Sorafenib-induced defective autophagy promotes cell death by necroptosis
Pedram Kharaziha1, Dimitris Chioureas1, George Baltatzis2
1Department of Oncology-Pathology, Cancer Centrum Karolinska, Karolinska Institutet and University Hospital, Stockholm, Sweden.
Abstract:
Autophagy is one of the main cytoprotective mechanisms that cancer cells deploy to withstand the cytotoxic stress and survive the lethal damage induced by anti-cancer drugs. However, under specific conditions, autophagy may, directly or indirectly, induce cell death. In our study, treatment of the Atg5-deficient DU145 prostate cancer cells, with the multi-tyrosine kinase inhibitor, sorafenib, induces mitochondrial damage, autophagy and cell death. Molecular inhibition of autophagy by silencing ULK1 and Beclin1 rescues DU145 cells from cell death indicating that, in this setting, autophagy promotes cell death. Re-expression of Atg5 restores the lipidation of LC3 and rescues DU145 and MEF atg5-/- cells from sorafenib-induced cell death. Despite the lack of Atg5 expression and LC3 lipidation, DU145 cells form autophagosomes as demonstrated by transmission and immuno-electron microscopy, and the formation of LC3 positive foci. However, the lack of cellular content in the autophagosomes, the accumulation of long-lived proteins, the presence of GFP-RFP-LC3 positive foci and the accumulated p62 protein levels indicate that these autophagosomes may not be fully functional. DU145 cells treated with sorafenib undergo a caspase-independent cell death that is inhibited by the RIPK1 inhibitor, necrostatin-1. Furthermore, treatment with sorafenib induces the interaction of RIPK1 with p62, as demonstrated by immunoprecipitation and a proximity ligation assay. Silencing of p62 decreases the RIPK1 protein levels and renders necrostatin-1 ineffective in blocking sorafenib-induced cell death. In summary, the formation of Atg5-deficient autophagosomes in response to sorafenib promotes the interaction of p62 with RIPK leading to cell death by necroptosis.
Insights
Autophagy can promote cancer cell death. In Atg5-deficient cells, sorafenib triggers non-functional autophagy and necroptosis via RIPK1 and p62 interaction, highlighting a novel cell death pathway.
Area of Science:
- Cell Biology
- Cancer Research
- Molecular Oncology
Background:
- Autophagy is a key cytoprotective mechanism utilized by cancer cells to survive anti-cancer drug treatments.
- While typically cytoprotective, autophagy can also induce cell death under specific circumstances.
- Understanding the dual role of autophagy in cancer therapy is crucial for developing effective treatments.
Purpose of the Study:
- To investigate the role of autophagy in sorafenib-induced cell death in Atg5-deficient prostate cancer cells.
- To elucidate the molecular mechanisms underlying this cell death pathway.
- To determine if autophagy can be therapeutically targeted to enhance anti-cancer drug efficacy.
Main Methods:
- Utilized Atg5-deficient DU145 prostate cancer cells and MEF atg5-/- cells.
- Treated cells with sorafenib, a multi-tyrosine kinase inhibitor.
- Performed molecular inhibition of autophagy (ULK1, Beclin1 silencing) and re-expression of Atg5.
- Employed transmission and immuno-electron microscopy, GFP-RFP-LC3 assays, and p62 protein level analysis.
- Investigated cell death pathways using necrostatin-1 and assessed RIPK1-p62 interactions via immunoprecipitation and proximity ligation assays.
Main Results:
- Sorafenib treatment induced mitochondrial damage, autophagy, and cell death in Atg5-deficient DU145 cells.
- Inhibition of autophagy (ULK1, Beclin1 silencing) rescued cells from death, indicating autophagy promotes cell death in this context.
- Re-expression of Atg5 restored LC3 lipidation and rescued cells from sorafenib-induced death.
- Atg5-deficient cells formed non-functional autophagosomes, evidenced by lack of content, protein accumulation (p62), and specific LC3 foci.
- Cell death was caspase-independent and sensitive to necrostatin-1, implicating RIPK1.
- Sorafenib induced RIPK1-p62 interaction, and p62 silencing reduced RIPK1 levels, rendering necrostatin-1 ineffective.
Conclusions:
- The formation of Atg5-deficient autophagosomes in response to sorafenib promotes cell death.
- This cell death occurs via necroptosis, mediated by the interaction between p62 and RIPK1.
- Targeting this specific autophagy-dependent necroptotic pathway could offer new therapeutic strategies against prostate cancer.
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