title: Protein tyrosine phosphatase SHP-1 sensitizes EGFR/HER-2 positive breast cancer cells to trastuzumab through modulating phosphorylation of EGFR and HER-2.
abstract: BACKGROUND
  Trastuzumab resistance in HER-2 positive breast cancer cells is closely related to overexpression of both epidermal growth factor receptor (EGFR) and human epidermal receptor (HER-2). SHP-1 has been demonstrated to downregulate tyrosine kinase activity including EGFR via its phosphatase function, but its effect on HER-2 activity is still unknown. Here, we examined the hypothesis that SHP-1 enhances the anticancer efficacy of trastuzumab in EGFR/HER-2 positive breast cancer cells through combining dual inhibition of EGFR and HER-2.
  
  
  METHODS
  Trastuzumab-resistant breast cancer SKBr-3 cells were generated by long-term in vitro culture of SKBr-3cells in the presence of trastuzumab. The SHP-1 was ectopically expressed by stable transfection. The activity and expression of EGFR, HER-2, and downstream signaling pathways were tested by Western blot. Cell viability was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and apoptosis was examined by flow cytometry. The binding between SHP-1 and EGFR/HER-2 was evaluated by immunoprecipitation assay and bimolecular fluorescence complementation. The effects of SHP-1 on tumorigenicity and trastuzumab sensitivity were confirmed via in vivo xenograft model.
  
  
  RESULTS
  Trastuzumab-resistant SKBr-3 cells showed aberrant co-expression of EGFR and HER-2. Introduction of wild-type SHP-1 inhibited cell proliferation, clone formation, and promoted the apoptosis induced by trastuzumab. Meanwhile, SHP-1 overexpression reduced phosphorylation levels of EGFR and HER-2 both in parental and trastuzumab-resistant SKBr-3 cells. In vivo study showed an increased antitumor effect of trastuzumab in SHP-1 overexpressed xenografts. At last, we discovered that SHP-1 can make complexes with both EGFR and HER-2, and both phospho-EGFR and phosphor-HER-2 levels in wild-type SHP-1 immunoprecipitates were less than those in phosphatase-inactive SHP-1 (C453S) immunoprecipitates, indicating that EGFR and HER-2 are potential substrates of SHP-1.
  
  
  CONCLUSIONS
  Taken together, we have demonstrated that the SHP-1 is a negative regulatory factor of the tyrosine kinase activity of HER-2 and EGFR through inhibiting phosphorylation. Dual targeting of EGFR and HER-2, by combining trastuzumab with SHP-1 overexpression, may improve response in HER-2 overexpressing breast cancer cells that also express high levels of EGFR.
authors.affiliation: Traditional Chinese Medicine-Integrated Hospital, Southern Medical University, Guangzhou, People's Republic of China ; Department of Oncology, Dongguan People's Hospital, Dongguan, People's Republic of China.
authors.forename: Yifen
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Department of Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, People's Republic of China.
authors.forename: Rong
authors.initials: R
authors.lastname: Li
authors.affiliation: Department of Oncology, Traditional Chinese Medicine-Integrated Hospital, Southern Medical University, Guangzhou, People's Republic of China.
authors.forename: Junyi
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Radiology, Dongguan People's Hospital, Dongguan, People's Republic of China.
authors.forename: Gang
authors.initials: G
authors.lastname: Wang
authors.affiliation: Second Affiliated Hospital of Guangzhou Medical College, Southern Medical University, Guangdong, People's Republic of China.
authors.forename: Bin
authors.initials: B
authors.lastname: Liu
authors.affiliation: College of Traditional Chinese medicine, Southern Medical University, Guangdong, People's Republic of China.
authors.forename: Xiaofang
authors.initials: X
authors.lastname: Huang
authors.affiliation: College of Traditional Chinese medicine, Southern Medical University, Guangdong, People's Republic of China.
authors.forename: Tao
authors.initials: T
authors.lastname: Zhang
authors.affiliation: Cancer Center, Traditional Chinese Medicine-Integrated Hospital, Southern Medical University, Guangzhou, Guangdong, People's Republic of China.
authors.forename: Rongcheng
authors.initials: R
authors.lastname: Luo
keywords: EGFR; HER-2; SHP-1; breast cancer; drug resistance; trastuzumab