The alteration of the cellular response to TGF-β is a key step in the development of prostate cancer (1
). The loss of the cellular response to specific BMPs is also implicated in prostate carcinogenesis (33
). Although the Smad pathway is the canonical signaling mechanism activated by these factors (3
), the present study provides evidence for a novel Smad-independent TGF-β effector that regulates cell migration via phosphorylation of endoglin by ALK2. We previously demonstrated that ALK1 directly phosphorylates endoglin in human umbilical vein endothelial cells and, as a consequence, the ALK1-dependent inhibition of cell adhesion and proliferation in these cells is inhibited (15
). We now show that endoglin is phosphorylated in prostate cancer cells and, as in endothelial cells, the phosphorylated residues are predominantly the five threonines in the CD. Importantly, this finding confirms that endoglin phosphorylation is not an isolated mechanism that operates only in endothelial cells but supports the view that it is a more broadly significant mechanism for the regulation of endoglin function.
Despite the foregoing similarities, we observed striking differences between endoglin phosphorylation in endothelial and prostate cancer cells. First, detection of endoglin phosphorylation in prostate cancer cell lines is much more technically demanding than for endothelial cells (15
), probably reflecting lower levels and higher rates of turnover of phosphorylated endoglin serine and threonine residues in non-endothelial cells. The lower levels of endoglin in PC3 cells also suggests a closer stoichiometric relationship between endoglin in TGF-β receptors in prostate cancer cells than is the case in endothelial cells, where endoglin is probably in excess of TGF-β ligand and receptor levels (6
). Second, in prostate cancer cells, we did not detect basal phosphorylation of endoglin on serine residues, suggesting that the turnover of serine-linked phosphates is higher in these cells than in endothelial cells. Third, different type I receptors phosphorylate endoglin in endothelial cells (ALK1) versus prostate cancer cells (ALK2 and ALK5). Finally, threonine phosphorylation of ΔPDZ-endoglin is dramatically enhanced in endothelial cells (15
), whereas it was virtually abolished for the same mutated protein in prostate cancer cells. This result suggests that the PDZ-binding motif, which has been implicated in endoglin interaction with other cellular proteins including β-arrestin (18
), is involved in cell type-specific processes.
In the present study, we determined that ALK2 and ALK5 phosphorylated endoglin in prostate cancer cells. Based on these results, and on the described Smad specificities of ALK2 and ALK5 (3
), we propose that upon TGF-β1 stimulation ALK5 phosphorylates Smad2 and 3 with a negative impact on ALK2-Smad1, 5 and 8 signaling. Therefore, our data suggest that ALK2 phosphorylates endoglin as an alternative substrate, and upon BMP7 stimulation, ALK2 phosphorylates endoglin without a requirement for ALK5 participation (depicted in ).
Proposed model for endoglin phosphorylation by TGF-β1 and BMP7 in prostate cancer cells and its effect in cell migration.
We demonstrate that endoglin phosphorylation affects prostate cancer cell migration. The results of our tumorigenicity experiments suggest that endoglin attenuates the progression of prostate carcinogenesis. Moreover, the tumorigenic potential of prostate cancer cells expressing an endoglin mutant that cannot be phosphorylated was the same as the tumorigenicity of endoglin-deficient cells. This result is consistent with the observations of Perez-Gomez et al.
). These authors demonstrated that L-endoglin inhibits keratinocyte-induced tumorigenicity in mice, whereas the short, S-endoglin isoform, which lacks the L-endoglin isoform CD, does not inhibit tumorigenicity, thus supporting the view that TGF-β receptor-dependent endoglin phosphorylation regulates tumorigenicity in vivo
Another functional consequence of endoglin phosphorylation is that phosphorylated endoglin no longer exerts its inhibitory effect on prostate cancer cell migration (2
). The present study supports a role for endoglin as a novel element in TGF-β1-dependent regulation of cancer cell migration. TGF-β1 stimulates cell migration via ALK5/Smad2 and 3 (2
). In addition, in response to TGF-β1, endoglin is phosphorylated and its inhibitory effect in cell migration is blocked ().
This work advances the hypothesis that ALK2 plays a dual role in prostate cancer cell migration depending on the available substrates for its kinase activity. When ALK2 phosphorylates Smad1, the net result is the inhibition of cell migration (2
). However, when ALK2 phosphorylates endoglin, cell migration is promoted. Thus, there is an implied balance between endoglin expression levels and phosphorylation, ALK2 activation and Smad1 availability, which may explain the differences between endoglin anti-invasive action in prostate cancer versus the observation that endoglin expression on metastatic breast cancer cells promotes their invasive character (37
). Therefore, additional effects of local ligand activation and availability, receptor-substrate affinities and potential interactions between the cells and the extracellular matrix remain to be elucidated.
The present study describes for the first time that TGF-β receptor-mediated phosphorylation of endoglin is a Smad-independent mechanism involved in the regulation of prostate cancer cell migration and tumor progression. We are currently exploring novel animal models to further examine how endoglin expression in tumor cells and their microenvironment affects prostate cancer progression.