Novel role of Giα2 in cell migration: Downstream of PI3-kinase-AKT and Rac1 in prostate cancer cells.

Tumor cell motility is the essential step in cancer metastasis. Previously, we showed that oxytocin and epidermal growth factor (EGF) effects on cell migration in prostate cancer cells require Giα2 protein. In the current study, we investigated the interactions among G-protein coupled receptor (GPCR), Giα2, PI3-kinase, and Rac1 activation in the induction of migratory and invasive behavior by diverse stimuli. Knockdown and knockout of endogenous Giα2 in PC3 cells resulted in attenuation of transforming growth factor ß1 (TGFß1), oxytocin, SDF-1α, and EGF effects on cell migration and invasion. In addition, knockdown of Giα2 in E006AA cells attenuated cell migration and overexpression of Giα2 in LNCaP cells caused significant increase in basal and EGF-stimulated cell migration. Pretreatment of PC3 cells with Pertussis toxin resulted in attenuation of TGFß1- and oxytocin-induced migratory behavior and PI3-kinase activation without affecting EGF-induced PI3-kinase activation and cell migration. Basal- and EGF-induced activation of Rac1 in PC3 and DU145 cells were not affected in cells after Giα2 knockdown. On the other hand, Giα2 knockdown abolished the migratory capability of PC3 cells overexpressing constitutively active Rac1. The knockdown or knockout of Giα2 resulted in impaired formation of lamellipodia at the leading edge of the migrating cells. We conclude that Giα2 protein acts at two different levels which are both dependent and independent of GPCR signaling to induce cell migration and invasion in prostate cancer cells and its action is downstream of PI3-kinase-AKT-Rac1 axis.

Differential role of PTEN in transforming growth factor ß (TGF-ß) effects on proliferation and migration in prostate cancer cells.

BACKGROUND: Transforming growth factor-ß (TGF-ß) acts as a tumor suppressor in normal epithelial cells but as a tumor promoter in advanced prostate cancer cells. PI3-kinase pathway mediates TGF-ß effects on prostate cancer cell migration and invasion. PTEN inhibits PI3-kinase pathway and is frequently mutated in prostate cancers. We investigated possible role(s) of PTEN in TGF-ß effects on proliferation and migration in prostate cancer cells. METHODS: Expression of PTEN mRNA and proteins were determined using RT-PCR and Western blotting in RWPE1 and DU145 cells. We also studied the role of PTEN in TGF-ß effects on cell proliferation and migration in DU145 cells after transient silencing of endogenous PTEN. Conversely, we determined the role of PTEN in cell proliferation and migration after over-expression of PTEN in PC3 cells which lack endogenous PTEN. RESULTS: TGF-ß1 and TGF-ß3 had no effect on PTEN mRNA levels but both isoforms increased PTEN protein levels in DU145 and RWPE1 cells indicating that PTEN may mediate TGF-ß effects on cell proliferation. Knockdown of PTEN in DU145 cells resulted in significant increase in cell proliferation which was not affected by TGF-ß isoforms. PTEN overexpression in PC3 cells inhibited cell proliferation. Knockdown of endogenous PTEN enhanced cell migration in DU145 cells, whereas PTEN overexpression reduced migration in PC3 cells and reduced phosphorylation of AKT in response to TGF-ß. CONCLUSION: We conclude that PTEN plays a role in inhibitory effects of TGF-ß on cell proliferation whereas its absence may enhance TGF-ß effects on activation of PI3-kinase pathway and cell migration.

TGF-ß Effects on Prostate Cancer Cell Migration and Invasion Require FosB.

BACKGROUND: Activator Protein-1 (AP-1) family (cJun, JunB, JunD, cFos, FosB, Fra1, and Fra2) plays a central role in the transcriptional regulation of many genes that are associated with cell proliferation, differentiation, migration, metastasis, and survival. Many oncogenic signaling pathways converge at the AP-1 transcription complex. Transforming growth factor beta (TGF-ß) is a multifunctional regulatory cytokine that regulates many aspects of cellular function, including cellular proliferation, differentiation, migration, apoptosis, adhesion, angiogenesis, immune surveillance, and survival. METHODS: This study investigated, the role of FOS proteins in TGF-ß signaling in prostate cancer cell proliferation, migration, and invasion. Steady state expression levels of FOS mRNA and proteins were determined using RT-PCR and western blotting analyses. DU145 and PC3 prostate cancer cells were exposed to TGF-ß1 at varying time and dosage, RT-PCR, western blot, and immunofluorescence analyses were used to determine TGF-ß1 effect on FOS mRNA and protein expression levels as well as FosB subcellular localization. Transient silencing of FosB protein was used to determine its role in cell proliferation, migration, and invasion. RESULTS: Our data show that FOS mRNA and proteins were differentially expressed in human prostate epithelial (RWPE-1) and prostate cancer cell lines (LNCaP, DU145, and PC3). TGF-ß1 induced the expression of FosB at both the mRNA and protein levels in DU145 and PC3 cells, whereas cFos and Fra1 were unaffected. Immunofluorescence analysis showed an increase in the accumulation of FosB protein in the nucleus of PC3 cells after treatment with exogenous TGF-ß1. Selective knockdown of endogenous FosB by specific siRNA did not have any effect on cell proliferation in PC3 and DU145 cells. However, basal and TGF-ß1- and EGF-induced cell migration was significantly reduced in DU145 and PC3 cells lacking endogenous FosB. TGF-ß1- and EGF-induced cell invasion were also significantly decreased after FosB knockdown in PC3 cells. CONCLUSION: Our data suggest that FosB is required for migration and invasion in prostate cancer cells. We also conclude that TGF-ß1 effect on prostate cancer cell migration and invasion may be mediated through the induction of FosB. Prostate 77:72-81, 2017. © 2016 Wiley Periodicals, Inc.

Inhibitor of differentiation 1 (Id1) and Id3 proteins play different roles in TGFß effects on cell proliferation and migration in prostate cancer cells.

BACKGROUND: In prostate cancer cells, transforming growth factor ß (TGFß) inhibits proliferation in earlier stages of the disease; however, the cancer cells become refractory to growth inhibitory effects in advanced stages where TGFß promotes cancer progression and metastasis. Inhibitor of differentiation (Id) family of closely related proteins (Id1-Id4) are dominant negative regulators and basic helix loop helix (bHLH) transcription factors and in general promote proliferation, and inhibit differentiation. In the present study, we have investigated the role of Id1 and Id3 proteins in the growth inhibitory effects of TGFß on prostate cancer cells. METHODS: The effect of TGF ß on proliferation and Id1 and Id3 expression were investigated in PZ-HPV7, DU145, and PC3 cells. Id1 silencing through siRNA was also used in DU145 and PC3 cells to examine its role in anti-proliferative and migratory effects of TGFß. RESULTS: TGFß increased expression of Id1 and Id3 in all cell lines followed by a later down regulation of Id1 in PZ-HPV7 expression and DU145 cells but not in PC3 cells. Id3 expression remained elevated in all three cell lines. This loss of Id1 protein correlated with an increase of CDKNI p21. Id1 knockdown in both DU145 and PC3 cells resulted in decreased proliferation. However, while TGFß caused a further decrease in proliferation of DU145, but had no further effects in PC3 cells. Knockdown of Id1 or Id3 inhibited TGFß1induced migration in PC3 cells. CONCLUSIONS: These findings suggest an essential role of Id1 and Id3 in TGFß1 effects on proliferation and migration in prostate cancer cells.

Follicle-stimulating hormone-induced aromatase in immature rat Sertoli cells requires an active phosphatidylinositol 3-kinase pathway and is inhibited via the mitogen-activated protein kinase signaling pathway.

Postnatal development and function of testicular Sertoli cells are regulated primarily by FSH. During this early period of development, estrogens play a role in proliferation of somatic cells, which contributes significantly to testicular development. Growth factors like epidermal growth factor (EGF) are produced in the testis and play a role in regulation of estradiol production and male fertility. Although these divergent factors modulate gonadal function, little is known about their mechanism of action in Sertoli cells. The present study investigates the intracellular events that take place down-stream of FSH and EGF receptors in Sertoli cells isolated from immature (10-d-old) rats, and examines which intracellular signals may be involved in their effects on aromatase activity and estradiol production in immature rat Sertoli cells. Primary cultures of rat Sertoli cells were treated with FSH in combination with EGF and signaling pathway-specific inhibitors. Levels of estradiol production, aromatase mRNA (Cyp19a1), and aromatase protein (CYP19A1) were determined. Western blot analysis was performed to determine the effects of FSH and EGF on levels of activated (phosphorylated) AKT1 and p42 ERK2 and p44 ERK1, also named MAPK1 and MAPK3, respectively. The stimulatory actions of FSH on aromatase mRNA, aromatase protein, and estradiol production were blocked by inhibition of the phosphatidylinositol 3-kinase/AKT1 signaling pathway. In contrast, inhibition of ERK signaling augmented the stimulatory effects of FSH on estradiol production, aromatase mRNA, and protein levels. Furthermore, EGF inhibited the expression of aromatase mRNA and protein in response to FSH, and these inhibitory effects of EGF were critically dependent on the activation of the ERK signaling pathway. We conclude that an active phosphatidylinositol 3-kinase /AKT signaling pathway is required for the stimulatory actions of FSH, whereas an active ERK/MAPK pathway inhibits estradiol production and aromatase expression in immature Sertoli cells.

Expression of TGFß3 and its effects on migratory and invasive behavior of prostate cancer cells: involvement of PI3-kinase/AKT signaling pathway.

Transforming growth factor-ß (TGFß) is a secreted cytokine implicated as a factor in cancer cell migration and invasion. Previous studies have indicated that TGFß isoforms may exert differential effects on cancer cells during different stages of the disease, however very little is known about the expression patterns and activity of the three isoforms in prostate cancer. Non-traditional signaling pathways including the PI3-Kinase have been associated with TGFß-mediated effects on cancer cell invasion. In the present study, we have carried out expression analysis of TGFß isoforms and signaling components in cell line models representing different stages of prostate cancer and studied the differential effects of specific isoforms on migratory and invasive behavior and induction of the PI3-kinase pathway. TGFß1 and TGFß3 were expressed in all cell lines, with TGFß3 expression increasing in metastatic cell lines. Both TGFß1 and TGFß3 induced motility and invasive behavior in PC3 cells, however, TGFß3 was significantly more potent than TGFß1. TGFßRI and Smad3 inhibitors blocked TGFß1 and TGFß3 induced motility and invasion. TGFß3 caused a significant increase in pAKT(ser473) in PC3 cells and PI3-kinase inhibitor LY294002 blocked TGFß3 induced migration, invasion and phosphorylation of AKT. Both TGFßRI and Smad3 inhibitors blocked TGFß3 induced pAKT(ser473). Based on these results, we conclude that TGFß3 is expressed in metastatic prostate cancer cell lines and is involved in induction of invasive behavior in these cells. Furthermore, these effects of TGFß3 are TGFßRI and Smad3 dependent and mediated via the PI3-kinase pathway.

TGF-ß effects on prostate cancer cell migration and invasion are mediated by PGE2 through activation of PI3K/AKT/mTOR pathway.

TGF-ß plays an important role in the progression of prostate cancer. It exhibits both tumor suppressor and tumor-promoting activities. Correlations between cyclooxygenase (COX)-2 overexpression and enhanced production of prostaglandin (PG)E2 have been implicated in cancer progression; however, there are no studies indicating that TGF-ß effects in prostate cancer cells involve PGE2 synthesis. In this study, we investigated TGF-ß regulation of COX-1 and COX-2 expression in prostate cancer cells and whether the effects of TGF-ß on cell proliferation and migration are mediated by PGE2. COX-1 protein was ubiquitously expressed in prostate cells; however, COX-2 protein levels were detected only in prostate cancer cells. TGF-ß treatment increased COX-2 protein levels and PGE2 secretion in PC3 cells. Exogenous PGE2 and PGF2α had no effects on cell proliferation in LNCaP, DU145, and PC3 cells whereas PGE2 and TGF-ß induced migration and invasive behavior in PC3 cells. Only EP2 and EP4 receptors were detected at mRNA levels in prostate cells. The EP4-targeting small interfering RNA inhibited PGE2 and TGF-ß-induced migration of PC3 cells. TGF-ß and PGE2 induce activation of PI3K/AKT/mammalian target of rapamycin pathway as indicated by increased AKT, p70S6K, and S6 phosphorylation. Rapamycin completely blocked the effects of TGF-ß and PGE2 on phosphorylation of p70S6K and S6 but not on AKT phosphorylation. PGE2 and TGF-ß induced phosphorylation of AKT, which was blocked by antagonists of PGE2 (EP4) receptors (L161982, AH23848) and PI3K inhibitor (LY294002) in PC3 cells. Pretreatment with L161982 or AH23848 blocked the stimulatory effects of PGE2 and TGF-ß on cell migration, whereas LY294002 or rapamycin completely eliminated PGE2, TGF-ß, and epidermal growth factor-induced migration in PC3 cells. We conclude that TGF-ß increases COX-2 levels and PGE2 secretion in prostate cancer cells which, in turn, mediate TGF-ß effects on cell migration and invasion through the activation of PI3K/AKT/mammalian target of rapamycin pathway.

Oxytocin induces the migration of prostate cancer cells: involvement of the Gi-coupled signaling pathway.

Expression of genes that encode oxytocin (OXT) and vasopressin (AVP) and their cognate receptors in normal and diseased prostates are only partially characterized. Reverse transcription and PCR were used to examine the expression of these genes in normal prostate epithelial and stromal cell lines, k-ras-transformed prostate epithelial cell lines, and in four prostate cancer cell lines. Secreted and cell-associated OXT peptide was measured by an enzyme immunoassay. OXT and its receptor (OXTR) were expressed in all eight prostate cell lines. Cell-associated OXT peptide was also found in all prostate epithelial cell lines except in DU145 cells. Neither AVP nor its cognate receptors (V1a receptor and V2 receptor) were expressed in any prostate cell line examined. These data point to the OXTR as the primary target of OXT and AVP, and suggest that OXT might be an autocrine/paracrine regulator in human prostate. We found that OXT induces the migration of PC3 and PC3M, but not DU145 prostate cancer cells. The effect of OXT is distinct from the epidermal growth factor (EGF)-induced migration of prostate cancer cells, in which ERK1/2 and EGF receptor kinase activities were required. When cells were pretreated with pertussis toxin, the effect of OXT, but not EGF, on cell migration was abolished. Pretreatment with the cyclic AMP analogue, 8-Br-cAMP, did not affect OXT-induced cell migration, which eliminated the nonspecific effect of pertussis toxin. We conclude that a Gi-dependent mechanism is involved in OXTR-mediated migration of prostate cancer cells, and indicates a role for OXTR in prostate cancer metastasis.