YAP increases response to Trastuzumab in HER2-positive Breast Cancer by enhancing P73-induced apoptosis

Abstract

The role of the Yes-associated protein (YAP) in oncogenesis and progression of breast cancer remains controversial. Meanwhile, development of therapeutic resistance to trastuzumab, a common breast cancer treatment administered after chemotherapy, is a significant challenge in the treatment of HER2-positive breast cancer. We, therefore, analyzed the role of YAP in trastuzumab resistance in HER2-positive-breast carcinoma cells in vitro and evaluated the status of YAP and related proteins in patient-derived breast carcinoma tissues by immunohistochemistry. YAP expression was observed in both BT474-TS (trastuzumab-sensitive) and BT474-TR (trastuzumab-resistant) cells. Treatment with trastuzumab increased expression of nuclear-YAP (N-YAP) in BT474-TS cells, whereas BT474-TR cells showed a decrease in N-YAP expression following trastuzumab treatment. YAP silencing significantly reduced trastuzumab-induced inhibitory effects in BT474-TS cells. YAP-silenced cells also showed decreased apoptosis and significantly lower p73 levels following trastuzumab treatment. Combined protein kinase B (AKT) inhibitor-trastuzumab treatment significantly inhibited BT474-TR cell proliferation, resulting in increased N-YAP and p73 expression, as well as apoptosis. In both paclitaxel, doxorubicin and cyclophosphamide (TAC)-treated, and docetaxel, carboplatin, and trastuzumab (TCbH)-treated groups; the pathological complete response (pCR) ratios were inversely correlated with p-AKT status in biopsy specimens, while YAP and p73 status were positively correlated with the pCR ratio in the biopsy specimens of the TCbH group. Our results show that YAP is involved in trastuzumab resistance in HER2-positive breast carcinoma cells and that YAP and AKT may be developed as prognostic markers of neoadjuvant trastuzumab therapy in patients with HER2-positive breast cancer.

Keywords: Breast cancer; Chemotherapy; Neoadjuvant therapy; Protein kinase B/AKT; Tumor progression.

Figure 1

YAP expression in various cell lines. Trastuzumab (Traz) treatment affected YAP expression in BT474-TS and BT474-TR cells. (A) YAP protein expression was detected by western blot in different cancer cells. (B) Total YAP expression after trastuzumab treatment in BT474-TS and BT474-TR cells. (C) Trastuzumab treatment significantly affected N-YAP levels in BT474-TS and BT474-TR cells. Data represent the mean ± standard deviation (SD) of three independent experiments. *p < 0.05, **p < 0.01, and *** p < 0.001.

Figure 2

YAP-knockdown altered trastuzumab (Traz) treatment effects and p73 expression in BT474-TS cells. (A) YAP-knockdown increased viability of BT474-TS cells after trastuzumab treatment. (B) YAP-knockdown altered apoptosis, measured by flow cytometry, after treatment in BT474-TS cells. (C) Expression of apoptotic proteins was examined by western blotting. (D) p73 protein expression was detected by western blotting in different cancer cells. Data represent the mean ± standard deviation (SD) of three independent experiments. *p < 0.05, **p < 0.01, and *** p < 0.001.

Figure 3

Trastuzumab (Traz) treatment affected AKT and p-AKT expression levels in BT474-TS and BT474-TR cells. Combination treatment of AKT inhibitor (GSK) and Traz in BT474-TR cells. (A) Trastuzumab treatment significantly affected AKT and p-AKT levels in BT474-TS and BT474-TR cells. (B) Trastuzumab plus GSK strongly affected the viability of BT474-TR cells. (C) Trastuzumab plus GSK increased apoptosis ratio of BT474-TR cells; (D) Expression of apoptotic proteins were examined by western blotting; (E) YAP, N-YAP, and p73 expression in BT474-TR cells after combination treatment. Data represent the mean ± standard (SD) of three independent experiments. *p < 0.05 and **p < 0.01.

Figure 4

Immunohistochemical detection of YAP, p73, AKT, and p-AKT expression in pre-treated HER2-positive breast cancer tissues. (A and B) Positive and negative expression of YAP in breast cancer tissues. (C and D) Positive and negative expression of p73 in breast cancer tissues. (E and F) Positive and negative expression of AKT in breast cancer tissues. (G and H) Positive and negative expression of p-AKT in breast cancer tissues (original magnification ×100).