Metabolic Stress Remodeling: Insights into the Role(s) of FOXO1 in Promoting Cancer-Like Changes in Systemic Sclerosis (SSc)

Objectives Systemic sclerosis (SSc) is a life-threatening autoimmune disease with limited treatment options. Autologous stem cell transplantation (ASCT) is the only disease-modifying therapy in SSc; however, its effects on fibroblasts are unknown. We have recently shown that dermal fibroblasts (DFs) from patients with diffuse cutaneous SSc (dSSc) develop a cancer-like phenotype characterized by genomic instability and increased double-stranded DNA breaks (DSBs) associated with resistance-to-apoptosis (Figure 1). In cancer, this is promoted by mitochondrial-dependent metabolic remodelling which is activated by the transcription factor forkhead Box 1 (FOXO1). We hypothesized that metabolic remodeling in dSSc DFs may promote resistance-to-apoptosis via FOXO1, which normalizes post-ASCT. Figure 1. Graphical abstract : In fibroblasts from patients with severe diffuse systemic sclerosis (dSSc), mitochondrial dysfunction results in excessive production of reactive oxygen species (ROS), leading to metabolic stress (1). The elevated ROS levels promotes DNA breaks (2). which activate the transcription factor FOXO1. Upon activation. FOXO1 translocates to the nucleus (3) and binds to the promoter of superoxide dismutase 2 (SOD2) and pyruvate dehydrogenase kinase 4 (PDK4). SOD2 then translocates to the mitochondria, where it reduces mitochondrial ROS into diffusible hydrogen peroxide (H2O2). FOXO1 also promotes metabolic remodelling through PDK4 to increase resistance to apoptosis, and further exacerbate mitochondrial dysfunction. Additionally, nuclear FOXO1 binds to the promoters of fibrotic mediators, driving fibrosis (4). This cycle of mitochondrial dysfunction and FOXOI activation ultimately promotes genomic Instability, senescence and increased ROS (all of which are key pathogenic mechanisms in progressive forms of SSc). This maladaptive mechanism is normalized following ASCT. Methods DFs were generated from healthy control volunteers (HC), limited cutaneous SSc (lSSc), dSSc and post-ASCT (~12 months) patients using 4 mm skin biopsies (N=6-10/group). We quantified the frequency of DSBs and resistance-to-apoptosis via γ-H2AX and/or TUNEL/cleaved caspase 3 (+/− treatment with cyclophosphamide or a FOXO1-inhibitor), respectively. FOXO1 activation was determined by measuring nuclear (active) FOXO1, and expression of its downstream mRNA targets SOD2 and PDK4. Mitochondrial morphology was assessed using confocal microscopy. We also assessed changes in mitochondrial functions, namely mitochondrial dynamics (fusion/fission, immunoblot), biogenesis (qRT-PCR), electron transport chain (ETC) genes (qRT-PCR), and oxygen consumption (XFlux Seahorse analysis). Results dSSc DF had the highest frequency of DSBs compared to HC, lSSc and post-ASCT. This was associated with increased resistance-to-apoptosis in response to cyclophosphamide, with associated activation of FOXO1. Notably, pharmacological inhibition of FOXO1 in dSSc DF resulted in decreased indicators associated with fibrosis and increased apoptosis. In addition, dSSc DF had an increased frequency of elongated mitochondria and indicators associated with mitochondrial fusion (e.g. OPA1, phospho-DRP1), mitochondrial biogenesis, ETC expression with reduced oxygen consumption rate. Overexpression of constitutively active FOXO1 in HC DF resulted in similar metabolic changes as seen in dSSc. Finally, post-ASCT DF did not have increased DSB, FOXO1 activation, or metabolic reprogramming - suggesting that ASCT may provide some of its beneficial effects by modulating this novel mitochondrial/FOXO1 axis. Conclusion Our study highlights the critical role of metabolic stress remodeling as a driver of the cancer-like phenotype in severe forms of SSc.[1] It also provides mechanistic insights related to how ASCT may impart some of its beneficial effects. Future studies targeting this pathway may implicate this novel mitochondrial/FOXO1 pathway as a novel therapeutic strategy in patients with SSc. [1.] Gniadecki R. J Autoimmun 2022;131:102847.

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