Our studies have elucidated a new step in the CDCP1 activation cascade: cCDCP1 homodimerization, which is critical for phosphorylation of PKC and migration of TNBC cells.Figure 7illustrates an updated model for CDCP1 activation, including its phosphorylation, cleavage and dimerization. We suggest that the cCDCP1 dimer acts as a docking station to facilitate PKC phosphorylation by Src: cCDCP1 dimer is important for binding of Src to one subunit and PKC to the other subunit, bringing Src and PKC into close proximity, allowing the phosphorylation of PKC by Src. This signaling cascade further leads to phospho-PKC-stimulated migration. Our model also explains the ability of flCDCP1 to bind to Src and PKC without stimulating PKC phosphorylation: Src and PKC are not bound to flCDCP1 at the same time. These data are in line withex vivodata in TNBC²andin vivodata in prostate cancer,^3,8showing that CDCP1 cleavage is required to induce cellular dissemination and multiple-organ metastasis. Although the positive and negative regulators of CDCP1 dimerization remain to be elucidated, we have shown that ECC represents a way to inhibit dimerization of cCDCP1, disrupting cCDCP1 pro-migratory signaling.

Figure 7.

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Proposed model of CDCP1 activation and signal transduction. (1) CDCP1 is cleaved on the extracellular side of the membrane between CUB1 and CUB2. Src family kinases (SFKs) (Src/Fyn/Yes) can phosphorylate both flCDCP1 and cCDCP1. (2) SFKs and PKC bind to flCDCP1 or cCDCP1 protein monomers, presumably to overlapping/close binding sites, but signal transduction from SFKs to PKC does not occur. (3) cCDCP1 is capable of forming a dimer, which allows SFKs and PKC to bind to each individual subunit of a dimer, leading to PKC phosphorylation by SFKs. Activated PKC induces pro-migratory signaling. ECC is capable of inhibiting cCDCP1 dimerization, PKC phosphorylation and migration of TNBC cells. We propose that targeting CDCP1 dimerization will lead to blockade of SFK/PKC signaling and inhibit CDCP1-mediated migration and invasion in TNBC. Black arrows, phosphorylation by SFKs; gray arrows, binding; blue, CUB domains; red, binding site; P in red circle, phosphorylation.

Our data in 3D showing an increase in ECC-expressing TNBC cell apoptosis are in line with previous studies demonstrating that CDCP1 knockdown by si/shRNA induces apoptosis in lung and breast cancer cells cultured in anchorage-free conditions,^4,39,40a study showing that inhibition of CDCP1 cleavagein vivoinduced apoptosis of prostate cancer cells⁸and studies demonstrating that a function-blocking antibody against CDCP1 inhibited cell invasiveness in a 3D culture system and tumor growth in a xenograft model of breast cancer.^41,42The induction of apoptosis in the presence of extracellular matrix components, collagen I and Matrigel (collagen IV, laminin, enactin/nidogen-1 and proteoglycans), may indicate that ECC inhibits stellate structure formation, which is indicative of cell binding and anchoring to these substrates during invasion. This explanation is supported by a study showing preferential and direct binding of ₁integrin to cCDCP1 over flCDCP1.³Our data suggest that cCDCP1 may promote ₁integrin activity, which is inhibited in the presence of ECC. It also remains to be investigated whether ECC is specific to the cCDCP1 homodimer or also inhibits cCDCP1 interactions with other proteins. The increased efficacy of ECC in 3D culture remains to be validated in an orthotopic mouse model of TNBC, where we expect the same accumulation of ECC extracellularly to inhibit cell-extracellular matrix adhesion, proliferation and induce apoptosis.

The ability of CDCP1 to form a dimer is supported by previous findings that treatment of NIH3T3 and MCF7 cells overexpressing exogenous CDCP1 with a bivalent anti-CDCP1 antibody stimulates CDCP1 activity, presumably by stimulating its clustering in lipid rafts.⁴¹Kollmorgenet al.²²proposed that CDCP1 forms a dimer through the transmembrane or intracellular dimer interface but they did not investigate the dependence of dimer formation on CDCP1 cleavage. Although we do not exclude the possibility that CDCP1 forms a dimer through transmembrane or intracellular domains as well, our data support dimer formation through the extracellular CUB domains, with the secreted ECC portion capable of inhibiting dimerization of cCDCP1. Our data show that cCDCP1 forms a dimer, although we do not exclude the possibility that it is capable of forming a tetramer or higher molecular complex. Further research is needed to test this possibility.

It remains to be investigated how flCDCP1 stimulates signaling through PKC in ccRCC. For instance, 786-0 cells express almost exclusively flCDCP1 and we have shown previously that flCDCP1 stimulates migration of ccRCC cells through PKC.¹⁰One striking difference between ccRCC and TNBC lies in abundance of lipid droplets, which is extremely high in ccRCC⁴³and rather low in TNBC.^44,45As CDCP1 is present in Triton X-100-insoluble lipid rafts,^25,41we propose that the high lipid content of ccRCC cells facilitates clustering of flCDCP1 at the membrane in lipid rafts, which leads to stimulation of PKC phosphorylation by Src. On the other hand, TNBC relies on CDCP1 cleavage to induce cCDPC1 dimerization to stimulate PKC phosphorylation by Src. In line with this, Casaret al.^3,8showed that PC3 and HEK 293T cells, which have low lipid content,^46,47are dependent on CDCP1 cleavage for cellular disseminationin vitro, in chick embryo and mouse models of metastasis.

Although we found that CDCP1 is not hypoxia inducible at the mRNA and protein levels in TNBC, the phosphorylation of cCDCP1 and PKC are hypoxia inducible. Importantly, we¹⁰and others¹¹have shown that CDCP1 is hypoxia inducible through the hypoxia inducible factor pathway at the mRNA and protein levels in ccRCC. Cell-type-specific hypoxic induction of hypoxia inducible factor target genes has been described previously,⁴⁸which could explain the discrepancy between ccRCC and TNBC. Emerlinget al.¹¹demonstrated that Src Y416 and CDCP1 Y734 phosphorylation are hypoxia inducible in ccRCC, which we replicated in TNBC. The mechanism of stimulation of these types of phosphorylation under hypoxia deserves further investigation.

The CDCP1 signaling cascade has not been investigated in depth in TNBC and other types of breast cancer. It was previously reported that cCDCP1 promotes TNBC migration by sequestering E-cadherin intracellularly²and TNBC invasion by stimulating membrane type 1 matrix metalloproteinase activity.²⁵Our studies suggest that cCDCP1 promotes TNBC migration through dimerization, which is important for PKC phosphorylation and potentially ERK1/2, and p38 mitogen-activated protein kinase phosphorylation (requires further investigation). In addition, it was recently shown that CDCP1 directly interacts with HER2 and stimulates progression of HER2-positive tumors.⁴⁹On the other hand, a correlation between estrogen and progesterone receptor status and CDCP1 expression has not been found.⁵⁰Thus, the dependence of CDCP1 activity on cleavage and dimerization in hormone receptor-positive breast cancers remains to be investigated.

Our data validate the previously demonstrated therapeutic potential of targeting the cleaved isoform of CDCP1 to inhibit cancer metastasis.³However, we provide novel insight into the CDCP1 activation mechanism and propose targeting a step downstream of CDCP1 cleavage: dimerization.