Iida Mari, Brand Toni M, Starr Megan M, Huppert Evan J, Luthar Neha, Bahrar Harsh, Coan John P, Pearson Hannah E, Salgia Ravi, Wheeler Deric L
Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, Wisconsin Institute for Medical Research, 1111 Highland Ave,, Madison, WI 53705, USA.
Mol Cancer. 2014 Oct 24;13:242. doi: 10.1186/1476-4598-13-242.
Cetuximab, an anti-EGFR monoclonal antibody, is used to treat several cancers. However, many patients who initially respond to cetuximab acquire resistance. To examine mechanisms of acquired resistance, we developed a series of cetuximab-resistant (Ctx(R)) clones derived from the cetuximab sensitive (CtxS) non-small cell lung cancer (NSCLC) cell line H226. Previous studies characterizing this model revealed that: 1) EGFR was robustly overexpressed in Ctx(R) clones due to decreased EGFR ubiquitination and degradation and 2) Ctx(R) clones expressed increased HER2 and HER3 activation resulting in constitutive activation of the PI3K/AKT signaling axis. These findings suggest that dual targeting HER family receptors would be highly beneficial in the Ctx(R) setting.
Since HER3 has been implicated in resistance to EGFR inhibitors, the efficacy of dually targeting both EGFR and HER3 in Ctx(R) models was evaluated. First, EGFR and HER3 expression were knocked down with siRNAs. Compared to the Ctx(S) parental cell line (HP), all Ctx(R) clones exhibited robust decreases in cell proliferation upon dual knockdown. Analysis of Ctx(R) clones indicated that neuregulin-1 was highly overexpressed compared to HP cells. Incubation of HP cells with neuregulin-1 rendered them resistant to cetuximab. Next, dual treatment of Ctx(R) clones with cetuximab and the HER3 neutralizing monoclonal antibody (mAb) U3-1287 led to potent anti-proliferative effects. Blockade of EGFR with cetuximab resulted in inactivation of MAPK, while blockade of HER3 with U3-1287 resulted in the inactivation of AKT. Treatment with both mAbs resulted in knockdown of both signaling pathways simultaneously. HER2 was also strongly inactivated upon dual mAb therapy, suggesting that this treatment regimen can diminish signaling from three HER family receptors. De novo CtxR H226 mouse xenografts were established to determine if dual therapy could overcome acquired resistance to cetuximab in vivo. Tumors that had acquired resistance to cetuximab were significantly growth delayed upon dual treatment of U3-1287 and cetuximab compared to those continued on cetuximab only. Combinatorial-treated xenograft tumors expressed decreased Ki67 and increased cleaved caspase-3 levels compared to tumors treated with either monotherapy.
These studies demonstrate that dually targeting HER family receptors with antibody-based therapies can overcome acquired resistance to cetuximab.
西妥昔单抗是一种抗表皮生长因子受体（EGFR）单克隆抗体，用于治疗多种癌症。然而，许多最初对西妥昔单抗有反应的患者会产生耐药性。为了研究获得性耐药的机制，我们从西妥昔单抗敏感（CtxS）的非小细胞肺癌（NSCLC）细胞系H226中衍生出了一系列西妥昔单抗耐药（Ctx(R)）克隆。此前对该模型的研究表明：1）由于EGFR泛素化和降解减少，Ctx(R)克隆中EGFR大量过表达；2）Ctx(R)克隆中HER2和HER3激活增加，导致PI3K/AKT信号轴的组成性激活。这些发现表明，双重靶向HER家族受体在Ctx(R)环境中可能非常有益。
由于HER3与EGFR抑制剂耐药有关，因此评估了在Ctx(R)模型中双重靶向EGFR和HER3的疗效。首先，用小干扰RNA（siRNA）敲低EGFR和HER3的表达。与Ctx(S)亲本细胞系（HP）相比，所有Ctx(R)克隆在双重敲低后细胞增殖均显著降低。对Ctx(R)克隆的分析表明，与HP细胞相比，神经调节蛋白-1高度过表达。用神经调节蛋白-1孵育HP细胞使其对西妥昔单抗产生耐药性。接下来，用西妥昔单抗和HER3中和单克隆抗体（mAb）U3-1287对Ctx(R)克隆进行双重处理，产生了强大的抗增殖作用。用西妥昔单抗阻断EGFR导致丝裂原活化蛋白激酶（MAPK）失活，而用U3-·1287阻断HER3导致AKT失活。两种单克隆抗体联合处理导致两条信号通路同时被敲低。双重单克隆抗体治疗后HER2也被强烈失活，表明该治疗方案可减少来自三种HER家族受体的信号传导。建立了新的CtxR H226小鼠异种移植模型，以确定双重疗法是否能在体内克服对西妥昔单抗的获得性耐药。与仅继续使用西妥昔单抗治疗的肿瘤相比，对西妥昔单抗产生耐药性的肿瘤在接受U3-1287和西妥昔单抗双重治疗后生长明显延迟。与单药治疗的肿瘤相比，联合治疗的异种移植肿瘤中Ki67表达降低，裂解的半胱天冬酶-3水平升高。
这些研究表明，基于抗体的疗法双重靶向HER家族受体可克服对西妥昔单抗的获得性耐药。
