NetrinA and Frazzled in regulation of wing disc epithelia

© 2017 Dr. Sofya GolenkinaThe chemotrophic factor Netrin and its receptors play a critical role during axon outgrowth, organogenesis and cancer progression. In my PhD I have found that Drosophila NetrinA and its receptor Frazzled also control epithelial-mesenchymal transition (EMT), a key process of embryogenesis, regeneration and tumor metastasis. Using wing imaginal disc eversion as a genetic model, I have demonstrated that both loss of netrinA and elevation of frazzled expression in the peripodial epithelium suppress zonula adherens dissociation and alter cytoskeleton organization, but do not affect basement membrane degradation. Loss-of-function and overexpression analysis of frazzled in the disc proper epithelium shown that Frazzled controls localisation of junctional proteins (E-Cadherin and β-catenin), F-actin polymerization and cellular contractility. A key question is whether the Frazzled signaling pathways that regulate cell motility are distinct from those that control epithelial phenotypes. To address this I have conducted a structure function analysis to determine which intracellular domains of Frazzled are required for inhibiting wing eversion, and which are required for regulating E-Cadherin, F-Actin and cell shape. I shown that P1 domain is responsible for eversion disruption, E-Cadherin delocalization and cellular contractility, but not for formation of the F-actin protrusions. P3 domain is required for cell motility. Finally, I performed an RNAi screen to identify components acting downstream of NetrinA/Frazzled signaling. I have found that the apical polarity protein Par6 is required for inhibiting EMT and partly for E-Cadherin delocalization. The results establish a new role for Netrin signaling in a developmental EMT and highlight the complexity of NetrinA/Frazzled pathway regulation of epithelial cells

A genome-wide Drosophila epithelial tumorigenesis screen identifies Tetraspanin 29Fb as an evolutionarily conserved suppressor of Ras-driven cancer.

Oncogenic mutations in the small GTPase Ras contribute to ~30% of human cancers. However, Ras mutations alone are insufficient for tumorigenesis, therefore it is paramount to identify cooperating cancer-relevant signaling pathways. We devised an in vivo near genome-wide, functional screen in Drosophila and discovered multiple novel, evolutionarily-conserved pathways controlling Ras-driven epithelial tumorigenesis. Human gene orthologs of the fly hits were significantly downregulated in thousands of primary tumors, revealing novel prognostic markers for human epithelial tumors. Of the top 100 candidate tumor suppressor genes, 80 were validated in secondary Drosophila assays, identifying many known cancer genes and multiple novel candidate genes that cooperate with Ras-driven tumorigenesis. Low expression of the confirmed hits significantly correlated with the KRASG12 mutation status and poor prognosis in pancreatic cancer. Among the novel top 80 candidate cancer genes, we mechanistically characterized the function of the top hit, the Tetraspanin family member Tsp29Fb, revealing that Tsp29Fb regulates EGFR signaling, epithelial architecture and restrains tumor growth and invasion. Our functional Drosophila screen uncovers multiple novel and evolutionarily conserved epithelial cancer genes, and experimentally confirmed Tsp29Fb as a key regulator of EGFR/Ras induced epithelial tumor growth and invasion