Taxane Resistance in Breast and Ovarian Cancer Cells

乳腺癌和卵巢癌细胞中的紫杉烷耐药性

• 批准号: 7656733
• 负责人: BRANIMIR I SIKIC
• 金额: $ 29.77万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7656733
• 关键词: ABCB1 gene; Accounting; Adverse effects; Affect; Antibodies; Apoptosis; Apoptosis Regulator; Apoptotic; Binding; Breast; Breast Cancer Cell; Breast Carcinoma; Cancer cell line; Candidate Disease Gene; Cell Line; Cell model; Cells; Cetuximab; Clinical; Clinical Markers; Collaborations; Cooperative Human Tissue Network; Data; Drug resistance; EGFR inhibition; Epidermal Growth Factor Receptor; Gefitinib; Gene Expression Profiling; Genes; Genetic; Genetic Markers; Genome; Genomics; Growth; Immunoblotting; Jordan; Kinetics; Lung; MAP4; MCF7 cell; Malignant Neoplasms; Malignant neoplasm of ovary; Mediator of activation protein; Microarray Analysis; Microtubule-Associated Proteins; Microtubules; Minority; Molecular Target; Mutation; Ovarian; Ovary; P-Glycoprotein; P-Glycoproteins; Paclitaxel; Pathway interactions; Patients; Pharmaceutical Preparations; Phosphorylation; Prostate; Protein Isoforms; Proteins; RNA Interference; Regulation; Relative (related person); Research Personnel; Resistance; SDZ-PSC-833; Small Interfering RNA; Specimen; Taxane Compound; Technology; Tetanus Helper Peptide; Tetracyclines; Tubulin; Tyrosine Kinase Inhibitor; Variant; cancer cell; cell growth; chemotherapeutic agent; docetaxel; drug sensitivity; inhibitor/antagonist; insight; interest; kinase inhibitor; malignant breast neoplasm; novel; overexpression; programs; resistance factors; resistance mechanism; response; tau Proteins; taxane; tumor; vector

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.

描述（由申请人提供）：该提案将研究乳腺癌和卵巢癌紫杉烷治疗的非 MDR1 遗传和细胞决定因素。我们已经从 4 种乳腺癌和 6 种卵巢癌细胞系中建立了 17 种紫杉烷抗性变体，我们假设这些不同的变体可能有助于深入了解紫杉烷类耐药的新机制。具体目标是： 1. 确定微管蛋白同种型含量和微管动态不稳定性的潜在改变。我们对六类 (3-微管蛋白亚型) 表达的分析揭示了许多紫杉烷抗性变体中 p-微管蛋白含量的改变。将与 Mary Ann Jordan 博士合作评估这些细胞中的微管组装。2. 研究改变的微管蛋白和微管相关蛋白基因对药物敏感性的影响。其中一些变体下调了 MAP4 并上调了 MAP Tau，我们假设这些改变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微管蛋白亚型的影响，并利用四环素调节的诱导载体将感兴趣的基因转染到亲本细胞中。 3. 探索细胞凋亡的调控因子（如Bcl-2、 Bcl-XL、Akt）和细胞生长（例如 EGFR-1、her2）作为紫杉烷类药物反应的决定因素。一些非 P-gp 变体具有改变的凋亡调节因子的表达。我们将使用反义和 RNAi 方法来研究这些调节剂的抑制对细胞对紫杉烷类敏感性的影响。我们还将确定生长途径的抑制对紫杉烷敏感性的影响 使用激酶抑制剂抑制 EGFR-1、her2、Akt 等基因。 4. 鉴定新的遗传标记来预测紫杉烷类药物的抗性。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，以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 使用我们制作的 MCF-7 Flp-ln 系进行 tet 调节表达。 5. 研究乳腺癌和卵巢癌临床标本中的紫杉烷耐药性。将对来自 NCI 人类组织合作网络的乳腺癌和卵巢癌肿瘤样本进行全基因组分析，并注释对紫杉烷治疗的反应。细胞模型中鉴定的耐药因子的表达也将在这些样本中得到验证。在 总之，该项目将使用卵巢癌和乳腺癌细胞系中对紫杉烷类药物的大量独特细胞耐药模型以及从接受紫杉烷类药物治疗的患者获得的临床标本，以：（1）研究已知耐药机制与这些药物的相关性，（2）探索新机制，以及（3）开发临床反应标志物。