Yes1 signaling mediates the resistance to Trastuzumab/Lap atinib in breast cancer

Abstract

Background: Overexpression of human epidermal growth factor receptor 2 (HER2) is observed in approximately 15-23% of breast cancers and these cancers are classified as HER2-positive breast cancer. Trastuzumab is the first-line targeted therapeutic drug for HER2-positive breast cancer and has improved patient overall survival. However, acquired resistance to trastuzumab is still a critical issue in breast cancer treatment. We previously established a trastuzumab-resistant breast cancer cell line (named as BT-474-R) from a trastuzumab-sensitive HER2-amplified cell line BT-474. Lapatinib is also a molecular-targeted drug for HER2-positive breast cancer, which acquired the resistance to trastuzumab. Acquired resistance to lapatinib is also an issue to be conquered.

Methods: We established trastuzumab/lapatinib-dual resistant cell line (named as BT-474-RL2) by additionally treating BT-474-R with lapatinib. We analyzed the mechanisms of resistance to trastuzumab and lapatinib. Besides, we analyzed the effect of the detected resistance mechanism in HER2-positive breast cancer patients.

Results: Proto-oncogene tyrosine-protein kinase Yes1, which is one of the Src family members, was amplified, overexpressed and activated in BT-474-R and BT-474-RL2. Silencing of Yes1 by siRNA induced both BT-474-R and BT-474-RL2 to restore the sensitivity to trastuzumab and lapatinib. Pharmaceutical inhibition of Yes1 by the Src inhibitor dasatinib was also effective to restore the sensitivity to trastuzumab and lapatinib in the two resistant cell lines. Combination treatment with dasatinib and trastuzumab induced down-regulation of signaling molecules such as HER2 and Akt. Moreover, the combination treatments induced G1-phase cell-cycle arrest and apoptosis. Consistent with cell line data, high expression of Yes1 mRNA was correlated with worse prognosis in patients with HER2-positive breast cancer.

Conclusion: Yes1 plays an important role in acquired resistance to trastuzumab and lapatinib in HER2-positive breast cancer. Our data suggest that pharmacological inhibition of Yes1 may be an effective strategy to overcome resistance to trastuzumab and lapatinib.

Fig 1. Establishment of trastuzumab-resistant cell line BT-474-R and trastuzumab/lapatinib-dual resistant cell line BT-474-RL2.

(A) Morphological changes in parental cell line (BT-474) and its sublines (BT-474-R and BT-474-RL2). (B) MTS assay evaluating cell viability of BT-474, BT-474-R and BT-474-RL2 upon treatment with various concentrations of trastuzumab or lapatinib for 72 hours. Data are shown as means ± standard errors (SE). The assay using trastuzumab was repeated five times, and the assay using lapatinib was repeated three times.

Fig 2. Expression of HER2 and its related proteins.

(A) Flow cytometric analysis of HER2 expression level on BT-474, BT-474-R and BT-474-RL2. (B) Protein expression profile of BT-474, BT-474-R and BT-474-RL2. Phosphorylation of major cell signaling pathways changes among BT-474, BT-474-R and BT-474-RL2.

Fig 3. Yes1, one of the Src family member, is a key molecule in acquired HER2-resistance.

(A) Comparison of mRNA expression of Src family members in BT-474, BT-474-R and BT-474-RL2 by q-RT-PCR. Fgr, Blk and Hck was not detected. Data are shown as means + SE. The assay was repeated three times. (B) Western blot analysis of Yes1 protein expression among BT-474, BT-474-R and BT-474-RL2. (C) Copy number assay of Yes1 in BT-474, BT-474-R and BT-474-RL2. Human Genomic DNA (HGD) was used as the control (2 copies). Data are shown as means + SE. The assay was repeated three times. (D) Protein expression profile after Yes1 knockdown by Yes1 siRNA in BT-474, BT-474-R and BT-474-RL2. (E) MTS assay assessing the sensitivity to trastuzumab or lapatinib among BT-474, BT-474-R and BT-474-RL2 after Yes1 knockdown. Cells (3,000/well) were seeded in 96-well plates and transfected with siRNA for 48 hours followed by the treatment of trastuzumab or lapatinib for 72 hours. Data are shown as means + SE. The assay was repeated three times.

Fig 4. Dasatinib, Src family inhibitor, overcomes HER2-resistance.

(A) MTS assay evaluating the effect of trastuzumab or lapatinib alone, dasatinib alone or the combination on BT-474, BT-474-R and BT-474-RL2. Data are shown as means + SE. The assay was repeated three times. (B) Clonogenic assay evaluating the effect of long-term exposure to trastuzumab alone, dasatinib alone or the combination of them on BT-474-R and BT-474-RL2. Data are shown as means + SE. The assay was repeated three times. (C) Soft agar colony formation assay in BT-474-R and BT-474-RL2. Data are shown as means + SE. The assay was repeated three times. (D) phosphorylation status of HER2 and Akt upon treatment with trastuzumab alone, dasatinib alone or the combination of them for 1 hour in BT-474, BT-474-R and BT-474-RL2.

Fig 5. Combination of trastuzumab and dasatinib induce cell-cycle arrest in G1 phase and apoptosis.

(A) Upper: Cell cycle analysis of BT-474-R upon treatment with trastuzumab alone, dasatinib alone or combination of them. Lower: G0-G1 population in BT-474, BT-474-R and BT-474-RL2. Data are shown as means + SE. The assay was repeated three times. (B) Apoptosis analysis by the detection of PARP cleavage in western blot analysis in BT-474, BT-474-R and BT-474-RL2.

Fig 6. Effect of the Yes1 mRNA expression on prognosis in HER2-positive breast cancer patients.

(A) In TP53 cohort (GSE3494) with higher expression of HER2, disease-specific survival (DSS) of the cases with higher Yes1 mRNA expression was significantly shorter than that of the cases with lower expression of Yes1 (P = 0.0133). (B) In metastasis cohort (GSE12276) with higher expression of HER2, relapse free survival (RFS) of the cases with higher expression of Yes1 was significantly shorter than that of the cases with lower expression of Yes1 (P = 0.0000546).