Osteoblast-derived factors promote metastatic potential in human prostate cancer cells, in part via non-canonical transforming growth factor ß (TGFß) signaling.

BACKGROUND: Transforming growth factor ß (TGFß) functions as a double-edged sword in prostate cancer tumorigenesis. In initial stages of the disease, TGFß acts as a growth inhibitor upon tumor cells, whereas it in later stages of disease rather promotes invasion and metastatic potential. One well-known cellular source of TGFß in the bone metastatic site is the bone-forming osteoblasts. Here we have studied the effects by osteoblast-derived factors on metastatic potential in several human prostate cancer cell lines. METHODS: Effects on metastatic potential in prostate cancer cells by osteoblast-derived factors were studied in vitro using several methods, including Transwell migration and evaluation of formation of pro-migratory protrusions. Confocal microscopy was used to evaluate possible changes in differentiation state in tumor cells by analysis of markers for epithelial-to-mesenchymal transition (EMT). The Matrigel-on-top 3D culture method was used for further assessment of metastatic characteristics in tumor cells by analysis of formation of filopodium-like protrusions (FLPs). RESULTS: Osteoblast-derived factors increased migration of PC-3U cells, an effect less prominent in cells overexpressing a mutated type I TGFß receptor (TßRI) preventing non-canonical TRAF6-dependent TGFß signaling. Osteoblast-derived factors also increased the formation of long protrusions and loss of cell-cell contacts in PC-3U cells, suggesting induction of a more aggressive phenotype. In addition, treatment with TGFß or osteoblast-derived factors of PC-3U cells in Matrigel-on-top 3D cultures promoted formation of FLPs, previously shown to be essential for metastatic establishment. CONCLUSIONS: These findings suggests that factors secreted from osteoblasts, including TGFß, can induce several cellular traits involved in metastatic potential of PC-3U cells, further strengthening the role for bone cells to promote metastatic tumor cell behavior.

Pro-invasive properties of Snail1 are regulated by sumoylation in response to TGFß stimulation in cancer.

Transforming growth factor ß (TGFß) is a key regulator of epithelial-to-mesenchymal transition (EMT) during embryogenesis and in tumors. The effect of TGFß, on ΕΜΤ, is conveyed by induction of the pro-invasive transcription factor Snail1. In this study, we report that TGFß stimulates Snail1 sumoylation in aggressive prostate, breast and lung cancer cells. Sumoylation of Snail1 lysine residue 234 confers its transcriptional activity, inducing the expression of classical EMT genes, as well as TGFß receptor I (TßRI) and the transcriptional repressor Hes1. Mutation of Snail1 lysine residue 234 to arginine (K234R) abolished sumoylation of Snail1, as well as its migratory and invasive properties in human prostate cancer cells. An increased immunohistochemical expression of Snail1, Sumo1, TßRI, Hes1, and c-Jun was observed in aggressive prostate cancer tissues, consistent with their functional roles in tumorigenesis.

TRAF6 promotes TGFß-induced invasion and cell-cycle regulation via Lys63-linked polyubiquitination of Lys178 in TGFß type I receptor.

Transforming growth factor ß (TGFß) can act either as a tumor promoter or a tumor suppressor in a context-dependent manner. High levels of TGFß are found in prostate cancer tissues and correlate with poor patient prognosis. We recently identified a novel TGFß-regulated signaling cascade in which TGFß type I receptor (TßRI) is activated by the E3 ligase TNF-receptor-associated factor 6 (TRAF6) via the Lys63-linked polyubiquitination of TßRI. TRAF6 also contributes to activation of TNF-α-converting enzyme and presenilin-1, resulting in the proteolytic cleavage of TßRI and releasing the intracellular domain of TßRI, which is translocated to the nucleus to promote tumor invasiveness. In this report, we provide evidence that Lys178 of TßRI is polyubiquitinated by TRAF6. Moreover, our data suggest that TRAF6-mediated Lys63-linked ubiquitination of the TßRI intracellular domain is a prerequisite for TGFß regulation of mRNA for cyclin D1 (CCND1), expression, as well as for the regulation of other genes controlling the cell cycle, differentiation, and invasiveness of prostate cancer cells.

TRAF6 stimulates the tumor-promoting effects of TGFß type I receptor through polyubiquitination and activation of presenilin 1.

Transforming growth factor-ß (TGFß) can be both a tumor promoter and suppressor, although the mechanisms behind the protumorigenic switch remain to be fully elucidated. The TGFß type I receptor (TßRI) is proteolytically cleaved in the ectodomain region. Cleavage requires the combined activities of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and TNF-α-converting enzyme (TACE). The cleavage event occurs selectively in cancer cells and generates an intracellular domain (ICD) of TßRI, which enters the nucleus to mediate gene transcription. Presenilin 1 (PS1), a γ-secretase catalytic core component, mediates intramembrane proteolysis of transmembrane receptors, such as Notch. We showed that TGFß increased both the abundance and activity of PS1. TRAF6 recruited PS1 to the TßRI complex and promoted lysine-63-linked polyubiquitination of PS1, which activated PS1. Furthermore, PS1 cleaved TßRI in the transmembrane domain between valine-129 and isoleucine-130, and ICD generation was inhibited when these residues were mutated to alanine. We also showed that, after entering the nucleus, TßRI-ICD bound to the promoter and increased the transcription of the gene encoding TßRI. The TRAF6- and PS1-induced intramembrane proteolysis of TßRI promoted TGFß-induced invasion of various cancer cells in vitro. Furthermore, when a mouse xenograft model of prostate cancer was treated with the γ-secretase inhibitor DBZ {(2S)-2-[2-(3,5-difluorophenyl)-acetylamino]-N-(5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)-propionamide}, generation of TßRI-ICD was prevented, transcription of the gene encoding the proinvasive transcription factor Snail1 was reduced, and tumor growth was inhibited. These results suggest that γ-secretase inhibitors may be useful for treating aggressive prostate cancer.

TRAF6 ubiquitinates TGFß type I receptor to promote its cleavage and nuclear translocation in cancer.

Transforming growth factor ß (TGFß) is a pluripotent cytokine promoting epithelial cell plasticity during morphogenesis and tumour progression. TGFß binding to type II and type I serine/threonine kinase receptors (TßRII and TßRI) causes activation of different intracellular signaling pathways. TßRI is associated with the ubiquitin ligase tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6). Here we show that TGFß, via TRAF6, causes Lys63-linked polyubiquitination of TßRI, promoting cleavage of TßRI by TNF-alpha converting enzyme (TACE), in a PKCζ-dependent manner. The liberated intracellular domain (ICD) of TßRI associates with the transcriptional regulator p300 to activate genes involved in tumour cell invasiveness, such as Snail and MMP2. Moreover, TGFß-induced invasion of cancer cells is TACE- and PKCζ- dependent and the TßRI ICD is localized in the nuclei of different kinds of tumour cells in tissue sections. Thus, our data reveal a specific role for TßRI in TGFß mediated tumour invasion.