BCAR1, a Human Homologue of the Adapter Protein p130Cas, and Antiestrogen Resistance in Breast Cancer Cells

Abstract

Overexpression of the BCAR1 gene confers antiestrogen resistance on human ZR-75-1 breast cancer cells. Overexpression of BCAR1 in retrovirus-mutated cells appears to result from activation of the gene's promoter. The isolation and characterization of this gene open new avenues to elucidating mechanisms by which the growth of human breast cancer becomes independent of estrogen.

The substrate domain of BCAR1 is essential for anti-estrogen-resistant proliferation of human breast cancer cells

“…All resulting plasmids were sequence verified and tested for production of full length protein using the in vitro transcription-translation assay (ITT, Promega, Leiden, The Netherlands). Expression cassettes were excised with the unique cutting restriction enzymes XhoI and EcoRI and inserted in the LZRS-IRES-Neo vector [12], and the resulting expression vectors characterized by detailed restriction enzyme analyses.…”

“…The culture medium was replaced twice a week. Cells were harvested using trypsin/EDTA at the indicated days, counted and reseeded at the original density as previously described [12]. Expression of the relevant proteins was verified by western blot analyses immediately before start of the assay or after the first passage.…”

“…Cultured cells were trypsinized, rinsed with PBS buffer, sonicated for 10 s and lysed in 1% SDS, 10 mM Tris-HCl pH 7.5 buffer for 10 min at 100°C [12]. Protein content in total cell extracts was determined using the BCA protein assay reagent (Pierce Chemical Co Rockford, IL).…”

“…The BCAR1 locus was the first retroviral integration site identified and demonstrated to contain a causative gene for the resistant phenotype [8,9]. Additional experimentation demonstrated that the BCAR1 gene was the human homologue of rat p130Cas, a prominent tyrosine phosphorylated protein in Src-and Crktransformed cells, and also confirmed the dominant role of BCAR1 in tamoxifen-resistant cell growth [10][11][12]. Recently, BCAR1 has also been implicated in resistance to the chemotherapeutic drug adriamycin [13].…”

“…These domains are mostly conserved among the family members NEDD9 (HEF1/Cas-L), EFS (Sin) and HEPL [21]. As a consequence of the multiple protein interaction sites, BCAR1 serves as a docking molecule and has been implicated in many cellular processes, including cell transformation, integrin signaling, estrogen signaling, cell death, cytoskeletal rearrangements, migration, proliferation, force sensing, and bacterial infection [7,12,19,20,[22][23][24][25]. The closely related family member NEDD9/HEF1/CAS-L (hereafter referred to as HEF1) is unable to substitute for BCAR1 in vivo, since p130Cas knock-out mice die in utero due to developmental cardiovascular defects and growth retardation [26].…”