Taxane Resistance in Breast and Ovarian Cancer Cells

描述(申请人提供)：这项提案将研究紫杉烷治疗乳腺癌和卵巢癌的非多药耐药基因和细胞决定因素。我们已经从4个乳腺癌细胞株和6个卵巢癌细胞系中建立了17个紫杉烷耐药变异体，我们假设这些不同的变异体可能对紫杉烷耐药的新机制有深入的了解。具体目的是：1.测定微管动态不稳定性中微管蛋白亚型的含量和潜在的变化。我们对6类(3-微管蛋白亚型)表达的分析表明，在许多紫杉烷耐药变异体中，p-微管蛋白含量发生了变化。这些细胞中的微管组装将与Mary Ann Jordan博士合作进行评估。2.研究微管蛋白和微管相关蛋白基因改变对药物敏感性的影响。一些变异体下调MAP4和上调MAP Tau，我们假设这些变化可能通过改变紫杉烷结合而对紫杉烷敏感性产生不利影响。我们计划使用siRNA技术来研究这些基因和特定的微管蛋白亚型的作用，并利用四环素调控的可诱导载体将感兴趣的基因导入亲本细胞。3.探讨细胞凋亡调节因子(如Bcl2、Bclxl、Akt)和细胞生长调节因子(如EGFR-1、HER2)作为紫杉烷类药物反应的决定因素。几个非P-gp变异体具有凋亡调节因子的表达变化。我们将使用反义和RNAi方法来研究这些调节因子的抑制对细胞对紫杉烷敏感性的影响。我们还将通过使用激酶抑制剂抑制EGFR-1、HER2、Akt和其他基因来确定抑制生长途径对紫杉烷敏感性的影响。4.寻找预测紫杉烷类药物抗性的新遗传标记。遗传(阵列CGH)和基因组(微阵列基因表达谱)方法将用于寻找新的遗传标记，用于预测紫杉烷治疗的敏感性和耐药性。候选基因将使用我们建立的MCF-7 FLP-ln系，通过聚合酶链式反应、免疫印迹和tet调节表达来验证。5.研究乳腺癌和卵巢癌临床标本对紫杉烷的耐药性。将对来自NCI合作人类组织网络的乳腺癌和卵巢癌肿瘤标本进行全基因组图谱分析，注释为对紫杉烷治疗的反应性。在细胞模型中确定的抗性因子的表达也将在这些标本中得到验证。综上所述，该项目将使用一大套卵巢和乳腺癌细胞对紫杉烷耐药的独特模型和从接受紫杉烷治疗的患者获得的临床标本：(1)研究已知耐药机制与这些药物的相关性，(2)探索新的机制，以及(3)开发临床反应标记。

DESCRIPTION (provided by applicant): This proposal will study non-MDR1 genetic and cellular determinants of taxane therapy in breast and ovarian cancers. We have established 17 taxane-resistant variants from 4 breast and 6 ovarian cancer cell lines, and we hypothesize that these diverse variants may yield insight into novel mechanisms of resistance to taxanes. The specific aims are: 1. To determine tubulin isoform content and potential alterations in microtubule dynamic instability. Our analyses of the expression of the six classes of (3- tubulin isoforms reveal altered p-tubulin content in many of the taxane-resistant variants. Microtubule assembly in these cells will be assessed in collaboration with Dr. Mary Ann Jordan. 2. To study the effects of altered tubulin and microtubule associated protein genes on drug sensitivity. Several of the variants have down-regulated MAP4 and up-regulated MAP Tau, and we hypothesize that these alterations may have an adverse effect on taxane sensitivity by altering taxane binding. We plan to use siRNA technology to study the effects of these genes and specific tubulin isoforms, and inducible vectors utilizing tetracycline regulation will be used to transfect genes of interest into parental cells. 3. To explore regulators of apoptosis (e.g. Bcl-2, Bcl-XL, Akt) and cell growth (e.g. EGFR-1, her2) as determinants of response to taxanes. Several of the non-P-gp variants possess altered expression of apoptotic regulators. We will use antisense and RNAi approaches to study the effects of inhibition of these regulators on cellular sensitivity to taxanes. We will also determine the effects of inhibition of growth pathways on taxane sensitivity by inhibiting EGFR-1, her2, Akt and other genes using kinase inhibitors. 4. To identify novel genetic markers to predict resistance to taxanes. Genetic (array CGH) and genomic (microarray gene expression profiling) approaches will be used to search for novel genetic markers for the prediction of sensitivity and resistance to taxane therapy. Candidate genes will be validated by PCR, immunoblotting, and tet-regulated expression using an MCF-7 Flp-ln line which we have made. 5. To study taxane resistance in clinical specimens of breast and ovarian cancer. Whole genome profiling of tumor specimens from breast and ovarian cancers from the NCI Cooperative Human Tissue Network, annotated for responsiveness to taxane therapy, will be performed. Expression of resistance factors identified in cellular models will also be validated in these specimens. In summary, this project will use a large set of unique models of cellular resistance to taxanes in ovarian and breast cancer cell lines and clinical specimens obtained from patients treated with taxanes to: (1) study the relevance of known resistance mechanisms to these drugs, (2) explore new mechanisms, and (3) develop clinical markers for response.