Taxane Resistance in Breast and Ovarian Cancer Cells

描述（由申请人提供）：该提案将研究紫杉烷治疗乳腺癌和卵巢癌的非mdr1遗传和细胞决定因素。我们已经从4种乳腺癌细胞系和6种卵巢癌细胞系中建立了17种紫杉烷耐药变异，我们假设这些不同的变异可能有助于了解紫杉烷耐药的新机制。具体目标是：1。测定微管异构体含量和微管动态不稳定性的潜在改变。我们对六类（3-微管蛋白异构体）表达的分析揭示了许多紫杉烷抗性变体中对微管蛋白含量的改变。这些细胞中的微管组装将与Mary Ann Jordan博士合作进行评估。目的：研究微管蛋白及微管相关蛋白基因改变对药物敏感性的影响。一些变体下调了MAP4和上调了MAP Tau，我们假设这些改变可能通过改变紫杉烷结合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微管蛋白亚型的作用，并利用四环素调控的诱导载体将感兴趣的基因转染到亲代细胞中。3. 探讨凋亡调节因子（如Bcl-2、Bcl-XL、Akt）和细胞生长调节因子（如EGFR-1、her2）作为紫杉烷反应的决定因素。一些非p -gp变异具有凋亡调节因子的表达改变。我们将使用反义和RNAi方法来研究这些调节因子的抑制对紫杉烷细胞敏感性的影响。我们还将通过使用激酶抑制剂抑制EGFR-1、her2、Akt等基因来确定抑制生长通路对紫杉烷敏感性的影响。4. 鉴定新的遗传标记以预测紫杉烷类药物的耐药性。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来预测紫杉烷治疗的敏感性和耐药性。候选基因将通过PCR、免疫印迹和我们制作的MCF-7 Flp-ln细胞系的tet调节表达来验证。5. 目的探讨乳腺癌和卵巢癌临床标本紫杉烷耐药情况。将对来自NCI合作人体组织网络的乳腺癌和卵巢癌肿瘤标本进行全基因组分析，并对紫杉烷治疗的反应性进行注释。在细胞模型中鉴定的耐药因子的表达也将在这些标本中得到验证。综上所述，本项目将利用卵巢癌和乳腺癌细胞系中大量独特的紫杉烷细胞耐药模型和紫杉烷治疗患者的临床标本：(1)研究已知耐药机制的相关性；(2)探索新的耐药机制；(3)开发临床反应标志物。

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.