Selective inactivation of a Fas-associated death domain protein (FADD)-dependent apoptosis and autophagy pathway in immortal epithelial cells - PubMed (original) (raw)

Selective inactivation of a Fas-associated death domain protein (FADD)-dependent apoptosis and autophagy pathway in immortal epithelial cells

Jacqueline Thorburn et al. Mol Biol Cell. 2005 Mar.

Abstract

Although evasion of apoptosis is thought to be required for the development of cancer, it is unclear which cell death pathways are evaded. We previously identified a novel epithelial cell death pathway that works in normal cells but is inactivated in tumor cells, implying that it may be targeted during tumor development. The pathway can be activated by the Fas-associated death domain (FADD) of the adaptor protein but is distinct from the known mechanism of FADD-induced apoptosis through caspase-8. Here, we show that a physiological signal (tumor necrosis factor-related apoptosis-inducing ligand) can kill normal epithelial cells through the endogenous FADD protein by using the novel FADD death domain pathway, which activates both apoptosis and autophagy. We also show that selective resistance to this pathway occurs when primary epithelial cells are immortalized and that this occurs through a mechanism that is independent of known events (telomerase activity, and loss of function of p53, Rb, INK4a, and ARF) that are associated with immortalization. These data identify a novel cell death pathway that combines apoptosis and autophagy and that is selectively inactivated at the earliest stages of epithelial cancer development.

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Figures

Figure 1.

Figure 1.

TRAIL can kill normal and cancerous epithelial cells by different mechanisms. (A) Time-lapse microscopy of normal prostate epithelial cells or prostate cancer cells treated with TRAIL plus AEBSF and zVAD.fmk. TRAIL kills both cell types, but zVAD.fmk alone can protect only the cancer cells; the combination of zVAD.fmk and AEBSF is required to protect normal cells from TRAIL-induced death. (B) The number of dead cells for each time point was determined by counting rounded cells in individual frames for each treatment. Treatment with zVAD.fmk in the normal cells altered the slope of the line, indicating that the caspase-independent cell death response in normal cells occurred more slowly than caspase-dependent cell death. (C) Normal or cancerous prostate cells were treated with TRAIL in the presence of the protease inhibitors and harvested for Western blot analysis of caspase-3 and PARP cleavage. In both cell types, caspase-3 was activated, leading to PARP cleavage, and the caspase inhibitor zVAD.fmk completely blocked the response. (D) MTS assays of TRAIL treated cells were performed. ZVAD.fmk only partially protected normal cells but completely protected cancer cells. The combination of zVAD.fmk and AEBSF completely protected normal cells.

Figure 2.

Figure 2.

TRAIL activates the FADD-DD pathway through the endogenous FADD protein. (A) Normal prostate epithelial cells were injected with YFP control, FADD-DD, or FADD-DD V108E expression constructs in the presence or absence of TRAIL as indicated, and cell death was determined by monitoring the response of each injected cell. In the absence of TRAIL, FADD-DD induced apoptosis but the V108E mutant did not. The V108E mutant blocked TRAIL-induced cell death. (B) Normal cells or cancer cells were infected with doxycycline-regulated adenoviruses expressing YFP, FADD-DD, or FADD-DD V108E as indicated and then treated with or without TRAIL. Cell survival was determined using an MTS assay. TRAIL killed both normal and cancer cells, and in the absence of TRAIL, FADD-DD could kill only normal cells. In cancer cells, both the V108E mutant and the wild-type FADD-DD were equally effective at inhibiting TRAIL-induced cell death. In the normal cells, wild-type FADD-DD plus TRAIL led to increased cell death compared with either FADD-DD or TRAIL alone, whereas the V108E mutant completely inhibited TRAIL-induced cell death. These data indicate that FADD-DD functions differently in normal and cancerous prostate cells and can cooperate with TRAIL to increase normal cell death.

Figure 3.

Figure 3.

FADD-DD can cause autophagic vesicle formation in normal epithelial cells. (A) Normal primary prostate epithelial cells or DU145 prostate cancer cells were treated with TRAIL or infected with adenoviruses expressing FADD-DD or V108E FADD-DD as indicated and analyzed by TEM. Large numbers of vesicular structures (arrows) were found in normal cells expressing FADD-DD or treated with TRAIL. Normal cells were treated with zVAD.fmk to prevent caspase-dependent signaling from obscuring any caspase-independent effects. Bars, 5 μm. (B), higher power images of autophagic vesicles from FADD-DD or TRAIL-treated normal prostate cells showing double membranes and cellular debris. Bar, 0.5 μm. (C) Cell area taken up by autophagic vesicles, indicating that FADD-DD and TRAIL increase the proportion of each normal cell that is vacuolated.

Figure 4.

Figure 4.

FADD-DD-induced autophagy in normal cells. (A) Normal prostate cells or DU145 cancer cells were injected with GFP-tagged LC3 plus FADD-DD or treated with TRAIL and followed by fluorescence microscopy. GFP-LC3 forms aggregates (arrows) in FADD-DD–expressing or TRAIL-treated normal cells but does not aggregate in cancer cells. (B) Normal prostate cell survival 24 h after injection with FADD-DD and treatment with zVAD.fmk or 3-MA alone or in combination. FADD-DD–induced cell death is not prevented by either inhibitor alone but is inhibited by the combined inhibitors.

Figure 5.

Figure 5.

FADD-DD–induced cell death is selectively inhibited in immortalized cells. (A) HMECs at different stages of immortalization and transformation were injected with YFP control or YFP-FADD-DD expression vectors, and the percentage of survival for fluorescent cells was determined. FADD-DD killed normal HMECs and TERT-expressing HME cells, but it did not kill HMEC expressing T antigen plus TERT (HMLcE) or any of the other cells. Panel B, HME cells and HMLcE cells were injected with control, FADD-DD, or a full-length FADD construct that can activate caspase-8. Both FADD-DD and FADD could kill HME cells, but only the FADD molecule capable of activating caspase-8 killed HMLcE cells. These data show that resistance to FADD-DD–induced cell death arises in response to expression of T antigen, which causes immortalization and that this resistance is specific to the FADD-DD pathway.

Figure 6.

Figure 6.

Inhibition of the FADD-DD pathway in immortal cells is not caused by inactivation of genes that are known to regulate immortalization. (A) Primary mouse mammary epithelial cells, spontaneously immortalized epithelial cells, or primary breast fibroblasts were injected with YFP control, FADD-DD, or FADD expression constructs, and cell survival was determined. All the cell types were killed by the FADD molecule that can activate caspase-8, but only the primary epithelial cells were killed by FADD-DD. (B) Mammary epithelial cells were isolated from p53 knockout mice and tested for sensitivity to FADD-DD after limited culture (passage 2–10) or extended in vitro culture (passage 18–28). FADD-DD killed the low passage number cells but could not kill the high passage number cells. The Western blot insert compares protein samples from the p53 knockout or wild-type animals showing that the cells lacked p53. (C) Primary MMECs were cultured from p21 and INK4a/ARF knockout animals or from animals with floxed Rb genes, which were subsequently infected with a Cre recombinase adenovirus and maintained for 3 d in culture at which time no detectable Rb protein was present (inset). All the low passage (< passage 10) primary cells underwent apoptosis in response to FADD-DD. (D) Low passage (passage 3–8) or high passage (passage 23–27) MMECs from INK4a/ARF -/- mice were injected with FADD-DD as indicated. Only the low passage cells were killed by FADD-DD.

Figure 7.

Figure 7.

FADD-DD-induced autophagy is lost in late passage epithelial cells. (A) Low or high passage MMECs from INK4a/ARF knockout animals were infected with FADD-DD or V108E FADD-DD adenoviruses in the presence of zVAD.fmk to inhibit caspase-dependent effects and analyzed by TEM for signs of autophagy. Large numbers of vesicles (arrows) were observed only in the low passage cells expressing wild-type FADD-DD. (B) Cell area taken up by vacuolated structures, indicating that FADD-DD but not the V108E mutant causes an increase in such structures in low passage cells.

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