PTEN loss increases efficiency of breast tumor metastasis

描述(申请人提供)：该项目的长期目标是研究PTEN缺失促进循环肿瘤细胞转移效率的分子机制。PTEN缺失可抵抗细胞凋亡，并在脱落时产生微触角(McTns)乳腺上皮细胞。与表达PTEN的同源亲本相比，McTN的频率、每细胞的数量和长度都增加了。这些新结构(McTns)在结构上不同于经典的基于肌动蛋白的贴壁细胞延伸，在对失巢凋亡耐药的乳腺肿瘤株中持续数天， 并有助于重新附着到基质或细胞单层。因此，PTEN缺失导致的细胞凋亡抵抗和McTns表达增强的结合可能对促进肿瘤细胞外渗和有效黏附新的部位具有重要的影响。我们将通过检测微触角(McTns)的形成和失巢细胞，以及扩散后的细胞存活，最终转移生长来确定PTEN在转移过程中的早期作用。我们将验证PTEN缺失通过耐失巢细胞抵抗、细胞骨架改变和细胞休眠的独特组合来增加乳腺癌细胞转移效率的假设。在第一个特定的目标中，将确定由于PTEN缺失而导致的异常信号通路，这些信号通路是McTns启动和休眠细胞生存所必需的。在目标2中，将确定McTns抑制和失巢凋亡所需的细胞区域和/或PDZ结合域。这两个目标都将使用同基因的乳腺上皮细胞MCF-10A和MCF-10A/PTEN-/-细胞与其他缺乏PTEN表达的人类转移细胞株相结合。在目标3中，我们将结合已知的癌基因ErbB2(Her2/Neu)，研究PTEN缺失导致的细胞扩散后存活、肿瘤细胞休眠和转移性生长的分子机制。很大一部分乳腺肿瘤携带导致ErbB2过度激活的癌基因突变，并且丢失PTEN的具有活性的ErbB2的肿瘤细胞对曲妥珠单抗具有耐药性，这构成了一个重要的临床问题，在每个特定目的中确定的信号机制可能为旨在防止转移的治疗提供新的靶点。应聘者的近期目标是通过使用共聚焦和活体成像的培训来补充细胞信号转导方面的广泛经验，更新和提高癌症遗传学学科的技能，并发展成功过渡到独立教员职位的关键技能。拟议的研究可以自由转让，并将为设立独立调查员奠定基础。PTEN等基因体细胞敲除模型，结合体外和体内成像，将为重点研究PTEN缺失促进转移扩散的分子机制提供独特的利基环境。

DESCRIPTION (provided by applicant): The long term objectives of this project are to investigate the molecular mechanisms by which PTEN loss promotes metastatic efficiency of circulating tumor cells. PTEN loss confers apoptotic resistance and production of microtentacles (McTNs) mammary epithelial cells upon detachment. The McTNs are increased in frequency, and number and length per cell as compared to their isogenic, PTEN expressing parental counterparts. These novel structures (McTNs) are structurally distinct from classical actin based extensions of adherent cells, persist for days in breast tumor lines that are resistant to anoikis, and aid in the reattachment to matrix or cell monolayers. Therefore, the combination of apoptotic resistance and enhanced McTNs expression due to PTEN loss may have important consequences for facilitating tumor cell extravasation and efficient adherence to new sites. We will determine the role of PTEN early in metastatic process by examining microtentacles (McTNs) formation and anoikis, as well as cell survival after dissemination, and finally metastatic outgrowth. We will test the hypothesis that PTEN loss increases metastatic efficiency of breast cancer cells through a unique combination of anoikis resistance, cytoskeletal alterations, and cell dormancy. In the first specific aim, the aberrant signaling pathways due to PTEN loss necessary for McTNs promotion and dormant cell survival will be identified. In Aim 2, the cellular locale and/or PDZ binding domain requirement for McTNs suppression and anoikis will be determined. Both Aims will employ the isogenic mammary epithelial MCF-10A and MCF- 10A/PTEN-/- cells in combination with other human metastatic cell lines that lack PTEN expression. In Aim 3, we will investigate the molecular mechanisms of cell survival after dissemination, tumor cell dormancy, and metastatic outgrowth due to PTEN loss in combination with the known oncogene ErbB2 (Her2/Neu). A large fraction of breast tumors carry oncogenic mutations that cause hyperactivation of ErbB2, and tumor cells with active ErbB2 that have lost PTEN are resistant to trastuzumab, posing a significant clinical problem Overall, the signaling mechanisms identified in each specific aim may provide new targets for therapies aimed at preventing metastasis. The immediate goals of the candidate are to supplement broad experience in cellular signal transduction with training in the use of confocal and in vivo imaging update and enhance skills in the discipline of cancer genetics, and develop critical skills for a successful transition to an independent faculty position. The proposed studies can be transferred freely and will provide a foundation for the establishment as an independent investigator. The PTEN isogenic somatic cell knock-out model, in combination with in vitro and in vivo imaging, should provide a unique niche for research focused on the molecular mechanisms by which PTEN loss promotes metastatic spread.