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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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Enhancement of TRAIL sensitivity by silencing autophagy regulatory genes(A & B) The indicated cell lines were transiently transfected with a scramble siRNA (siCtrl) as a negative control or siRNA against ATG7 for 72 h, followed by incubation with rhTRAIL (100 ng/ml) for an additional 24 h. The resultant cells were analyzed by flow cytometry for apoptosis (A) or immunoblotting for caspase cleavage (B). (C & D) BT474 cells were transiently transfected with a control siRNA (siCtrl) or siRNA specific to Beclin 1, LC3 or in combination. After 48 h post-transfection, cells were analyzed for apoptosis and caspase activation. *p<0.0001.
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Figure 5: Enhancement of TRAIL sensitivity by silencing autophagy regulatory genes(A & B) The indicated cell lines were transiently transfected with a scramble siRNA (siCtrl) as a negative control or siRNA against ATG7 for 72 h, followed by incubation with rhTRAIL (100 ng/ml) for an additional 24 h. The resultant cells were analyzed by flow cytometry for apoptosis (A) or immunoblotting for caspase cleavage (B). (C & D) BT474 cells were transiently transfected with a control siRNA (siCtrl) or siRNA specific to Beclin 1, LC3 or in combination. After 48 h post-transfection, cells were analyzed for apoptosis and caspase activation. *p<0.0001.

Mentions: Suppression of autophagosome formation sensitizes TRAIL-resistant cells to TRAIL induced apoptosis. To determine whether the basal autophagy is actively involved in the development of TRAIL resistance, we tested the effects of autophagy inhibition on TRAIL induced cytotoxicity. First, TRAIL-resistant cell lines AU565 and BT474 were pretreated with 3-MA followed by TRAIL treatment. While blocking autophagosome formation (Fig. 3A), 3-MA treatment also induced a marked decrease in cell viability in both cell lines in response to TRAIL (Fig. 4A). The reduction in cell viability directly correlated with an increased apoptosis index and appearance of cleaved caspase-8 and caspase-3 (Fig. 4B). To rule out the off-target effects of 3-MA, we further tested the effects of silencing the key autophagy regulatory genes, including ATG7, Beclin 1 and LC3. As shown in Fig. 3B, transfection of siRNA against the individual autophagic genes abolished autophagosome formation in TRAIL-resistant cells. Under similar conditions, knockdown of ATG7 increased TRAIL-induced apoptosis (Fig. 5A) and caspase activation (Fig. 5B). Similar results were obtained by silencing Beclin 1 or LC3 (Fig. 5C & D). Collectively, these data demonstrate that basal autophagy is actively involved in the development of inherent resistance of breast cancer cells to TRAIL induced apoptosis.


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)

Enhancement of TRAIL sensitivity by silencing autophagy regulatory genes(A & B) The indicated cell lines were transiently transfected with a scramble siRNA (siCtrl) as a negative control or siRNA against ATG7 for 72 h, followed by incubation with rhTRAIL (100 ng/ml) for an additional 24 h. The resultant cells were analyzed by flow cytometry for apoptosis (A) or immunoblotting for caspase cleavage (B). (C & D) BT474 cells were transiently transfected with a control siRNA (siCtrl) or siRNA specific to Beclin 1, LC3 or in combination. After 48 h post-transfection, cells were analyzed for apoptosis and caspase activation. *p<0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Enhancement of TRAIL sensitivity by silencing autophagy regulatory genes(A & B) The indicated cell lines were transiently transfected with a scramble siRNA (siCtrl) as a negative control or siRNA against ATG7 for 72 h, followed by incubation with rhTRAIL (100 ng/ml) for an additional 24 h. The resultant cells were analyzed by flow cytometry for apoptosis (A) or immunoblotting for caspase cleavage (B). (C & D) BT474 cells were transiently transfected with a control siRNA (siCtrl) or siRNA specific to Beclin 1, LC3 or in combination. After 48 h post-transfection, cells were analyzed for apoptosis and caspase activation. *p<0.0001.
Mentions: Suppression of autophagosome formation sensitizes TRAIL-resistant cells to TRAIL induced apoptosis. To determine whether the basal autophagy is actively involved in the development of TRAIL resistance, we tested the effects of autophagy inhibition on TRAIL induced cytotoxicity. First, TRAIL-resistant cell lines AU565 and BT474 were pretreated with 3-MA followed by TRAIL treatment. While blocking autophagosome formation (Fig. 3A), 3-MA treatment also induced a marked decrease in cell viability in both cell lines in response to TRAIL (Fig. 4A). The reduction in cell viability directly correlated with an increased apoptosis index and appearance of cleaved caspase-8 and caspase-3 (Fig. 4B). To rule out the off-target effects of 3-MA, we further tested the effects of silencing the key autophagy regulatory genes, including ATG7, Beclin 1 and LC3. As shown in Fig. 3B, transfection of siRNA against the individual autophagic genes abolished autophagosome formation in TRAIL-resistant cells. Under similar conditions, knockdown of ATG7 increased TRAIL-induced apoptosis (Fig. 5A) and caspase activation (Fig. 5B). Similar results were obtained by silencing Beclin 1 or LC3 (Fig. 5C & D). Collectively, these data demonstrate that basal autophagy is actively involved in the development of inherent resistance of breast cancer cells to TRAIL induced apoptosis.

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