Ting Song

Herein, we developed a dual-factor recognition activatable probe, S1-F, which accurately lights up Bcl-2 and GSH dual-overexpressed cancer tissue with a superior tumor-to-normal tissue (T/N) ratio, highlighting the potential for this probe to be used in the visual diagnosis and imaging-guided surgery of cancer.

The process of degrading unwanted or damaged mitochondria by autophagy, called mitophagy, is essential for mitochondrial quality control together with mitochondrial apoptosis. In mammalian cells, pan-Bcl-2 family members including conical Bcl-2 members and non-conical ones are involved in and govern the two processes. We have illustrated recently the BH3 receptor Hsp70 interacts with Bim to mediate both apoptosis and mitophagy. However, whether similar pathways exist in lower eukaryotes where conical Bcl-2 members are absent remained unclear. Here, a specific inhibitor of the Hsp70-Bim PPI, S1g-10 and its analogs were used as chemical tools to explore the role of yeast Bxi1/Ybh3 in regulating mitophagy and apoptosis. Using Om45-GFP processing assay, we illustrated that yeast Ybh3 mediates a ubiquitin-related mitophagy pathway in both yeast and mammalian cells through association with Hsp70, which is in the same manner with Bim. Moreover, by using Bax/Bak double knockout MEF cells, Ybh3 was identified to induce apoptosis through forming oligomerization to trigger mitochondrial outer membrane permeabilization (MOMP) like Bax. We not only illustrated a conserved ubiquitin-related mitophagy pathway in yeast but also revealed the multi-function of Ybh3 which combines the function of BH3-only protein and multi-domain Bax protein as one.

Introduction: Estrogen receptor (ER) positive patients compromise about 70% of breast cancers. Tamoxifen, an antagonist of ERα66 (the classic ER), is the most effective and the standard first-line drug. However, its efficacy is limited by the development of acquired resistance. Methods: A specific inhibitor of Hsp70-Bim protein–protein interaction (PPI), S1g-2, together with an inhibitor of Hsp70-Bag3 PPI, MKT-077 and an ATP-competitive inhibitor VER155008, were used as chemical tools. Cell viability assays, co-immunoprecipitation and gene knockdown were used to investigate the role of Hsp70 in tamoxifen resistance. A xenograft model was established in which tamoxifen-resistant breast cancer (MCF-7/TAM-R) cells maintained in the presence of 5 μM tamoxifen were subcutaneously inoculated. The anti-tumor efficiency of S1g-2 was measured after a daily injection of 0.8 mg/kg for 14 days. Results: It was revealed that Hsp70-Bim PPI protects ERα-positive breast cancer from tamoxifen-induced apoptosis through binding and stabilizing ERα36, rather than ERα66, resulting in sustained EGFR mRNA and protein expression. Disruption of Hsp70-Bim PPI and downregulation of ERα36 expression in tumor samples are consistent with the in vitro functions of S1g-2, resulting in about a three-fold reduction in tumor volume. Conclusions: The in vivo activity and safety of S1g-2 illustrated that it is a potential strategy for Hsp70-Bim disruption to overcome tamoxifen-resistant ER-positive breast cancer.

Background and Purpose: Although chemotherapeutics or molecular targeted drugs often elicit profound initial responses, fractional killing capable of driving acquired resistance can persist. Identifying stress‐induced negative feedback or an incoherent feedforward loop (IFFL), which may contribute to fractional killing, is urgently needed.Experimental Approach: Mathematical modelling was used to identify how and to what extent a recently reported Hsp70‐Bim protein–protein interaction (PPI) contributes to the adaptation of the Bcl‐2 network. Experimental validation was made by using a specific inhibitor of Hsp70‐Bim PPI, S1g‐2, as chemical tool. Bifurcation analysis and stochastic simulation were used for the theoretical study of the impact of Hsp70‐Bim PPI on cell‐fate heterogeneity and factional killing.Key Results: The Hsp70‐Bim‐AKT circuit forms an IFFL that greatly contributes to the adaptation of the Bcl‐2‐regulated apoptosis network, thus leading to fractional killing. This adaptive programme enhances noise‐induced cell‐fate heterogeneity by shifting from a saddle‐node to a saddle‐collision transition scenario.Conclusion and Implications: Hsp70‐Bim IFFL serves as a molecular pathway induced by DNA damaging drugs or tyrosine kinase inhibitors that enabled fractional killing, whereby acquired resistance emerges. A synergistic strategy is unveiled for overcoming fractional killing by suppressing Hsp70‐Bim PPI.

Mitophagy, a form of selective autophagy, plays an essential role to maintain a population of healthy and functional mitochondria for normal cellular metabolism. Acting mainly as one of the B-cell lymphoma 2 (Bcl-2) family pro-apoptotic members, Bim (also known as BCL2L11) was identified to be a co-chaperone of Hsp70 to promote mitophagy in mammalian cells. Herein, with the help of a specific Hsp70/Bim disruptor and Om45-GFP processing assay, we illustrated that ectopic BimEL is able to promote mitophagy through Hsp70/Bim interaction in yeast, where Bax/Bak is absent. The Hsp70/Bim-mediated mitophagy is conserved in eukaryotes, from yeast to humans.

Background: For cancer therapy, the identification of both selective autophagy targets and small molecules that specifically regulate autophagy is greatly needed. Heat shock protein 70 (Hsp70) is a recently discovered BH3 receptor that forms a protein‒protein interaction (PPI) with Bcl-2-interacting mediator of cell death (Bim). Herein, a specific inhibitor of the Hsp70-Bim PPI, S1g-2, and its analog S1, which is a Bcl-2-Bim disruptor, were used as chemical tools to explore the role of Hsp70-Bim PPI in regulating mitophagy. Methods: Co-immunoprecipitation and immunofluorescence assays were used to determine protein interactions and colocalization patterns. Organelle purification and immunodetection of LC3-II/LC3-I on mitochondria, endoplasmic reticulum (ER) and Golgi were applied to identify specific types of autophagy. Cell-based and in vitro ubiquitination studies were used to study the role of the Hsp70-Bim PPI in parkin-mediated ubiquitination of outer mitochondrial membrane 20 (TOMM20). Results: We found that after the establishment of their PPI, Hsp70 and Bim form a complex with parkin and TOMM20, which in turn facilitates parkin translocation to mitochondria, TOMM20 ubiquitination and mitophagic flux independent of Bax/Bak. Moreover, S1g-2 selectively inhibits stress-induced mitophagy without interfering with basal autophagy. Conclusions: The findings highlight the dual protective function of the Hsp70-Bim PPI in regulating both mitophagy and apoptosis. S1g-2 is thus a newly discovered antitumor drug candidate that drives both mitophagy and cell death via apoptosis. Graphical

Background: Inhibitors of B‐cell CLL/lymphoma 2 (Bcl‐2) family proteins have shown hope as antitumor drugs. While the notion that it is efficient to coordinate, balance, and neutralize both arms of the anti‐apoptotic Bcl‐2 family has been validated in many cancer cells, the weights of the two arms contributing to apoptosis inhibition have not been explored. This study analyzed the best combination ratio for different Bcl‐2 selective inhibitors.Methods: We used a previously established mathematical model to study the weights of Bcl‐2 (representing both Bcl‐2 and Bcl‐xL in this study) and myeloid cell leukemia‐1 (Mcl‐1). Correlation and single‐parameter sensitivity analysis were used to find the major molecular determinants for Bcl‐2 and Mcl‐1 dependency, as well as their weights. Biological experiments were used to verify the mathematical model.Results: Bcl‐2 protein level and Mcl‐1 protein level, production, and degradation rates were the major molecular determinants for Bcl‐2 and Mcl‐1 dependency. The model gained agreement with the experimental assays for ABT‐737/A‐1210477 and ABT‐737/compound 5 combination effect in MCF‐7 and MDA‐MB‐231. Two sets of equations composed of Bcl‐2 and Mcl‐1 levels were obtained to predict the best combination ratio for Bcl‐2 inhibitors with Mcl‐1 inhibitors that stabilize and downregulate Mcl‐1, respectively.Conclusions: The two sets of equations can be used as tools to bypass time‐consuming and laborious experimental screening to predict the best drug combination ratio for treatment.