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- Title
ITCH-dependent proteasomal degradation of c-FLIP induced by the anti-HER3 antibody 9F7-F11 promotes DR5/caspase 8-mediated apoptosis of tumor cells.
- Authors
Le Clorennec, Christophe; Lazrek, Yassamine; Dubreuil, Olivier; Sampaio, Carla; Larbouret, Christel; Lanotte, Romain; Poul, Marie-Alix; Barret, Jean-Marc; Prost, Jean-François; Pèlegrin, André; Chardès, Thierry
- Abstract
Background: HER3/ErbB3 receptor deletion or blockade leads to tumor cell apoptosis, whereas its overexpression confers anti-cancer drug resistance through upregulation of protective mechanisms against apoptosis. We produced the anti-HER3 antibody 9F7-F11 that promotes HER3 ubiquitination and degradation via JNK1/2-dependent activation of the E3 ubiquitin ligase ITCH, and that induces apoptosis of cancer cells. Cellular FLICE-like inhibitory protein (c-FLIP) is a key regulator of apoptotic pathways. Here, we wanted to determine the mechanisms underlying the pro-apoptotic effect of 9F7-F11. Methods: Anti-HER3 antibody-induced apoptosis was assessed by western blot, and by flow cytometry measurement of Annexin V/7-AAD-labelled tumor cells (BxPC3, MDA-MB-468 and DU145 cell lines). c-FLIP/ITCH interaction and subsequent degradation/ubiquitination were investigated by co-immunoprecipitation of ITCH-silenced vs scramble control cells. The relationship between ITCH-mediated c-FLIP degradation and antibody-induced apoptosis was examined by western blot and flow cytometry of tumor cells, after ITCH RNA interference or by pre-treatment with ITCH chemical inhibitor chlorimipramine (CI). Results: Following incubation with 9F7-F11, cancer cell apoptosis occurs through activation of caspase-8, − 9 and − 3 and the subsequent cleavage of poly (ADP-ribose) polymerase (PARP). Moreover we showed that ubiquitination and proteasomal degradation of the anti-apoptotic protein c-FLIP was mediated by USP8-regulated ITCH recruitment. This effect was abrogated by ITCH- and USP8-specific RNA interference (siRNA), or by the ITCH chemical inhibitor CI. Specifically, ITCH silencing or CI blocked 9F7-F11-induced caspase-8-mediated apoptosis of tumor cells, and restored c-FLIP expression. ITCH-silencing or CI concomitantly abrogated HER3-specific antibody-induced apoptosis of Annexin V/7-AAD-labelled BxPC3 cells. 9F7-F11 favored the extrinsic apoptosis pathway by inducing TRAIL-R2/DR5 upregulation and TRAIL expression that promoted the formation of death-inducing signaling complex (DISC), leading to caspase-8-mediated apoptosis. Incubation with 9F7-F11 also induced BID cleavage, BAX upregulation and BIM expression, which initiated the caspase-9/3-mediated mitochondrial death pathway. The anti-HER3 antibody pro-apoptotic effect occurred concomitantly with downregulation of the pro-survival proteins c-IAP2 and XIAP. Conclusions: The allosteric non-neuregulin competing modulator 9F7-F11, sensitizes tumor cells to DR5/caspase-8-mediated apoptosis through ITCH-dependent downregulation of c-FLIP.
- Subjects
UBIQUITINATION; APOPTOSIS; RNA interference; CANCER cells; PROTEOLYSIS; CHEMICAL inhibitors; UBIQUITIN ligases
- Publication
Cell Communication & Signaling, 2019, Vol 17, Issue 1, pN.PAG
- ISSN
1478-811X
- Publication type
Article
- DOI
10.1186/s12964-019-0413-8