Ana C. Millena

TGF-β Effects on Prostate Cancer Cell Migration and Invasion Are Mediated by PGE2 through Activation of PI3K/AKT/mTOR Pathway

TGF-β 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-β effects in prostate cancer cells involve PGE2 synthesis. In this study, we investigated TGF-β regulation of COX-1 and COX-2 expression in prostate cancer cells and whether the effects of TGF-β 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-β 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-β 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-β-induced migration of PC3 cells. TGF-β 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-β and PGE2 on phosphorylation of p70S6K and S6 but not on AKT phosphorylation. PGE2 and TGF-β 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-β on cell migration, whereas LY294002 or rapamycin completely eliminated PGE2, TGF-β, and epidermal growth factor-induced migration in PC3 cells. We conclude that TGF-β increases COX-2 levels and PGE2 secretion in prostate cancer cells which, in turn, mediate TGF-β 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. Mol Cancer Res; 8(8); 1164–72. ©2010 AACR.

Autocrine regulation of steroidogenic function of Leydig cells by transforming growth factor-α

We have determined the effects of LH on the expression of transforming growth factor-alpha (TGFalpha) and epidermal growth factor receptor (EGFR) system in rat Leydig cells and investigated its role in steroidogenesis. LH and TGFalpha/epidermal growth factor (EGF) significantly increased the levels of TGFalpha mRNA and protein, and the levels of EGFR protein in immature rat Leydig cells (ILC). Treatment with TGFalpha or EGF for 24h resulted in significant increase in androgen production in ILC. The increase in androgen production in response to TGFalpha was associated with increased mRNA levels of SR-BI, steroidogenic acute regulatory (StAR) and P450scc but not of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and P450c17. TGFalpha also caused a marked increase in the levels StAR protein in ILC. EGFR inhibitor (AG1478) blocked the effects of TGFalpha while MEK-inhibitor (PD98059) potentiated TGFalpha or LH effects on steroidogenesis. A PKA inhibitor (H89) blocked both TGFalpha and LH effects on steroidogenesis. We conclude that TGFalpha plays an autocrine role in LH dependent development and function of Leydig cells.

Expression of TGFβ3 and its effects on migratory and invasive behavior of prostate cancer cells: involvement of PI3-kinase/AKT signaling pathway

Transforming growth factor-β (TGFβ) is a secreted cytokine implicated as a factor in cancer cell migration and invasion. Previous studies have indicated that TGFβ 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 TGFβ-mediated effects on cancer cell invasion. In the present study, we have carried out expression analysis of TGFβ 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. TGFβ1 and TGFβ3 were expressed in all cell lines, with TGFβ3 expression increasing in metastatic cell lines. Both TGFβ1 and TGFβ3 induced motility and invasive behavior in PC3 cells, however, TGFβ3 was significantly more potent than TGFβ1. TGFβRI and Smad3 inhibitors blocked TGFβ1 and TGFβ3 induced motility and invasion. TGFβ3 caused a significant increase in pAKTser473 in PC3 cells and PI3-kinase inhibitor LY294002 blocked TGFβ3 induced migration, invasion and phosphorylation of AKT. Both TGFβRI and Smad3 inhibitors blocked TGFβ3 induced pAKTser473. Based on these results, we conclude that TGFβ3 is expressed in metastatic prostate cancer cell lines and is involved in induction of invasive behavior in these cells. Furthermore, these effects of TGFβ3 are TGFβRI and Smad3 dependent and mediated via the PI3-kinase pathway.

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

In prostate cancer cells, transforming growth factor β (TGFβ) inhibits proliferation in earlier stages of the disease; however, the cancer cells become refractory to growth inhibitory effects in advanced stages where TGFβ 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 TGFβ on prostate cancer cells.

JunD Is Required for Proliferation of Prostate Cancer Cells and Plays a Role in Transforming Growth Factor-β (TGF-β)-induced Inhibition of Cell Proliferation

TGF-β inhibits proliferation of prostate epithelial cells. However, prostate cancer cells in advanced stages become resistant to inhibitory effects of TGF-β. The intracellular signaling mechanisms involved in differential effects of TGF-β during different stages are largely unknown. Using cell line models, we have shown that TGF-β inhibits proliferation in normal (RWPE-1) and prostate cancer (DU145) cells but does not have any effect on proliferation of prostate cancer (PC3) cells. We have investigated the role of Jun family proteins (c-Jun, JunB, and JunD) in TGF-β effects on cell proliferation. Jun family members were expressed at different levels and responded differentially to TGF-β treatment. TGF-β effects on JunD protein levels, but not mRNA levels, correlated with its effects on cell proliferation. TGF-β induced significant reduction in JunD protein in RWPE-1 and DU145 cells but not in PC3 cells. Selective knockdown of JunD expression using siRNA in DU145 and PC3 cells resulted in significant reduction in cell proliferation, and forced overexpression of JunD increased the proliferation rate. On the other hand, knockdown of c-Jun or JunB had little, if any, effect on cell proliferation; overexpression of c-Jun and JunB decreased the proliferation rate in DU145 cells. Further studies showed that down-regulation of JunD in response to TGF-β treatment is mediated via the proteasomal degradation pathway. In conclusion, we show that specific Jun family members exert differential effects on proliferation in prostate cancer cells in response to TGF-β, and inhibition of cell proliferation by TGF-β requires degradation of JunD protein.

Differential role of PTEN in transforming growth factor β (TGF‐β) effects on proliferation and migration in prostate cancer cells

Transforming growth factor‐β (TGF‐β) acts as a tumor suppressor in normal epithelial cells but as a tumor promoter in advanced prostate cancer cells. PI3‐kinase pathway mediates TGF‐β 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‐β effects on proliferation and migration in prostate cancer cells.

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

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 β1 (TGFβ1), 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 TGFβ1‐ 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.

Abstract 4073: Proteasomal degradation of JunD is required for TGF-β induced inhibition of proliferation in prostate cancer cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Transforming growth factor-β (TGF-β) inhibits proliferation of prostate epithelial cells. However, prostate cancer cells in advanced stages become resistant to TGF-β effects on proliferation. In this study, we have investigated the role of AP-1 family of transcription factors, specifically the Jun family members (c-Jun, JunB and JunD) in TGF-β1 effects on prostate cancer cell proliferation. Basal expression of individual Jun family members was determined by RT-PCR and Western blot analysis in four prostate cell lines: RWPE1, LNCaP, DU145 and PC3 cells. Jun levels were also examined in response to TGF-β1. c-Jun was expressed in all cell lines and its expression was up-regulated by TGF-β1. Levels of JunB were higher in normal cells compared to those in cancer cells and TGF-β1caused an increase in JunB levels. JunD levels were higher in all cell lines; TGF-β1 had differential effects on JunD protein levels. While it caused a significant decrease in JunD levels in RWPE-1 and DU145 cells, it did not affect JunD levels in PC3 cells. These differential effects on JunD levels correlated with differential effects of TGF-β on cell proliferation. Individual Jun family members were overexpressed in DU145 cells and effects on cell proliferation were determined. Over-expression of c-Jun and JunB decreased proliferation rate in DU145 cells; however, overexpression of JunD increased proliferation in these cells. Interestingly, cells over-expressing JunD still succumb to inhibitory effect of TGF-β1 after six to eight days of incubation. Furthermore, silencing JunD by siRNA decreased proliferation in both DU145 and PC3 cells. To determine the molecular mechanism of TGF-β-induced down-regulation of JunD, we pretreated cells over-expressing JunD with a proteosomal inhibitor, MG132. Pretreatment of MG132 blocked the degradation of JunD in in DU145 cells over-expressing JunD. In conclusion, our studies show that specific Jun family exerts differential effects on proliferation in prostate cancer cells in response to TGF-β1. While c-Jun and Jun B mediate the inhibitory effects of TGF-β1 on proliferation, JunD counteracts these effects. TGF-β1 causes inhibition in proliferation by degrading JunD via proteasomal degradation. Acknowledgements: These studies were supported by the NIH/NIMHD/RCMI grant #G12MD007590, NIH/NIMHD #5P20MD002285, and DOD grant # W8I-08-1-0077. Citation Format: Ana C. Millena, BaoHan T. Vo, Nicole Strong, Lindsey Walker, Yang Cao, Natalya Klueva, Curt Pfarr, Shafiq Khan. Proteasomal degradation of JunD is required for TGF-β induced inhibition of proliferation in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4073. doi:10.1158/1538-7445.AM2013-4073

Abstract 4080: TGF-β Effects on prostate cancer cell migration are mediated by PGE2 through activation of PI3K/AKT/mTOR pathway.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Transforming growth factor-beta (TGF-β) plays an important role in the progression of prostate cancer. It exhibits both tumor suppressor and tumor promoting activities. Correlations between COX-2 overexpression and enhanced production of PGE2 have been implicated in cancer progression; however, there are no studies indicating that TGF-β effects in prostate cancer cells involve PGE2 synthesis. In this study, we investigated TGF-β regulation of COX-1 and COX-2 expression in prostate cancer cells and whether the effects of TGF-β on cell proliferation and migration are mediated by PGE2. COX-1 protein was ubiquitously expressed in prostate cells; however, COX-2 protein levels were only detected in prostate cancer cells. TGF-β 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 while PGE2 and TGF-β induced migratory behavior only in PC3 cells. Only EP2 and EP4 receptors were detected at mRNA levels in prostate cells. The EP4-targeting siRNA inhibited PGE2 and TGF-β-induced migration of PC3 cells. PGE2 and TGF-β induced phosphorylation of AKT which was blocked by antagonists of PGE2 (EP4) receptors (L161982, AH23848) and PI3-kinase inhibitor (LY294002) in PC3 cells. Pretreatment with L161982 or AH23848 blocked the stimulatory effects of PGE2 and TGF-β on cell migration, while LY294002 or rapamycin completely eliminated PGE2, TGF-β, and EGF-induced migration PC3 cells. We conclude that TGF-β increases COX-2 levels and PGE2 secretion in prostate cancer cells which, in turn, mediates TGF-β effects on cell migration through the activation of PI3K/AKT/mTOR pathway. Acknowledgements: These studies were supported by the NIH/NIMHD/RCMI grant #G12MD007590, NIH/NIMHD #5P20MD002285, and DOD grant # W8I-08-1-0077. Citation Format: Baohan T. Vo, Derrick Morton, Shravan Komaragiri, Ana C. Millena, Shafiq A. Khan. TGF-β Effects on prostate cancer cell migration are mediated by PGE2 through activation of PI3K/AKT/mTOR pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4080. doi:10.1158/1538-7445.AM2013-4080