Prostate Cancer Cell Lines 22Rv1 Research Articles

Gene Expression Profiles of Prostate Cancer Stem Cells Isolated by Aldehyde Dehydrogenase Activity Assay

Prostate cancer cells include a small population of cancer stem-like/cancer initiating cells, which have roles in cancer initiation and progression. Recently aldehyde dehydrogenase activity was used to isolate stem cells of various cancer and normal cells. We evaluated the aldehyde dehydrogenase activity of the human prostate cancer cell line 22Rv1 (ATCC®) with the ALDEFLUOR® assay and determined its potency as prostate cancer stem-like/cancer initiating cells. The human prostate cancer cell line 22Rv1 was labeled with ALDEFLUOR reagent and analyzed by flow cytometry. ALDH1(high) and ALDH1(low) cells were isolated and tumorigenicity was evaluated by xenograft transplantation into NOD/SCID mice. Tumor sphere forming ability was evaluated by culturing in a floating condition. Invasion capability was evaluated by the Matrigel™ invasion assay. Gene expression profiling was assessed by microarrays and reverse transcriptase-polymerase chain reaction. ALDH1(high) cells were detected in 6.8% of 22Rv1 cells, which showed significantly higher tumorigenicity than ALDH1(low) cells in NOD/SCID mice (p < 0.05). Gene expression profiling revealed higher expression of the stem cell related genes PROM1 and NKX3-1 in ALDH1(high) cells than in ALDH1(low) cells. ALDH1(high) cells also showed higher invasive capability and sphere forming capability than ALDH1(low) cells. Results indicate that cancer stem-like/cancer initiating cells are enriched in the ALDH1(high) population of the prostate cancer cell line 22Rv1. This approach may provide a breakthrough to further clarify prostate cancer stem-like/cancer initiating cells. To our knowledge this is the first report of cancer stem-like/cancer initiating cells of 22Rv1 using the aldehyde dehydrogenase activity assay.

Abstract 3762: Second generation PIM inhibitors exhibit improved activity in solid tumor models

Abstract The proto-oncogene PIM kinase family (PIM-1, -2 and -3) includes constitutively active serine/threonine kinases upregulated in multiple cancer indications, including lymphomas, leukemias, multiple myeloma, prostate and bladder cancers. Overexpression of one or more PIM family members in patient tumors frequently correlates with poor prognosis. The PIM kinases function by inhibiting apoptosis in MYC-driven tumors, and promoting tumor cell survival and proliferation. In the HEK-293T cell line, enhanced PIM kinase substrate BAD phosphorylation (pBAD) was observed following PIM and BAD overexpression. Enhancement of pBAD was inhibited by SGI-1776, a well-described PIM inhibitor, and more effectively by second generation PIM inhibitors exhibiting 4-10 fold improved potency against the PIM kinase family. The current PIM inhibitors display sub-µM activity in pharmacodynamic marker, proliferation and 2D colony formation assays using the UM-UC-3 human bladder cancer cell line. PIM1 and PIM2 overexpression models were established in the human prostate cancer cell line 22RV-1 and the non-tumorigenic mouse NIH-3T3 cell background. Overexpression of PIM kinases led to enhanced cell growth and tumorigenicity in both NIH-3T3 and 22RV-1 cell lines. In vivo xenograft studies using both PIM overexpression models and a clinically relevant solid tumor model facilitated identification of a lead candidate with demonstrated efficacy and favorable toxicity. IND-enabling studies with a lead candidate are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3762. doi:1538-7445.AM2012-3762

Abstract 2573: Carnosol and carnosic acid, from rosemary, promote endoplasmic reticulum stress and androgen receptor degradation in prostate cancer cells

Abstract Background: Prostate cancer is one of the most prevalent forms of cancer among men, especially in the United States. Previously, we have shown that carnosol, a dietary diterpene from rosemary (Rosmarinus officinalis), has a unique function in that it is a dual disruptor of androgen and estrogen receptor alpha. Herein, we examined how carnosol and another dietary diterpene from rosemary, carnosic acid, promote endoplasmic reticulum (ER) stress and modulate the activity and expression of androgen receptor (AR). Materials and Methods: Experiments were performed using the prostate cancer cell lines 22Rv1 and LNCaP cells. Cell proliferation and cell viability were studied using BrdU and MTT assay respectively. Western blotting, ELISA and siRNA were used to evaluate the relationship between ER stress and androgen receptor degradation. Results: Both, carnosic acid and carnosol, inhibited growth and decreased the cell viability of 22Rv1 and LNCaP cells in a dose-dependent manner. The anti-proliferative effect was associated with apoptosis as evidenced by a significant increase in levels of cleaved caspase-3 protein. Interestingly, these diterpenes modulated proteins associated with ER stress including BiP, IRE1 and CHOP. Furthermore, we saw a dose-dependent decrease in expression of AR in 22Rv1 and LNCaP cells when treated with either carnosol or carnosic acid. The proteasome has been shown to be regulated by multiple proteins including proteins associated with ER stress. The AR expression increased in presence of proteasome inhibitor, MG-132, suggesting that decrease in AR expression was due to proteasomal degradation. Carnosic acid and carnosol, also showed a decrease in expression of prostate-specific antigen (PSA) levels in a dose-dependent manner. Conclusion: Both, carnosic acid and carnosol, induce ER stress-mediated apoptotic cell death in prostate cancer cell lines, 22Rv1and LNCaP cells, leading to disruption of AR expression. Our data suggests that these natural diterpenes possess strong anticancer activity and should be evaluated further as potential chemopreventive and/or chemotherapeutic agents for controlling prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2573. doi:1538-7445.AM2012-2573

Abstract 538: ROS differentially regulates prostate cancer cell survival

Abstract In vitro and in vivo studies have shown that ROS accumulation correlates with increasing aggressiveness of prostate cancer cells, and increased expression of oncogenic molecules, such as CXCR4. CXCR4 is a G-Protein Coupled Receptor that is heavily implicated in the metastatic dissemination of prostate tumor cells to bone marrow. In cancer and other cell models, CXCR4 expression and activity increases in response to hypoxic environments and/or oxidative stress. Cells react by inducing expression of a HIF1α transcription factor, which enhances expression of targeted genes involved in adjusting to oxidative stress and cell survival. HIF1α has been shown to directly induce the expression of CXCR4 in prostate cancer cells. Therefore, generation of ROS in tumor tissues may critically influence CXCR4-mediated expression and functions, ultimately encouraging cancer progression. In this study, we elucidate a mechanism by which ROS induces CXCR4 expression and function in prostate cancer cells. We initially investigated the role of ROS on the phosphorylation of AKT (p-AKT) and ERK1/2 (p-ERK1/2) in DU145 and 22Rv1 prostate cancer cell lines. Using H2O2 as our model of ROS, we found that H2O2 induced a gradual expression of p-AKT and p-ERK1/2 by western blot analysis, which was abrogated by the ROS scavenger N-acetyl cysteine (NAC). It has been shown that PTEN is a negative regulator of AKT and ERK1/2 signaling, and that ROS accumulation inactivates PTEN phosphatase function; hence, we determined whether the increase in p-AKT and p-ERK1/2 expression was due to loss of PTEN function. By performing a phosphatase assay, we found that H2O2 inhibited PTEN catalytic activity compared to untreated cells, while NAC abrogated these effects in both cell lines. The increase in ROS and loss of PTEN has been shown to correlate with CXCR4-mediated function; therefore, we determined the role of H2O2 on CXCR4 expression. We found that H2O2 induced CXCR4 expression in DU145 cells, but not in 22Rv1 cells. Furthermore, we investigated whether ROS-mediated induction of CXCR4 was HIF1α dependent. Interestingly, H2O2 induced expression of HIF1α in DU145 cells, which was abrogated by siRNA for HIF1α. HIF1α expression decreased in 22Rv1 cells, which correlated with apoptosis, as confirmed by Annexin-V analysis. Finally, we observed that the H2O2-mediated increase in CXCR4 enhanced transendothelial invasion and migration in DU145 cells in a CXCR4 dependent manner. Our study suggests that ROS differentially regulates CXCR4 expression and function in prostate cancer cells, suggesting that ROS plays a differential role on heterogeneous tumor mass. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 538. doi:1538-7445.AM2012-538

Abstract 4858: Identification of ZDHHC14 as a novel tumor suppressor gene commonly downregulated in human cancers

Abstract Tumor suppressor genes (TSGs) play critical roles in preventing tumorigenesis and they are frequently inactivated in tumours. Recently developed high-density microarrays can detect subchromosomal deletions, recurrence of which usually indicates the location of TSGs within the deleted region. We analyzed testicular germ cell tumour (TGCT) clinical samples using SNP arrays and found a frequent small deletion on the region 6q25.3 containing only one known gene, ZDHHC14. While its cellular function is unknown, ZDHHC14 belongs to the recently discovered DHHC family, which are predicted to be involved in protein palmitoylation, a reversible lipid modification that regulates membrane tethering for key proteins in cell signaling, cancer, neuronal transmission, and membrane trafficking. Consistently, we found a dramatic under-expression of ZDHHC14 mRNA and protein in TGCTs, and this associated with chemoresistance. Oncomine database mining showed that ZDHHC14 is also under-expressed in lymphoma, liposarcoma, brain, kidney, lung and colorectal cancers. Thus, it appears that ZDHHC14 downregulation may be involved in other cancers. We studied ZDHHC14 expression in prostate cancer (PCa), detecting a decrease at mRNA and protein level. We also detected that ZDHHC14 mRNA was downregulated in a pilot study on breast cancer samples. As genomic loss of the ZDHHC14 region was only detected in a small number of PCa samples, we checked whether promoter hypermethylation was the cause for ZDHHC14 downregulation. However, no changes in methylation status were found. We then sequenced the whole genomic region surrounding ZDHHC14 by next generation sequencing in TGCTs and PCa and found several mutations in the promoter, the coding region, as well as in intronic regions. Finally, we tested the function of ZDHHC14 in cell-based studies. We generated a 293 T-REx tetracycline inducible ZDHHC14 overexpressing stable cell line, which showed that ZDHHC14 overexpression decreased cell viability. The induction of apoptosis by ZDHHC14 overexpression was detected both by FACS and caspase 7 and PARP cleavage analyses. This was confirmed by transient ZDHHC14 overexpression in the PCa cell line 22RV1. In vivo we xenografted mice using both tetracycline inducible ZDHHC14 overexpressing 293 T-REx cells and control cells transfected with the empty vector. ZDHHC14 expression was induced by tetracycline at the beginning of inoculation and we detected that ZDHHC14 overexpression blocked tumour initiation completely. In conclusion, these results implicate ZDHHC14 as a tumour suppressor gene commonly inactivated in human cancers, indicating that it might exert its tumor suppressor role through the induction of programmed cell death. This is the first study showing the involvement of ZDHHC14 in a specific pathway, the classic caspase-dependent apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4858. doi:1538-7445.AM2012-4858

Abstract A62: Human endogenous retrovirus activation in prostate cancer: Association with disease progression

Abstract Introduction: In recent years scientists have observed that several tumor types, including prostate cancer, show increased expression of human endogenous retrovirus (HERV) when compared to normal tissue. The HERVs originated from germ cell infections by exogenous retroviruses during the course of evolution and became incorporated into the human genome. These elements are widely dispersed throughout the genome and are estimated to comprise of greater than &amp;gt;8% of genomic material. The HERV-K family, evolutionarily the youngest HERV, is the only HERV family with complete open reading frames for all viral genes, and thus are the most likely to be biologically active and potentially pathogenic. The HERV-K provirus encodes for the gag, pol and env genes flanked by two long terminal repeat regions. Both HERV-K type I and HERV-K type II proviruses exist: Type II proviruses are complete (and a such are chronological “older” with regard to their insertion into the genome), while Type I proviruses contain a 292-bp deletion at the boundary of the pol and env genes. Hypothesis: We hypothesize that HERV-K expression is activated with advancing prostate cancer progression and that as a result of this, HERV-K leads to a sustained inflammatory response and subsequent promotion of tumor progression. We believe that HERV-K is suitable target of therapeutic intervention. Experimental Procedures: RNA sequences for type II HERV-K108 (AF074086) were blasted against type I HERV-K102 (AF164610). Sequences were 99% homologous between nucleotides 1-6492 (K108) &amp; 1-1691 (K102) (5′LTR-gag-pro-pol region), the type 1 del in K102 occurred between nucleotides 6493-6793 of K108, subsequent nucleotides 6794-9472 (K108) &amp; 6501-9178 (K102) were 99% homologous (env-3′LTR region). For the detection of HERV-K gag, pol and env mRNA proteins were designed in Primer3, and verified for specificity in Primer-BLAST. Levels of HERV-K gag, pol and env type I or II mRNA were quantified in RWPE1 normal immortalized prostate cells, and CRW22 androgen dependent prostate cancer cell lines, and 22Rv1, PC-3 and DU145 androgen independent prostate cancer cell lines (22Rv1 AI variant of CRW22, PC-3 and DU145 derived from prostate cancer metastasis). HERV-K gag and env protein expression was detected in cell lines by western blotting and in prostate cancer tissue microarrays of hyperplasia and adenocarcinoma using anti-HERV-K gag monoclonal antibody (MAb) and anti-HERV-K env MAb. Effects of HERV-K env inhibition in prostate cancer cells were examined by inhibiting HERV-K env using a HERV-K env targeted MAb, and assessing its effect on cell proliferation and cell invasion using the xCelligence Real-Time Cell Analyser. Results: We found that HERV-K is activated in CRW22, 22Rv1, PC3 and DU145 prostate cancer cell lines, but not in RWPE1 normal prostate cells. Additionally, we observed the HERV-K levels were highest in the bone metastasis derived PC3 and brain metastasis derived DU145 cell lines. We also found that HERV-K was differentially expressed between prostate hyperplasia and adenocarcinoma. Inhibition of HERV-K env resulted in inhibition of PC3 and DU145 cell proliferation and cell invasion. Conclusions: HERV-K is activated in prostate cancer and represents a potential therapeutic target for metastatic prostate cancer. Citation Format: Ronan Downey, Amy Burke, Francis J. Giles, Frank Sullivan, Feng Wang-Johanning, Blanaid Mee, Eoin Gaffney, Sharon Anneve Glynn. Human endogenous retrovirus activation in prostate cancer: Association with disease progression [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A62.

Evidence for a dual function of EphB4 as tumor promoter and suppressor regulated by the absence or presence of the ephrin‐B2 ligand

Overexpression of the receptor tyrosine kinase EphB4 is common in epithelial cancers and linked to tumor progression by promoting angiogenesis, increasing survival and facilitating invasion and migration. However, other studies have reported loss of EphB4 suggesting a tumor suppressor function in some cancers. These opposing roles may be regulated by (i) the presence of the primary ligand ephrin-B2 that regulates pathways involved in tumor suppression or (ii) the absence of ephrin-B2 that allows EphB4 signaling via ligand-independent pathways that contribute to tumor promotion. To explore this theory, EphB4 was overexpressed in the prostate cancer cell line 22Rv1 and the mammary epithelial cell line MCF-10A. Overexpressed EphB4 localized to lipid-rich regions of the plasma membrane and confirmed to be ligand-responsive as demonstrated by increased phosphorylation of ERK1/2 and internalization. EphB4 overexpressing cells demonstrated enhanced anchorage-independent growth, migration and invasion, all characteristics associated with an aggressive phenotype, and therefore supporting the hypothesis that overexpressed EphB4 facilitates tumor promotion. Importantly, these effects were reversed in the presence of ephrin-B2 which led to a reduction in EphB4 protein levels, demonstrating that ligand-dependent signaling is tumor suppressive. Furthermore, extended ligand stimulation caused a significant decrease in proliferation that correlated with a rise in caspase-3/7 and -8 activities. Together, these results demonstrate that overexpression of EphB4 confers a transformed phenotype in the case of MCF-10A cells and an increased metastatic phenotype in the case of 22Rv1 cancer cells and that both phenotypes can be restrained by stimulation with ephrin-B2, in part by reducing EphB4 levels.

Targeting of distinct signaling cascades and cancer-associated fibroblasts define the efficacy of Sorafenib against prostate cancer cells

Sorafenib, a multi-tyrosine kinase inhibitor, kills more effectively the non-metastatic prostate cancer cell line 22Rv1 than the highly metastatic prostate cancer cell line PC3. In 22Rv1 cells, constitutively active STAT3 and ERK are targeted by sorafenib, contrasting with PC3 cells, in which these kinases are not active. Notably, overexpression of a constitutively active MEK construct in 22Rv1 cells stimulates the sustained phosphorylation of Bad and protects from sorafenib-induced cell death. In PC3 cells, Src and AKT are constitutively activated and targeted by sorafenib, leading to an increase in Bim protein levels. Overexpression of constitutively active AKT or knockdown of Bim protects PC3 cells from sorafenib-induced killing. In both PC3 and 22Rv1 cells, Mcl-1 depletion is required for the induction of cell death by sorafenib as transient overexpression of Mcl-1 is protective. Interestingly, co-culturing of primary cancer-associated fibroblasts (CAFs) with 22Rv1 or PC3 cells protected the cancer cells from sorafenib-induced cell death, and this protection was largely overcome by co-administration of the Bcl-2 antagonist, ABT737. In summary, the differential tyrosine kinase profile of prostate cancer cells defines the cytotoxic efficacy of sorafenib and this profile is modulated by CAFs to promote resistance. The combination of sorafenib with Bcl-2 antagonists, such as ABT737, may constitute a promising therapeutic strategy against prostate cancer.

MicroRNAs and zinc metabolism-related gene expression in prostate cancer cell lines treated with zinc(II) ions

MicroRNAs (miRNAs) are a large class of single-stranded RNA molecules involved in post-transcriptional gene silencing. miRNAs not only regulate various developmental and physiological processes but also are involved in cancer development. Additionally, they can be considered as biomarkers of some pathological processes. The aim of this study was to determine the expression levels of selected miRNA and zinc(II)-related genes (ZIP-1, BAX, MT2A and MT1A) in the non-tumor PNT1A prostate cell line in comparison with the prostate cancer cell lines 22Rv1, PC-3 and LNCaP after zinc(II) treatment. Using bioinformatic approaches we selected miRNAs with putative binding sites in the 3'UTR regions in Metallothionein 1A and 2A as miRNA23a, 141, 224, 296-3p, 320, 375 and 376. We observed significantly higher expression of miRNA23a in all tumor lines compared to non-tumor PNT1A (13.6-fold in 22Rv1, 7.3-fold in PC-3, 8.3-fold in LNCaP, p<0.01). We also observed that the 22Rv1 cell line has significantly higher expression of miRNA224 in comparison to other cell lines. In addition, all tumor cell lines expressed significantly higher levels of miRNA375 in comparison to non-tumor PNT1A (87.1‑fold in 22Rv1, nearly 2,000-fold in PC-3, 56.3‑fold in LNCaP, p<0.01). Nevertheless, miRNA375 and 23a expression levels strongly suggest their potential to contribute to the diagnosis of prostate cancer and miRNA224 eventually may be suitable for classification of primary tumors. The expression of miRNA224 in 22Rv1 cell line was negatively correlated with increasing zinc(II) concentration only. Our experiments revealed significant negative correlation of miRNA376 and MT2A in 22Rv1 and a negative correlation between miRNA 224 and MT1A in PC-3 cells which may denote possible direct regulation of MT genes by specific miRNAs in prostate cancer.

Abstract C200: Second-generation PIM inhibitors exhibit improved activity in solid tumor models in vitro.

Abstract The proto-oncogene PIM kinase family (PIM-1, -2 and -3) comprises constitutively active serine/threonine kinases upregulated in multiple cancer indications, including lymphoma, leukemia, multiple myeloma, prostate, bladder, gastric and head &amp; neck cancers. Overexpression of one or more PIM family members in patient tumors frequently correlates with poor prognosis. The PIM kinases function by inhibiting apoptosis in MYC-driven tumors, promoting tumor cell survival and proliferation. PIM-1 and PIM-2 overexpression models were obtained in the human prostate cancer cell lines PC-3M and 22RV-1 and the non-tumorigenic mouse NIH-3T3 background. Overexpression of PIM kinases led to enhanced cell growth and tumorigenicity in both NIH-3T3 and 22RV-1 cell lines. In the PC-3M cell line, enhanced phosphorylation of the PIM kinase substrate BAD (pBAD) was observed following PIM overexpression. Enhancement of pBAD was inhibited by SGI-1776, a well-described PIM inhibitor, as well as next generation PIM inhibitors exhibiting 4–10 fold improved potency against the PIM kinase family. The current PIM inhibitors display sub-μM activity in pharmacodynamic marker, proliferation and 2D colony formation assays using the PC-3M prostate cancer cell line, the UM-UC-3 bladder cancer cell line, and the HSC3 head &amp; neck cancer cell line. The second generation PIM inhibitors possess favorable hERG and CYP inhibition profiles compared with SGI-1776, and demonstrate excellent oral bioavailability. In vivo xenograft studies using both PIM overexpression models and clinically relevant solid tumor models will facilitate identification of a clinical candidate. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C200.

GPRC6A regulates prostate cancer progression

GPRC6A is a nutrient sensing GPCR that is activated in vitro by a variety of ligands, including amino acids, calcium, zinc, osteocalcin (OC), and testosterone. The association between nutritional factors and risk of prostate cancer, the finding of increased expression of OC in prostate cancer cells, and the association between GPRC6A and risk of prostate cancer in Japanese men implicates a role of GPRC6A in prostate cancer. We examined if GPRC6A is expressed in human prostate cancer cell lines and used siRNA-mediated knockdown GPRC6A expression in prostate cancer cells to explore the function of GPRC6A in vitro. To assess the role of GPRC6A in prostate cancer progression in vivo, we intercrossed Gprc6a(-/-) mice onto the TRAMP mouse prostate cancer model. GPRC6A transcripts were markedly increased in prostate cancer cell lines 22Rv1, PC-3, and LNCaP, compared to the normal prostate RWPE-1 cell line. In addition, a panel of GPRC6A ligands, including calcium, OC, and arginine, exhibited in prostate cancer cell lines a dose-dependent stimulation of ERK activity, cell proliferation, chemotaxis, and prostate specific antigen and Runx2 gene expression. These responses were inhibited by siRNA-mediated knockdown of GPRC6A. Finally, transfer of Gprc6a deficiency onto a TRAMP mouse model of prostate cancer significantly retarded prostate cancer progression and improved survival of compound Gprc6a(-/-) /TRAMP mice. GPRC6A is a novel molecular target for regulating prostate growth and cancer progression. Increments in GPRC6A may augment the ability of prostate cancer cells to proliferate in response to dietary and bone derived ligands.

Abstract 3060: Target genes of chromosomal 9p13.3 amplification in prostate cancer

Abstract The development and progression of cancer is caused by alterations that accumulate in the genome of malignant cells. We are studying genetic alterations occurring in prostate cancer, the most common male malignancy in Western countries. We have previously identified a frequent (39%) gain at 9p13-q21 in 13 prostate cancer xenografts and 5 cell lines by aCGH (Saramäki et al., Int J Cancer 119:1322-1329, 2006). Preliminary results indicate that in clinical prostatectomy samples, gains and amplifications are found in approximately 30% and 10% of the cases, respectively, and that the amplification frequency seems to increase with disease progression to hormone-refractory stage. The 9p13.3 amplification is also found from several breast cancer cell lines, raising the possibility that the amplification target gene(s) in this area may take part in breast cancer formation in addition to prostate cancer. We aim at identifying the target gene of the 9p13.3 amplification in prostate cancer. A minimal region for the 9p13.3 amplification, determined by fluorescence in situ hybridisation (FISH), spans nearly 3.5 Mb and contains more than 40 known and hypothetical protein-coding genes. We initially screened mRNA expression levels by quantitative RT-PCR and chromosomal copy number alterations by FISH for these genes in 7 prostate cancer cell lines and 19 prostate cancer xenografts. Thus, we narrowed down the list of amplification target candidates to eight protein-coding genes (C9orf25, GALT, PIGO, UBAP1, UBAP2, UBE2R2, UNC13B and VCP). We have performed siRNA-mediated downregulation of these genes in cell lines with either a normal copy number status or a gain/amplification in 9p13.3 (prostate cancer cell lines PC-3 and 22Rv1; breast cancer cell lines MCF-7 and BT-474). Downregulation of several of these genes decrease the proliferation rate of the cells significantly (GALT, PIGO, UBAP1, UBAP2, VCP). As decreased levels of VCP induce cell death in PC-3 cells, downregulation of UBAP1 induces a cell cycle arrest in the G2/M phase. In addition, invasion of PC-3 cells in Boyden chamber assay is affected by knock-down of GALT and PIGO. Genes exhibiting alterations in the siRNA assays have been further analyzed for their expression in clinical prostate cancer tumor material by qRT-PCR and immunohistochemistry. These genes are also studied by overexpressing their cDNAs in cell lines with normal 9p13.3 copy number status and screening for altered cellular growth, survival and invasion in vitro. We aim at identifying a novel diagnostic and/or prognostic marker for prostate cancer, and potentially also a target for new cancer therapies, from the 9p13.3 region. 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 3060. doi:10.1158/1538-7445.AM2011-3060

Abstract 4561: Preclinical characterization of MT112/BAY 2010112, a novel PSMA/CD3-bispecific BiTE antibody for the treatment of prostate cancer

Abstract Prostate-specific membrane antigen (PSMA) has been frequently selected as target antigen for antibody-based therapy of prostate cancer. Here, we recombinantly constructed a PSMA/CD3-bispecific BiTE antibody, called MT112/BAY 2010112. The BiTE antibody was produced in Chinese hamster ovary cells as a secreted protein of 55 kDa, showing high serum and thermal stability. MT112/BAY 2010112, purified as homogenous monomeric protein, bound at low nanomolar concentrations to defined epitopes on PSMA and CD3 antigens of human and macaque origin, respectively. In cell culture studies bispecific binding of MT112/BAY 2010112 selectively redirected human T cells against several PSMA-positive human prostate cancer cell lines as well as PSMA cDNA-transfected cell lines and potently induced specific target cell lysis with EC50 values ranging from 1 to 50 pM using non-stimulated human peripheral blood mononuclear cells as effector cells. EC50 values correlated with the number of PSMA molecules on the cell surface ranging from 37,000-500,000 molecules per cell among the cell lines analyzed. The anti-tumor activity of MT112/BAY 2010112 was assessed in several SCID mouse models bearing subcutaneous xenografts derived from the human prostate cancer cell lines LNCaP, PC3 and 22Rv1. The BiTE antibody completely inhibited growth of tumors at doses as low as 0.005 mg/kg administered daily intravenously when human T cells and tumor cells were co-inoculated. Transient regression of tumors were observed in a model where human T cells were adoptively transferred into the peritoneal space of mice with subcutaneously established tumors. These data suggest that the PSMA/CD3-bispecific antibody MT112/BAY 2010112 has high therapeutic potential for treatment of prostate cancer. Its cross-reactivity with human and macaque PSMA and CD3 antigens will greatly facilitate assessment of pharmacology, pharmacokinetics and safety during further pre-clinical development. 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 4561. doi:10.1158/1538-7445.AM2011-4561

624 PROTEOMICS AND GENOMICS INVESTIGATIONS OF DIETHYLSTILBESTROL ACTION ON A PROSTATE CANCER CELL LINE 22RV1

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 2011624 PROTEOMICS AND GENOMICS INVESTIGATIONS OF DIETHYLSTILBESTROL ACTION ON A PROSTATE CANCER CELL LINE 22RV1 Pierre Bigot, Kevin Mouzat, Souhil Lebdai, Catherine Guette, Géraldine Cancel-tassin, Abdel-Rahmène Azzouzi, and Olivier Cussenot Pierre BigotPierre Bigot Angers, France More articles by this author , Kevin MouzatKevin Mouzat Paris, France More articles by this author , Souhil LebdaiSouhil Lebdai Angers, France More articles by this author , Catherine GuetteCatherine Guette Angers, France More articles by this author , Géraldine Cancel-tassinGéraldine Cancel-tassin Paris, France More articles by this author , Abdel-Rahmène AzzouziAbdel-Rahmène Azzouzi Angers, France More articles by this author , and Olivier CussenotOlivier Cussenot Paris, France More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1477AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Diethylstilbestrol (DES) is a synthetic estrogen used for prostate cancer (PCa) treatment. Its action is independent from the estrogen receptors and is preserved after a first line hormonotherapy treatment. By using 2-Dimensional-Differential in-Gel Electrophoresis (2D-DIGE), we presented the expression variations of 14 proteins in the prostate cancer cell line 22RV1 after DES exposure. Our objective was to study the action of DES on the prostate cancer cell line 22RV1 by using a complementary quantitative proteomic approach by isobaric tagging iTRAQ followed by an assessment of variations of the identified protein of interest by RT-PCR. METHODS The prostate cancer 22RV1 cells were exposed to standard and DES (10 and 20 μM) enriched medium. After extraction, trypsine digestion and iTRAQ labeling, peptides were fractionated using offgel expression levels. Each iTRAQ set was analyzed by matrix assisted laser desorption-time-of-flight (MALDI-TOF/TOF) mass spectrometry. Statistical analysis was performed by iQuantitator. RNA were also extracted and quantitative RT-PCR was performed. RESULTS After proteomic analysis, 895 constitutive proteins of the 22RV1 proteome were identified. Among these proteins, 66 had a modified expression due to DES exposure (23 over-expressed, 46 under-expressed). Most of them were implicated in apoptosis and redox processes and had a mitochondrial expression. Gene expression analysis confirmed that there estrogens receptors ER1 and ER2 were not expressed in our model. We assessed by RT-PCR the expression of ornithine aminotransferase (OAT), Heat shock protein beta-1 (HSPB1) and Insulin-like growth factor-binding protein 2 (IGFB2), which are implicated in natural history of PCa. Their expression was changed by DES exposure according to proteomic analysis. These analyses confirmed the over-expression of OAT (p=0.006) and HSBP1 (p=0.046). However, the variation of IGFB2 was contradictory (it was over-expressed according to iTRAQ analysis and under-expressed according to RT-PCR (p=0,011)), suggesting that DES exerts both transcriptional and post-transcriptional effects on 22RV1 cells. CONCLUSIONS The proteomic complementary approach by 2D-DIGE method and iTRAQ analysis allowed identifying 67 proteins with an expression variation depending on the presence of DES. Among them, several mitochondrial proteins were identified. Thus a hypothesis on the apoptosis consecutive to an oxidative stress induced by DES can be formulated. The over expression of OAT and HSBP1 has been confirmed by genomic and proteomic analysis. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e251 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Pierre Bigot Angers, France More articles by this author Kevin Mouzat Paris, France More articles by this author Souhil Lebdai Angers, France More articles by this author Catherine Guette Angers, France More articles by this author Géraldine Cancel-tassin Paris, France More articles by this author Abdel-Rahmène Azzouzi Angers, France More articles by this author Olivier Cussenot Paris, France More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

736 PRO-INFLAMMATORY AND MITOGENIC ACTIVITY OF PERIPROSTATIC ADIPOSE TISSUE IN PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 2011736 PRO-INFLAMMATORY AND MITOGENIC ACTIVITY OF PERIPROSTATIC ADIPOSE TISSUE IN PROSTATE CANCER David S. Finley, Colette Galet, Joyce Yamashiro, Evelyn Kono, Chau Tran, Alan Pantuck, Jean Dekernion, William Aronson, and Robert Reiter David S. FinleyDavid S. Finley Los Angeles, CA More articles by this author , Colette GaletColette Galet Los Angeles, CA More articles by this author , Joyce YamashiroJoyce Yamashiro Los Angeles, CA More articles by this author , Evelyn KonoEvelyn Kono Los Angeles, CA More articles by this author , Chau TranChau Tran Los Angeles, CA More articles by this author , Alan PantuckAlan Pantuck Los Angeles, CA More articles by this author , Jean DekernionJean Dekernion Los Angeles, CA More articles by this author , William AronsonWilliam Aronson Los Angeles, CA More articles by this author , and Robert ReiterRobert Reiter Los Angeles, CA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1705AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Obesity has been characterized as a state of chronic, low-grade inflammation. There is evidence linking obesity with prostate cancer (CaP) aggressiveness. Visceral adipose tissue has recently been recognized as a bioactive endocrine organ that secretes a host of cytokines and growth factors, many of which are pro-inflammatory and may modulate the tumor microenvironment. The objective of this pilot study was to characterize inflammatory gene expression in various adipose tissue depots and its effect on prostate cancer cell growth. METHODS Periprostatic (PAT) and subcutaneous adipose control tissue (SAT) were harvested from patients with prostate cancer undergoing radical prostatectomy or from control patients with BPH undergoing simple prostatectomy. Adipose was directly analyzed by inflammatory PCR array (n=8) or used to generate conditioned medium (CM) for cell proliferation assays using a 22Rv1 prostate cancer cell line (n=9). RESULTS A host of inflammatory genes (e.g. IL8RA, ILRAB, CXCL2, CCL8) were modestly upregulated (5-6X) in high-grade (Gleason 9) CaP associated-PAT compared with SAT (Figure 1). Several genes including CCL21 and TOLLIP were upregulated 5.9 and 4.8X, respectively, in high-grade CaP associated-PAT compared with BPH associated-PAT while CCL2,CCL4 and CXCL1-3 were down-regulated 4-9.9X, respectively. In both BRDU and MTS assays, high-grade CaP associated PAT-CM stimulated 22Rv1 cell proliferation more than SAT, low-grade or BPH-associated PAT (Figure 2). CONCLUSIONS In this pilot study, PAT from CaP patients demonstrated upregulation of pro-inflammatory gene expression. Conditioned media generated from cancer associated PAT stimulated 22Rv1 cell growth and proliferation more than BPH associated PAT or SAT. In addition, the magnitude of the effect was greatest for high grade cancer associated PAT. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e295-e296 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information David S. Finley Los Angeles, CA More articles by this author Colette Galet Los Angeles, CA More articles by this author Joyce Yamashiro Los Angeles, CA More articles by this author Evelyn Kono Los Angeles, CA More articles by this author Chau Tran Los Angeles, CA More articles by this author Alan Pantuck Los Angeles, CA More articles by this author Jean Dekernion Los Angeles, CA More articles by this author William Aronson Los Angeles, CA More articles by this author Robert Reiter Los Angeles, CA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

RNAi-Mediated Knock-Down of Arylamine N-acetyltransferase-1 Expression Induces E-cadherin Up-Regulation and Cell-Cell Contact Growth Inhibition

Arylamine N-acetyltransferase-1 (NAT1) is an enzyme that catalyzes the biotransformation of arylamine and hydrazine substrates. It also has a role in the catabolism of the folate metabolite p-aminobenzoyl glutamate. Recent bioinformatics studies have correlated NAT1 expression with various cancer subtypes. However, a direct role for NAT1 in cell biology has not been established. In this study, we have knocked down NAT1 in the colon adenocarcinoma cell-line HT-29 and found a marked change in cell morphology that was accompanied by an increase in cell-cell contact growth inhibition and a loss of cell viability at confluence. NAT1 knock-down also led to attenuation in anchorage independent growth in soft agar. Loss of NAT1 led to the up-regulation of E-cadherin mRNA and protein levels. This change in E-cadherin was not attributed to RNAi off-target effects and was also observed in the prostate cancer cell-line 22Rv1. In vivo, NAT1 knock-down cells grew with a longer doubling time compared to cells stably transfected with a scrambled RNAi or to parental HT-29 cells. This study has shown that NAT1 affects cell growth and morphology. In addition, it suggests that NAT1 may be a novel drug target for cancer therapeutics.

Amplification and overexpression of vinculin are associated with increased tumour cell proliferation and progression in advanced prostate cancer

Androgen withdrawal is the standard treatment for advanced prostate cancer. Although this therapy is initially effective, nearly all prostate cancers become refractory to it. Approximately 15% of these castration-resistant prostate cancers harbour a genomic amplification at 10q22. The aim of this study was to explore the structure of the 10q22 amplicon and to determine the major driving genes. Application of high-resolution array-CGH using the 244k Agilent microarrays to cell lines with 10q22 amplification allowed us to narrow down the common amplified region to a region of 5.8 megabases. We silenced each of the genes of this region by an RNAi screen in the prostate cancer cell lines PC-3 and 22Rv1. We selected genes with a significant growth reduction in the 10q22 amplified cell line PC-3, but not in the non-amplified 22Rv1 cells, as putative target genes of this amplicon. Immunohistochemical analysis of the protein expression of these candidate genes on a tissue microarray enriched for 10q22 amplified prostate cancers revealed vinculin as the most promising target of this amplicon. We found a strong association between vinculin gene amplification and overexpression (p < 0.001). Further analysis of 443 specimens from across all stages of prostate cancer progression showed that vinculin expression was highest in castration-resistant prostate cancers, but negative or very low in benign prostatic hyperplasia (p < 0.0001). Additionally, high tumour cell proliferation measured by Ki67 expression was significantly associated with high vinculin expression in prostate cancer (p < 0.0001). Our data suggest that vinculin is a major driving gene of the 10q22 amplification in prostate cancer and that vinculin overexpression might contribute to prostate cancer progression by enhancing tumour cell proliferation.

SLUG promotes prostate cancer cell migration and invasion via CXCR4/CXCL12 axis

BackgroundSLUG is a zinc-finger transcription factor of the Snail/Slug zinc-finger family that plays a role in migration and invasion of tumor cells. Mechanisms by which SLUG promotes migration and invasion in prostate cancers remain elusive.MethodsExpression level of CXCR4 and CXCL12 was examined by Western blot, RT-PCR, and qPCR analyses. Forced expression of SLUG was mediated by retroviruses, and SLUG and CXCL12 was downregulated by shRNAs-expressing lentiviruses. Migration and invasion of prostate cancer were measured by scratch-wound assay and invasion assay, respectively.ResearchWe demonstrated that forced expression of SLUG elevated CXCR4 and CXCL12 expression in human prostate cancer cell lines PC3, DU145, 22RV1, and LNCaP; conversely, reduced expression of SLUG by shRNA downregulated CXCR4 and CXCL12 expression at RNA and protein levels in prostate cancer cells. Furthermore, ectopic expression of SLUG increased MMP9 expression and activity in PC3, 22RV1, and DU-145 cells, and SLUG knockdown by shRNA downregulated MMP9 expression. We showed that CXCL12 is required for SLUG-mediated MMP9 expression in prostate cancer cells. Moreover, we found that migration and invasion of prostate cancer cells was increased by ectopic expression of SLUG and decreased by SLUG knockdown. Notably, knockdown of CXCL12 by shRNA impaired SLUG-mediated migration and invasion in prostate cancer cells. Lastly, our data suggest that CXCL12 and SLUG regulate migration and invasion of prostate cancer cells independent of cell growth.ConclusionWe provide the first compelling evidence that upregulation of autocrine CXCL12 is a major mechanism underlying SLUG-mediated migration and invasion of prostate cancer cells. Our findings suggest that CXCL12 is a therapeutic target for prostate cancer metastasis.

Docetaxel maintains its cytotoxic activity under hypoxic conditions in prostate cancer cells

ObjectiveThe efficacy of docetaxel has recently been shown to be increased under hypoxic conditions through the down-regulation of hypoxia-inducible-factor 1α (HIF1A). Overexpression of the hypoxia-responsive gene class III β-tubulin (TUBB3) has been associated with docetaxel resistance in a number of cancer models. We propose that administration of docetaxel to prostate patients has the potential to reduce the hypoxic response through HIF1A down-regulation and that TUBB3 down-regulation participates in sensitivity to docetaxel. MethodsThe cytotoxic effect of docetaxel was determined in both 22Rv1 and DU145 prostate cancer cell lines and correlated with HIF1A expression levels under aerobic and hypoxic conditions. Hypoxia-induced chemoresistance was investigated in a pair of isogenic docetaxel-resistant PC3 cell lines. Basal and hypoxia-induced TUBB3 gene expression levels were determined and correlated with methylation status at the HIF1A binding site. ResultsProstate cancer cells were sensitive to docetaxel under both aerobic and hypoxic conditions. Hypoxic cytotoxicity of docetaxel was consistent with a reduction in detected HIF1A levels. Sensitivity correlated with reduced basal and hypoxia-induced HIF1A and TUBB3 expression levels. The TUBB3 HIF1A binding site was hypermethylated in prostate cell lines and tumor specimens, which may exclude transcription factor binding and induction of TUBB3 expression. However, acquired docetaxel resistance was not associated with TUBB3 overexpression. ConclusionThese data suggest that the hypoxic nature of a tumor may have relevance as regard to their response to docetaxel. Further investigation into the nature of this relationship may allow identification of novel targets to improve tumor control in prostate cancer patients.

Differential Protein Expression Profiling by iTRAQ-Two-dimensional LC-MS/MS of Human Bladder Cancer EJ138 Cells Transfected with the Metastasis Suppressor KiSS-1 Gene

KiSS-1 is a metastasis suppressor gene reported to be involved in the progression of several solid neoplasias. The loss of KiSS-1 gene expression has been shown to be inversely correlated with increasing tumor stage, distant metastases, and poor overall survival in bladder tumors. To identify the molecular pathways associated with the metastasis suppressor role of KiSS-1 in bladder cancer, we carried out a proteomics analysis of bladder cancer cells (EJ138) transiently transfected with a vector encompassing the full-length KiSS-1 gene using an iTRAQ (isobaric tags for relative and absolute quantitation) approach. Protein extracts collected after 24- and 48-h transfection were fractionated and cleaved with trypsin, and the resulting peptides were labeled with iTRAQ reagents. The labeled peptides were separated by strong cation exchange and reversed phase LC and analyzed by MALDI-TOF/TOF MS. Three software packages were utilized for data analysis: ProteinPilot for identification and quantification of differentially expressed proteins, Protein Center for gene ontology analysis, and Ingenuity Pathways Analysis to provide insight into biological networks. Comparative analysis among transfected, mock, and empty vector-exposed cells identified 1529 proteins with high confidence (>99%) showing high correlation rates among replicates (70%). The involvement of the identified proteins in biological networks served to characterize molecular pathways associated with KiSS-1 expression and to select critical candidates for verification analyses by Western blot using independent transfected replicates. As part of complementary clinical validation strategies, immunohistochemical analyses of proteins regulated by KiSS-1, such as Filamin A, were performed on bladder tumors spotted onto tissue microarrays (n = 280). In summary, our study not only served to uncover molecular mechanisms associated with the metastasis suppressor role of KiSS-1 in bladder cancer but also to reveal the biomarker role of Filamin A in bladder cancer progression and clinical outcome.