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

Claerhout S, Dutta B, Bossuyt W, Zhang F, Nguyen-Charles C, Dennison JB, Yu Q, Yu S, Balázsi G, Lu Y, Mills GB - PLoS ONE (2012)

Bottom Line: The goal of this study was to identify modulators of the autophagic cell death pathway and elucidate their effects on cellular signaling and function.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America. SAClaerhout@mdanderson.org

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.

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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.
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pone-0039400-g004: 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.

Mentions: Because depletion of COPI complex members caused cell death and autophagosome formation, we set out to test whether abortive autophagy was the mechanism. To differentiate between abortive and productive autophagy we analyzed p62, which is preferentially degraded during autophagy, but levels remain constant or increased upon the induction of abortive autophagy [21]. As determined by immunoblot analysis of different cancer cell lines (Fig. 4A) and immunofluorescence analysis in MDA-MB-231 cells (Fig. 4B,C), siCOPB2 did not reduce p62 levels, as would be expected of productive autophagy, but increased the levels of p62 as compared to the control levels. In contrast, p62 levels were decreased in MCF10A (Fig. S9D), although COPB2 protein levels were significantly decreased (Fig. S9C). In addition, we used Bafilomycin A1 (BafA1), a lysosome-specific inhibitor of protein degradation and the end stages of the autophagy process to inhibit autophagic flux [18], [22]. BafA1 treatment has been shown to increase LC3-II levels in cells undergoing productive autophagy but has limited effect on LC3-II levels in cells undergoing abortive autophagy [20]. In MDA-MB-231 cells with knockdown of COPB2, BafA1 treatment did not further increase the LC3-II levels (Fig. 4D) in contrast to COPG2 depleted or control cells, indicating impaired degradation of the autophagosomal content by the lysosomes after COPB2 depletion. We confirmed this observation in U2OS cancer cells treated with either BafA1 or rapamycin (Fig. S5). Impaired degradation of the inner luminal content of autophagosomes by lysosomes can result from: 1) failed autophagosome/lysosome fusion; 2) impaired degradation; or 3) incomplete recycling of the autolysosomal components [18], [23]. To determine which of these possibilities was responsible for the increase in LC3-II and p62 in cells with COPI depletion, we first analyzed fusion of autophagosomes (GFP-LC3) and lysosomes (LAMP2) after knockdown of COPI in MDA-MB-231 cells stably transfected with GFP-LC3 (Fig. 5A). Confocal analysis revealed colocalization of GFP-LC3 and LAMP2 in cancer cells treated with siRNA against COPB2 (Fig. 5A), suggesting fusion was not completely blocked by depletion of COPI. We further tested the effect of COPB2 siRNA on autophagosomal degradation using mRFP-GFP tandem fluorescent tagged LC3 (tfLC3; [15]). In cells transiently transfected with tfLC3, GFP−/RFP-positive puncta represent autophagosomes not fused to lysosomes, whereas RFP-positive/GFP-negative puncta represent autolysosomes as GFP is more rapidly quenched by low lysosomal pH [15]. MDA-MB-231 cells transiently transfected with tfLC3 showed colocalization of the mRFP and GFP signal after COPI depletion or BafA1 treatment (Fig. 5B, row 2 and 3), indicative of impaired maturation of the autophagosomes. Treatment of MDA-MB-231 cells with imatinib resulted in RFP-positive/GFP-negative puncta representing the presence of autolysosomes and thus autophagic flux (Fig. 5B, last row). Similar results were obtained in U2OS cancer cells (Fig. S6). In addition, COPB2 depleted cells showed large LAMP2 positive organelles (Fig. S4D). Together, these results indicate that disruption of COPI induces impaired, abortive autophagy possibly through a failure in the maturation and recycling process.


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

Claerhout S, Dutta B, Bossuyt W, Zhang F, Nguyen-Charles C, Dennison JB, Yu Q, Yu S, Balázsi G, Lu Y, Mills GB - PLoS ONE (2012)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3383753&req=5

pone-0039400-g004: 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.
Mentions: Because depletion of COPI complex members caused cell death and autophagosome formation, we set out to test whether abortive autophagy was the mechanism. To differentiate between abortive and productive autophagy we analyzed p62, which is preferentially degraded during autophagy, but levels remain constant or increased upon the induction of abortive autophagy [21]. As determined by immunoblot analysis of different cancer cell lines (Fig. 4A) and immunofluorescence analysis in MDA-MB-231 cells (Fig. 4B,C), siCOPB2 did not reduce p62 levels, as would be expected of productive autophagy, but increased the levels of p62 as compared to the control levels. In contrast, p62 levels were decreased in MCF10A (Fig. S9D), although COPB2 protein levels were significantly decreased (Fig. S9C). In addition, we used Bafilomycin A1 (BafA1), a lysosome-specific inhibitor of protein degradation and the end stages of the autophagy process to inhibit autophagic flux [18], [22]. BafA1 treatment has been shown to increase LC3-II levels in cells undergoing productive autophagy but has limited effect on LC3-II levels in cells undergoing abortive autophagy [20]. In MDA-MB-231 cells with knockdown of COPB2, BafA1 treatment did not further increase the LC3-II levels (Fig. 4D) in contrast to COPG2 depleted or control cells, indicating impaired degradation of the autophagosomal content by the lysosomes after COPB2 depletion. We confirmed this observation in U2OS cancer cells treated with either BafA1 or rapamycin (Fig. S5). Impaired degradation of the inner luminal content of autophagosomes by lysosomes can result from: 1) failed autophagosome/lysosome fusion; 2) impaired degradation; or 3) incomplete recycling of the autolysosomal components [18], [23]. To determine which of these possibilities was responsible for the increase in LC3-II and p62 in cells with COPI depletion, we first analyzed fusion of autophagosomes (GFP-LC3) and lysosomes (LAMP2) after knockdown of COPI in MDA-MB-231 cells stably transfected with GFP-LC3 (Fig. 5A). Confocal analysis revealed colocalization of GFP-LC3 and LAMP2 in cancer cells treated with siRNA against COPB2 (Fig. 5A), suggesting fusion was not completely blocked by depletion of COPI. We further tested the effect of COPB2 siRNA on autophagosomal degradation using mRFP-GFP tandem fluorescent tagged LC3 (tfLC3; [15]). In cells transiently transfected with tfLC3, GFP−/RFP-positive puncta represent autophagosomes not fused to lysosomes, whereas RFP-positive/GFP-negative puncta represent autolysosomes as GFP is more rapidly quenched by low lysosomal pH [15]. MDA-MB-231 cells transiently transfected with tfLC3 showed colocalization of the mRFP and GFP signal after COPI depletion or BafA1 treatment (Fig. 5B, row 2 and 3), indicative of impaired maturation of the autophagosomes. Treatment of MDA-MB-231 cells with imatinib resulted in RFP-positive/GFP-negative puncta representing the presence of autolysosomes and thus autophagic flux (Fig. 5B, last row). Similar results were obtained in U2OS cancer cells (Fig. S6). In addition, COPB2 depleted cells showed large LAMP2 positive organelles (Fig. S4D). Together, these results indicate that disruption of COPI induces impaired, abortive autophagy possibly through a failure in the maturation and recycling process.

Bottom Line: The goal of this study was to identify modulators of the autophagic cell death pathway and elucidate their effects on cellular signaling and function.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America. SAClaerhout@mdanderson.org

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.

Show MeSH
Related in: MedlinePlus