IDENTIFICATION OF MOLECULAR MECHANISMS RESPONSIBLE FOR DEGRADATION OF THE TUMOR SUPPRESSOR PROTEIN NUMB IN CANCER.

ABSTRACT NUMB was initially described as a cell fate determinant involved in neurogenesis. More recently, NUMB has been implicated in different types of human cancers, in which it has a tumor suppressor role. In particular, data from our laboratory revealed that loss of NUMB protein occurred in approximately 50% of breast cancers and 30% of non-small cell lung cancers, and leads to increased oncogenic NOTCH activity and decreased p53 tumor suppressor function. Mechanistically, loss of NUMB in human breast cancers is due to its deregulated ubiquitination and ensuing proteasomal degradation, as witnessed by the restoration of physiological NUMB levels in NUMB-deficient primary breast tumor cells upon proteasome inhibition with MG-132. Therefore, the molecular mechanism underlying NUMB degradation in cancer most likely involves deregulation of components of the cellular machinery normally regulating the ubiquitination/phosphorylation status of the NUMB protein, such as E3-ubiquitin ligases/kinases. In this thesis, we devised a high-throughput phenotypic screening to identify the molecular determinants responsible for NUMB loss among E3 ligase family. The screening assay measures restoration of NUMB expression upon siRNA-mediated silencing of candidate enzymes, in a NUMB-deficient model-system. We identified the breast cancer epithelial cell line MDA-MB-361, as a suitable cell model system for the screening assay as it recapitulates the phenotype of NUMB-deficient primary tumor cells. Indeed, NUMB protein levels in these cells are restored to physiological levels by MG-132 treatment. For the high-throughput phenotypic assay, we developed and optimized for a miniaturized format, a NUMB capture ELISA assay. Using the high-throughput screening platform, we assessed the involvement of over 600 E3 ligases in NUMB downregulation, and identified 21 candidate E3 ligases. We then went through the validation of these 21 candidate hits, which is the topic of this thesis. Upon validation of the top six candidates of E3 ligases list, we confirmed that the E3 ligase, RBX1 (RING-Box 1), mediates the downregulation of NUMB in both MDA-MB-361 cells and human primary NUMB-deficient breast and lung tumor cells. Indeed, we demonstrated that silencing RBX1 in these cells restores NUMB protein levels, while no effect was observed in NUMB-proficient cell lines or primary tumor cells. Moreover, we also established a physical interaction between NUMB and RBX1 in MDA-MB-361 cells indicating that RBX1 directly mediates NUMB degradation. RBX1 belongs to the tetrameric E3 ligase complex, Skp1/Cullin1/F-box (SCF), in which the specificity for substrates is mediated by the F-box protein. Intriguingly, among the 21 candidates from the high-throughput screening, we identified the F-box protein, FBXW8 (F-box and WD repeat domain containing 8), which has been described to form a complex with RBX1. We, therefore, assessed the role of FBXW8 in NUMB donwregulation in high-resolution studies in MDA-MB-361 cells and confirmed its involvement. We are currently validating FBXW8 also in primary tumors cells from human breast and lung cancers. In conclusion, our data indicate that an SCF E3 ligase complex involving RBX1 and FBXW8, likely mediates NUMB hyperdegradation in human cancers. This result has potential translational ramifications as RBX1 and FBXW8 could represent novel molecular targets for therapeutic intervention in NUMB-deficient cancers.