Expression of Mitochondrial Dysfunction-Related Genes And

Research Article Molecular Pain Volume 14: 1–16 Expression of mitochondrial ! The Author(s) 2018 Article reuse guidelines: dysfunction-related genes and sagepub.com/journals-permissions DOI: 10.1177/1744806918816462 pathways in paclitaxel-induced peripheral journals.sagepub.com/home/mpx neuropathy in breast cancer survivors Kord M Kober1 , Adam Olshen2, Yvettte P Conley3, Mark Schumacher2, Kimberly Topp2, Betty Smoot2, Melissa Mazor1, Margaret Chesney2, Marilyn Hammer4, Steven M Paul1, Jon D Levine2, and Christine Miaskowski1 Abstract Background: Paclitaxel is one of the most commonly used drugs to treat breast cancer. Its major dose-limiting toxicity is paclitaxel-induced peripheral neuropathy (PIPN). PIPN persists into survivorship and has a negative impact on patient’s mood, functional status, and quality of life. No interventions are available to treat PIPN. A critical barrier to the development of efficacious interventions is the lack of understanding of the mechanisms that underlie PIPN. Mitochondrial dysfunction has been evaluated in preclinical studies as a hypothesized mechanism for PIPN, but clinical data to support this hypothesis are limited. The purpose of this pilot study was to evaluate for differential gene expression and perturbed pathways between breast cancer survivors with and without PIPN. Methods: Gene expression in peripheral blood was assayed using RNA-seq. Differentially expressed genes (DEG) and pathways associated with mitochondrial dysfunction were identified between survivors who received paclitaxel and did (n ¼ 25) and did not (n ¼ 25) develop PIPN. Results: Breast cancer survivors with PIPN were significantly older; more likely to be unemployed; reported lower alcohol use; had a higher body mass index and poorer functional status; and had a higher number of lower extremity sites with loss of light touch, cold, and pain sensations and higher vibration thresholds. No between-group differences were found in the cumulative dose of paclitaxel received or in the percentage of patients who had a dose reduction or delay due to PIPN. Five DEGs and nine perturbed pathways were associated with mitochondrial dysfunction related to oxidative stress, iron homeostasis, mitochondrial fission, apoptosis, and autophagy. Conclusions: This study is the first to provide molecular evidence that a number of mitochondrial dysfunction mechanisms identified in preclinical models of various types of neuropathic pain including chemotherapy-induced peripheral neuropathy are found in breast cancer survivors with persistent PIPN and suggest genes for validation and as potential therapeu- tic targets. Keywords Taxanes, mitochondria, neuropathy, breast cancer, survivor, paclitaxel, differential gene expression, pathway analysis Date Received: 29 June 2018; revised: 12 October 2018; accepted: 16 October 2018 1School of Nursing, University of California, San Francisco, San Francisco, CA, USA 2School of Medicine, University of California, San Francisco, San Francisco, CA, USA Introduction 3School of Nursing, University of Pittsburgh, Pittsburgh, PA, USA 4 Paclitaxel is one of the most commonly used drugs to Department of Nursing, Mount Sinai Medical Center, New York, NY, USA treat breast, ovarian, and lung cancers.1 Its major dose- Corresponding Author: Kord M Kober, Department of Physiological Nursing, University of limiting toxicity is paclitaxel-induced peripheral neurop- California, 2 Koret Way-N631Y, San Francisco, CA 94143, USA. athy (PIPN). Prevalence rates for PIPN are extremely Email: [email protected] Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution- NonCommercial 4.0 License (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us. sagepub.com/en-us/nam/open-access-at-sage). 2 Molecular Pain high, ranging from 59% to 87%.2,3 PIPN is character- Materials and methods ized by paresthesias and dysesthesias that occur in a “stocking-glove” distribution. PIPN persists into the Survivors and settings cancer survivorship period and has a negative impact The methods for this study, which is part of a larger on individuals’ mood, functional status, and quality of 14 4 study, are described in detail elsewhere. In brief, sur- life (QOL). In particular, the negative impact of PIPN vivors were recruited from throughout the San Francisco on breast cancer (BC) survivors has been identified as a 5,6 Bay area and met prespecified inclusion and exclusion gap in QOL for BC patients. criteria (see Supplemental Material). The National Paclitaxel exerts its primary antitumor effects by Coalition for Cancer Survivorship’s definition of binding to beta-tubulin in microtubules, forming cancer survivor was used in this study (i.e., a person is extremely stable and nonfunctional microtubules that 7 a cancer survivor from the moment of diagnosis through results in apoptosis. In terms of its neurotoxic effects, the balance of life).15 Of the 1450 survivors who were the exact mechanisms that underlie the development of screened, 754 were enrolled and 623 completed the self- PIPN remain unclear. However, several lines of evidence report questionnaires and the study visit. Data from a suggest that interruption of microtubule function in randomly selected sample of BC survivors with (n ¼ 25) neuron impairs axonal transport and results in a dying and without (n ¼ 25) chronic PIPN were used in back neuropathy. In addition, paclitaxel alters mito- this analysis. chondrial function through the depletion of mRNAs that encode the mitochondrial fission/fusion machinery in distal axons.8 This toxic effect leads to a deficit in Study procedures axonal energy supply and subsequent axonal Research nurses screened and consented the survivors degeneration.9,10 over the phone, sent and asked them to complete the Using the National Cancer Institute’s Common self-report questionnaires prior to their study visit, and Terminology Criteria for Adverse Events, PIPN of scheduled the in-person assessment. At this assessment, grades 2 to 4 occurs at cumulative doses of between written informed consent was obtained, questionnaires 715 and 1500 mg/m2.11 However, not all patients who were reviewed for completeness, and objective measure- receive this dose of paclitaxel develop PIPN. The wide ments were done. Blood samples were drawn, processed, range in occurrence rates suggests that genetic factors and stored for subsequent molecular analyses in may play a role in the development of PIPN. While PAXgeneVR Blood RNA tubes (Qiagen, Inc.). This preclinical studies suggest that a number of mecha- study was approved by the institutional review board nisms are involved in the development of PIPN,9,10,12 of the University of California, San Francisco. the majority of the genetic association studies of PIPN evaluated for polymorphisms in candidate Study measures genes that influence the metabolism and transport of neurotoxic drugs. As noted in a recent systematic Demographic and clinical characteristics. Survivors provided review and meta-analysis of the molecular genetics of information on demographic characteristics and completed the Alcohol Use Identification Test (AUDIT),16 chemotherapy-induced peripheral neuropathy 17–19 13 Karnofsky Performance Status (KPS) Scale, and the (CIPN), notable methodological issues, including 20,21 lack of standardization and detail in the phenotype Self-Administered Comorbidity Questionnaire. definition and acknowledgment of potential confound- ing factors, prevented the authors from identifying any Pain measures. Survivors with PIPN rated the intensity of candidate genes that were associated with CIPN in their pain using a 0 to 10 numeric rating scale and com- general or PIPN specifically. pleted the pain interference scale from the Brief Pain 22 No studies were identified that evaluated for associ- Inventory. The qualities of PIPN were evaluated 23,24 ations between changes in the expression of genes using the Pain Quality Assessment Scale. involved in mitochondrial function and chronic PIPN in cancer survivors. Given the preclinical evidence that Objective measures of sensation. Light touch was evaluated paclitaxel alters mitochondrial function in primary using Semmes Weinstein monofilaments.25 Cold sensa- afferent neurons,9,10 the purpose of this study was to tion was evaluated using the Tiptherm Rod.26,27 Pain identify differentially expressed mitochondrial dysfunc- sensation was evaluated using the Neurotip.27 tion (MD)-related genes and perturbed pathways in BC Vibration threshold was assessed using a vibrometer.28 survivors with (n ¼ 25) and without (n ¼ 25) chron- For all of the measures of sensation, both the upper and ic PIPN. lower extremities on the dominant side were tested. Kober et al. 3 Balance. Self-report questions from the CIPN basecall files (bclfiles) were demultiplexed and converted Assessment Tool were used to assess the balance.29 into an FASTQ file format using the bcl2fastq v2.17 The objective measures of balance were the timed get software (Illumina Inc., San Diego, CA). Data were up and go test (TUG)30 and the Fullerton Advanced posted and retrieved from a secured site hosted by the Balance (FAB) test.31,32 Core Facility. Phenotypic data analysis RNA-seq alignment, data processing, and Data were analyzed using SPSS version 23.33 Descriptive quality control

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