Evaluation of CUB-Domain-Containing Protein (CDCP1)-Expression as Predictive Marker of Adhesion-Independant Cell Survival in Breast Cancer Cell Lines.
Authors: J. Seidel, C. Bruemann, K. Kunc, K. Possinger, D. Lueftner
Journal: Cancer Research (2009)

Abstract Background: A crucial step in the metastatic cascade is to overcome anoikis, a type of programmed cell death after loss of cell-cell contact. CDCP1 has recently been indentified to be an essential prerequisite for anoikis resistance in lung adenocarcinoma cell lines [Uekita et al. 2007]. CDCP1 is overexpressed in distinct tumor tissues and suspected to signal via Src family kinases (SFK). Since CDCP1 overexpression has been detected in a variety of primary breast cancers and cell lines [Bühring et al. 2004], this study aimed at investigating the correlation between CDCP1 expression and anoikis-resistance in breast cancer cell lines and the involvement of SFKs.Material and Methods: Various breast cancer cell lines were tested for their CDCP1 status by FACS analysis and immunoblotting. CDCP1-negative breast cancer cell line MCF7 and primary normal breast cells were compared to the CDCP1-high cell lines MDA-MB-231 (∼90% positive cells (p.c.)), T47D (∼65% p.c.), SKBR3 (∼52% p.c.) and HBL100 (∼60% p.c.) when cultured in ultra low-adhesion culture plates. Apoptosis rate was evaluated measuring the nucleosome enrichment factor (EF). SFK-involvement was tested via proliferation analysis after treatment with SKF-inhibitor PP2 and its inactive derivate PP3. Since various types of breast cancer were influenced by the presence or absence of steroids, MDA-MB-231 cells and HBL100 cells were stimulated with ß17-Estradiol and CDCP1 expression was measured using relative quantification in qRT-PCR.Results: When grown in solution on ultra-low adhesion plates, CDCP1-negative primary normal breast cells underwent apoptosis (EF:5,29). CDCP1-high expressing MDA-MB-231 cells (EF:1,75) were less apoptotic than T47D (EF:3,68:) and SKBR3 cells (EF:4,35). However, CDCP1-negative MCF7 breast cancer cells were relatively resistant towards loss of anchorage (EF:2,03). When grown in suspension, proliferation rate of MDA-MB-231 is 6-fold lower than in normal tissue plates, both cultured with or without SFK-inhibitor PP2. Proliferation rate of CDCP1-negative MCF7 cells grown in solution in presence and absence of PP2 is even 24-fold reduced. MDA-MB-231 cells show a significant 50% reduction in RNA-levels of CDCP1 when cultured 48h in presence of ß17-ER (10-8M). However, in HBL100 cells, no change of RNA-level of CDCP1 was detectable.Conclusions: Anoikis resistance seems to directly correlate with the expression level of CDCP1, when cells are cultured in suspension. However, CDCP1-negative cell line MCF7 also shows relative resistance towards loss of anchorage. Thus, anoikis resistance may not be a monocausal phenomenon. Nonetheless, high level of CDCP1-expression also promotes cell proliferation in absence of anchorage to an extracellular matrix. RNAi-mediated knock-down will elucidate if CDCP1 is also involved in cell migration or invasion. Our studies show, that CDCP1-expression level has strong impact on the capacity of breast cancer cell lines to grow and survive anchorage independent - an essential prerequisite for metastatic potential. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6167.