MUSCULOSKELETAL DISEASE Smad7 gene delivery prevents muscle wasting associated with cancer cachexia in mice Catherine E. Winbanks, 1 Kate T. Murphy, 2 Bianca C. Bernardo, 1 Hongwei Qian, 1 Yingying Liu, 1 Patricio V. Sepulveda, 1 Claudia Beyer, 1 Adam Hagg, 1 Rachel E. Thomson, 1 Justin L. Chen, 1,3 Kelly L. Walton, 3 Kate L. Loveland, 4 Julie R. McMullen, 1,5,6 Buel D. Rodgers, 7 Craig A. Harrison, 3,4,6 Gordon S. Lynch, 2 Paul Gregorevic 1,2,4,8 * Patients with advanced cancer often succumb to complications arising from striated muscle wasting associated with cachexia. Excessive activation of the type IIB activin receptor (ActRIIB) is considered an important mech- anism underlying this wasting, where circulating procachectic factors bind ActRIIB and ultimately lead to the phosphorylation of SMAD2/3. Therapeutics that antagonize the binding of ActRIIB ligands are in clinical devel- opment, but concerns exist about achieving efficacy without off-target effects. To protect striated muscle from harmful ActRIIB signaling, and to reduce the risk of off-target effects, we developed an intervention using re- combinant adeno-associated viral vectors (rAAV vectors) that increase expression of Smad7 in skeletal and car- diac muscles. SMAD7 acts as an intracellular negative regulator that prevents SMAD2/3 activation and promotes degradation of ActRIIB complexes. In mouse models of cachexia, rAAV:Smad7 prevented wasting of skeletal muscles and the heart independent of tumor burden and serum levels of procachectic ligands. Mechanistically, rAAV:Smad7 administration abolished SMAD2/3 signaling downstream of ActRIIB and inhibited expression of the atrophy-related ubiquitin ligases MuRF1 and MAFbx. These findings identify muscle-directed Smad7 gene delivery as a potential approach for preventing muscle wasting under conditions where excessive ActRIIB signaling occurs, such as cancer cachexia. INTRODUCTION Cachexia, a state of pronounced weight loss, frailty, and fatigue that is characterized by severe atrophy of muscle and fat, affects up to 80% of patients with advanced solid cancers (1). Cachexia is a strong indicator of poor prognosis and reduced survival, because progressively debili- tating frailty ultimately deprives patients of independent movement and respiratory function and also reduces tolerance for aggressive chemotherapy regimens (2). Current care measures for cachexia large- ly focus on nutritional supplementation, prescription of appetite stim- ulants and anti-inflammatory glucocorticoids (although these drugs can cause muscle wasting), and physical therapy to promote ac- tivity (2). However, although anorexia and reduced activity contribute to the exacerbation of cachexia in chronically ill individuals, tumor- and host-derived factors that drive catabolic signaling in muscle and adipose tissue are considered the primary cause of cachexia onset and progression (3). Consequently, developing interventions that target the effects of procachectic ligands is considered the best prospect for preventing or reversing cachexia. Of the various secreted factors associated with cachexia to date, much interest has developed around the therapeutic prospects of inhibiting type IIB activin receptor (ActRIIB) ligands—in particular, myostatin, activins, and growth/differentiation factor 11 (GDF11)— because this pathway stimulates muscle catabolism, and expression of ActRIIB ligands is elevated under conditions associated with muscle wasting (3–5). Antibodies against myostatin have shown some capac- ity to ameliorate muscle wasting in animal studies, although effects are modest, likely because they do not inhibit the activity of other ActRIIB ligands that can be simultaneously elevated in cachectic patients’ ser- um and tissues. As a strategy to antagonize multiple ligands, admin- istration of ligand traps, such as soluble forms of modified ActRIIB, has been shown to reverse muscle wasting and increase life span in animal models of cachexia, despite elevated circulating levels of pro- cachectic cytokines (6). However, in 2011, phase 2 clinical trials of a soluble ActRIIB receptor–based intervention that targeted circulating ActRIIB ligands (to combat frailty associated with a form of muscular dystrophy) were terminated owing to safety concerns (7). Although the significance of the associated off-target effects remains a subject of discussion, it is generally acknowledged that targeting the interac- tion between circulating ligands and ActRIIB receptors may disrupt critical processes in many organ systems, including vascular remodel- ing, inflammatory regulation, and reproduction (8, 9). Thus, findings to date suggest that interventions that prevent ActRIIB signaling could prove instrumental in treating muscle wasting such as that associated with cachexia, but need to target signaling selectively in muscle to minimize the probability of evoking off-target effects. Once activated, ActRIIB recruits type I activin receptors (ActRI, also known as ALK4/7, encoded for by ACVR1b/1c) to form an activated ActRIIB:ActRI complex that phosphorylates SMAD2/3 (8). These re- ceptor SMAD proteins repress protein synthesis by inhibiting the Akt/mTOR (mammalian target of rapamycin) signaling pathway (10) and also translocate to the nucleus in complex with SMAD4 to pro- mote a transcriptional program that increases protein degradation. The transcriptional response also up-regulates expression of the inhibitory 1 Baker IDI Heart and Diabetes Institute, Melbourne, Victoria 3004, Australia. 2 Depart- ment of Physiology, The University of Melbourne, Melbourne, Victoria 3010, Australia. 3 Hudson Institute of Medical Research, Clayton, Victoria 3168, Australia. 4 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Aus- tralia. 5 Department of Medicine, Monash University, Clayton, Victoria 3800, Australia. 6 Department of Physiology, Monash University, Clayton, Victoria 3800, Australia. 7 De- partment of Animal Sciences, Washington State University, Pullman, WA 99164, USA. 8 Department of Neurology, The University of Washington School of Medicine, Seattle, WA 98195, USA. *Corresponding author. Email: paul.gregorevic@bakeridi.edu.au RESEARCH ARTICLE www.ScienceTranslationalMedicine.org 20 July 2016 Vol 8 Issue 348 348ra98 1 by guest on May 31, 2020 http://stm.sciencemag.org/ Downloaded from