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Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is required for induction of autophagy during lumen formation in vitro.

ABSTRACT: The molecular events regulating the elimination of cells to create a hollow lumen during tissue development are poorly understood. By using an in vitro morphogenesis model in which MCF-10A human mammary epithelial cells form hollow acini-like structures, we have observed both caspase-mediated apoptosis and autophagy associated with cells that are lost during lumen formation. Here, we show that the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates induction of autophagic processes associated with lumen formation. TRAIL is up-regulated during morphogenesis of MCF-10A mammary epithelial cells in 3D basement-membrane cultures and inhibition of TRAIL signaling during morphogenesis blocks the formation of autophagic vacuoles. In addition, treatment with exogenous TRAIL induces extensive autophagy in monolayer and 3D cultures. When combined with inhibition of caspase 3 activity (by Bcl-X(L) overexpression), inhibition of TRAIL-induced autophagy results in luminal filling. Thus, TRAIL regulates an autophagic program during acinar morphogenesis, which together with caspase-mediated apoptotic events, results in lumen formation during MCF-10A morphogenesis.

SUBMITTER: Mills KR

PROVIDER: S-EPMC373480 | biostudies-literature | 2004 Mar

REPOSITORIES: biostudies-literature

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Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is required for induction of autophagy during lumen formation in vitro.

Mills Kenna R KR Reginato Mauricio M Debnath Jayanta J Queenan Bridget B Brugge Joan S JS

Proceedings of the National Academy of Sciences of the United States of America 20040301 10

The molecular events regulating the elimination of cells to create a hollow lumen during tissue development are poorly understood. By using an in vitro morphogenesis model in which MCF-10A human mammary epithelial cells form hollow acini-like structures, we have observed both caspase-mediated apoptosis and autophagy associated with cells that are lost during lumen formation. Here, we show that the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates induction of autophagic pr ...[more]

PMID: 14993595

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Project description:IntroductionTumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to its receptors, TRAIL-receptor 1 (TRAIL-R1) and TRAIL-receptor 2 (TRAIL-R2), leading to apoptosis by activation of caspase-8 and the downstream executioner caspases, caspase-3 and caspase-7 (caspase-3/7). Triple-negative breast cancer (TNBC) cell lines with a mesenchymal phenotype are sensitive to TRAIL, whereas other breast cancer cell lines are resistant. The underlying mechanisms that control TRAIL sensitivity in breast cancer cells are not well understood. Here, we performed small interfering RNA (siRNA) screens to identify molecular regulators of the TRAIL pathway in breast cancer cells.MethodsWe conducted siRNA screens of the human kinome (691 genes), phosphatome (320 genes), and about 300 additional genes in the mesenchymal TNBC cell line MB231. Forty-eight hours after transfection of siRNA, parallel screens measuring caspase-8 activity, caspase-3/7 activity, or cell viability were conducted in the absence or presence of TRAIL for each siRNA, relative to a negative control siRNA (siNeg). A subset of genes was screened in cell lines representing epithelial TNBC (MB468), HER2-amplified breast cancer (SKBR3), and estrogen receptor-positive breast cancer (T47D). Selected putative negative regulators of the TRAIL pathway were studied by using small-molecule inhibitors.ResultsThe primary screens in MB231 identified 150 genes, including 83 kinases, 4 phosphatases, and 63 nonkinases, as potential negative regulators of TRAIL. The identified genes are involved in many critical cell processes, including apoptosis, growth factor-receptor signaling, cell-cycle regulation, transcriptional regulation, and DNA repair. Gene-network analysis identified four genes (PDPK1, IKBKB, SRC, and BCL2L1) that formed key nodes within the interaction network of negative regulators. A secondary screen of a subset of the genes identified in additional cell lines representing different breast cancer subtypes and sensitivities to TRAIL validated and extended these findings. Further, we confirmed that small-molecule inhibition of SRC or BCL2L1, in combination with TRAIL, sensitizes breast cancer cells to TRAIL-induced apoptosis, including cell lines resistant to TRAIL-induced cytotoxicity.ConclusionsThese data identify novel molecular regulators of TRAIL-induced apoptosis in breast cancer cells and suggest strategies for the enhanced application of TRAIL as a therapy for breast cancer.

Dataset Information

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is required for induction of autophagy during lumen formation in vitro.

Publications

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is required for induction of autophagy during lumen formation in vitro.

Similar Datasets

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