Institut de Recherche en Cancérologie de Montpellier (IRCM), INSERM U1194, Université de Montpellier, Institut régional du Cancer de Montpellier (ICM), F-34298, Montpellier, France.
Present Address: UCSD School of Medicine, Moores Cancer Center, La Jolla, CA, 92093-0815, USA.
Cell Commun Signal. 2019 Aug 23;17(1):106. doi: 10.1186/s12964-019-0413-8.
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.
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).
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.
The allosteric non-neuregulin competing modulator 9F7-F11, sensitizes tumor cells to DR5/caspase-8-mediated apoptosis through ITCH-dependent downregulation of c-FLIP.
HER3/ErbB3 受体缺失或阻断会导致肿瘤细胞凋亡，而其过表达则通过上调抗细胞凋亡的保护机制赋予抗癌药物耐药性。我们生产了抗 HER3 抗体 9F7-F11，它通过 JNK1/2 依赖性激活 E3 泛素连接酶 ITCH 促进 HER3 泛素化和降解，并诱导癌细胞凋亡。细胞 FLICE 样抑制剂（c-FLIP）是凋亡途径的关键调节剂。在这里，我们想确定 9F7-F11 促凋亡作用的机制。
通过 Western blot 和 Annexin V/7-AAD 标记的肿瘤细胞（BxPC3、MDA-MB-468 和 DU145 细胞系）的流式细胞术测量评估抗 HER3 抗体诱导的细胞凋亡。通过 ITCH 沉默与对照细胞的共免疫沉淀研究 c-FLIP/ITCH 相互作用及随后的降解/泛素化。通过 ITCH RNA 干扰或用 ITCH 化学抑制剂氯咪帕明（CI）预处理肿瘤细胞，通过 Western blot 和流式细胞术检测 ITCH 介导的 c-FLIP 降解与抗体诱导的细胞凋亡之间的关系。
用 9F7-F11 孵育后，通过 caspase-8、-9 和 -3 的激活以及随后的多聚（ADP-核糖）聚合酶（PARP）的切割，导致癌细胞凋亡。此外，我们表明，抗凋亡蛋白 c-FLIP 的泛素化和蛋白酶体降解是由 USP8 调节的 ITCH 募集介导的。这种效应被 ITCH 和 USP8 的特异性 RNA 干扰（siRNA）或 ITCH 化学抑制剂 CI 所消除。具体而言，ITCH 沉默或 CI 阻断了 9F7-F11 诱导的肿瘤细胞 caspase-8 介导的凋亡，并恢复了 c-FLIP 的表达。ITCH 沉默或 CI 同时消除了 9F7-F11 诱导的 Annexin V/7-AAD 标记的 BxPC3 细胞中 TRAIL-R2/DR5 的上调和 TRAIL 的表达，促进了死亡诱导信号复合物（DISC）的形成，从而导致 caspase-8 介导的凋亡。用 9F7-F11 孵育还诱导了 BID 切割、BAX 上调和 BIM 的表达，从而启动了 caspase-9/3 介导的线粒体死亡途径。抗 HER3 抗体的促凋亡作用与抗生存蛋白 c-IAP2 和 XIAP 的下调同时发生。
变构非神经调节素竞争调节剂 9F7-F11 通过 ITCH 依赖性下调 c-FLIP，使肿瘤细胞对 DR5/caspase-8 介导的凋亡敏感。
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