RESUMEN
Human cancer cells display extensive heterogeneity in their sensitivities to the proteasome inhibitor bortezomib (Velcade). The molecular mechanisms underlying this heterogeneity remain unclear, and strategies to overcome resistance are limited. Here, we discover that inherent differences in eIF2α phosphorylation among a panel of ten human pancreatic cancer cell lines significantly impacts bortezomib sensitivity, and implicate the HRI (heme-regulated inhibitor) eIF2α kinase as a novel therapeutic target. Within our panel, we identified a subset of cell lines with defective induction of eIF2α phosphorylation, conferring a high degree of sensitivity to bortezomib. These bortezomib-sensitive cells exhibited impaired translation attenuation followed by toxic accumulation of protein aggregates and reactive oxygen species (ROS), whereas the bortezomib-resistant cell lines displayed increased phosphorylation of eIF2α, decreased translation, few protein aggregates, and minimal ROS production. Importantly, we identified HRI as the primary bortezomib-activated eIF2α kinase, and demonstrated that HRI knockdown promoted cell death in the bortezomib-resistant cells. Overall, our data implicate inducible HRI-mediated phosphorylation of eIF2α as a central cytoprotective mechanism following exposure to bortezomib and provide proof-of-concept for the development of HRI inhibitors to overcome proteasome inhibitor resistance.
Asunto(s)
Bortezomib/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Inhibidores de Proteasoma/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Factor 2 Eucariótico de Iniciación/genética , Humanos , Fosforilación/efectos de los fármacos , Fosforilación/genética , Biosíntesis de Proteínas/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) are a promising human cardiac model system for drug development and toxicity screening, along with cell therapy and mechanistic research. The scalable differentiation of human PSCs into CMs provides a renewable cell source that overcomes species differences present in rodent primary CMs. In addition, induced pluripotent stem cell (iPSC) technology allows for development of patient-specific CMs, representing a valuable tool that may lead to better prediction, prevention, and treatment of cardiovascular diseases in this new era of precision medicine. However, the utility of PSC-CMs as an in vitro model is currently limited by their immature phenotype when compared to adult CMs. Recent work has identified microRNAs (miRNAs) as critical regulators of heart development and function. These studies have shown that miRNAs are essential to key processes that span the life cycle of a cardiomyocyte, including proliferation, hypertrophy, beating rhythm, and apoptosis. Importantly, emerging evidence strongly suggests that modulation of select miRNAs can enhance the maturation of PSC-CMs. Here, we review key miRNAs associated with heart development and function, and discuss strategies to promote PSC-CM maturation, focusing on current knowledge surrounding miRNA-based approaches and the application of PSC-CMs with respect to drug screening and disease models. Ultimately, it is likely that combinations of both miRNA and non-miRNA maturation strategies may collectively provide the best path forward for producing mature cardiomyocytes in vitro.
Asunto(s)
Cardiotoxicidad , Diferenciación Celular , MicroARNs/genética , Modelos Cardiovasculares , Miocitos Cardíacos/citología , Células Madre Pluripotentes/citología , Adulto , Humanos , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/metabolismoRESUMEN
The proteasome inhibitor bortezomib (Velcade) is a promising new agent for bladder cancer therapy, but inducible cytoprotective mechanisms may limit its potential efficacy. We used whole genome mRNA expression profiling to study the effects of bortezomib on stress-induced gene expression in a panel of human bladder cancer cell lines. Bortezomib induced strong upregulation of the inducible HSP70 isoforms HSPA1A and HSPA1B isoforms of Hsp72 in 253J B-V and SW780 (HSPA1A(high)) cells, but only induced the HSPA1B isoform in UM-UC10 and UM-UC13 (HSPA1A(low)) cells. Bortezomib stimulated the binding of heat shock factor-1 (HSF1) to the HSPA1A promoter in 253JB-V but not in UM-UC13 cells. Methylation-specific PCR revealed that the HSPA1A promoter was methylated in the HSPA1A(low) cell lines (UM-UC10 and UM-UC13), and exposure to the chromatin demethylating agent 5-aza-2'-deoxycytidine restored HSPA1A expression. Overexpression of Hsp72 promoted bortezomib resistance in the UM-UC10 and UM-UC13 cells, whereas transient knockdown of HSPA1B further sensitized these cells to bortezomib, and exposure to the chemical HSF1 inhibitor KNK-437 promoted bortezomib sensitivity in the 253J B-V cells. Finally, shRNA-mediated stable knockdown of Hsp72 in 253J B-V promoted sensitivity to bortezomib in vitro and in tumor xenografts in vivo. Together, our results provide proof-of-concept for using Hsp72 inhibitors to promote bortezomib sensitivity in bladder cancers and suggest that selective targeting of HSPA1B could produce synthetic lethality in tumors that display HSPA1A promoter methylation.