ESCRT-0 complex modulates Rbf-mutant cell survival by regulating Rhomboid endosomal trafficking and EGFR signaling

Abstract

The Rb tumor suppressor is conserved in Drosophila, and its inactivation can lead to cell proliferation or death depending on the specific cellular context. Therefore, identifying genes that affect the survival of Rb-mutant cells can potentially identify novel targets for therapeutic intervention in cancer. From a genetic screen in Drosophila, we identified synthetic lethal interactions between mutations of fly Rb (rbf ) and the ESCRT-0 components stam and hrs. We show that inactivation of ESCRT-0 sensitizes rbf-mutant cells to undergo apoptosis through inhibition of EGFR signaling and accumulation of Hid protein. Mutation of stam inhibits EGFR signaling upstream of secreted Spi and downstream of Rhomboid expression, and causes Rhomboid protein to accumulate in the abnormal endosomes labeled with both the early and late endosomal markers Rab5 and Rab7. These results reveal that ESCRT-0 mutants inhibit EGFR signaling by disrupting Rhomboid endosomal trafficking in the ligand-producing cells. Because ESCRT-0 also plays crucial roles in EGFR downregulation after ligand binding, this study provides new insights into how loss of ESCRT-0 function can either increase or decrease EGFR signaling.

NatB modulates Rb mutant cell death and tumor growth by regulating EGFR/MAPK signaling through the N-end rule pathways

“…This is consistent with the reported direct regulation of Hid by MAPK [15]. Taken together, these results suggest that rbf mutant cells are more sensitive to reduced level of EGFR/MAPK signaling and that the rbf synthetic lethal screens [13,16,17] can identify additional modulators of EGFR signaling. In this study, we report new alleles of NatB subunits in rbf synthetic lethal screen and identify a novel mechanism by which NatB modulates EGFR signaling by regulating the levels of positive and negative components of the pathway coordinately through the two branches of the N-end rule pathway.…”

“…Immunostaining was performed as previously described [13]. For dpErk staining, dissected discs were fixed with 8% formaldehyde in PBS for 1 hr.…”

“…Our previous studies suggest that mutations such as TSC2 and axin induce synergistic cell death with rbf most strongly in anterior eye discs due to induction of excessive cellular stress [16,17,21]. In contrast, mutations that cause deficiency in EGFR/MAPK signaling induce synergistic cell death with rbf mainly in posterior eye discs [13]. The observed psid, rbf synergistic cell death in posterior eye discs prompted us to determine whether psid mutation affect EGFR/MAPK signaling.…”

“…EGFR signaling is activated in the MF and posterior region of the developing eye discs and plays critical roles in preventing cell death in addition to its role in regulating cell cycle and differentiation [12]. Interestingly, reducing EGFR signaling by mutations that function upstream of MAPK preferentially increased apoptosis of rbf mutant cells in posterior eye discs [11,13]. In contrast, mutation of rno, which functions downstream of MAPK and regulates the nuclear output of EGFR signaling [14], did not affect apoptosis of rbf mutant cells.…”

“…; psd-D1(BL41122, BL indicating Bloomington Drosophila stock center); psd-D4 (BL41123), UAS-Rbf RNAi (BL36744), scrib 673 (BL41175), UAS-NAA20 RNAi (BL36899), UAS-Drk RNAi (BL41692), Drk-PZ(BL12378), UAS-Poe RNAi (BL32945), UAS-PP2AC RNAi (BL 27723), UAS-Cnot4 RNAi (BL42513), UAS-Cnot4 (BL22246), UAS-p35 (BL5072, BL5073), UAS-MAPK RNAi (BL34855), UAS-Ubr5 RNAi (BL32352), UAS-Ubr1 RNAi (BL31374), UAS Psid-WT [19], UAS-Psid-S678Dand UAS-Psid-S678A[20], aos-lacz and UAS-EGFR CA[13], UAS-ras v12 (BL64196), UAS-raf gof (BL2033), UAS-GFP (BL5431), CoinFLP-Gal4-UAS-GFP (BL58751), UAS-Dcr2 (BL58757), UAS-sprouty RNAi (BL36709), and UAS-sprouty[33].…”