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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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TRAIL resistance correlates with the expression of autophagosome marker LC3-II in breast cancer cell lines(A) Differential sensitivities of the indicated cell lines to TRAIL-induced cytotoxicity. Cells were grown onto 96-well plates and treated with serial doses of rhTRAIL (5 to 100 ng/mL) for 24 h. Cell viability was determined by a colorimetric assay after MTT staining. GI50 values were derived from the dose-response curves and were used to group the cell lines into TRAIL-resistant (GI50 >500 nM) or TRAIL-sensitive (GI50 <100 nM). (B) LC3 protein expressions in cells grown under healthy conditions. Individual cell lines were cultured in nutrient-rich medium. At 80% confluence, cells were harvested and analyzed by western blotting. LC3 immunoblots detect two bands: LC3-I at an apparent mobility of 18 kDa and the lipidated form LC3-II at 16 kDa (moves faster than LC3-I on SDS-PAGE). LC3-II is known to associate with autophagosomes and therefore serves a marker of active autophagy. β-actin was detected as a loading control. (C) Densitometry analysis of the immunoblots in (B) yielded the relative levels of LC3-II to LC3-I in individual cell lines. Shown are means and standard deviations of three independent experiments. TRAIL-sensitive cells are characterized by a LC3-II/LC3-I ratio less than 1 arbitrary unit. Statistical difference in LC3-II/LC3-I ratios between TRAIL-resistant and TRAIL-sensitive cell lines was determined by Fisher's PLSD test.
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Figure 1: TRAIL resistance correlates with the expression of autophagosome marker LC3-II in breast cancer cell lines(A) Differential sensitivities of the indicated cell lines to TRAIL-induced cytotoxicity. Cells were grown onto 96-well plates and treated with serial doses of rhTRAIL (5 to 100 ng/mL) for 24 h. Cell viability was determined by a colorimetric assay after MTT staining. GI50 values were derived from the dose-response curves and were used to group the cell lines into TRAIL-resistant (GI50 >500 nM) or TRAIL-sensitive (GI50 <100 nM). (B) LC3 protein expressions in cells grown under healthy conditions. Individual cell lines were cultured in nutrient-rich medium. At 80% confluence, cells were harvested and analyzed by western blotting. LC3 immunoblots detect two bands: LC3-I at an apparent mobility of 18 kDa and the lipidated form LC3-II at 16 kDa (moves faster than LC3-I on SDS-PAGE). LC3-II is known to associate with autophagosomes and therefore serves a marker of active autophagy. β-actin was detected as a loading control. (C) Densitometry analysis of the immunoblots in (B) yielded the relative levels of LC3-II to LC3-I in individual cell lines. Shown are means and standard deviations of three independent experiments. TRAIL-sensitive cells are characterized by a LC3-II/LC3-I ratio less than 1 arbitrary unit. Statistical difference in LC3-II/LC3-I ratios between TRAIL-resistant and TRAIL-sensitive cell lines was determined by Fisher's PLSD test.

Mentions: TRAIL resistance correlates with an accumulation of autophagosomes in breast cancer cell lines. We determined the activity of TRAIL in inducing apoptosis in a panel of 11 breast cancer cell lines (Fig. 1A and Ref. [50]). The obtained 50% growth inhibition (GI50) values allowed us to separate the cell lines into TRAIL-resistant lines (GI50 > 500 nM; AU565, BT474, HCC1428, MCF-7 and MDA-MB-453) and TRAIL-sensitive lines (GI50 < 100 nM; MDA-MB-231, BT549, HCC38, HCC1954, MDA-MB-157 and Hs578T). To determine the basal levels of autophagosomes, individual cell lines were grown in nutrient-rich media per ATCC recommendations. At 80% confluence, cells were harvested and analyzed by three complementary approaches following the guidelines for the use of assays for monitoring autophagy [51]. We first performed immunoblot analysis for the microtubule associated protein 1 light chain 3 (LC3). LC3 protein is involved in the formation of autophagosomes and its turnover from a cytosolic form LC3-I to a lipidated form LC3-II (i.e. addition of phosphatidylethanolamine at Gly120 residue of LC3-I) has been widely used as a molecular marker of autophagosomes. The results showed distinct patterns of LC3 expression between TRAIL-resistant and TRAIL-sensitive cell lines (Fig. 1B & C). In TRAIL-sensitive cell lines, LC3 existed primarily in its cytosolic form, LC3-I. By contrast, TRAIL-resistant cell lines were characterized by an increased level of the lipidated form, LC3-II. To rule out the possibility of cell density effects, we cultured the cells at 20%, 50%, and 80% confluence and observed a similar pattern in LC3 expression of two representative cell lines (BT474 and MDA-MB-231) (Supplement I). Notably, the high ratios of LC3-II/LC3-I correlated with the observed TRAIL resistance in the cell lines examined.


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)

TRAIL resistance correlates with the expression of autophagosome marker LC3-II in breast cancer cell lines(A) Differential sensitivities of the indicated cell lines to TRAIL-induced cytotoxicity. Cells were grown onto 96-well plates and treated with serial doses of rhTRAIL (5 to 100 ng/mL) for 24 h. Cell viability was determined by a colorimetric assay after MTT staining. GI50 values were derived from the dose-response curves and were used to group the cell lines into TRAIL-resistant (GI50 >500 nM) or TRAIL-sensitive (GI50 <100 nM). (B) LC3 protein expressions in cells grown under healthy conditions. Individual cell lines were cultured in nutrient-rich medium. At 80% confluence, cells were harvested and analyzed by western blotting. LC3 immunoblots detect two bands: LC3-I at an apparent mobility of 18 kDa and the lipidated form LC3-II at 16 kDa (moves faster than LC3-I on SDS-PAGE). LC3-II is known to associate with autophagosomes and therefore serves a marker of active autophagy. β-actin was detected as a loading control. (C) Densitometry analysis of the immunoblots in (B) yielded the relative levels of LC3-II to LC3-I in individual cell lines. Shown are means and standard deviations of three independent experiments. TRAIL-sensitive cells are characterized by a LC3-II/LC3-I ratio less than 1 arbitrary unit. Statistical difference in LC3-II/LC3-I ratios between TRAIL-resistant and TRAIL-sensitive cell lines was determined by Fisher's PLSD test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: TRAIL resistance correlates with the expression of autophagosome marker LC3-II in breast cancer cell lines(A) Differential sensitivities of the indicated cell lines to TRAIL-induced cytotoxicity. Cells were grown onto 96-well plates and treated with serial doses of rhTRAIL (5 to 100 ng/mL) for 24 h. Cell viability was determined by a colorimetric assay after MTT staining. GI50 values were derived from the dose-response curves and were used to group the cell lines into TRAIL-resistant (GI50 >500 nM) or TRAIL-sensitive (GI50 <100 nM). (B) LC3 protein expressions in cells grown under healthy conditions. Individual cell lines were cultured in nutrient-rich medium. At 80% confluence, cells were harvested and analyzed by western blotting. LC3 immunoblots detect two bands: LC3-I at an apparent mobility of 18 kDa and the lipidated form LC3-II at 16 kDa (moves faster than LC3-I on SDS-PAGE). LC3-II is known to associate with autophagosomes and therefore serves a marker of active autophagy. β-actin was detected as a loading control. (C) Densitometry analysis of the immunoblots in (B) yielded the relative levels of LC3-II to LC3-I in individual cell lines. Shown are means and standard deviations of three independent experiments. TRAIL-sensitive cells are characterized by a LC3-II/LC3-I ratio less than 1 arbitrary unit. Statistical difference in LC3-II/LC3-I ratios between TRAIL-resistant and TRAIL-sensitive cell lines was determined by Fisher's PLSD test.
Mentions: TRAIL resistance correlates with an accumulation of autophagosomes in breast cancer cell lines. We determined the activity of TRAIL in inducing apoptosis in a panel of 11 breast cancer cell lines (Fig. 1A and Ref. [50]). The obtained 50% growth inhibition (GI50) values allowed us to separate the cell lines into TRAIL-resistant lines (GI50 > 500 nM; AU565, BT474, HCC1428, MCF-7 and MDA-MB-453) and TRAIL-sensitive lines (GI50 < 100 nM; MDA-MB-231, BT549, HCC38, HCC1954, MDA-MB-157 and Hs578T). To determine the basal levels of autophagosomes, individual cell lines were grown in nutrient-rich media per ATCC recommendations. At 80% confluence, cells were harvested and analyzed by three complementary approaches following the guidelines for the use of assays for monitoring autophagy [51]. We first performed immunoblot analysis for the microtubule associated protein 1 light chain 3 (LC3). LC3 protein is involved in the formation of autophagosomes and its turnover from a cytosolic form LC3-I to a lipidated form LC3-II (i.e. addition of phosphatidylethanolamine at Gly120 residue of LC3-I) has been widely used as a molecular marker of autophagosomes. The results showed distinct patterns of LC3 expression between TRAIL-resistant and TRAIL-sensitive cell lines (Fig. 1B & C). In TRAIL-sensitive cell lines, LC3 existed primarily in its cytosolic form, LC3-I. By contrast, TRAIL-resistant cell lines were characterized by an increased level of the lipidated form, LC3-II. To rule out the possibility of cell density effects, we cultured the cells at 20%, 50%, and 80% confluence and observed a similar pattern in LC3 expression of two representative cell lines (BT474 and MDA-MB-231) (Supplement I). Notably, the high ratios of LC3-II/LC3-I correlated with the observed TRAIL resistance in the cell lines examined.

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