Aberrant overexpression of Mcl-1 has been associated with poor prognosis and resistance to chemotherapy in a variety of human cancers [36
]. Sensitizing tumor cells to apoptosis induction by selectively targeting Mcl-1, in combination with conventional chemotherapy, has emerged as an attractive therapeutic strategy [3
]. In this study, we presented evidence that elevated Mcl-1 expression is associated with clinical PCa progression, particularly bone metastasis. We further showed that activation of VEGF-NRP1-c-MET signaling is responsible for Mcl-1 expression, which may confer survival advantages allowing PCa cells to evade apoptosis and progress towards invasive states (Figure ).
Only limited information are available on Mcl-1 expression profile in PCa. An early study found that Mcl-1 expression was increased at tumors (81%) compared with only 38% in PIN. The percentage of Mcl-1-positive cells was typically higher in Gleason grade 8-10 tumors and metastasis than lower grade tumors, but there was no significant difference in Mcl-1 immunointensity between high grade tumors (47%) and metastasis in lymph node (38%) and bone (50) [5
]. Our present study confirmed elevated Mcl-1 expression in high grade (≥ 7) PCa, though the difference between Gleason score 7 and 8-10 tumors is not statistically significant. Intriguingly, our results revealed a remarkable increase in Mcl-1 immunointensity in bone metastasis compared to primary tumors, indicating that Mcl-1 overexpression is positively correlated to PCa progression towards metastatic status in clinical situation.
Accumulating evidence suggests that the function of VEGF in tumor progression may not be limited to angiogenesis [38
]. Numerous tumor cells express significant levels of VEGF-Rs, which could engage VEGF and initiate multiple signaling responses involved in cell proliferation, survival and migration. VEGF autocrine signaling confers a degree of self-sufficiency that may be crucial to metastasis as the microenvironment becomes increasingly hostile. Nonetheless, it remains controversial whether VEGF has significant autocrine effects in PCa cells, since the "classical" VEGF-Rs, i.e., VEGF-R1 and -R2, are undetectable in most established PCa cell lines [11
]. Previously we reported that serum VEGF is positively associated with bone metastatic status in PCa patients, and recapitulated this close association in the ARCaP model [18
]. In this study, we investigated whether VEGF could affect PCa cell behavior in an autocrine manner. Intriguingly, VEGF165
was found to be capable of inducing Mcl-1 expression within a non-saturating range, suggesting it may act as a survival factor in PCa cells. Moreover, NRP1 was found to be highly expressed by PCa cell lines and displayed a positive association with invasiveness, suggesting that it may be the primary receptor responsible for VEGF autocrine effects in PCa cells. Gene transfer experiments supported an indispensible role of NRP1 in mediating VEGF165
regulation of Mcl-1 in metastatic PCa cells. Importantly, a positive association between NRP1 expression and in vivo
bone metastatic potential was found in ARCaPM
xenografts and further confirmed in clinical PCa specimens. These data collectively support a novel role of NRP1 in PCa progression and metastasis, presumably by transmitting the VEGF autocrine survival signal and enabling PCa cells to evade apoptosis.
NRP1 is not a typical "signaling receptor" since it lacks sequences predicted to have kinase receptor activities [24
]. Direct interaction between NRP1 and VEGF-Rs or plexin A is essential to its function in prioritizing differential signals from VEGF165
or semaphorin 3 in endothelial cells and nerve cells [24
]. Recent studies found that the NRP1 intracellular domain, especially the C-terminal three amino acids (SEA-COOH), may be required for the interaction between NRP1 and its binding partners, including NRP1-interacting protein (NIP; or RGS-GAIP-interacting protein, GIPC) and VEGF-R2 [41
]. However, the molecular effects and mechanism for NRP1-mediated signaling in tumor cells, especially in the absence of VEGF-Rs, remain elusive. It has been proposed that NRP1 may store or sequester VEGF165
and attract endothelial cells towards tumor, contributing to angiogenesis via
juxtacrine or paracrine mechanisms [9
]. In this study, we investigated whether NRP1 is required for VEGF165
autocrine signaling in PCa cells lacking VEGF-Rs. We presented evidence that NRP1 and c-MET are physically associated on plasma membrane, and in response to VEGF165
stimulation, their interaction may significantly facilitate further recruitment and activation of c-MET. Though the structural and molecular basis for interaction between the two receptors needs to be further investigated, this study indicates for the first time that c-MET activity can be modulated by VEGF, and the NRP1-c-MET interaction may be a critical component in transmitting the VEGF survival signal in PCa cells. It may be plausible to assign c-MET phosphorylation as an indicator for activation of NRP1 autocrine signaling in tumor cells. Using human PCa tissue specimens as the "gold standard", we observed that NRP1 and p-c-MET were both significantly increased with progression of primary PCa, and further elevated in bone metastatic PCa, suggesting that activation of NRP1-c-MET signaling may be positively associated with clinical PCa progression. It is worth noting that these results need cautious interpretation since a limited number of patient specimens was examined, and the polyclonal antibody against p-c-MET has not been fully validated for immunohistochemical analysis in tissue sections.
In human primary hepatocytes, HGF transcriptionally induced Mcl-1 expression, but not Bcl-2 or Bcl-x(L) [27
]. Our studies found that, however, HGF treatment did not significantly affect Mcl-1 protein expression in ARCaPM
cells, suggesting HGF activation of c-MET signaling may not be sufficient to increase Mcl-1 expression. We postulated that VEGF165
may utilize a different mechanism to regulate Mcl-1 expression in PCa cells. An important observation supporting this hypothesis is that VEGF165
only rapidly induced Stat3 phosphorylation at Tyr705, but not altering Ser727 phosphorylation in ARCaPM
cells. In hepatocytes, however, HGF triggered Ser727, but not Tyr705, phosphorylation of Stat3 [27
]. It will be intriguing to investigate the role of NRP1-c-MET interaction in differentiating extracellular ligands, i.e., VEGF165
or HGF, and activating distinct downstream cascades leading to Mcl-1 expression in a highly cell context-dependent fashion.
Mcl-1 is a protein with very short half-life and is highly regulated at multiple levels in response to survival and differentiation signals. Rapid turnover of Mcl-1 protein can be initiated by stress (such as serum withdrawal) through caspase-mediated and proteasome-dependent degradation in tumor cells [3
]. Previous study found that VEGF protects multiple myeloma cells against apoptosis by up-regulating Mcl-1 protein in a time-dependent manner, peaking at 6 h and returning to baseline after 24 h; change in Mcl-1 mRNA expression was not reported [43
]. In ARCaPM
cells, however, VEGF165
treatment rapidly induced Mcl-1 mRNA expression and significantly increased Mcl-1 protein level at 72 h. Though whether VEGF165
may also regulate Mcl-1 expression at other levels needs to be further investigated, the data presented here suggest that transcriptional activation, presumably mediated by Stat3, is an important mechanism for VEGF165
induction of Mcl-1 in PCa cells.
Interrupting NRP1 functions with mimetic peptides and monoclonal antibodies is being developed in xenograft models of human cancers [44
]. NRP1 monoclonal antibody has been shown to effectively inhibit tumor vascular remodeling, rendering vessels more susceptible to anti-VEGF therapy [45
]. If the VEGF165
-NRP1-c-MET pathway is required for Mcl-1 expression and survival in PCa cells, it would be intriguing to evaluate whether targeting NRP1 could interrupt both NRP1-c-MET signaling in tumor cells (survival) and NRP1-VEGF-Rs signaling in endothelial cells (angiogenesis). Inhibition of NRP1 signaling may be a promising strategy alone or in combination with other therapeutic approaches for treating PCa.