Department of Radiation Oncology, Rush University Medical Center, Chicago, Illinois 60612, USA.
J Biol Chem. 2011 Jan 28;286(4):2864-76. doi: 10.1074/jbc.M110.143271. Epub 2010 Nov 17.
Endocrine therapy with tamoxifen (TAM) significantly improves outcomes for patients with estrogen receptor-positive breast cancer. However, intrinsic (de novo) or acquired resistance to TAM occurs in a significant proportion of treated patients. To identify genes involved in resistance to TAM, we introduced full-length cDNA expression library into estrogen receptor-positive MCF7 cells and exposed them to a cytotoxic dose of 4-hydroxytamoxifen (4OHTAM). Four different library inserts were isolated from surviving clones. Re-introduction of the genes individually into naive MCF7 cells made them resistant to 4OHTAM. Cells overexpressing these genes had an increase in acidic autophagic vacuoles induced by 4OHTAM, suggesting their role in autophagy. One of them, prolylcarboxypeptidase (PRCP), was investigated further. Overexpression of PRCP increased cell proliferation, boosted several established markers of autophagy, including expression of LC3-2, sequestration of monodansylcadaverine, and proteolysis of BSA in an ER-α dependent manner, and increased resistance to 4OHTAM. Conversely, knockdown of endogenous PRCP in MCF7 cells increased cell sensitivity to 4OHTAM and at the same time decreased cell proliferation and expression of LC3-2, sequestration of monodansylcadaverine, and proteolysis of BSA. Inhibition of enzymatic activity of PRCP enhanced 4OHTAM-induced cytotoxicity in MCF7 cells. Cells with acquired resistance to 4OHTAM exhibited increased PRCP activity, although inhibition of PRCP prevented development of 4OHTAM resistance in parental MCF7 cells and restored response to 4OHTAM in MCF7 cells with acquired resistance to 4OHTAM. Thus, we have for the first time identified PRCP as a resistance factor for 4OHTAM resistance in estrogen receptor-positive breast cancer cells.
他莫昔芬（TAM）的内分泌治疗显著改善了雌激素受体阳性乳腺癌患者的预后。然而，在相当一部分接受治疗的患者中，存在 TAM 的内在（原发）或获得性耐药。为了鉴定与 TAM 耐药相关的基因，我们将全长 cDNA 表达文库导入雌激素受体阳性 MCF7 细胞，并使其暴露于细胞毒性剂量的 4-羟基他莫昔芬（4OHTAM）下。从存活的克隆中分离出了 4 个不同的文库插入物。将这些基因单独重新引入到幼稚的 MCF7 细胞中，使它们对 4OHTAM 产生耐药性。过表达这些基因的细胞中，酸性自噬空泡在 4OHTAM 诱导下增加，表明它们在自噬中的作用。其中之一，脯氨酰羧肽酶（PRCP），进一步进行了研究。PRCP 的过表达增加了细胞增殖，促进了几个已建立的自噬标志物的表达，包括 LC3-2 的表达、单丹磺酰尸胺的隔离和 BSA 的蛋白水解，这一切均依赖于 ER-α，并增加了对 4OHTAM 的耐药性。相反，在 MCF7 细胞中敲低内源性 PRCP 增加了细胞对 4OHTAM 的敏感性，同时降低了细胞增殖和 LC3-2 的表达、单丹磺酰尸胺的隔离和 BSA 的蛋白水解。PRCP 的酶活性抑制增强了 MCF7 细胞中 4OHTAM 诱导的细胞毒性。对 4OHTAM 获得耐药性的细胞表现出 PRCP 活性增加，尽管抑制 PRCP 可防止亲本 MCF7 细胞中 4OHTAM 耐药的发展，并恢复对 4OHTAM 的反应在对 4OHTAM 获得耐药性的 MCF7 细胞中。因此，我们首次鉴定出 PRCP 是雌激素受体阳性乳腺癌细胞中 4OHTAM 耐药的耐药因子。
