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.

Molecular heterogeneity and CXorf67 alterations in posterior fossa group A (PFA) ependymomas.

Of nine ependymoma molecular groups detected by DNA methylation profiling, the posterior fossa type A (PFA) is most prevalent. We used DNA methylation profiling to look for further molecular heterogeneity among 675 PFA ependymomas. Two major subgroups, PFA-1 and PFA-2, and nine minor subtypes were discovered. Transcriptome profiling suggested a distinct histogenesis for PFA-1 and PFA-2, but their clinical parameters were similar. In contrast, PFA subtypes differed with respect to age at diagnosis, gender ratio, outcome, and frequencies of genetic alterations. One subtype, PFA-1c, was enriched for 1q gain and had a relatively poor outcome, while patients with PFA-2c ependymomas showed an overall survival at 5 years of > 90%. Unlike other ependymomas, PFA-2c tumors express high levels of OTX2, a potential biomarker for this ependymoma subtype with a good prognosis. We also discovered recurrent mutations among PFA ependymomas. H3 K27M mutations were present in 4.2%, occurring only in PFA-1 tumors, and missense mutations in an uncharacterized gene, CXorf67, were found in 9.4% of PFA ependymomas, but not in other groups. We detected high levels of wildtype or mutant CXorf67 expression in all PFA subtypes except PFA-1f, which is enriched for H3 K27M mutations. PFA ependymomas are characterized by lack of H3 K27 trimethylation (H3 K27-me3), and we tested the hypothesis that CXorf67 binds to PRC2 and can modulate levels of H3 K27-me3. Immunoprecipitation/mass spectrometry detected EZH2, SUZ12, and EED, core components of the PRC2 complex, bound to CXorf67 in the Daoy cell line, which shows high levels of CXorf67 and no expression of H3 K27-me3. Enforced reduction of CXorf67 in Daoy cells restored H3 K27-me3 levels, while enforced expression of CXorf67 in HEK293T and neural stem cells reduced H3 K27-me3 levels. Our data suggest that heterogeneity among PFA ependymomas could have clinicopathologic utility and that CXorf67 may have a functional role in these tumors.

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.

Localized high-risk prostate cancer harbors an androgen receptor low subpopulation susceptible to HER2 inhibition.

Patients diagnosed with localized high-risk prostate cancer have higher rates of recurrence, and the introduction of neoadjuvant intensive hormonal therapies seeks to treat occult micrometastatic disease by their addition to definitive treatment. Sufficient profiling of baseline disease has remained a challenge in enabling the in-depth assessment of phenotypes associated with exceptional vs. poor pathologic responses after treatment. In this study, we report comprehensive and integrative gene expression profiling of 37 locally advanced prostate tumors prior to six months of androgen deprivation therapy (ADT) plus the androgen receptor (AR) inhibitor enzalutamide prior to radical prostatectomy. A robust transcriptional program associated with HER2 activity was positively associated with poor outcome and opposed AR activity, even after adjusting for common genomic alterations in prostate cancer including PTEN loss and expression of the TMPRSS2:ERG fusion. Patients experiencing exceptional pathologic responses demonstrated lower levels of HER2 and phospho-HER2 by immunohistochemistry of biopsy tissues. The inverse correlation of AR and HER2 activity was found to be a universal feature of all aggressive prostate tumors, validated by transcriptional profiling an external cohort of 121 patients and immunostaining of tumors from 84 additional patients. Importantly, the AR activity-low, HER2 activity-high cells that resist ADT are a pre-existing subset of cells that can be targeted by HER2 inhibition alone or in combination with enzalutamide. In summary, we show that prostate tumors adopt an AR activity-low prior to antiandrogen exposure that can be exploited by treatment with HER2 inhibitors.

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.

Snail transcription factor negatively regulates maspin tumor suppressor in human prostate cancer cells.

BACKGROUND: Maspin, a putative tumor suppressor that is down-regulated in breast and prostate cancer, has been associated with decreased cell motility. Snail transcription factor is a zinc finger protein that is increased in breast cancer and is associated with increased tumor motility and invasion by induction of epithelial-mesenchymal transition (EMT). We investigated the molecular mechanisms by which Snail increases tumor motility and invasion utilizing prostate cancer cells. METHODS: Expression levels were analyzed by RT-PCR and western blot analyses. Cell motility and invasion assays were performed, while Snail regulation and binding to maspin promoter was analyzed by luciferase reporter and chromatin immunoprecipitation (ChIP) assays. RESULTS: Snail protein expression was higher in different prostate cancer cells lines as compared to normal prostate epithelial cells, which correlated inversely with maspin expression. Snail overexpression in 22Rv1 prostate cancer cells inhibited maspin expression and led to increased migration and invasion. Knockdown of Snail in DU145 and C4-2 cancer cells resulted in up-regulation of maspin expression, concomitant with decreased migration. Transfection of Snail into 22Rv1 or LNCaP cells inhibited maspin promoter activity, while stable knockdown of Snail in C4-2 cells increased promoter activity. ChIP analysis showed that Snail is recruited to the maspin promoter in 22Rv1 cells. CONCLUSIONS: Overall, this is the first report showing that Snail can negatively regulate maspin expression by directly repressing maspin promoter activity, leading to increased cell migration and invasion. Therefore, therapeutic targeting of Snail may be useful to re-induce expression of maspin tumor suppressor and prevent prostate cancer tumor progression.

Expression of nodal and nodal receptors in prostate stem cells and prostate cancer cells: autocrine effects on cell proliferation and migration.

BACKGROUND: Nodal, a TGFß like growth factor, functions as an embryonic morphogen that maintains the pluripotency of embryonic stem cells. Nodal has been implicated in cancer progression; however, there is no information on expression and functions of Nodal in prostate cancer. In this study, we have investigated the expression of Nodal, its receptors, and its effects on proliferation and migration of human prostate cells. METHODS: RT-PCR, qPCR, and Western blot analyses were performed to analyze expression of Nodal and Nodal receptors and its effects on phosphorylation of Smad2/3 in prostate cells. The effects on proliferation and migration were determined by (3) H-Thymidine incorporation and cell migration assays in the presence or absence of Nodal receptor inhibitor (SB431542). RESULTS: Nodal was highly expressed in WPE, DU145, LNCaP, and LNCaP-C81 cells with low expression in RWPE1 and RWPE2 cells, but not in PREC, PC3 and PC3M cells. Nodal receptors are expressed at varying levels in all prostate cells. Treatment with exogenous Nodal induced phosphorylation of Smad2/3 in WPE, DU145, and PC3 cells, which was blocked by SB431542. Nodal dose-dependently inhibited proliferation of WPE, RWPE1 and DU145 cells, but not LNCaP and PC3 cells. Nodal induced cell migration in PC3 cells, which was inhibited by SB431542; Nodal had no effect on cell migration in WPE and DU145 cells. The effects of Nodal on cell proliferation and migration are mediated via ALK4 and ActRII/ActRIIB receptors and Smad 2/3 phosphorylation. CONCLUSIONS: Nodal may function as an autocrine regulator of proliferation and migration of prostate cancer cells.

An ABC Transporter Drives Medulloblastoma Pathogenesis by Regulating Sonic Hedgehog Signaling.

Mutations in Sonic hedgehog (SHH) signaling promote aberrant proliferation and tumor growth. SHH-medulloblastoma (MB) is among the most frequent brain tumors in children less than 3 years of age. Although key components of the SHH pathway are well-known, we hypothesized that new disease-modifying targets of SHH-MB might be identified from large-scale bioinformatics and systems biology analyses. Using a data-driven systems biology approach, we built a MB-specific interactome. The ATP-binding cassette transporter ABCC4 was identified as a modulator of SHH-MB. Accordingly, increased ABCC4 expression correlated with poor overall survival in patients with SHH-MB. Knockdown of ABCC4 expression markedly blunted the constitutive activation of the SHH pathway secondary to Ptch1 or Sufu insufficiency. In human tumor cell lines, ABCC4 knockdown and inhibition reduced full-length GLI3 levels. In a clinically relevant murine SHH-MB model, targeted ablation of Abcc4 in primary tumors significantly reduced tumor burden and extended the lifespan of tumor-bearing mice. These studies reveal ABCC4 as a potent SHH pathway regulator and a new candidate to target with the potential to improve SHH-MB therapy. SIGNIFICANCE: These findings identify ABCC4 transporter as a new target in SHH-MB, prompting the development of inhibitors or the repurporsing of existing drugs to target ABCC4.

Thermostable llama single domain antibodies for detection of botulinum A neurotoxin complex.

Immunoglobulins from animals of the Camelidae family boast unique forms that do not incorporate light chains. Antigen binding in these unconventional heavy-chain homodimers is mediated through a single variable domain. When expressed recombinantly these variable domains are termed single domain antibodies (sdAb) and are among the smallest naturally IgG-derived antigen binding units. SdAb possess good solubility, thermostability, and can refold after heat and chemical denaturation making them promising alternative recognition elements. We have constructed a library of phage-displayed sdAb from a llama immunized with a cocktail of botulinum neurotoxin (BoNT) complex toxoids and panned the library for binders for BoNT A complex toxoid. Six unique binders were isolated and found to specifically bind BoNT A complex in toxoid and untoxoided forms and when used in optimal combinations in buffer and milk could detect 100 pg/mL untoxoided complex. All sdAb retained their ability to specifically bind target after heating to 85 degrees C for 1 h, in contrast to conventional polyclonal sera. All of the sdAb were highly specific for subtype A1 rather than A2 and demonstrated binding to the 33 kDa hemagglutinin, potentially to a somewhat overlapping linear epitope. The unique properties of these sdAb may provide advantages for many diagnostic applications where long-term storage and in-line monitoring require very rugged yet highly specific recognition elements.

Mouse medulloblastoma driven by CRISPR activation of cellular Myc.

MYC-driven Group 3 (G3) medulloblastoma (MB) is the most aggressive of four molecular subgroups classified by transcriptome, genomic landscape and clinical outcomes. Mouse models that recapitulate human G3 MB all rely on retroviral vector-induced Myc expression driven by viral regulatory elements (Retro-Myc tumors). We used nuclease-deficient CRISPR/dCas9-based gene activation with combinatorial single guide RNAs (sgRNAs) to enforce transcription of endogenous Myc in Trp53-null neurospheres that were orthotopically transplanted into the brains of naïve animals. Three combined sgRNAs linked to dCas9-VP160 induced cellular Myc expression and large cell anaplastic MBs (CRISPR-Myc tumors) which recapitulated the molecular characteristics of mouse and human G3 MBs. The BET inhibitor JQ1 suppressed MYC expression in a human G3 MB cell line (HD-MB03) and CRISPR-Myc, but not in Retro-Myc MBs. This G3 MB mouse model in which Myc expression is regulated by its own promoter will facilitate pre-clinical studies with drugs that regulate Myc transcription.

Inactivation of Ezh2 Upregulates Gfi1 and Drives Aggressive Myc-Driven Group 3 Medulloblastoma.

The most aggressive of four medulloblastoma (MB) subgroups are cMyc-driven group 3 (G3) tumors, some of which overexpress EZH2, the histone H3K27 mono-, di-, and trimethylase of polycomb-repressive complex 2. Ezh2 has a context-dependent role in different cancers as an oncogene or tumor suppressor and retards tumor progression in a mouse model of G3 MB. Engineered deletions of Ezh2 in G3 MBs by gene editing nucleases accelerated tumorigenesis, whereas Ezh2 re-expression reversed attendant histone modifications and slowed tumor progression. Candidate oncogenic drivers suppressed by Ezh2 included Gfi1, a proto-oncogene frequently activated in human G3 MBs. Gfi1 disruption antagonized the tumor-promoting effects of Ezh2 loss; conversely, Gfi1 overexpression collaborated with Myc to bypass effects of Trp53 inactivation in driving MB progression in primary cerebellar neuronal progenitors. Although negative regulation of Gfi1 by Ezh2 may restrain MB development, Gfi1 activation can bypass these effects.

The Interaction of Myc with Miz1 Defines Medulloblastoma Subgroup Identity.

Four distinct subgroups of cerebellar medulloblastomas (MBs) differ in their histopathology, molecular profiles, and prognosis. c-Myc (Myc) or MycN overexpression in granule neuron progenitors (GNPs) induces Group 3 (G3) or Sonic Hedgehog (SHH) MBs, respectively. Differences in Myc and MycN transcriptional profiles depend, in part, on their interaction with Miz1, which binds strongly to Myc but not MycN, to target sites on chromatin. Myc suppresses ciliogenesis and reprograms the transcriptome of SHH-dependent GNPs through Miz1-dependent gene repression to maintain stemness. Genetic disruption of the Myc/Miz1 interaction inhibited G3 MB development. Target genes of Myc/Miz1 are repressed in human G3 MBs but not in other subgroups. Therefore, the Myc/Miz1 interaction is a defining hallmark of G3 MB development.

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.

The phytoalexin camalexin mediates cytotoxicity towards aggressive prostate cancer cells via reactive oxygen species.

Camalexin is a phytoalexin that accumulates in various cruciferous plants upon exposure to environmental stress and plant pathogens. Besides moderate antibacterial and antifungal activity, camalexin was reported to also exhibit antiproliferative and cancer chemopreventive effects in breast cancer and leukemia. We studied the cytotoxic effects of camalexin treatment on prostate cancer cell lines and whether this was mediated by reactive oxygen species (ROS) generation. As models, we utilized LNCaP and its aggressive subline, C4-2, as well as ARCaP cells stably transfected with empty vector (Neo) control or constitutively active Snail cDNA that represents an epithelial to mesenchymal transition (EMT) model and displays increased cell migration and tumorigenicity. We confirmed previous studies showing that C4-2 and ARCaP-Snail cells express more ROS than LNCaP and ARCaP-Neo, respectively. Camalexin increased ROS, decreased cell proliferation, and increased apoptosis more significantly in C4-2 and ARCaP-Snail cells as compared to LNCaP and ARCaP-Neo cells, respectively, while normal prostate epithelial cells (PrEC) were unaffected. Increased caspase-3/7 activity and increased cleaved PARP protein shown by Western blot analysis was suggestive of increased apoptosis. The ROS scavenger N-acetyl cysteine (NAC) antagonized the effects of camalexin, whereas the addition of exogenous hydrogen peroxide potentiated the effects of camalexin, showing that camalexin is mediating its effects through ROS. In conclusion, camalexin is more potent in aggressive prostate cancer cells that express high ROS levels, and this phytoalexin has a strong potential as a novel therapeutic agent for the treatment of especially metastatic prostate cancer.

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.

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.