Figure 7. Erlotinib and Gefitinib Induce a Sec5-Dependent Role for Inactive EGFR in Autophagy Initiation.

(A) The EGFR TKIs, erlotinib and gefitinib, induce autophagy in MDA-MB-231 cells. Cells were treated with DMSO or 2 μM of erlotinib or gefitinib for 24 h, followed by 2 h of 80 μM chloroquine (CQ) treatment as indicated, and whole cell lysates were analyzed for LC3 levels.

(B) Knockdown of EGFR blocks gefitinib-induced autophagy in MDA-MB-231 cells. Control or EGFR knockdown cells were treated with DMSO or 2 μM gefitinib for 24 h, followed by 2 h of CQ treatment as indicated.

(C) Quantification of LC3-II levels in (B); mean + SD, n = 3; ** P < 0.01.

(D) Erlotinib/gefitinib inhibits EGFR but not Akt phosphorylation in MDA-MB-231 cells. Cells were treated with DMSO or 2 μM of erlotinib or gefitinib for 4 h or 24 h, followed by whole cell lysate harvest and Western blot analysis of indicated proteins. Specific antibodies recognizing pEGFR (Y1068) and pAkt (S473) were used.

(E) Erlotinib/gefitinib inhibits EGFR association with LAPTM4B. HEK293 cells co-transfected with EGFR and Flag-LAPTM4B were treated with MDSO or 2 μM erlotinib or gefitinib for 2 h and whole cell lysates were harvested for co-immunoprecipitation (co-IP) assay.

(F) Gefitinib treatment stimulates EGFR accumulation at endosomes. MDA-MB-231 cells treated with DMSO or 2 μM of gefitinib for 24 h were fixed for co-staining of endogenous LAPTM4B (red) and EGFR (green). Bar, 10 μm.

(G and H) Erlotinib/gefitinib (2 μM) treatment enhances EGFR association with Sec5 and Rubicon in HEK 293 cells co-transfected with EGFR and Sec5 or Rubicon.

(I) Quantification of relative EGFR association with indicated proteins in (E), (G) and (H); mean + SD, n = 3.

(J) Erlotinib/gefitinib (2 μM) induces disassociation of Rubicon from Beclin 1 in MDA-MB-231 cells. Asterisk indicates a nonspecific band.

(K) Gefitinib induced autophagy requires Sec5 but not LAPTM4B. MDA-MB-231 cells were transfected with indicated siRNAs, treated with DMSO or 2 μM gefitinib for 24 h, followed with or without 2 h of CQ treatment. Whole cell lysate were harvested for Western blot analysis of LC3-II.

(L) Quantification of LC3-II levels in (K); mean + SD, n = 3; ** P < 0.01; *** P < 0.001; NS, not significant.

(M) A model for the role of inactive EGFR in autophagy initiation. Serum starvation enhances EGFR interaction with LAPTM4B, resulting in increased accumulation of inactive EGFR at endosomes where EGFR also gains stronger interaction with Sec5. LAPTM4B and Sec5 facilitate Rubicon association with EGFR, which promotes the release of Beclin 1 from Rubicon for autophagy initiation. The EGFR TKIs, erlotinib and gefitinib, stimulate LAPTM4B-independent EGFR accumulation at endosomes where EGFR still gains enhanced Sec5 and Rubicon interaction so as to release Rubicon-free Beclin 1 for autophagy initiation.

WCL, whole cell lysate; See also Figure S7.