Project Summary Cyclin E, a key regulatory protein controlling the G1 to S phase transition in mammalian cells, is post- translationally modified by neutrophil elastase mediated proteolytic cleavage to generate the low molecular weight isoforms of cyclin E (LMW-E) that are detected in many cancer types. Our laboratory has elucidated several distinct oncological attributes of LMW-E versus full length cyclin E (EL) in breast cancer, using in vitro and in vivo model systems. In this application, using a robust inducible murine transgenic model of LMW-E mediated tumorigenesis, we have mapped some of the early events in the pre-neoplastic mammary gland that gives rise to aggressive tumors with high metastatic potential. These LMW-E oncogenic events permit the induction of sustained tumorigenesis even in the absence of LMW-E expression. These events include induction of DNA damage, upregulation of several genes involved in unregulated DNA replication and G2/M transition, and specific mutations in genes, such as ALK, that is readily targetable. These preliminary results have led to the following three testable hypotheses: (1) expression of LMW-E early in the pre-invasive breast cancer (i.e. ductal carcinoma in situ) results in induction of genomic alteration leading to an invasive carcinoma, (2) LMW-E in a cyclin E knockout model will result in a more aggressive phenotype than overexpression of EL, resulting in increased genomic instability, centrosome amplification and transformability in hMECs, (3) Inhibition of ALK, a secondary oncogenic event to LMW-E induction, early in the neoplastic process can inhibit tumorigenesis and also be used as a target for the treatment of triple negative breast cancers (TNBC) expressing LMW-E. The following aims are designed to test each aspect of these 3 hypotheses: Aim 1:Examine the role of cytoplasmic cyclin E in differentiating indolent versus high-risk ductal carcinoma in situ (DCIS). Aim 2: Investigate the mechanism of LMW-E mediated DNA damage response and centrosome amplification in the absence of endogenous cyclin E in somatic hMEC models. Aim 3: Investigate the role of ALK as a mediator of LMW-E mediated mammary tumorigenesis and as a therapeutic target in TNBC. These studies have the potential to identify LMW-E-induced early oncogenic events and provide the rationale to use LMW-E as a biomarker to identify the DCIS cases which are at high risk for developing invasive cancer. Our studies will show if ALK can be a viable target for the LMW-E overexpressing TNBC patients. Since there are already several ALK inhibitors, which have undergone Phase I-III clinical trials in malignancies other than breast, the translational of these pre-clinical studies to TNBC patients could occur readily.