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.
The majority of breast cancer patients that succumb to the disease die from complications of metastasis, a process by which cells escape a site of primary tumor growth and colonize distant tissues. There is currently tremendous clinical interest in dormant tumor cells, since their presence in the bloodstream strongly predicts poor patient outcome in breast cancer; emphasized by the observation that breast tumor patients who are diagnosed early with no detectable regional metastases have over a 30% chance of recurrence when followed for 10-15 years. Recent data supports a model in which PTEN loss increases dormant cell survival and microtentacle (McTN) formation in detached or circulating tumor cells to promote seeding of bloodborne metastatic disease. Additionally, a large fraction of breast tumors carry oncogenic mutations that cause hyperactivation of ErbB2 (Her2/Neu). Tumor cells with active ErbB2 that have lost PTEN are resistant to trastuzumab, the leading adjuvant and metastatic treatment for ErbB2 positive patients, thus posing a significant clinical problem. Elucidating the mechanism by which PTEN loss enhances dormant cell survival and McTNs formation may prove to be invaluable for the design of novel therapeutic strategies that reduce metastasis by selectively targeting the survival of circulating tumor cells.
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