Novel potential of tunicamycin as an activator of the aryl hydrocarbon receptor – dioxin responsive element signaling pathway

AbstractTunicamycin is a well-known inhibitor of protein glycosylation and used as an inducer of endoplasmic reticulum (ER) stress. We found that tunicamycin induced expression of cytochrome P450 1A1 in a dose-dependent manner. Like dioxin, the transcriptional induction was associated with dose-dependent activation of the dioxin responsive element (DRE). This effect was independent of inhibition of protein glycosylation or induction of ER stress. Pharmacological and genetic inhibition of the aryl hydrocarbon receptor (AhR) significantly attenuated activation of DRE by tunicamycin. These results elucidated the novel potential of tunicamycin as an activator of the AhR – DRE signaling pathway

M-COPA suppresses endolysosomal Kit-Akt oncogenic signalling through inhibiting the secretory pathway in neoplastic mast cells

<div><p>Gain-of-function mutations in Kit receptor tyrosine kinase result in the development of a variety of cancers, such as mast cell tumours, gastrointestinal stromal tumours (GISTs), acute myeloid leukemia, and melanomas. The drug imatinib, a selective inhibitor of Kit, is used for treatment of mutant Kit-positive cancers. However, mutations in the Kit kinase domain, which are frequently found in neoplastic mast cells, confer an imatinib resistance, and cancers expressing the mutants can proliferate in the presence of imatinib. Recently, we showed that in neoplastic mast cells that endogenously express an imatinib-resistant Kit mutant, Kit causes oncogenic activation of the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the signal transducer and activator of transcription 5 (STAT5) but only on endolysosomes and on the endoplasmic reticulum (ER), respectively. Here, we show a strategy for inhibition of the Kit-PI3K-Akt pathway in neoplastic mast cells by M-COPA (2-methylcoprophilinamide), an inhibitor of this secretory pathway. In M-COPA-treated cells, Kit localization in the ER is significantly increased, whereas endolysosomal Kit disappears, indicating that M-COPA blocks the biosynthetic transport of Kit from the ER. The drug greatly inhibits oncogenic Akt activation without affecting the association of Kit with PI3K, indicating that ER-localized Kit-PI3K complex is unable to activate Akt. Importantly, M-COPA but not imatinib suppresses neoplastic mast cell proliferation through inhibiting anti-apoptotic Akt activation. Results of our M-COPA treatment assay show that Kit can activate Erk not only on the ER but also on other compartments. Furthermore, Tyr568/570, Tyr703, Tyr721, and Tyr936 in Kit are phosphorylated on the ER, indicating that these five tyrosine residues are all phosphorylated before mutant Kit reaches the plasma membrane (PM). Our study provides evidence that Kit is tyrosine-phosphorylated soon after synthesis on the ER but is unable to activate Akt and also demonstrates that M-COPA is efficacious for growth suppression of neoplastic mast cells.</p></div

In neoplastic mast cells, Kit phosphorylation at Tyr568/570, Tyr703, Tyr721, and Tyr936 occurs on the ER.

<p>(<b>A</b>) Tyrosine phosphorylation sites in human Kit. (<b>B</b>-<b>F</b>) Cells were treated with vehicle or (<b>B</b>-<b>D</b>) 5 μM M-COPA for 16 hours or (<b>E</b> and <b>F</b>) 250 nM monensin for 24 hours. Anti-Kit immunoprecipitates (<b>B</b> and <b>E</b>) and cell lysates (<b>C</b>, <b>D</b>, and <b>F</b>) were immunoblotted with anti-Kit, anti-phospho-Kit^Tyr568/570 (anti-pKit^Tyr568/570), anti-pKit^Tyr703, anti-pKit^Tyr721, and anti-pKit^Tyr936. Note that ER-localized Kit was phosphorylated at Tyr568/570, Tyr703, Tyr721 and Tyr936 in neoplastic mast cells.</p

Model of the effect of M-COPA on Kit signalling and on autophosphorylation.

<p>(<b>A</b>) In neoplastic mast cells, mutant Kit is trafficked to the PM along the secretory pathway and then moves to endolysosomes through endocytosis. Kit activates Akt only on endolysosomes as previously described (Obata et al., 2014). Note that M-COPA inhibits the activation of Akt through blocking ER export of Kit. STAT5 activation is enhanced by M-COPA because it is activated by ER-localized Kit. HM, high mannose; CG, complex glycosylation. (<b>B</b>) Tyr568/570, Tyr703, Tyr721, and Tyr936 in Kit are phosphorylated on the ER in neoplastic mast cells.</p

In neoplastic mast cells, M-COPA inhibits Kit trafficking from the ER, resulting in a decrease in endolysosomal Kit.

<p>(<b>A</b>) RCM cells were immunostained with anti-Kit (green) and anti-cathepsin D (endolysosome marker, red). Insets indicate magnified images of the boxed area. Bar, 10 μm. (<b>B</b>) RCM cells were treated with vehicle or 0.1~5 μM M-COPA for 16 hours, then stained for Kit (green) and calnexin (ER marker, red). Insets indicate magnified images of the boxed area. Bars, 10 μm. (<b>C</b>) Pearson’s R correlation coefficients were calculated by intensity analysis of Kit <i>vs</i>. calnexin. Results are means ± SD (<i>n</i> = 11~36). Data were subjected to one-way ANOVA with Dunnett’s multiple comparison <i>post-hoc</i> test. ***<i>P</i> < 0.001; NS, not significant. Note that in RCM cells, co-localization of Kit with calnexin was significantly increased by M-COPA treatment.</p

In neoplastic mast cells, M-COPA inhibits Kit-dependent Akt activation through blocking the biosynthetic transport of Kit from the ER.

<p>(<b>A</b>-<b>C</b>) RCM (<b>A</b>) and HMC-1.2 (<b>B</b>) were treated for 16 hours with vehicle (0) or 0.1~5 μM M-COPA. Cell lysates were immunoblotted with anti-Kit, anti-phospho-Kit^Tyr721 (anti-pKit^Tyr721), anti-Akt, anti-pAkt, anti-STAT5, anti-pSTAT5, and anti-cleaved caspase-3. The graphs show the levels of pSTAT5 (open circles) or pAkt (closed circles) expressed relative to lysate from vehicle-treated cells. CG, complex-glycosylated form; HM, high mannose form. (<b>C</b>) RCM and HMC-1.2 were treated with 5 μM M-COPA for 16 hours. Anti-Kit immunoprecipitates were immunoblotted with anti-Kit and anti-PI3K p85. Note that M-COPA inhibited Akt activation without affecting the phosphorylation of Kit at Tyr721 or the association of Kit with PI3K p85. (<b>D</b>) RCM (left) and HMC-1.2 (right) were treated with vehicle (0), M-COPA (open circles) or imatinib (closed circles) for 24 hours. Proliferation was assessed by [³H]-thymidine incorporation. Results (c.p.m.) are means ± SD (<i>n</i> = 3).</p

Effect of M-COPA treatment on Kit-dependent Erk activation in RCM cells.

<p>(<b>A</b> and <b>B</b>) RCM cells were treated with vehicle (0), (<b>A</b>) 1 μM PKC412 for 24 hours, or (<b>B</b>) 0.2~5 μM M-COPA for 16 hours. Cell lysates were immunoblotted with anti-Erk and anti-phospho-Erk (anti-pErk).</p