Cytokeratin 5/Beta-catenin interaction and crosstalk in hormone regulated breast cancer stem cells

激素调节的乳腺癌干细胞中细胞角蛋白 5/β-连环蛋白的相互作用和串扰

• 批准号: 9760721
• 负责人: Olivia F McGinn
• 金额: $ 3.49万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-20 至 2022-02-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9760721
• 关键词: 14-3-3 Family; Adherens Junction; Affect; Biological Assay; Breast Cancer Cell; Breast Cancer Patient; Breast Cancer cell line; Cancer Etiology; Cell Fractionation; Cell Maintenance; Cell membrane; Cells; Cellular Stress; Cessation of life; Clustered Regularly Interspaced Short Palindromic Repeats; Co-Immunoprecipitations; Complex; Confocal Microscopy; Cytokeratin; Data; Dependence; Development; Drug resistance; Endocrine; Epithelial Cells; Estrogen Receptors; Estrogen receptor positive; Estrogens; Face; Fatty acid glycerol esters; Genetic Transcription; Goals; Hormones; Immunoprecipitation; Implant; Individual; Intermediate Filament Proteins; Knock-out; Laboratories; Lead; Malignant neoplasm of lung; Mammary Neoplasms; Mammary gland; Mammospheres; Mass Spectrum Analysis; Measurement; Measures; Mediating; Membrane; Modeling; Mus; Neoplasm Metastasis; Nuclear; Pathway Analysis; Pathway interactions; Play; Population; Progesterone; Property; Proteins; RNA; Recurrence; Reporter; Reporting; Residual state; Resistance; Risk; Role; Scaffolding Protein; Signal Transduction; Small Interfering RNA; Stains; Stem cells; Stimulus; Structural Protein; Testing; Tumor Initiators; WNT Signaling Pathway; Woman; Xenograft procedure; beta catenin; breast cancer survival; cancer cell; cancer stem cell; deprivation; design; experimental study; in vivo; inhibitor/antagonist; knock-down; malignant breast neoplasm; member; novel; overexpression; prevent; progesterone receptor positive; protein protein interaction; protein structure; response; self-renewal; single-cell RNA sequencing; small hairpin RNA; stem cell population; therapy resistant; transcription factor; transcriptome; transcriptome sequencing; tumor; tumor growth; tumor initiation; tumor progression

Project Summary Breast cancer is the second-leading cause of cancer-related deaths in women. Over two thirds of breast cancers are estrogen receptor (ER) and progesterone receptor (PR) positive (termed luminal), and account for the majority of breast cancer deaths. Following endocrine treatments, residual luminal breast cancer cells can become dormant and pose a risk of recurrence that can last up to 20 years. The existence of a rare population of cancer stem cells (CSCs) that are more quiescent, self-renewing, invasive, drug resistant, and tumor initiating than non-CSCs may explain the prolonged risk of recurrence in luminal breast cancer. Our laboratory has previously identified the intermediate filament protein cytokeratin 5 (CK5) as a marker of luminal breast CSCs. These cells are relatively rare in luminal breast cancers but can be expanded by progesterone or estrogen deprivation. CK5 knockdown, knockout, and overexpression in luminal breast cancer cell lines has indicated that CK5 is both necessary and sufficient, respectively, for mammosphere formation, a measurement of cancer cell self-renewal. This suggests that CK5 may have a functional role in maintaining a luminal breast CSC population, but how it accomplishes this is still unknown. Although cytokeratins are largely regarded as structural proteins that protect epithelial cells from stress, recent studies have identified that cytokeratins affect cell signaling through protein-protein interactions. We performed an unbiased immunoprecipitation (IP)-mass spectrometry screen to identify novel CK5 interacting proteins in luminal breast cancer cells. We identified β- catenin, the key transcription factor of the Wnt signaling pathway and essential component of adherens junctions, as an interactor of CK5 and confirmed this interaction in luminal and basal breast cancer cell lines and a PDX model. We found that in addition to increasing mammosphere formation and CK5 expression, progesterone was capable of increasing β-catenin transcriptional activity, which was abrogated by CK5 CRISPR knockout. Furthermore, both CK5 overexpression and progesterone treatment caused loss of membrane β-catenin which could have implications in the invasive potential of these cells. Several members of the 14-3-3 family of scaffolding proteins were identified by IP-MS and confirmed by co-IP. These proteins are known to interact with β-catenin. Therefore, this proposal tests the hypothesis that progesterone induced CK5+ CSCs rely on altered β-catenin dynamics mediated by 14-3-3 proteins to promote luminal breast tumor progression. The specific aims of this proposal are to 1) evaluate the dependence of progesterone induced CSCs on CK5 and β-catenin dynamics, and 2) determine if the CK5/14-3-3 interaction is necessary for altered β-catenin dynamics. These studies will define novel protein interactions that contribute to luminal breast CSC maintenance, which could ultimately lead to development of new strategies to prevent recurrence.

项目摘要 乳腺癌是妇女癌症相关死亡的第二大原因。超过三分之二的乳房 癌症是雌激素受体（ER）和孕激素受体（PR）阳性（称为管腔性）， 大多数乳腺癌死亡。内分泌治疗后，残留的管腔乳腺癌细胞可以 潜伏并构成复发的风险，可持续长达20年。稀有种群的存在 癌症干细胞（CSCs）更安静，自我更新，侵袭性，耐药性和肿瘤 与非CSC相比，启动的肿瘤细胞的数量可能解释了腔型乳腺癌复发风险的延长。本实验室 先前已经确定了中间丝蛋白细胞角蛋白5（CK5）作为管腔乳腺癌的标志物， CSC。这些细胞在管腔型乳腺癌中相对罕见，但可以通过孕酮或 雌激素剥夺。CK5敲低、敲除和过表达在管腔型乳腺癌细胞系中的作用 表明CK5对于乳腺球的形成是必要的，也是足够的， 癌细胞的自我更新。这表明，CK5可能在维持管腔乳房中起功能性作用。 CSC人口，但它如何实现这一点仍然是未知的。虽然细胞角蛋白在很大程度上被认为是 保护上皮细胞免受应激的结构蛋白，最近的研究已经确定细胞角蛋白影响 通过蛋白质-蛋白质相互作用的细胞信号传导。我们进行了无偏免疫沉淀（IP）-质量 光谱法筛选以鉴定管腔乳腺癌细胞中的新型CK5相互作用蛋白。我们发现β- catenin是Wnt信号通路的关键转录因子，也是粘附分子的重要组成部分 连接，作为CK5的相互作用，并证实了这种相互作用在管腔和基底乳腺癌细胞系 PDX模型。我们发现，除了增加乳腺球形成和CK5表达外， 孕酮能够增加β-catenin的转录活性，而CK5则可以抑制这种活性 CRISPR敲除。此外，CK5过表达和孕酮治疗均导致了细胞凋亡的丧失。 膜β-连环蛋白，这可能与这些细胞的侵袭潜力有关。的几名成员 14 - 3 - 3家族的支架蛋白通过IP-MS鉴定并通过co-IP确认。这些蛋白质 与β-连环蛋白相互作用因此，该提议检验了孕酮诱导CK5+的假设， CSC依赖于由14 - 3 - 3蛋白介导的β-catenin动力学改变来促进乳腺腔肿瘤 进展该建议的具体目的是：1）评估孕酮诱导的依赖性 CSCs对CK5和β-连环蛋白动力学的影响，以及2）确定CK5/14 - 3 - 3相互作用是否是改变CSCs对CK5和β-连环蛋白动力学的影响所必需的。 β-连环蛋白动力学。这些研究将确定有助于管腔乳腺CSC的新型蛋白质相互作用 这可能最终导致制定新的战略，以防止复发。