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

Abstract

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.

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

“…All cells were cultured in the recommended growth media at 37°C with 5% CO 2 and 100% humidity as described previously (29,64).…”

“…4 l of the diluted cDNA were added to separate RT-PCR mixtures following established procedures. All gene-specific primers were designed with Beacon-Designer version 5.0 as described previously (29). The following primers were used: c-Jun forward, 5Ј-TGGAAACG-ACCTTCTATGACGA-3Ј; c-Jun reverse, 5Ј-GTTGCT-GGACTGGATTATCAGG-3Ј; JunB forward, 5Ј-TACCACG-ACGACTCATACACA-3Ј; JunB reverse, 5Ј-CGCTTTGAGA-CTCCGGTAGG-3Ј; JunD forward, 5Ј-CAAACCCTGCCTT-TCCTTTAC-3Ј; JunD reverse, 5Ј-GGCGAACCAAGGAT-TACAAA-3Ј; L19 forward, 5Ј-GAAATCGCCAATGCCAA-CTC-3Ј; L19 reverse 5Ј-TCTTAGACCTGCGAGCCTCA-3Ј.…”

“…On the other hand, TGF-␤ induces cell migration and invasion in PC3 cells but does not affect migration and invasion in DU145 cells (16,29,64). Because both DU145 and PC3 cells express functional TGF-␤ receptors and Smad signaling (15,29) but exhibit differential responses to TGF-␤ treatment, we have exploited these cell lines to understand differences in the signaling mechanism that may act either downstream or parallel to Smad signaling and are responsible for their differential responses to TGF-␤. Our results show that TGF-␤ induces a reduction in JunD protein levels in DU145 cells but not in PC3 cells.…”

“…It has been well established that extensive interactions exist between the TGF-␤ signaling pathway and other major signaling pathways, including Wnt, Notch, Hedgehog, JNK, MAPK, and AKT/PI3K (20 -24). It is also becoming apparent that TGF-␤ signaling intersects with several transcription factors and regulators, such as GL1, SOX4, Tieg3/Klf11, Id, and AP-1 proteins (25)(26)(27)(28)(29). Many studies have implicated AP-1 proteins in TGF-␤ signaling (30 -32).…”

Chlamydia pneumoniae Infection Promotes Vascular Smooth Muscle Cell Migration through a Toll-Like Receptor 2-Related Signaling Pathway

“…LY294002 can also inhibit fibroblast growth factor 2-stimulated migration of the periodontal ligament cells through suppressing Akt activation (32). Recently, Walker et al (47) reported that transforming growth factor ␤3 significantly induced motility of human prostate cancer PC3 cells by increasing the level of phosphorylated Akt (Ser 473), and LY294002 blocked transforming growth factor ␤3-stimulated migration via the suppression of Akt phosphorylation at Ser 473. These studies revealed that Akt phosphorylation is closely associated with cell migration, supporting our hypothesis that C. pneumoniae infection promotes VSMC migration possibly by inducing Akt phosphorylation.…”