Abortive autophagy induces endoplasmic reticulum stress and cell death in cancer cells

Abstract

Autophagic cell death or abortive autophagy has been proposed to eliminate damaged as well as cancer cells, but there remains a critical gap in our knowledge in how this process is regulated. The goal of this study was to identify modulators of the autophagic cell death pathway and elucidate their effects on cellular signaling and function. The result of our siRNA library screenings show that an intact coatomer complex I (COPI) is obligatory for productive autophagy. Depletion of COPI complex members decreased cell survival and impaired productive autophagy which preceded endoplasmic reticulum stress. Further, abortive autophagy provoked by COPI depletion significantly altered growth factor signaling in multiple cancer cell lines. Finally, we show that COPI complex members are overexpressed in an array of cancer cell lines and several types of cancer tissues as compared to normal cell lines or tissues. In cancer tissues, overexpression of COPI members is associated with poor prognosis. Our results demonstrate that the coatomer complex is essential for productive autophagy and cellular survival, and thus inhibition of COPI members may promote cell death of cancer cells when apoptosis is compromised.

Figure 1. Coatomer proteins modulate cell viability.

(A) MDA-MB-231, MDA-MB-468, and SKOV3 cells were transfected with siRNA targeting independent members of COPI. Knockdown of PLK1 was used as positive control for cell death. Cell number after 72 h was measured by crystal violet staining. (B,C) Representative light microscopy pictures of MDA-MB-231 cells treated with RF or siRNA against COPB2. (D) Clonogenic replating efficiency of MDA-MB-231 breast cancer cells after decreased expression of COPA, COPB2 or COPG2 compared to cells treated with risc-free (RF). Results are the mean ± SD from triplicates of two independent experiments. Clonogenic replating efficiency of U2OS cancer cells after treatment with siRNA against COPB2 or COPG2 (E) or rapamycin (F). Results are the mean ± SD from triplicates of two independent experiments. **, p<0.01; ***, p<0.001.

Figure 2. Coatomer proteins modulate autophagy.

(A) MDA-MB-231, MDA-MB-468, and SKOV3 cells stably overexpressing GFP-LC3 were transfected with siRNA targeting independent members of COPI for 72 h, and autophagosome formation was analyzed using the IN Cell analyzer 1000 system. Imatinib was used as a positive control for autophagosome formation. Results are the mean ± SD from triplicates. Representative graph of two independent experiments is shown. (B,C) Representative epifluorescent microscopy images (IN Cell Analyzer 1000) of GFP-LC3–positive vesicles in control MDA-MB-231 cells or after COPB2 depletion. (D) Immunoblot analysis of LC3 in MDA-MB-231 cells 72 h after siRNA mediated depletion of independent members of COPI. β-actin was used as a loading control. (E) MDA-MB-468 and U2OS cells treated with RF or COPB2 siRNA were assayed for protein levels of LC3. (F) MDA-MB-231 cells were transfected with siRNA against COPB2, COPG2 or risc-free (RF) and grown for the indicated times, lysed, and immunoblotted with antibodies to LC3, COPB2, COPG2 and β-actin. *, aspecific band.

Figure 3. COPI depletion increases autophagosome formation.

(A–F) Transmission electron microscopy analysis of autophagy in MDA-MB-231 cells treated with RF or siRNA against COPB2 for 48 h or imatinib for 24 h. White arrowheads indicating autophagosomes are shown in panels C and E. (B, D, F) High magnification images of boxed areas. Scale bars indicate 2 µm (A, C, E) and 500 nm (B, D, F). (G) Number of autophagosomes per cell was calculated by counting the number of double membrane organelles in 20 or more individual cells. (H) Percentage autophagosomal area was calculated by measuring area covered by double membrane organelles in the cytoplasm of 20 or more individual cells. **, p<0.01; ****, p<0.0001,

Figure 4. COPI depletion induces abortive autophagy.

(A) Indicated cancer cell lines were treated with control siRNA (RF) or COPB2 siRNA for 72 h and p62 level was analyzed by immunoblotting. Quantification of p62 protein levels is shown as the mean ± SD from four independent experiments. **, p<0.01. (B,C) MDA-MB-231 cells were treated as in (A) and p62 was analyzed by immunofluorescence microscopy. (D) COPB2 or COPG2 depleted MDA-MB-231 cells were analyzed for LC3 accumulation in the absence or presence of 50 nM BafA1.

Figure 5. COPI depletion inhibits complete degradation of autophagosomes.

(A) GFP-LC3-expressing MDA-MB-231 cells were transfected with risc free (RF) control or with siRNA against COPB2 for 72 h, processed to assess the colocalization between GFP-LC3 and LAMP2 by confocal microscopy. Arrow, colocalization of GFP-LC3 and LAMP2. (B) tfLC3-expressing MDA-MB-231 cells were treated with control siRNA (RF) or COPB2 siRNA for 72 h and BafA1 (50 nM) or imatinib (10 µM) for 24 h. Colocalization of GFP and mRFP was assessed by confocal microscopy. Arrow, RFP-positive/GFP-positive puncta (autophagosome); arrow with open arrowhead: RFP-positive/GFP-negative puncta (autolysosome). Scale bar first column: 50 µM; scale bar last column: 25 µM.

Figure 6. Loss of coatomer subunits promotes ER stress and activates UPR.

(A) Independent COPI subunits were depleted in MDA-MB-231 cells and levels of calnexin, BiP, and Ire1α were measured by westernblot after 72 h. *, a non-specific band. (B–E) Transmission electron microscopy analysis of MDA-MB-231 cells after 48 h treatment with non-targeting siRNA (RF) (B), siCOPB2 (C) or overnight treatment with imatinib (10 µM) (D) or BafA1 (50 nM) (E). Electron micrograph showing the dilated ER (white arrow heads in C and E). M: mitochondria, N: nucleus and G: Golgi apparatus. (F) COPB2 or COPG2 depleted U2OS cells were analyzed for BiP levels. (G) LC3, p62 and BiP levels were analyzed by immunoblot after depletion of COPB2 and BFA treatment with or without BiP siRNA. (H) MDA-MB-231 cells were incubated with control siRNA or COPB2 siRNA, either alone or in combination with siRNA targeting the Atg5/Atg12 complex. Images from different parts of one gel were grouped. (I) MDA-MB-231 cancer cells were depleted of COPB2 or COPG2, either alone or with BafA1, and Ire1α and BiP levels were analyzed by immunoblot. BFA was used as a positive control for ER stress.

Figure 7. RPPA analysis identifies signaling pathways altered during abortive autophagy.

(A) RPPA analysis was performed on lysates of MDA-MB-231 cells depleted from COPB2 for 48 h or 72 h. The heatmap represents values of triplicates normalized to a pool of controls (untransfected, mock transfected and RF transfected cells). Significant changes in proteins related to depletion of COPG2 were excluded. (B,C) Phosphorylated and total levels of the indicated proteins were validated in MDA-MB-231, MDA-MB-468 and U2OS cells. Actin was used as a loading control. M: marker.

Figure 8. COPI members are overexpressed in cancer.

Expression level of COPB2 in (A) ovarian cancer tissue compared with benign tissue and (B) different subtypes of breast cancer. (C) Survival fraction related to high or low COPB2 levels. The cutoff for high and low COPB2 expression is 9.58 (RMA preprocessed log value); this cutoff is optimized to achieve the lowest p value. RMA, Robust Multi-array Average.

Figure 9. Schematic representation of the role of COPI in the regulation of productive and abortive autophagy.

COPI is required for productive autophagy under basal conditions. Depletion of the COPI complex results in abortive autophagy, ER stress and decreased PI3K/AKT signaling.