Research paperProteomic analysis of human iPSC-derived sensory neurons implicates cell stress and microtubule dynamics dysfunction in bortezomib-induced peripheral neurotoxicity
Introduction
Chemotherapy-induced peripheral neuropathy (CIPN) affects over 1/3 of the patient population undergoing treatment for cancer and its prevalence is anticipated to rise with improvements in survival rates (Shah et al. 2018). Symptoms of CIPN typically begin during the first 2 months of treatment and become more severe throughout its duration. Countermeasures are limited to dose reduction or a discontinuation of chemotherapy treatment, underscoring the need both to better understand the central pathobiology and to improve our repertoire of therapeutic agents (Staff et al., 2017).
The majority of CIPN signs and symptoms arise from damage to dorsal root ganglion (DRG) neurons or their axons in the form of a dying-back neuropathy, seemingly initiated through defects in axon transport, altered mitochondrial function, or altered calcium homeostasis (Staff et al., 2017), (Meregalli 2015). Although there are different mechanisms by which chemotherapeutic agents cause this effect, it is not entirely clear how proteasome inhibitors such as bortezomib facilitate axonal degeneration. Bortezomib inhibits the activity of the 26S proteasome, which recognizes ubiquinated proteins and selectively targets them for degradation in order to maintain cellular homeostasis. In tumorigenic cells, proteasome inhibitors lead to the accumulation of damaged and unfolded proteins, which ultimately results in cell cycle arrest and the upregulation of pro-apoptotic signaling proteins (Dou and Zonder 2014). Nontumorigenic cells rely on protein kinase B (Akt) upregulation, the endoplasmic reticulum (ER) stress response, the unfolded protein response (UPR), and autophagy to mitigate bortezomib's cytotoxic effects, and the degree to which any of these mechanisms are applicable to bortezomib neurotoxicity remains to be established.
Preclinical studies that yielded encouraging results for the prevention of CIPN failed in clinical trials and reinforced the need to refine our understanding of chemotherapeutic response (Fukuda et al. 2017). Towards that end, technological advancements in the stem cell field now allow for us to create iPSCs (induced pluripotent stem cells) from patients, and the directed differentiation of these cells into various tissue-specific subtypes has reliably recapitulated many disease phenotypes as well as drug intolerances that are not as readily detected in rodents or other species.
Due to the multifaceted impact of proteasome inhibition on cellular function, we employed a proteomics study to identify key processes responsible for bortezomib-induced neurotoxicity. We opted to investigate the proteomics signature due the discordance between RNA and protein levels (Wiita et al. 2013), (Rendleman et al. 2018). The study was performed on human iPSC-derived sensory neurons (iSNs) exposed to a clinically relevant dose of bortezomib to create a proteomics dataset that resembles the in vivo proteotoxicity of bortezomib.
Section snippets
Human iPSC cell culture and maintenance, iSN differentiation, and bortezomib treatment
Three iPSC lines created from the skin biopsies of individuals with no known health concerns were acquired from the Mayo Clinic Biotrust according to IRB approved guidelines. Fibroblasts were reprogrammed using Cytotune-iPS 2.0 (courtesy of Regen Theranostics) and karyotypically normal iPSC clones for each line that passed quality control testing for pluripotency and germ layer differentiation were selected. The lines used in this study were from 2 females (21-year-old, 6BT1; 77-year-old, 1BT1)
Bortezomib induces neurotoxic effects in iSNs
To ensure that the dose and duration of bortezomib exposure was appropriate for this model system, we compared the iSN model to our previous studies in rat E15 DRG neurons. A 24 h bortezomib exposure led to the accumulation of β3-tubulin (Tuj-1) proximal to the plasma membrane in the somata of rat E15 DRG neurons (Staff et al., 2013). This same effect was observed in iSNs at both 24 h and 48 h (Fig. 1A). Another similarity shared by both models was that bortezomib exposure had no effect on
Discussion
The central focus of this study was to better characterize the molecular underpinnings that predispose sensory neurons to bortezomib-induced neurotoxicity. Our approach was to use a model system based on iPSCs to recapitulate bortezomib's effects on human iSNs. Although iPSC-derived models possess immature characteristics, they have proven to be a useful model system for studying chemotherapeutic drug responses. Our iSN model reproduced a cascade of effects that have been observed in other
Acknowledgements
This publication was made possible by support from the Medical Genome Facility Proteomics Core, Mayo Clinic. The core is a shared resource of the Mayo Clinic Cancer Center (NCI P30 CA15083). We thank Dr. Cristine Charlesworth for her guidance on sample preparation for MS analysis.
NPS and SA conceived the study. NPS, SA, SCLH, and RFH contributed to experimental design. Experiments were completed by SA, RFH, SCLH, BA, JPK, BN, and IS. SCLH, RAM, NPS, and SD completed analyses of the
Funding
This work was supported by the National Institutes of Health [NPS: R01 CA211887] and the Mayo Foundation for Medical Education and Research.
Disclosure statement
The authors have no conflicting interests.
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