Department of Surgery, Taipei Veterans General Hospital, No. 201 Sec. 2 Shih-Pai Road, Taipei 112, Taiwan.
Breast Cancer Res. 2012 Apr 26;14(2):R68. doi: 10.1186/bcr3175.
Triple negative breast cancer (TNBC) is very aggressive and currently has no specific therapeutic targets, such as hormone receptors or human epidermal growth factor receptor type 2 (HER2); therefore, prognosis is poor. Bortezomib, a proteasome inhibitor, may exert efficacy in TNBC through its multiple cellular effects. Here, we tested the efficacy of bortezomib and examined the drug mechanism in breast cancer cells.
Five breast cancer cell lines: TNBC HCC-1937, MDA-MB-231, and MDA-MB-468; HER2-overexpressing MDA-MB-453; and estrogen receptor positive MCF-7 were used for in vitro studies. Apoptosis was examined by both flow cytometry and Western Blot. Signal transduction pathways in cells were assessed by Western Blot. Gene silencing was done by small interfering RNA (siRNA). In vivo efficacy of bortezomib was tested in nude mice with breast cancer xenografts. Immunohistochemical study was performed on tumor tissues from patients with TNBC.
Bortezomib induced significant apoptosis, which was independent of its proteasome inhibition, in the three TNBC cell lines, but not in MDA-MB-453 or MCF-7 cells. Furthermore, cancerous inhibitor of protein phosphatase 2A (CIP2A), a cellular inhibitor of protein phosphatase 2A (PP2A), mediated the apoptotic effect of bortezomib. We showed that bortezomib inhibited CIP2A in association with p-Akt downregulation in a dose- and time-dependent manner in all sensitive TNBC cells, whereas no alterations in CIP2A expression and p-Akt were noted in bortezomib-resistant cells. Overexpression of CIP2A upregulated p-Akt and protected MDA-MB-231 and MDA-MB-468 cells from bortezomib-induced apoptosis, whereas silencing CIP2A by siRNA overcame the resistance to bortezomib-induced apoptosis in MCF-7 cells. In addition, bortezomib downregulated CIP2A mRNA but did not affect the degradation of CIP2A protein. Furthermore, bortezomib exerted in vivo antitumor activity in HCC-1937 xenografted tumors, but not in MCF-7 tumors. Bortezomib downregulated CIP2A expression in the HCC-1937 tumors but not in the MCF-7 tumors. Importantly, CIP2A expression is readily detectable in tumor samples from TNBC patients.
CIP2A is a major determinant mediating bortezomib-induced apoptosis in TNBC cells. CIP2A may thus be a potential therapeutic target in TNBC.
三阴性乳腺癌（TNBC）非常具有侵袭性，目前尚无特定的治疗靶点，如激素受体或人表皮生长因子受体 2（HER2）；因此，预后较差。硼替佐米是一种蛋白酶体抑制剂，可能通过其多种细胞作用在 TNBC 中发挥疗效。在这里，我们测试了硼替佐米的疗效，并研究了该药在乳腺癌细胞中的作用机制。
我们使用了五种乳腺癌细胞系：TNBC HCC-1937、MDA-MB-231 和 MDA-MB-468；HER2 过表达 MDA-MB-453；以及雌激素受体阳性 MCF-7 进行体外研究。通过流式细胞术和 Western Blot 检测细胞凋亡。通过 Western Blot 评估细胞内信号转导通路。通过小干扰 RNA（siRNA）进行基因沉默。在具有乳腺癌异种移植的裸鼠中测试硼替佐米的体内疗效。对三阴性乳腺癌患者的肿瘤组织进行免疫组织化学研究。
硼替佐米在三种 TNBC 细胞系中诱导了显著的凋亡，而与蛋白酶体抑制无关，但在 MDA-MB-453 或 MCF-7 细胞中没有诱导凋亡。此外，癌性蛋白磷酸酶 2A 抑制剂（CIP2A），一种蛋白磷酸酶 2A（PP2A）的细胞抑制剂，介导了硼替佐米的凋亡作用。我们表明，硼替佐米以剂量和时间依赖的方式与 p-Akt 下调相关联抑制 CIP2A 在所有敏感的 TNBC 细胞中，而在硼替佐米耐药细胞中未观察到 CIP2A 表达和 p-Akt 的改变。CIP2A 的过表达上调了 p-Akt，并保护 MDA-MB-231 和 MDA-MB-468 细胞免受硼替佐米诱导的凋亡，而 CIP2A 的 siRNA 沉默则克服了 MCF-7 细胞对硼替佐米诱导的凋亡的耐药性。此外，硼替佐米下调了 CIP2A 的 mRNA，但不影响 CIP2A 蛋白的降解。此外，硼替佐米在 HCC-1937 异种移植肿瘤中具有体内抗肿瘤活性，但在 MCF-7 肿瘤中没有。硼替佐米下调了 HCC-1937 肿瘤中的 CIP2A 表达，但未下调 MCF-7 肿瘤中的 CIP2A 表达。重要的是，CIP2A 在三阴性乳腺癌患者的肿瘤样本中可轻易检测到。
CIP2A 是介导硼替佐米诱导 TNBC 细胞凋亡的主要决定因素。因此，CIP2A 可能是 TNBC 的一个潜在治疗靶点。
