| Breast cancer is the most frequently diagnosed and one of the most life-threatening cancers in women around the world. The American cancer society released statistics show that the incidence of breast cancer ranks first and the mortality rate ranks second in female cancer in2014. The epidemiological statistics of our country also show that female breast cancer incidence and mortality increased year by year, and the onset of age tend to be younger. Breast cancer is a highly heterogeneous malignancy; there are great differences in the organization form, immune phenotype, biological behaviour and response to treatment Several intrinsic breast cancer subtypes have been identified by gene expression profiling method. Triple negative breast cancer (TNBC), one of the subtypes, accounting for10%-15%of invasive breast cancers, is characterized by the absence of estrogen receptor (ER) and progesterone receptor (PR) and no over-expression of human epidermal growth factor receptor2(HER2). Therefore, patients with TNBC cannot be treated with endocrine therapy or therapies targeted to HER2. As a group, they have a worse prognosis and tend to relapse early compared with other subtypes of breast cancers. Hence, there is a compelling need to find more effective treatments. It has been reported that the over-expression of epidermal growth factor receptor (EGFR) was seen in approximately80%of TNBC. This discovery led to the investigation of the EGFR inhibitors (EGFRI). However, both the anti-EGFR monoclonal antibody cetuximab and the small molecular tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib seem to be ineffective according to phase II studies reports. Thus, more researches are needed to answer the question of why EGFR inhibitors failed in treatment of those EGFR over-expressed breast cancers.TAZ (transcriptional coactivator with PDZ-binding domain; also known as WWTR1) and its paralog YAP (YES associated protein) are the two main downstream effectors of the Hippo signaling pathway, which plays a major role in cell differentiation,organ size control, and tumorigenesis across species. Functionally, when the TAZ or YAP translocated into the nucleus, they will act as transcription coactivators to promote proliferation-associated genes expression.It has been reported that over-expression of TAZ induced the activation of EGFR signaling, and one of the EGFR ligands, amphiregulin (AREG), is a target of TAZ. AREG functions in a non-cell-autonomous manner to mediate EGF-independent growth and malignant behavior of mammary epithelial cells. YAP increases EGFR expression at the level of transcription when binding the EGFR promoter. Most importantly, exogenous induction of YAP induces resistance to5-FU and docetaxcel, while knockdown of YAP sensitizes esophageal cancer cells to these cytotoxics. Knockdown of YAP significantly increased EGFRI erlotinib sensitivity in ovarian cancer cell lines.Purpose:To investigate the relationship between the expression of TAZ/YAP and epidermal growth factor receptor inhibitor (EGFRI) resistance, and observe the effect of TAZ/YAP knockdown on cell apoptosis and proliferation in triple negative breast cancer (TNBC) cells.Methods:Detection the expression of TAZ and YAP in4kinds of human breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-468, BT-549) by Western blot and RT-qPCR. Knockdown of TAZ in TNBC cell lines (BT-549and MDA-MB-231) was made by transfection with TAZ-specific short hairpin RNA (shRNA). IC50of EGFR inhibitors (gefitinib, AG-1478) and cell proliferation rates were determined by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The cell apoptosis was assessed by flow cytometry (FCM). Knockdown of YAP in MDA-MB-231cells by YAP specific siRNA, and study the mechanism of the apoptosis inducing effect by Western blot and RT-qPCR methods.Results:TAZ expressions in TNBC cell lines were higher than that of hormone receptor-positive MCF-7cells. Transfection of TAZ-specific shRNA successfully silenced TAZ gene expression in transfected cells. TAZ knockdown up-regulated EGFRI sensitivity in BT-549cells, but down-regulated that in MDA-MB-231cells. TAZ knockdown increased BT-549cells proliferation suppression and apoptosis, but the expression of YAP has no change; however, TAZ knockdown induced inhibition of cell apoptosis and proliferation, as well as YAP up-regulated in MDA-MB-231cells. In order to study the relation between YAP expression and EGFR inhibitors resistance in MDA-MB-231/sh-TAZ cells, we further knocked down YAP by YAP specific siRNA, and the results show co-knockdown TAZ and YAP induced cell apoptosis and proliferation inhibition, as well as restored the EGFR inhibitors sensitivity in MDA-MB-231cells. These results suggest that the effect of TAZ knockdown on improving the drug sensitivity of breast cancer cells have cell specificity, for breast cancer cells which co-expression of TAZ and YAP, simultaneously inhibit the TAZ and YAP is needed. Knockdown of YAP in MDA-MB-231cells will result in cell proliferation suppression, apoptosis increase, PTEN express up-regulation, AKT pathway inhibition, caspase3and PARP activation. Application of PTEN specific inhibitor BPV-Hopic can partially rescue the cell proliferation suppression and apoptosis induction by YAP knockdown. These results suggest that YAP may be through the PTEN/AKT pathway to influence cell proliferation and apoptosis.Conclusions:the current study highlights the potential for TAZ to be a therapeutic target in breast cancers, as reducing TAZ levels can partially reverted resistance to EGFR inhibitors. In addition, for the first time, we found up-regulation of YAP could be induced by TAZ inhibition in certain breast cancer cell line, which leads to EGFRI resistance. For patients with high expression of both TAZ and YAP, anti-YAP drugs are needed to be added. Therefore, develop new therapeutic agents that can simultaneously target TAZ and YAP is needed. We believe that a specific inhibitor to TAZ/YAP combined with anti-EGFR therapy may improve the therapeutic efficacy in TNBC treatment. |
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