Hypoxia stabilizes GAS6/Axl signaling in metastatic prostate cancer

The receptor tyrosine kinase Axl is overexpressed in a variety of cancers and is known to play a role in proliferation and invasion. Previous data from our laboratory indicate that Axl and its ligand growth arrest-specific 6 (GAS6) may play a role in establishing metastatic dormancy in the bone marrow microenvironment. In the current study, we found that Axl is highly expressed in metastatic prostate cancer cell lines PC3 and DU145 and has negligible levels of expression in a nonmetastatic cancer cell line LNCaP. Knockdown of Axl in PC3 and DU145 cells resulted in decreased expression of several mesenchymal markers including Snail, Slug, and N-cadherin, and enhanced expression of the epithelial marker E-cadherin, suggesting that Axl is involved in the epithelial-mesenchymal transition in prostate cancer cells. The Axl-knockdown PC3 and DU145 cells also displayed decreased in vitro migration and invasion. Interestingly, when PC3 and DU145 cells were treated with GAS6, Axl protein levels were downregulated. Moreover, CoCl(2), a hypoxia mimicking agent, prevented GAS6-mediated downregulation of Axl in these cell lines. Immunochemical staining of human prostate cancer tissue microarrays showed that Axl, GAS6, and hypoxia-inducible factor-1α (Hif-1α; indicator of hypoxia) were all coexpressed in prostate cancer and in bone metastases compared with normal tissues. Together, our studies indicate that Axl plays a crucial role in prostate cancer metastasis and that GAS6 regulates the expression of Axl. Importantly, in a hypoxic tumor microenvironment Axl expression is maintained leading to enhanced signaling.     

Regulation of G protein-mediated signal transduction by RGS proteins

RGS proteins form a new family of regulatory proteins of G protein signaling. They contain homologous core domains (RGS domains) of about 120 amino acids. RGS domains interact with activated Galpha subunits. Several RGS proteins have been shown biochemically to act as GTPase activating proteins (GAPs) for their interacting Galpha subunits. Other than RGS domains, RGS proteins differ significantly in size, amino acid sequences, and tissue distribution. In addition, many RGS proteins have other protein-protein interaction motifs involved in cell signaling. We have shown that p115RhoGEF, a newly identified GEF(guanine nucleotide exchange factor) for RhoGTPase, has a RGS domain at its N-terminal region and this domain acts as a specific GAP for Galpha12 and Galpha13. Furthermore, binding of activated Galpha13 to this RGS domain stimulated GEF activity of p115RhoGEF. Activated Galpha12 inhibited Galpha13-stimulated GEF activity. Thus p115RhoGEF is a direct link between heterotrimeric G protein and RhoGTPase and it functions as an effector for Galpha12 and Galpha13 in addition to acting as their GAP. We also found that RGS domain at N-terminal regions of G protein receptor kinase 2 (GRK2) specifically interacts with Galphaq/11 and inhibits Galphaq-mediated activation of PLC-beta, apparently through sequestration of activated Galphaq. However, unlike other RGS proteins, this RGS domain did not show significant GAP activity to Galphaq. These results indicate that RGS proteins have far more diverse functions than acting simply as GAPs and the characterization of function of each RGS protein is crucial to understand the G protein signaling network in cells.     

Matrigel® invasion by the prostate cancer cell lines, PC3 and DU145, and cathepsin L+B activity

Cathepsins L and B are lysosomal cysteine proteinases whose activities and cellular location are altered in many types of cancers and cancer cell lines. Cathepsins L and B play an unspecified role in cancer invasion and metastasis. The purpose of our study was to determine whether cathepsins L and B are important for the ability of two prostate cancer cell lines, PC3 and DU 145, to invade the basement membrane-like preparation, Matrigel®. Exposure of PC3 and DU145 to the irreversible cysteine proteinase inhibitor, E64, decreases the invasive ability of DU145, but not PC3. PC3 and DU145 were treated with the phorbol ester analogue, phorbol 12-myristate 13-acetate (PMA), a known tumor promoter that activates protein kinase C and contributes to the metastatic phenotype. PMA increased secreted cathepsin L+B activity and the invasive ability of PC3 and DU145; co-exposure to E64 and PMA decreased both cathepsin L+B activity and invasion. We conclude that DU145 requires cathepsin L+B activity more than PC3 for the invasion of the Matrigel®. When the amount of secreted cathepsin L+B activity is increased by PMA treatment, however, PC3 becomes dependent on cathepsin L+B for invasion. Our study demonstrates that modulation of the amount of secreted cathepsin L+B activity influences the invasive phenotype of PC3 and DU145.     

Matrigel invasion by the prostate cancer cell lines, PC3 and DU145, and cathepsin L+B activity.

Cathepsins L and B are lysosomal cysteine proteinases whose activities and cellular location are altered in many types of cancers and cancer cell lines. Cathepsins L and B play an unspecified role in cancer invasion and metastasis. The purpose of our study was to determine whether cathepsins L and B are important for the ability of two prostate cancer cell lines, PC3 and DU 145, to invade the basement membrane-like preparation, Matrigel. Exposure of PC3 and DU145 to the irreversible cysteine proteinase inhibitor, E64, decreases the invasive ability of DU145, but not PC3. PC3 and DU145 were treated with the phorbol ester analogue, phorbol 12-myristate 13-acetate (PMA), a known tumor promoter that activates protein kinase C and contributes to the metastatic phenotype. PMA increased secreted cathepsin L+B activity and the invasive ability of PC3 and DU145; co-exposure to E64 and PMA decreased both cathepsin L+B activity and invasion. We conclude that DU145 requires cathepsin L+B activity more than PC3 for the invasion of the Matrigel. When the amount of secreted cathepsin L+B activity is increased by PMA treatment, however, PC3 becomes dependent on cathepsin L+B for invasion. Our study demonstrates that modulation of the amount of secreted cathepsin L+B activity influences the invasive phenotype of PC3 and DU145.     

Matrigel® invasion by the prostate cancer cell lines,PC3 and DU145, and cathepsin L+B activity

Cathepsins L and B are lysosomal cysteine proteinases whose activities and cellular location are altered in many types of cancers and cancer cell lines. Cathepsins L and B play an unspecified role in cancer invasion and metastasis. The purpose of our study was to determine whether cathepsins L and B are important for the ability of two prostate cancer cell lines, PC3 and DU 145, to invade the basement membrane-like preparation, Matrigel®. Exposure of PC3 and DU145 to the irreversible cysteine proteinase inhibitor, E64, decreases the invasive ability of DU145, but not PC3. PC3 and DU145 were treated with the phorbol ester analogue, phorbol 12-myristate 13-acetate (PMA), a known tumor promoter that activates protein kinase C and contributes to the metastatic phenotype. PMA increased secreted cathepsin L+B activity and the invasive ability of PC3 and DU145; co-exposure to E64 and PMA decreased both cathepsin L+B activity and invasion. We conclude that DU145 requires cathepsin L+B activity more than PC3 for the invasion of the Matrigel®. When the amount of secreted cathepsin L+B activity is increased by PMA treatment, however, PC3 becomes dependent on cathepsin L+B for invasion. Our study demonstrates that modulation of the amount of secreted cathepsin L+B activity influences the invasive phenotype of PC3 and DU145.     

Protein kinase Cdelta signaling downstream of the EGF receptor mediates migration and invasiveness of prostate cancer cells

Tumor progression to the invasive phenotype occurs secondary to upregulated signaling from growth factor receptors that drive key cellular responses like proliferation, migration, and invasion. We hypothesized that Protein kinase Cdelta (PKCdelta)-mediated transcellular contractility is required for migration and invasion of prostate tumor cells. Two invasive human prostate cancer cell lines, DU145 cells overexpressing wildtype human EGFR (DU145WT) and PC3 cells, were studied. PKCdelta is overexpressed in these cells relative to normal prostate epithelial cells, and is activated downstream of EGFR leading to cell motility via modulation of myosin light chain activity. Abrogation of PKCdelta using Rottlerin and specific siRNA significantly decreased migration and invasion of both cell lines in vitro. Both PKCdelta and phosphorylated PKCdelta protein levels were higher in human prostate cancer tissue relative to normal donor prostate as assessed by Western blotting and immunohistochemistry. Thus, we conclude that PKCdelta inhibition can limit migration and invasion of prostate cancer cells.     

Mechanisms for reversible regulation between G13 and Rho exchange factors.

The heterotrimeric G proteins, G(12) and G(13), mediate signaling between G protein-coupled receptors and the monomeric GTPase, RhoA. One pathway for this modulation is direct stimulation by Galpha(13) of p115 RhoGEF, an exchange factor for RhoA. The GTPase activity of both Galpha(12) and Galpha(13) is increased by the N terminus of p115 Rho guanine nucleotide exchange factor (GEF). This region has weak homology to the RGS box sequence of the classic regulators of G protein signaling (RGS), which act as GTPase-activating proteins (GAP) for G(i) and G(q). Here, the RGS region of p115 RhoGEF is shown to be distinctly different in that sequences flanking the predicted "RGS box" region are required for both stable expression and GAP activity. Deletions in the N terminus of the protein eliminate GAP activity but retain substantial binding to Galpha(13) and activation of RhoA exchange activity by Galpha(13). In contrast, GTRAP48, a homolog of p115 RhoGEF, bound to Galpha(13) but was not stimulated by the alpha subunit and had very poor GAP activity. Besides binding to the N-terminal RGS region, Galpha(13) also bound to a truncated protein consisting only of the Dbl homology (DH) and pleckstrin homology (PH) domains. However, Galpha(13) did not stimulate the exchange activity of this truncated protein. A chimeric protein, which contained the RGS region of GTRAP48 in place of the endogenous N terminus of p115 RhoGEF, was activated by Galpha(13). These results suggest a mechanism for activation of the nucleotide exchange activity of p115 RhoGEF that involves direct and coordinate interaction of Galpha(13) to both its RGS and DH domains.     

Anti-proliferative effects of evodiamine on human prostate cancer cell lines DU145 and PC3

Prostate carcinoma is one of the most common malignant tumors and has become a more common cancer in men. Previous studies demonstrated that evodiamine (EVO) exhibited anti-tumor activities on several cancers, but its effects on androgen-independent prostate cancer are unclear. In the present study, the action mechanisms of EVO on the growth of androgen-independent prostate cancer cells (DU145 and PC3 cells) were explored. EVO dramatically inhibited the growth and elevated cytotoxicity of DU145 and PC3 cells. The flow cytometric analysis of EVO-treated cells indicated a block of G2/M phase and an elevated level of DNA fragmentation. The G2/M arrest was accompanied by elevated Cdc2 kinase activity, an increase in expression of cyclin B1 and phosphorylated Cdc2 (Thr 161), and a decrease in expression of phosphorylated Cdc2 (Tyr 15), Myt-1, and interphase Cdc25C. TUNEL examination showed that EVO-induced apoptosis was observed at 72 h. EVO elevated the activities of caspase 3, 8, and 9 in DU145 cells, while in PC3 cells only the activities of caspase 3 and 9 were elevated. EVO also triggered the processing of caspase 3 and 9 in both DU145 and PC3 cells. We demonstrate that roscovitine treatment result in the reversion of G2/M arrest in response to EVO in both DU145 and PC3. However, inhibitory effect of roscovitine on EVO-induced apoptosis could only be observed in DU145 rather than PC3. In DU145, G2/M arrest might be a signal for initiation of EVO-triggered apoptosis. Whereas EVO-triggered PC3 apoptosis might be independent of G2/M arrest. These results suggested that EVO inhibited the growth of prostate cancer cell lines, DU145 and PC3, through an accumulation at G2/M phase and an induction of apoptosis.     

Selective amino acid restriction differentially affects the motility and directionality of DU145 and PC3 prostate cancer cells

We previously found that selective restriction of amino acids inhibits invasion of two androgen-independent human prostate cancer cell lines, DU145 and PC3. Here we show that the restriction of tyrosine (Tyr) and phenylalanine (Phe), methionine (Met) or glutamine (Gln) modulates the activity of G proteins and affects the balance between two actin-binding proteins, cofilin and profilin, in these two cell lines. Selective amino acid restriction differentially reduces G protein binding to GTP in DU145 cells. Tyr/Phe deprivation reduces the amount of Rho-GTP and Rac1-GTP. Met deprivation reduces the amount of Ras-GTP and Rho-GTP, and Gln deprivation decreases Ras-GTP, Rac-GTP, and Cdc42-GTP. Restriction of these amino acids increases the amount of profilin, cofilin and phosphorylation of cofilin-Ser(3). Increased PAK1 expression and phosphorylation of PAK1-Thr(423), and Ser(199/204) are consistent with the increased phosphorylation of LIMK1-Thr(508). In PC3 cells, Tyr/Phe or Gln deprivation reduces the amount of Ras-GTP, and all of the examined amino acid restrictions reduce the amount of profilin. PAK1, LIMK1 and cofilin are not significantly altered. These data reveal that specific amino acid deprivation differentially affects actin dynamics in DU145 and PC3. Modulation on Rho, Rac, PAK1, and LIMK1 likely alter the balance between cofilin and profilin in DU145 cells. In contrast, profilin is inhibited in PC3 cells. These effects modulate directionality and motility to inhibit invasion.     

Targeted inhibition of the epidermal growth factor receptor-tyrosine kinase by ZD1839 ('Iressa') induces cell-cycle arrest and inhibits proliferation in prostate cancer cells

The epidermal growth factor (EGF) plays a role in the development of prostate cancer, which becomes essential after androgen resistance has emerged. The EGF receptor (EGFR) is therefore a potential target for anticancer therapy. We evaluated the effects of ZD1839 ('Iressa'), an orally active EGFR-tyrosine kinase inhibitor, on prostate cancer cell lines. The effects of ZD1839 were evaluated on the anchorage dependent and independent growth of androgen-responsive (LNCaP) and androgen-independent (DU145 and PC3) cells by a cell proliferation assay, cell counting, and soft agar analysis. Flow cytometric analysis and Western blotting were used to assess the effects on the cell-cycle and on protein expression levels, respectively. ZD1839 caused a dose- and time-dependent growth inhibition in all three cell lines. A dose-dependent supra-additive increase in growth inhibition was observed when ZD1839 was combined with the antiandrogen flutamide or ionizing radiation (IR). The antiproliferative effect of ZD1839 was mainly cytostatic and associated with a block in the G(0)/G(1) phase of the cell-cycle, evident after about 12 h of treatment. In the DU145 cells this block was associated with an increase in expression of the CDK inhibitor p27(Kip1), both in the cytoplasmic and nuclear fractions. The increase in p27(Kip1) was not evident in the LNCaP and PC3 cells. No changes were observed in the expression of cyclin D1 protein. These results demonstrate the antiproliferative effects of ZD1839 on the growth of prostate cancer cells and suggest that inhibition of EGFR-associated signal transduction pathway might represent a promising novel therapeutic strategy for the treatment of prostate cancer.     

p53-induced gene 3 mediates cell death induced by glutathione peroxidase 3

Expression of glutathione peroxidase 3 (GPx3) is down-regulated in a variety of human malignancies. Both methylation and deletion of GPx3 gene underlie the alterations of GPx3 expression in prostate cancer. A strong correlation between the down-regulation of GPx3 expression and progression of prostate cancer and the suppression of prostate cancer xenografts in SCID mice by forced expression of GPx3 suggests a tumor suppression role of GPx3 in prostate cancer. However, the mechanism of GPx3-mediated tumor suppression remains unclear. In this report, GPx3 was found to interact directly with p53-induced gene 3 (PIG3). Forced overexpression of GPx3 in prostate cancer cell lines DU145 and PC3 as well as immortalized prostate epithelial cells RWPE-1 increased apoptotic cell death. Expression of GPx3(x73c), a peroxidase-negative OPAL codon mutant, in DU145 and PC3 cells also increased cell death. The induced expression of GPx3 in DU145 and PC3 cells resulted in an increase in reactive oxygen species and caspase-3 activity. These activities were abrogated by either knocking down PIG3 or mutating the PIG3 binding motif in GPx3 or binding interference from a peptide corresponding to PIG3 binding motif in GPx3. In addition, UV-treated RWPE-1 cells underwent apoptotic death, which was partially prevented by knocking down GPx3 or PIG3, suggesting that GPx3-PIG3 signaling is critical for UV-induced apoptosis. Taken together, these results reveal a novel signaling pathway of GPx3-PIG3 in the regulation of cell death in prostate cancer.     

miR-105 Inhibits Prostate Tumour Growth by Suppressing CDK6 Levels

A significant role for micro (mi)RNA in the regulation of gene expression in tumours has been recently established. In order to further understand how miRNA expression may contribute to prostate tumour growth and progression, we evaluated expression of miRNA in two invasive prostate tumour lines, PC3 and DU145, and compared it to that in normal prostate epithelial cells. Although a number of miRNAs were differentially expressed, we focused our analysis on miR-105, a novel miRNA not previously linked to prostate cancer. miR-105 levels were significantly decreased in both tumour cell lines in comparison to normal prostate epithelial cells. To determine its potential role in prostate cancer pathogenesis, we overexpressed miR-105 in both PC3 and DU145 cells and determined its effect on various tumourigenic properties. miR-105 overexpression inhibited tumour cell proliferation, tumour growth in anchorage-independent three-dimensional conditions and tumour invasion in vitro, properties of highly aggressive tumour cells. Of potential clinical significance, miR-105 overexpression inhibited tumour growth in vivo in xenograft models using these cell lines. We further identified CDK6 as a putative target of miR-105 which is likely a main contributor to the inhibition of tumour cell growth observed in our assays. Our results suggest that miR-105 inhibits tumour cell proliferation and hence may represent a novel therapeutically relevant cellular target to inhibit tumour growth or a marker of aggressive tumours in prostate cancer patients.     

Proteomic exploration of the impacts of pomegranate fruit juice on the global gene expression of prostate cancer cell

Prostate cancer has been known to be the second highest cause of death in cancer among men. Pomegranate is rich in polyphenols with the potent antioxidant activity and inhibits cell proliferation, invasion, and promotes apoptosis in various cancer cells. This study demonstrated that pomegranate fruit juice could effectively hinder the proliferation of human prostate cancer DU145 cell. The results of apoptotic analyses implicated that fruit juice might trigger the apoptosis in DU145 cells via death receptor signaling and mitochondrial damage pathway. In this study, we exploited 2DE‐based proteomics to compare nine pairs of the proteome maps collected from untreated and treated DU145 cells to identify the differentially expressed proteins. Comparative proteomics indicated that 11 proteins were deregulated in affected DU145 cells with three upregulated and eight downregulated proteins. These dys‐regulated proteins participated in cytoskeletal functions, antiapoptosis, proteasome activity, NF‐κB signaling, cancer cell proliferation, invasion, and angiogenesis. Western immunoblotting were implemented to confirm the deregulated proteins and the downstream signaling proteins. The analytical results of this study help to provide insight into the molecular mechanism of inducing prostate cancer cell apoptosis by pomegranate fruit juice and to develop a novel mechanism‐based chemopreventive strategy for prostate cancer.     

Thrombin and lysophosphatidic acid receptors utilize distinct rhoGEFs in prostate cancer cells

Thrombin and lysophosphatidic acid (LPA) receptors play important roles in vascular biology, development, and cancer. These receptors activate rho via G(12/13) family heterotrimeric G proteins, which are known to directly activate three distinct rho guanine nucleotide exchange factors (rhoGEFs) that contain a regulator of G protein signaling (RGS) domain (RGS-rhoGEFs). However, it is not known which, if any, of these RGS-rhoGEFs (LARG (leukemia-associated rhoGEF), p115rhoGEF, or PDZrhoGEF) plays a role in G protein-coupled receptor-stimulated rho signaling. Using oligonucleotide small interfering RNAs that suppress specific RGS-rhoGEF expression, we show that thrombin receptor stimulation of rho is primarily mediated by LARG in HEK293T and PC-3 prostate cancer cell lines. In contrast, the LPA-stimulated rho response in PC-3 cells is dependent on PDZrhoGEF expression. Suppression of p115rhoGEF had no effect. Thus different rhoGEFs (LARG and PDZrhoGEF) mediate downstream rho signaling by the thrombin and LPA receptors.     

Focal adhesion kinase controls aggressive phenotype of androgen-independent prostate cancer

Overexpression of focal adhesion kinase (FAK) has been well correlated with tumor development and/or the maintenance of tumor phenotype. In addition, inappropriate activation of the extracellular regulated kinase (ERK) signaling pathway is common to many human cancers. In the present study, we investigated the interplay between FAK and ERK in androgen-independent prostate cancer cells (PC3 and DU145 cells). We observed that suppression of FAK expression using small interfering RNA-mediated knockdown decreased the clonogenic activity, whereas overexpression of FAK increased it. We also observed that detachment of PC3 and DU145 cells from their substrate induced tyrosine phosphorylation of FAK. ERK knockdown diminished FAK protein levels and tyrosine phosphorylation of FAK as well as FAK promoter-reporter activity. We also tested the effect of MEK inhibitors and small interfering RNA-mediated knockdown of ERK1 and/or ERK2 on cell proliferation, invasiveness, and growth in soft agar of PC3 and DU145 cells. Inhibition of ERK signaling grossly impaired clonogenicity as well as invasion through Matrigel. However, inhibition of ERK signaling resulted in only a modest inhibition of 3H-thymidine incorporation and no effect on overall viability of the cells or increased sensitivity to anoikis. Taken together, these data show, for the first time, a requirement for FAK in aggressive phenotype of prostate cancer cells; reveal interdependence of FAK and ERK1/2 for clonogenic and invasive activity of androgen-independent prostate cancer cells; suggest a role for ERK regulation of FAK in substrate-dependent survival; and show for the first time, in any cell type, the regulation of FAK expression by ERK signaling pathway.     

Human cytomegalovirus-encoded G protein-coupled receptor US28 mediates smooth muscle cell migration through Galpha12

Coupling of G proteins to ligand-engaged chemokine receptors is the paramount event in G-protein-coupled receptor signal transduction. Previously, we have demonstrated that the human cytomegalovirus-encoded chemokine receptor US28 mediates human vascular smooth muscle cell (SMC) migration in response to either RANTES or monocyte chemoattractant protein 1. In this report, we identify the G proteins that couple with US28 to promote vascular SMC migration and identify other signaling molecules that play critical roles in this process. US28-mediated cellular migration was enhanced with the expression of the G-protein subunits Galpha12 and Galpha13, suggesting that US28 may functionally couple to these G proteins. In correlation with this observation, US28 was able to activate RhoA, a downstream effector of Galpha12 and Galpha13 in cell types with these G proteins but not in those without them and activation of RhoA was dependent on US28 stimulation with RANTES. In addition, inactivation of RhoA or the RhoA-associated kinase p160ROCK with a dominant-negative mutant of RhoA or the small molecule inhibitor Y27632, respectively, abrogated US28-induced SMC migration. The data presented here suggest that US28 functionally signals through Galpha12 family G proteins and RhoA in a ligand-dependent manner and these signaling molecules are important for the ability of US28 to induce cellular migration.     

Mitostatin is down-regulated in human prostate cancer and suppresses the invasive phenotype of prostate cancer cells

MITOSTATIN, a novel putative tumor suppressor gene induced by decorin overexpression, is expressed in most normal human tissues but is markedly down-regulated in advanced stages of mammary and bladder carcinomas. Mitostatin negatively affects cell growth, induces cell death and regulates the expression and activation levels of Hsp27. In this study, we demonstrated that ectopic expression of Mitostatin in PC3, DU145, and LNCaP prostate cancer cells not only induced a significant reduction in cell growth, but also inhibited migration and invasion. Moreover, Mitostatin inhibited colony formation in soft-agar of PC3 and LNCaP cells as well as tumorigenicity of LNCaP cells in nude mice. Conversely, targeting endogenous Mitostatin by siRNA and anti-sense strategies in PC3 and DU145 prostate cancer cells enhanced the malignant phenotype in both cell lines. In agreement of these anti-oncogenic roles, we discovered that Mitostatin was absent in ∼35% (n = 124) of prostate tumor samples and its overall reduction was associated with advanced cancer stages. Collectively, our findings indicate that MITOSTATIN may acts as a tumor suppressor gene in prostate cancer and provide a novel cellular and molecular mechanism to be further exploited and deciphered in our understanding of prostate cancer progression.