Promotes Prostate Tumor Growth Research Articles

The Therapeutic Efficacy and Mechanism of Action of Gnetin C, a Natural Compound from the Melinjo Plant, in a Preclinical Mouse Model of Advanced Prostate Cancer.

The metastasis-associated protein 1/protein kinase B (MTA1/AKT) signaling pathway has been shown to cooperate in promoting prostate tumor growth. Targeted interception strategies by plant-based polyphenols, specifically stilbenes, have shown great promise against MTA1-mediated prostate cancer progression. In this study, we employed a prostate-specific transgenic mouse model with MTA1 overexpression on the background of phosphatase and tensin homolog (Pten) null (R26MTA1; Ptenf/f) and PC3M prostate cancer cells which recapitulate altered molecular pathways in advanced prostate cancer. Mechanistically, the MTA1 knockdown or pharmacological inhibition of MTA1 by gnetin C (dimer resveratrol) in cultured PC3M cells resulted in the marked inactivation of mammalian target of rapamycin (mTOR) signaling. In vivo, mice tolerated a daily intraperitoneal treatment of gnetin C (7 mg/kg bw) for 12 weeks without any sign of toxicity. Treatment with gnetin C markedly reduced cell proliferation and angiogenesis and promoted apoptosis in mice with advanced prostate cancer. Further, in addition to decreasing MTA1 levels in prostate epithelial cells, gnetin C significantly reduced mTOR signaling activity in prostate tissues, including the activity of mTOR-target proteins: p70 ribosomal protein S6 kinase (S6K) and eukaryotic translational initiation factor 4E (elF4E)-binding protein 1 (4EBP1). Collectively, these findings established gnetin C as a new natural compound with anticancer properties against MTA1/AKT/mTOR-activated prostate cancer, with potential as monotherapy and as a possible adjunct to clinically approved mTOR pathway inhibitors in the future.

Uridine Diphosphate Glucuronosyl Transferase 2B28 (UGT2B28) Promotes Tumor Progression and Is Elevated in African American Prostate Cancer Patients.

Prostate cancer (PCa) is the second most diagnosed cancer in the United States and is associated with metabolic reprogramming and significant disparities in clinical outcomes among African American (AA) men. While the cause is likely multi-factorial, the precise reasons for this are unknown. Here, we identified a higher expression of the metabolic enzyme UGT2B28 in localized PCa and metastatic disease compared to benign adjacent tissue, in AA PCa compared to benign adjacent tissue, and in AA PCa compared to European American (EA) PCa. UGT2B28 was found to be regulated by both full-length androgen receptor (AR) and its splice variant, AR-v7. Genetic knockdown of UGT2B28 across multiple PCa cell lines (LNCaP, LAPC-4, and VCaP), both in androgen-replete and androgen-depleted states resulted in impaired 3D organoid formation and a significant delay in tumor take and growth rate of xenograft tumors, all of which were rescued by re-expression of UGT2B28. Taken together, our findings demonstrate a key role for the UGT2B28 gene in promoting prostate tumor growth.

Non-enzymatic glycoxidation linked with nutrition enhances the tumorigenic capacity of prostate cancer epithelia through AGE mediated activation of RAGE in cancer associated fibroblasts

The molecular implications of food consumption on cancer etiology are poorly defined. The rate of nutrition associated non-enzymatic glycoxidation, a reaction that occurs between reactive carbonyl groups on linear sugars and nucleophilic amino, lysyl and arginyl groups on fats and proteins, is rapidly increased by food cooking and manufacturing processes. In this study, we assign nutrition-associated glycoxidation with significant oncogenic potential, promoting prostate tumor growth, progression, and metastasis in vivo. Advanced glycation end products (AGEs) are the final irreversible product of non-enzymatic glycoxidation. Exogenous treatment of prostate tumor cells with a single AGE peptide replicated glycoxidation induced tumor growth in vivo. Mechanistically, receptor for AGE (RAGE) deficiency in the stroma inhibited AGE mediated tumor growth. Functionally, AGE treatment induced RAGE dimerization in activated fibroblasts which sustained and increased the migratory potential of tumor epithelial cells. These data identify a novel nutrition associated pathway that can promote a tissue microenvironment conducive for aggressive tumor growth. Targeted and/or interventional strategies aimed at reducing AGE bioavailability as a consequence of nutrition may be viewed as novel chemoprevention initiatives.

Nutritional AGEing and RAGEing as a Regulator of the Tumor Microenvironment

Abstract Objectives Advanced glycation end products (AGEs) are reactive metabolites formed endogenously by glyoxidative, oxidative and lipoxidative stresses. Foods associated with modern dietary habits are particularly AGE laden but despite increasing epidemiological evidence for oncogenic potential, cause and effect relationships are lacking. The objective was to provide detailed mechanistic insight and in vivo confirmation that AGEs found in the diet are oncogenic drivers of tumorigenesis. Methods We used the heat driven formation of glyoxidative, oxidative and lipoxidative stresses in experimental mouse chow to reproduce the wide spectrum of the AGEs found in vivo. Syngeneic xenograft and spontaneous prostate and breast cancer mouse models were then fed the AGE specific diets and the effects of chronic AGE consumption on tumor growth assessed. To gain mechanistic insight, human and mouse two compartment co-culture models using primary fibroblasts and matched tumor epithelial cells were then used to assess the effects of AGEs on extracellular crosstalk in the TME. Results A high impact finding from our research is that consumption of AGEs found in our diet promotes prostate tumor growth, aggression and metastasis by functioning as ligand to the transmembrane receptor for AGE (RAGE). Dietary-AGEs promoted neoplastic growth by functioning as ligand to RAGE expressed in the prostate tumor stroma not tumor epithelium. Dietary-AGE activation of stromal RAGE caused a regulatory program of ‘activated fibroblasts’ defined by the increased expression of cancer associated fibroblast markers, NFkB, MYC and pro-tumorigenic paracrine secretion. Fibroblast activation was accompanied by decreased expression of androgen receptor (AR) and the increased expression of neuroendocrine differentiation markers in tumor epithelial cells. AGE exposed primary fibroblasts isolated from patient tissue conferred tumor promoting abilities when cultured with patient matched tumor epithelial cells. Conclusions For the first time these data demonstrate a direct cause and effect relationship between dietary-AGEs and neoplastic growth. This may lay the foundation for strategic self-management strategies aimed at reducing AGE exposure in the diet to reduce cancer incidence and mortality. Funding Sources NIH/NCI; ACS.

Neuronal calcitonin gene-related peptide promotes prostate tumor growth in the bone microenvironment

Advanced stage of prostate cancer cells preferentially metastasizes to varying bones of prostate cancer patients, resulting in incurable disease with poor prognosis and limited therapeutical treatment options. Calcitonin gene-related peptide (CGRP), a neuropeptide produced by prostate gland, is known to play a pivotal role in facilitating tumor growth and metastasis of numerous human cancers. In this study, we aim to investigate the clinical relevance of CGRP in prostate cancer patients and the effects of CGRP and CGRP antagonists on prostate tumor growth in the mouse model. The prostate tumor-bearing mice were received either CGRP or CGRP antagonist treatment, and the tumor growth was monitored by quantification of luminescence intensities. We found that the CGRP+ nerve fiber density and serum CGRP levels were substantially upregulated in the bone or serum specimens from advanced prostate cancer patients as well as in prostate tumor-bearing mice. Administration of CGRP promoted, whereas treatment of CGRP antagonists inhibited prostate tumor growth in the femurs of mice. In addition, CGRP treatment activated extracellular signal-regulated kinases (ERKs)/ Signal transducer and activator of transcription 3 (STAT3) signaling in prostate cancer cells. Targeting CGRP may serve as a potential therapeutic strategy for advanced prostate cancer patients.

Abstract B047: Advanced glycation end products promote prostate tumor growth and are a potential biologic consequence of lifestyle factors contributing to cancer disparity

Abstract Advanced glycation end-products (AGEs), are reactive metabolites produced endogenously as a consequence of glucose uptake during glycolysis. AGEs accumulate in tissues and organs as we grow older to promote multiple chronic disease phenotypes. AGE pathogenic effects are mediated through modification of protein function, genetic fidelity, stress responses and cellular signaling pathways. Critically, cancer disparity factors such as a sedentary lifestyle, obesity and an unhealthy diet are external influences that also contribute to the AGE accumulation pool in the body. This research group examined circulating and tumor AGE levels in clinical specimens of prostate cancer and identified a race specific, tumor-dependent pattern of accumulation. AGE levels were highest in aggressive tumors, especially those from men with African ancestry. Increased AGE levels correlated with an upregulation in the receptor for advanced glycation end products (RAGE) and activated NFkB. In a syngeneic subcutaneous prostate cancer mouse model, chronic consumption of AGE resulted in a 3-fold increase in tumor growth. Strikingly, dietary-AGE mediated increases in tumor growth were accompanied by a cytoplasmic accumulation of AR, elevation in MYC, RAGE, and AGE as well as increased cell proliferation. Cytoplasmic accumulation of AR is elevated in CRPC tissue and is an independent predictor of biochemical recurrence. Our data show that treatment of prostate cancer cells with AGEs induces a neuroendocrine differentiated phenotype by promoting a more mesenchymal morphology and increasing the expression of associated marker genes including ENO2, MYC and SYP and the prostate cancer stem cell marker CD44 as well as the downregulation of AR. The aberrant activation and recruitment of immune cells is a major pathogenic consequence of AGE accumulation, and a series of studies have highlighted the tumor-associated immune response as a critical pathway contributing to cancer disparity. Using patient-derived primary tumor cells, the investigators found that AGEs released into the extracellular matrix may recapitulate the tumor-associated immune response observed in ancestry-specific prostate tumor tissues. Further preliminary studies indicate that AGE treatment of prostate cancer cells can alter how cancer cells metabolize glucose to promote an aggressive phenotype. The investigators' studies support the concept that AGE metabolites represent a biologic consequence of the socioeconomic and environmental factors that promote cancer health disparity. As our understanding of tumor biology advances, it is becoming increasingly clear that these inter-related lifestyle factors have distinct molecular consequences on the biologic makeup of tumors, altering cell signaling events and gene expression profiles to contribute to cancer disparity outcomes such as its earlier development or its progression to more aggressive disease. Citation Format: Pamela Woods, Bradley A. Krisanits, Dion Foster, Lourdes M. Nogueira, Laura Spruill, Marvella E. Ford, Michael B. Lilly, Victoria J. Findlay, David P. Turner. Advanced glycation end products promote prostate tumor growth and are a potential biologic consequence of lifestyle factors contributing to cancer disparity [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B047.

Abstract C083: Consumption of lifestyle-associated advanced glycation end products promotes prostate tumor growth by creating a tumor-enhancing stromal microenvironment

Abstract Our research has demonstrated that advanced glycation end products (AGEs) derived from the diet can directly impact neoplastic growth by creating a tumor-enhancing micro-environment. Most people are unaware of what AGEs are or the damage they can cause, but we are exposed to them every day through the lives we lead and the foods that we eat. The Western diet together with more sedentary habits means that lifestyle-associated AGEs are accumulating in our bodies at a faster rate than ever before. Changes in the AGE equilibrium due to lifestyle cause protein dysfunction, reduced genetic fidelity, and aberrant cell signaling activation which we believe contribute to cancer disparity outcomes. Disparity populations defined by AGE-associated risk factors such as diet, smoking, drinking and physical inactivity often bear a greater cancer burden when compared to the general population (reviewed by the PI, Cancer Research 2015). Lifestyle associated AGEs therefore may represent a unifying biological consequence of the social, demographic and environmental risk factors that contribute to the increased cancer incidence and mortality associated with cancer disparity. An important discovery from our work is that consumption of a diet high in AGEs accelerates prostate tumor growth in syngeneic xenograft prostate cancer (PCa) models as well as disease progression in spontaneous PCa models. Critically, dietary-AGE mediated effects on prostate tumor growth were dependent upon the stromal activation of RAGE. An activated stroma is a critical pathway impacting prostate cancer outcomes in African American men. Our studies show that dietary-AGE alters cytokine profiles, increases the activation of cancer associated fibroblasts (CAFs) and increases immune cell recruitment to the tumor microenvironment. Tumor associated immune cells adopt distinct metabolic patterns which function to maintain the energy requirements needed for cell differentiation and functionality. Pathway analysis of expression data from excised tumors shows that AGE consumption significantly impacts energy metabolism through the aberrant expression of MYC regulated transcriptional targets. Our studies also show that AGEs are highest in African American men with prostate cancer. Dietary-AGE mediated activation of tumor stroma therefore may align with the ancestry specific stromal and immune profiles observed in African American men with prostate cancer. Due to their links with lifestyle, both pharmacological and/or interventional strategies aimed at reducing the AGE accumulation pool may be viewed as universal cancer preventive and/or therapeutic initiatives. This may be an attractive option for populations where lifestyle change is not feasible due to poverty, inability, illness, treatment side effects, time, apathy and depression. Citation Format: Bradley Krisanits, Callen Fry, Lourdes M Nogueira, Reid Schuster, Marvella El Ford, Mark T Hamann, Michaell B Lilly, Mahtabuddin Ahmed, Victoria J Findlay, David P Turner. Consumption of lifestyle-associated advanced glycation end products promotes prostate tumor growth by creating a tumor-enhancing stromal microenvironment [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr C083.

Consumption of Dietary Advanced Glycation End Products Promotes Prostate Tumor Growth by Creating a Tumor Enhancing Stromal Microenvironment

Abstract Objectives The literature regarding the role of advanced glycation end products (AGEs) on tumor biology has shown only moderate promise reflected by increases in cell growth, migration and invasion in vitro which is not supported by increased tumor growth in vivo14-16– A caveat to these studies is that they are centered upon a single AGE peptide and a subsequent assessment of their molecular effects on tumor epithelial cells. The objective is to show that by feeding mice a high AGE diet we can recapitulate a microenvironment comprising of a wide spectrum of AGEs which can influence neoplastic growth. Methods We recapitulated a dietary-AGE induced microenvironment in syngeneic xenograft and spontaneous breast and prostate mouse cancer models and the effects on tumor growth assessed. The mechanistic consequences of dietary-AGEs on the tumor microenvironment were further defined using mouse and human primary and immortalized two-compartment co-culture ex vivo culture models. Results Dietary-AGE consumption in breast and prostate tumor models significantly accelerated tumor growth by functioning as ligand to the transmembrane receptor for AGE (RAGE). Our studies demonstrate that AGEs promote neoplastic growth by functioning as ligand to RAGE expressed in the tumor stroma not the tumor epithelial cells. Dietary-AGE activation of RAGE in both breast and prostate tumors caused a regulatory program of ‘activated fibroblasts’ defined by increased expression of cancer associated fibroblast markers, NFkB and MYC upregulation, and pro-tumorigenic paracrine secretion. Complementary to this, our published studies show that high intake of dietary AGE after BCa diagnosis increases risk of mortality in postmenopausal women. Conclusions These data demonstrate, for the first time, the oncogenic potential of dietary-AGEs in promoting neoplastic growth. This lays the foundation for strategic changes aimed at reducing cancer incidence and mortality as pharmacological, educational and/or interventional strategies aimed at reducing the dietary-AGE accumulation pool may one day be viewed as universal cancer preventative and/or therapeutic initiatives especially when combined with existing therapies. Funding Sources David P. Turner was supported by grants from the NIH/NCI, R21 CA194469 and U54 CA21096..

Cancer-associated fibroblasts promote prostate tumor growth and progression through upregulation of cholesterol and steroid biosynthesis

BackgroundAndrogen receptor targeted therapies have emerged as an effective tool to manage advanced prostate cancer (PCa). Nevertheless, frequent occurrence of therapy resistance represents a major challenge in the clinical management of patients, also because the molecular mechanisms behind therapy resistance are not yet fully understood. In the present study, we therefore aimed to identify novel targets to intervene with therapy resistance using gene expression analysis of PCa co-culture spheroids where PCa cells are grown in the presence of cancer-associated fibroblasts (CAFs) and which have been previously shown to be a reliable model for antiandrogen resistance.MethodsGene expression changes of co-culture spheroids (LNCaP and DuCaP seeded together with CAFs) were identified by Illumina microarray profiling. Real-time PCR, Western blotting, immunohistochemistry and cell viability assays in 2D and 3D culture were performed to validate the expression of selected targets in vitro and in vivo. Cytokine profiling was conducted to analyze CAF-conditioned medium.ResultsGene expression analysis of co-culture spheroids revealed that CAFs induced a significant upregulation of cholesterol and steroid biosynthesis pathways in PCa cells. Cytokine profiling revealed high amounts of pro-inflammatory, pro-migratory and pro-angiogenic factors in the CAF supernatant. In particular, two genes, 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2 (HMGCS2) and aldo-keto reductase family 1 member C3 (AKR1C3), were significantly upregulated in PCa cells upon co-culture with CAFs. Both enzymes were also significantly increased in human PCa compared to benign tissue with AKR1C3 expression even being associated with Gleason score and metastatic status. Inhibiting HMGCS2 and AKR1C3 resulted in significant growth retardation of co-culture spheroids as well as of various castration and enzalutamide resistant cell lines in 2D and 3D culture, underscoring their putative role in PCa. Importantly, dual targeting of cholesterol and steroid biosynthesis with simvastatin, a commonly prescribed cholesterol synthesis inhibitor, and an inhibitor against AKR1C3 had the strongest growth inhibitory effect.ConclusionsFrom our results we conclude that CAFs induce an upregulation of cholesterol and steroid biosynthesis in PCa cells, driving them into AR targeted therapy resistance. Blocking both pathways with simvastatin and an AKR1C3 inhibitor may therefore be a promising approach to overcome resistances to AR targeted therapies in PCa.3iMBuBncLfXKMeSSDowVrVVideo abstract

Abstract 5745: Effect of activator of G-protein signaling 3 (AGS3) on prostate tumorigenesis

Abstract Prostate cancer (PCa) is a leading cause of cancer death among men, with greater prevalence of the disease among the African American population in the US. Activator of G-protein Signaling 3 (AGS3/GPSM1), a receptor-independent activator of G-protein signaling has been shown to affect different cellular processes and cell cycle activity as well as tumor growth and development. AGS3 contains seven tetratricopeptide (TPR) repeats in its N-terminal half and four G-protein regulatory (GPR) motifs in its C-terminal half. The aim of this study is to assess the role of AGS3 in prostate cancer development and metastasis as well as to understand the molecular dynamics involved. To that end, the metastatic PCa cell lines LNCaP, PC3, MDA PCa 2b and DU-145 were analysed for AGS3 expression relative to RWPE-1, a non-metastatic Pca cell line. Quantitative RT-PCR and western blot analysis have shown that AGS3 expression varies in the PCa cell lines relative to control RWPE-1. Overexpression of AGS3 in PC3 and LNCaP cells significantly enhanced tumor progression in nude mice xenografts. Interestingly, expression of the TPR, not the GPR, repeats in LNCaP promoted tumor growth as well as the full length AGS3. Preliminary studies with a Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice deficient in AGS3 expression (TRAMP+/AGS3-/-) displayed decrease prostate tumorigenesis. Taken together, these results indicate that AGS3 expression promotes prostate tumor growth. The data also suggest that the effect of AGS3 in prostate tumorigenesis is mediated via its TPR, not GPR motif. Citation Format: Timothy O. Adekoya, Nikia Smith, Tonelia Mowatt, Temilade Aladeniyi, Ricardo M. Richardson. Effect of activator of G-protein signaling 3 (AGS3) on prostate tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5745.

Bone marrow-derived mesenchymal stem cells induced by inflammatory cytokines produce angiogenetic factors and promote prostate cancer growth

BackgroundProstate is susceptible to infection and pro-inflammatory agents in a man’s whole life. Chronic inflammation might play important roles in the development and progression of prostate cancer. Mesenchymal stem cells (MSCs) are often recruited to the tumor microenvironment due to local inflammation. We have asked whether stimulation of MSCs by pro-inflammatory cytokines could promote prostate tumor growth. The current study investigated the possible involvement of MSCs stimulated by pro-inflammatory cytokines in promotion and angiogenesis of prostate cancer through relative pathway in vitro and in vivo.MethodsA syngeneic mouse model of C57 was established. The murine prostate cancer cells (RM-1) mixing with MSCs treated with tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) or vehicle were subcutaneously injected into C57 mice. Tumor volume of C57 mouse model was estimated and serum level of platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) was test by Enzyme-linked Immunosorbent Assay (ELISA). A hen egg test-chorioallantoic membrane (HET-CAM) assay was applied to test the effect of conditioned media of stimulated MSCs in chorioallantoic membrane angiogenesis. Short interfering RNA (siRNA) knocked down either hypoxia-inducible factor-1alpha (HIF-1α) or nuclear factor-erythroid-2-related factor 2 (NRF2) were employed. mRNA of PDGF and VEGF in MSCs, as well as NRF2 and HIF-1α was test by Real time polymerase chain reaction (PCR) analyses. Protein expression levels of PDGF and VEGF from conditioned medium, NRF2, HIF-1α, as well as PDGF and VEGF in MSCs were detected by Western blot analysis.ResultsMSCs treated with TNF-α and IFN-γ promote tumor growth in C57 syngeneic mouse model, correlating with increased serum level of PDGF, VEGF. HET-CAM assay shows the angiogenic effect of conditioned medium of MSCs pre-treated with the pro-inflammatory cytokines. mRNA and protein levels of two pro-angiogenic factors (PDGF and VEGF) and key hypoxia regulators (HIF-1α and NRF2) in MSCs were induced after MSCs’ pretreatment. siRNA knockdown either HIF-1α or NRF2 results reduction of PDGF and VEGF expression.ConclusionsMSCs stimulated by pro-inflammatory cytokines increase the expression of PDGF and VEGF via the NRF2-HIF-1α pathway and accelerate prostate cancer growth in mice.

A Unique Cellular and Molecular Microenvironment Is Present in Tertiary Lymphoid Organs of Patients with Spontaneous Prostate Cancer Regression.

Multiple solid cancers contain tertiary lymphoid organs (TLO). However, it is unclear whether they promote tumor rejection, facilitate tumor evasion, or simply whether they are a byproduct of chronic inflammation. We hypothesize that although chronic inflammation induces TLO formation, the tumor milieu can modulate TLO organization and functions in prostate cancer. Therefore, our study seeks to elucidate the cellular and molecular signatures in unique prostatectomy specimens from evanescent carcinoma patients to identify markers of cancer regression, which could be harnessed to modulate local immunosuppression or potentially enhance TLO function. We used multicolor immunofluorescence to stain prostate tissues, collected at different stages of cancer progression (prostatic intraepithelial neoplasia, intermediate and advanced cancer) or from patients with evanescent prostate carcinoma. Tissues were stained with antibodies specific for pro-inflammatory molecules (cyclooxygenase 2, CXCL10, IL17), tumor-infiltrating immune cells (mature DC-LAMP+ dendritic cells, CD3+ T cells, CD3+Foxp3+ regulatory T cells (Treg), T bet+ Th1 cells, granzyme B+ cytotoxic cells), and stromal cell populations (lymphatic vessels, tumor neovessels, high endothelial venules (HEV), stromal cells), which promote prostate tumor growth or are critical components of tumor-associated TLO. Generally, inflammatory cells are located at the margins of tumors. Unexpectedly, we found TLO within prostate tumors from patients at different stages of cancer and in unique samples from patients with spontaneous cancer remission. In evanescent prostate carcinomas, accumulation of Treg was compromised, while Tbet+ T cells and CD8 T cells were abundant in tumor-associated TLO. In addition, we found a global decrease in tumor neovascularization and the coverage by cells positive for cyclooxygenase 2 (COX2). Finally, consistent with tumor regression, prostate stem cell antigen was considerably reduced in TLO and tumor areas from evanescent carcinoma patients. Collectively, our results suggest that COX2 and Treg are attractive therapeutic targets that can be harnessed to enhance TLO-driven tumor immunity against prostate cancer. Specially, the presence of HEV and lymphatics indicate that TLO can be used as a platform for delivery of cell-based and/or COX2 blocking therapies to improve control of tumor growth in prostate cancer.

Structurally related odorant ligands of the olfactory receptor OR51E2 differentially promote metastasis emergence and tumor growth.

Olfactory receptors are G protein-coupled receptors. Some of them are expressed in tumor cells, such as the OR51E2 receptor overexpressed in LNCaP prostate cancer cells. It is considered a prostate tumor marker. We previously demonstrated that this receptor is able to promote LNCaP cell invasiveness in vitro upon stimulation with its odorant agonist β-ionone, leading to increased generation of metastases in vivo. In the present study, we show that even a relatively short exposure to β-ionone is sufficient to promote metastasis emergence. Moreover, α-ionone, considered an OR51E2 antagonist, in fact promotes prostate tumor growth in vivo. The combination of α-ionone with β-ionone triggers a higher increase in the total tumor burden than each molecule alone. To support the in vivo results, we demonstrate in vitro that α-ionone is a real agonist of OR51E2, mainly sustaining LNCaP cell growth, while β-ionone mainly promotes cell invasiveness. So, while structurally close, α-ionone and β-ionone appear to induce different cellular effects, both leading to increased tumor aggressiveness. This behaviour could be explained by a different coupling to downstream effectors, as it has been reported for the so-called biased ligands of other G protein-coupled receptors.

Prostate Cancer-Associated Kallikrein-Related Peptidase 4 Activates Matrix Metalloproteinase-1 and Thrombospondin-1.

Prostate cancer metastasis to bone is terminal; thus, novel therapies are required to prevent end-stage disease. Kallikrein-related peptidase 4 (KLK4) is a serine protease that is overproduced in localized prostate cancer and is abundant in prostate cancer bone metastases. In vitro, KLK4 induces tumor-promoting phenotypes; however, the underlying proteolytic mechanism is undefined. The protein topography and migration analysis platform (PROTOMAP) was used for high-depth identification of KLK4 substrates secreted by prostate cancer bone metastasis-derived PC-3 cells to delineate the mechanism of KLK4 action in advanced prostate cancer. Thirty-six putative novel substrates were determined from the PROTOMAP analysis. In addition, KLK4 cleaved the established substrate, urokinase-type plasminogen activator, thus validating the approach. KLK4 activated matrix metalloproteinase-1 (MMP1), a protease that promotes prostate tumor growth and metastasis. MMP1 was produced in the tumor compartment of prostate cancer bone metastases, highlighting its accessibility to KLK4 at this site. KLK4 further liberated an N-terminal product, with purported angiogenic activity, from thrombospondin-1 (TSP1) and cleaved TSP1 in an osteoblast-derived matrix. This is the most comprehensive analysis of the proteolytic action of KLK4 in an advanced prostate cancer model to date, highlighting KLK4 as a potential multifunctional regulator of prostate cancer progression.

Thrombin and Factor XII Drive Prostate Tumor Growth In Vivo

Several hemostatic factors are known to strongly influence the metastatic process by promoting the survival of early metastases. Despite this consistent phenomenon observed across multiple tumor cell types and multiple procoagulant components of the hemostatic system, the role of hemostasis in the establishment and growth of primary tumors is highly context dependent. One clinically relevant context where coagulation appears to be important for tumor development/outgrowth is prostate cancer. Men on long-term anticoagulation appear to be at significantly lower risk for developing prostate cancer compared to men on short-term anticoagulation or those not on anticoagulation. Here, we report that multiple hemostatic proteases drive prostate tumor growth in vivo. To test the hypothesis that the central hemostatic protease, thrombin, promotes prostate tumor growth, mice with a life-long genetically-imposed diminution in circulating prothrombin levels (fIILox/-), or mice administered a prothrombin-specific antisense oligonucleotide (ASO) gapmer that limits prothrombin expression, were inoculated subcutaneously with the murine-derived prostatic adenocarcinoma cell line TRAMP-C2Re3 in parallel with appropriate controls. Genetically or pharmacologically lowering prothrombin expression significantly impeded prostate tumor growth. In complementary experiments, treatment with the prothrombin-specific ASO gapmer significantly impeded the growth of human-derived prostate cancer cells (PC3) in immunodeficient mice. Detailed histological analysis revealed that tumors harvested from mice with diminished prothrombin expression had significantly lower mitotic indices, suggesting that thrombin promotes tumor proliferation in vivo. Lowering prothrombin levels did not affect apoptosis, vascular density, or the presence of tumor-associated macrophages. Previous studies have indicated that tumor cell-associated tissue factor expression is one important mechanism by which cancer cells can locally generate thrombin. However, the relative importance of the contact pathway of coagulation system activation in cancer progression is less defined. In order to determine whether fXII plays a role in prostate cancer growth, mice challenged with prostate cancer were treated with a fXII-specific ASO gapmer or a control oligonucleotide with no homology in the murine genome in parallel. Remarkably, tumors from anti-fXII ASO gapmer treated mice were ~3-fold smaller than those harvested from control animals at the end of the ~3 week experiment. To our knowledge, this finding is the first demonstration that the contact system protease fXII plays a significant role in tumor growth. In sum, these data show that thrombin promotes the growth of prostate cancer, and suggest that contact activation of the coagulation cascade may be a critical factor in prostate cancer growth. DisclosuresMonia:Isis Pharmaceuticals: Employment, Other: Shareholder. Revenko:SIS Pharmaceuticals Inc.: Employment.

Abstract 612: Endostatin is a novel inhibitor of androgen receptor function in prostate cancer

Abstract Abnormalities in androgen-androgen receptor (AR) axis have long been recognized for their roles in promoting prostate tumor growth and metastasis. Conventional therapies targeting androgen ablation show transient tumor regression, however, when the tumor recurs during castration-refractory metastatic stage, the current treatment options are very limited. Although the precise mechanism by which prostate cancer (PCa) cells circumvent the androgen deprivation is unclear, recurring tumors have been shown to retain AR activity and upregulation of AR-target gene expression such as prostate specific antigen (PSA), suggesting that targeting AR remains a key component of developing novel therapeutic strategies. Here, we show a profound effect of endostatin, an endogenous angiogenesis inhibitor, on proliferation and invasion of AR-positive PCa cells by targeting AR function. The present study identified that intracellular trafficking of endostatin and direct interaction with AR disrupts AR nuclear translocation and the consequent transcriptional activation of PSA gene. In addition, our structural modeling and site-directed mutagenesis studies suggest that the binding mechanism may include the interaction of bulky side chains (F31 and F34) present in endostatin with the co-activator binding interface (AF-2) in AR ligand-binding domain (LBD). Regarding drug resistance to the second-generation androgen antagonists in patients with metastatic PCa, recent studies revealed that a missense mutation in AR LBD (F876L) and the consequential antagonist-to-agonist switch confers continued AR activity. In this context, our study suggests that endostatin can be recognized as an endogenous AR inhibitor where its molecular interaction with AR may prevent from agonistic switch of AR function by fully masking the AF-2 subdomain. Overall, the current finding provides new insights into endostatin whose anti-cancer activity is not only limited to inhibiting angiogenesis, but can also be expanded to suppressing AR-mediated PCa progression by disrupting AR transactivation. Citation Format: Joo Hyoung Lee, Tatyana Isayeva, Matthew Larson, Diptiman Chanda, Igor Chesnokov, Selvarangan Ponnazhagan. Endostatin is a novel inhibitor of androgen receptor function in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 612. doi:10.1158/1538-7445.AM2014-612

GRK3 is essential for metastatic cells and promotes prostate tumor progression

The biochemical mechanisms that regulate the process of cancer metastasis are still poorly understood. Because kinases, and the signaling pathways they comprise, play key roles in regulation of many cellular processes, we used an unbiased RNAi in vitro screen and a focused cDNA in vivo screen against human kinases to identify those with previously undocumented roles in metastasis. We discovered that G-protein-coupled receptor kinase 3 (GRK3; or β-adrenergic receptor kinase 2) was not only necessary for survival and proliferation of metastatic cells, but also sufficient to promote primary prostate tumor growth and metastasis upon exogenous expression in poorly metastatic cells in mouse xenograft models. Mechanistically, we found that GRK3 stimulated angiogenesis, at least in part through down-regulation of thrombospondin-1 and plasminogen activator inhibitor type 2. Furthermore, GRK3 was found to be overexpressed in human prostate cancers, especially in metastatic tumors. Taken together, these data suggest that GRK3 plays an important role in prostate cancer progression and metastasis.

PTEN loss mediated Akt activation promotes prostate tumor growth and metastasis via CXCL12/CXCR4 signaling

IntroductionThe chemokine CXCL12, also known as SDF-1, and its receptor, CXCR4, are overexpressed in prostate cancers and in animal models of prostate-specific PTEN deletion, but their regulation is poorly understood. Loss of the tumor suppressor PTEN (phosphatase and tensin homolog) is frequently observed in cancer, resulting in the deregulation of cell survival, growth, and proliferation. We hypothesize that loss of PTEN and subsequent activation of Akt, frequent occurrences in prostate cancer, regulate the CXCL12/CXCR4 signaling axis in tumor growth and bone metastasis.MethodsMurine prostate epithelial cells from PTEN+/+, PTEN+/−, and PTEN−/− (prostate specific knockdown) mice as well as human prostate cancer cell lines C4-2B, PC3, and DU145 were used in gene expression and invasion studies with Akt inhibition. Additionally, HA-tagged Akt1 was overexpressed in DU145, and tumor growth in subcutaneous and intra-tibia bone metastasis models were analyzed.ResultsLoss of PTEN resulted in increased expression of CXCR4 and CXCL12 and Akt inhibition reversed expression and cellular invasion. These results suggest that loss of PTEN may play a key role in the regulation of this chemokine activity in prostate cancer. Overexpression of Akt1 in DU145 resulted in 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. Akt1 overexpression reversed the osteosclerotic phenotype associated with DU145 cells to an osteolytic phenotype and enhanced intra-osseous tumor growth.ConclusionsThese 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. Akt1-associated CXCL12/CXCR4 signaling promotes tumor growth, suggesting that Akt inhibitors may potentially be employed as anticancer agents to target expansion of PC bone metastases.

Abstract 308: δ-Catenin/NPRAP/Neurojungin mutation promotes prostate tumorigenesis in mice overexpressing Myc oncogene .

Abstract Prostate cancer is the most common non-cutaneous malignant neoplasm in men in Western countries. In humans, prostate cancer progresses from precursor lesions, termed prostatic intraepithelial neoplasia (PIN), to overt carcinoma confined to the prostate, and finally to metastatic disease that often results in lethality. Previous studies in our laboratory have shown that δ-catenin/NPRAP/Neurojungin (gene designation: CTNND2), a primarily neural-enriched protein in the brain of healthy individuals, is overexpressed corresponding to human prostate cancer progression. This finding has led us to investigate the roles of δ-catenin in prostate cancer progression. We utilized a novel transgenic model of mouse prostate cancer with overexpression of human Myc oncogene driven by rat-probasin promoter as the initiating event, which has been proven to mimic human disease conditions. These Myc overexpressing mice were further crossbred with δ-catenin mutant (δ-cat−/−) mice whose exon 9 of δ-catenin gene was disrupted. In this study, we examined the histological differences of mouse prostate in wild type and Myc overexpression with δ-catenin mutant mice (Myc/δ-cat−/−). We observed that morphological alterations characteristic of high grade PIN developed in luminal epithelial cells of transgenic mice by 6 weeks. These changes include an increase of nuclear size, multi-layering, and cribriform formation. Surprisingly, Myc/δ-cat−/- mice at 6 months of age displayed the dramatically increased tumor size and progression from PIN to adenocarcinomas. We also discovered that Myc/δ-cat−/- mice increased in both number of proliferating cells and cells undergoing apoptosis compared with that of wild type animals. However, in Myc/δ-cat−/- mice, proliferative index was greater than apoptotic index by 3.7 fold. Collectively, our findings suggest that mutation of δ-catenin may contribute to tumorigenesis of mouse prostate by enhancing the effects of Myc oncogene resulting in an increase of cell proliferation and thereby promoting prostate tumor growth. Supported by NIHCA111891 and NIHCA165202. Citation Format: Jongdee Nopparat, Jiao Zhang, Yan-Hua Chen, Qun Lu. δ-Catenin/NPRAP/Neurojungin mutation promotes prostate tumorigenesis in mice overexpressing Myc oncogene . [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 308. doi:10.1158/1538-7445.AM2013-308

Abstract 4088: PTEN loss mediated Akt activation promotes prostate tumor growth via CXCL12/CXCR4 signaling.

Abstract 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