C-Cbl reverses HER2-mediated tamoxifen resistance in human breast cancer cells.
BACKGROUND
Tamoxifen is a frontline therapy for estrogen receptor (ER)-positive breast cancer in premenopausal women. However, many patients develop resistance to tamoxifen, and the mechanism underlying tamoxifen resistance is not well understood. Here we examined whether ER-c-Src-HER2 complex formation is involved in tamoxifen resistance.


METHODS
MTT and colony formation assays were used to measure cell viability and proliferation. Western blot was used to detect protein expression and protein complex formations were detected by immunoprecipitation and immunofluorescence. SiRNA was used to examine the function of HER2 in of BT474 cells. An in vivo xenograft animal model was established to examine the role of c-Cbl in tumor growth.


RESULTS
MTT and colony formation assay showed that BT474 cells are resistant to tamoxifen and T47D cells are sensitive to tamoxifen. Immunoprecipitation experiments revealed ER-c-Src-HER2 complex formation in BT474 cells but not in T47D cells. However, ER-c-Src-HER2 complex formation was detected after overexpressing HER2 in T47D cells and these cells were more resistant to tamoxifen. HER2 knockdown by siRNA in BT474 cells reduced ER-c-Src-HER2 complex formation and reversed tamoxifen resistance. ER-c-Src-HER2 complex formation was also disrupted and tamoxifen resistance was reversed in BT474 cells by the c-Src inhibitor PP2 and HER2 antibody trastuzumab. Nystatin, a lipid raft inhibitor, reduced ER-c-Src-HER2 complex formation and partially reversed tamoxifen resistance. ER-c-Src-HER2 complex formation was disrupted by overexpression of c-Cbl but not by the c-Cbl ubiquitin ligase mutant. In addition, c-Cbl could reverse tamoxifen resistance in BT474 cells, but the ubiquitin ligase mutant had no effect. The effect of c-Cbl was validated in BT474 tumor-bearing nude mice in vivo. Immunofluorescence also revealed ER-c-Src-HER2 complex formation was reduced in tumor tissues of nude mice with c-Cbl overexpression.


CONCLUSIONS
Our results suggested that c-Cbl can reverse tamoxifen resistance in HER2-overexpressing breast cancer cells by inhibiting the formation of the ER-c-Src-HER2 complex.
['Li|Wei|W|', 'Xu|Ling|L|', 'Che|Xiaofang|X|', 'Li|Haizhou|H|', 'Zhang|Ye|Y|', 'Song|Na|N|', 'Wen|Ti|T|', 'Hou|Kezuo|K|', 'Yang|Yi|Y|', 'Zhou|Lu|L|', 'Xin|Xing|X|', 'Xu|Lu|L|', 'Zeng|Xue|X|', 'Shi|Sha|S|', 'Liu|Yunpeng|Y|', 'Qu|Xiujuan|X|', 'Teng|Yuee|Y|http://orcid.org/0000-0002-9198-9795']
D000818:Animals D001943:Breast Neoplasms D000081247:CSK Tyrosine-Protein Kinase D045744:Cell Line, Tumor D019008:Drug Resistance, Neoplasm D005260:Female D064593:Heterografts D006801:Humans D051379:Mice D008819:Mice, Nude D050721:Proto-Oncogene Proteins c-cbl D018719:Receptor, ErbB-2 D011960:Receptors, Estrogen D020845:Selective Estrogen Receptor Modulators D013629:Tamoxifen D019061:src-Family Kinases
C-Cbl Tamoxifen Resistance Breast cancer HER2