A high-throughput screening approach to discover potential colorectal cancer chemotherapeutics: Repurposing drugs to disrupt 14-3-3 protein-BAD interactions.

Inducing apoptosis in different types of cancer cells is an effective therapeutic strategy. However, the success of existing chemotherapeutics can be compromised by tumor cell resistance and systemic off-target effects. Therefore, the discovery of pro-apoptotic compounds with minimal systemic side-effects is crucial. 14-3-3 proteins are molecular scaffolds that serve as important regulators of cell survival. Our previous study demonstrated that 14-3-3ζ can sequester BAD, a pro-apoptotic member of the BCL-2 protein family, in the cytoplasm and prevent its translocation to mitochondria to inhibit the induction of apoptosis. Despite being a critical mechanism of cell survival, it is unclear whether disrupting 14-3-3 protein:BAD interactions could be harnessed as a chemotherapeutic approach. Herein, we established a BRET-based high-throughput drug screening approach (Z'-score= 0.52) capable of identifying molecules that can disrupt 14-3-3ζ:BAD interactions. An FDA-approved drug library containing 1971 compounds was used for screening, and the capacity of identified hits to induce cell death was examined in NIH3T3-fibroblasts and colorectal cancer cell lines, HT-29 and Caco-2. Our in vitro results suggest that terfenadine, penfluridol, and lomitapide could be potentially repurposed for treating colorectal cancer. Moreover, our screening method demonstrates the feasibility of identifying pro-apoptotic agents that can be applied towards conditions where aberrant cell growth or function are key determinants of disease pathogenesis.

A single-cell atlas of de novo ß-cell regeneration reveals the contribution of hybrid ß/δ-cells to diabetes recovery in zebrafish.

Regeneration-competent species possess the ability to reverse the progression of severe diseases by restoring the function of the damaged tissue. However, the cellular dynamics underlying this capability remain unexplored. Here, we have used single-cell transcriptomics to map de novo ß-cell regeneration during induction and recovery from diabetes in zebrafish. We show that the zebrafish has evolved two distinct types of somatostatin-producing δ-cells, which we term δ1- and δ2-cells. Moreover, we characterize a small population of glucose-responsive islet cells, which share the hormones and fate-determinants of both ß- and δ1-cells. The transcriptomic analysis of ß-cell regeneration reveals that ß/δ hybrid cells provide a prominent source of insulin expression during diabetes recovery. Using in vivo calcium imaging and cell tracking, we further show that the hybrid cells form de novo and acquire glucose-responsiveness in the course of regeneration. The overexpression of dkk3, a gene enriched in hybrid cells, increases their formation in the absence of ß-cell injury. Finally, interspecies comparison shows that plastic δ1-cells are partially related to PP cells in the human pancreas. Our work provides an atlas of ß-cell regeneration and indicates that the rapid formation of glucose-responsive hybrid cells contributes to the resolution of diabetes in zebrafish.

Leader ß-cells coordinate Ca2+ dynamics across pancreatic islets in vivo.

Pancreatic ß-cells form highly connected networks within isolated islets. Whether this behaviour pertains to the situation in vivo, after innervation and during continuous perfusion with blood, is unclear. In the present study, we used the recombinant Ca2+ sensor GCaMP6 to assess glucose-regulated connectivity in living zebrafish Danio rerio, and in murine or human islets transplanted into the anterior eye chamber. In each setting, Ca2+ waves emanated from temporally defined leader ß-cells, and three-dimensional connectivity across the islet increased with glucose stimulation. Photoablation of zebrafish leader cells disrupted pan-islet signalling, identifying these as likely pacemakers. Correspondingly, in engrafted mouse islets, connectivity was sustained during prolonged glucose exposure, and super-connected 'hub' cells were identified. Granger causality analysis revealed a controlling role for temporally defined leaders, and transcriptomic analyses revealed a discrete hub cell fingerprint. We thus define a population of regulatory ß-cells within coordinated islet networks in vivo. This population may drive Ca2+ dynamics and pulsatile insulin secretion.