Abstract 1634: Cyclin D1 induces Akt1 activity in RB deficient prostate cancer

Prostate cancer is one of the major causes of cancer-related death in the western world. Androgen-deprivation therapy (ADT) for the suppression of androgens binding to the androgen receptor (AR) has been the norm of prostate cancer treatment. Despite early success to suppress prostate tumor growth, ADT eventually fails leading to recurrent tumor growth in a hormone-refractory manner, even though AR remains to function in hormone-refractory prostate cancer. Interestingly, some prostate cancer survivors who received androgen replacement therapy had improved quality of life without adverse effect on their cancer progression. These contrasting clinical data suggest that differential androgen/AR signals in individual cells of prostate tumors can exist in the same or different patients, and may be used to explain why ADT of prostate cancer fails. Such a hypothesis is supported by the results obtained from transgenic mice with selective knockout of AR in prostatic stromal vs epithelial cells and orthotopic transplants of various human prostate cancer cell lines with AR over-expression or knockout. These studies concluded that AR functions as a stimulator for prostate cancer proliferation and metastasis in stromal cells, as a survival factor of prostatic cancer epithelial luminal cells, and as a suppressor for prostate cancer basal intermediate cell growth and metastasis. These dual yet opposite functions of the stromal and epithelial AR may challenge the current ADT to battle prostate cancer and should be taken into consideration when developing new AR-targeting therapies in selective prostate cancer cells.

Isolation and characterization of the major form of human MUC18 cDNA gene and correlation of MUC18 over-expression in prostate cancer cell lines and tissues with malignant progression

Structure-activity relationship studies of 1,7-diheteroarylhepta-1,4,6-trien-3-ones with two different terminal rings in prostate epithelial cell models

Recent studies have highlighted tumor microenvironment as important in carcinogenesis and progression. Especially in prostate cancer (PC), it has been suggested that tumor microenvironment plays an important role in progression, acquisition of androgen independence, and distant metastasis. However, little is known about the genetic basis of human PC microenvironment. The prognosis of PC is related to invasion and metastasis. Epithelial-to-mesenchymal transition (EMT) has gained considerable attention as a conceptual paradigm to explain invasive and metastatic behavior during cancer progression. EMT is a normal physiologic process by which cells of epithelial origin convert into cells with mesenchymal characteristics. It has been proposed that EMT is induced by cancer cells during metastatic dissemination from a primary organ to secondary sites, but how EMT occurs during PC invasion and metastasis remains uncertain. To clarify the mechanism of PC progression and metastasis, we investigated cross-interaction of PC epithelial cells and stromal cells by analyzing genome-wide gene expression profiles. We hypothesized that PC cells could affect on stromal cells to change their characteristics into so-called cancer-associated fibroblasts (CAFs), which might contribute to cancer invasion and metastasis. Likewise, CAFs could affect on surrounding normal epithelial cells to change their characteristics into PC cells. We first analyzed three of genome-wide gene expression profiles; normal human prostate stromal cells (PrSC) co-cultured with human prostate cancer cells (LNCaP), LNCaP co-cultured with PrSC, and human prostate epithelial cells (PrEC) co-cultured with cancer-associated PrSC which is PrSC previously co-cultured with LNCaP. Before microarray analysis, we confirmed the overexpression of alpha-smooth muscle actin in PrSC co-cultured with LNCaP, which means the transition from normal stromal cells to CAFs. We then analyzed networks among PC cells, stromal cells, and surrounding normal epithelial cells by analyzing gene expression profiles and validated our data using real-time PCR, immunohistochemistry, and bioinformatics. These data provide clues to the molecular mechanisms underlying PC invasion and metastasis, and suggest candidate genes the products of which might serve as molecular targets for the treatment of PC. 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 4282. doi:1538-7445.AM2012-4282

Introduction: Chemokines are a family of cytokines known to regulate the migration of cells. The chemokine CXCL12, also known as SDF-1, and its receptor CXCR4 are associated with prostate cancer bone metastasis. The tumor suppressor PTEN (phosphatase and tensin homolog) is a critical regulator of growth factors and inhibitor of PI3K. Loss of PTEN is frequently observed in cancer, resulting in the deregulation of cell survival, growth, and proliferation. Previous studies have found that PTEN is lost or mutated in 30-80% of primary prostate cancer, and 50% of prostate cancer bone metastases. In mouse models of prostate cancer, it has been shown that loss of PTEN is critical for tumor initiation, and the level of PTEN expression is inversely associated with prostate tumorigenesis. Murine epithelial cells from PTEN-deficient prostate tumors also display increased expression of CXCR4 and CXCL12. We hypothesize that loss of PTEN and subsequent activation of Akt, frequent occurrences in prostate cancer, regulate the CXCL12/CXCR4 signaling axis in prostate cancer progression. Methods: Prostate-specific deletion of floxed exon 5 of PTEN was achieved by Cre recombinase expressed under the control of an androgen-responsive probasin promoter. Stable cell lines were developed from PTEN+/+, PTEN+/−, and PTEN−/− murine prostate epithelial cells isolated from prostates of corresponding mice at 8 weeks of age. PTEN status in these cell lines was confirmed by genotyping and immunoblot analyses. Human prostate cancer cell lines BPH-1, C4-2B, PC3, and DU145 were also utilized. Gene expression and invasion were analyzed in the presence or absence of Akt inhibitor. Additionally, DU145 with overexpressed HA-tagged Akt1 was also utilized in a subcutaneous injection model. Results: Loss of PTEN resulted in increased expression of CXCR4 and CXCL12, suggesting that loss of PTEN may play a key role in the regulation of these chemokines in prostate cancer. Upon treatment of PTEN-null cells with Akt inhibitor, the induction of CXCL12/CXCR4 was reversed, further demonstrating the role of the PTEN regulated pathway in the expression of these genes. Akt inhibition also resulted in decreased invasion in response to CXCL12. Overexpression of Akt1 in DU145 resulted increased CXCR4 expression, as well as increased proliferation and cell cycle progression. Subcutaneous injection of these cells also resulted in increased tumor growth as compared to neo controls. Conclusions: These results suggest the basis for activation of CXCL12 signaling through CXCR4 in prostate cancer driven by the loss of PTEN and subsequent activation of Akt. Citation Format: Katie Conley-Lacomb, Allen Saliganan, Yong Q. Chen, Hyeong-Reh C. Kim, Michael L. Cher, Sreenivasa R. Chinni. PTEN loss mediated Akt activation promotes prostate tumor growth via CXCL12/CXCR4 signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4088. doi:10.1158/1538-7445.AM2013-4088

Objective To observe the level of eleven-nineteen lysine-rich leukemia gene 2 (ELL2) in human prostate cancer tissues and its effect on proliferation of prostate cancer cell C4-2. Methods The expression of ELL2 in human prostate cancer tissues was analyzed by immunohistochemical method, and the relationship between ELL2 and malignancy degree of prostate cancer was evaluated. The human prostate cancer cell line C4-2 was stimulated with synthetic androgen R1881 to detect its response to androgen at different time points. ELL2-small interfering RNA (siRNA) and overexpression of ELL2-bearing plasmid were transfected into C4-2 cells respectively, then cell proliferation was evaluated by methyl thiazol tetrazolium (MTT) assay. The DNA synthesis ability of cells was assessed by 5-Bromo-2’-deoxy-uridine (BrdU) incorporation assay. Results The expression of ELL2 was significantly lower in human prostate cancer than that of benign specimen, and decreased with the increase of malignancy of prostate cancer. The correlation analysis between ELL2 and prostate cancer Gleason score reveal a negative correlation result (r=0.836, P=0.000). The protein expression of ELL2 was significantly increased in 48 h and increased by (38.27±10.11)% (t=7.573, P=0.005) and (58.34±13.70)% (t=8.519, P=0.003) at 24 h and 48 h, respectively. The expression of ELL2 protein in the two RNA interference groups were significantly decreased, and the decreased rate were (56.38±11.26)% (t=10.010, P=0.002) and (65.27±12.10)% (t=10.790, P=0.002), respectively. While the ELL2 protein expression in the overexpression group was significantly increased at (80.23±20.94)% (t=7.662, P=0.005). The proliferation ability of C4-2 cells was significantly increased (39.28±8.52)% (t=11.300, P=0.000) and (48.2±7.87)% (t=15.000, P=0.000) in the two RNA interference groups, respectively, compared with the control group. While its proliferation ability decreased (42.74±10.38)% (t=10.090, P=0.000) in ELL2-bearing plasmid. Transfection of ELL2-specific interfering RNA increased the ability of DNA synthesis, while the transfection of ELL2-bearing plasmid cells had lower DNA synthesis ability. Conclusion ELL2 is lowly expressed in human prostate cancer tissues and negatively correlated with the malignant degree. ELL2 can inhibit the proliferation of prostate cancer cells to a certain extent. Key words: Prostate cancer; Eleven-nineteen lysine-rich leukemia gene 2; Proliferation

Research into the mechanisms of prostate cancer progression has been limited by the lack of suitable in vitro systems. A hurdle in understanding the molecular genetic changes in prostate cancer has been the difficulty in establishing premalignant lesions and primary prostate tumors as in vitro cell cultures. Primary prostate epithelial cells grow for a finite life span and then senesce. Immortalization is defined by continuous growth of otherwise senescing cells and is believed to represent an early stage in tumor progression. To examine these early stages, we and others have developed in vitro models of prostate epithelial cell immortalization. Generation of primary human prostate epithelial (HPE) cells has been achieved using the serum-free condition. Retrovirus containing human telomerase reverse transcriptase (hTERT) was successfully used for the immortalization of primary HPE cells. Putative stem cell markers CD133 and CXCR4 were further identified in hTERT-immortalized primary nonmalignant and malignant tumor-derived HPE lines. In addition, an hTERT-immortalized nonmalignant HPE cell were found to retain the properties of multipotent stem cells. These in vitro prostate cell culture models should be useful for the study of carcinogenesis and of normal and cancer stem cells. Prostate cancer is the most common male cancer in the Western World and second leading cause of male cancer death in the United States [1]. The therapy most widely used against advanced disease is androgen ablation and, initially, it almost always produces objective clinical responses. However, most patients eventually relapse with ablation-resistant prostate cancer and develop metastatic disease; currently, there is no treatment that will cure progressive hormone-refractory metastatic prostate cancer. The mechanisms of progression of prostate cancer have been extensively studied, yet are poorly understood. One of the concepts that has been evolved is that cancer arises from the neoplastic transformation of normal prostate epithelial stem cells or transit amplifying cells. Understanding normal stem cells and cancer stem cells (CSCs) may provide insight into the origin of and new therapeutics for prostate cancer. However, research in this field is limited by the lack of suitable in vitro systems.

14-3-3σ is down-regulated in human prostate cancer

Emerging evidence suggests that the SAM pointed domain containing ETS transcription factor (SPDEF) plays a significant role in tumorigenesis in prostate, breast, colon, and ovarian cancer. However, there are no in vivo studies with respect to the role of SPDEF in tumor metastasis. The present study examined the effects of SPDEF on tumor cell metastasis using prostate tumor cells as a model. Utilizing two experimental metastasis models, we demonstrate that SPDEF inhibits cell migration and invasion in vitro and acts a tumor metastasis suppressor in vivo. Using stable expression of SPDEF in PC3-Luc cells and shRNA-mediated knockdown of SPDEF in LNCaP-Luc cells, we demonstrate for the first time that SPDEF diminished the ability of disseminated tumors cells to survive at secondary sites and establish micrometastases. These effects on tumor metastasis were not a result of the effect of SPDEF on cell growth as SPDEF expression had no effect on cell growth in vitro or subcutaneous tumor xenograft-growth in vivo. Transcriptional analysis of several genes associated with tumor metastasis, invasion, and the epithelial-mesenchymal transition demonstrated that SPDEF expression selectively down-regulated MMP9 and MMP13 in prostate cancer cells. Further analysis indicated that forced MMP9 or MMP13 expression rescued the invasive phenotype in SPDEF expressing PC3 cells in vitro, suggesting that the effects of SPDEF on tumor invasion are mediated, in part, through the suppression of MMP9 and MMP13 expression. These results demonstrate for the first time, in any system, that SPDEF functions as a tumor metastasis suppressor in vivo.

Vaccinium myrtillus berry extract (VME) and a recombined standard mixture (RSM) of its main native phenolic compounds were investigated for cell growth inhibition and pro-apoptotic activity on hormone-dependent (LNCaP) and hormone-independent (PC3 and DU-145) prostate cancer (PCa) cell lines. Normal prostate epithelial cells (PrEC) were also studied in comparison. VME hindered anchorage-dependent PCa cell proliferation in a dose-dependent manner, that is, at 1/800 (v/v) dilution for LNCaP and PC3, and 1/100 (v/v) dilution for DU-145 (corresponding to 14.15 and 113.2 μg cyanidin-3-O-glucoside equivalents per ml of culture medium), respectively. VME had a growth inhibitory effect towards PrEC at the same dilution of DU-145 cells although the IC50 values indicated that PrEC are more resistant than PCa cell lines. VME also reduced the anchorage-independent growth of PCa cells. The study of the apoptotic profile (i.e., non-apoptotic, early apoptotic, late apoptotic and necrotic cells) evidenced that the apoptotic rate (early+late) was statistically higher in all three cell lines exposed to VME compared to control. Anchorage-dependent and anchorage-independent growth inhibition of RSM was very similar to that displayed by VME. Moreover, RSM exerted its growth inhibitory effect also under hypoxia, the latter representing a biological condition known to sustain PCa proliferation and aggressiveness.

BackgroundThe human TSPY is the putative gene for the gonadoblastoma locus on the Y chromosome (GBY). Various molecular, pathological and transgenic mouse studies suggest that TSPY is a Y-located proto-oncogene contributing to the initiation/progression in human cancers, including germ cell tumors and various somatic cancers, such as prostate and liver cancer, and melanoma. The TgTSPY9 transgenic mouse line harbors a 8.2-kb human TSPY structural gene, which is tandemly integrated in the mouse Y chromosome, and expressed in a similar pattern as that of the endogenous gene in the human genome. This mouse model of human TSPY gene offers an opportunity to examine its behavior and potential contribution in various mouse models of human diseases, such as human cancers. We had investigated the expression of such TSPY-transgene in the LADY mouse model of prostate cancer, harboring a SV40 T antigen gene directed by a rat probasin promoter; and compared the expression pattern with those of endogenous TSPY gene and biomarkers in human prostate cancer specimens.ResultsBy introducing the Y-located TSPY-transgene to the LADY mice, we had examined the expression pattern of the human TSPY during prostatic oncogenesis in this mouse model of prostate cancer. Our results showed that the TSPY-transgene was activated in selected areas of the hypercellular stroma but not in the intraepithelial cells/neoplasia in the prostates of TgTSPY9/LADY mice. Using a specific biomarker, FOXA1, for epithelial cells, we demonstrated that TSPY-positive cells proliferated exclusively in the cancerous stroma in the LADY model at late stages of tumorigenesis. In contrast, in the human situation, TSPY was predominantly co-expressed with FOXA1 in the epithelial cells of PIN lesions and FOXA1 and another cancer biomarker, AMACR, in the adenocarcinoma cells in clinical prostate cancer samples of various degrees of malignancy.ConclusionsOur data show that human TSPY could be abnormally activated during prostatic oncogenesis, and could possibly contribute to the heterogeneity of prostate cancer. The differential expression patterns of the human TSPY between the LADY mouse model and clinical prostate cancer suggest potential limitations of current mouse models for studies of either TSPY behavior in diseased conditions or prostate cancer development.

MP64-02 THERAPEUTIC TARGETING OF TRANSCRIPTIONAL REPRESSOR BCL-6 FOR CASTRATION-RESISTANT PROSTATE CANCER THROUGH REPRESSING ANDROGEN RECEPTOR VARIANTS.

Interferon regulatory factor-5 (IRF5) is a transcription factor and has essential cellular mechanisms as a tumour suppressor gene. IRF5 protein function is irregular in various human cancers, and its role in prostate cancer is also unknown. This study presents the first evidence that IRF5 expression is controlled with androgen receptor (AR) signalling interaction and stem cell factors (Nanog, Oct4, Sox2) in prostate cancer. Human prostate cancer cell lines (PC3, DU145 and LNCaP) were transfected plasmids and assessed for cellular localization of IRF5 and AR interaction with IF-staining. Co-immunoprecipitation and ChIP assay were used to detect the IRF5 and AR protein-protein interaction and IRF5 stem cell factors protein-gene interaction. The target relation between IRF5, AR, CREB, p300, ISRE, ARE and NF-кB was tested by luciferase assay. IRF5 was low expressed in androgen-dependent prostate cancer cells and tissues. The analysis of human prostate cancer clinical samples supports the interaction of IRF5 and AR in a pathological role, as IRF5 expression is down-regulated in the tumours' advanced stages. Tumour suppression mechanism of IRF5 and SOX2 levels in cells reduces and causes AR acetylation. Those affect the prostate cancer mechanism by modifying the cellular response in the signal pathway. IRF5 can be promising for treating androgen-dependent prostate cancers and is a therapeutic protein for new drug studies.

Preferential metastasis of human prostate cancer cells to bone marrow contributes to disease mortality and morbidity. MicroRNAs (miRNAs) are endogenous ~22nt RNAs that play important regulatory roles in animals and plants by binding to specific messenger RNA (mRNA) targets to either block their translation or trigger their degradation. Accumulating evidence indicates that miRNAs are involved in human cancer. Here we used the human prostate cancer cell line, LNCaP, and its derivative bone metastatic subline, C4‐2B, as a model system to study the roles of miRNAs in prostate cancer progression. We sequenced more than two million small RNAs from LNCaP and C4‐2B cell lines using a 454 library. Known miRNAs and new small candidate miRNAs with expression differences between these two cell lines were selected for analysis. Various bioinformatics tools and gene databases were used to identify potential targets for both novel and known microRNAs. Prostate cancer gene expression microarray data were used to further narrow the prospective target genes for known microRNAs. Biobase (http://bkl.biobase.de/cgi‐bin/bkl/idb/1.0/searchengine/start.cgi) software was used to place these gene targets in relevant cellular pathways. Ongoing studies seek to explore the potential targets of miRNAs involved in these pathways. These data provide us with important information about how miRNAs are involved in prostate cancer including identification of new pathways for modulation of gene expression during progression to bone. Understanding the targets and functions of these miRNAs will translate into new means to inhibit prostate cancer metastasis and growth in bone. (Supported by NCI P01 CA098912 and University of Delaware Science and Engineering Scholars Cancer and Genetics Fellowship.)

Targeting TCTP as a New Therapeutic Strategy in Castration-resistant Prostate Cancer