A marine-derived small molecule induces immunogenic cell death against triple-negative breast cancer through ER stress-CHOP pathway.

Although triple-negative breast cancer (TNBC) is the most refractory subtype among all breast cancers, it has been shown to have higher immune infiltration than other subtypes. We identified the marine-derived small molecule MHO7, which acts as a potent immunogenic cell death (ICD) inducer through the endoplasmic reticulum (ER) stress-C/EBP-homologous protein (CHOP) pathway, to treat TNBC. MHO7 exerted cytostatic and cytotoxic effects on TNBC cells at an IC50 of 0.96-1.75 µM and suppressed tumor growth with an approximately 80% inhibition rate at a dose of 60 mg/kg. In 4T1 cell tumor-bearing mice, 30 mg/kg MHO7 inhibited pulmonary metastasis with an efficacy of 70.26%. Transcriptome analyses revealed that MHO7 changed the transcription of genes related to ribosome and protein processes in the ER. MHO7 also triggered reactive oxygen species (ROS) generation and attenuated glutathione (GSH) levels, which caused excessive oxidative stress and ER stress via the PERK/eIF2α/AFT4/CHOP pathway and led to cell apoptosis. ER stress and ROS production facilitated the release of ICD-related danger-associated molecular patterns (DAMPs) from TNBC cells, which activated the immune response in vivo, as indicated by the release of antitumor cytokines such as IL-6, IL-1ß, IFN-γ, and TNF-α, increases in CD86+ and MHC-II dendritic cells and CD4+ and CD8+ T cells and a decrease in regulatory T cells (Tregs). These results reveal that MHO7 triggers an aggressive stress response to amplify tumor immunogenicity and induce a robust immune response. This synergistic effect inhibits primary breast cancer growth and spontaneous metastasis in TNBC, providing a new strategy for TNBC treatment.

Synthesis and structure-activity relationship study of a potent MHO7 analogue as potential anti-triple negative breast cancer agent.

Triple-negative breast cancer (TNBC) is the most aggressive, high recurrence and metastatic breast cancer subtype. There are few safe and effective therapeutic drugs for treatment of TNBC. The marine natural product MHO7 has been determined to be a potential antitumor agent. However, its moderate activity and complex structure hampered its clinical application. In this study, a series of novel derivatives with modification on C24 of MHO7 were first synthesized. Some of the analogues were significantly more potent than MHO7 against all selected breast cancer cell lines. Among them, compound 4m had the best activity, and its IC50 value against TNBC was up to 0.51 µM. A whole-genome transcriptomic analysis shown that the mechanism of compound 4m against TNBC cells was similar with that of parent compound MHO7. Subsequent cellular mechanism studies showed that compound 4m could induce apoptosis of MDA-MB-231 cells through mitochondria pathway and cause G1 phase arrest. Moreover, 4m could disrupt the expressions of MAPK/Akt pathway-associated proteins (p-p38 and p-Akt) and remarkably increase the ratio of Bax to Bcl-2 and activate cleaved caspase 3/9/PARP. Importantly, 4m could influence the expression of Smad 7, and p-Smad 3 to inhibit TNBC cells metastasis. Stability assays in rat plasma and liver microsomes indicated that 4m still have room for further optimization. And the results of the online molinspiration software predicted that 4m has desirable physicochemical properties but some properties still have violation from the Lipinski rule of five. Overall, the modification on C24 of MHO7 was a promising way for developing novel anti-TNBC agents with considerable potential for optimization.

The Biosynthesis and Transport of Ophiobolins in Aspergillus ustus 094102.

Ophiobolins are a group of sesterterpenoids with a 5-8-5 tricyclic skeleton. They exhibit a significant cytotoxicity and present potential medicinal prospects. However, the biosynthesis and transport mechanisms of these valuable compounds have not been fully resolved. Herein, based on a transcriptome analysis, gene inactivation, heterologous expression and feeding experiments, we fully explain the biosynthesis pathway of ophiobolin K in Aspergillus ustus 094102, especially proved to be an unclustered oxidase OblCAu that catalyzes dehydrogenation at the site of C16 and C17 of both ophiobolin F and ophiobolin C. We also find that the intermediate ophiobolin C and final product ophiobolin K could be transported into a space between the cell wall and membrane by OblDAu to avoid the inhibiting of cell growth, which is proved by a fluorescence observation of the subcellular localization and cytotoxicity tests. This study completely resolves the biosynthesis mechanism of ophiobolins in strain A. ustus 094102. At the same time, it is revealed that the burden of strain growth caused by the excessive accumulation and toxicity of secondary metabolites is closely related to compartmentalized biosynthesis.

Design and synthesis of marine sesterterpene analogues as novel estrogen receptor α degraders for breast cancer treatment.

Targeted protein degradation using small molecules is an intriguing strategy for drug development. The marine sesterterpene compound MHO7 had been reported to be a potential ERα degradation agent. In order to further improve its biological activity, two series of novel MHO7 derivatives with long side chains were designed and identified as novel selective estrogen receptor down-regulators (SERDs). The growth inhibition activity of the novel SERD compounds were significantly affected by the type and length of the side chain. Most of the derivatives were significantly more potent than MHO7 against both drug-sensitive and drug-resistant breast cancer cells. Among them, compound 16a, with IC50 values of 0.41 µM against MCF-7 cell lines and 9.6-fold stronger than MHO7, was the most potential molecule. A whole-genome transcriptomic analysis of MCF-7 cells revealed that the mechanism of 16a against MCF-7 cell was similar with that of MHO7. The estrogen signaling pathway was the most affected among the disturbed genes, but the ERα degradation activity of 16a was observed higher than that of MHO7. Other effects of 16a were confirmed similar with MHO7, which means that the basic mechanisms of the derivatives are the same with the ophiobolin backbone, i.e. the degradation of ERα is mediated via proteasome-mediated process, the induction of apoptosis and the cell cycle arrest at the G1 phase. Meanwhile, a decrease of mitochondrial membrane potential and an increase of cellular ROS were also detected. Based on these results, as a novel modified ophiobolin derived compound, 16a may warrant further exploitation as a promising SERD candidate agent for the treatment of breast cancer.

Genome Mining Reveals a Multiproduct Sesterterpenoid Biosynthetic Gene Cluster in Aspergillus ustus.

Genome mining of Aspergillus ustus 094102 enabled the discovery of a multiproduct bifunctional terpene synthase (BTS), AuAS. Heterologous expression of AuAS led to the discovery of five new sesterterpenes, and coexpression of the upstream CYP450 monooxygenase (AuAP450) generated four new sesterterpene alcohols. Additionally, aspergilol A showed cytotoxic activities against MCF-7, MDA-MB231, and HepG2 cancer cells (IC50 21.20-48.76 µM), and aspergilol B exhibited a cytotoxic effect on MCF-7 cells (IC50 27.41 µM).