Gαi2 Protein Inhibition Blocks Chemotherapy- and Anti-Androgen-Induced Prostate Cancer Cell Migration.

We have previously shown that heterotrimeric G-protein subunit alphai2 (Gαi2) is essential for cell migration and invasion in prostate, ovarian and breast cancer cells, and novel small molecule inhibitors targeting Gαi2 block its effects on migratory and invasive behavior. In this study, we have identified potent, metabolically stable, second generation Gαi2 inhibitors which inhibit cell migration in prostate cancer cells. Recent studies have shown that chemotherapy can induce the cancer cells to migrate to distant sites to form metastases. In the present study, we determined the effects of taxanes (docetaxel), anti-androgens (enzalutamide and bicalutamide) and histone deacetylase (HDAC) inhibitors (SAHA and SBI-I-19) on cell migration in prostate cancer cells. All treatments induced cell migration, and simultaneous treatments with new Gαi2 inhibitors blocked their effects on cell migration. We concluded that a combination treatment of Gαi2 inhibitors and chemotherapy could blunt the capability of cancer cells to migrate and form metastases.

Alterations in TGFß signaling during prostate cancer progression.

During prostate cancer progression, TGF-ß acts as both a tumor suppressor and tumor promoter. TGF-ß inhibits cell proliferation in normal and early-stage prostate cancer cells, but during later stages of the disease the cancer cells develop resistance to inhibitory effects on cell proliferation. In these cells, TGF-ß promotes cancer progression due to its effects on epithelial to mesenchymal transition (EMT), cell migration and invasion, and immune suppression. The intracellular mechanisms involved in the development of resistance to TGF-ß effects on cell proliferation are largely unknown. In this review, we summarized the roles of several intracellular proteins including PTEN, Id1 and JunD, which may play a role in this transition. The role of Ski/SnoN proteins in inhibition of Smad2/3 signaling is highlighted.

Small Molecule Inhibitors Targeting Gαi2 Protein Attenuate Migration of Cancer Cells.

Heterotrimeric G-proteins are ubiquitously expressed in several cancers, and they transduce signals from activated G-protein coupled receptors. These proteins have numerous biological functions, and they are becoming interesting target molecules in cancer therapy. Previously, we have shown that heterotrimeric G-protein subunit alphai2 (Gαi2) has an essential role in the migration and invasion of prostate cancer cells. Using a structure-based approach, we have synthesized optimized small molecule inhibitors that are able to prevent specifically the activation of the Gαi2 subunit, keeping the protein in its inactive GDP-bound state. We observed that two of the compounds (13 and 14) at 10 µΜ significantly inhibited the migratory behavior of the PC3 and DU145 prostate cancer cell lines. Additionally, compound 14 at 10 µΜ blocked the activation of Gαi2 in oxytocin-stimulated prostate cancer PC3 cells, and inhibited the migratory capability of DU145 cells overexpressing the constitutively active form of Gαi2, under basal and EGF-stimulated conditions. We also observed that the knockdown or inhibition of Gαi2 negatively regulated migration of renal and ovarian cancer cell lines. Our results suggest that small molecule inhibitors of Gαi2 have potential as leads for discovering novel anti-metastatic agents for attenuating the capability of cancer cells to spread and invade to distant sites.

Androgen attenuates the inactivating phospho-Ser-127 modification of yes-associated protein 1 (YAP1) and promotes YAP1 nuclear abundance and activity.

The transcriptional coactivator YAP1 (yes-associated protein 1) regulates cell proliferation, cell-cell interactions, organ size, and tumorigenesis. Post-transcriptional modifications and nuclear translocation of YAP1 are crucial for its nuclear activity. The objective of this study was to elucidate the mechanism by which the steroid hormone androgen regulates YAP1 nuclear entry and functions in several human prostate cancer cell lines. We demonstrate that androgen exposure suppresses the inactivating post-translational modification phospho-Ser-127 in YAP1, coinciding with increased YAP1 nuclear accumulation and activity. Pharmacological and genetic experiments revealed that intact androgen receptor signaling is necessary for androgen's inactivating effect on phospho-Ser-127 levels and increased YAP1 nuclear entry. We also found that androgen exposure antagonizes Ser/Thr kinase 4 (STK4/MST1) signaling, stimulates the activity of protein phosphatase 2A, and thereby attenuates the phospho-Ser-127 modification and promotes YAP1 nuclear localization. Results from quantitative RT-PCR and CRISPR/Cas9-aided gene knockout experiments indicated that androgen differentially regulates YAP1-dependent gene expression. Furthermore, an unbiased computational analysis of the prostate cancer data from The Cancer Genome Atlas revealed that YAP1 and androgen receptor transcript levels correlate with each other in prostate cancer tissues. These findings indicate that androgen regulates YAP1 nuclear localization and its transcriptional activity through the androgen receptor-STK4/MST1-protein phosphatase 2A axis, which may have important implications for human diseases such as prostate cancer.

Differential roles and activation of mammalian target of rapamycin complexes 1 and 2 during cell migration in prostate cancer cells.

BACKGROUND: Mammalian target of rapamycin (mTOR) is a downstream substrate activated by PI3K/AKT pathway and it is essential for cell migration. It exists as two complexes: mTORC1 and mTORC2. mTORC1 is known to be regulated by active AKT, but the activation of mTORC2 is poorly understood. In this study, we investigated the roles and differential activation of the two mTOR complexes during cell migration in prostate cancer cells. METHODS: We used small interfering RNA to silence the expression of Rac1 and the main components of mTOR complexes (regulatory associated protein of mTOR [RAPTOR] and rapamycin-insensitive companion of mTOR [RICTOR]) in LNCaP, DU145, and PC3 prostate cancer cell lines. We performed transwell migration assay to evaluate the migratory capability of the cells, and Western blot analysis to study the activation levels of mTOR complexes. RESULTS: Specific knockdown of RAPTOR and RICTOR caused a decrease of cell migration, suggesting their essential role in prostate cancer cell movement. Furthermore, epidermal growth factor (EGF) treatments induced the activation of both the mTOR complexes. Lack of Rac1 activity in prostate cancer cells blocked EGF-induced activation of mTORC2, but had no effect on mTORC1 activation. Furthermore, the overexpression of constitutively active Rac1 resulted in significant increase in cell migration and activation of mTORC2 in PC3 cells, but had no effect on mTORC1 activation. Active Rac1 was localized in the plasma membrane and was found to be in a protein complex, with RICTOR, but not RAPTOR. CONCLUSION: We suggest that EGF-induced activation of Rac1 causes the activation of mTORC2 via RICTOR. This mechanism plays a critical role in prostate cancer cell migration.

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.

Ethnic differences in TGFß-signaling pathway may contribute to prostate cancer health disparity.

Epidemiological studies show that the incidence and mortality rates of prostate cancer (PCa) are significantly higher in African-American (AA) men when compared with Caucasian (CA) men in the United States. Transforming growth factor ß (TGFß) signaling pathway is linked to health disparities in AAs. Recent studies suggest a role of TGFß3 in cancer metastases and its effect on the migratory and invasive behavior; however, its role in PCa in AA men has not been studied. We determined the circulating levels of TGFß3 in AA and CA men diagnosed with PCa using ELISA. We analyzed serum samples from both AA and CA men diagnosed with and without PCa. We show that AA PCa patients had higher levels of TGFß3 protein compared with AA controls and CA patients. In fact, TGFß3 protein levels in serum were higher in AA men without PCa compared with the CA population, which may correlate with more aggressive disease seen in AA men. Studies on AA-derived PCa cell lines revealed that TGFß3 protein levels were also higher in these cells compared with CA-derived PCa cell lines. Our studies also reveal that TGFß does not inhibit cell proliferation in AA-derived PCa cell lines, but it does induce migration and invasion through activation of PI3K pathway. We suggest that increased TGFß3 levels are responsible for development of aggressive PCa in AA patients as a consequence of development of resistance to inhibitory effects of TGFß on cell proliferation and induction of invasive metastatic behavior.

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.

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

TGF-ß inhibits proliferation of prostate epithelial cells. However, prostate cancer cells in advanced stages become resistant to inhibitory effects of TGF-ß. The intracellular signaling mechanisms involved in differential effects of TGF-ß during different stages are largely unknown. Using cell line models, we have shown that TGF-ß 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-ß effects on cell proliferation. Jun family members were expressed at different levels and responded differentially to TGF-ß treatment. TGF-ß effects on JunD protein levels, but not mRNA levels, correlated with its effects on cell proliferation. TGF-ß 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-ß 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-ß, and inhibition of cell proliferation by TGF-ß requires degradation of JunD protein.

JAK/STAT pathway interacts with intercellular cell adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) while prostate cancer stem cells form tumor spheroids.

PURPOSE: JAK/STAT is an evolutionarily conserved pathway and very important for second messenger system. This pathway is important in malignant transformation and accumulated evidence indicates that this pathway is involved in tumorigenesis and progression of several cancers. It was possible to assume that activation of JAK/STAT pathway is associated with increase in the expressions of ICAM/1 and VCAM-1. In this study we hypothesized that when cells were maintained as spheroids or monolayers, the structure of cancer stem cells (CSCs) could show differentiation when compared with non-CSCs. METHODS: DU-145 human prostate cancer cells were cultured using the Ege University molecular embryology laboratory medium supplemented with 10% fetal bovine serum. Clusters of differentiation 133 (CD133)(+high)/CD44(+high) prostate CSCs were isolated from the DU145 cell line by using BD FACSAria. CD133//CD44+ CSCs were cultured until confluent with 3% noble agar. The expression of these proteins in CSCs and non-CSCs was analyzed by immunohistochemistry. RESULTS: Different expression profiles were observed in the conventional two-dimensional (2D) and three-dimensional (3D) experimental model system when CSCs and non-CSCs were compared. Human prostate CSCs exhibited intense ICAM-1 and VCAM-1 immunoreaction when compared with non-CSCs. These findings were supported by the fact that VCAM-1 on the surface of cancer cells binds to its counterreceptor, the α4ß1 integrin (also known as very-late antigen, VLA-4), on metastasis-associated macrophages, triggering VCAM-1-mediated activation of the phosphoinositide 3-kinase growth and survival pathway in cancer cells. CONCLUSIONS: The results of this study showed that changes in JAK/STAT pathway are related with adhesion molecules and could affect cancer progression.

Nano-Snowflower of Gold Nanoparticles-Ruthenium Metallopolymer-Carbon Nanotubes Binding Anti-DNP IgE Antibody.

Gold nanoparticles (AuNPs) have been chemically functionalized onto multiwalled carbon nanotubes (MWCNT) through a metallopolymer linker-bis (2,2':6'2"-terpyridine) ruthenium(II)-connected diblock poly(N-isopropyacryamide). A "nano-snowflower" pattern was formed by self-assembly MWCNT-AuNP nanocomposite with anti-DNP IgE antibody. MWCNT-AuNP nanohybrid has unique biocompatibility and electronic current-voltage properties. This nanohybrid shows the potential application for IgE biosensor to diagnose cancer cells. We represent a step towards building complex electronic circuits response by providing molecular recognition properties.

p66Shc longevity protein regulates the proliferation of human ovarian cancer cells.

p66Shc functions as a longevity protein in murine and exhibits oxidase activity in regulating diverse biological activities. In this study, we investigated the role of p66Shc protein in regulating ovarian cancer (OCa) cell proliferation. Among three cell lines examined, the slowest growing OVCAR-3 cells have the lowest level of p66Shc protein. Transient transfection with p66Shc cDNA expression vector in OVCAR-3 cells increases cell proliferation. Conversely, knock-down of p66Shc by shRNA in rapidly growing SKOV-3 cells results in decreased cell growth. In estrogen (E2)-treated CaOV-3 cells, elevated p66Shc protein level correlates with ROS level, ErbB-2 and ERK/MAPK activation, and cell proliferation. Further, the E2-stimulated proliferation of CaOV-3 cells was blocked by antioxidants and ErbB-2 inhibitor. Additionally, in E2-stimulated cells, the tartrate-sensitive, but not the tartrate-resistant, phosphatase activity decreases; concurrently, the tyrosine phosphorylation of ErbB-2 increases. Conversely, inhibition of phosphatase activity by L(+)-tartrate treatment increases p66Shc protein level, ErbB-2 tyrosine phosphorylation, ERK/MAPK activation, and cell growth. Further, inhibition of the ERK/MAPK pathway by PD98059 blocks E2-induced ERK/MAPK activation and cell proliferation in CaOV-3 cells. Moreover, immunohistochemical analyses showed that the p66Shc protein level was significantly higher in cancerous cells than in noncancerous cells in archival OCa tissues (n = 76; P = 0.00037). These data collectively indicate that p66Shc protein plays a critical role in up-regulating OCa progression.

Antiproliferative activity of novel imidazopyridine derivatives on castration-resistant human prostate cancer cells.

Metastatic prostate cancer (mPCa) relapses after a short period of androgen deprivation therapy and becomes the castration-resistant prostate cancer (CR PCa); to which the treatment is limited. Hence, it is imperative to identify novel therapeutic agents towards this patient population. In the present study, antiproliferative activities of novel imidazopyridines were compared. Among three derivatives, PHE, AMD and AMN, examined, AMD showed the highest inhibitory activity on LNCaP C-81 cell proliferation, following dose- and time-dependent manner. Additionally, AMD exhibited significant antiproliferative effect against a panel of PCa cells, but not normal prostate epithelial cells. Further, when compared to AMD, its derivative DME showed higher inhibitory activities on PCa cell proliferation, clonogenic potential and in vitro tumorigenicity. The inhibitory activity was apparently in part due to the induction of apoptosis. Mechanistic studies indicate that AMD and DME treatments inhibited both AR and PI3K/Akt signaling. The results suggest that better understanding of inhibitory mechanisms of AMD and DME could help design novel therapeutic agents for improving the treatment of CR PCa.

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.

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.

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.

The essential role of Giα2 in prostate cancer cell migration.

Cell- and receptor-specific regulation of cell migration by Gi/oα-proteins remains unknown in prostate cancer cells. In the present study, oxytocin (OXT) receptor was detected at the protein level in total cell lysates from C81 (an androgen-independent subline of LNCaP), DU145 and PC3 prostate cancer cells, but not in immortalized normal prostate luminal epithelial cells (RWPE1), and OXT-induced migration of PC3 cells. This effect of OXT has been shown to be mediated by Gi/oα-dependent signaling. Accordingly, OXT inhibited forskolin-induced luciferase activity in PC3 cells that were transfected with a luciferase reporter for cyclic AMP activity. Although mRNAs for all three Giα isoforms were present in PC3 cells, Giα2 was the most abundant isoform that was detected at the protein level. Pertussis toxin (PTx) inhibited the OXT-induced migration of PC3 cells. Ectopic expression of the PTx-resistant Giα2-C352G, but not wild-type Giα2, abolished this effect of PTx on OXT-induced cell migration. The Giα2-targeting siRNA was shown to specifically reduce Giα2 mRNA and protein in prostate cancer cells. The Giα2-targeting siRNA eliminated OXT-induced migration of PC3 cells. These data suggest that Giα2 plays an important role in the effects of OXT on PC3 cell migration. The Giα2-targeting siRNA also inhibited EGF-induced migration of PC3 and DU145 cells. Expression of the siRNA-resistant Giα2, but not wild type Giα2, restored the effects of EGF in PC3 cells transfected with the Giα2-targeting siRNA. In conclusion, Giα2 plays an essential role in OXT and EGF signaling to induce prostate cancer cell migration.

Differential role of Sloan-Kettering Institute (Ski) protein in Nodal and transforming growth factor-beta (TGF-ß)-induced Smad signaling in prostate cancer cells.

Transforming growth factor-beta (TGF-ß) signaling pathways contain both tumor suppressor and tumor promoting activities. We have demonstrated that Nodal, another member of the TGF-ß superfamily, and its receptors are expressed in prostate cancer cells. Nodal and TGF-ß exerted similar biological effects on prostate cells; both inhibited proliferation in WPE, RWPE1 and DU145 cells, whereas neither had any effect on the proliferation of LNCaP or PC3 cells. Interestingly, Nodal and TGF-ß induced migration in PC3 cells, but not in DU145 cells. TGF-ß induced predominantly phosphorylation of Smad3, whereas Nodal induced phosphorylation of only Smad2. We also determined the expression and differential role of Ski, a corepressor of Smad2/3, in Nodal and TGF-ß signaling in prostate cancer cells. Similar levels of Ski mRNA were found in several established prostate cell lines; however, high levels of Ski protein were only detected in prostate cancer cells and prostate cancer tissue samples. Exogenous Nodal and TGF-ß had no effects on Ski mRNA levels. On the other hand, TGF-ß induced a rapid degradation of Ski protein mediated by the proteasomal pathway, whereas Nodal had no effect on Ski protein. Reduced Ski levels correlated with increased basal and TGF-ß-induced Smad2/3 phosphorylation. Knockdown of endogenous Ski reduced proliferation in DU145 cells and enhanced migration of PC3 cells. We conclude that high levels of Ski expression in prostate cancer cells may be responsible for repression of TGF-ß and Smad3 signaling, but Ski protein levels do not influence Nodal and Smad2 signaling.

Vascular endothelial growth factor A, secreted in response to transforming growth factor-ß1 under hypoxic conditions, induces autocrine effects on migration of prostate cancer cells.

Hypoxia and transforming growth factor-ß1 (TGF-ß1) increase vascular endothelial growth factor A (VEGFA) expression in a number of malignancies. This effect of hypoxia and TGF-ß1 might be responsible for tumor progression and metastasis of advanced prostate cancer. In the present study, TGF-ß1 was shown to induce VEGFA(165) secretion from both normal cell lines (HPV7 and RWPE1) and prostate cancer cell lines (DU145 and PC3). Conversely, hypoxia-stimulated VEGFA(165) secretion was observed only in prostate cancer cell lines. Hypoxia induced TGF-ß1 expression in PC3 prostate cancer cells, and the TGF-ß type I receptor (ALK5) kinase inhibitor partially blocked hypoxia-mediated VEGFA(165) secretion. This effect of hypoxia provides a novel mechanism to increase VEGFA expression in prostate cancer cells. Although autocrine signaling of VEGFA has been implicated in prostate cancer progression and metastasis, the associated mechanism is poorly characterized. VEGFA activity is mediated via VEGF receptor (VEGFR) 1 (Flt-1) and 2 (Flk-1/KDR). Whereas VEGFR-1 mRNA was detected in normal prostate epithelial cells, VEGFR-2 mRNA and VEGFR protein were expressed only in PC3 cells. VEGFA(165) treatment induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in PC3 cells but not in HPV7 cells, suggesting that the autocrine function of VEGFA may be uniquely associated with prostate cancer. Activation of VEGFR-2 by VEGFA(165) was shown to enhance migration of PC3 cells. A similar effect was also observed with endogenous VEGFA induced by TGF-ß1 and hypoxia. These findings illustrate that an autocrine loop of VEGFA via VEGFR-2 is critical for the tumorigenic effects of TGF-ß1 and hypoxia on metastatic prostate cancers.