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Anomanolide C suppresses tumor progression and metastasis by ubiquitinating GPX4-driven autophagy-dependent ferroptosis in triple negative breast cancer.

ABSTRACT: Anomanolide C (AC), a natural withanolide isolated from Tubocapsicum anomalum, has been reported to have exhibits remarkable anti-tumour activities in several types of human cancers, particularly triple-negative breast cancer (TNBC). However, its intricate mechanisms still remain need to be clarified. Here, we evaluated whether AC could inhibit cell proliferation and the role of AC in ferroptosis induction and autophagy activation. Subsequently, the anti-migration potential of AC was found via autophagy-dependent ferroptosis. Additionally, we found that AC reduced the expression of GPX4 by ubiquitination and inhibited TNBC proliferation and metastasis in vitro and in vivo. Moreover, we demonstrated that AC induced autophagy-dependent ferroptosis, and led to Fe²⁺ accumulation via ubiquitinating GPX4. Moreover, AC was shown to induce autophagy-dependent ferroptosis as well as to inhibit TNBC proliferation and migration via GPX4 ubiquitination. Together, these results demonstrated that AC inhibited the progression and metastasis of TNBC by inducing autophagy-dependent ferroptosis via ubiquitinating GPX4, which might shed light on exploiting AC as a new drug candidate for the future TNBC therapy.

SUBMITTER: Chen YM

PROVIDER: S-EPMC10197885 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Anomanolide C suppresses tumor progression and metastasis by ubiquitinating GPX4-driven autophagy-dependent ferroptosis in triple negative breast cancer.

Chen Yan-Mei YM Xu Wei W Liu Yang Y Zhang Jia-Hui JH Yang Yuan-Yuan YY Wang Zhi-Wen ZW Sun De-Juan DJ Li Hua H Liu Bo B Chen Li-Xia LX

International journal of biological sciences 20230508 8

Anomanolide C (AC), a natural withanolide isolated from <i>Tubocapsicum anomalum</i>, has been reported to have exhibits remarkable anti-tumour activities in several types of human cancers, particularly triple-negative breast cancer (TNBC). However, its intricate mechanisms still remain need to be clarified. Here, we evaluated whether AC could inhibit cell proliferation and the role of AC in ferroptosis induction and autophagy activation. Subsequently, the anti-migration potential of AC was foun ...[more]

PMID: 37215985

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Project description:Patients with triple-negative breast cancer (TNBC) often have a poor prognosis largely due to lack of effective targeted therapy. Using a library of seleno-purines coupled to a high-throughput biochemical enzymatic assays we identified a potent pharmacological enhancer of autophagy (referred herein as SLLN-15) that selectively activated cytostatic macroautophagy/autophagy in TNBC preclinical models. SLLN-15 induced a dose-dependent anti-proliferative activity in the TNBC cell lines MDA-MB-231 and BT-20 via induction of autophagy and autophagic flux. This induction was associated with a selective inhibition of AKT-MTOR signaling. Conversely, rapamycin, a known autophagy inducer and MTOR inhibitor, was unable to duplicate SLLN-15's effect on TNBC cells. Inhibition of autophagy by siRNA-mediated targeting of the autophagy regulators, BECN1, ATG5 and ATG7 or using 3-methyladenine (3-MA), significantly protected against SLLN-15-induced inhibition of cell viability, further supporting that SLLN-15-induced inhibition of cancer cell proliferation was autophagy-dependent. SLLN-15-induced autophagy in TNBC cells was also associated with decreased AURKA expression, decreased AKT phosphorylation and subsequent blockage of the AKT-MTOR pathway. In vivo, oral SLLN-15 revealed a potent anticancer and anti-metastatic activity in mice bearing TNBC. Altogether, this study describes a novel regulator of mammalian autophagy, with potential utility as an experimental therapeutic for TNBCs. Abbreviations: 3-MA: 3-methyladenine; ATG5: autophagy related 5; ATG7: autophagy related 7; AURKA: aurora kinase A; AURKB: aurora kinase B; BECN1: beclin 1; CQ: chloroquine; DMSO: dimethyl sulfoxide; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; ERBB2: erb-b2 receptor tyrosine kinase 2; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PARP1: poly(ADP-ribose) polymerase 1; PI: propidium iodide; SQSTM1/p62: sequestosome 1; TNBC: triple-negative breast cancer.

Project description:Gankyrin is found in high levels in triple-negative breast cancer (TNBC) and has been established to form a complex with the E3 ubiquitin ligase MDM2 and p53, resulting in the degradation of p53 in hepatocarcinoma cells. Therefore, this study sought to determine whether gankyrin could inhibit ferroptosis through this mechanism in TNBC cells. The expression of gankyrin was investigated in relation to the prognosis of TNBC using bioinformatics. Co-immunoprecipitation and GST pull-down assays were then conducted to determine the presence of a gankyrin and MDM2 complex. RT-qPCR and immunoblotting were used to examine molecules related to ferroptosis, such as gankyrin, p53, MDM2, SLC7A11, and GPX4. Additionally, cell death was evaluated using flow cytometry detection of 7-AAD and a lactate dehydrogenase release assay, as well as lipid peroxide C11-BODIPY. Results showed that the expression of gankyrin is significantly higher in TNBC tissues and cell lines, and is associated with a poor prognosis for patients. Subsequent studies revealed that inhibiting gankyrin activity triggered ferroptosis in TNBC cells. Additionally, silencing gankyrin caused an increase in the expression of the p53 protein, without altering its mRNA expression. Co-immunoprecipitation and GST pull-down experiments indicated that gankyrin and MDM2 form a complex. In mouse embryonic fibroblasts lacking both MDM2 and p53, this gankyrin/MDM2 complex was observed to ubiquitinate p53, thus raising the expression of molecules inhibited by ferroptosis, such as SLC7A11 and GPX4. Furthermore, silencing gankyrin in TNBC cells disrupted the formation of the gankyrin/MDM2 complex, hindered the degradation of p53, increased SLC7A11 expression, impeded cysteine uptake, and decreased GPX4 production. Our findings suggest that TNBC cells are able to prevent cell ferroptosis through the gankyrin/p53/SLC7A11/GPX4 signaling pathway, indicating that gankyrin may be a useful biomarker for predicting TNBC prognosis or a potential therapeutic target.

Project description:Long noncoding RNAs (lncRNAs) are a novel class of noncoding RNAs that have emerged as critical regulators and biomarkers in various cancers. Nevertheless, the expression profile and mechanistic function of lncRNAs in cholangiocarcinoma (CCA) remain unclear. Herein, we examined the expression levels of linc00976 in clinical specimens and cell lines using reverse transcription-quantitative PCR. In total, 50 patients with CCA were enrolled to analyze the correlation between linc00976 expression and clinical characteristics of CCA. Loss- and gain-of-function experiments were performed to investigate the biological effects of linc00976 on proliferation, ferroptosis, migration, and invasion of CCA cells in vitro and in vivo. In situ hybridization, RNA immunoprecipitation, bioinformatic databases, RNA pull-down assay, a dual-luciferase reporter assay, mRNA sequencing, chromatin immunoprecipitation-PCR, and rescue experiments were performed to elucidate the underlying mechanisms of linc00976-induced competitive endogenous RNA regulatory networks. We characterized a novel and abundant lncRNA, linc00976, that functions as a pro-oncogenic regulator of CCA progression. Compared with normal controls, linc00976 was dramatically upregulated in CCA tissue samples and cell lines. Patients with CCA exhibiting high linc00976 expression had a highly advanced clinical stage, substantial lymph node metastasis, and poor overall survival. Knockdown of linc00976 significantly repressed proliferation and metastasis and promoted ferroptosis of CCA cells both in vitro and in vivo, whereas linc00976 overexpression exerted the opposite effect. Mechanistically, linc00976 competitively interacted with miR-3202 to upregulate GPX4 expression, thus contributing to the malignant biological behavior of CCA cells. Moreover, we demonstrated that JUND specifically interacts with the linc00976 promoter and activates linc00976 transcription. Accordingly, JUND promotes linc00976 transcription, and linc00976 plays a crucial role in accelerating CCA tumorigenesis and metastasis and inhibiting ferroptosis by modulating the miR-3202/GPX4 axis. These findings suggest that targeting linc00976 may afford a promising therapeutic strategy for patients with CCA.

Dataset Information

Anomanolide C suppresses tumor progression and metastasis by ubiquitinating GPX4-driven autophagy-dependent ferroptosis in triple negative breast cancer.

Publications

Anomanolide C suppresses tumor progression and metastasis by ubiquitinating GPX4-driven autophagy-dependent ferroptosis in triple negative breast cancer.

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