Abstract

Activating molecule in Beclin1-regulated autophagy1 (Ambra1) protein discovered in 2007 is an essential regulator in autophagy; it is also involved in the development of the nervous system, regulating normal cell survival, and, proliferation. Its role in autophagy has been extensively studied. However; its role in cell proliferation is less understood. Ambra1 has been related to proliferative disorders like cancer but, the underlying mechanisms by which Ambra1 can regulate cell proliferation in normal and pathologic conditions remain largely unclear. Melanoma is the most deadly type of skin cancers and, there is a continuous need to develop early biomarkers as well as treatments to improve the survivability of patients.
This study explores the role of Ambra1 in different cellular processes including cell proliferation with a focus on Melanoma. The research was designed to use a systems based “omics” approach to investigate novel roles of Ambra1. An interactomic approach was carried on to identify novel Ambra1 protein binding partners using yeast two-hybrid assays and Ambra1 differentially expressing A375 melanoma cell lines were utilized for cell proliferation assays, proteomics, metabolomics and transcriptomics approaches to investigate the impacts of Ambra1 overexpression and knockdown on different cellular processes..
Yeast two-hybrid assays performed in this study identified novel Ambra1 binding partners. Analyses of these interactors provide evidence for new roles for Ambra1 in different cellular processes. Additional analysis, with data from a recent large scale interactome screening project suggests that Ambra is a key component of a larger network of proteins than previous evidence suggests. Results from the cell proliferation assays, proteomic, metabolomic and transcriptomic analyses suggest that Ambra1 overexpression in nutrient rich media has little additional effect on proliferation or any other pathways. Transcriptomic analysis of the knock-down of Ambra1 however, was shown to result in significant dysregulation of a significant number of transcripts. This appears to have identified a number of novel roles for Ambra 1 in a range of cellular pathways; some of these are hallmarks of cancer signaling including, cell cycle, angiogenesis, tissue growth factor, axon guidance and Wnt signaling. The work shows that the Ambra1 knockdown appears to upregulate metastatic genes/proteins and supports previous studies demonstrating that the loss of Ambra1 is associated with poor prognosis in melanoma.