Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, CT 06520, USA.
Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
Gynecol Oncol. 2019 Apr;153(1):158-164. doi: 10.1016/j.ygyno.2019.01.002. Epub 2019 Jan 7.
Aberrant expression of HER2/neu and PIK3CA gene products secondary to amplification/mutations are common in high-grade-serous-endometrial (USC) and ovarian-cancers (HGSOC). Because scant information is currently available in the literature on the potential negative effect of PIK3CA mutations on the activity of afatinib, in this study we evaluate for the first time the role of oncogenic PIK3CA mutations as a potential mechanism of resistance to afatinib in HGSOC and USC overexpressing HER2/neu.
We used six whole-exome-sequenced primary HGSOC/USC cell-lines and three xenografts overexpressing HER2/neu and harboring mutated or wild-type PIK3CA/PIK3R1 genes to evaluate the role of PI3K-mutations as potential mechanism of resistance to afatinib, an FDA-approved pan-c-erb-inhibitor in clinical trials in USC. Primary-USC harboring wild-type-PIK3CA gene was transfected with plasmids encoding oncogenic PIK3CA-mutations (H1047R/E545K). The effect of afatinib on HER2/PI3K/AKT/mTOR pathway was evaluated by immunoblotting.
We found PI3K wild-type cell-lines to be significantly more sensitive (lower IC) than PI3K-mutated cell-lines p = 0.004). In vivo, xenografts of primary cell-line USC-ARK2, transfected with the PIK3CA-H1047R or E545K hotspot-mutations, exhibited significantly more rapid tumor growth when treated with afatinib, compared to mice harboring ARK2-tumors transfected with wild-type-PIK3CA (p = 0.041 and 0.001, respectively). By western-blot, afatinib effectively reduced total and phospho-HER2 proteins in all cell-lines. However, H1047R/E545K-PIK3CA-transfected-ARK2-cells demonstrated a greater compensatory increase in phosphorylated-AKT proteins after afatinib exposure when compared to controls ARK2.
Oncogenic PI3K mutations may represent a major mechanism of resistance to afatinib. Combinations of c-erb with PIK3CA, AKT or mTOR inhibitors may be necessary to more efficiently block the PIK3CA/AKT/mTOR pathway.
HER2/neu 和 PIK3CA 基因产物的扩增/突变导致的异常表达在高级别浆液性子宫内膜（USC）和卵巢癌（HGSOC）中很常见。由于目前文献中关于 PIK3CA 突变对 afatinib 活性的潜在负面影响的信息很少，因此在这项研究中，我们首次评估了致癌性 PIK3CA 突变作为 afatinib 在过表达 HER2/neu 的 HGSOC 和 USC 中耐药的潜在机制。
我们使用了六个全外显子组测序的原发性 HGSOC/USC 细胞系和三个过表达 HER2/neu 并携带突变或野生型 PIK3CA/PIK3R1 基因的异种移植物，以评估 PI3K 突变作为 afatinib 耐药的潜在机制，afatinib 是一种 FDA 批准的临床试验中用于 USC 的泛 c-erb 抑制剂。携带野生型 PIK3CA 基因的原发性 USC 被转染编码致癌 PIK3CA 突变（H1047R/E545K）的质粒。通过免疫印迹评估 afatinib 对 HER2/PI3K/AKT/mTOR 通路的影响。
我们发现 PI3K 野生型细胞系比 PI3K 突变型细胞系更敏感（更低的 IC；p=0.004）。在体内，用 afatinib 治疗过表达 PIK3CA-H1047R 或 E545K 热点突变的原发性细胞系 USC-ARK2 的异种移植物时，与转染野生型 PIK3CA 的 ARK2 肿瘤小鼠相比，肿瘤生长明显加快（分别为 p=0.041 和 0.001）。通过 Western blot，afatinib 有效地降低了所有细胞系中总和磷酸化 HER2 蛋白的水平。然而，与对照 ARK2 相比，在用 afatinib 暴露后，H1047R/E545K-PIK3CA 转染的 ARK2 细胞中磷酸化 AKT 蛋白的代偿性增加更大。
致癌性 PI3K 突变可能是 afatinib 耐药的主要机制。为了更有效地阻断 PI3KA/AKT/mTOR 通路，可能需要将 c-erb 与 PIK3CA、AKT 或 mTOR 抑制剂联合使用。
