Table_2_Discovery and Pharmacological Evaluation of STEAP4 as a Novel Target for HER2 Overexpressing Breast Cancer.xlsx

Breast cancer (BC) is a highly heterogeneous disease encompassing multiple subtypes with different molecular and histopathological features, disease prognosis, and therapeutic responses. Among these, the Triple Negative BC form (TNBC) is an aggressive subtype with poor prognosis and therapeutic outcome. With respect to HER2 overexpressing BC, although advanced targeted therapies have improved the survival of patients, disease relapse and metastasis remains a challenge for therapeutic efficacy. In this study the aim was to identify key membrane-associated proteins which are overexpressed in these aggressive BC subtypes and can serve as potential biomarkers or drug targets. We leveraged on the development of a membrane enrichment protocol in combination with the global profiling GeLC-MS/MS technique, and compared the proteomic profiles of a HER2 overexpressing (HCC-1954) and a TNBC (MDA-MB-231) cell line with that of a benign control breast cell line (MCF-10A). An average of 2300 proteins were identified from each cell line, of which approximately 600 were membrane-associated proteins. Our global proteomic methodology in tandem with invigoration by Western blot and Immunofluorescence analysis, readily detected several previously-established BC receptors like HER2 and EPHA2, but importantly STEAP4 and CD97 emerged as novel potential candidate markers. This is the first time that the mitochondrial iron reductase STEAP4 protein up-regulation is linked to BC (HER2+ subtype), while for CD97, its role in BC has been previously described, but never before by a global proteomic technology in TNBC. STEAP4 was selected for further detailed evaluation by the employment of Immunohistochemical analysis of BC xenografts and clinical tissue microarray studies. Results showed that STEAP4 expression was evident only in malignant breast tissues whereas all the benign breast cases had no detectable levels. A functional role of STEAP4 intervention was established in HER2 overexpressing BC by pharmacological studies, where blockage of the STEAP4 pathway with an iron chelator (Deferiprone) in combination with the HER2 inhibitor Lapatinib led to a significant reduction in cell growth in vitro. Furthermore, siRNA mediated knockdown of STEAP4 also suppressed cell proliferation and enhanced the inhibition of Lapatinib in HER2 overexpressing BC, confirming its potential oncogenic role in BC. In conclusion, STEAP4 may represent a novel BC related biomarker and a potential pharmacological target for the treatment of HER2 overexpressing BC.

乳腺癌（Breast Cancer, BC）是一种高度异质性疾病，涵盖多种具有不同分子与组织病理学特征、疾病预后及治疗应答的亚型。其中，三阴性乳腺癌（Triple Negative Breast Cancer, TNBC）是一类侵袭性强、预后与治疗效果均欠佳的亚型。针对人类表皮生长因子受体2（Human Epidermal Growth Factor Receptor 2, HER2）过表达乳腺癌，尽管先进的靶向治疗已改善了患者的生存情况，但疾病复发与转移仍是制约治疗疗效的核心难题。本研究旨在鉴定在这类侵袭性乳腺癌亚型中过表达的关键膜相关蛋白，以作为潜在的生物标志物或药物靶点。
本研究依托膜富集实验方案的开发成果，结合全局蛋白质组学分析技术GeLC-MS/MS（Gel Elution Liquid Chromatography-coupled Tandem Mass Spectrometry），对比了HER2过表达细胞系（HCC-1954）、三阴性乳腺癌细胞系（MDA-MB-231）与良性乳腺细胞系（MCF-10A）的蛋白质组谱。每条细胞系平均可鉴定出约2300种蛋白质，其中约600种为膜相关蛋白。
本研究的全局蛋白质组学方法联合蛋白质免疫印迹（Western Blot）与免疫荧光（Immunofluorescence）分析验证，成功检测到多种已被证实的乳腺癌受体，如HER2与EPHA2；尤为关键的是，STEAP4与CD97作为新型潜在候选标志物被成功筛选出来。
这是首次将线粒体铁还原酶STEAP4的上调与乳腺癌（HER2阳性亚型）建立关联；而CD97在乳腺癌中的作用此前已有报道，但从未通过全局蛋白质组学技术在三阴性乳腺癌中开展相关研究。
研究选取STEAP4进行进一步的详细评估，通过乳腺癌异种移植模型的免疫组织化学（Immunohistochemical）分析及临床组织芯片研究完成实验。结果显示，STEAP4仅在恶性乳腺组织中存在表达，而所有良性乳腺病例均未检测到其表达。
药理学研究证实了STEAP4在HER2过表达乳腺癌中的功能作用：采用铁螯合剂去铁酮（Deferiprone）联合HER2抑制剂拉帕替尼（Lapatinib）阻断STEAP4通路，可在体外显著抑制细胞增殖。此外，通过小干扰RNA（small interfering RNA, siRNA）介导的STEAP4敲低同样可抑制细胞增殖，并增强拉帕替尼对HER2过表达乳腺癌的抑制效果，证实了STEAP4在乳腺癌中潜在的致癌作用。
综上，STEAP4有望成为一种新型乳腺癌相关生物标志物，以及治疗HER2过表达乳腺癌的潜在药理学靶点。