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Accumulation of autophagosomes in breast cancer cells induces TRAIL resistance through downregulation of surface expression of death receptors 4 and 5.

Di X, Zhang G, Zhang Y, Takeda K, Rivera Rosado LA, Zhang B - Oncotarget (2013)

Bottom Line: In certain cancer cells, DR4 and DR5 were found to be mislocalized in intracellular compartments yet to be characterized.We found high levels of basal autophagosomes in TRAIL resistant breast cancer cell lines (e.g. BT474 and AU565) and relevant mouse xenograft models under nutrition-rich conditions.The results also provide a rationale for future non-clinical and clinical studies testing TRAIL agonists in combination with agents that directly inhibit autophagosome assembly.

View Article: PubMed Central - PubMed

Affiliation: Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, MD, United States.

ABSTRACT
TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis through death receptors (DRs) 4 and/or 5 expressed on the surface of target cells. We have previously shown that deficiency of DR4 and DR5 on the surface membrane is a critical mechanism of cancer cell resistance to the recombinant human TRAIL and its receptor agonistic antibodies, which are being evaluated clinically for treating cancers. In certain cancer cells, DR4 and DR5 were found to be mislocalized in intracellular compartments yet to be characterized. Here, we report a novel role of autophagy in the regulation of dynamics of TRAIL death receptors. We first assessed basal levels of autophagosomes in a panel of 11 breast cancer cell lines using complementary approaches (LC3 immunoblotting, RFP-LC3 fluorescence microscopy, and electron microscopy). We found high levels of basal autophagosomes in TRAIL resistant breast cancer cell lines (e.g. BT474 and AU565) and relevant mouse xenograft models under nutrition-rich conditions. Notably, DR4 and DR5 co-localized with LC3-II in the autophagosomes of TRAIL-resistant cells. Disruption of basal autophagosomes successfully restored the surface expression of the death receptors which was accompanied by sensitization of TRAIL-resistant cells to TRAIL induced apoptosis. By contrast, TRAIL-sensitive cell lines (MDA-MB-231) are characterized by high levels of surface DR4/DR5 and an absence of basal autophagosomes. Inhibition of lysosomal activity induced an accumulation of autophagosomes and a decrease in surface DR4 and DR5, and the cells became less sensitive to TRAIL-induced apoptosis. These findings demonstrate a novel role for the basal autophagosomes in the regulation of TRAIL death receptors. Further studies are warranted to explore the possibility of using autophagosome markers such as LC3-II/LC3-I ratios for prediction of tumor resistance to TRAIL related therapies. The results also provide a rationale for future non-clinical and clinical studies testing TRAIL agonists in combination with agents that directly inhibit autophagosome assembly.

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Inhibition of lysosomal activity induces differentially accumulation of autophagosome in MDA-MB-231 and TRAIL-resistant cell lines(A) Cells were treated with bafilomycin A1 (BafA1) at 100 nM for 8 or 24 h, and the resultant whole cell extracts were analyzed by immunoblotting for LC3, p62, DR4 and DR5. Actin was used as a loading control. (B) Relative protein levels were estimated by densitometry analysis of the blots in A, and normalized to the corresponding actin intensity. The level of each protein in the untreated cells (time 0) was arbitrarily set as 1. Shown are representatives of two independent experiments. (C) MDA-MB-231/RFP-LC3 cells and BT474/RFP-LC3, both stably express RFP-LC3 protein, were treated with BafA1 (100nM) for the indicated times, countered stained with Hoechst 33342 (blue), and analyzed by confocal microscopy. Scale bar, 10 μm. MDA-MB-231 cells accumulated punctate structures at a much higher rate compared to BT474 cells.
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Figure 7: Inhibition of lysosomal activity induces differentially accumulation of autophagosome in MDA-MB-231 and TRAIL-resistant cell lines(A) Cells were treated with bafilomycin A1 (BafA1) at 100 nM for 8 or 24 h, and the resultant whole cell extracts were analyzed by immunoblotting for LC3, p62, DR4 and DR5. Actin was used as a loading control. (B) Relative protein levels were estimated by densitometry analysis of the blots in A, and normalized to the corresponding actin intensity. The level of each protein in the untreated cells (time 0) was arbitrarily set as 1. Shown are representatives of two independent experiments. (C) MDA-MB-231/RFP-LC3 cells and BT474/RFP-LC3, both stably express RFP-LC3 protein, were treated with BafA1 (100nM) for the indicated times, countered stained with Hoechst 33342 (blue), and analyzed by confocal microscopy. Scale bar, 10 μm. MDA-MB-231 cells accumulated punctate structures at a much higher rate compared to BT474 cells.

Mentions: Inhibition of lysosomal activity induces an accumulation of autophagosomes and a loss of surface DR4 and DR5 in MDA-MB-231 cells. As autophagy is a highly dynamic process that involves multiple steps, it is therefore possible that the accumulation of autophagosomes could result from a genetic background with upregulated induction of autophagy or, alternatively, reflect a block in the later stages of the process, such as impaired autophagosome fusion and lysosomal degradation [50]. To address this issue, we examined autophagic flux in these cell lines using lysosomal protease inhibitors (bafilomycin or chloroquine). As expected, lysosomal inhibition induced a time-dependent accumulation of LC3-II protein in all three cell lines (Fig. 7A & B). Surprisingly, the rate of LC3-II accumulation is much higher in MDA-MB-231 cells compared to BT474 and AU565 cells. A similar accumulation pattern was also observed for endogenous p62, DR4, and DR5 proteins. Using stable cell lines that express fluorescent RFP-LC3, we further showed that MDA-MB-231 cells rapidly accumulated punctuate structures upon bafilomycin treatment (Fig. 7C). These data support a low level of autophagic flux activity in BT474 and AU565 cells, although they both contain high degrees of autophagosomes. This observation is suggestive of an insufficient fusion and autolysosome formation or lack of lysosomal activity or both. MDA-MB-231 cells appear to undergo a rapid lysosomal turnover, which may explain the lack of basal autophagosomes. However, these results do not rule out the possibility of differences in the regulatory proteins upstream of autophagosome formation between TRAIL-resistant and TRAIL-sensitive cells. Additional studies are required to determine the molecular basis of autophagosome accumulation in TRAIL-resistant cancer cells.


Accumulation of autophagosomes in breast cancer cells induces TRAIL resistance through downregulation of surface expression of death receptors 4 and 5.

Di X, Zhang G, Zhang Y, Takeda K, Rivera Rosado LA, Zhang B - Oncotarget (2013)

Inhibition of lysosomal activity induces differentially accumulation of autophagosome in MDA-MB-231 and TRAIL-resistant cell lines(A) Cells were treated with bafilomycin A1 (BafA1) at 100 nM for 8 or 24 h, and the resultant whole cell extracts were analyzed by immunoblotting for LC3, p62, DR4 and DR5. Actin was used as a loading control. (B) Relative protein levels were estimated by densitometry analysis of the blots in A, and normalized to the corresponding actin intensity. The level of each protein in the untreated cells (time 0) was arbitrarily set as 1. Shown are representatives of two independent experiments. (C) MDA-MB-231/RFP-LC3 cells and BT474/RFP-LC3, both stably express RFP-LC3 protein, were treated with BafA1 (100nM) for the indicated times, countered stained with Hoechst 33342 (blue), and analyzed by confocal microscopy. Scale bar, 10 μm. MDA-MB-231 cells accumulated punctate structures at a much higher rate compared to BT474 cells.
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Figure 7: Inhibition of lysosomal activity induces differentially accumulation of autophagosome in MDA-MB-231 and TRAIL-resistant cell lines(A) Cells were treated with bafilomycin A1 (BafA1) at 100 nM for 8 or 24 h, and the resultant whole cell extracts were analyzed by immunoblotting for LC3, p62, DR4 and DR5. Actin was used as a loading control. (B) Relative protein levels were estimated by densitometry analysis of the blots in A, and normalized to the corresponding actin intensity. The level of each protein in the untreated cells (time 0) was arbitrarily set as 1. Shown are representatives of two independent experiments. (C) MDA-MB-231/RFP-LC3 cells and BT474/RFP-LC3, both stably express RFP-LC3 protein, were treated with BafA1 (100nM) for the indicated times, countered stained with Hoechst 33342 (blue), and analyzed by confocal microscopy. Scale bar, 10 μm. MDA-MB-231 cells accumulated punctate structures at a much higher rate compared to BT474 cells.
Mentions: Inhibition of lysosomal activity induces an accumulation of autophagosomes and a loss of surface DR4 and DR5 in MDA-MB-231 cells. As autophagy is a highly dynamic process that involves multiple steps, it is therefore possible that the accumulation of autophagosomes could result from a genetic background with upregulated induction of autophagy or, alternatively, reflect a block in the later stages of the process, such as impaired autophagosome fusion and lysosomal degradation [50]. To address this issue, we examined autophagic flux in these cell lines using lysosomal protease inhibitors (bafilomycin or chloroquine). As expected, lysosomal inhibition induced a time-dependent accumulation of LC3-II protein in all three cell lines (Fig. 7A & B). Surprisingly, the rate of LC3-II accumulation is much higher in MDA-MB-231 cells compared to BT474 and AU565 cells. A similar accumulation pattern was also observed for endogenous p62, DR4, and DR5 proteins. Using stable cell lines that express fluorescent RFP-LC3, we further showed that MDA-MB-231 cells rapidly accumulated punctuate structures upon bafilomycin treatment (Fig. 7C). These data support a low level of autophagic flux activity in BT474 and AU565 cells, although they both contain high degrees of autophagosomes. This observation is suggestive of an insufficient fusion and autolysosome formation or lack of lysosomal activity or both. MDA-MB-231 cells appear to undergo a rapid lysosomal turnover, which may explain the lack of basal autophagosomes. However, these results do not rule out the possibility of differences in the regulatory proteins upstream of autophagosome formation between TRAIL-resistant and TRAIL-sensitive cells. Additional studies are required to determine the molecular basis of autophagosome accumulation in TRAIL-resistant cancer cells.

Bottom Line: In certain cancer cells, DR4 and DR5 were found to be mislocalized in intracellular compartments yet to be characterized.We found high levels of basal autophagosomes in TRAIL resistant breast cancer cell lines (e.g. BT474 and AU565) and relevant mouse xenograft models under nutrition-rich conditions.The results also provide a rationale for future non-clinical and clinical studies testing TRAIL agonists in combination with agents that directly inhibit autophagosome assembly.

View Article: PubMed Central - PubMed

Affiliation: Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, MD, United States.

ABSTRACT
TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis through death receptors (DRs) 4 and/or 5 expressed on the surface of target cells. We have previously shown that deficiency of DR4 and DR5 on the surface membrane is a critical mechanism of cancer cell resistance to the recombinant human TRAIL and its receptor agonistic antibodies, which are being evaluated clinically for treating cancers. In certain cancer cells, DR4 and DR5 were found to be mislocalized in intracellular compartments yet to be characterized. Here, we report a novel role of autophagy in the regulation of dynamics of TRAIL death receptors. We first assessed basal levels of autophagosomes in a panel of 11 breast cancer cell lines using complementary approaches (LC3 immunoblotting, RFP-LC3 fluorescence microscopy, and electron microscopy). We found high levels of basal autophagosomes in TRAIL resistant breast cancer cell lines (e.g. BT474 and AU565) and relevant mouse xenograft models under nutrition-rich conditions. Notably, DR4 and DR5 co-localized with LC3-II in the autophagosomes of TRAIL-resistant cells. Disruption of basal autophagosomes successfully restored the surface expression of the death receptors which was accompanied by sensitization of TRAIL-resistant cells to TRAIL induced apoptosis. By contrast, TRAIL-sensitive cell lines (MDA-MB-231) are characterized by high levels of surface DR4/DR5 and an absence of basal autophagosomes. Inhibition of lysosomal activity induced an accumulation of autophagosomes and a decrease in surface DR4 and DR5, and the cells became less sensitive to TRAIL-induced apoptosis. These findings demonstrate a novel role for the basal autophagosomes in the regulation of TRAIL death receptors. Further studies are warranted to explore the possibility of using autophagosome markers such as LC3-II/LC3-I ratios for prediction of tumor resistance to TRAIL related therapies. The results also provide a rationale for future non-clinical and clinical studies testing TRAIL agonists in combination with agents that directly inhibit autophagosome assembly.

Show MeSH
Related in: MedlinePlus