Promotes Prostate Cancer Growth Research Articles

The intriguing role of fibroblasts and c-Jun in the chemopreventive and therapeutic effect of finasteride on xenograft models of prostate cancer

In a large clinical trial, finasteride reduced the rate of low-grade prostate cancer (PCa) while increasing the incidence of high-grade cancer. Whether finasteride promotes the development of high-grade tumors remains controversial. We demonstrated the role of fibroblasts and c-Jun in chemopreventive and therapeutic effect of finasteride on xenograft models of PCa. LNCaP (PC3) cells or recombinants of cancer cells and fibroblasts were implanted in male athymic nude mice treated with finasteride. Tumor growth, cell proliferation, apoptosis, p-Akt, and p-ERK1/2 were evaluated. In LNCaP (PC3) mono-grafted models, finasteride did not change the tumor growth. In recombinant-grafted models, fibroblasts and c-Jun promoted tumor growth; finasteride induced proliferation of LNCaP cells and repressed PC3 cell apoptosis. When c-Jun was knocked out, fibroblasts and/or finasteride did not promote the tumor growth. Finasteride inhibited p-Akt and p-ERK1/2 in mono-culture cancer cells while stimulating the same signaling molecules in the presence of fibroblasts. Reduced p-Akt and p-ERK1/2 were noted in the presence of c-Jun−/− fibroblasts. Fibroblasts and c-Jun promote PCa growth; finasteride further stimulates tumor growth with promoted proliferation, repressed apoptosis, and up-regulated pro-proliferative molecular pathway in the presence of fibroblasts and c-Jun. Stromal-epithelial interactions play critical roles in finasteride's therapeutic effects on PCa. Our findings have preliminary implications in using finasteride as a chemopreventive or therapeutic agent for PCa patients.

Non-Coding RNAs in Castration-Resistant Prostate Cancer: Regulation of Androgen Receptor Signaling and Cancer Metabolism.

Hormone-refractory prostate cancer frequently relapses from therapy and inevitably progresses to a bone-metastatic status with no cure. Understanding of the molecular mechanisms conferring resistance to androgen deprivation therapy has the potential to lead to the discovery of novel therapeutic targets for type of prostate cancer with poor prognosis. Progression to castration-resistant prostate cancer (CRPC) is characterized by aberrant androgen receptor (AR) expression and persistent AR signaling activity. Alterations in metabolic activity regulated by oncogenic pathways, such as c-Myc, were found to promote prostate cancer growth during the development of CRPC. Non-coding RNAs represent a diverse family of regulatory transcripts that drive tumorigenesis of prostate cancer and various other cancers by their hyperactivity or diminished function. A number of studies have examined differentially expressed non-coding RNAs in each stage of prostate cancer. Herein, we highlight the emerging impacts of microRNAs and long non-coding RNAs linked to reactivation of the AR signaling axis and reprogramming of the cellular metabolism in prostate cancer. The translational implications of non-coding RNA research for developing new biomarkers and therapeutic strategies for CRPC are also discussed.

ACSL4 promotes prostate cancer growth, invasion and hormonal resistance.

Increases in fatty acid metabolism have been demonstrated to promote the growth and survival of a variety of cancers, including prostate cancer (PCa). Here, we examine the expression and function of the fatty acid activating enzyme, long-chain fatty acyl-CoA synthetase 4 (ACSL4), in PCa. Ectopic expression of ACSL4 in ACSL4-negative PCa cells increases proliferation, migration and invasion, while ablation of ACSL4 in PCa cells expressing endogenous ACSL4 reduces cell proliferation, migration and invasion. The cell proliferative effects were observed both in vitro, as well as in vivo. Immunohistochemical analysis of human PCa tissue samples indicated ACSL4 expression is increased in malignant cells compared with adjacent benign epithelial cells, and particularly increased in castration-resistant PCa (CRPC) when compared with hormone naive PCa. In cell lines co-expressing both ACSL4 and AR, proliferation was independent of exogenous androgens, suggesting that ACSL4 expression may lead to CRPC. In support for this hypothesis, ectopic ACSL4 expression induced resistance to treatment with Casodex, via decrease in apoptosis. Our studies further indicate that ACSL4 upregulates distinct pathway proteins including p-AKT, LSD1 and β-catenin. These results suggest ACSL4 could serve as a biomarker and potential therapeutic target for CRPC.

Autocrine Activation of CHRM3 Promotes Prostate Cancer Growth and Castration Resistance via CaM/CaMKK-Mediated Phosphorylation of Akt.

Although a previous study reported nerve ending-derived acetylcholine promoted prostate cancer invasion and metastasis by regulating the microenvironment of cancer cells, the present study aims to determine whether there is autocrine cholinergic signaling in prostate epithelial cells that promotes prostate cancer growth and castration resistance. In this study, IHC was performed to detect protein expression in mouse prostate tissue sections and human prostate cancer tissue sections. Subcutaneously and orthotopically xenografted tumor models were established to evaluate the functions of autocrine cholinergic signaling in regulating prostate cancer growth and castration resistance. Western blotting analysis was performed to assess the autocrine cholinergic signaling-induced signaling pathway. We found the expression of choline acetyltransferase (ChAT), the secretion of acetylcholine and the expression of CHRM3 in prostate epithelial cells, supporting the presence of autocrine cholinergic signaling in the prostate epithelium. In addition, we found that CHRM3 was upregulated in clinical prostate cancer tissues compared with adjacent non-cancer tissues. Overexpression of CHRM3 or activation of CHRM3 by carbachol promoted cell proliferation, migration, and castration resistance. On the contrary, blockading CHRM3 by shRNA or treatment with darifenacin inhibited prostate cancer growth and castration resistance both in vitro and in vivo. Furthermore, we found that autocrine cholinergic signaling caused calmodulin/calmodulin-dependent protein kinase kinase (CaM/CaMKK)-mediated phosphorylation of Akt. These findings suggest that blockade of CHRM3 may represent a novel adjuvant therapy for castration-resistant prostate cancer.

241 Fibroblast growth factor 23 (FGF23) promotes prostate cancer growth

241 Fibroblast growth factor 23 (FGF23) promotes prostate cancer growth

Abstract 4960: ERp46 (thioredoxin domain-containing protein 5, TXND5) promotes prostate cancer growth in vitro and in vivo

Abstract We have recently demonstrated that endoplasmic reticulum protein ERp46, a member of the protein disulfide isomerase family of oxidoreductases, TXND5, is overexpressed in human metastatic renal cell carcinoma. The expression and function of ERp46 in prostate cancer has not been studied. Using both in vitro and in vivo approaches, we explore the suitability of ERp46 as a potential therapeutic target in prostate cancer. Tissue microarray containing normal prostate epithelium (n = 9) and prostate cancer specimens from 57 patients was stained for ERp46 and the staining intensity (H-score) was determined. Human prostate adenocarcinoma 22Rv1 cells were used to generate gain- and loss-of-function models by stable ERp46 shRNA knockdown and ERp46 overexpression, respectively. In vitro, the doubling time and PSA production were determined. In vivo, xenografts of each subclone were established in nude mice (n = 10/group) to determine the longitudinal tumor growth and serum PSA values. Gene expression profiling of RNA isolated from 22Rv1 xenografts was performed using human whole genome HT-12 V4 BeadChip array (Illumina). Our results demonstrated that human prostate carcinoma samples of Gleason scores ≥7 showed strong cytoplasmic ERp46 staining which was significantly increased compared to normal prostatic tissue (p = 0.02). ERp46 staining in prostate tumors of Gleason scores ≤6, however, was not different compared to normal prostate tissue. The stably transfected human prostate carcinoma 22Rv1 cells expressed 89% knockdown of ERp46 protein expression (shERp46) or a 4-fold increase in ERp46 protein expression (ERp46+) compared to the respective control cells. In vitro, shERp46 cells proliferated slower, whereas ERp46+ cells exhibited accelerated growth compared to corresponding control cells (p<0.05). Similarly, the tumor volume of subcutaneously growing shERp46 cells in nude mice led to significantly slower tumor growth (p<0.0005, ANOVA). Vice versa, tumors from ERp46+ cells were significantly larger than the tumor volume of shControl-cell injected mice (p = 0.02, ANOVA). Gene expression analysis confirmed the downregulation and upregulation of ERp46 in the corresponding xenografts and also showed several candidate genes, including NAAA, SH3BP4 and ID1 that were reciprocally up-and downregulated. In conclusion, this is the first report to suggest a role for ERp46 as an oncogenic protein and potential therapeutic target in prostate cancer given its expression profile in human prostate cancer samples and its effect on prostate cancer cell growth. Funding was provided by Prostate Cancer Canada and McMaster Surgical Associates (JHP and WCMD) Citation Format: Jehonathan H. Pinthus, Sarah N. Hopmans, Stephanie Federov, Wilhelmina C. Duivenvoorden. ERp46 (thioredoxin domain-containing protein 5, TXND5) promotes prostate cancer growth in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4960. doi:10.1158/1538-7445.AM2015-4960

838 Non-canonical Wnt signaling promotes prostate cancer growth by increasing growth factors from stromal cells and plays a key role on stromal-epithelial cell communication

838 Non-canonical Wnt signaling promotes prostate cancer growth by increasing growth factors from stromal cells and plays a key role on stromal-epithelial cell communication

Insulin-like growth factor-I induces CLU expression through Twist1 to promote prostate cancer growth

Clusterin (CLU) is cytoprotective molecular chaperone that is highly expressed in castrate-resistant prostate cancer (CRPC). CRPC is also characterized by increased insulin-like growth factor (IGF)-I responsiveness which induces prostate cancer survival and CLU expression. However, how IGF-I induces CLU expression and whether CLU is required for IGF-mediated growth signaling remain unknown. Here we show that IGF-I induced CLU via STAT3–Twist1 signaling pathway. In response to IGF-I, STAT3 was phosphorylated, translocated to the nucleus and bound to the Twist1 promoter to activate Twist1 transcription. In turn, Twist1 bound to E-boxes on the CLU promoter and activated CLU transcription. Inversely, we demonstrated that knocking down Twist1 abrogated IGF-I induced CLU expression, indicating that Twist1 mediated IGF-I-induced CLU expression. When PTEN knockout mice were crossed with lit/lit mice, the resultant IGF-I deficiency suppressed Twist1 as well as CLU gene expression in mouse prostate glands. Moreover, both Twist1 and CLU knockdown suppressed prostate cancer growth accelerated by IGF-I, suggesting the relevance of this signaling not only in an in vitro, but also in an in vivo. Collectively, this study indicates that IGF-I induces CLU expression through sequential activation of STAT3 and Twist1, and suggests that this signaling cascade plays a critical role in prostate cancer pathogenesis.

Roles of Serine Protease Inhibitor Kazal type 1 (SPINK1) in Prostate Cancer

Altered genes that play a driving role in cancer development can often serve as specific diagnostic markers, criteria of molecular classification and therefore potential therapeutic targets. Serine protease inhibitor Kazal type 1 (SPINK1), also known as pancreatic secretory trypsin inhibitor or tumor-associated trypsin inhibitor, encodes a 56 amino acid secreted peptide, and its normal function is thought to be the inhibition of serine proteases such as trypsin. Recent studies have indicated marked overexpression of SPINK1 defines an aggressive molecular subtype of ETS (erythroblastosis virus E26 transformation-specific) fusion-negative prostate cancer ((PCa) patients. SPINK1 may act as an autocrine growth factor and promotes PCa growth and invasion. Most recently, we suggested that SPINK1 induces epithelial-mesenchymal transition (EMT) through EGFR signaling pathway in PCa. The association between SPINK1 overexpression and poor prognosis in PCa has been reported. Notably, SPINK1 might be a novel extracellular therapeutic target in a subset of high-grade PCa patients. In this review, we will summarize the current understanding of SPINK1 involving its role in PCa biology, association with prognosis as well as perspective in therapy from the pathologist's point of view.

Activation of the Wnt Pathway through AR79, a GSK3β Inhibitor, Promotes Prostate Cancer Growth in Soft Tissue and Bone

Due to its bone anabolic activity, methods to increase Wnt activity, such as inhibitors of dickkopf-1 and sclerostin, are being clinically explored. Glycogen synthase kinase (GSK3β) inhibits Wnt signaling by inducing β-catenin degradation, and a GSK3β inhibitor, AR79, is being evaluated as an osteoanabolic agent. However, Wnt activation has the potential to promote tumor growth; therefore, the goal of this study was to determine if AR79 has an impact on the progression of prostate cancer. Prostate cancer tumors were established in subcutaneous and bone sites of mice followed by AR79 administration, and tumor growth, β-catenin activation, proliferation, and apoptosis were assessed. Additionally, prostate cancer and osteoblast cell lines were treated with AR79, and β-catenin status, proliferation (with β-catenin knockdown in some cases), and proportion of ALDH(+)CD133(+) stem-like cells were determined. AR79 promoted prostate cancer tumor growth, decreased phospho-β-catenin, increased total and nuclear β-catenin, and increased tumor-induced bone remodeling. Additionally, AR79 treatment decreased caspase-3 and increased Ki67 expression in tumors and increased bone formation in normal mouse tibiae. Similarly, AR79 inhibited β-catenin phosphorylation, increased nuclear β-catenin accumulation in prostate cancer and osteoblast cell lines, and increased proliferation of prostate cancer cells in vitro through β-catenin. Furthermore, AR79 increased the ALDH(+)CD133(+) cancer stem cell-like proportion of the prostate cancer cell lines. In conclusion, AR79, while being bone anabolic, promotes prostate cancer cell growth through Wnt pathway activation. These data suggest that clinical application of pharmaceuticals that promote Wnt pathway activation should be used with caution as they may enhance tumor growth.

Steroidogenic Enzyme AKR1C3 Is a Novel Androgen Receptor-Selective Coactivator that Promotes Prostate Cancer Growth

Castration-resistant prostate cancer (CRPC) may occur by several mechanisms including the upregulation of androgen receptor (AR), coactivators, and steroidogenic enzymes, including aldo keto reductase 1C3 (AKR1C3). AKR1C3 converts weaker 17-keto androgenic precursors to more potent 17-hydroxy androgens and is consistently the major upregulated gene in CRPC. The studies in the manuscript were undertaken to examine the role of AKR1C3 in AR function and CRPC. LNCaP cells stably transfected with AKR1C3 and VCaP cells endogenously expressing AKR1C3 were used to understand the effect of AKR1C3 on prostate cancer cell and tumor growth in nude mice. Chromatin immunoprecipitation, confocal microscopy, and co-immunoprecipitation studies were used to understand the recruitment of AKR1C3, intracellular localization of AKR1C3 and its interaction with AR in cells, tumor xenograft, and in Gleason sum 7 CRPC tissues. Cells were transiently transfected for AR transactivation. Novel small-molecule AKR1C3-selective inhibitors were synthesized and characterized in androgen-dependent prostate cancer and CRPC models. We identified unique AR-selective coactivator- and prostate cancer growth-promoting roles for AKR1C3. AKR1C3 overexpression promotes the growth of both androgen-dependent prostate cancer and CRPC xenografts, with concomitant reactivation of androgen signaling. AKR1C3 interacted with AR in prostate cancer cells, xenografts, and in human CRPC samples and was recruited to the promoter of an androgen-responsive gene. The coactivator and growth-promoting functions of AKR1C3 were inhibited by an AKR1C3-selective competitive inhibitor. AKR1C3 is a novel AR-selective enzymatic coactivator and may represent the first of more than 200 known nuclear hormone receptor coactivators that can be pharmacologically targeted.

IGF-IR Promotes Prostate Cancer Growth by Stabilizing α5β1 Integrin Protein Levels

Dynamic crosstalk between growth factor receptors, cell adhesion molecules and extracellular matrix is essential for cancer cell migration and invasion. Integrins are transmembrane receptors that bind extracellular matrix proteins and enable cell adhesion and cytoskeletal organization. They also mediate signal transduction to regulate cell proliferation and survival. The type 1 insulin-like growth factor receptor (IGF-IR) mediates tumor cell growth, adhesion and inhibition of apoptosis in several types of cancer. We have previously demonstrated that β1 integrins regulate anchorage-independent growth of prostate cancer (PrCa) cells by regulating IGF-IR expression and androgen receptor-mediated transcriptional functions. Furthermore, we have recently reported that IGF-IR regulates the expression of β1 integrins in PrCa cells. We have dissected the mechanism through which IGF-IR regulates β1 integrin expression in PrCa. Here we report that IGF-IR is crucial for PrCa cell growth and that β1 integrins contribute to the regulation of proliferation by IGF-IR. We demonstrate that β1 integrin regulation by IGF-IR does not occur at the mRNA level. Exogenous expression of a CD4 - β1 integrin cytoplasmic domain chimera does not interfere with such regulation and fails to stabilize β1 integrin expression in the absence of IGF-IR. This appears to be due to the lack of interaction between the β1 cytoplasmic domain and IGF-IR. We demonstrate that IGF-IR stabilizes the β1 subunit by protecting it from proteasomal degradation. The α5 subunit, one of the binding partners of β1, is also downregulated along with β1 upon IGF-IR knockdown while no change is observed in the expression of the α2, α3, α4, α6 and α7 subunits. Our results reveal a crucial mechanistic role for the α5β1 integrin, downstream of IGF-IR, in regulating cancer growth.

Antisense now makes sense: dual modulation of androgen-dependent transcription by CTBP1-AS

EMBO J 32 12: 1665–1680 doi:10.1038/emboj.2013.99; published online May032013 Recent advances in genomic approaches for exploring the transcriptome have led to the identification of thousands of novel non-coding RNA (ncRNA) transcripts (Djebali et al, 2012). While some of these ncRNAs have been implicated in the development and progression of cancer and other human diseases, their underlying mechanisms of action remain largely unknown (Esteller, 2011). Takayama et al (2013) now report the identification of CTBP1-AS, a novel androgen-regulated long ncRNA that promotes prostate cancer growth through sense–antisense repression of the transcriptional co-regulator CTBP1, as well as through the global epigenetic regulation of tumour suppressor genes.

Abstract 3698: Inhibition of cholesterol uptake with ezetimibe reduces intra-tumoral testosterone levels and slows tumor growth in transgenic mouse models of prostate cancer.

Abstract Introduction: Western countries have 6 fold higher incidence of prostate cancer (PC). Dietary composition, in addition to other factors, may contribute to this geographic disparity. Cholesterol, the precursor for de novo steroid hormone synthesis, has been hypothesized to promote PC growth via multiple pathways including intratumoral steroidogenesis. While lowering serum cholesterol slows xenograft PC growth, this has not been tested in transgenic models, which allow testing of PC prevention as well as modulating PC growth in an orthotopic position. We determined the effect of cholesterol uptake inhibition, using ezetimibe, on PC growth in Hi-Myc and PTEN transgenic mice. Methods: We randomized 50 c-myc/wt (Hi-Myc) mice and 30 PTENloxP/loxP-Cre+ (PTEN) to one of two diets: high fat high cholesterol (HFHC; 40% fat, 17% protein, 43% carbohydrate, 1.25% cholesterol), or HFHC with ezetimibe (30 mg/kg food) (HFHC+Z). Mice were fed ad libitum starting right after weaning and body weights measured twice weekly. Prostate, liver, spleen, adipose tissue and serum were harvested from Hi-Myc and PTEN mice at age 6 and 4 months, respectively. Serum cholesterol and testosterone (T) and tumor T were measured in Hi-Myc mice and are ongoing in PTEN mice. Results: In Hi-Myc mice, body weights were significantly greater in the HFHC+Z group at sacrifice, relative to the HFHC group (p=0.04), but there were no differences in prostate weight between groups with adjustment for body weight (p=0.84). In PTEN mice, there was no difference in body weights between groups, but prostate weight was significantly lower in the HFHC+Z group, relative to the HFHC group (p=0.046). In Hi-Myc mice, while serum T levels did not vary between groups (p=0.93), tumor T, dihydrotestosterone (DHT) and androstenedione concentrations were significantly reduced in the HFHC+Z group, relative to the HFHC group (p=0.01, p=0.01, p=0.006, respectively). Discussion: Reduction of serum cholesterol levels using cholesterol uptake inhibitor, ezetimibe, significantly reduced prostate weight in PTEN mice consuming a HFHC diet. Furthermore, supplementation of the HFHC diet with ezetimibe significantly reduced tumor concentrations of T, DHT and androstenedione in Hi-Myc mice, although there was no effect on prostate weight. These findings highlight a potentially critical role for cholesterol in PC progression by providing evidence that reduction of hypercholesterolemia may reduce intratumoral de novo steroidogenesis and slow PC growth. This study may provide new insight into the use of cholesterol-lowering drugs for PC prevention and treatment. Citation Format: Emma H. Allott, Elizabeth M. Masko, Alexa Choy, Alexis R. Gaines, Keith R. Solomon, Salvatore V. Pizzo, Stephen J. Freedland. Inhibition of cholesterol uptake with ezetimibe reduces intra-tumoral testosterone levels and slows tumor growth in transgenic mouse models of prostate cancer. [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 3698. doi:10.1158/1538-7445.AM2013-3698

Roles of microRNAs during prostatic tumorigenesis and tumor progression

Prostate cancer (PCa) is considered to be a frequently diagnosed cancer in males with high mortality worldwide, but the molecular mechanism responsible for prostate tumorigenesis and progression remains unclear. Increasing evidence has shown that microRNAs (miRNAs) play an important role in PCa. In this review, we focus on the current advances about the role of miRNAs in regulating tumorigenesis and progression of PCa, mainly in suppressing or promoting PCa growth and metastasis, and maintaining the pluripotency of PCa stem cells (PCSC). More studies on miRNAs will provide a better understanding of their regulatory mechanisms in PCa.

Increased PrLZ-mediated androgen receptor transactivation promotes prostate cancer growth at castration-resistant stage.

Most advanced prostate cancers (PCa) will develop into the castration-resistant stage following androgen deprivation therapy, yet the molecular mechanisms remain unclear. In this study, we found PrLZ, a newly identified Prostate Leucine Zipper gene that is highly expressed in PCa could interact with the androgen receptor (AR) directly leading to enhance AR transactivation in the castration-resistant condition. PrLZ might enhance AR transactivation via a change of AR conformation that leads to promotion of AR nuclear translocation and suppression of AR degradation via modulating the proteasome pathway, which resulted in increased prostate-specific antigen expression and promoted PCa growth at the castration-resistant stage. Clinical PCa sample survey from same-patient paired specimens found increased PrLZ expression in castration-resistant PCa following the classical androgen deprivation therapy. Targeting the AR-PrLZ complex via ASC-J9® or PrLZ-siRNA resulted in suppression of PCa growth in various human PCa cells and in vivo mouse PCa models. Together, these data not only strengthen PrLZ roles in the transition from androgen dependence to androgen independence during the castration-resistant stage, but they may also provide a new potential therapy to battle PCa at the castration-resistant stage.

Nuclear Stat5a/b predicts early recurrence and prostate cancer–specific death in patients treated by radical prostatectomy

There is an urgent need for reliable markers to identify patients whose prostate cancer (PCa) will recur after initial therapy and progress to lethal disease. Gleason score (GS) is considered the most accurate predictive marker for disease-specific mortality after primary treatment of localized PCa. Most PCas cluster into groups of GS 6 and 7 with considerable variation in the disease recurrence and disease-specific death. In preclinical PCa models, Stat5a/b promotes PCa growth and progression. Stat5a/b is critical for PCa cell viability in vitro and for tumor growth in vivo and promotes metastatic dissemination of cancer in nude mice. Here, we analyzed the predictive value of high nuclear Stat5a/b protein levels in 2 cohorts of PCas: Material I (n = 562) PCas treated by radical prostatectomy (RP), and Material II (n = 106) PCas treated by deferred palliative therapy. In intermediate GS PCas treated by radical prostatectomy, high levels of nuclear Stat5a/b predicted both early recurrence (univariable analysis; P < .0001, multivariable analysis; HR = 1.82, P = .017) and early PCa-specific death (univariable analysis; P = .028). In addition, high nuclear Stat5a/b predicted early disease recurrence in both univariable (P < .0001) and multivariable (HR = 1.61; P = .012) analysis in the entire cohort of patients treated by RP regardless of the GS. Patients treated by deferred palliative therapy, elevated nuclear Stat5a/b expression was associated with early PCa-specific death by univariable Cox regression analysis (HR = 1.59; 95% CI = [1.04, 2.44]; P = .034). If confirmed in future prospective studies, nuclear Stat5a/b may become a useful independent predictive marker of recurrence of lethal PCa after RP for intermediate GS PCas.

Abstract 1940: Anti-tumor action of adenosine A3 receptor in prostate cancer involves suppression of micro RNA-21

Abstract Introduction: Adenosine-based drugs have shown promising results against various solid tumors including prostate cancer. These drugs exert their effects via adenosine receptors (ARs), and in prostate cancer, the A3AR subtype appears to play a major role in its anti-tumor action. MicroRNAs (miRs) are small non-coding RNAs that regulate the coding RNAs at post-transcriptional level. One of these, miR-21, has been shown to promote prostate cancer growth and invasiveness. In the present study, we hypothesized that A3AR agonists suppressed prostate tumor growth by reducing miR-21 expression. Methods: In vivo studies were performed in xenograft model of prostate cancer in severe combined immunodeficient (SCID) mice. Mice were injected subcutaneously with PC3-MM human prostate cancer cells, treated with vehicle, the agonist N’-(3-iodobenzyl) adenosine-5′-N-methyluronamide (IB-MECA), the antagonist 9-Chloro-2-(2-furanyl)-5-((phenylacetyl)amino)-[1,2,4]triazolo[1,5-c]quinazoline (MRS1220) or MRS1220 + IB-MECA, on alternate day for 2-3 weeks, and tumor growth was measured. Primary tumors were then excised and used to determine miR-21 (by TaqMan assay) and maspin, PDCD4 and TPM1 (by Western blotting and RT-PCR). In vitro studies were done in PC3-MM cells grown in cell culture plates. The levels of miR-21 were also detected in RNA samples obtained from human prostate cancer tissues (OriGene Technologies), while the levels of maspin and PDCD4 were determined in the formalin-fixed paraffin-embedded (FFPE) prostate tumor sections by immunohistochemistry. Results: IB-MECA suppressed tumor growth in SCID mice. This effect was blunted in presence of the antagonist, MRS1220. Reductions in tumor volume were associated with reduced expression of miR-21 in the primary tumors. Furthermore, we observed up-regulation of the tumor suppressor proteins maspin, PDCD4 and TPM1, which are known to be negatively regulated by miR-21. Similar findings were obtained in in vitro cell culture studies. In human prostate cancer RNA samples from patients with different stages of prostate cancer there was high expression of miR-21. This finding co-related with down-regulation of its target proteins, maspin and PDCD4, in human prostate tumor sections. Conclusions: Based on the data, we conclude that activation of the A3AR reduces the prostate cancer growth by suppressing miR-21 expression and up-regulating tumor suppressor proteins such as maspin, PDCD4 and TPM1. These data support the use of A3AR agonists as a viable therapeutic option against the advanced forms of 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 1940. doi:1538-7445.AM2012-1940

Abstract 1499: Periprostatic fat from obese patients promotes prostate cancer growth

Abstract Background: Obesity, particularly visceral obesity, increases the risk of prostate cancer (PCa) progression. However, the mechanism of this remains unclear. Here, we hypothesized that the visceral periprostatic fat (PPF) from obese PCa patients stimulates progression as compared to subcutaneous fat (SQF) or PPF from lean patients. Methods: PPF and SQF were collected from PCa patients. MTT assays were performed on endothelial and PC-3 PCa cells to test proliferative activity in explant culture tissue conditioned media (CM). Angiogenesis was evaluated ex vivo using MR to generate T2 relaxation times from a series of T2-weighted spin-echo images (Bruker 14.1 T imager). Results: PPF CM from an obese patient increased endothelial cell proliferation ∼5 times that of PPF CM from a lean patient (P&amp;lt;0.01). In contrast, SQF CM, from lean or obese patients, showed only minimal activity. When tested on PC-3 cells, SQF CM from obese or lean patients only weakly promoted proliferation. In contrast, PPF CM significantly induced proliferation; however, obese PPF CM was significantly higher than that of lean PPF CM (P&amp;lt;0.05). Interestingly, the MR data revealed key differences between PPF and SQF and between lean and obese fat (Table 1). In all patients, the PPF T2 was lower than that of the SQF (P&amp;lt;0.01). And, the PPF T2 from obese patients was lower than that of lean patients. A lower T2 reflects elevated hemoglobin levels and thus could serve as an indirect measure of vascularity. And, our data suggest that vascularity is increased in obese PPF as compared to SQF and lean PPF tissues. Conclusions: Our data reveal that PPF has increased angiogenic and tumorigenic capacity as compared to SQF and this potential is markedly increased by the presence of obesity. Adipokines and/or other molecules expressed by the PPF may modify the PCa microenvironment to potentiate tumor growth. Our results also support the potential use of MR clinically as a non-invasive test of the angiogenic potential of PPF in PCa patients. 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 1499. doi:1538-7445.AM2012-1499

Chemokine and Chemokine Receptor Expression in Kidney Tumors: Molecular Profiling of Histological Subtypes and Association With Metastasis

Molecular characterization of renal cell carcinoma may help differentiate benign oncocytoma from malignant renal cell carcinoma subtypes and predict metastasis. Chemokines, eg IL-8 and chemokine receptors such as CXCR4 and 7, promote inflammation and metastasis. SDF-1 is a CXCR4 and 7 ligand with 6 known isoforms. We evaluated the expression of these chemokines and chemokine receptors in kidney specimens. Using quantitative polymerase chain reaction we measured mRNA levels of IL-8, CXCR4 and 7, and SDF1 isoforms α, β and γ in a total of 166 specimens from 86 patients, including 86 tumor samples and 80 matched normal kidney samples. Mean ± SD followup was 18.9 ± 12 months (median 19.5). Renal cell carcinoma specimens included the clear cell, papillary and chromophobe subtype in 65, 10 and 5 cases, respectively, and oncocytoma in 6. A total of 17 cases were positive for metastasis. Median CXCR4 and 7, and SFD1-γ levels were increased twofold to tenfold. SDF1-α and β were unchanged or lower in clear cell renal cell carcinoma and papillary tumors than in normal tissue. Median SDF1-γ, IL-8, and CXCR4 and 7 were increased threefold to fortyfold in chromophobe tumors compared to oncocytoma. CXCR4 and 7 were increased in tumors less than 4 cm (mean 3,057 ± 2,230 and 806 ± 691) compared to oncocytoma (336 ± 325 and 201 ± 281, respectively, p ≤0.016). On multivariate analysis CXCR4 (p = 0.01), CXCR7 (p = 0.02) and SDF1-β (p = 0.005) were independently associated with metastasis. Combined CXCR7 plus SDF1-α and CXCR7 plus IL-8 markers showed the highest sensitivity (71% to 81%) and specificity (75% to 80%) of all individual or combined markers. Chemokines and chemokine receptors differentiate renal cell carcinoma and oncocytoma. Combined SDF1-α plus CXCR7 and IL-8 plus CXCR7 markers have about 80% accuracy for predicting renal cell carcinoma metastasis.