Metastatic CaP Cell Research Articles

Abstract 5014: Identifying and treating robo1−ve/dock1+ve prostate cancer: An aggressive cancer type highly prevalent in African-American patients

Abstract Grant Funding: R01CA193739 Metastasis is the principal cause of death in prostate cancer (CaP) patients. The prognosis is very poor for metastatic CaP. On comparison, African-American patients exhibit high mortality rates than Caucasian counterparts. The failure of therapies for metastatic CaP is observed very high in patients belonging to African-American race. There is a need to identify new target-based therapies which could be either (i) beneficial to patients regardless of race or (ii) treat high-risk patients such as African-Americans who fail standard therapies compared to Caucasians. This requires identification of molecular pathways which are either (i) preferentially active in African-Americans or (ii) highly active in both races. We posit that the loss of ROBO1 in advanced stage CaP contributes to the disparity observed between African-Americans and Caucasian. We conducted a comprehensive analysis of race-based primary and metastatic tumors for ROBO1 and its downstream targets (RAC1, DOCK1, E-Cadherin, and β-catenin). We show that ROBO1 expression is progressively lost with the increasing stage of CaP in African-American patients. Next, in order to explore the underlying mechanism that leads to ROBO1 loss, we analyzed matching tumor DNA from patients and found that the ROBO1 promoter is methylated in the majority of African-American cases in agreement with the CaP stage. In addition, we found that ROBO1 loss is associated to increasing RAC1 activation in tumors and representative cell models. We show that loss of ROBO1 results in increased DOCK1 activity that in turn induces Rac1 signaling in CaP cells. We hypothesized that targeting DOCK1/Rac1 network could be an ideal strategy to treat metastatic CaP, particularly in ROBO1−/- cases. To test the hypothesis, we tested the therapeutic efficacy of a selective inhibitor of DOCK1/Rac1 interaction in ROBO1+ve and ROBO1−ve CaP cells. We observed that DOCK1-inhibitor treatment significantly decreased the RAC1 activation and β-catenin activity in metastatic CaP cells. Notably, inhibition of DOCK1/Rac1 was observed to significantly decrease the motility and invasiveness of aggressive metastatic CaP cells. Furthermore, DOCK1-inhibitor significantly blocked the potential of metastatic CaP cells to extravasate (cross-over) through the endothelial lining (constituted of HUVEC cells). These data suggested the significance of DOCK1/Rac1 pathway in a metastasis of CaP particularly in African-Americans. We conclude that the ROBO1/RAC1 ratio in tumor biopsies could be developed as an early biomarker of metastasis in African-American patients. We suggest that DOCK1-inhibitor therapy would be more beneficial to patients who exhibit ROBO1− tumor, and African-American patients are such as group who likely to benefit this therapy. Citation Format: Marina Graciela Ferrari, Arsheed Ganaie, Adrian P. Mansini. Identifying and treating robo1−ve/dock1+ve prostate cancer: An aggressive cancer type highly prevalent in African-American patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5014.

Label-free electrochemical quantification of microRNA-375 in prostate cancer cells

Clinical applicability of a label-free electrochemical method was investigated, where prostate cancer (CaP) biomarkers miR-107 and miR-375 were examined in two different groups of prostate cell lines, metastatic CaP cells (PC-3, DU145, and LNCaP) and immortalized prostate epithelial cells (267B1, X/267B1, and Ki/267B1). First, the expression levels of miR-107 and miR-375 in these cells were analyzed by quantitative real-time polymerase chain reaction (PCR), resulting in useful discernable expression patterns from miR-375. Choosing miR-375 as a target CaP biomarker, a label-free electrochemical analysis was performed based on the optimized conditions of 100nmol/mL miR-375 capture probe for surface immobilization and 30-minute hybridization. Standard curves were prepared both in buffer and in 100-fold diluted serum resulting in lower detection limit of 11.7aM and 12.4aM respectively. The regression equations for buffer and diluted serum were I=−15.1349 log c+250.9735 and I=−11.8179 log c+193.1639, respectively, where I is the peak current in μA and c is the concentration of miR-375 in fM. The selectivity of the sensor was confirmed by a one-base mismatch test in 3 different concentrations of miR-375 under diluted serum, 10aM, 10fM, and 10pM. Finally, miR-375 expression in the metastatic CaP cells was quantified to range between 10 and 30fM, while its expression in the prostate epithelial cells was negligible, demonstrating close agreement with the results from quantitative real-time PCR, and thereby potential viability of the electrochemical evaluation method in clinical environment.

Abstract 1999: Study of CD44 variant 6 (CD44v6) in prostate cancer chemo-/radio resistance in vivo

Abstract Background: Chemo-/radio-resistance is an important reason for prostate cancer (CaP) progression and metastasis. CD44 is a well-documented cancer stem cell (CSC) biomarker, and one of its variants, CD44 variant 6 (CD44v6) is closely associated with aggressive behaviour and correlates with poor prognosis in a variety of human cancers. Our previous study has demonstrated increased expressions of CD44v6 in metastatic CaP cell lines and human CaP tissues, which was associated with CaP progression and chemo-/radio-resistance in vitro. However, the role of CD44v6 in CaP progression and therapeutic resistance in vivo is still uncertain. Aim: The aim of this study was to investigate the role of CD44v6 in CaP development and chemo-/radioresistance as well as underlying pathways in vivo, and find whether it is a suitable therapeutic target for CaP therapy. Methods: CD44v6 gene was knocked down (KD) in PC-3M CaP cell line using short hairpin RNA (shRNA). Subcutaneous (s.c.) and orthotopic CaP animal models were established using PC-3M-CD44v6-KD and PC-3M-CD44v6-scramble (scr) control cells, to assess the effect of CD44v6 on CaP tumourigenesis, chemotherapy (docetaxel) response and radiation response. Signal transduction proteins in PI3K/Akt/mTOR pathway (Akt, p-Akt, mTOR and p-mTOR) as well as proliferation and apoptosis makers were assessed by immunohistochemistry in xenografts from both animal models. Results: Both s.c. and orthotopic xenografts from PC-3M-CD44v6-KD cells displayed supressed tumour growth and increased responsiveness to docetaxel (40mg/kg) and radiation (2Gy/day for 4 consecutive days) compared to control xenografts from PC-3M-CD44v6-scr cells in NOD/SCID male mice. Down-regulation of the PI3K/Akt/mTOR pathway proteins (p-Akt and p-mTOR), proliferation marker (Ki-67) and angiogenesis marker (CD31), as well as up-regulation of apoptotic responses (cleaved Caspase-3) to chemotherapy, DNA damage responses (γ-H2AX) to radiation were found in PC-3M-CD44v6-KD s.c. xenografts, respectively. Conclusions: CD44v6 is actively involved in CaP tumorigenesis and treatment responses to chemo-/radio-therapy via PI3K/Akt/mTOR signalling pathway in in vivo mouse models and holds promise as a therapeutic target for the treatment of CaP. Key words: CD44v6, prostate cancer, chemo-/radio-resistance, animal model, metastasis Citation Format: Jie Ni, Paul Cozzi, Julia Beretov, Joseph Bucci, Peter Graham, Yong Li. Study of CD44 variant 6 (CD44v6) in prostate cancer chemo-/radio resistance in vivo [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 1999.

Abstract 2833: Epithelial cell adhesion molecule (EpCAM) is associated with prostate cancer progression and chemo-/radio-resistance in vitro and in vivo

Abstract Aims: Prostate cancer (CaP) is the most common cancer in males in Australia which caused more than 3000 deaths in 2015. EpCAM is a transmembrane protein that is expressed at low levels in a variety of human epithelial tissues, but overexpressed in most solid tumors. Our previous study indicated that EpCAM was strongly expressed in metastatic CaP cell lines, primary human CaP tissues and lymph node metastasis and is a biomarker involved in CaP progression, and chemo-/radio-resistance. However, the role of EpCAM in CaP progression and therapeutic resistance is still uncertain. The aim of this study was to investigate the role of EpCAM in CaP progression and chemo-/radio-resistance as well as underlying mechanisms in vitro and in vivo. Methods: EpCAM gene was knocked down (KD) in PC-3, DU145 and LNCaP CaP cell lines using shRNA. Proliferation assay, colony formation assay, docetaxel (DTX) and radiation dose-response assay were carried out to evaluate the effect of KD of EpCAM on proliferation and therapeutic response of CaP cells in vitro. Subcutaneous (s.c) and orthotopic CaP animal models were established using PC-3-EpCAM-KD and PC-3-EpCAM-scramble (scr) control cells in NOD/SCID mice, to assess the effect of EpCAM on CaP tumourigenecity, chemotherapy (DTX) and radiation response. Signal transduction proteins in PI3K/Akt/mTOR signaling pathway, as well as proliferation, apoptotic and radiation response markers were evaluated by immunohistochemistry in xenografts. Results: KD of EpCAM reduced CaP proliferative potential and enhanced DTX and radiation sensitivity in three CaP cell lines. Both s.c and orthotopic EpCAM-KD xenografts displayed suppressed tumor growth and increased DTX and radiation responsiveness compared to EpCAM-scr control xenografts in NOD/SCID mice. Marked down-regulation of PI3K/Akt/mTOR pathway proteins (p-Akt and p-mTOR) and proliferation marker (Ki-67) and significant up-regulation of apoptotic (Caspase-3) and radiation (γ-H2AX) responses to chemo-/radio-therapies were found in EpCAM-KD xenografts, compared with control xenografts. In addition, Kaplan-Meier curve analysis demonstrated that KD of EpCAM improved median survival (MS) of tumor-bearing mice by 21.5 days compared with the control group (HR=26.94, CI95% 4.317-168.1, p=0.0004) and that KD of EpCAM improved MS of tumor-bearing mice which received docetaxel (50mg/kg, single dose, i.p.) by 11 days (HR=20.95, CI95% 3.599-121.9, p=0.0007), and radiotherapy (2Gy/day for 4 days) by 12 days (HR=11.00, CI95% 2.11-57.36, p=0.0044) compared with the control group, respectively. Conclusions: EpCAM plays an important role in CaP progression and chemo-/radio-resistance via PI3K/Akt/mTOR signaling pathway in vitro and in vivo and it is a promising therapeutic target for the treatment of CaP. EpCAM targeted therapy combined with chemo-/radio-therapy could be a novel modality for treatment-resistant CaP. Citation Format: Jie Ni, Paul Cozzi, Julia Beretov, Junli Deng, Joseph Bucci, Peter Graham, Yong Li. Epithelial cell adhesion molecule (EpCAM) is associated with prostate cancer progression and chemo-/radio-resistance in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2833. doi:10.1158/1538-7445.AM2017-2833

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.

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

ROBO1, a tumor suppressor and critical molecular barrier for localized tumor cells to acquire invasive phenotype: study in African-American and Caucasian prostate cancer models.

High-risk populations exhibit early transformation of localized prostate cancer (CaP) disease to metastasis which results in the mortality of such patients. The paucity of knowledge about the molecular mechanism involved in acquiring of metastatic behavior by primary tumor cells and non-availability of reliable phenotype-discriminating biomarkers are stumbling blocks in the management of CaP disease. Here, we determine the role and translational relevance of ROBO1 (an organogenesis-associated gene) in human CaP. Employing CaP-progression models and prostatic tissues of Caucasian and African-American patients, we show that ROBO1 expression is localized to cell-membrane and significantly lost in primary and metastatic tumors. While Caucasians exhibited similar ROBO1 levels in primary and metastatic phenotype, a significant difference was observed between tumor phenotypes in African-Americans. Epigenetic assays identified promoter methylation of ROBO1 specific to African-American metastatic CaP cells. Using African-American CaP models for further studies, we show that ROBO1 negatively regulates motility and invasiveness of primary CaP cells, and its loss causes these cells to acquire invasive trait. To understand the underlying mechanism, we employed ROBO1-expressing/ROBO1-C2C3-mutant constructs, immunoprecipitation, confocal-microscopy and luciferase-reporter techniques. We show that ROBO1 through its interaction with DOCK1 (at SH3-SH2-domain) controls the Rac-activation. However, loss of ROBO1 results in Rac1-activation which in turn causes E-Cadherin/β-catenin cytoskeleton destabilization and induction of cell migration. We suggest that ROBO1 is a predictive biomarker that has potential to discriminate among CaP types, and could be exploited as a molecular target to inhibit the progression of disease as well as treat metastasis in high-risk populations such as African-Americans.

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.

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.

Constitutive and treatment-induced CXCL8-signalling selectively modulates the efficacy of anti-metabolite therapeutics in metastatic prostate cancer.

BackgroundThe current study was undertaken to characterize the effect of anti-metabolites on inducing CXCL8 signaling and determining whether the constitutive and/or drug-induced CXCL8 signaling in metastatic prostate cancer (CaP) cells modulates their sensitivity to this class of agent.MethodsThe response of metastatic CaP cells to 5-Fluorouracil (5-FU), Pemetrexed or Tomudex was determined using cell count assays, flow cytometry and PARP cleavage analysis. Quantitative-PCR, ELISA and immunoblots were employed to determine effects of drugs or CXCL8 administration on target gene/protein expression.ResultsAdministration of 5-FU but not pemetrexed potentiated CXCL8 secretion and increased CXCR1 and CXCR2 gene expression in metastatic PC3 cells. Consistent with this, the inhibition of CXCL8 signaling using a CXCR2 antagonist, AZ10397767, increased the cytotoxicity of 5-FU by 4-fold (P<0.001), and increased 5-FU-induced apoptosis in PC3 cells (P<0.01). In contrast, while administration of AZ10397767 had no effect on the sensitivity of pemetrexed, the CXCR2 antagonist exerted the greatest effect in increasing the sensitivity of PC3 cells to Tomudex, a directed thymidylate synthase (TS) inhibitor. Subsequent experiments confirmed that administration of recombinant human CXCL8 increased TS expression, a response mediated in part by the CXCR2 receptor. Moreover, siRNA-mediated knockdown of the CXCL8-target gene Bcl-2 increased the sensitivity of PC3 cells to 5-FU.ConclusionsCXCL8 signaling provides a selective resistance of metastatic prostate cancer cells to specific anti-metabolites by promoting a target-associated resistance, in addition to underpinning an evasion of treatment-induced apoptosis.

Effect of Wnt signaling protein (Wisp2/CCN5) on angiogenesis and invasion in prostate cancer.

227 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 IL-8 is a major effecter of inflammatory pathway, we investigated the physiological consequence of Wisp-2 modulation in CaP cells. We hypothesised that Wisp-2 is a down-stream effector of IL-8 induced angiogenesis and invasion. Methods: Quantitative PCR (qPCR) 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 (HUVEC). Invasive activity and their attributes were determined using gelatin-zymography for matrix metalloproteinases (MMPs), and Matrigel invasion 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.

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.

RHAMM (CD168) Is Overexpressed at the Protein Level and May Constitute an Immunogenic Antigen in Advanced Prostate Cancer Disease

Localized prostate cancer (CaP) can be cured using several strategies. However, the need to identify active substances in advanced tumor stages is tremendous, as the outcome in such cases is still disappointing. One approach is to deliver human tumor antigen-targeted therapy, which is recognized by T cells or antibodies. We used data mining of the Cancer Immunome Database (CID), which comprises potential immunologic targets identified by serological screening of expression libraries. Candidate antigens were screened by DNA microarrays. Genes were then validated at the protein level by tissue microarrays, representing various stages of CaP disease. Of 43 targets identified by CID, 10 showed an overexpression on the complementary DNA array in CaP metastases. The RHAMM (CD168) gene, earlier identified by our group as an immunogenic antigen in acute and chronic leukemia, also showed highly significant overexpression in CaP metastases compared with localized disease and benign prostatic hyperplasia. At the protein level, RHAMM was highest in metastatic tissue samples and significantly higher in neoplastic localized disease compared with benign tissue. High RHAMM expression was associated with clinical parameters known to be linked to better clinical outcome. Patients with high RHAMM expression in the primaries had a significantly lower risk of biochemical failure. The number of viable cells in cell cultures was reduced in blocking experiments using hormone-sensitive and hormone-insensitive metastatic CaP cell lines. Acknowledging the proven immunogenic effects of RHAMM in leukemia, this antigen is intriguing as a therapeutic target in far-advanced CaP.

The role of extracellular matrix metalloproteinase inducer protein in prostate cancer progression.

Extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) is a multifunctional membrane glycoprotein overexpressed in many solid tumors, and involved in tumor invasion and angiogenesis. We investigated EMMPRIN expression in human prostate cancer (CaP) tissues and cells, and evaluated whether EMMPRIN expression is related to tumor progression and matrix metalloproteinase (MMPs) expression in human CaP. An immunohistochemical study using tissue microarrays of 120 primary CaPs of different grades and 20 matched lymph node metastases from untreated patients was performed. The association of EMMPRIN expression with clinicopathological parameters was evaluated. Co-immunolocalization for EMMPRIN and MMP-1, MMP-2 or MMP-9 in primary tumors was examined using confocal microscopy. Flow cytometry and immunoblotting were used to examine EMMPRIN expression in 11 metastatic CaP cell lines. Heterogeneous expression of EMMPRIN was found in 78/120 (65%) CaPs, correlated significantly with progression parameters including pre-treatment PSA level (P < 0.05) and increased with progression of CaP (Gleason score, P < 0.05; pathological stage, P < 0.01; nodal involvement, P < 0.05 and surgical margin, P < 0.05). Heterogeneous cytoplasmic MMP-1, MMP-2 and MMP-9 associated with EMMPRIN immunolabeling was observed, particularly in tumors with Gleason scores >3 + 4. Metastatic CaP cell lines, except DuCaP, expressed abundant EMMPRIN protein, indicating highly ( approximately 45 to approximately 65 kDa) and less ( approximately 30 kDa) glycosylated forms, although with no relationship to cells being either androgen responsive or nonresponsive. Our results suggest that EMMPRIN may regulate MMPs and be involved in CaP progression, and as such, could provide a target for treating metastatic CaP disease.

Direct evidence of lipid translocation between adipocytes and prostate cancer cells with imaging FTIR microspectroscopy

Various epidemiological studies show a positive correlation between high intake of dietary FAs and metastatic prostate cancer (CaP). Moreover, CaP metastasizes to the bone marrow, which harbors a rich source of lipids stored within adipocytes. Here, we use Fourier transform infrared (FTIR) microspectroscopy to study adipocyte biochemistry and to demonstrate that PC-3 cells uptake isotopically labeled FA [deuterated palmitic acid (D(31)-PA)] from an adipocyte. Using this vibrational spectroscopic technique, we detected subcellular locations in a single adipocyte enriched with D(31)-PA using the upsilon(as+s)(C-D)(2+3) (D(31)-PA): upsilon(as+s)(C-H)(2+3) (lipid hydrocarbon) signal. In addition, larger adipocytes were found to consist of a higher percentage of D(31)-PA of the total lipid found within the adipocyte. Following background subtraction, the upsilon(as)(C-D)(2+3) signal illuminated starved PC-3 cells cocultured with D(31)-PA-loaded adipocytes, indicating translocation of the labeled FA. This study demonstrates lipid-specific translocation between adipocytes and tumor cells and the use of FTIR microspectroscopy to characterize various biomolecular features of a single adipocyte without the requirement for cell isolation and lipid extraction.

Biomolecular profiling of metastatic prostate cancer cells in bone marrow tissue using FTIR microspectroscopy: a pilot study

Prostate cancer (CaP) cells preferentially metastasise to the bone marrow, a microenvironment that plays a substantial role in the sustenance and progression of the CaP tumour. Here we use a combination of FTIR microspectroscopy and histological stains to increase molecular specificity and probe the biochemistry of metastatic CaP cells in bone marrow tissue derived from a limited source of paraffin-embedded biopsies of different patients. This provides distinction between the following dominant metabolic processes driving the proliferation of the metastatic cells in each of these biopsies: glycerophospholipid synthesis from triacylglyceride, available from surrounding adipocytes, in specimen 1, through significantly high (p < or = 0.05) carbohydrate (8.23 +/- 1.44 cm(-1)), phosphate (6.13 +/- 1.5 cm(-1)) and lipid hydrocarbon (24.14 +/- 5.9 cm(-1)) signals compared with the organ-confined CaP control (OC CaP), together with vacuolation of cell cytoplasm; glycolipid synthesis in specimen 2, through significantly high (p < or = 0.05) carbohydrate (5.51 +/- 0.04 cm(-1)) and high lipid hydrocarbon (17.91 +/- 2.3 cm(-1)) compared with OC CaP, together with positive diastase-digested periodic acid Schiff staining in the majority of metastatic CaP cells; glycolysis in specimen 3, though significantly high (p < or = 0.05) carbohydrate (8.86 +/- 1.78 cm(-1)) and significantly lower (p < or = 0.05) lipid hydrocarbon (11.67 +/- 0.4 cm(-1)) than OC CaP, together with negative diastase-digested periodic acid Schiff staining in the majority of metastatic CaP cells. Detailed understanding of the biochemistry underpinning the proliferation of tumour cells at metastatic sites may help towards refining chemotherapeutic treatment.

Lack of Noggin Expression by Cancer Cells Is a Determinant of the Osteoblast Response in Bone Metastases

Prostate and mammary cancer bone metastases can be osteoblastic or osteolytic, but the mechanisms determining these features are unclear. Bone morphogenetic and Wnt proteins are osteoinductive molecules. Their activity is modulated by antagonists such as noggin and dickkopf-1. Differential expression analysis of bone morphogenetic and Wnt protein antagonists in human prostate and mammary cancer cell lines showed that osteolytic cell lines constitutively express in vitro noggin and dickkopf-1 and at least one of the osteolytic cytokines parathyroid hormone-related protein, colony-stimulating factor-1, and interleukin-8. In contrast, osteoinductive cell lines express neither noggin nor dickkopf-1 nor osteolytic cytokines in vitro. The noggin differential expression profile observed in vitro was confirmed in vivo in prostate cancer cell lines xenografted into bone and in clinical samples of bone metastasis. Forced noggin expression in an osteoinductive prostate cancer cell line abolished the osteoblast response induced in vivo by its intraosseous xenografts. Basal bone resorption and tumor growth kinetics were marginally affected. Lack of noggin and possibly dickkopf-1 expression by cancer cells may be a relevant mechanism contributing to the osteoblast response in bone metastases. Concomitant lack of osteolytic cytokines may be permissive of this effect. Noggin is a candidate drug for the adjuvant therapy of bone metastasis.

Conditional expression of PTEN alters the androgen responsiveness of prostate cancer cells

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is lost as a function of prostate tumor androgen dependence. While the transcriptional activity of the androgen receptor (AR) is inhibited by PTEN in androgen sensitive prostate cancer (CaP), the role of PTEN in androgen disease is unclear. We developed a system where PTEN can be conditionally re-expressed at physiologic levels into a PTEN null metastatic human CaP cell line, C4-2, and androgen responsiveness examined. PTEN induction reduces cell growth and blocks the growth effect of synthetic androgen R1881. The anti-androgen Casodex enhances the growth-inhibitory action of PTEN and this effect is independent of Akt phosphorylation. Combined PTEN induction and Casodex, result in a further decrease in prostate specific antigen promoter activity compared to PTEN but not Casodex alone. PTEN induction confers androgen independent CaP cells enhanced responsiveness to the anti-proliferative effects of anti-androgens and this action may involve non-AR mediated effects.