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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 downregulates DR4 and DR5 from the surface of MDA-MB-231 cells(A) Cells were treated with BafA1 (100 nM) at 37°C for 0, 2, 4 or 24 h, stained with PE-conjugated antibodies specific to DR4, DR5 or transferrin receptor (TfR), and analyzed by flow cytometry as in Fig. 6. Shown are representatives from three independent measurements. (B) Cells were pretreated with BafA1 (100 nM) for 24 h and followed by rhTRAIL (20 ng/mL) for additional 5, 15 or 30 min. A delay in cleavage of caspase 8 (C-8) and C-3) was detected in BafA-1 treated cells. RhoGDI, a caspase-resistant protein, was used as a loading control.
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Figure 8: Inhibition of lysosomal activity downregulates DR4 and DR5 from the surface of MDA-MB-231 cells(A) Cells were treated with BafA1 (100 nM) at 37°C for 0, 2, 4 or 24 h, stained with PE-conjugated antibodies specific to DR4, DR5 or transferrin receptor (TfR), and analyzed by flow cytometry as in Fig. 6. Shown are representatives from three independent measurements. (B) Cells were pretreated with BafA1 (100 nM) for 24 h and followed by rhTRAIL (20 ng/mL) for additional 5, 15 or 30 min. A delay in cleavage of caspase 8 (C-8) and C-3) was detected in BafA-1 treated cells. RhoGDI, a caspase-resistant protein, was used as a loading control.

Mentions: We further determined DR4/DR5 expression in MDA-MB-231 cells in response to lysosomal inhibition. Strikingly, both receptors were downregulated from surface membrane in a time-dependent fashion (Fig. 8A). The resultant cells became less sensitive to TRAIL-induced apoptosis, as indicated by a delay in the cleavage of caspase 8 and 3 immediately after TRAIL treatment (Fig. 8B). We wished to determine a decrease in apoptosis index after bafilomycin treatment, but this effort was hindered by the cytotoxicity of inhibitor itself. Collectively, both inherently occurring and induced autophagosomes appear to negatively regulate the surface expression of TRAIL death receptors.


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 downregulates DR4 and DR5 from the surface of MDA-MB-231 cells(A) Cells were treated with BafA1 (100 nM) at 37°C for 0, 2, 4 or 24 h, stained with PE-conjugated antibodies specific to DR4, DR5 or transferrin receptor (TfR), and analyzed by flow cytometry as in Fig. 6. Shown are representatives from three independent measurements. (B) Cells were pretreated with BafA1 (100 nM) for 24 h and followed by rhTRAIL (20 ng/mL) for additional 5, 15 or 30 min. A delay in cleavage of caspase 8 (C-8) and C-3) was detected in BafA-1 treated cells. RhoGDI, a caspase-resistant protein, was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824535&req=5

Figure 8: Inhibition of lysosomal activity downregulates DR4 and DR5 from the surface of MDA-MB-231 cells(A) Cells were treated with BafA1 (100 nM) at 37°C for 0, 2, 4 or 24 h, stained with PE-conjugated antibodies specific to DR4, DR5 or transferrin receptor (TfR), and analyzed by flow cytometry as in Fig. 6. Shown are representatives from three independent measurements. (B) Cells were pretreated with BafA1 (100 nM) for 24 h and followed by rhTRAIL (20 ng/mL) for additional 5, 15 or 30 min. A delay in cleavage of caspase 8 (C-8) and C-3) was detected in BafA-1 treated cells. RhoGDI, a caspase-resistant protein, was used as a loading control.
Mentions: We further determined DR4/DR5 expression in MDA-MB-231 cells in response to lysosomal inhibition. Strikingly, both receptors were downregulated from surface membrane in a time-dependent fashion (Fig. 8A). The resultant cells became less sensitive to TRAIL-induced apoptosis, as indicated by a delay in the cleavage of caspase 8 and 3 immediately after TRAIL treatment (Fig. 8B). We wished to determine a decrease in apoptosis index after bafilomycin treatment, but this effort was hindered by the cytotoxicity of inhibitor itself. Collectively, both inherently occurring and induced autophagosomes appear to negatively regulate the surface expression of TRAIL death receptors.

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