In vivo cleaved CDCP1 promotes early tumor dissemination via complexing with activated β1 integrin and induction of FAK/PI3K/Akt motility signaling

Abstract

Specific cleavage of the transmembrane molecule, CUB domain-containing protein-1 (CDCP1), by plasmin-like serine proteases induces outside–in signal transduction that facilitates early stages of spontaneous metastasis leading to tumor cell intravasation, namely cell escape from the primary tumor, stromal invasion and transendothelial migration. We identified active β1 integrin as a biochemical and functional partner of the membrane-retained 70-kDa CDCP1 fragment, newly generated from its full-length 135-kDa precursor though proteolytic cleavage by serine proteases. Both in cell cultures and in live animals, active β1 integrin complexed preferentially with functionally activated, phosphorylated 70-kDa CDCP1. Complexing of β1 integrin the 70-kDa with CDCP1 fragment induced intracellular phosphorylation signaling, involving focal adhesion kinase-1 (FAK) and PI3 kinase (PI3K)-dependent Akt activation. Thus, inhibition of FAK/PI3K activities by specific inhibitors as well as short-hairpin RNA downregulation of β1 integrin significantly reduced FAK/Akt phosphorylation under conditions where CDCP1 was processed by serine proteases, indicating that FAK/PI3K/Akt pathway operates downstream of cleaved CDCP1 complexed with β1 integrin. Furthermore, this complex-dependent signaling correlated positively with high levels of tumor cell intravasation and dissemination. Correspondingly, abrogation in vivo of CDCP1 cleavage either by unique cleavage-blocking monoclonal antibody 10-D7 or by inhibition of proteolytic activity of plasmin-like serine proteases with aprotinin prevented β1 integrin/CDCP1 complexing and downstream FAK/Akt signaling concomitant with significant reduction of stromal invasion and spontaneous metastasis. Therefore, β1 integrin appears to serve as a motility-regulating partner mediating cross-talk between proteolytically cleaved, membrane-retained CDCP1 and members of FAK/PI3K/Akt pathway. This CDCP1 cleavage-induced signaling cascade constitutes a unique mechanism, independent of extracellular matrix remodeling, whereby a proteolytically cleaved CDCP1 regulates in vivo locomotion and metastasis of tumor cells through β1 integrin partnering. Our findings indicate that CDCP1 cleavage, occurring at the apex of a β1 integrin/FAK/PI3K/Akt signaling cascade, may represent a therapeutic target for CDCP1-positive cancers.

Elevated CDCP1 predicts poor patient outcome and mediates ovarian clear cell carcinoma by promoting tumor spheroid formation, cell migration and chemoresistance

“…At the time of killing on day 24, mice treated with combined 10D7 and of OCC patients, and our finding that CDCP1 is expressed by the vast majority of OCC tumors but not by normal ovaries is encouraging that this protein could be selectively targeted in OCC without impacting on normal cells and structures present within the tumor environment. CDCP1 has previously been shown to protect cancer cells that metastasize via the vasculature, [18][19][20] and our data are the first to show that this protein is critical in processes essential for the peritoneal metastasis characteristic of OCC. In particular, for this distinct type of metastasis it is a key mediator for formation and growth of the tumor spheroids that promote the anchorage-independent survival, and seeding of cancer cells on peritoneal organs and structures that is essential for intraperitoneal dissemination of OCC.…”

“…Based on this analysis, TOV21G, KOC7C, OVTOKO and RMG-I cells were selected for reduction of CDCP1 expression, and KK cells for overexpression. Stable lentiviral-mediated shRNA silencing reduced CDCP1 levels by 490% in TOV21G, KOC7C (Figure 2b As pro-survival signaling involving proteolytic conversion of CDCP1 to 70 kDa requires Akt signaling, 18,20 we assessed whether activation of this kinase is altered in the three cell lines modulated for CDCP1 expression. As shown in Figure 2b, western blot analysis showed that modulation of CDCP1 was sufficient to robustly impact levels of pAkt in these OCC cell lines, and this was independent of cleavage of CDCP1.…”

“…Consistent with CDCP1 having a role in sphere formation, western blot analysis showed that CDCP1 levels were markedly higher in SFCs compared with NSFCs ( Figure 5b, non-adhered). In addition, analysis of Akt, Src and ERK (extracellular-signal-regulated kinase) pathways that are important in CDCP1-mediated signaling, 18,20,[23][24][25] demonstrated that each is activated in SFCs compared with NSFCs ( Figure 5b, non-adhered). Interestingly, when both SFCs and NSFCs were replated onto cell culture dishes for adherent growth, levels of CDCP1 and activation of Akt, Src and ERK returned to basal levels (Figure 5b, adhered).…”

“…18,20 In these in vivo models, proteolytic conversion of 135 kDa CDCP1 to 70 kDa p-CDCP1-Y734 initiated docking of this cleaved and phosphorylated form of CDCP1 to β1 integrin, and the focal adhesion kinase 1 and PI3K-dependent activation of Akt is critical for cell survival during extravasation. [18][19][20] Our data indicate a novel mechanism of CDCP1-mediated activation of Akt in OCC that is distinct to the serine protease-mediated conversion of 135 to 70 kDa p-CDCP1-Y734 that promotes hematogenous metastasis via pAkt. Rather than proteolysis of CDCP1 being the initiator of Akt activation, increased CDCP1 expression was sufficient to induce pAkt-S473, and this was independent of both Src and ERK signaling.…”

EGF inhibits constitutive internalization and palmitoylation-dependent degradation of membrane-spanning procancer CDCP1 promoting its availability on the cell surface

“…Based on published analysis of patient cohorts and animal models, antibody targeting of CDCP1 has been proposed for cancer treatment (9,10,18,20). This is supported by molecular analysis demonstrating that CDCP1 is pro-cancerous in vivo by promoting cell survival via pathways involving Src, PKCδ, FAK, PI3K and Akt (14,18,19), and that anti-CDCP1 antibodies efficiently block survival, inducing apoptosis (18)(19)(20)22). It is also supported by a recent mechanistic study showing that an anti-CDCP1 monoclonal antibody that induces lipid raft translocation, internalization and degradation of CDCP1, blocked subcutaneous growth in mice of established human cell line xenograft tumors (21).…”