Promotes Prostate Cancer Cell Invasion Research Articles

TM4SF3 and AR: A Nuclear Complex that Stabilizes Both Proteins

Transmembrane 4 superfamily 3 (TM4SF3) was identified as a novel androgen-regulated gene in prostate cancer (PCa) cells. Our data demonstrate that TM4SF3 exhibits androgen-induced repression of the mRNA but up-regulation of the protein. The androgen positive effect on the TM4SF3 protein is of significant interest in view of the procancer functions of both androgens and tetraspanin proteins. Androgen positively regulates TM4SF3 protein stability by inhibiting its proteasome-dependent degradation. This androgen stabilization of TM4SF3 is involved in promoting PCa cell invasion and migration of both androgen-dependent and androgen-independent PCa cells. Although confirming androgen up-regulation of the TM4SF3 protein, we observed that TM4SF3 is localized not only to the membrane, but also, surprisingly, the nuclei of PCa cells. This novel nuclear localization of TM4SF3 depends on androgen-induced nuclear localization of androgen receptor (AR) in both androgen-dependent and androgen-independent PCa cell lines. TM4SF3 interacts with AR both in PCa cell types and in vitro, strongly suggesting a direct interaction. This direct interaction is required for the stabilization of not only TM4SF3, but also remarkably AR, because down-regulation of TM4SF3 resulted in reduced AR protein levels. As expected of an important AR regulator, TM4SF3 regulates androgen-dependent gene expression in and proliferation of PCa cells. Importantly, a direct correlation between AR and TM4SF3 protein levels and nuclear colocalization were also observed in prostate tumors, strongly suggesting that the mutual stabilization resulting from the AR-TM4SF3 interaction is found in tumors and that this interaction is important in PCa biology.

S1P promotes breast cancer progression by angiogenesis and lymphangiogenesis.

S1P promotes breast cancer progression by angiogenesis and lymphangiogenesis.

PAX2 promoted prostate cancer cell invasion through transcriptional regulation of HGF in an in vitro model

Elucidating the mechanism of prostate cancer cell invasion may lead to the identification of novel therapeutic strategies for its treatment. Paired box 2 (PAX2) and hepatocyte growth factor (HGF) proteins are promoters of prostate cancer cell invasion. We found that PAX2 protein activated the HGF gene promoter through histone H3 acetylation and upregulated HGF gene expression. Deletion analysis revealed that the region from −637 to −314 of the HGF gene was indispensable for HGF promoter activation by PAX2. This region contains consensus PAX2 binding sequences and mutations of the sequences attenuated HGF promoter activation. Using an in vitro invasion model, we found that PAX2 and HGF promoted prostate cancer cell invasion in the same pathway. Knockdown of HGF expression attenuated the cells' invasive capacity. Moreover, in tissue samples of human prostate cancers, HGF and PAX2 expression levels were positively correlated. These results suggested that upregulation of HGF gene expression by PAX2 enhanced the invasive properties of prostate cancer cells. The PAX2/HGF pathway in prostate cancer cells may be a novel therapeutic target in prostate cancer patients.

P2Y2 Receptor and EGFR Cooperate to Promote Prostate Cancer Cell Invasion via ERK1/2 Pathway.

As one member of G protein-coupled P2Y receptors, P2Y2 receptor can be equally activated by extracellular ATP and UTP. Our previous studies have proved that activation of P2Y2 receptor by extracellular ATP could promote prostate cancer cell invasion and metastasis in vitro and in vivo via regulating the expressions of some epithelial-mesenchymal transition/invasion-related genes (including IL-8, E-cadherin, Snail and Claudin-1), and the most significant change in expression of IL-8 was observed after P2Y2 receptor activation. However, the signaling pathway downstream of P2Y2 receptor and the role of IL-8 in P2Y2-mediated prostate cancer cell invasion remain unclear. Here, we found that extracellular ATP/UTP induced activation of EGFR and ERK1/2. After knockdown of P2Y2 receptor, the ATP -stimulated phosphorylation of EGFR and ERK1/2 was significantly suppressed. Further experiments showed that inactivation of EGFR and ERK1/2 attenuated ATP-induced invasion and migration, and suppressed ATP-mediated IL-8 production. In addition, knockdown of IL-8 inhibited ATP-mediated invasion and migration of prostate cancer cells. These findings suggest that P2Y2 receptor and EGFR cooperate to upregulate IL-8 production via ERK1/2 pathway, thereby promoting prostate cancer cell invasion and migration. Thus blocking of the P2Y2-EGFR-ERK1/2 pathway may provide effective therapeutic interventions for prostate cancer.

The endogenous cell-fate factor dachshund restrains prostate epithelial cell migration via repression of cytokine secretion via a cxcl signaling module.

Prostate cancer is the second leading form of cancer-related death in men. In a subset of prostate cancer patients, increased chemokine signaling IL8 and IL6 correlates with castrate-resistant prostate cancer (CRPC). IL8 and IL6 are produced by prostate epithelial cells and promote prostate cancer cell invasion; however, the mechanisms restraining prostate epithelial cell cytokine secretion are poorly understood. Herein, the cell-fate determinant factor DACH1 inhibited CRPC tumor growth in mice. Using Dach1(fl/fl)/Probasin-Cre bitransgenic mice, we show IL8 and IL6 secretion was altered by approximately 1,000-fold by endogenous Dach1. Endogenous Dach1 is shown to serve as a key endogenous restraint to prostate epithelial cell growth and restrains migration via CXCL signaling. DACH1 inhibited expression, transcription, and secretion of the CXCL genes (IL8 and IL6) by binding to their promoter regulatory regions in chromatin. DACH1 is thus a newly defined determinant of benign and malignant prostate epithelium cellular growth, migration, and cytokine abundance in vivo.

OTUB1 de-ubiquitinating enzyme promotes prostate cancer cell invasion in vitro and tumorigenesis in vivo.

BackgroundUbiquitination is a highly dynamic and reversible process with a central role in cell homeostasis. Deregulation of several deubiquitinating enzymes has been linked to tumor development but their specific role in prostate cancer progression remains unexplored.MethodsRNAi screening was used to investigate the role of the ovarian tumor proteases (OTU) family of deubiquitinating enzymes on the proliferation and invasion capacity of prostate cancer cells. RhoA activity was measured in relation with OTUB1 effects on prostate cancer cell invasion. Tumor xenograft mouse model with stable OTUB1 knockdown was used to investigate OTUB1 influence in tumor growth.ResultsOur RNAi screening identified OTUB1 as an important regulator of prostate cancer cell invasion through the modulation of RhoA activation. The effect of OTUB1 on RhoA activation is important for androgen-induced repression of p53 expression in prostate cancer cells. In localized prostate cancer tumors OTUB1 was found overexpressed as compared to normal prostatic epithelial cells. Prostate cancer xenografts expressing reduced levels of OTUB1 exhibit reduced tumor growth and reduced metastatic dissemination in vivo.ConclusionsOTUB1 mediates prostate cancer cell invasion through RhoA activation and promotes tumorigenesis in vivo. Our results suggest that drugs targeting the catalytic activity of OTUB1 could potentially be used as therapeutics for metastatic prostate cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-014-0280-2) contains supplementary material, which is available to authorized users.

Exogenous fatty acid binding protein 4 promotes human prostate cancer cell progression.

Epidemiologic studies have found that obesity is associated with malignant grade and mortality in prostate cancer. Several adipokines have been implicated as putative mediating factors between obesity and prostate cancer. Fatty acid binding protein 4 (FABP4), a member of the cytoplasmic fatty acid binding protein multigene family, was recently identified as a novel adipokine. Although FABP4 is released from adipocytes and mean circulating concentrations of FABP4 are linked with obesity, effects of exogenous FABP4 on prostate cancer progression are unclear. In this study, we examined the effects of exogenous FABP4 on human prostate cancer cell progression. FABP4 treatment promoted serum-induced prostate cancer cell invasion in vitro. Furthermore, oleic acid promoted prostate cancer cell invasion only if FABP4 was present in the medium. These promoting effects were reduced by FABP4 inhibitor, which inhibits FABP4 binding to fatty acids. Immunostaining for FABP4 showed that exogenous FABP4 was taken up into DU145 cells in three-dimensional culture. In mice, treatment with FABP4 inhibitor reduced the subcutaneous growth and lung metastasis of prostate cancer cells. Immunohistochemical analysis showed that the number of apoptotic cells, positive for cleaved caspase-3 and cleaved PARP, was increased in subcutaneous tumors of FABP4 inhibitor-treated mice, as compared with control mice. These results suggest that exogenous FABP4 might promote human prostate cancer cell progression by binding with fatty acids. Additionally, exogenous FABP4 activated the PI3K/Akt pathway, independently of binding to fatty acids. Thus, FABP4 might be a key molecule to understand the mechanisms underlying the obesity-prostate cancer progression link.

Eotaxin-1 promotes prostate cancer cell invasion via activation of the CCR3-ERK pathway and upregulation of MMP-3 expression

Chemokines have been reported to play crucial roles in tumor progression. Eotaxin-1 (CCL11), a member of the CC chemokine family, is elevated in many types of human cancer. Here, to reveal the molecular mechanisms of eotaxin-1 in prostate cancer cell invasion, the expression of eotaxin-1 receptors [CC chemokine receptor (CCR)2, CCR3 and CCR5] were silenced by small interfering RNA (siRNA). The ERK pathway was inhibited by the specific MEK inhibitor U0126. The role of eotaxin-1 and the CCR3-ERK pathway in prostate cancer cell invasion was assessed by invasion and migration assays. MMP-3 expression was detected by real-time PCR and ELISA assay. The results demonstrated that eotaxin-1 promoted the invasion and migration of DU-145 cells, and increased ERK1/2 activation and MMP-3 expression. Knockdown of CCR3 inhibited the invasion and migration of prostate cancer cells, and attenuated the eotaxin-1-induced ERK1/2 activation and MMP-3 expression. Furthermore, inactivation of the ERK pathway suppressed the eotaxin‑1-promoted invasion and migration, and decreased MMP-3 expression in the prostate cancer cells. Together, the present study suggests that eotaxin-1 increases MMP-3 expression via the CCR3-ERK pathway, thereby promoting prostate cancer cell invasion and migration. Thus, therapies that block eotaxin-1 and CCR3 may be effective interventions for prostate cancer.

Copper Modulates Zinc Metalloproteinase-Dependent Ectodomain Shedding of Key Signaling and Adhesion Proteins and Promotes the Invasion of Prostate Cancer Epithelial Cells

A disintegrin and metalloproteinases (ADAMs) and matrix metalloproteinases (MMPs) are zinc metalloproteinases (ZMPs) that catalyze the "ectodomain shedding" of a range of cell surface proteins including signaling and adhesion molecules. These "sheddases" are associated with the invasion and metastasis of a range of cancers. Increased serum and tumor tissue levels of copper are also observed in several cancers, although little is known about how the metal might promote disease progression at the molecular level. In the current study, we investigated whether copper might regulate the ectodomain shedding of two key cell surface proteins implicated in the invasion and metastasis of prostate cancer, the Notch ligand Jagged1 and the adhesion molecule E-cadherin, and whether the metal was able to influence the invasion of the prostate cancer epithelial cell line PC3. Physiological copper concentrations stimulated the ZMP-mediated proteolysis of Jagged1 and E-cadherin in cell culture models, whereas other divalent metals had no effect. Copper-mediated Jagged1 proteolysis was also observed following the pretreatment of cells with cycloheximide and in a cell-free membrane system, indicating a posttranslational mechanism of sheddase activation. Finally, the concentrations of copper that stimulated ZMP-mediated protein shedding also enhanced PC3 invasion; an effect that could be negated using a sheddase inhibitor or copper chelators. Collectively, these data implicate copper as an important factor in promoting prostate cancer cell invasion and indicate that the selective posttranslational activation of ZMP-mediated protein shedding might play a role in this process.

Abstract 5330: Androgen independent promotion of prostate cancer cell invasion by HOXB13-mediated zinc regulation

Abstract A healthy prostate maintains the high level of zinc while intracellular zinc is diminished during prostate cancer (PCa) developments. However, down-regulation of intracellular zinc in PCa are not fully understood, particularly in development of hormone refractory PCa (HRPC). Our previous studies showed that HOXB13 was overexpressed in HRPC. In our attempt to profile androgen-independent HOXB13 target genes in LNCaP PCa cells by DNA microarray, most strikingly regulated gene was ZnTs, a group of zinc output transporters. HOXB13 drove PCa cells to decrease intracellular zinc concentration. In the absence of androgen, HOXB13 dramatically promoted NF-κB-mediated signaling by reduction of inhibitor of NF-κB alpha (IκBα) and correspondingly stimulated nuclear translocation of RelA/p65. HOXB13-mediated NF-κB activation was nearly eliminated by suppression of ZnT4. At the same time, prostate tumors strongly indicated inverse correlation of HOXB13 and IκBα expression. Correspondingly, HOXB13 stimulated an invasive property of LNCaP cells, which can be negated by suppression of ZnT4 in an androgen-free environment. Taken together, these results demonstrate that HOXB13 promoted PCa cell invasion in an androgen-deprived condition by decreasing intracellular zinc concentration to further stimulate NF-κB-mediated transactivation, indicating that HOXB13 overexpression contributes to the malignant biological behavior of HRPC. 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 5330. doi:1538-7445.AM2012-5330

PKD2 and PKD3 Promote Prostate Cancer Cell Invasion via uPA by Shifting Balance Between NF-κB and HDAC1

Although protein kinase D3 (PKD3) has been shown to contribute to prostate cancer cell growth and survival, the role of PKD in prostate cancer cell motility remains unclear. Here, we show that PKD2 and PKD3 promote nuclear factor kappa B (NF-κB) signaling and urokinase-type plasminogen activator (uPA) expression/activation, which are crucial for prostate cancer cell invasion. Silencing of endogenous PKD2 and/or PKD3 markedly decreased prostate cancer cell migration and invasion, reduced uPA and uPA receptor (uPAR) expression and increased plasminogen activator inhibitor-2 (PAI-2) expression. These results were further substantiated by the finding that PKD2 and PKD3 promoted the activity of uPA and matrix metalloproteinase 9 (MMP9). Furthermore, depletion of PKD2 and/or PKD3 decreased the level of binding of the p65 subunit of NF-κB to the promoter of the gene encoding uPA (PLAU), suppressing transcriptional activation of uPA. Endogenous PKD2 and PKD3 interacted with inhibitor of NF-κB (IκB) kinase β (IKKβ); PKD2 mainly regulated the phosphorylated IKK (pIKK)-phosphorylated IκB (pIκB)-IκB degradation cascade, p65 nuclear translocation, and phosphorylation of Ser276 on p65, whereas PKD3 was responsible for the phosphorylation of Ser536 on p65. Conversely, inhibition of uPA transactivation by PKD3 silencing was rescued by constitutive Ser536 p65 phosphorylation, and reduced tumor cell invasion resulting from PKD2 or PKD3 silencing was rescued by ectopic expression of p65. Interestingly, PKD3 interacted with histone deacetylase 1 (HDAC1), suppressing HDAC1 expression and decreasing its binding to the uPA promoter. Moreover, depletion of HDAC1 resulted in recovery of uPA transactivation in PKD3-knockdown cells. Taken together, these data suggest that PKD2 and PKD3 coordinate to promote prostate cancer cell invasion through p65 NF-κB- and HDAC1-mediated expression and activation of uPA.

Antibody Microarray Analysis of Signaling Networks Regulated by Cxcl13 and Cxcr5 in Prostate Cancer

Advanced prostate cancer (PCa) often spreads to distant organs, leading to increased morbidity and mortality. It is now well established that chemokines and their cognate receptors play a crucial role in the multi-step process of metastasis. We have previously identified CXCR5 to be highly expressed by PCa tissues and cell lines and its specific ligand, CXCL13, is significantly elevated in the serum of patients with PCa and differentiated PCa cases with other benign prostatic diseases. CXCR5:CXCL13 interactions promote PCa cell invasion, migration, and differential matrix metalloproteinase (MMP) expression. Thus, it is important to understand the molecular and cellular processes that mediate these events. In this study, we quantified changes in apoptosis, cell cycle, and cytoskeleton rearrangement biological pathways from CXCL13-treated hormone refractory PCa cell line (PC3) to better elucidate the signaling pathways activated by CXCL13:CXCR5 interaction. Using antibody arrays that displayed 343 different protein- and phosphorylation-specific antibodies, regulatory networks that control cancer progression signaling cascades were identified. Three regulatory networks were dramatically induced by CXCL13: Akt1/2-cyclin-dependent kinases (Cdk1/2)-Cdk inhibitor 1B (CDKN1B), Integrinβ3-focal adhesion kinase (Fak)/Src-Paxillin(PXN), and Akt-Jun-cAMP response-element binding protein (CREB1). In general, phosphoinositide-3 kinase (PI3K)/Akt and stress-activated protein kinase (SAPK)/c-jun kinase (JNK) were the major signaling pathways modulated by CXCL13 in PCa cells. This cluster analysis revealed proteins whose activation patterns can be attributed to CXCL13:CXCR5 interaction in the androgen-independent PC3 cell line. Taken together, these results suggest that CXCL13 contributes to cell-signaling cascades that regulate advanced PCa cell invasion, growth, and/or survival.

ADAM17 targets MMP-2 and MMP-9 via EGFR-MEK-ERK pathway activation to promote prostate cancer cell invasion

ADAM17, also known as tumor necrosis factor-α converting enzyme (TACE), is involved in proteolytic ectodomain shedding of cell surface molecules and cytokines. Although aberrant expression of ADAM17 has been shown in various malignancies, the function of ADAM17 in prostate cancer has not been clarified. In the present study, we sought to elucidate whether ADAM17 contributes to prostate cancer cell invasion, as well as the mechanism involved in the process. The expression pattern of ADAM17 was investigated in human prostate cancer cells. The results showed that ADAM17 expression levels are correlated with the invasive ability of androgen-independent prostate cancer cell lines. Further, ADAM17 was overexpressed in cells showing high invasion characteristics, activation of the EGFR-MEK-ERK pathway, up-regulation of MMP-2, MMP-9, and an increased TGF-α release into the supernatant. However, AG1478, PD98059 and antibody against TGF-α deactivating the EGFR-MEK-ERK signaling pathway, abolished up-regulation of MMP-2, MMP-9 and prevented cell invasion. In addition, cells with knockdown of ADAM17 by siRNA exhibited low invasive ability, deactivated EGFR-MEK-ERK signaling pathway, reduced TGF-α released and down-regulation of MMP-2, MMP-9. However, these effects could be reversed by simultaneous addition of TGF-α. These data demonstrated that ADAM17 contributes to androgen-independent prostate cancer cell invasion by shedding of EGFR ligand TGF-α, which subsequently activates the EGFR-MEK-ERK signaling pathway, leading finally to overexpression of MMP-2 and MMP-9. This study suggests that the ADAM17 expression level may be a new predictive biomarker of invasion and metastasis of prostate cancer, and ADAM17 could provide a target for treating metastatic PCa.

Cyr61 is regulated by cAMP‐dependent protein kinase with serum levels correlating with prostate cancer aggressiveness

Cysteine-rich angiogenic inducer 61 (Cyr61) is an extracellular matrix protein involved in the transduction of growth factor and hormone signaling. Previously, we demonstrated that Cyr61 was highly expressed in prostate cancer (PCa) but that the expression levels were associated with a lower risk of PCa recurrence. In the present study, we demonstrate that serum Cyr61 is a potential biomarker that correlates with PCa aggressiveness. Furthermore, we also explore the potential mechanism underlying the changes in Cyr61 expression during PCa progression. Cyr61 concentrations in the medium from PCa cell lines and in serum samples obtained from PCa patients were measured by sandwich ELISA. Serum Cyr61 levels were correlated with disease characteristics and the association between Cyr61 expression changes by several types of stimulation or stress and cAMP/cAMP-dependent protein kinase (PKA) pathway were examined. There was a positive correlation between Cyr61 levels in cell supernatants and mRNA expression in these cell lines. Serum Cyr61 levels were significantly higher in non-organ-confined PCa patients (116.3 ± 140.2 ng/ml) than in organ-confined PCa patients (79.7 ± 56.1 ng/ml) (P = 0.031). Cyr61 expression was up-regulated in response to both lysophosphatidic acid and androgen treatments which promoted PCa cell invasion. Serum starvation and phosphoinositide-3-kinase inhibition also resulted in Cyr61 up-regulation; however, they suppressed cell proliferation. Cyr61 up-regulation was correlated with an increase in cAMP and suppressed by PKA inhibition. These findings suggest that Cyr61 expression in PCa is regulated by the cAMP/PKA pathway and that circulating Cyr61 levels are a potential serum-based biomarker for characterizing PCa.

Estrogens Promote Invasion of Prostate Cancer Cells in a Paracrine Manner through Up-Regulation of Matrix Metalloproteinase 2 in Prostatic Stromal Cells

Accumulating evidence suggests an enhancing effect of estrogens on prostate cancer (PCa) progression. Matrix metalloproteinase 2 (MMP2), which plays an important role in prostate cancer invasion, is mainly expressed in prostatic stromal cells (PrSC). Here we show that estradiol (E(2)) treatment up-regulates MMP2 production in PrSC, which promotes PCa cell invasion in a paracrine manner. Conditioned medium (CM) was collected from E(2)-treated prostatic stromal cell line WPMY-1 and primary PrSC. The CM of E(2)-treated WPMY-1 and PrSC promoted invasion of PCa cells, as measured by Matrigel transwell assays. Treatment with E(2) and 1,3,5-Tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole, an estrogen receptor-alpha (ERα) specific agonist, significantly up-regulated MMP2 expression in WPMY-1 and PrSC cells at both mRNA and protein levels. The CM treated with an anti-MMP2 antibody lost the stimulatory effect on invasion of PCa cells. The ER inhibitor ICI 182,780, as well as a TGFβ1 neutralizing antibody and ERα-specific small interfering RNA effectively suppressed E(2)-induced MMP2 expression in WPMY-1 cells. Mechanistic studies showed that E(2) up-regulated MMP2 in an indirect manner: E(2) induced TGFβ1 expression via ERα; TGFβ1 stimulated MMP2 expression in PrSC; the invasion of PCa cells were stimulated by elevated MMP2 expression induced by E(2) in a paracrine manner. Our data show that E(2) induces MMP2 expression in WPMY-1 and PrSC cells, which was mediated by TGFβ1. The effect of E(2) on invasion of PCa cells is mediated by up-regulation of MMP2 in a paracrine mechanism.

PI3Kp110-, Src-, FAK-dependent and DOCK2-independent migration and invasion of CXCL13-stimulated prostate cancer cells

BackgroundMost prostate cancer (PCa)-related deaths are due to metastasis, which is mediated in part by chemokine receptor and corresponding ligand interaction. We have previously shown that PCa tissue and cell lines express high levels of the chemokine receptor CXCR5, than compared to their normal counterparts, and interaction of CXCR5 with its specific ligand (CXCL13) promoted PCa cell invasion, migration, and differential matrix metalloproteinase (MMP) expression. This study dissects some of the molecular mechanisms following CXCL13-CXCR5 interaction that mediate PCa cell migration and invasion.ResultsUsing Western blot analysis, kinase-specific cell-based ELISAs, and migration and invasion assays, we show that PCa cell lines differentially express phosphoinositide-3 kinase (PI3K) catalytic subunit isoforms and dedicator of cytokinesis 2 (DOCK2). Specifically, we show that PC3 and normal prostatic epithelial (RWPE-1), but not LNCaP cell lines expressed DOCK2, while RWPE, PC3, and LNCaP cell lines expressed PI3K-p110α and -p110β. Moreover, PC3 selectively expressed PI3K-p110γ, but LNCaP and RWPE cell lines expressed PI3Kp110δ. CXCL13 caused CXCR5-dependent activation of the PI3Kp85α in LNCaP cells, and p85α as well as -p101 in PC3 cells. CXCL13-CXCR5 interaction regulated LNCaP and PC3 cell migration and invasion through extracellular signal-regulated kinase 1/2 (ERK1/2) activation that was primarily dependent on the PI3Kp110 isoform(s), Src, and focal adhesion kinase (FAK), but not DOCK2.ConclusionsWhile additional studies will be needed to determine the PI3K-independent (i.e., DOCK2-mediated) and -dependent events that dictate PCa cell responsiveness to CXCL13, these data provide evidence of the existence of cell type- and stimulus-specific signaling events that support migration and invasion of PCa cells.

Loss of BRCA2 promotes prostate cancer cell invasion through up‐regulation of matrix metalloproteinase‐9

BRCA2 is a multifunctional tumor suppressor protein which plays critical roles in DNA repair, transcription, and cell proliferation, and the loss of which has been linked to the biology of several types of cancers. Here, on prostate adenocarcinoma specimens from 80 patients, we demonstrate that BRCA2 protein is lost in carcinoma cells compared to normal and hyperplastic prostate epithelium. Using highly metastatic prostate cancer PC-3 cells, we show that while BRCA2 depletion by small-interfering RNA promoted migration onto the extracellular matrix proteins fibronectin, laminin, and collagens, as well as invasion through the reconstituted basement membrane matrix Matrigel by more than 140%, recombinant BRCA2 overexpression decreased both phenomena by 57-80% and changed cell morphology from angular and spindle to round and compact. The BRCA2 inhibitory effect on cancer cell migration and invasion resulted from down-regulation of matrix metalloproteinase (MMP)-9 protein levels due to increased MMP-9 proteolysis, and was signaled through inhibition of PI3-kinase/AKT and activation of MAPK/ERK pathway. In BRCA2-overexpressing PC-3 cells, transient transfection with a constitutively active PI3-kinase mutant or treatment with the MAPK/ERK inhibitor PD98059 rescued MMP-9 levels and restored the migratory and invasive capabilities. Consistently, PI3-kinase inhibition with a dominant-negative mutant or MAPK/ERK activation with a gain-of-function mutant reduced MMP-9 levels and prevented migration and invasion in wild-type PC-3 cells. These results provide novel evidence showing that a functional BRCA2 protein may limit the metastatic potential of neoplastic cells by down-regulating MMP-9 production through inhibition of PI3-kinase/AKT and activation of MAPK/ERK, effectively hindering cancer cell migration and invasion.

Activation of PI3K‐Akt signaling pathway promotes prostate cancer cell invasion

Activated phosphoinositide 3-kinase (PI3K) and its downstream target Akt/PKB are important signaling molecules and key survival factors involved in the control of cell proliferation, apoptosis and oncogenesis. We investigated the role of the PI3K-Akt signaling pathway in the invasion of prostate cancer cell lines and activation of this pathway in primary human prostate tumors. Treatment of human prostate cancer cells viz. LNCaP, PC-3 and DU145 with PI3K pharmacological inhibitor, LY294002, potentially suppressed the invasive properties in each of these cell lines. Restoration of the PTEN gene to highly invasive prostate cancer PC-3 cells or expression of a dominant negative version of the PI3K target, Akt also significantly inhibited invasion and downregulated protein expression of urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMP)-9, markers for cell invasion, indicating a central role of the PI3K-Akt pathway in this process. Immunoblot analysis of PI3K and total/activated levels of Akt showed increased protein levels of catalytic (p110alpha/beta) and regulatory (p85) subunits of PI3K and constitutive Akt activation in high-grade tumors compared to low-grade tumor and benign tissue. Immunohistochemical analyses further confirmed a progressive increase in p-Akt (p-Ser473) levels but not of total-Akt (Akt1/2) in cancer tissues compared to benign specimens. A successive increase in p-Akt expression was further noted in specimens serially obtained from individuals with time-course disease progression. Taken together, these results suggest that aberrant activation of PI3K-Akt pathway may contribute to increased cell invasiveness and facilitate prostate cancer progression.

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.

β1 Integrins Modulate Cell Adhesion by Regulating Insulin-Like Growth Factor-II Levels in the Microenvironment

The interactions between cancer cells and the extracellular matrix (ECM) regulate cancer progression. The beta1C and beta1A integrins, two cytoplasmic variants of the beta1 integrin subfamily, are differentially expressed in prostate cancer. Using gene expression analysis, we show here that the beta1C variant, an inhibitor of cell proliferation, which is down-regulated in prostate cancer, up-regulates insulin-like growth factor-II (IGF-II) mRNA and protein levels. In contrast, beta1A does not affect IGF-II levels. We provide evidence that beta1C-mediated up-regulation of IGF-II levels increases adhesion to Laminin-1, a basement membrane protein down-regulated in prostate cancer, and that the beta1C cytoplasmic domain contains the structural motif sufficient to increase cell adhesion to Laminin-1. This autocrine mechanism that locally supports cell adhesion to Laminin-1 via IGF-II is selectively regulated by the beta1 cytoplasmic domain via activation of the growth factor receptor binding protein 2-associated binder-1/SH2-containing protein-tyrosine phosphatase 2/phosphatidylinositol 3-kinase pathway. Thus, the concurrent local loss of beta1C integrin, of its ligand Laminin-1, and of IGF-II in the tumor microenvironment may promote prostate cancer cell invasion and metastasis by reducing cancer cell adhesive properties. It is, therefore, conceivable that reexpression of beta1C will be sufficient to revert a neoplastic phenotype to a nonproliferative and highly adherent normal phenotype.