Subtractive immunization using highly metastatic human tumor cells identifies SIMA135/CDCP1, a 135 kDa cell surface phosphorylated glycoprotein antigen

Abstract

We have previously used a subtractive immunization (SI) approach to generate monoclonal antibodies (mAbs) against proteins preferentially expressed by the highly metastatic human epidermoid carcinoma cell line, M + HEp3. Here we report the immunopurification, identification and characterization of SIMA135/CDCP1 (subtractive immunization M + HEp3 associated 135 kDa protein/CUB domain containing protein 1) using one of these mAbs designated 41-2. Protein expression levels of SIMA135/CDCP1 correlated with the metastatic ability of variant HEp3 cell lines. Protein sequence analysis predicted a cell surface location and type I orientation of SIMA135/CDCP1, which was confirmed directly by immunocytochemistry. Analysis of deglycosylated cell lysates indicated that up to 40 kDa of the apparent molecular weight of SIMA135/CDCP1 is because of N-glycosylation. Western blot analysis using a antiphosphotyrosine antibody demonstrated that SIMA135/ CDCP1 from HEp3 cells is tyrosine phosphorylated. Selective inhibitor studies indicated that an Src kinase family member is involved in the tyrosine phosphorylation of the protein. In addition to high expression in M + HEp3 cells, the SIMA135/CDCP1 protein is expressed to varying levels in 13 other human tumor cell lines, manifesting only a weak correlation with the reported metastatic ability of these tumor cell lines. The protein is not detected in normal human fibroblasts and endothelial cells. Northern blot analysis indicated that SIMA135/ CDCP1 mRNA has a restricted expression pattern in normal human tissues with highest levels of expression in skeletal muscle and colon. Immunohistochemical analysis indicated apical and basal plasma membrane expression of SIMA135/CDCP1 in epithelial cells in normal colon. In colon tumor, SIMA135/CDCP1 expression appeared dysregulated showing extensive cell surface as well as cytoplasmic expression. Consistent with in vitro shedding experiments on HEp3 cells, SIMA135/CDCP1 was also detected within the lumen of normal and cancerous colon crypts, suggesting that protein shedding may occur in vivo. Thus, specific immunodetection followed by proteomic analysis allows for the identification and partial characterization of a heretofore uncharacterized human cell surface antigen.

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

“…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).…”

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

“…Quantitative densitometric analysis showed a shift from only ~20% of CDCP1 being present in lipid raft-poor fraction 4 from unstimulated Caov3, to about 65% in EGF-stimulated cells. As the kinase Src, which is the key mediator of tyrosine phosphorylation of CDCP1 (7,14,32), is found in lipid rafts and differentially signals within and outside these structures (33), we were interested to examine the impact of EGF on phosphotyrosine-CDCP1 (pY-CDCP1). CaOV3 and OVCA420 cells were incubated with EGF for 6, 12 and 24 h before immunoprecipitation of CDCP1 and detection of pY-CDCP1 using an anti-phosphotyrosine antibody.…”

CDCP1 Regulates the Function of MT1-MMP and Invadopodia-Mediated Invasion of Cancer Cells

“…Complement C1r/C1s, Uegf, Bmp1 (CUB) domaincontaining protein 1 (CDCP1), also described as SIMA135 and TRASK (1,2), is a type 1 transmembrane protein with 3 CUB domains as the extracellular domains and several tyrosine residues that can be phosphorylated by Src family kinases (SFK) in the intracellular domain (1)(2)(3)(4)(5)(6). The expression of CDCP1 was reported in several human malignancies, such as colon and breast cancers (3,7).…”