and 81 +/- 5% growth inhibition, respectively. Greatest growth suppression was observed in bevacizumab/5-FU treatment. Bevacizumab/5-FU-induced growth suppression was associated with reduction in microvessel density, inhibition of cell proliferation; up-regulation of phosphatase and tensin homologue, p21(Cip1/Waf1), p16(INK4a), and p27(Kip1); hypophosphorylation of retinoblastoma protein; and inhibition of Akt/mammalian target of rapamycin pathway. Our data indicate that bevacizumab/5-FU effectively inhibits angiogenesis and cell cycle progression and suggest that bevacizumab/5-FU may represent an alternative treatment for patients with prostate cancer.

The significance of LMO2 expression in the progression of prostate cancer

LIM domain only 2 (LMO2) proteins are important regulators in determining cell fate and controlling cell growth and differentiation. This study has investigated LMO2 expression in human prostatic tissue specimens, prostate cancer cell lines, and xenografts; and has assessed the possible role and mechanism of LMO2 in prostate carcinogenesis. Immunohistochemical analysis on a tissue microarray consisting of 91 human prostate specimens, including normal, prostatic hyperplasia, high-grade prostatic intraepithelial neoplasia, and invasive carcinoma, revealed that overexpression of LMO2 was significantly associated with advanced tumour stage, as measured by Gleason score (p = 0.012), as well as with the development of distant metastasis (p = 0.018). These data were supported by quantitative real-time PCR experiments, where LMO2 mRNA levels were found to be significantly higher in prostate tumour specimen than in normal epithelium (p = 0.037). The expression of LMO2 in cell lines and xenografts representing androgen-dependent (AD) and androgen-independent (AI) prostate cancer stages was further studied. Consistent with the in vivo data, LMO2 mRNA and protein were found to be overexpressed in the more aggressive AI cells (PC3, DU145, and AI CWR22 xenografts) compared with less aggressive AD cells (LNCaP and AD CWR22 xenografts). Furthermore, stable introduction of LMO2 into LNCaP cells conferred enhanced cell motility and invasiveness in vitro, accompanied by down-regulation of E-cadherin expression. Taken together, these findings provide the first evidence to support the hypothesis that LMO2 may play an important role in prostate cancer progression, possibly via repression of E-cadherin expression.

Imaging Mitogen-Activated Protein Kinase Function in Xenograft Models of Prostate Cancer

Mitogen-activated protein kinases (MAPK) play important roles in malignancy. The ability to detect and quantitate MAPKs in live animal models of cancer will facilitate an understanding of disease progression. We have developed a gene expression-based imaging system that detects and quantifies MAPK activity in prostate cancer tumors implanted into severe combined immunodeficient mice. The imaging technology uses a modified version of two-step transcriptional amplification (TSTA). The tissue specificity of gene expression is imparted by an enhanced version of the prostate-specific antigen regulatory region that expresses GAL4-ELK1. GAL4-ELK1 confers MAPK specificity by activating a firefly luciferase (FLuc) reporter gene when the Ets-like transcription factor (ELK) 1 activation domain is phosphorylated by MAPK. FLuc activity in live animals was detected using the Xenogen In vivo Imaging System. We validated the TSTA-ELK1 system by analyzing its response to epidermal growth factor treatment in transfected tissue culture cells and in adenovirus (AdTSTA-ELK1)-injected prostate cancer xenograft tumors. We measured MAPK activity in two well-characterized xenograft models, CWR22 and LAPC9. Although no significant differences in MAPK levels were detected between androgen-dependent and androgen-independent xenografts, the CWR22 models display significantly higher levels of AdTSTA-ELK1 activity versus LAPC9. Western blots of tumor extracts showed that the elevated imaging signal in CWR22 xenografts correlated with elevated levels of phosphorylated extracellular signal-regulated kinase 1/2 but not p38 or c-Jun NH(2)-terminal kinase. We conclude that a gene expression-based optical imaging system can accurately detect and quantify MAPK activity in live animals.

ACTR/AIB1/SRC-3 and androgen receptor control prostate cancer cell proliferation and tumor growth through direct control of cell cycle genes

Co-factor ACTR is frequently overexpressed and/or amplified in multiple types of tumors. The mechanism of its function in prostate cancer (CaP) is still unclear. The effects of ACTR and androgen receptor (AR) depletion on cell proliferation and gene expression and their functions were analyzed in a panel of androgen-dependent and -independent CaP cells and CWR22 xenograft. ACTR and AR, but not TIF2, are required for proliferation of androgen-dependent and -independent cells, and for tumor growth. While AR depletion inhibited the expression of cyclin D1, cyclin B, and cdc2, ACTR depletion reduced the expression of cyclin E and cdk2. In response to serum stimulation, AR and ACTR are recruited to the corresponding target gene promoters to activate their expression in androgen-independent manner. These findings suggest that AR and ACTR may play important roles in androgen ablation resistance by controlling key cell cycle gene expression.

Longitudinally Quantitative 2-Deoxy-2-[18F]fluoro-d-glucose Micro Positron Emission Tomography Imaging for Efficacy of New Anticancer Drugs: A Case Study with Bortezomib in Prostate Cancer Murine Model

The aim of this study was to validate quantitative metabolic response of tumors to a treatment measured by longitudinal 2-deoxy-2-[(18)F]fluoro-D-glucose (FDG) micro positron emission tomography (microPET) as a robust tool for preclinical evaluation of new anticancer agents. Severe combined immunodeficiency mice with CWR22 xenografts were intravenously treated with bortezomib (Velcade) at 0.8 mg/kg on days 0, 3, 7, 10, and 14 and imaged with FDG microPET before, during and after treatment. Quantitative indices of tumor FDG uptake were developed. FDG microPET images successfully revealed the gradual reduction of tumor FDG uptake on day 4 onward despite no absolute tumor shrinkage. The standardized uptake values of FDG in tumors was reduced to 43% of the baseline values. Using the total tumor FDG uptake as the viable tumor burden, we found 86% tumor inhibition, compared to a 55% tumor growth inhibition in tumor volume measurement. FDG microPET imaging can provide an additional dimension of the efficacy of anticancer therapies that may otherwise be underestimated by tumor volume measurement.

Androgen Receptor Remains Critical for Cell-Cycle Progression in Androgen-Independent CWR22 Prostate Cancer Cells

The majority of prostate cancers (PCa) that relapse after androgen deprivation therapy (androgen-independent PCa) continue to express androgen receptor (AR). To study the functional importance of AR in these tumors, we derived androgen-independent CWR22 PCa xenografts in castrated mice and generated a cell line from one of these xenografts (CWR22R3). Similarly to androgen-independent PCa in patients, the relapsed xenografts and cell line expressed AR and were resistant to treatment with bicalutamide. However, expression of the AR-regulated PSA gene in the CWR22R3 cell line was markedly decreased compared to the relapsed xenografts in vivo. Transfections with androgen-regulated reporter genes further indicated that the cells lacked androgen-independent AR transcriptional activity and were not hypersensitive to low androgen concentrations despite constitutive activation of the Erk/MAP kinases. Nonetheless, AR remained essential for androgen-independent growth because retroviral shRNA-mediated AR down-regulation resulted in marked long-term growth suppression. This was associated with increased levels of p27(kip1) and hypophosphorylation of retinoblastoma protein but not with decreases in D-type cyclin levels or MAP kinase activation. These results reveal a potentially critical function of AR in androgen-independent PCa that is distinct from its previously described transcriptional or nontranscriptional functions.