The purpose of this study was to establish the precise mechanism by which KAI1 (CD82), a metastasis-suppressor gene initially discovered in prostate cancer, inhibits Src and affects the metastasis of prostate carcinoma. Clinical cancer studies have linked a high level of KAI1 expression with a good prognosis. Conversely, the loss of KAI1 expression is correlated with metastasis in tumor progression [3, 5]. However, how KAI1 functions to suppress metastasis has not yet been clearly established. Tetraspanins, including KAI1, may play a role in the plasma membrane as organizers of multi-molecular complexes that contain not only tetraspanins but also numerous cell membrane components. The TEM has been suggested to serve as a signal-networking platform that shifts the characteristics of a cancer between primary tumor and a metastatic phenotype [30, 31]. Thus, it is of special interest to define the constituents of this special domain and their interrelationships.
Current studies have proposed that KAI1 functions to inhibit Src kinase activity, but the identified mechanisms have been limited to associations with integrins, EGFR, and c-Met . It has been proposed that KAI1 attenuates EGFR signaling and inhibits cell motility in breast cancer . However, other study reported no alteration of EGFR signaling in response to KAI1 expression in DU145 and PC3 prostate cancer cell lines . It is generally accepted that c-Met can play a role in the metastasis-suppression activity of KAI1 in prostate cancer [33, 34]; however, inhibition of this pathway and Src kinase by KAI1 expression appear to be independent of one another . Similarly, and consistent with a previous report , we found no connection between integrin complexes and Src inhibition, showing that depletion of integrin β1, the major component of integrin complexes, had no specific effect on Src phosphorylation status but decrease of phosphorylated FAK and p130CAS, downstream effector of integrin complexes, in PC3 cells (Figure 2A). Therefore, the absence of a connection between these KAI1 targets and inhibition of Src kinase activity prompted us to investigate another target of KAI1.
Together with KAI1, CDCP1 has been reported to be a component of the TEM in colon cancer, but its function in this context has remained poorly characterized . CDCP1 is a transmembrane protein that primarily associates with Src and PKCδ in a phosphorylation-dependent manner and may act as a scaffold for various interacting proteins in the plasma membrane . Generally, CDCP1 is highly phosphorylated and functionally activated in metastatic cancer; dysregulated expression of CDCP1 is associated with tumor malignancy [14, 36–38]. Tyrosine phosphorylated CDCP1 has been linked to cell detachment from the extracellular matrix and subsequent cell migration . The kinase activity of Src is essential for the initial CDCP1 phosphorylation; Src-activated CDCP1, in turn, further potentiates Src kinase activity [25, 40], although the mechanism for this latter effect is not clear. Our data showed that KAI1-induced inhibition of CDCP1 protein resulted in an almost complete elimination of Src phosphorylation (Figure 2F). This observation suggests that the function of CDCP1 protein, which acts as a positive regulator of Src kinase, may be lost due to KAI1 expression in prostate cancer, raising the question of the possible existence of a feedback control mechanism between CDCP1 and Src.
CDCP1 gene regulation has been investigated in various cancer cell lines. PC3 cells show abundant expression of CDCP1 protein and a low frequency of methylation in transcription-initiation sites . We found that both HMW and LMW forms of CDCP1 are expressed in PC3 cells (Figure 2). Although the precise mechanism by which CDCP1 is cleaved has not been defined, proteolytic processing by a serine protease results in the generation of the LMW form of CDCP1 and a subsequent increase in LMW phosphorylation . Our data clearly showed that KAI1 expression decreased the level of both HMW and LMW forms without altering CDCP1 mRNA levels (Figure 2), suggesting that the KAI 1-induced decrease in CDCP1 in PC3 cells reflects the operation of a posttranscriptional mechanism. To test this, we initially treated PC3 cells with cytochalasin D, which blocks cytoskeletal movement, with the goal of determining whether KAI1 affected the endocytic trafficking of CDCP1. We found no effect of this actin-depolymerizing reagent on the KAI1-induced decrease in CDCP1 (data not shown). As demonstrated in previously reported proteomic analyses, CDCP1 and KAI1 are colocalized with various proteases in the TEM , raising the possible involvement of KAI1 in protease activity. Since serine protease-induced proteolysis of CDCP1 has been documented , we treated PC3 cells with protease inhibitors and monitored the levels of CDCP1 under conditions of KAI1 over-expression, but found no significant change in either HMW or LMW forms of CDCP1 (data not shown). Finally, to test the involvement of proteasome-dependent degradation in the process of CDCP1 down-regulation, we treated PC3 cells with the proteasome inhibitor, MG132, and found that inhibiting the proteasome prevented the KAI1-dependent reduction in CDCP1 levels (Figure 3A). Because CDCP1 is reported to localize in the TEM of colon cancer by proteomic analysis, we could not exclude a possible interaction between KAI1 and CDCP1. In experiments designed to determine whether these two membrane proteins were co-localized in PC3 cells, it was hard to detect an interaction of CDCP1 with KAI1 due to KAI1-induced loss of CDCP1 protein level. Our observations indicate that KAI1-induced down-regulation of CDCP1 reflects effects on CDCP1 protein stability, but unknown mechanisms may also participate in this phenomenon.
Angiogenesis is an indispensible step in the progression of prostate cancer, and VEGF has emerged as a critical proangiogenic growth factor in prostate carcinogenesis [22, 42, 43]. Hypoxia is the environmental factor best known for its ability to induce cancer metastasis; it also stabilizes HIF-1α and up-regulates the expression of VEGF, which, in turn, induces the formation of tumor-feeding vessels . Moreover, it is known that Src kinase not only affects cell proliferation and migration, it also control angiogenesis via up-regulation of VEGF expression . However, few studies have focused on the role of KAI1 activity in VEGF expression and vasculature formation in tumors. Thus, given the critical role of KAI1 in inhibiting Src kinase and our motivation to identify a plausible mechanism to account for KAI1 effects in carcinogenesis, we examined HIF-1α and VEGF expression in the context of KAI1 expression. We found that HIF-1α and VEGF expression were dramatically inhibited upon restoration of KAI1 expression (Figures 4 and 5). These findings were supported by the results obtained using an in vivo xenograft model, which showed that KAI1-expressing tumor volumes were significantly reduced compared with those of controls (Figure 6). Moreover, whereas CDCP1 and HIF-1α expression were reduced in KAI1-expressing tumor tissue, VHL expression was clearly augmented. Thus, our data provide a mechanistic basis for the clear correlation between the loss of KAI1 in cancer and poor prognosis. It has recently been demonstrated that Src can promote oncogenesis through destabilization of the VHL tumor suppressor . Importantly, functional inactivation of VHL, including through germline mutations, has been well documented in highly vascularized tumors such as renal cell carcinomas, hemangiosarcomas, and pheochromocytomas . The tumor-suppressive function of VHL is best viewed in the context of its role as an E3 ubiquitin ligase that targets various substrates, including HIF-1α and atypical PKC . Two forms of VHL, approximately 30 and 18 kDa, exist, but the significance of the two forms is unknown . It has been suggested that VHL is extremely unstable when not complexed with components of ubiquitin ligase complexes, such as elongin B and elongin C . This observation may account for the difficulty in detecting VHL protein expression and may help to explain why most VHL functions have been examined by overexpressing or immunoprecipitating VHL. We observed that VHL protein levels were increased by KAI1 expression (Figure 3B, C). How KAI1 negatively regulates CDCP1 remains uncertain, but our data clearly suggest that this effect of KAI1 involves the regulation of CDCP1 protein stability. Intriguingly, VHL over-expression decreased CDCP1 levels; this result was confirmed by treating cells with siRNA against VHL, which had the opposite effect on CDCP1 levels (Figure 3D). VHL protein is expressed in various normal and cancer tissues, where it is localized in the cytosol or membrane . In human renal cell carcinoma, its presence in the membrane is significantly associated with a missense mutation [10, 47]. These reports prompted us to further consider changes in subcellular localization of VHL in the process of tumorigenesis. In addition, we observed the existence of VHL in both of cytosol and membrane fractions in VHL overexpressing PC3 cells (data not shown). This observation raises the possibility of recruitment of VHL from cytosol to plasma membrane and ubiquitination of unknown substrates located in the membrane. Although we could not observe co-localization of CDCP1, VHL and KAI1 in cell membrane or TEM, these proteins would be expected to coexist in the plasma membrane during the process of prostate carcinogenesis.