Molecular mechanisms of resistance to HER2 inhibitors in breast cancer

描述(由申请人提供)：本提案描述了一项为期五年的培训计划，旨在发展一个独立的翻译学术癌症研究计划。应聘者接受过研究生研究培训和内科住院医生培训，完成了血液学/肿瘤学的团契培训，并继续在有指导的研究环境中担任教员。拟议的计划将促进候选人在高级癌症生物学、癌症小鼠模型和转化性乳腺癌研究方面的进一步培训。候选人的导师是乳腺癌基础科学和转化研究方面公认的领导者，并致力于监督PI的研究培训，并促进他向独立的过渡。培训将在积极的乳腺癌临床实践和基础科学研究的环境中进行，并为拟议的研究提供出色的资源，并为培养和发展内科科学家提供机构支持。 这项拟议的研究将侧重于了解乳腺癌对HER2抑制剂耐药的分子机制。HER2癌基因扩增发生在25%的人类乳腺癌中。曲妥珠单抗是HER2的抗体，拉帕替尼是HER2和EGFR的酪氨酸激酶抑制剂，对HER2+乳腺癌患者是有效的治疗方法，但大多数接受这些药物治疗的患者最终会复发，这表明肿瘤获得或本质上具有逃避HER2抑制的机制。磷脂酰肌醇-3-激酶(PI3K)-Akt轴的异常激活正在成为抗HER2治疗耐药的重要机制，拉帕替尼耐药细胞系模型系统的初步数据支持这一点。我们推测，作为选择性治疗压力的结果，对HER2抑制的抵抗是由于PI3K-Akt轴通过上调交替的代偿信号和/或PI3K途径的突变而重新激活的结果。抑制这些替代途径应该提供治疗靶点，以防止或克服对抗HER2治疗的耐药性。其具体目标是1)确定在对拉帕替尼耐药的HER2过表达的乳腺癌细胞中PI3K-Akt异常激活的分子机制；2)确定PI3K突变导致耐药细胞中PI3K-Akt通路激活的机制；以及3)确定PI3K的治疗性抑制剂是否恢复对HER2抑制剂的敏感性，以及PI3K通路激活是否预测对HER2抑制剂的临床反应。AIM 1的实验将使用对拉帕替尼耐药的细胞系模型来确定HER3在激活PI3K方面的作用，或确定PI3K途径的其他候选激活物。将测试几种方法，包括RNAi击倒候选蛋白质，免疫沉淀PI3K亚基以识别结合伙伴，以及对可疑耐药途径的药物操纵。目的2通过用质谱仪定量耐药细胞中突变的PI3K亚型，以及通过敲除PI3K亚基来测试突变对PI3K信号的作用，来了解PI3K突变的作用。利用异种移植小鼠模型，AIM 3将测试PI3K突变是否会在体内产生耐药性，以及PI3K抑制和HER2抑制剂的结合是否提供了一种克服HER2抑制剂耐药性的策略。此外，PIK3CA突变或PTEN缺失激活PI3K通路预测临床对HER2抑制剂的耐药性的假设将在拉帕替尼新辅助临床试验的肿瘤样本中进行测试。 与公共卫生相关：用靶向疗法治疗HER2阳性乳腺癌是成功的，但尽管这些疗法有效，大多数患者在治疗后最终进展。这项研究将集中于了解肿瘤如何对HER2指导的治疗产生耐药性。这项工作还将测试克服这种耐药性的潜在策略，通过改善HER2阳性乳腺癌患者的预后来造福公众健康。

DESCRIPTION (provided by applicant): This proposal describes a five year training plan for the development of an independent translational academic cancer research program. The candidate has had research training as a graduate student and residency training in internal medicine, has completing fellowship training in hematology/oncology, and continues as faculty in a mentored research environment. The proposed program will promote the candidate's further training in advanced cancer biology, mouse models of cancer, and translational breast cancer research. The candidate's mentor is a recognized leader in breast cancer basic science and translational research, and is committed to supervising the PI's research training and fostering his transition to independence. Training will occur in an environment of active clinical practice and basic science investigation in breast cancer, coupled with outstanding resources for the proposed research and with institutional support for the nurturing and development of physician-scientists. The proposed research will focus on understanding molecular mechanisms of resistance to HER2 inhibitors in breast cancer. Amplification of the HER2 oncogene occurs in 25% of human breast cancers. Trastuzumab, an antibody against HER2, and lapatinib, a tyrosine kinase inhibitor of HER2 and EGFR, are effective therapies for patients with HER2+ breast cancer, but most patients treated with these agents eventually relapse, suggesting that tumors acquire or intrinsically possess mechanisms for escape from HER2 inhibition. Aberrant activation of the phosphoinositide-3-kinase (PI3K)-Akt axis is emerging as an important mechanism of resistance to anti-HER2 therapies, supported by preliminary data from cell line model systems of lapatinib resistance. We hypothesize that as a result of the selective pressure of treatment, resistance to HER2 inhibition results from reactivation of the PI3K-Akt axis through upregulation of alternate compensatory signaling and/or mutations in the PI3K pathway. Inhibition of these alternate pathways should provide therapeutic targets to prevent or overcome resistance to anti-HER2 treatment. Specific aims are 1) To determine the molecular mechanisms of aberrant PI3K-Akt activation in lapatinib-resistant HER2-overexpressing breast cancer cells; 2) To determine the mechanisms whereby PI3K mutations contribute to PI3K-Akt pathway activation in drug-resistant cells; and 3) To determine whether therapeutic inhibitors of PI3K restore sensitivity to HER2 inhibitors and whether PI3K pathway activation predicts clinical response to HER2 inhibitors. Experiments in Aim 1 will use cell line models of lapatinib resistance to determine the role of HER3 in activating PI3K, or to identify other candidate activators of the PI3K pathway. Several approaches including RNAi knockdown of candidate proteins, immunoprecipitation of PI3K subunits to identify binding partners, and pharmacologic manipulation of suspected resistance pathways will be tested. Aim 2 will focus on understanding the role of PI3K mutations by using mass spectrometry to quantitate mutant PI3K isoforms in resistant cells, and by knockdown of PI3K subunits to test the role of mutations on PI3K signaling. Using xenograft mouse models, Aim 3 will test whether PI3K mutations confer resistance in vivo, and whether combination of PI3K inhibition with HER2 inhbitors provides a strategy to overcome HER2 inhibitor resistance. Further, the hypothesis that activation of the PI3K pathway by PIK3CA mutation or PTEN loss predicts for clinical resistance to HER2 inhibitors will be tested in tumor samples from a neoajuvant clinical trial of lapatinib. PUBLIC HEALTH RELEVANCE: Treatment of HER2-positive breast cancer with targeted therapies has been successful, but despite the effectiveness of these therapies, most patients eventually progress after treatment. This research will focus on understanding how tumors develop resistance to HER2-directed therapies. This work will also test potential strategies for overcoming that resistance, to benefit public health by improving the outcome of patients with HER2-positive breast cancer.