Chemotherapy impairs skeletal muscle mitochondrial homeostasis in early breast cancer patients

Joris Mallard, Elyse Hucteau, Anne-Laure Charles, Laura Bender, Claire Baeza, Mathilde Pélissie, Philippe Trensz, Carole Pflumio, Michal Kalish-Weindling, Bernard Gény, Roland Schott, Fabrice Favret, Xavier Pivot, Thomas J Hureau, Allan F Pagano, Joris Mallard, Elyse Hucteau, Anne-Laure Charles, Laura Bender, Claire Baeza, Mathilde Pélissie, Philippe Trensz, Carole Pflumio, Michal Kalish-Weindling, Bernard Gény, Roland Schott, Fabrice Favret, Xavier Pivot, Thomas J Hureau, Allan F Pagano

Abstract

Background: Chemotherapy is extensively used to treat breast cancer and is associated with skeletal muscle deconditioning, which is known to reduce patients' quality of life, treatment efficiency, and overall survival. To date, skeletal muscle mitochondrial alterations represent a major aspect explored in breast cancer patients; nevertheless, the cellular mechanisms remain relatively unknown. This study was dedicated to investigating overall skeletal muscle mitochondrial homeostasis in early breast cancer patients undergoing chemotherapy, including mitochondrial quantity, function, and dynamics.

Methods: Women undergoing (neo)adjuvant anthracycline-cyclophosphamide and taxane-based chemotherapy participated in this study (56 ± 12 years). Two muscle biopsies were collected from the vastus lateralis muscle before the first and after the last chemotherapy administration. Mitochondrial respiratory capacity, reactive oxygen species production, and western blotting analyses were performed.

Results: Among the 11 patients, we found a decrease in key markers of mitochondrial quantity, reaching -52.0% for citrate synthase protein levels (P = 0.02) and -38.2% for VDAC protein levels (P = 0.04). This mitochondrial content loss is likely explained by reduced mitochondrial biogenesis, as evidenced by a decrease in PGC-1α1 protein levels (-29.5%; P = 0.04). Mitochondrial dynamics were altered, as documented by a decrease in MFN2 protein expression (-33.4%; P = 0.01), a key marker of mitochondrial outer membrane fusion. Mitochondrial fission is a prerequisite for mitophagy activation, and no variation was found in either key markers of mitochondrial fission (Fis1 and DRP1) or mitophagy (Parkin, PINK1, and Mul1). Two contradictory hypotheses arise from these results: defective mitophagy, which probably increases the number of damaged and fragmented mitochondria, or a relative increase in mitophagy through elevated mitophagic potential (Parkin/VDAC ratio; +176.4%; P < 0.02). Despite no change in mitochondrial respiratory capacity and COX IV protein levels, we found an elevation in H2 O2 production (P < 0.05 for all substrate additions) without change in antioxidant enzymes. We investigated the apoptosis pathway and found an increase in the protein expression of the apoptosis initiation marker Bax (+72.0%; P = 0.04), without variation in the anti-apoptotic protein Bcl-2.

Conclusions: This study demonstrated major mitochondrial alterations subsequent to chemotherapy in early breast cancer patients: (i) a striking reduction in mitochondrial biogenesis, (ii) altered mitochondrial dynamics and potential mitophagy defects, (iii) exacerbated H2 O2 production, and (iv) increased initiation of apoptosis. All of these alterations likely explain, at least in part, the high prevalence of skeletal muscle and cardiorespiratory deconditioning classically observed in breast cancer patients.

Keywords: Apoptosis; H2O2; Mitochondrial biogenesis; Mitochondrial dynamics; Mitophagy; Skeletal muscle deconditioning.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

© 2022 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.

Figures

Figure 1
Figure 1
Flow chart of the participants.
Figure 2
Figure 2
Changes in key markers of mitochondrial quantity and mitochondrial biogenesis. Citrate synthase (A), VDAC (B), and PGC‐1α1 (C) protein levels from vastus lateralis muscle biopsies taken before (pre‐CT) and after (post‐CT) breast cancer chemotherapy (CT). Above each panel, western blots from two representative subjects are displayed. All values are expressed as the mean ± SD (N = 11). *P < 0.05.
Figure 3
Figure 3
Changes in protein markers of mitochondrial dynamics and mitophagy. MFN2 (A); MFN1 85 and 71 kDa isoforms (B); OPA1 isoforms: L, S1, S2, and S3 (C); Fis1 (D); p‐DRP1 (Ser616)/DRP1 (E); Mul1 (F); Parkin (G); PINK1 (H); and Parkin/VDAC (I) protein levels from vastus lateralis muscle biopsies taken before (pre) and after (post) breast cancer chemotherapy (CT). Above each panel, western blots from two representative subjects are displayed. All values are expressed as the mean ± SD (N = 11). *P < 0.05.
Figure 4
Figure 4
Changes in mitochondrial respiration, reactive oxygen species production, and redox signalling protein markers. Mitochondrial respiratory capacity quantification (A), and associated mean representative curves (B), for CI‐linked substrate state, OXPHOS by CI, OXPHOS by CI&CII, and OXPHOS by CII on permeabilized fibres from vastus lateralis muscle biopsies taken before (pre) and after (post) breast cancer chemotherapy (CT). COX IV (C) protein levels in pre‐CT vs. post‐CT. H2O2 production quantification (D), and associated mean representative curves (E), for CI‐linked substrate state, OXPHOS by CI, OXPHOS by CI&CII, and OXPHOS by CII in pre‐CT vs. post‐CT permeabilized fibres. Superoxide anion measurement (F) through electron paramagnetic resonance in pre‐CT vs. post‐CT minced muscle samples. 4‐HNE (G), total carbonylated (H), SOD2 (I), catalase (J), GPx1 (K), p‐eIF2α (Ser51)/eIF2α (L), p‐p38 MAPK (Thr180/Tyr182)/p38 MAPK (M) and p‐p53 (Ser15)/p53 (N) protein levels in pre‐CT vs. post‐CT. Above each panel with western blot analyses, two representative subjects are displayed. All values are expressed as the mean ± SD (N = 11). *P < 0.05; **P < 0.01; ***P < 0.001; CI: complex I; CII: complex II; OXPHOS: oxidative phosphorylation.
Figure 5
Figure 5
Changes in protein markers of apoptosis induction. Bax (A), Bcl‐2 (B), and active/pro‐caspase 3 (C) protein levels from vastus lateralis muscle biopsies taken before (pre) and after (post) breast cancer chemotherapy (CT). Above each panel, western blots from two representative subjects are displayed. All values are expressed as the mean ± SD (N = 11). *P < 0.05.
Figure 6
Figure 6
Summary of the mitochondrial alterations observed in early breast cancer patients undergoing chemotherapy. Through the analysis of vastus lateralis muscle biopsies taken before (pre) and after (post) breast cancer chemotherapy (CT), we identified an increased mitochondrial loss (grey box) that may be explained by a decrease in PGC‐1α1 protein levels, reflecting a reduced mitochondrial biogenesis process (in blue). Mitochondrial dynamics (in red) were also impaired, as evidenced by a dysregulation in the fusion process, mediated by a decrease in the mitochondrial outer membrane fusion marker MFN2, without any change in MFN1 and OPA1. No change was observed in fission markers (Fis1 and DRP1), indicating a dysregulation in the mitochondrial fusion/fission balance. These alterations are likely leading to the accumulation of fragmented and damaged mitochondria (rose‐coloured mitochondria), corroborated by the accumulation of S3‐OPA1 isoform. As a consequence, fragmented and damaged mitochondria accumulate in skeletal muscle and produce excessive amounts of reactive oxygen species such as H2O2 (in orange). As mentioned above, we found no change in mitochondrial fission, a prerequisite step for the activation of mitophagy. Despite the absence of change in the expression of different mitophagy markers (Parkin, PINK1, and Mul1; in green), two contradictory hypotheses arise from these results: defective mitophagy (Hypothesis 1) or a relative increase in mitophagy (increased mitophagic potential reflected by the Parkin/VDAC ratio; Hypothesis 2). Antioxidant enzyme (in orange), lipid peroxidation (in brown), and carbonylated protein levels (in yellow) were unchanged. However, we found a large increase in Bax protein levels, a proapoptotic protein that initiates apoptosis, and no change was observed for Bcl‐2, its antiapoptotic counterpart (in black). While several pre‐clinical studies found increased levels in the active (cleaved)‐caspase 3/pro‐caspase 3 ratio, the slight increase observed in our study did not reach significance (in black dotted line).

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