CaP Metastasis Research Articles

Abstract 2418: PTEN regulated PI3K-p110 and AKT isoform plasticity controls metastatic prostate cancer progression

Abstract Background: The loss of PTEN function, one of the most frequent mutations in prostate cancer (CaP), is presumed to drive disease progression through AKT activation. However, two transgenic mouse CaP models, Pten/RbPE:-/- and Akap12-/-;RbPE:-/-, exhibited different CaP and metastasis development even though both shared Rb loss plus Akt activation, based on increased relative AKTpoS473 levels. Experimental Procedures: We analyzed whether PI3K-p110 and/or AKT isoform reliance, using isoform-specific RNAi or small molecule inhibitors, played a role in the different CaP outcomes. Findings: In comparison to Akap12/Rb-null high-grade prostatic intraepithelial neoplasias, Pten/Rb-null adenocarcinomas had increased protein and activation levels of AKT2 and decreased relative levels of Smad4, a known CaP metastasis suppressor. Comparison of isogenic PTEN +/- pairs of human or mouse CaP cell lines indicated that PTEN-deficiency correlated with dependence on both p110β and AKT2 for survival or oncogenic growth/invasiveness, whereas PTEN expression correlated with greater dependence on AKT1 plus p110α. Inhibition of AKT2 in PTEN-null CaP cells induced higher SMAD4 levels and decreased chemotaxis and Matrigel invasiveness. Given that PTEN likely regulates non-redundant roles of the AKT isoforms during CaP progression, we sought to characterize AKT isoform substrates by phosphoproteomics analysis. Specifically, after endogenous kinases were inactivated in mouse Pten/Rb-null adenocarcinoma cell lysates using 5'-fluorosulfonylbenzoyl-5'-adenosine (FSBA), dialyzed samples were subjected to in vitro kinase reactions using enzymatically-active, full-length baculovirus-expressed AKT1, AKT2 or AKT3, or as a negative control, a heat-inactivated mixture of all three isoforms. Tryptic peptides isolated on TiO2 beads were subjected to LC-MS/MS. Data will be shown on the role of AKT isoform-shared vs. -preferred substrates in specific parameters of CaP progression controlled by PTEN. Conclusion: Our data suggest that in the context of RB1 loss, PTEN controls a signaling plasticity and drug sensitivity through specific pairings of PI3K-p110 and AKT isoforms. Citation Format: Karina Miller, Seamus Degan, Yanqing Wang, Sheng-Yu Ku, David W. Goodrich, Irwin H. Gelman. PTEN regulated PI3K-p110 and AKT isoform plasticity controls metastatic prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2418.

MP79-09 ANTI-ANDROGEN THERAPY OVERCOMES THE TIME-DELAY IN INITIATION OF SALVAGE RADIATION THERAPY AND RESCUES ONCOLOGICAL OUTCOMES IN MEN WITH RECURRENT PROSTATE CANCER AFTER RADICAL PROSTATECTOMY

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) IV (MP79)1 Apr 2020MP79-09 ANTI-ANDROGEN THERAPY OVERCOMES THE TIME-DELAY IN INITIATION OF SALVAGE RADIATION THERAPY AND RESCUES ONCOLOGICAL OUTCOMES IN MEN WITH RECURRENT PROSTATE CANCER AFTER RADICAL PROSTATECTOMY Akshay Sood*, Jacob Keeley, Deepansh Dalela, Sohrab Arora, Isaac Palma-Zamora, Marcus Jamil, Natalija Kovacevic, Guillaume Farah, Philip Olson, Wooju Jeong, Quoc-Dien Trinh, Craig G. Rogers, James O. Peabody, Mani Menon, and Firas Abdollah Akshay Sood*Akshay Sood* More articles by this author , Jacob KeeleyJacob Keeley More articles by this author , Deepansh DalelaDeepansh Dalela More articles by this author , Sohrab AroraSohrab Arora More articles by this author , Isaac Palma-ZamoraIsaac Palma-Zamora More articles by this author , Marcus JamilMarcus Jamil More articles by this author , Natalija KovacevicNatalija Kovacevic More articles by this author , Guillaume FarahGuillaume Farah More articles by this author , Philip OlsonPhilip Olson More articles by this author , Wooju JeongWooju Jeong More articles by this author , Quoc-Dien TrinhQuoc-Dien Trinh More articles by this author , Craig G. RogersCraig G. Rogers More articles by this author , James O. PeabodyJames O. Peabody More articles by this author , Mani MenonMani Menon More articles by this author , and Firas AbdollahFiras Abdollah More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000971.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: To examine whether the addition of anti-androgen therapy (AAT) to ‘late’ salvage radiation therapy (sRT) can lead to oncological outcomes similar to that of early sRT in men with recurrent prostate cancer after radical prostatectomy. METHODS: Data on 670 men who participated in the RTOG 9601 trial and experienced biochemical recurrence were extracted using the NCTN data archive platform. Patients were stratified into four treatment groups: early sRT (pre-sRT PSA <0.7 ng/mL) with/without concomitant AAT and late sRT (pre-sRT PSA ³0.7 ng/mL) with/without concomitant AAT, based on the cut-offs reported in the original trial. Cox proportional hazards and Fine-Gray competing-risk regression analyses assessed the adjusted hazards of overall mortality, CaP specific mortality and metastatic/local disease progression among the four treatment groups. RESULTS: Patients were well-matched in baseline characteristics. The median follow-up was 12 years. At 10-years, for patients treated with early sRT, early sRT with AAT, late sRT and late sRT with AAT, the overall mortality, CaP specific mortality and metastasis rates were 14%, 16%, 31% and 17% (Log-rank p=0.0001), 7%, 3%, 16% and 4% (Gray’s p=0.0001) and 12%, 9%, 26% and 12% (Gray’s p=0.0003), respectively (Figure). Adjusted analysis demonstrated increased hazards of overall mortality in patients receiving delayed sRT versus early sRT, HR 1.62 (95% CI: 1.11 - 2.37). However, no difference remained after addition of concomitant AAT to late sRT ¾ referent early sRT, HR 0.89 (95% CI: 0.58 - 1.39). Similarly, the hazards of cancer-specific mortality and metastatic progression were worse for late sRT, when compared to early sRT, but were no different after addition of AAT to late sRT. CONCLUSIONS: Poorer outcomes associated with late sRT may be rescued with delivery of concomitant AAT. Source of Funding: None © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e1218-e1219 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Akshay Sood* More articles by this author Jacob Keeley More articles by this author Deepansh Dalela More articles by this author Sohrab Arora More articles by this author Isaac Palma-Zamora More articles by this author Marcus Jamil More articles by this author Natalija Kovacevic More articles by this author Guillaume Farah More articles by this author Philip Olson More articles by this author Wooju Jeong More articles by this author Quoc-Dien Trinh More articles by this author Craig G. Rogers More articles by this author James O. Peabody More articles by this author Mani Menon More articles by this author Firas Abdollah More articles by this author Expand All Advertisement PDF downloadLoading ...

GABARAPL1 suppresses metastasis by counteracting PI3K/Akt pathway in prostate cancer.

Metastasis remains the primary cause of prostate cancer (CaP)-related death. Using a genome wide shRNA screen, we identified GABARAPL1 as a potential CaP metastasis suppressor. GABARAPL1 mRNA levels inversely correlate with the invasive potential of a panel of human CaP cell lines. Lower mRNA levels correlate with higher Gleason scores in clinical CaP tumor samples. Moreover, Kaplan-Meier curves analysis showed that GABARAPL1 down-regulation in cancer tissues is associated with decreased disease-free survival in CaP patients. Knockdown of GABARAPL1 in human LNCaP cells results in increased invasion in vitro and lymph node metastasis in vivo. Vice versa, ectopic expression of GABARAPL1 decreases the invasiveness of CWR22Rv1 cells. Our previous in vitro shRNA screening identified FOXO4, a PI3K/Akt-inactivating downstream target, as a potential CaP metastasis suppressor. We show here that silencing FOXOs leads to reduced GABARAPL1 expression and enhanced invasion in LNCaP cells. Transfection of constitutively-activated Akt (myr-Akt) increased the invasion of LNCaP cells, which is associated with the inactivation of FOXOs and decreased GABARAPL1 expression. Indeed, forced expression of GABARAPL1 reversed the increased invasiveness of LNCaP/myr-Akt cells. Finally, immunohistochemistry analysis shows that Akt phosphorylation is negatively correlated with GABARAPL1 expression in human CaP tissues. Taken together, our data indicate that the suppression of FOXOs-GABARAPL1 signaling by Akt is an important mechanism for CaP progression and metastasis.

EphA6 promotes angiogenesis and prostate cancer metastasis and is associated with human prostate cancer progression

Metastasis is the primary cause of prostate cancer (CaP)-related death. We investigate the molecular, pathologic and clinical outcome associations of EphA6 expression and CaP metastasis. The expression profiling of Eph receptors (Ephs) and their ephrin ligands was performed in parental and metastatic CaP cell lines. Among Ephs and ephrins, only EphA6 is consistently overexpressed in metastatic CaP cells. Metastatic potential of EphA6 is assessed by RNAi in a CaP spontaneous metastasis mouse model. EphA6 knock-down in human PC-3M cells causes decreased invasion in vitro and reduced lung and lymph node metastasis in vivo. In addition, knock-down of EphA6 decreases tube formation in vitro and angiogenesis in vivo. EphA6 mRNA expression is higher in 112 CaP tumor samples compared with benign tissues from 58 benign prostate hyperplasia patients. Positive correlation was identified between EphA6 expression and vascular invasion, neural invasion, PSA level, and TNM staging in CaP cases. Further, genome-wide gene expression analysis in EphA6 knock-down cells identified a panel of differentially regulated genes including PIK3IPA, AKT1, and EIF5A2, which could contribute to EphA6-regulated cancer progression. These findings identify EphA6 as a potentially novel metastasis gene which positively correlates with CaP progression. EphA6 may be a therapeutic target in metastatic CaP.

Differential requirement for Src family tyrosine kinases in the initiation, progression, and metastasis of prostate cancer.

Prostate cancer (CaP) recurrence after androgen ablation therapy remains a significant cause of mortality in aging men. Malignant progression and metastasis are typically driven by genetic and epigenetic changes controlled by the androgen receptor (AR). However, evidence suggests that activated nonreceptor tyrosine kinases, including those of the Src family kinases (SFK), directly phosphorylate AR, thereby activating its transcriptional activity in the absence of serum androgen levels. To ascertain whether CaP progression and metastasis require SFK members, an autochthonous transgenic adenocarcinoma (AD) of the mouse prostate (TRAMP) model was crossed into Src-, Lyn- or Fyn-null backgrounds. Primary-site CaP formation was dependent on Src, to a lesser extent, Lyn, but not Fyn. Only Src(-) (/) (-);TRAMP prostate tumors were marked by reactive stroma. SFK deficiency did not affect progression to neuroendocrine (NE) disease, although there were fewer new cancer cases initiating after 34 weeks in the SFK(-/-);TRAMP mice compared with TRAMP controls. Of note, 15% to 21% of older (>33 weeks) Lyn- or Fyn-null TRAMP mice lacking primary-site tumors suffered from aggressive metastatic AD growths, compared with 3% of TRAMP mice. Taken with the data that TRAMP mice lacking Src or Lyn exhibited fewer macroscopic metastases compared with Fyn(-) (/) (-);TRAMP and TRAMP controls, this suggests that SFK can either promote or suppress specific parameters of metastatic growth, possibly depending on cross-talk with primary tumors. These data identify critical, yet potentially opposing roles played by various SFKs in the initiation and metastatic potential of CaP using the TRAMP model. Genetically defined mouse models indicate a critical role for Src tyrosine kinase in CaP initiation and metastatic progression.

Abstract 4005: CD44 isoform variant 6 is associated with prostate cancer progression, metastasis and chemo-/radio-resistance via PI3K/Akt/mTOR and Wnt/β-catenin signaling pathways in vitro

Abstract Aim: The role of CD44 variant 6 (CD44v6) in prostate cancer (CaP) metastasis and progression remains unclear. The aim of this study was 1) to examine the expression of CD44v6 in metastatic CaP cell lines, and normal, benign hyperplastic, and CaP tissues in different stages and lymph node metastases; 2) to investigate the role of CD44v6 in CaP progression, metastasis and therapy resistance as well as underlying pathways in vitro for a potential therapeutic target. Methods: Immunohistochemical/immunocytochemical analysis using an anti-CD44V6 monoclonal antibody (MAb) was performed on metastatic CaP cell lines and paraffin embedded specimens from patients (n=10) who underwent radical prostatectomy (RP), lymph node metastases (n=10), benign prostatic hyperplasia (BPH) (n=10), and normal prostate tissues (n=10). The immunostaining results from CaP cell lines were further confirmed by Western blot. The functional roles of CD44v6 in CaP metastasis and chemo- and radio-sensitivity were examined by knocking down (KD) CD44v6 gene using siRNAs in PC-3M, DU145 and LNCaP CaP cells, respectively and then performing cell invasion, colony formation, proliferation rate, sphere formation, chemo- and radio-sensitivity assays. Results: The expression of CD44v6 was found moderately to strongly positive in androgen-responsive (LNCaP, LNCaP-LN3) and androgen-nonresponsive (PC-3, PC-3M, DU145 and LNCaP-C4B2) metastatic CaP cell lines. The expression of CD44v6 was found negative in normal prostate and BPH tissues, and moderately to strongly positive primary CaP tissues and lymph node metastases. Three CD44v6-KD cell lines showed notable decreases in functions of invasion, colony formation, proliferation, sphere formation, and an increase in chemodrug and radiation sensitivity, compared to the parental cell lines, respectively. Obvious down-regulation of PI3K/Akt/mTOR and Wnt/β-catenin signaling pathways were observed in three CD44v6-KD cell lines by Western Blot. Conclusions: CD44v6 is a biomarker involved in CaP progression and metastasis, associated with CaP chemo-/radio-resistance via PI3K/Akt/mTOR and Wnt/β-catenin signaling pathways and could be an ideal potential therapeutic target for the treatment of castrate-resistant prostate cancer (CRPC). The in vivo study of CD44v6 functions in CaP animal models is ongoing in our laboratory. Citation Format: Jie Ni, Paul Cozzi, Jingli Hao, Julia Beretov, Lei Chang, Wei Duan, Warick Delprado, Peter Graham, Joseph Bucci, John Kearsley, Yong Li. CD44 isoform variant 6 is associated with prostate cancer progression, metastasis and chemo-/radio-resistance via PI3K/Akt/mTOR and Wnt/β-catenin signaling pathways in vitro. [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 4005. doi:10.1158/1538-7445.AM2014-4005

A genome-wide RNAi screen identifies FOXO4 as a metastasis-suppressor through counteracting PI3K/AKT signal pathway in prostate cancer.

Activation of the PI3K/AKT signal pathway is a known driving force for the progression to castration-recurrent prostate cancer (CR-CaP), which constitutes the major lethal phenotype of CaP. Here, we identify using a genomic shRNA screen the PI3K/AKT-inactivating downstream target, FOXO4, as a potential CaP metastasis suppressor. FOXO4 protein levels inversely correlate with the invasive potential of a panel of human CaP cell lines, with decreased mRNA levels correlating with increased incidence of clinical metastasis. Knockdown (KD) of FOXO4 in human LNCaP cells causes increased invasion in vitro and lymph node (LN) metastasis in vivo without affecting indices of proliferation or apoptosis. Increased Matrigel invasiveness was found by KD of FOXO1 but not FOXO3. Comparison of differentially expressed genes affected by FOXO4-KD in LNCaP cells in culture, in primary tumors and in LN metastases identified a panel of upregulated genes, including PIP, CAMK2N1, PLA2G16 and PGC, which, if knocked down by siRNA, could decrease the increased invasiveness associated with FOXO4 deficiency. Although only some of these genes encode FOXO promoter binding sites, they are all RUNX2-inducible, and RUNX2 binding to the PIP promoter is increased in FOXO4-KD cells. Indeed, the forced expression of FOXO4 reversed the increased invasiveness of LNCaP/shFOXO4 cells; the forced expression of FOXO4 did not alter RUNX2 protein levels, yet it decreased RUNX2 binding to the PIP promoter, resulting in PIP downregulation. Finally, there was a correlation between FOXO4, but not FOXO1 or FOXO3, downregulation and decreased metastasis-free survival in human CaP patients. Our data strongly suggest that increased PI3K/AKT-mediated metastatic invasiveness in CaP is associated with FOXO4 loss, and that mechanisms to induce FOXO4 re-expression might suppress CaP metastatic aggressiveness.

CD44 variant 6 is associated with prostate cancer metastasis and chemo‐/radioresistance

Prostate cancer (CaP) is the second leading malignancy in older men in Western countries. The role of CD44 variant 6 (CD44v6) in CaP progression and therapeutic resistance is still uncertain. Here, we investigated the roles of CD44v6 in CaP metastasis and chemo/radioresistance. Expression of CD44v6 in metastatic CaP cell lines, human primary CaP tissues and lymph node metastases was assessed using immunofluorescence and immunohistochemistry, respectively. Knock down (KD) of CD44v6 was performed in PC-3M, DU145, and LNCaP cells using small interfering RNA (siRNA), and confirmed by confocal microscope, Western blot and quantitative real time polymerase chain reaction (qRT-PCR). Cell growth was evaluated by proliferation and colony formation assays. The adhesive ability and invasive potential were assessed using a hyaluronic acid (HA) adhesion and a matrigel chamber assay, respectively. Tumorigenesis potential and chemo-/radiosensitivity were measured by a sphere formation assay and a colony assay, respectively. Over-expression of CD44v6 was found in primary CaP tissues and lymph node metastases including cancer cells and surrounding stromal cells. KD of CD44v6 suppressed CaP proliferative, invasive and adhesive abilities, reduced sphere formation, enhanced chemo-/radiosensitivity, and down-regulated epithelial-mesenchymal transition (EMT), PI3K/Akt/mTOR, and Wnt/β-catenin signaling pathway proteins in vitro. Our findings demonstrate that CD44v6 is an important cancer stem cell-like marker associated with CaP proliferation, invasion, adhesion, metastasis, chemo-/radioresistance, and the induction of EMT as well as the activation PI3K/Akt/mTOR and Wnt signaling pathways, suggesting that CD44v6 is a novel therapeutic target to sensitize CaP cells to chemo/radiotherapy.

Emerging roles of radioresistance in prostate cancer metastasis and radiation therapy

Radiation therapy (RT) continues to be one of the most popular treatment options for localized prostate cancer (CaP). Local CaP recurrence after RT is a pattern of treatment failure attributable to radioresistance of cancer cells. One major obstacle to RT is that there is a limit to the amount of radiation that can be safely delivered to the target organ. Recent results indicate that phosphoinositide 3-kinase (PI3K)/Akt/phosphatase and tensin homolog (PTEN)/mammalian target of rapamycin (mTOR) signaling pathway, autophagy, epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) are involved in CaP metastasis and radioresistance. Emerging evidence also suggests that combining a radiosensitizer with RT increases the efficacy of CaP treatment. Understanding the mechanisms of radioresistance will help to overcome recurrence after RT in CaP patients and prevent metastasis. In this review, we discuss the novel findings of PI3K/Akt/PTEN/mTOR signaling pathway, autophagy, EMT and CSCs in the regulation of CaP metastasis and radioresistance, and focus on combination of radiosensitizers with RT in the treatment of CaP in preclinical studies to explore novel approaches for future clinical trials.

Epithelial cell adhesion molecule (EpCAM) is associated with prostate cancer metastasis and chemo/radioresistance via the PI3K/Akt/mTOR signaling pathway

Prostate cancer (CaP) is the second leading malignancy in men. The role of epithelial cell adhesion molecule (EpCAM), also known as CD326, in CaP progression and therapeutic resistance is still uncertain. Here, we aimed to investigate the roles of EpCAM in CaP metastasis and chemo/radioresistance. Expression of EpCAM in CaP cell lines and human CaP tissues was assessed using immunofluorescence and immunohistochemistry, respectively. EpCAM was knocked down (KD) in PC-3, DU145 and LNCaP-C4-2B cells using small interfering RNA (siRNA), and KD results were confirmed by confocal microscope, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Cell growth was evaluated by proliferation and colony formation assays. The invasive potential was assessed using a matrigel chamber assay. Tumorigenesis potential was measured by a sphere formation assay. Chemo-/radiosensitivity were measured using a colony formation assay. Over-expression of EpCAM was found in primary CaP tissues and lymph node metastases including cancer cells and surrounding stromal cells. KD of EpCAM suppressed CaP proliferation and invasive ability, reduced sphere formation, enhanced chemo-/radiosensitivity, and down-regulated E-cadherin, p-Akt, p-mTOR, p-4EBP1 and p-S6K expression in CaP cells. Our findings suggest that EpCAM plays an important role in CaP proliferation, invasion, metastasis and chemo-/radioresistance associated with the activation of the PI3K/Akt/mTOR signaling pathway and is a novel therapeutic target to sensitize CaP cells to chemo-/radiotherapy.

Induction of reactive oxygen species generation inhibits epithelial–mesenchymal transition and promotes growth arrest in prostate cancer cells

Oxidative stress is one causative factor of the pathogenesis and aggressiveness of most of the cancer types, including prostate cancer (CaP). A moderate increase in reactive oxygen species (ROS) induces cell proliferation whereas excessive amounts of ROS promote apoptosis. In this study, we explored the pro-oxidant property of 3,9-dihydroxy-2-prenylcoumestan (psoralidin [pso]), a dietary agent, on CaP (PC-3 and C4-2B) cells. Pso greatly induced ROS generation (more than 20-fold) that resulted in the growth inhibition of CaP cells. Overexpression of anti-oxidant enzymes superoxide dismutase 1 (SOD1), SOD2, and catalase, or pretreatment with the pharmacological inhibitor N-acetylcysteine (NAC) significantly attenuated both pso-mediated ROS generation and pso-mediated growth inhibition in CaP cells. Furthermore, pso administration significantly inhibited the migratory and invasive property of CaP cells by decreasing the transcription of β-catenin, and slug, which promote epithelial-mesenchymal transition (EMT), and by concurrently inducing E-cadherin expression in CaP cells. Pso-induced ROS generation in CaP cells resulted in loss of mitochondrial membrane potential, cytochrome-c release, and activation of caspase-3 and -9 and poly (ADP-ribose) polymerase (PARP), which led to apoptosis. On the other hand, overexpression of anti-oxidants rescued pso-mediated effects on CaP cells. These findings suggest that increasing the threshold of intracellular ROS could prevent or treat CaP growth and metastasis.

Effect of Wnt-1 induced signaling protein-2 (Wisp-2/CCN5) on angiogenesis and invasion in prostate cancer.

164 Background: The Wnt-Induced Signaling Protein-2 (Wisp-2/CCN5) is a secreted protein implicated in modification of extracellular matrix, invasion, and angiogenesis. The regulation of Wisp-2 and its function in CaP is poorly explored although it is highly expressed in advanced CaP cells. We discovered recently that a pro-inflammatory chemokine, Interleukin-8 (IL-8) strongly modulates Wisp2 expression in CaP cells. Since chronic inflammation is thought to be significant in CaP metastasis and IL-8 is a major effecter of inflammatory pathway, we investigated the physiological consequence of Wisp-2 modulation in CaP cells. Methods: Quantitative PCR was used to determine the expression of Wisp-2 mRNA and other mRNA in prostate epithelial and cancer cells. Wisp-2 mediated-VEGF secretion was determined by ELISA and endothelial tube formation assay using human vascular endothelial cells. Invasive activity and their attributes were determined using gelatin-zymography for matrix metalloproteinases (MMPs), Matrigel invasion assay and chemotaxis assay. Results: Wisp2 RNA was strongly expressed in two highly metastatic CaP cell lines, DU145 and PC-3 and moderately in less invasive LAPC4 and LNCaP cells. Constitutive autocrine expression or external addition of IL-8 increased Wisp-2 mRNA by ≥15.2 times in LNCaP, and 7.4 times in LAPC4 cells. Depleting Wisp-2 by RNA interference caused 82% reduction of Wisp2 in DU145, and 75% in PC-3. Depletion of Wisp2 did not affect cell proliferation in DU145 and PC3 but it reduced expression of VEGF mRNA by 64% in DU145 and 43% in PC3. Secreted VEGF protein in cultures of DU145 and PC-3 cells showed a decrease of 62% in DU145 and 30% in PC3. Condition medium of Wisp-2 depleted DU145 cell cultures showed 20% decrease in proliferation and vessel formation activity in HUVEC cells. Further, Wisp-2 depletion in DU145 cells resulted in significant decrease in chemoinvasion activity (28%) and secretion of MMP-9 (25%). Conclusions: These results show that Wisp2 is down stream effector of IL-8 and is an important modulator, affecting extracellular matrix to stimulate angiogenesis and invasiveness in CaP cells. Suppression of Wisp-2 in CaP may reduce their metastatic potential.

In Vitro and In Vivo Prostate Cancer Metastasis and Chemoresistance Can Be Modulated by Expression of either CD44 or CD147

CD44 and CD147 are associated with cancer metastasis and progression. Our purpose in the study was to investigate the effects of down-regulation of CD44 or CD147 on the metastatic ability of prostate cancer (CaP) cells, their docetaxel (DTX) responsiveness and potential mechanisms involved in vitro and in vivo. CD44 and CD147 were knocked down (KD) in PC-3M-luc CaP cells using short hairpin RNA (shRNA). Expression of CD44, CD147, MRP2 (multi-drug resistance protein-2) and MCT4 (monocarboxylate tranporter-4) was evaluated using immunofluorescence and Western blotting. The DTX dose-response and proliferation was measured by MTT and colony assays, respectively. The invasive potential was assessed using a matrigel chamber assay. Signal transduction proteins in PI3K/Akt and MAPK/Erk pathways were assessed by Western blotting. An in vivo subcutaneous (s.c.) xenograft model was established to assess CaP tumorigenecity, lymph node metastases and DTX response. Our results indicated that KD of CD44 or CD147 decreased MCT4 and MRP2 expression, reduced CaP proliferation and invasive potential and enhanced DTX sensitivity; and KD of CD44 or CD147 down-regulated p-Akt and p-Erk, the main signal modulators associated with cell growth and survival. In vivo, CD44 or CD147-KD PC-3M-luc xenografts displayed suppressed tumor growth with increased DTX responsiveness compared to control xenografts. Both CD44 and CD147 enhance metastatic capacity and chemoresistance of CaP cells, potentially mediated by activation of the PI3K and MAPK pathways. Selective targeting of CD44/CD147 alone or combined with DTX may limit CaP metastasis and increase chemosensitivity, with promise for future CaP treatment.

Role of the EpCAM (CD326) in prostate cancer metastasis and progression

Despite significant advances in surgery, radiotherapy and chemotherapy to treat prostate cancer (CaP), many patients die of secondary disease (metastases). Current therapeutic approaches are limited, and there is no cure for metastatic castration-resistant prostate cancer (CRPC). Epithelial cell adhesion molecule (EpCAM, also known as CD326) is a transmembrane glycoprotein that is highly expressed in rapidly proliferating carcinomas and plays an important role in the prevention of cell-cell adhesion, cell signalling, migration, proliferation and differentiation. Stably and highly expressed EpCAM has been found in primary CaP tissues, effusions and CaP metastases, making it an ideal candidate of tumour-associated antigen to detect metastasis of CaP cells in the circulation as well as a promising therapeutic target to control metastatic CRPC disease. In this review, we discuss the implications of the newly identified roles of EpCAM in terms of its diagnostic and metastatic relevance to CaP. We also summarize EpCAM expression in human CaP and EpCAM-mediated signalling pathways in cancer metastasis. Finally, emerging and innovative approaches to the management of the disease and expanding potential therapeutic applications of EpCAM for targeted strategies in future CaP therapy will be explored.

Abstract 616: A cumulative molecular model for the mechanisms of green tea catechins in the chemoprevention of prostate cancer

Abstract Background: Prostate cancer (CaP) is the most commonly diagnosed cancer in men. It's long latency, slow progression, and high incidence rates make it ideal for targeting chemoprevention therapies. Studies suggest that green tea may be suitable candidate for CaP chemoprevention. The green tea catechin (GTC) chemopreventive mechanisms against CaP cells, however, are not completely clear. Understanding and refining models of the fundamental molecular pathways by which GTCs modulate prostate carcinogenesis is essential to most appropriately utilize green tea for CaP prevention in clinical settings. Objective: The objective of the study was to review the current literature and develop a model that attempts to recreate the molecular mechanisms and pathways by which GTCs modulate prostate carcinogenesis. Methods: Laboratory studies, clinical studies, and clinical trials focused on GTC chemoprevention of CaP were critically analyzed and reviewed for mechanisms of GTC chemoprevention. The most prevalent mechanisms were combined into a multi-mechanistic model that attempts to recreate the cumulative events that occur as GTCs exert anti-cancer activity on prostate cancer cells and tissues. Results: GTCs exert anti-cancer actions on CaP cells and tissue through six major mechanisms: proteasome inhibition, cell cycle arrest, inhibition of cell proliferation, induction of apoptosis, suppression of carcinogenesis/progression, and inhibition of metastasis. We have proposed a novel model in which GTCs exert chemopreventive effects on CaP though these six major mechanisms that work simultaneously and dependently, largely driven by proteasome inhibition-induced regulation of the NFκB pathway. The cumulative effect of these mechanisms ultimately leads to the inhibition of CaP cell growth, progression, and metastasis. Conclusions: Several mechanisms of GTC chemopreventive activity on CaP cells and tissues have been identified in laboratory and clinical studies. Although GTCs act through distinct cell cycle regulative pathways, the cumulative chemopreventative effect appears to be attributed to their well-coordinated ensemble, rather than a single pathway. Additionally, transitioning to the use of standardized GTC preparations may more accurately reflect human tea consumption, deliver the maximum amount of GTCs, and allow for more generalizable results in epidemiological, laboratory, and clinical studies. We are currently testing and refining our novel cumulative model to fully elucidate the complex mechanism of GTC chemoprevention of prostate cancer in its entirety. 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 616. doi:1538-7445.AM2012-616

Abstract C56: The combined loss of SSeCKS/Gravin/AKAP12 and Rb in the prostate promotes spontaneous lymph node metastases with basal-luminal transitional phenotypes

Abstract Introduction: Previous data indicated that the loss of SSeCKS/Gravin/AKAP12 (“SSeCKS”) or Rb correlates with prostate cancer (CaP) progression and that their deletion results in increased prostatic hyperplasia (1,2). SSeCKS re-expression in MAT-LyLu prostate cancer cells has little effect on the growth potential of primary-site tumors and on the ability of metastatic cells to colonize distal sites, yet it suppresses the growth of macrometastases by suppressing tumor-encoded vascular endothelial growth factor expression and neovascularization of the metastatic niche(3). We demonstrated recently that SSeCKS-null (Akap12−/−) mouse embryo fibroblasts (KO-MEF) suffer from an Rb-dependent premature senescence, and that the hyperplastic prostates in KO mice exhibit markers of increased senescence (4). Experimental Procedures: In order to determine whether SSeCKS and Rb act synergistically to promote CaP progression, KO mice were crossed to mice with prostate-specific conditional Rb deletion (Pb-Cre;Rbfl/fl). Results: Compared to controls (WT, Akap12−/− or Pb-Cre;Rbfl/fl mice), 100% of Akap12−/−;Pb-Cre;Rbfl/fl male mice showed evidence of low grade prostatic intraepithelial neoplasia (LG-PIN), with a smaller percentage (22%) displaying high grade (HG) PIN by 10 months of age. These lesions were marked by increased levels of cytoplasmic E-cadherin (compared to the cell-cell junctional staining in normal or hyperplastic luminal prostate layers) in LGPIN and by the loss of E-cadherin expression in HG-PIN. Unexpectedly, Akap12−/−;Pb-Cre;Rbfl/fl males showed increased numbers (1.8- to 2.5-fold) of p63-positive basal cells in all four lobes compared with control mouse prostates. Importantly, whereas control mice showed neither primary-site prostate cancer nor lymph node (LN) metastasis, &amp;gt;/80% of the Akap12−/−;Pb-Cre;Rbfl/fl males displayed metastases in pelvic or inguinal LN. These metastases did not stain for E-cadherin, but were positive for both basal (5 and 14) and luminal (8) cytokeratins, and low levels of androgen receptor. Foci within these lesions were positive for the basal marker, p63, and for the mesenchymal marker, vimentin. The LN lesions contained deletions of their floxed Rb loci, as shown by nested PCR assays, corroborating that they derived from the transgenic prostate cells. Conclusions: The combined loss of SSeCKS and Rb leads to early CaP initiation marked by increased basal cell proliferation in all four lobes, as well as the marked increase in LN metastasis of tumor cells with a basal-luminal transitional phenotype. These data strongly suggest that SSeCKS suppresses parameters of CaP initiation and metastasis. Citation Format: Hyun-Kyung Ko, Shin Akakura, Jennifer Peresie, Barbara Foster, Irwin H. Gelman. The combined loss of SSeCKS/Gravin/AKAP12 and Rb in the prostate promotes spontaneous lymph node metastases with basal-luminal transitional phenotypes [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C56.

Abstract 5159: Wnt signaling protein (Wisp2/CCN5) stimulates angiogenesis and invasion in prostate cancer

Abstract Introduction: Angiogenesis and invasion are essential attributes of metastatic cancer cells. The Wnt-Induced Signaling Protein-2 (Wisp-2 /CCN5) is a secreted protein implicated in modification of extracellular matrix, invasion, and angiogenesis. The regulation of Wisp-2 and its function in CaP is poorly explored although it is highly expressed in advanced CaP cells. We discovered recently that a pro-inflammatory chemokine, Interleukin-8 (IL-8) strongly modulates Wisp2 expression in CaP cells. Since chronic inflammation is thought to be significant in CaP metastasis and IL-8 is a major effecter of inflammatory pathway, we investigated the physiological consequence of Wisp-2 modulation in CaP cells. Hypothesis; Wisp-2 is a down-stream effector of IL-8 induced angiogenesis and invasion, and is essential for metastatic progression of prostate cancer. Methods: Quantitative PCR (qPCR) was used to determine the expression of Wisp-2 mRNA and other mRNA in prostate epithelial and cancer cells (e.g. RWPE-1, LNCaP, LAPC-4, DU145, and PC3). Wisp-2 mediated-VEGF secretion was determined by ELISA and endothelial tube formation assay using human vascular endothelial cells (HUVEC cells). Invasive activity and their attributes were determined using gelatin-zymography for matrix metalloproteinases (MMPs), Matrigel invasion assay and chemotaxis assay. Results: Wisp2 RNA was strongly expressed in two highly metastatic CaP cell lines, DU145 and PC-3 and moderately in less invasive LAPC4 and LNCaP cells. Constitutive autocrine expression or external addition of IL-8 increased Wisp-2 mRNA by ≥15.2 times in LNCaP, and 7.4 times in LAPC4 cells. Depleting Wisp-2 by RNA interference caused 82% reduction of Wisp2 in DU145, and 75% in PC-3. Depletion of Wisp2 did not affect cell proliferation in DU145 and PC3 but it reduced expression of VEGF mRNA by 64% in DU145 and 43% in PC3. Secreted VEGF protein in cultures of DU145 and PC-3 cells showed a decrease of 62% in DU145 and 30% in PC3. Condition medium of Wisp-2 depleted DU145 cell cultures showed 20% decrease in proliferation and vessel formation activity in HUVEC cells. Further, Wisp-2 depletion in DU145 cells resulted in significant decrease in chemo-invasion activity (28%) and secretion of MMP-9 (25%). Conclusions: These results show that Wisp2 is down stream effector of IL-8 and is an important modulator, affecting extracellular matrix to stimulate angiogenesis and invasiveness in CaP cells. Suppression of Wisp-2 in CaP may reduce their metastatic potential. (Grant Support; VA MERIT Review, VA5312.01 (BLL)) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5159. doi:10.1158/1538-7445.AM2011-5159

Co-expression of CD147 (EMMPRIN), CD44v3-10, MDR1 and monocarboxylate transporters is associated with prostate cancer drug resistance and progression

Background:The aim of this study is to seek an association between markers of metastatic potential, drug resistance-related protein and monocarboxylate transporters in prostate cancer (CaP).Methods:We evaluated the expression of invasive markers (CD147, CD44v3-10), drug-resistance protein (MDR1) and monocarboxylate transporters (MCT1 and MCT4) in CaP metastatic cell lines and CaP tissue microarrays (n=140) by immunostaining. The co-expression of CD147 and CD44v3-10 with that of MDR1, MCT1 and MCT4 in CaP cell lines was evaluated using confocal microscopy. The relationship between the expression of CD147 and CD44v3-10 and the sensitivity (IC50) to docetaxel in CaP cell lines was assessed using MTT assay. The relationship between expression of CD44v3-10, MDR1 and MCT4 and various clinicopathological CaP progression parameters was examined.Results:CD147 and CD44v3-10 were co-expressed with MDR1, MCT1 and MCT4 in primary and metastatic CaP cells. Both CD147 and CD44v3-10 expression levels were inversely related to docetaxel sensitivity (IC50) in metastatic CaP cell lines. Overexpression of CD44v3-10, MDR1 and MCT4 was found in most primary CaP tissues, and was significantly associated with CaP progression.Conclusions:Our results suggest that the overexpression of CD147, CD44v3-10, MDR1 and MCT4 is associated with CaP progression. Expression of both CD147 and CD44v3-10 is correlated with drug resistance during CaP metastasis and could be a useful potential therapeutic target in advanced disease.

Interactions between human osteoblasts and prostate cancer cells in a novel 3D in vitro model

Cell-cell and cell-matrix interactions play a major role in tumor morphogenesis and cancer metastasis. Therefore, it is crucial to create a model with a biomimetic microenvironment that allows such interactions to fully represent the pathophysiology of a disease for an in vitro study. This is achievable by using three-dimensional (3D) models instead of conventional two-dimensional (2D) cultures with the aid of tissue engineering technology. We are now able to better address the complex intercellular interactions underlying prostate cancer (CaP) bone metastasis through such models. In this study, we assessed the interaction of CaP cells and human osteoblasts (hOBs) within a tissue engineered bone (TEB) construct. Consistent with other in vivo studies, our findings show that intercellular and CaP cell-bone matrix interactions lead to elevated levels of matrix metalloproteinases, steroidogenic enzymes and the CaP biomarker, prostate specific antigen (PSA); all associated with CaP metastasis. Hence, it highlights the physiological relevance of this model. We believe that this model will provide new insights for understanding of the previously poorly understood molecular mechanisms of bone metastasis, which will foster further translational studies, and ultimately offer a potential tool for drug screening.

CD147/EMMPRIN and CD44 are Potential Therapeutic Targets for Metastatic Prostate Cancer

Prostate cancer (CaP) is a major health problem in males in Western countries. Current therapeutic approaches are limited and many patients die of secondary disease (metastases). There is no cure for metastatic castration-resistant prostate cancer (CRPC). Targeting tumor-associated antigens is fast emerging as an area of promise to treat late stage and recurrent CaP. Extracellular matrix metalloproteinase inducer, EMMPRIN (CD147) is a multifunctional glycoprotein that can modify the tumor microenvironment by activating proteinases, inducing angiogenic factors in tumor and stromal cells, and regulating growth and survival of anchorage-independent tumor cells (micrometastases) and multidrug resistance (MDR). CD44 is a multifunctional protein involved in cell adhesion, migration and drug resistance, and is a primary receptor for hyaluronan (HA), a major component of the extracellular matrix (ECM) with a critical role in cell signaling and cell-ECM interactions in cancer. Our recent studies indicate both CD147 and CD44 are involved in cancer drug resistance and play very important roles in CaP metastasis. Thus, CD147 and CD44 may be ideal therapeutic targets to control metastatic and CRPC disease. This review will discuss their putative roles in CaP metastasis and MDR, and give an overview of literature regarding their expression on human CaP tissues. Additional focus will be on the potential of therapeutic strategies targeting CD147 and CD44 to prevent CaP metastasis and overcome drug resistance.