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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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TEM images reveals autophagosome structures in TRAIL-resistant cell lines and xenograft tumor models(A) Electron microscopy (EM) images show the ultrastructural features of untreated cells or cells treated with 3-methyladenine (3-MA) at 10 mM for 24 h (Bar =1 μm). Arrows denote the autophagosome ultrastructures in cytoplasm. Lower panel shows the average number of autophagosome structures per view (371 μm2) obtained by examining at least 50 images per testing sample. *p<0.0001. (B) EM images of parental cells and cells transfected with siRNA specific to the autophagy regulatory genes ATG7, Beclin 1, and LC3, respectively. Images are representatives of at least 50 captures. Lower panel shows the average number of autophagosome structures as determined in (B) for individual samples. *p<0.0001. (C) Nude mice were injected s.c. with BT474 or MDA-MB-231 cells per the protocol described in the Materials and Methods. When tumors reached 0.6 cm3 in size, tumor tissues were harvested and analyzed by EM imaging. Bar =1 μm. Lower panel shows the quantification of autophagosome numbers in the respective tissues.
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Figure 3: TEM images reveals autophagosome structures in TRAIL-resistant cell lines and xenograft tumor models(A) Electron microscopy (EM) images show the ultrastructural features of untreated cells or cells treated with 3-methyladenine (3-MA) at 10 mM for 24 h (Bar =1 μm). Arrows denote the autophagosome ultrastructures in cytoplasm. Lower panel shows the average number of autophagosome structures per view (371 μm2) obtained by examining at least 50 images per testing sample. *p<0.0001. (B) EM images of parental cells and cells transfected with siRNA specific to the autophagy regulatory genes ATG7, Beclin 1, and LC3, respectively. Images are representatives of at least 50 captures. Lower panel shows the average number of autophagosome structures as determined in (B) for individual samples. *p<0.0001. (C) Nude mice were injected s.c. with BT474 or MDA-MB-231 cells per the protocol described in the Materials and Methods. When tumors reached 0.6 cm3 in size, tumor tissues were harvested and analyzed by EM imaging. Bar =1 μm. Lower panel shows the quantification of autophagosome numbers in the respective tissues.

Mentions: We confirmed the high basal levels of autophagosomes in TRAIL-resistant cell lines using fluorescence microscopy and electron microscopy. Two representatives of TRAIL-resistant cell lines (BT474 and AU565) were compared to a TRAIL-sensitive cell line (MDA-MB-231). To facilitate fluorescence microscopy, cells were transiently transfected with a plasmid that expresses RFP-LC3 fusion protein. Compared to MDA-MB-231 cells, which showed an evenly distributed staining of RFP-LC3 red fluorescence, both BT474 and AU565 cells exhibited punctuate structures that are typical features of autophagosomes (Fig. 2). Further, electron microscopy images clearly showed the presence of a large number of autophagosomes in BT474 and AU565 cells but not in MDA-MB-231 cells (Fig. 3A). The number of autophagosomes decreased to baseline levels when the cells were treated with 3-methyladenine (3-MA), a pharmacological inhibitor of autophagy. Consistently, silencing of key autophagy regulatory genes (ATG7, LC3, or Beclin1) effectively abrogated the accumulation of autophagosomes in BT474 and AU565 cells (Fig. 3B). These data demonstrate that there are upregulated levels of basal autophagy in certain breast cancer cell lines even under healthy growing conditions, which correlates with the observed resistance to TRAIL-induced apoptosis. To assess the relevance of the high basal autophagy in a tumor setting, we established mouse xenograft models using BT474 and MDA-MB-231 cells. When tumor reached similar sizes (~0.6 cm3), tumor tissues were harvested and analyzed by electron microscopy. Consistent with the cell line data, a large number of autophagosomes were visualized in the tumors derived from BT474 cells but were virtually undetected in MDA-MB-231 xenografts (Fig. 3C).


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)

TEM images reveals autophagosome structures in TRAIL-resistant cell lines and xenograft tumor models(A) Electron microscopy (EM) images show the ultrastructural features of untreated cells or cells treated with 3-methyladenine (3-MA) at 10 mM for 24 h (Bar =1 μm). Arrows denote the autophagosome ultrastructures in cytoplasm. Lower panel shows the average number of autophagosome structures per view (371 μm2) obtained by examining at least 50 images per testing sample. *p<0.0001. (B) EM images of parental cells and cells transfected with siRNA specific to the autophagy regulatory genes ATG7, Beclin 1, and LC3, respectively. Images are representatives of at least 50 captures. Lower panel shows the average number of autophagosome structures as determined in (B) for individual samples. *p<0.0001. (C) Nude mice were injected s.c. with BT474 or MDA-MB-231 cells per the protocol described in the Materials and Methods. When tumors reached 0.6 cm3 in size, tumor tissues were harvested and analyzed by EM imaging. Bar =1 μm. Lower panel shows the quantification of autophagosome numbers in the respective tissues.
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Figure 3: TEM images reveals autophagosome structures in TRAIL-resistant cell lines and xenograft tumor models(A) Electron microscopy (EM) images show the ultrastructural features of untreated cells or cells treated with 3-methyladenine (3-MA) at 10 mM for 24 h (Bar =1 μm). Arrows denote the autophagosome ultrastructures in cytoplasm. Lower panel shows the average number of autophagosome structures per view (371 μm2) obtained by examining at least 50 images per testing sample. *p<0.0001. (B) EM images of parental cells and cells transfected with siRNA specific to the autophagy regulatory genes ATG7, Beclin 1, and LC3, respectively. Images are representatives of at least 50 captures. Lower panel shows the average number of autophagosome structures as determined in (B) for individual samples. *p<0.0001. (C) Nude mice were injected s.c. with BT474 or MDA-MB-231 cells per the protocol described in the Materials and Methods. When tumors reached 0.6 cm3 in size, tumor tissues were harvested and analyzed by EM imaging. Bar =1 μm. Lower panel shows the quantification of autophagosome numbers in the respective tissues.
Mentions: We confirmed the high basal levels of autophagosomes in TRAIL-resistant cell lines using fluorescence microscopy and electron microscopy. Two representatives of TRAIL-resistant cell lines (BT474 and AU565) were compared to a TRAIL-sensitive cell line (MDA-MB-231). To facilitate fluorescence microscopy, cells were transiently transfected with a plasmid that expresses RFP-LC3 fusion protein. Compared to MDA-MB-231 cells, which showed an evenly distributed staining of RFP-LC3 red fluorescence, both BT474 and AU565 cells exhibited punctuate structures that are typical features of autophagosomes (Fig. 2). Further, electron microscopy images clearly showed the presence of a large number of autophagosomes in BT474 and AU565 cells but not in MDA-MB-231 cells (Fig. 3A). The number of autophagosomes decreased to baseline levels when the cells were treated with 3-methyladenine (3-MA), a pharmacological inhibitor of autophagy. Consistently, silencing of key autophagy regulatory genes (ATG7, LC3, or Beclin1) effectively abrogated the accumulation of autophagosomes in BT474 and AU565 cells (Fig. 3B). These data demonstrate that there are upregulated levels of basal autophagy in certain breast cancer cell lines even under healthy growing conditions, which correlates with the observed resistance to TRAIL-induced apoptosis. To assess the relevance of the high basal autophagy in a tumor setting, we established mouse xenograft models using BT474 and MDA-MB-231 cells. When tumor reached similar sizes (~0.6 cm3), tumor tissues were harvested and analyzed by electron microscopy. Consistent with the cell line data, a large number of autophagosomes were visualized in the tumors derived from BT474 cells but were virtually undetected in MDA-MB-231 xenografts (Fig. 3C).

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