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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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Schematic representation of the basal autophagosome mediated cellular resistance to TRAIL induced apoptosisIn MDA-MB-231 cells, TRAIL binds DR4 and/or DR5 expressed on cell surface, thereby recruiting adaptor protein Fas-associated death domain (FADD) and pro-caspase 8 into a death inducing signaling complex (DISC). Within the DISC, caspase 8 undergoes self-cleavage and activation which triggers the caspase cascade, cleavage of structural proteins, and eventually apoptosis. Both BT474 and AU565 cells are characterized by high basal level of autophagosomes that sequester DR4 and DR5, which may contribute to their deficiency on cell surface. Disruption of autophagosome structures (e.g. by 3-MA or siATG7) restores the surface expression of DR4 and DR5 which make the cells susceptible to TRAIL induced apoptosis.
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Figure 9: Schematic representation of the basal autophagosome mediated cellular resistance to TRAIL induced apoptosisIn MDA-MB-231 cells, TRAIL binds DR4 and/or DR5 expressed on cell surface, thereby recruiting adaptor protein Fas-associated death domain (FADD) and pro-caspase 8 into a death inducing signaling complex (DISC). Within the DISC, caspase 8 undergoes self-cleavage and activation which triggers the caspase cascade, cleavage of structural proteins, and eventually apoptosis. Both BT474 and AU565 cells are characterized by high basal level of autophagosomes that sequester DR4 and DR5, which may contribute to their deficiency on cell surface. Disruption of autophagosome structures (e.g. by 3-MA or siATG7) restores the surface expression of DR4 and DR5 which make the cells susceptible to TRAIL induced apoptosis.

Mentions: Overcoming tumor resistance is the key to success of development of TRAIL receptor targeted therapies for cancer treatment. Our laboratory investigates the mechanisms underlying TRAIL resistance with aims at identifying biomarkers for prediction of tumor resistance to the targeted therapies and also identifying novel molecular targets for therapeutic intervention for improved anticancer efficacy [12, 13, 29, 30, 50]. We have previously shown an aberrant expression of TRAIL death receptors in certain cancer cell lines in which these receptors are mainly localized in intracellular compartments such as nucleus and others yet to be characterized [29, 30]. Here we report a novel role of autophagy in regulation of subcellular localization of TRAIL receptors. Using complementary approaches, we show an upregulated formation of autophagosomes in a panel of TRAIL-resistant breast cancer cell lines and relevant animal models. We also provide evidence that the presence of basal autophagosomes is actively involved in the regulation of surface expression of TRAIL death receptors. Pharmacological inhibition of autophagosomes effectively restored the surface expression of DR4 and DR5 without altering their total protein levels. Importantly, blockage of basal autophagosome formation sensitized TRAIL-resistant cells (e.g. AU565 and BT474) to TRAIL induced apoptosis (Fig. 9). These findings highlight an important role of basal autophagosomes in the regulation of apoptotic response to TRAIL agonists. The molecular markers of autophagosomes (e.g. LC3-II/LC3-I ratio) could be evaluated as an indicator of inherent TRAIL resistance in tumor cells, and blockade of the basal autophagosomes may have a potential to improve the clinical efficacy of TRAIL receptor targeted therapies.


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)

Schematic representation of the basal autophagosome mediated cellular resistance to TRAIL induced apoptosisIn MDA-MB-231 cells, TRAIL binds DR4 and/or DR5 expressed on cell surface, thereby recruiting adaptor protein Fas-associated death domain (FADD) and pro-caspase 8 into a death inducing signaling complex (DISC). Within the DISC, caspase 8 undergoes self-cleavage and activation which triggers the caspase cascade, cleavage of structural proteins, and eventually apoptosis. Both BT474 and AU565 cells are characterized by high basal level of autophagosomes that sequester DR4 and DR5, which may contribute to their deficiency on cell surface. Disruption of autophagosome structures (e.g. by 3-MA or siATG7) restores the surface expression of DR4 and DR5 which make the cells susceptible to TRAIL induced apoptosis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Schematic representation of the basal autophagosome mediated cellular resistance to TRAIL induced apoptosisIn MDA-MB-231 cells, TRAIL binds DR4 and/or DR5 expressed on cell surface, thereby recruiting adaptor protein Fas-associated death domain (FADD) and pro-caspase 8 into a death inducing signaling complex (DISC). Within the DISC, caspase 8 undergoes self-cleavage and activation which triggers the caspase cascade, cleavage of structural proteins, and eventually apoptosis. Both BT474 and AU565 cells are characterized by high basal level of autophagosomes that sequester DR4 and DR5, which may contribute to their deficiency on cell surface. Disruption of autophagosome structures (e.g. by 3-MA or siATG7) restores the surface expression of DR4 and DR5 which make the cells susceptible to TRAIL induced apoptosis.
Mentions: Overcoming tumor resistance is the key to success of development of TRAIL receptor targeted therapies for cancer treatment. Our laboratory investigates the mechanisms underlying TRAIL resistance with aims at identifying biomarkers for prediction of tumor resistance to the targeted therapies and also identifying novel molecular targets for therapeutic intervention for improved anticancer efficacy [12, 13, 29, 30, 50]. We have previously shown an aberrant expression of TRAIL death receptors in certain cancer cell lines in which these receptors are mainly localized in intracellular compartments such as nucleus and others yet to be characterized [29, 30]. Here we report a novel role of autophagy in regulation of subcellular localization of TRAIL receptors. Using complementary approaches, we show an upregulated formation of autophagosomes in a panel of TRAIL-resistant breast cancer cell lines and relevant animal models. We also provide evidence that the presence of basal autophagosomes is actively involved in the regulation of surface expression of TRAIL death receptors. Pharmacological inhibition of autophagosomes effectively restored the surface expression of DR4 and DR5 without altering their total protein levels. Importantly, blockage of basal autophagosome formation sensitized TRAIL-resistant cells (e.g. AU565 and BT474) to TRAIL induced apoptosis (Fig. 9). These findings highlight an important role of basal autophagosomes in the regulation of apoptotic response to TRAIL agonists. The molecular markers of autophagosomes (e.g. LC3-II/LC3-I ratio) could be evaluated as an indicator of inherent TRAIL resistance in tumor cells, and blockade of the basal autophagosomes may have a potential to improve the clinical efficacy of TRAIL receptor targeted therapies.

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