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Background Patients diagnosed with triple negative breast cancer (TNBC) have limited treatment options and often suffer from resistance and toxicity due to chemotherapy. We previously found that depleting calcium and integrin-binding protein 1 (CIB1) induces cell death selectively in TNBC cells, while sparing normal cells. Therefore, we asked whether CIB1 depletion further enhances tumor-specific killing when combined with either the commonly used chemotherapeutic, docetaxel, or the cell death-inducing ligand, TRAIL. Methods We targeted CIB1 by RNA interference in MDA-MB-436, MDA-MB-231, MDA-MB-468, docetaxel-resistant MDA-MB-436 TNBC cells and ME16C normal breast epithelial cells alone or combination with docetaxel or TRAIL. Cell death was quantified via trypan blue exclusion using flow cytometry and cell death mechanisms were analyzed by Western blotting. Cell surface levels of TRAIL receptors were measured by flow cytometry analysis. Results CIB1 depletion combined with docetaxel significantly enhanced tumor-specific cell death relative to each treatment alone. The enhanced cell death strongly correlated with caspase-8 activation, a hallmark of death receptor-mediated apoptosis. The death receptor TRAIL-R2 was upregulated in response to CIB1 depletion, which sensitized TNBC cells to the ligand TRAIL, resulting in a synergistic increase in cell death. In addition to death receptor-mediated apoptosis, both combination treatments activated a non-apoptotic mechanism, called paraptosis. Interestingly, these combination treatments also induced nearly complete death of docetaxel-resistant MDA-MB-436 cells, again via apoptosis and paraptosis. In contrast, neither combination treatment induced cell death in normal ME16C cells. Conclusion Novel combinations of CIB1 depletion with docetaxel or TRAIL selectively enhance naive and docetaxel-resistant TNBC cell death while sparing normal cell. Therefore, combination therapies that target CIB1 could prove to be a safe and durable strategy for treatment of TNBC and potentially other cancers. Electronic supplementary material The online version of this article (10.1186/s12935-019-0740-2) contains supplementary material, which is available to authorized users.

Bacterial β-glucuronidase (GUS) enzymes cause drug toxicity by reversing Phase II glucuronidation in the gastrointestinal tract. While many human gut microbial GUS enzymes have been examined with model glucuronide substrates like p-nitrophenol-β-D-glucuronide (pNPG), the GUS orthologs that are most efficient at processing drug-glucuronides remain unclear. Here we present the crystal structures of GUS enzymes from human gut commensals Lactobacillus rhamnosus, Ruminococcus gnavus, and Faecalibacterium prausnitzii that possess an active site loop (Loop 1; L1) analogous to that found in E. coli GUS, which processes drug substrates. We also resolve the structure of the No Loop GUS from Bacteroides dorei. We then compare the pNPG and diclofenac glucuronide processing abilities of a panel of twelve structurally diverse GUS proteins, and find that the new L1 GUS enzymes presented here process small glucuronide substrates inefficiently compared to previously characterized L1 GUS enzymes like E. coli GUS. We further demonstrate that our GUS inhibitors, which are effective against some L1 enzymes, are not potent towards all. Our findings pinpoint active site structural features necessary for the processing of drug-glucuronide substrates and the inhibition of such processing.

Modular type I polyketide synthases (PKSs) produce some of the most chemically complex metabolites in nature through a series of multienzyme modules. Each module contains a variety of catalytic domains to selectively tailor the growing molecule. PKS O-methyltransferases ( O-MTs) are predicted to methylate β-hydroxyl or β-keto groups, but their activity and structure have not been reported. We determined the domain boundaries and characterized the catalytic activity and structure of the StiD and StiE O-MTs, which methylate opposite β-hydroxyl stereocenters in the myxobacterial stigmatellin biosynthetic pathway. Substrate stereospecificity was demonstrated for the StiD O-MT. Key catalytic residues were identified in the crystal structures and investigated in StiE O-MT via site-directed mutagenesis and further validated with the cyanobacterial CurL O-MT from the curacin biosynthetic pathway. Initial structural and biochemical analysis of PKS O-MTs supplies a new chemoenzymatic tool, with the unique ability to selectively modify hydroxyl groups during polyketide biosynthesis.

The current standard of care for triple-negative breast cancer (TNBC) patients is limited to chemotherapy, radiation, and/or surgery. Therefore, there is a pressing need for new targeted therapies to improve clinical efficacy, reduce toxicity, and overcome resistance. We previously found that CIB1 depletion induces cell death selectively in TNBC cells, while sparing normal cells. The purpose of this study was to ask whether depleting CIB1 further enhances tumor-specific killing when combined with either the commonly used chemotherapeutic, docetaxel, or the cell death-inducing ligand, TRAIL. To address this, we targeted CIB1 by RNA interference in MDA-MB-436, MDA-MB-231, MDA-MB-468 TNBC cells and ME16C normal breast epithelial cells alone or combination with docetaxel or TRAIL. Cell death was quantified via trypan blue exclusion using FACS and cell death mechanisms were analyzed by Western blotting. We found that the combination of CIB1 depletion with docetaxel significantly enhanced tumor-specific cell death relative to each treatment alone. The enhanced cell death strongly correlated with caspase-8 activation, a hallmark of death receptor-mediated apoptosis. Because CIB1 depletion also upregulated the death receptor, TRAIL-R2, we tested for sensitization to TRAIL. While TRAIL alone had no effect on cell viability, CIB1 depletion profoundly sensitized tumor cells to TRAIL, resulting in a synergistic increase in cell death. In addition to death receptor-mediated apoptosis, both combination treatments activated a non-apoptotic mechanism, called paraptosis. Interestingly, these combination treatments also induced nearly complete death of docetaxel-resistant MDA-MB-436 cells, again via apoptosis and paraptosis. In contrast, neither combination treatment induced cell death in normal ME16C cells. In conclusion, our results demonstrate that the novel combinations of CIB1 depletion with docetaxel or TRAIL significantly enhance naive and docetaxel-resistant TNBC cell death via increased death receptor-mediated apoptosis and paraptosis, suggesting that targeting CIB1 may also overcome resistance to chemotherapeutics. Taken together, combination therapies that target CIB1 could prove to be a safe and durable strategy for treatment of TNBC and potentially other cancers. Citation Format: Alexander H. Chung, Tina M. Leisner, Gabrielle Dardis, Marissa Bivins, Leslie V. Parise. Potent treatment combinations with CIB1 depletion in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3967.