Non-transformed Prostate Epithelial Cells Research Articles

Enhanced Cytotoxicity from Deoxyguanosine-Enriched T-oligo in Prostate Cancer Cells

Prostate cancer represents approximately 10 percent of all cancer cases in men and accounts for more than a quarter of all cancer types. Advances in understanding the molecular mechanisms of prostate cancer progression, however, have not translated well to the clinic. Patients with metastatic and hormone-refractory disease have only palliative options for treatment, as chemotherapy seldom produces durable or complete responses, highlighting the need for novel therapeutic approaches. T-oligo, a single-stranded deoxyribonucleic acid with partial sequence homology to human telomeric DNA, has elicited cytostatic and/or cytotoxic effects in multiple cancer cell types. In contrast, normal primary cells of varying tissue types are resistant to cytotoxic actions of T-oligo, underscoring its potential utility as a novel targeted cancer therapeutic. Mechanistically, T-oligo is hypothesized to interfere with normal telomeric structure and form G-quadruplex structures, thereby inducing genomic stress in addition to aberrant upregulation of DNA damageresponse pathways. Here, we present data demonstrating the enhanced effectiveness of a deoxyguanosine-enriched sequence of T-oligo, termed (GGTT)4, which elicits robust cytotoxic effects in prostate cancer cells at lower concentrations than the most recent T-oligo sequence (5'-pGGT TAG GTG TAG GTT T 3') described to date and used for comparison in this study, while exerting no cytotoxic actions on nontransformed human prostate epithelial cells. Additionally, we provide evidence supporting the T-oligo induced activation of cJun N-terminal kinase (JNK) signaling in prostate cancer cells consistent with G-quadruplex formation, thereby significantly advancing the understanding of the T-oligo mechanism of action.

Abstract 463: MAP4K4 (HGK) plays a key role in tumor progression

Abstract MAP4K4/HGK is an STE kinase that was identified in an siRNA screen for modulators of tumor cell motility and in a five-gene signature predicting metastasis and survival in colon cancer. Interestingly, its catalytic function is required for anchorage-independent growth in 3D soft agar (but not in 2D monolayers) of 3T3 fibroblasts and rat intestinal epithelial cells. The aim of the present study was to evaluate MAP4K4 expression in a range of human tumor cell lines and further explore its role in cell motility and invasion. Using western blotting analysis, MAP4K4 protein expression was measured in a panel of approximately 50 human tumor cell lines and 3 non-transformed cell lines. Selected high-expressing MAP4K4 cell lines were used for genetic manipulation. Lentiviral shRNA inducible (TRIPZ-RFP) and stable (GIPZ-GFP) knockdowns (KD) permitted the selection of isogenic cell lines for functional studies. Novel high-throughput 96-well plate-based 3D assays were used to analyze: (i) 3D spheroid growth, (ii) tumor cell dissemination over gelatin or collagen type I and (iii) invasion into Matrigel™. A fully automated image analysis system was validated against standard microscopy-based methods and applied to these studies. Cell viability was assessed in 2D and 3D by the Cell Titer Glo assay. Pharmacodynamic biomarkers were analyzed in KD and control cells to correlate MAP4K4 expression to the activation/inhibition status of putative downstream signalling pathways. MAP4K4 protein expression levels were high to moderate in 60% of the cell lines tested (including glioma, neuroblastoma, hepatocellular, prostate, colorectal, ovarian, squamous and breast carcinomas) and undetectable in PNT2 and MCF10A non-transformed prostate and breast epithelial cells. It was also expressed in activated human endothelial cells. The SF188 pediatric glioma cell line was prioritized for further study based on its strong expression of MAP4K4 (and activated downstream signalling pathways), the ability to generate reproducible tumor spheroids and the highly invasive nature of this cancer type. Additional confirmatory studies utilized the metastatic triple-negative human breast cancer cell line MDA MB 231. Both stable and inducible KD were achieved as described above. Functional studies showed that MAP4K4 KD induced between 30-50% inhibition of cell viability, motility and/or invasion. Inhibition of MAP4K4 expression in SF188 cells also down-regulated key downstream signalling molecules such as pJNK, pSTAT3, pTAK1 and MMP2. In MDA MB 231, MAP4K4 was upregulated when cells were cultured in 3D spheroids and again, stable knockdown inhibited 3D migration by ∼ 50%. Conclusions: In the present study we provide evidence that MAP4K4 plays an important role in functions associated with tumor progression (ability to grow in 3D, migration and invasion) in two cancer types where invasion and/or metastasis contribute to their notoriously poor prognosis. 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 463. doi:1538-7445.AM2012-463

Abstract B52: Huntingtin interacting protein 1 (HIP1) mediates prostate carcinogenesis and progression through effects on signal transducers and activators of transcription (STATs)

Abstract Background: HIP1 is an adaptor protein classically involved in clathrin mediated endocytosis by acting as a scaffolding factor. Its overexpression has been linked to a multitude of epithelial cancers including prostate cancer (CaP)(1). FGFR4 stabilisation by HIP1 in non-transformed PNT1 a cells was implicated in its role in CaP(2). The molecular mechanisms that mediate the effects of HIP1 overexpression however, remain largely unknown. Methods: PNT1a- a non-transformed prostate epithelial cell line stably overexpressing HIP1 (PNT1a-HIP1) or empty vector (control) was used in anchorage independent growth, and migration assays to investigate functional effects; IIIumina bead arrays to determine gene expression; Human Phospho-Kinase Array(R&D systems) to detect site-specific phosphorylation of 46 protein kinase sites; iCys imaging for quantification of IF to determine nuclear STAT protein; rtPCR and western blots for mRNA and protein respectively. Results: Analysis of downstream effects of FGFR4 stabilisation by HIP1 using the Human Phospho-Kinase Array revealed significant differences in STAT phosphorylation. Knocking down (KD) HIP1 in PNT1a-HIP1 cells reversed these phosphorylation events. Since STATs undergo nuclear translocation to cause transcription we assessed nuclear STAT levels and observed significantly increased nuclear STATs in PNT1a-HIP1. We also observed increased nuclear STAT in cells treated with FGF2 following serum starvation indicating FGFR mediated STAT phosphorylation. To test whether JAKs acting downstream of FGFR mediated STAT phosphorylation, we used a JAK-STAT kinase inhibitor and observed reduction in phospho-STATs and nuclear STAT proteins. Cellular transformation of PNT1 a-HIP1-the ability to form colonies in soft agar, was only partially abrogated by FGFR4 KD (2) in previous studies. However, inhibitors of STAT phosphorylation completely abrogated soft agar colony formation, while also significantly reducing cell migration of the PNT1a-HIP1. Indeed, PNT1a-HIP1 were significantly more sensitive (EVIC50 > HIP1 IC50) to STAT inhibition. We tested this in DU145, a highly transformed, invasive androgen-receptor negative CaP cell line, which has constitutively high levels of STAT phosphorylation. Interestingly, HIP1 knockdown in DU145 caused a reduction in STAT phosphorylation, soft agar colony formation, and cell migration. Using unbiased gene expression arrays and stringent cut-offs (p <0.01; −0.05<log FC>0.6) to generate a list of differentially expressed genes, we observed a significant upregulation of TGFβ superfamily ligands which have been implicated to have paracrine effects on CaP growth and progression(3). To test these paracrine effects on classical androgen receptor positive cell lines, we cultured LNCaPs with conditioned media from PNT1 a-HIP1 and observed a significant increase in growth. Taken together, phospho-STATs affect TGFβ signalling which contributes to CaP progression. Conclusion: We believe HIP1 overexpression mediates its effects through constitutive activation of STATs, which contributes to cancer progression, and drug resistance through activation of autocrine and paracrine TGFβ signalling molecules. Future work will assess HIP1 as a predictor of chemoresistance and a potential role for STAT inhibitors as an adjuvant for treatment of aggressive drug resistant CaP. 1. D. S. Rao et al., J Clin Invest 110, 351 (Aug, 2002). 2. J. Wang, W. Yu, Y. Cai, C. Ren, M. M. Ittmann, Neoplasia 10, 847 (Aug, 2008). 3. A. R. Bauskin et al., Cancer Res 66, 4983 (May 15, 2006). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B52.

Matriptase is inhibited by extravascular antithrombin in epithelial cells but not in most carcinoma cells

Antithrombin, a major anticoagulant, is robustly transported into extravascular compartments where its target proteases are largely unknown. This serpin was previously detected in human milk as complexes with matriptase, a membrane-bound serine protease broadly expressed in epithelial and carcinoma cells, and under tight regulation by hepatocyte growth factor activator inhibitor (HAI)-1, a transmembrane Kunitz-type serine protease inhibitor that forms heat-sensitive complexes with active matriptase. In the current study, we detect, in addition to matriptase-HAI-1 complexes, heat-resistant matriptase complexes generated by nontransformed mammary, prostate, and epidermal epithelial cells that we show to be matriptase-antithrombin complexes. These findings suggest that in addition to HAI-1, interstitial antithrombin participates in the regulation of matriptase activity in epithelial cells. This physiological mechanism appears, however, to largely be lost in cancer cells since matriptase-antithrombin complexes were not detected in all but two of a panel of seven breast, prostate, and ovarian cancer cell lines. Using purified active matriptase, we further characterize the formation of matriptase-antithrombin complex and show that heparin can significantly potentiate the inhibitory potency of antithrombin against matriptase. Second-order rate constants for the inhibition were determined to be 3.9 × 10(3) M(-1)s(-1) in the absence of heparin and 1.2 × 10(5) M(-1)s(-1) in the presence of heparin, a 30-fold increase, consistent with the established role of heparin in activating antithrombin function. Taken together these data suggest that normal epithelial cells employ a dual mechanism involving HAI-1 and antithrombin to control matriptase and that the antithrombin-based mechanism appears lost in the majority of carcinoma cells.

Abstract 3417: Class III HDAC induces epithelial-to-mesenchymal transition in prostate cancer cells

Abstract The epithelial-to-mesenchymal transition (EMT) is a crucial program for initiating invasion and metastasis in many epithelial tumors. EMT is characterized by loss of cell adhesion and increased cell mobility; however, the mechanism underlying this transition is not fully understood. Here we identified SIRT1, a class III histone deacetylase, as a critical regulator of EMT. We show that forced expression of SIRT1 disrupts the epithelial morphology in non-transformed prostate epithelial cells, concomitant with a decrease of epithelial marker E-cadherin, and an increase of mesenchymal protein N-cadherin, and fibronection expression, indicating EMT. In contrast, silencing SIRT1 expression in metastatic prostate tumor cells restores cell to cell adhesion and induces changes in morphology characteristic of epithelial cells, concomitant with an increase of E-cadherin and a decrease of N-cadherin and fibronection expressions. In addition, we show that SIRT1 represses the activity of the E-cadherin proximal promoter with an E-box dependent mechanism, indicating a connection between SIRT1 and the zinc-finger EMT transcription factor, which needs the E-box to suppress E-cadherin transcription. Moreover, we performed ChIP assays to show that SIRT1 and ZEB1 bind to the same DNA fragment on the E-cadherin promoter, and SIRT1 occupancy in the E-cadherin promoter requires ZEB1. In addition, we show that acetylated H3K9 levels were inversely related to occupancy of SIRT1 and ZEB1. Finally, our results show that SIRT1 knockdown reduces prostate cancer cell migration and invasion. Thus, in this study we identified SIRT1 as an important regulator of EMT and demonstrated a new transcriptional mechanism regulating E-cadherin expression by the cooperation of SIRT1 with ZEB1. These findings suggest that overexpressed SIRT1 in prostate cancer cells may be targeted to reverse EMT, and SIRT1 could be a potential target in the treatment of prostate cancer progression. 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 3417. doi:10.1158/1538-7445.AM2011-3417

Aberrant Promoter CpG Methylation Is a Mechanism for Impaired PHD3 Expression in a Diverse Set of Malignant Cells

BackgroundThe prolyl-hydroxylase domain family of enzymes (PHD1-3) plays an important role in the cellular response to hypoxia by negatively regulating HIF-α proteins. Disruption of this process can lead to up-regulation of factors that promote tumorigenesis. We observed decreased basal expression of PHD3 in prostate cancer tissue and tumor cell lines representing diverse tissues of origin. Furthermore, some cancer lines displayed a failure of PHD3 mRNA induction when introduced to a hypoxic environment. This study explores the mechanism by which malignancies neither basally express PHD3 nor induce PHD3 under hypoxic conditions.Methodology/Principal FindingsUsing bisulfite sequencing and methylated DNA enrichment procedures, we identified human PHD3 promoter hypermethylation in prostate, breast, melanoma and renal carcinoma cell lines. In contrast, non-transformed human prostate and breast epithelial cell lines contained PHD3 CpG islands that were unmethylated and responded normally to hypoxia by upregulating PHD3 mRNA. Only treatment of cells lines containing PHD3 promoter hypermethylation with the demethylating drug 5-aza-2′-deoxycytidine significantly increased the expression of PHD3.Conclusions/SignificanceWe conclude that expression of PHD3 is silenced by aberrant CpG methylation of the PHD3 promoter in a subset of human carcinoma cell lines of diverse origin and that this aberrant cytosine methylation status is the mechanism by which these cancer cell lines fail to upregulate PHD3 mRNA. We further show that a loss of PHD3 expression does not correlate with an increase in HIF-1α protein levels or an increase in the transcriptional activity of HIF, suggesting that loss of PHD3 may convey a selective advantage in some cancers by affecting pathway(s) other than HIF.

Epidermal growth factor receptor transactivation is implicated in IL-6-induced proliferation and ERK1/2 activation in non-transformed prostate epithelial cells

Epidermal growth factor receptor (EGF-R) is a receptor tyrosine kinase that can be activated by molecules other than its cognate ligands. This form of crosstalk called transactivation is frequently observed in both physiological and pathological cellular responses, yet it involves various mechanisms. Using the RWPE-1 cell line as a model of non-transformed prostate epithelial progenitor cells, we observed that interleukin-6 (IL-6) is able to promote cell proliferation and ERK1/2 activation provided that EGF-R kinase activity is not impaired. Treatment with GM6001, a general matrix metalloprotease inhibitor, indicated that IL-6 activates EGF-R through cleavage and release of membrane-anchored EGF-R ligands. Several inhibitors were used to test implication of “a disintegrin and metalloprotease” ADAM10 and ADAM17. GW280264X that targets both ADAM10 and ADAM17 blocked IL-6-induced proliferation and ERK1/2 phosphorylation with same potency as GM6001. However, ADAM10 inhibitor GI254023X and ADAM17 inhibitor TAPI-2 were less efficient in inhibiting response of RWPE-1 cells to IL-6, indicating possible cooperation of ADAM17 with ADAM10 or other metalloproteases. Accordingly, our findings suggest that IL-6 stimulates shedding of EGF-R ligands and transactivation of EGF-R in normal prostate epithelial cells, which may be an important mechanism to promote cell proliferation in inflammatory prostate.

FZD4 as a Mediator of ERG Oncogene–Induced WNT Signaling and Epithelial-to-Mesenchymal Transition in Human Prostate Cancer Cells

TMPRSS2-ERG and other gene fusions involving ETS factors and genes with strong promoter elements are common in prostate cancer. Although ERG activation has been linked to invasive properties of prostate cancers, the precise mechanisms and pathways of ERG-mediated oncogenesis remain poorly understood. Here, we show that ERG knockdown in VCaP prostate cancer cells causes an activation of cell adhesion, resulting in strongly induced active beta(1)-integrin and E-cadherin expression as well as changes in WNT signaling. These observations were corroborated by data from ERG-overexpressing nontransformed prostate epithelial cells as well as gene expression data from clinical prostate cancer samples, which both indicated a link between ERG and epithelial-to-mesenchymal transition (EMT). Upregulation of several WNT pathway members was seen in ERG-positive prostate cancers, with frizzled-4 (FZD4) showing the strongest overexpression as verified by both reverse transcription-PCR and immunostaining. Both ERG knockin and knockdown modulated the levels of FZD4 expression. FZD4 silencing could mimic the ERG knockdown phenotype by inducing active beta(1)-integrin and E-cadherin expression, whereas FZD4 overexpression reversed the phenotypic effects seen with ERG knockdown. Taken together, our results provide mechanistic insights to ERG oncogenesis in prostate cancer, involving activation of WNT signaling through FZD4, leading to cancer-promoting phenotypic effects, including EMT and loss of cell adhesion.

A Comprehensive Panel of Three-Dimensional Models for Studies of Prostate Cancer Growth, Invasion and Drug Responses

Prostate epithelial cells from both normal and cancer tissues, grown in three-dimensional (3D) culture as spheroids, represent promising in vitro models for the study of normal and cancer-relevant patterns of epithelial differentiation. We have developed the most comprehensive panel of miniaturized prostate cell culture models in 3D to date (n = 29), including many non-transformed and most currently available classic prostate cancer (PrCa) cell lines. The purpose of this study was to analyze morphogenetic properties of PrCa models in 3D, to compare phenotypes, gene expression and metabolism between 2D and 3D cultures, and to evaluate their relevance for pre-clinical drug discovery, disease modeling and basic research. Primary and non-transformed prostate epithelial cells, but also several PrCa lines, formed well-differentiated round spheroids. These showed strong cell-cell contacts, epithelial polarization, a hollow lumen and were covered by a complete basal lamina (BL). Most PrCa lines, however, formed large, poorly differentiated spheroids, or aggressively invading structures. In PC-3 and PC-3M cells, well-differentiated spheroids formed, which were then spontaneously transformed into highly invasive cells. These cell lines may have previously undergone an epithelial-to-mesenchymal transition (EMT), which is temporarily suppressed in favor of epithelial maturation by signals from the extracellular matrix (ECM). The induction of lipid and steroid metabolism, epigenetic reprogramming, and ECM remodeling represents a general adaptation to 3D culture, regardless of transformation and phenotype. In contrast, PI3-Kinase, AKT, STAT/interferon and integrin signaling pathways were particularly activated in invasive cells. Specific small molecule inhibitors targeted against PI3-Kinase blocked invasive cell growth more effectively in 3D than in 2D monolayer culture, or the growth of normal cells. Our panel of cell models, spanning a wide spectrum of phenotypic plasticity, supports the investigation of different modes of cell migration and tumor morphologies, and will be useful for predictive testing of anti-cancer and anti-metastatic compounds.

1,25 dihydroxyvitamin D-mediated orchestration of anticancer, transcript-level effects in the immortalized, non-transformed prostate epithelial cell line, RWPE1

BackgroundProstate cancer is the second leading cause of cancer mortality among US men. Epidemiological evidence suggests that high vitamin D status protects men from prostate cancer and the active form of vitamin D, 1α,25 dihydroxyvitamin D3 (1,25(OH)2D) has anti-cancer effects in cultured prostate cells. Still, the molecular mechanisms and the gene targets for vitamin D-mediated prostate cancer prevention are unknown.ResultsWe examined the effect of 1,25(OH)2D (+/- 100 nM, 6, 24, 48 h) on the transcript profile of proliferating RWPE1 cells, an immortalized, non-tumorigenic prostate epithelial cell line that is growth arrested by 1,25(OH)2D (Affymetrix U133 Plus 2.0, n = 4/treatment per time and dose). Our analysis revealed many transcript level changes at a 5% false detection rate: 6 h, 1571 (61% up), 24 h, 1816 (60% up), 48 h, 3566 (38% up). 288 transcripts were regulated similarly at all time points (182 up, 80 down) and many of the promoters for these transcripts contained putative vitamin D response elements. Functional analysis by pathway or Gene Set Analysis revealed early suppression of WNT, Notch, NF-kB, and IGF1 signaling. Transcripts related to inflammation were suppressed at 6 h (e.g. IL-1 pathway) and suppression of proinflammatory pathways continued at later time points (e.g. IL-17 and IL-6 pathways). There was also evidence for induction of anti-angiogenic pathways and induction of transcripts for protection from oxidative stress or maintenance of cell redox homeostasis at 6 h.ConclusionsOur data reveal of large number of potential new, direct vitamin D target genes relevant to prostate cancer prevention. In addition, our data suggests that rather than having a single strong regulatory effect, vitamin D orchestrates a pattern of changes within prostate epithelial cells that limit or slow carcinogenesis.

Adipose Tissue–derived Mesenchymal Stem Cells Expressing Prodrug-converting Enzyme Inhibit Human Prostate Tumor Growth

The ability of human adipose tissue-derived mesenchymal stem cells (AT-MSCs), engineered to express the suicide gene cytosine deaminase::uracil phosphoribosyltransferase (CD::UPRT), to convert the relatively nontoxic 5-fluorocytosine (5-FC) into the highly toxic antitumor 5-fluorouracil (5-FU) together with their ability to track and engraft into tumors and micrometastases makes these cells an attractive tool to activate prodrugs directly within the tumor mass. In this study, we tested the feasibility and efficacy of these therapeutic cells to function as cellular vehicles of prodrug-activating enzymes in prostate cancer (PC) therapy. In in vitro migration experiments we have shown that therapeutic AT-MSCs migrated to all the prostate cell lines tested. In a pilot preclinical study, we observed that coinjections of human bone metastatic PC cells along with the transduced AT-MSCs into nude mice treated with 5-FC induced a complete tumor regression in a dose dependent manner or did not even allow the establishment of the tumor. More importantly, we also demonstrated that the therapeutic cells were effective in significantly inhibiting PC tumor growth after intravenous administration that is a key requisite for any clinical application of gene-directed enzyme prodrug therapies.

Leukocytic promotion of prostate cellular proliferation

Histological evidence of pervasive inflammatory infiltrate has been noted in both benign prostatic hyperplasia/hypertrophy (BPH) and prostate cancer (PCa). Cytokines known to attract particular leukocyte subsets are secreted from prostatic stroma consequent to aging and also from malignant prostate epithelium. Therefore, we hypothesized that leukocytes associated with either acute or chronic inflammation attracted to the prostate consequent to aging or tumorigenesis may promote the abnormal cellular proliferation associated with BPH and PCa. An in vitro system designed to mimic the human prostatic microenvironment incorporating prostatic stroma (primary and immortalized prostate stromal fibroblasts), epithelium (N15C6, BPH-1, LNCaP, and PC3 cells), and inflammatory infiltrate (HL-60 cells, HH, and Molt-3 T-lymphocytes) was developed. Modified Boyden chamber assays were used to test the ability of prostate stromal and epithelial cells to attract leukocytes and to test the effect of leukocytes on prostate cellular proliferation. Antibody arrays were used to identify leukocyte-secreted cytokines mediating prostate cellular proliferation. Leukocytic cells migrated towards both prostate stromal and epithelial cells. CD4+ T-lymphocytes promoted the proliferation of both transformed and non-transformed prostate epithelial cell lines tested, whereas CD8+ T-lymphocytes as well as dHL-60M macrophagic and dHL-60N neutrophilic cells selectively promoted the proliferation of PCa cells. The results of these studies show that inflammatory cells can be attracted to the prostate tissue microenvironment and can selectively promote the proliferation of non-transformed or transformed prostate epithelial cells, and are consistent with differential role(s) for inflammatory infiltrate in the etiologies of benign and malignant proliferative disease in the prostate.

ADAM‐mediated amphiregulin shedding and EGFR transactivation

The ectodomain shedding of epidermal growth factor receptor (EGFR) ligands, such as amphiregulin (AREG), by ADAMs (A Disintegrin And Metalloproteases) can be stimulated by G protein-coupled receptor (GPCR) agonists. Interactions between the CXCR4 GPCR and the CXCL12 chemokine have been shown to mediate gene transcription and cellular proliferation in non-transformed and transformed prostate epithelial cells, as well as motility/invasiveness in transformed cells. In this report, we investigated the ability of CXCL12 to stimulate amphiregulin ectodomain shedding in non-transformed and transformed prostate epithelial cells that respond proliferatively to sub-nanomolar levels of CXCL12 and amphiregulin. Non-transformed N15C6 and transformed PC3 prostate epithelial cells were assessed for amphiregulin shedding, ADAM activation, Src phosphorylation and EGFR activation using ELISA, immunoblot, and immunoprecipitation techniques, and for proliferation using cell counting after stimulation with CXCL12 or vehicle. The results of these studies identify CXCL12 as a novel inducer of amphiregulin ectodomain shedding and show that both basal and CXCL12-mediated amphiregulin shedding are ADAM10- and Src kinase-dependent in non-transformed N15C6 cells. In contrast, amphiregulin shedding is not amplified subsequent to stimulation with exogenous CXCL12, and is not reduced subsequent to metalloprotease- or Src kinase-inhibition, in highly aggressive PC3 prostate cancer cells. These data also show that CXCL12-mediated cellular proliferation requires EGFR transactivation in a Src- and ADAM-dependent manner in non-transformed prostate epithelial cells. However, these same mechanisms are dysfunctional in highly transformed prostate cancer cells, which secrete amphiregulin in an autocrine manner that cannot be repressed through metalloprotease- or Src kinase inhibition. These findings show that non-transformed and transformed prostate epithelial cells may employ different mechanisms to activate EGFR ligands and thereby utilize the EGFR axis to promote cellular proliferation.

Intestinal inflammation is coordinated by the metalloprotease ADAM17

Epidermal growth factor receptor (EGF-R) is a receptor tyrosine kinase that can be activated by molecules other than its cognate ligands. This form of crosstalk called transactivation is frequently observed in both physiological and pathological cellular responses, yet it involves various mechanisms. Using the RWPE-1 cell line as a model of non-transformed prostate epithelial progenitor cells, we observed that interleukin-6 (IL-6) is able to promote cell proliferation and ERK1/2 activation provided that EGF-R kinase activity is not impaired. Treatment with GM6001, a general matrix metalloprotease inhibitor, indicated that IL-6 activates EGF-R through cleavage and release of membrane-anchored EGF-R ligands. Several inhibitors were used to test implication of “a disintegrin and metalloprotease” ADAM10 and ADAM17. GW280264X that targets both ADAM10 and ADAM17 blocked IL-6-induced proliferation and ERK1/2 phosphorylation with same potency as GM6001. However, ADAM10 inhibitor GI254023X and ADAM17 inhibitor TAPI-2 were less efficient in inhibiting response of RWPE-1 cells to IL-6, indicating possible cooperation of ADAM17 with ADAM10 or other metalloproteases. Accordingly, our findings suggest that IL-6 stimulates shedding of EGF-R ligands and transactivation of EGF-R in normal prostate epithelial cells, which may be an important mechanism to promote cell proliferation in inflammatory prostate.

Redox regulation of anoikis resistance of metastatic prostate cancer cells: key role for Src and EGFR-mediated pro-survival signals

Resistance to detachment-induced apoptosis, a process commonly referred as anoikis, is emerging as a hallmark of metastatic malignancies, mainly because it can ensure anchorage-independent growth and survival during organ colonization. Besides, a sustained oxidative stress has been associated with several steps of carcinogenesis, including transformation and achievement of a motile mesenchymal phenotype. Here, we demonstrate that metastatic prostate carcinoma cells, undergoing a constitutive deregulated production of reactive oxygen species due to sustained activation of 5-lipoxygenase, lack suicidal pathways in response to lack of matrix contact. These amplified and persistent redox signals in PC3 cells leads to maintenance of Src oxidation and activation in the absence of adhesion, thereby sustaining a ligand-independent phosphorylation of epidermal growth factor receptor. This leads to chronic activation of pro-survival signals, culminating in degradation of the pro-apoptotic protein Bim, thereby promoting cell survival even in the absence of proper adhesion. Anoikis sensitivity of metastatic cells is restored with antioxidant intervention or genetic manipulation of the redox-mediated pro-survival pathway, as well as exposure to a pro-oxidant environment strongly increases anoikis resistance in non-transformed prostate epithelial cells. Hence, our results allow new insight into the aetiology of the molecular mechanisms granting anoikis resistance of metastatic cancers, opening new avenues to pharmacological intervention for antioxidant-sensitive invasive tumours.

Anti-Androgen Receptor Signaling and Prostate Cancer Inhibitory Effects of Sucrose- and Benzophenone-Compounds

Novel agents that target multiple aspects of androgen receptor (AR) signaling are desirable for chemoprevention and treatment of prostate cancer (PCa). We aimed to identify compounds isolated from medicinal herbs as such drug candidates. In the LNCaP human androgen sensitive PCa cell model, we tested five compounds purified from Lindera fruticosa Hemsley in the range of 10-50 microM for growth inhibition and AR-prostate specific antigen (PSA) suppressing potency. We determined the relationship between these activities and P53 tumor suppressor protein activation and apoptotic cleavage of PARP. We compared these compounds to the anti-androgen drug Casodex/bicalutamide to identify mechanistic novelty. Among 3 sucrose compounds, beta-D-(3,4-di-sinapoyl)fructofuranosyl-alpha-D-(6-sinapoyl)glucopyranoside decreased AR and PSA mRNA and protein levels in LNCaP cells and inhibited androgen-stimulated AR translocation from the cytosol to the nucleus. This compound also increased P53 Ser(15) phosphorylation and PARP cleavage in LNCaP cells, but required higher dosage than for suppressing AR-PSA. Interestingly, this compound did not inhibit the growth of RWPE-1 non-transformed prostate epithelial cells. The benzophenone compound 2-methoxy-3,4-(methylenedioxy)benzophenone suppressed PSA and AR in LNCaP cells without apoptosis. Our data support novel anti-AR actions of these herbal compounds distinct from Casodex and merit further investigation as drug candidates.

Prostate stromal cells produce CXCL-1, CXCL-2, CXCL-3 and IL-8 in response to epithelia-secreted IL-1

It is well accepted that tumor microenvironment is essential for tumor cells survival, cancer progression and metastasis. However, the mechanisms by which tumor cells interact with their surrounding at early stages of cancer development are largely unidentified. The aim of this study was to identify specific molecules involved in stromal-epithelial interactions that might contribute to early stages of prostate tumor formation. Here, we show that conditioned medium (CM) from immortalized non-transformed prostate epithelial cells stimulated immortalized prostate stromal cells to express cancer-related molecules. CM obtained from epithelial cells triggered stromal cells to express and secrete CXCL-1, CXCL-2, CXCL-3 and interleukin (IL)-8 chemokines. This effect was predominantly mediated by the cytokines of the IL-1 family secreted by the epithelial cells. Thus, prostate epithelial cells induced the secretion of proinflammatory and cancer-promoting chemokines by prostate stromal cells. Such interactions might contribute to prostatic inflammation and progression at early stages of prostate cancer formation.

CXCL12 Activates a Robust Transcriptional Response in Human Prostate Epithelial Cells

CXCL12 is a CXC-type chemokine that plays important roles in hematopoiesis, development, and organization of the immune system and supports the survival or growth of a variety of normal or malignant cell types. Our laboratory recently showed that CXCL12 is secreted by aging stromal fibroblast cells and is a major paracrine factor that specifically stimulates the proliferation of prostate epithelial cells. The current study shows that this CXCL12-mediated proliferative response may be either ERK-dependent or ERK-independent. Moreover, CXCL12 initiates a previously undefined and complex global transcriptional response in prostate epithelial cells. This CXCL12-mediated transcriptional response directly stimulates the expression of genes encoding proteins that are involved in the promotion of cellular proliferation and progression through the cell cycle, tumor metastasis, and cellular motility, and directly represses the transcription of genes encoding proteins involved in cell-cell adhesion and resistance to apoptosis. Thus, CXCL12 may play a major role in the etiology of benign proliferative disease in the context of an aging tissue microenvironment.

A Role for the G12 Family of Heterotrimeric G Proteins in Prostate Cancer Invasion

Many studies have suggested a role for the members of the G12 family of heterotrimeric G proteins (Galpha12 and Galpha13) in oncogenesis and tumor cell growth. However, few studies have examined G12 signaling in actual human cancers. In this study, we examined the role of G12 signaling in prostate cancer. We found that expression of the G12 proteins is significantly elevated in prostate cancer. Interestingly, expression of the activated forms of Galpha12 or Galpha13 in the PC3 and DU145 prostate cancer cell lines did not promote cancer cell growth. Instead, expression of the activated forms of Galpha12 or Galpha13 in these cell lines induced cell invasion through the activation of the RhoA family of G proteins. Furthermore, inhibition of G12 signaling by expression of the RGS domain of the p115-Rho-specific guanine nucleotide exchange factor (p115-RGS) in the PC3 and DU145 cell lines did not reduce cancer cell growth. However, inhibition of G12 signaling with p115-RGS in these cell lines blocked thrombin- and thromboxane A2-stimulated cell invasion. These observations identify the G12 family proteins as important regulators of prostate cancer invasion and suggest that these proteins may be targeted to limit invasion- and metastasis-induced prostate cancer patient mortality.

The Endocrine-Gland-Derived Vascular Endothelial Growth Factor (EG-VEGF)/Prokineticin 1 and 2 and Receptor Expression in Human Prostate: Up-Regulation of EG-VEGF/Prokineticin 1 with Malignancy

A new family of angiogenic factors named endocrine-gland-derived vascular endothelial growth factors (EG-VEGF)/prokineticins (PK) have been recently described as predominantly expressed in steroidogenic tissues. Whether the normal and malignant epithelial prostate cells and tissues express EG-VEGF/PK1 and PK2 and their receptors is still unknown. We studied the expression of EG-VEGF/PK1 and PK2 and their receptors (PK-R1 and PK-R2) in human prostate and their involvement in cancer. Using immunohistochemistry, Western blot, and RT-PCR, we determined the expression of EG-VEGF/PK1 in normal prostate (NP) and malignant prostate tissues (PCa), in epithelial cell primary cultures from normal prostate (NPEC) and malignant prostate (CPEC) and in a panel of prostate cell lines. In NPEC, CPEC, and in EPN, a nontransformed human prostate epithelial cell line, EG-VEGF/PK1, PK2, PK-R1, and PK-R2 mRNA levels were evaluated by quantitative RT-PCR. EG-VEGF/PK1 transcript was found in PCa, in CPEC, in EPN, and in LNCaP, whereas it was detected at low level in NP and in NPEC. EG-VEGF/PK1 was absent in androgen-independent PC3 and DU-145 cell lines. Immunochemistry confirmed that EG-VEGF/PK1 protein expression was restricted to hyperplastic and malignant prostate tissues, localized in the glandular epithelial cells, and progressively increased with the prostate cancer Gleason score advancement. EG-VEGF/PK1 and PK2 were weakly expressed in NPEC and EPN. On the other hand, their transcripts were highly detected in CPEC. PK-R1 and PK-R2 were found in NPEC, EPN, and CPEC. Interestingly, CPEC showed a significantly (P < 0.05) higher expression of EG-VEGF/PK1, PK2, PK-R1, and PK-R2 compared with NPEC and EPN. We demonstrated that PKs and their receptors are expressed in human prostate and that their levels increased with prostate malignancy. It may imply that EG-VEGF/PK1 could be involved in prostate carcinogenesis, probably regulating angiogenesis. Thus, the level of EG-VEGF/PK1 could be useful for prostate cancer outcome evaluation and as a target for prostate cancer treatment in the future.