Research ArticleMUSCULOSKELETAL DISEASE

Smad7 gene delivery prevents muscle wasting associated with cancer cachexia in mice

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Science Translational Medicine  20 Jul 2016:
Vol. 8, Issue 348, pp. 348ra98
DOI: 10.1126/scitranslmed.aac4976

Taking action against cachexia

An unfortunate morbidity associated with cancer is muscle wasting, known as cachexia, where healthy cells erode in the face of malignancy. Cachexia has been difficult to treat, and the most promising new therapies inhibiting ActRIIB ligands, such as myostatin, a protein that promotes muscle breakdown, have been pulled after clinical trials indicated safety issues. Targeting ActRIIB ligands may still be possible—just in a different way, to avoid toxicity. Winbanks et al. demonstrated that gene therapy could be used to block ActRIIB ligands’ catabolic signaling. Delivering the gene Smad7 to mice with tumors prevented muscle atrophy and preserved muscle mass and force production by inhibiting ActRIIB signaling. The Smad7 gene therapy did not affect other organs, suggesting that safely targeting ActRIIB signaling is possible.

Abstract

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 mechanism 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 development, 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 recombinant adeno-associated viral vectors (rAAV vectors) that increase expression of Smad7 in skeletal and cardiac 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 debilitating frailty ultimately deprives patients of independent movement and respiratory function and also reduces tolerance for aggressive chemotherapy regimens (2). Current care measures for cachexia largely focus on nutritional supplementation, prescription of appetite stimulants and anti-inflammatory glucocorticoids (although these drugs can cause muscle wasting), and physical therapy to promote activity (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 (35). Antibodies against myostatin have shown some capacity 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’ serum and tissues. As a strategy to antagonize multiple ligands, administration 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 procachectic 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 interaction between circulating ligands and ActRIIB receptors may disrupt critical processes in many organ systems, including vascular remodeling, 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 receptor 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 promote a transcriptional program that increases protein degradation. The transcriptional response also up-regulates expression of the inhibitory protein SMAD7 as a form of intracellular negative feedback that prevents SMAD2/3 phosphorylation and promotes ActRIIB complex degradation (11). We hypothesized that using recombinant adeno-associated viral vectors (rAAV vectors) to genetically increase SMAD7 abundance in striated muscle could ameliorate muscle wasting by attenuating ActRIIB signaling, without the known side effects of soluble ligand traps. rAAV vectors have been demonstrated to be useful tools for therapeutic gene delivery, owing to their propensity for achieving efficacious and targeted delivery of transgenes to the skeletal muscles of mammals (12), including humans (13, 14), that can sustain gene expression for more than a decade after a single treatment (14). Our preclinical studies demonstrate that the overexpression of Smad7 in the striated muscle of mice enhances muscle mass and function and prevents wasting in rodent models of cancer cachexia.

RESULTS

Increasing expression of Smad7 promotes skeletal muscle hypertrophy in mice

We chose rAAV6 for delivering Smad7 because it has strong tropism for striated muscle (12). Injection of the tibialis anterior (TA) hindlimb muscles of C57BL/6 mice with rAAV6 vectors (12, 15) carrying a Smad7 expression cassette (rAAV6:Smad7) elicited a ~45% increase in TA muscle mass within 28 days of injection (Fig. 1A), concomitant with increases in SMAD7 protein expression that considerably exceeded endogenous SMAD7 abundance (Fig. 1B) and increases in myofiber diameter (Fig. 1C). Systemic administration of rAAV6:Smad7 promoted hypertrophy of skeletal muscles body-wide (Fig. 1D), which recapitulated the increases in myofiber diameter observed after local injection of rAAV6:Smad7 (fig. S1A). As expected, owing to rAAV6 vector tropism (12), SMAD7 was expressed in skeletal muscles and the heart (Fig. 1E), but not visceral organs (fig. S1B). We have previously modulated the transforming growth factor–β (TGFβ) network using interventions that are more potent than Smad7 (for example, follistatin isoforms) and found no changes in myofiber number, or evidence of altered nonmuscle content to support the occurrence of remodeling events in adult muscle (16). Thus, we did not evaluate those outcomes in this study.

Fig. 1. Smad7 gene delivery promotes skeletal muscle hypertrophy in healthy mice.

Mouse TA hindlimb muscles were injected intramuscularly with rAAV6:Smad7. (A and B) TA muscle mass (A) and SMAD7 protein abundance (B) were measured at 7, 14, and 28 days after injection. Data are means ± SEM (n = 5, 7, and 4 at 7, 14, and 28 days, respectively). GAPDH, glyceraldehyde phosphate dehydrogenase. (C) Representative hematoxylin and eosin (H&E)–stained cross sections of TA myofibers and myofiber diameter at 28 days after injection (image scale, 100 μm). Data are means ± SEM (n = 3 muscles per condition and ~500 myofibers counted per muscle). (D) Body-wide muscle hypertrophy and individual muscle masses associated with increased abundance of SMAD7 in striated musculature examined 28 days after intravenous administration of rAAV6:Smad7. Images are representative of n = 3. Quad, quadriceps; Tri, triceps. (E) Western blot of SMAD7 in skeletal muscles and the heart in systemically treated mice. (F) Western blot of phosphorylated SMAD3 (pSMAD3) and corresponding quantification in muscles examined at specified time points after local injection of rAAV6:Smad7. Data are means ± SEM (n = 5, 4, and 4 at 7, 14, and 28 days, respectively). (G) Mass of mouse TA muscles administered rAAV6:Smad7 and/or rAAV6:Smad3-CA (S3-CA) or control vector. Data are means ± SEM at day 28 after injection (n = 3 for Smad3-CA, n = 5 for Smad7, n = 5 for codelivery of Smad7 and Smad3-CA). (H) H&E-stained sections demonstrate myofiber size in muscles examined 28 days after receiving rAAV6:Smad7 alone or with rAAV6:Smad3-CA. Scale bar, 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001 by t test (A to F) or one-way analysis of variance (ANOVA) (G).

SMAD3S432/435 phosphorylation was potently suppressed in muscles administered rAAV6:Smad7 (Fig. 1F), indicating marked inhibition of SMAD2/3 signaling when SMAD7 is increased. Moreover, the hypertrophic effects of rAAV6:Smad7 treatment were abolished when muscles were coadministered rAAV6:Smad7 with rAAV6 expressing a constitutively active (CA) form of SMAD3, rAAV6:Smad3-CA (Fig. 1, G and H) (17). The coadministration of vectors did not affect expression of transgenes, compared with administration of individual vectors (fig. S1C).

To investigate the mechanisms of Smad7-mediated muscle growth, we examined processes associated with protein synthesis and degradation. SMAD7 overexpression in muscles increased fractional protein synthesis rates (fig. S2A) concomitant with increased phosphorylation of S6 ribosomal protein (S6RPS235/236) and eukaryotic translation initiation factor 4E–binding protein 1 (4EBP1T37/46)—key modulators of protein synthesis that are responsive to mTOR activation (fig. S2B). However, we did not detect altered Akt phosphorylation or activity (fig. S2, B and C). Repeated treatment of mice with rapamycin (an inhibitor of mTOR) did not suppress muscle hypertrophy induced by rAAV6:Smad7 administration (fig. S2D) but prevented phosphorylation of S6RP and 4EBP1 in Smad7-treated muscles (fig. S2E).

Subsequently, we investigated whether the anabolic effects of rAAV6:Smad7 were associated with altered proteolysis. Muscles administered rAAV6:Smad7 exhibited attenuated transcription of MuRF1 and MAFbx (fig. S2F), two E3 ubiquitin ligases that have been identified as key mediators of proteasome-based protein degradation in muscle. Furthermore, the overexpression of Smad7 prevented Smad3-mediated transcription of MuRF1 and MAFbx (fig. S2G). Together, these data demonstrate that administering rAAV6:Smad7 to healthy muscles promotes hypertrophy by shifting the balance between protein synthesis and degradation independent of mTOR activity.

Smad7 delivery prevents myostatin- and activin-induced muscle atrophy

We hypothesized that SMAD7 promotes skeletal muscle hypertrophy in part by suppressing the activation of SMAD2/3 by the ActRIIB ligands myostatin and activin. Administering rAAV6:Smad7 to the TA muscles of both wild-type and myostatin-null mice (Mstn−/−) increased TA muscle mass (Fig. 2A) and myofiber size (Fig. 2B) and reduced SMAD3 phosphorylation relative to muscles receiving control vector (Fig. 2C). However, the relative increase in muscle mass as a consequence of treatment was tempered in Mstn−/− mice, relative to wild-type littermates (Fig. 2D). The comparatively diminished hypertrophic action of rAAV6:Smad7 in mice lacking myostatin is consistent with the fact that additional ActRIIB ligands (for example, activin and GDF11) act in concert with myostatin to negatively regulate muscle mass (18, 19).

Fig. 2. Smad7 gene delivery prevents myostatin- and activin-induced muscle atrophy.

Myostatin-null mice (Mstn−/−) and littermate controls [wild-type (WT)] were examined 14 and 28 days after intramuscular administration of one dose of rAAV6:Smad7. (A) TA muscle mass. Data are means ± SEM (n = 5 to 8). (B) H&E-stained sections demonstrate myofiber size. Scale bar, 100 μm. (C) Western blot of phosphorylated SMAD3S432/435 in the TA muscle 28 days after local injection of rAAV6:Smad7. Data are means ± SEM (n = 4). (D) Relative gains in TA muscle mass 14 and 28 days after vector administration. Data are means ± SEM (n = 5 to 8). (E and F) TA muscle mass examined 28 days after intramuscular injection of rAAV:Smad7 in conjunction with rAAV6:Myostatin (n = 7 to 15) (E) or rAAV6:Activin A (Act A) (n = 3 to 6) (F), with transcription of myostatin (E) and activin (F) in muscles administered rAAV:Smad7 or control vector. Data are means ± SEM. (G)Western blot of phosphorylated SMAD3S432/435 in muscles in (E). Data are means ± SEM (n = 4 to 6). (H) Western blot of phosphorylated SMAD3S432/435 in muscles in (F). Data are means ± SEM. (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001 by two-way ANOVA [plus post hoc test in (A)].

Because elevated levels of the ActRIIB ligands myostatin and activin have been shown to cause muscle wasting in mice by driving excessive SMAD2/3 signaling (20), we examined whether increased expression of Smad7 could protect muscles from myostatin- and activin-induced atrophy. Direct injection of mouse limb muscles with rAAV6 vectors expressing either myostatin (Mstn) or activin A (Act A) reduced TA mass (Fig. 2, E and F) concomitant with increased phosphorylation of SMAD3 (Fig. 2, G and H). Muscles overexpressing myostatin or activin were protected from atrophy and excessive SMAD2/3 signaling when coadministered rAAV6:Smad7 (Fig. 2, E and F). These data demonstrate that SMAD7 can inhibit the actions of different ActRIIB ligands acting upon skeletal muscles.

Smad7 delivery prevents muscle wasting associated with cancer cachexia

Because ActRIIB signaling is hypothesized to contribute to the development and progression of cancer cachexia (6, 21, 22), we examined whether rAAV6:Smad7 could prevent muscle wasting in mice bearing cachexia-inducing Colon-26 (C-26) carcinomas. Body mass, skeletal muscle mass, and heart and fat mass were all reduced in tumor-bearing mice, compared with tumor-free mice (fig. S2H). Administering rAAV6:Smad7 to the TA muscles of tumor-bearing mice preserved muscle mass, peak isometric tetanic force-producing capacity, and myofiber cross-sectional area relative to healthy control values (Fig. 3A). Muscles from mice bearing tumors had a reduced proportion of myofibers that expressed the type IIa myosin heavy chain (MHCIIa) isoform compared with tumor-free controls (Fig. 3, B and C). Administration of rAAV6:Smad7 to the muscles of mice at the time of tumor implantation prevented the shift in myofiber-type proportions (Fig. 3, B and C).

Fig. 3. Smad7 gene delivery prevents cancer cachexia.

(A) SMAD7 expression, mass, force-producing capacity, and myofiber cross-sectional area (CSA) for TA muscles of mice injected intramuscularly (i.m.) with rAAV6:Smad7 at the time of subcutaneous flank implantation of cachexia-inducing C-26 tumors. Data are means ± SEM (n = 11 to 14 for TA mass and peak force, n = 8 for myofiber cross-sectional area). (B) TA muscles labeled for laminin and MHCIIa. Scale bar, 100 μm. (C) Proportion (left) and cross-sectional area (right) of type IIa myofibers or type IIx/b fibers in TA muscles. Data are means ± SEM (n = 8). (D) TUNEL stain of TA muscles. Scale bar, 100 μm. (E) TA muscle mass in tumor-free mice and C-26 tumor–bearing mice administered rAAV6:Smad7 or control vector 7 or 14 days after tumor implantation (when tumor growth is evident). Data are means ± SEM (n = 5 to 7). (F) Tumor volume reported for individual animals, with mean (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001 by two-way ANOVA (A, C, and E) or t test (F).

Differences in myofiber size between muscles were consistent with the proportional differences in muscle mass. No difference was observed in the frequency of TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling)–positive nuclei (as a marker of apoptosis) in muscles receiving rAAV6:Smad7 or control vector in tumor-free or tumor-bearing mice (Fig. 3D), indicating that the tumor-mediated loss of muscle mass was due to myofiber atrophy, which was prevented by rAAV6:Smad7 administration. Because administering therapies for cachexia before muscle wasting develops may not always be possible, we examined whether injecting muscles with rAAV6:Smad7 at 7 or 14 days after tumor establishment was also protective. Delayed rAAV6:Smad7 still significantly ameliorated TA muscle atrophy, even as tumors increased in volume by two orders of magnitude (Fig. 3, E and F).

Because cancer cachexia causes wasting of muscles throughout the body, we next tested whether rAAV6:Smad7 could prevent muscle atrophy at a systemic level. Intravenous injection of rAAV6:Smad7 robustly increased SMAD7 expression throughout the striated musculature of tumor-bearing mice (Fig. 4A and fig. S3A). Systemic rAAV6:Smad7 administration also preserved the mass of skeletal muscles in these mice, without affecting tumor mass, to the extent that the mass of specific muscles in treated tumor-bearing mice did not differ from that of untreated tumor-free mice (Fig. 4, B to D, and fig. S3, B and C). The relative change in muscle mass of healthy versus tumor-bearing mice systemically administered rAAV6:Smad7 appeared greater in the former group, although this was likely due to the up-regulation of catabolic processes in tumor-bearing mice before the expression of Smad7 reaching therapeutic levels. Systemic rAAV6:Smad7 also prevented cardiac atrophy in tumor-bearing mice (Fig. 4, E and F) and did not elicit increases in atrial mass or gross lung weight as typically associated with heart failure (Fig. 4F) (23).

Fig. 4. Systemic Smad7 prevents cancer cachexia.

Tumor-free and C-26 tumor–bearing BALB/c mice were examined 21 days after tumor implantation and tail vein [intravenous (i.v.)] administration of rAAV6:Smad7 or control vector. (A and B) SMAD7 Western blot of heart, quadriceps muscles, and TA muscles (A) and masses of specific muscles (Ga, gastrocnemius; Quad/Qd, quadriceps; Tr, triceps) (B). Data are means ± SEM (n = 4 to 14). α-Tub, α-tubulin. (C) Images at day 21 of tumor-free mice and C-26 tumor–bearing mice administered a systemic dose of rAAV6:Smad7 or control vector. (D) H&E-stained sections of TA muscles from the mice in (C). Scale bar, 100 μm. (E) Gross heart appearance at day 21 for tumor-free mice and C-26 tumor–bearing mice systemically administered rAAV6:Smad7 or control vector. Scale bar, 2 mm. (F) Heart, atrial, and lung masses relative to tibia length (TL). Data are means ± SEM (n = 4 to 11). *P < 0.05, **P < 0.01, ***P < 0.001 by two-way ANOVA.

Despite the protection of muscles from atrophy in tumor-bearing mice that had received rAAV6:Smad7, the animals had reduced body mass compared with untreated tumor-free mice (fig. S3D). We hypothesized that the discrepancy between prevention of muscle atrophy and incomplete conservation of body mass was attributed to cachectic effects on other tissues (3), which still presented evidence of reduced mass in treated tumor-bearing mice (fig. S3D). This observation was supported by the finding that systemic rAAV6:Smad7 did not increase SMAD7 expression in the nonmuscle tissues of healthy mice (fig. S1B). Moreover, systemic rAAV6:Smad7 administration did not alter the expression of SMAD2/3 gene targets (PAI1, COL1A1, CTGF, and FN1) in a range of nonmuscle tissues (lung, liver, spleen, and kidney) examined from healthy or C-26 tumor–bearing mice (fig. S4A).

Clinical studies have shown that antagonizing ActRIIB signaling using systemic ligand traps can produce pathological remodeling of vascular endothelium, which shares some similarities with the vascular changes associated with hereditary hemorrhagic telangiectasia (HHT) (7, 9). We therefore assessed blood vessel integrity in the musculature and organs of healthy mice and C-26 tumor–bearing mice administered rAAV6:Smad7 or control vector. No evidence of HHT-like pathology was observed by histological analysis or gross morphological assessment of muscles and tissues from mice receiving rAAV6:Smad7 (fig. S4B).

To confirm that the therapeutic effects of rAAV6:Smad7 in the setting of cachexia were not unique to the model used, we tested the efficacy of our rAAV6:Smad7 intervention in inhibin-α knockout mice (Inha−/−), which exhibit profound cachexia subsequent to the development of gonadal tumors (Fig. 5, A and B) (6, 22, 24). Consistent with results obtained from mice bearing C-26 tumors, intramuscular administration of rAAV6:Smad7 to the muscles of Inha−/− mice (Fig. 5C) prevented muscle atrophy (Fig. 5, D to F), thus demonstrating that rAAV6:Smad7 can prevent cachexia independent of tumor origin.

Fig. 5. Smad7 administration prevents cancer cachexia in inhibin α–null mice.

(A) Mean total body, lean, and fat masses for inhibin α–null mice (Inha−/−) and WT controls from 6 weeks of age. Data are means ± SEM (n = 4). (B) Gonadal tumor mass in 12-week-old Inha−/− mice versus WT mice. (C to F) Western blot of SMAD7 abundance (C), muscle mass (D), muscle fiber diameter (E), and H&E-stained sections (F) of TA muscles from 12-week-old cachectic Inha−/− mice and controls (WT) examined 4 weeks after local injection of rAAV6:Smad7 or control vector (n = 4 per cohort). Scale bar, 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001 by two-way ANOVA (A, D, and E) and t test (B).

Smad7 gene delivery prevents transcription of ubiquitin ligases involved in skeletal muscle proteolysis

In the cachectic mouse models, SMAD7 protected striated musculature from wasting despite circulating levels of activins A and B remaining elevated (Fig. 6A). Application of serum from C-26 tumor–bearing mice to cultures of mouse myogenic C2C12 cells stimulated Smad3 signaling (Fig. 6B). We attribute the protective effects of rAAV6:Smad7 in cachectic mice to the suppression of SMAD3 phosphorylation (Fig. 6, C and D), and also reduced expression of Smad3 (Fig. 6E), that would diminish SMAD3-mediated effects on target gene transcription in striated musculature.

Fig. 6. Smad7 gene delivery regulates the canonical TGFβ signaling pathway and E3 ubiquitin ligase expression to prevent muscle atrophy in cancer cachexia.

(A) Serum levels of activins A and B (by enzyme-linked immunosorbent assay) in cachectic mice 21 days after implanting C-26 tumors (and, in the case of activin A, also administering rAAV6:Smad7 or control vector). Data are means ± SEM (n = 4 to 11). (B) SMAD3-responsive luciferase expression in cultured C2C12 cells exposed to serum from tumor-free or C-26 tumor–bearing mice. Data are means ± SEM (n = 3 per condition, experiment repeated three times). (C) Western blot of phosphorylated SMAD3S432/435 in TA muscles from tumor-free mice and C-26 tumor–bearing mice examined 21 days after tumor implantation and administration of rAAV6:Smad7 or control vector. Data are means ± SEM (n = 8 to 13). (D) SMAD3S432/435 phosphorylation in TA muscles from Inha−/− mice and WT controls examined 4 weeks after local injection of rAAV6:Smad7. Data are means ± SEM (n = 4). (E to L) Parameters examined in tumor-free mice and C-26 tumor–bearing mice 21 days after tumor implantation and administration of rAAV6:Smad7 or control vector. (E) Smad3 transcription in TA muscles [assessed by reverse transcription polymerase chain reaction (RT-PCR)] (n = 6 to 8; two-way ANOVA). (F) Serum levels of IL-6 in tumor-free mice and tumor-bearing mice 21 days after implanting C-26 tumors and administering rAAV6:Smad7 or control vector. UD, undetectable. Data are means ± SEM (n = 4 to 5). (G to J) Western blots of phosphorylated STAT3, p65, and FOXO1/3. Data are means ± SEM (n = 5 to 11). (J) Western blots of FOXO1 and FOXO3. Data are means ± SEM (n = 5 to 6). (K and L) Transcription of Foxo1 and Foxo3 (K) and MuRF1 and MAFbx (L). Data are means ± SEM (n = 4 to 8). *P < 0.05, **P < 0.01, ***P < 0.001 by t test (A), two-way ANOVA (B to I, K, and L), and two-way repeated-measures ANOVA (J).

However, because tumor- and host-derived interleukin-6 (IL-6) is also implicated in the etiology of cachexia (3, 25), we examined whether the protective effects of Smad7 were associated with altered IL-6 signaling. Administration of rAAV6:Smad7 did not alter circulating levels of IL-6 (Fig. 6F) or the phosphorylation of interleukin-responsive STAT3 (signal transducer and activator of transcription 3) in the muscles of tumor-bearing mice (Fig. 6G), nor did treatment affect phosphorylation of p65, a key regulator of nuclear factor κB signaling engaged by procachectic cytokines (Fig. 6H). Administration of rAAV6:Smad7 to muscles did not alter phosphorylation of FOXO1 or FOXO3, which are transcriptional regulators of the muscle-specific E3 ubiquitin ligases MurRF1 and MAFbx (Fig. 6I). However, in the muscles of tumor-bearing mice, treatment reduced FOXO1/3 abundance (Fig. 6J) and transcription (Fig. 6K) and suppressed MuRF1 and MAFbx expression (Fig. 6L).

DISCUSSION

Muscle wasting is a leading predictor of poor prognosis in cancer patients (2). Here, we demonstrate that attenuating ActRIIB intramuscular signaling with an AAV-based gene therapeutic is an effective means to prevent muscle wasting associated with cancer cachexia. Smad7 gene therapy improved muscle mass in three established mouse models of cachexia, including instances of treatment commencing after implantation of tumors, and did not affect other organs, suggesting high specificity and low toxicity. Because other AAV-based “gene therapies” have proven safe and functional in clinical trials for neuromuscular disorders and nonmuscle-related diseases (13, 26), the strategy proposed herein, although only tested in mice, may have potential to help reduce cancer-related morbidity and mortality.

Alternative approaches to modulating ActRIIB signaling in muscle have targeted the circulating factors that bind and activate ActRIIB, which include myostatin, activin and GDF11 (3, 6, 27, 28), because these factors can induce striated muscle atrophy and are often associated with muscle wasting in various disease states (35, 21, 22, 2933). However, the feasibility of such ligand traps and ActRIIB receptor antagonists remains in question because of the potential for serious off-target effects caused by inhibiting pleiotropic actions of the multiple ligands involved (79). Moreover, strategies that target only one ActRIIB ligand, such as myostatin (and not activins) (27), may be of limited use owing to the redundant actions of other ActRIIB ligands that also circulate at increased levels with cancer (20).

Overexpressing Smad7, an endogenous inhibitor of ActRIIB and SMAD2/3 signaling (11, 34), specifically in striated muscle avoids the off-target effects of ligand traps, enhances muscle mass and function, and prevents cancer cachexia in mice. rAAV6:Smad7 markedly increased Smad7 expression only in striated muscle and prevented muscle atrophy when challenged by experimental overexpression of the ActRIIB ligands myostatin or activin A. The amelioration of systemic muscle wasting was independent of tumor burden and occurred despite elevated circulating levels of procachectic ligands. Thus, these results demonstrate the utility of our approach for dissociating such ligands from their effects on intracellular signaling processes that promote muscle wasting.

The results reported here are comparable to those reported by others testing the efficacy of soluble ActRIIB ligand traps in cachectic mice (6), although our approach, which targets intracellular signaling specifically in striated muscle, is conceivably safer. Soluble ActRIIB ligand traps sequester not only ligands that cause muscle wasting but also other TGFβ superfamily members with different functions in nonmuscle tissues (7, 9). The most notable side effects of such ligand traps are bleeding from mucous membranes and other symptoms associated with HHT. HHT results from mutations in either of two transmembrane receptors, endoglin or activin-like kinase-1, which ultimately impair TGFβ signaling in endothelial cells, compromising microvasculature and inducing hemorrhage (35). Such off-target effects were reported in clinical trials of an ActRIIB ligand trap (36) and are evident in mouse models of HHT. No signs of adverse microvascular remodeling or hemorrhage were observed in our mice administered rAAV6:Smad7, nor was increased Smad7 expression or markers of SMAD7 bioactivity evident in nonmuscle tissues. The combined specificity of muscle-directed Smad7 gene delivery and lack of side effects suggest that rAAV6:Smad7 is an efficacious and more specific alternative to ActRIIB ligand traps.

Mechanistically, excessive ActRIIB signaling promotes muscle catabolism by repressing muscle protein synthesis and promoting its degradation (10, 37). Our studies demonstrate that overexpressing Smad7 increases S6K/S6RP signaling, but not Akt and mTOR activation, which are key regulators of protein synthesis. Others have similarly demonstrated upstream regulation of S6RP independent of mTOR (38). Overexpressing Smad7 in cachectic muscles also reduced transcription of the E3 ubiquitin ligases MuRF1 and MAFbx (Atrogin-1), which facilitate protein degradation in skeletal muscle. These combined effects on both anabolic and catabolic signaling required suppression of SMAD2/3 phosphorylation as did the attenuation of myostatin and activin A overexpression. Thus, increased SMAD7 expression represents a potent method to inhibit excessive ActRIIB signaling and the resulting increase in SMAD2/3 phosphorylation and muscle catabolism, by shifting the balance from protein degradation in favor of protein synthesis.

Similar to studies where cachectic mice received soluble ActRIIB (6), fat mass was not preserved in tumor-bearing mice treated systemically with rAAV6:Smad7. These results are not surprising, because the muscle-targeted approach of rAAV6:Smad7 would not be expected to directly influence adipose physiology. Ameliorating lipolysis in cachexia may be more effectively achieved by jointly targeting other cachexia-associated cytokines, such as IL-6 and TWEAK (25, 39), although conserving lean mass and muscle function is arguably the primary goal for interventions aimed at prolonging survival (2, 6, 40). Of particular note, the systemic administration of rAAV6:Smad7 also prevented cardiac atrophy in tumor-bearing mice, because rAAV6 vectors administered via the circulation can transduce cardiac muscle. Increased expression of myostatin and activin A is associated with heart disease (30, 41, 42), whereas conversely, physiological cardiac hypertrophy, enhanced cardiac contractility, and protection from cardiac atrophy occur in myostatin-null mice and cardiac muscle (4245). Therefore, the administration of rAAV6:Smad7 to the heart could help prevent cardiac cachexia and preserve cardiac performance in cancer patients.

Because ActRIIB is expressed in many nonmuscle tissues and several TGFβ superfamily ligands engage this receptor to exert diverse tissue-specific actions, ligand-trapping approaches pose significant risk for causing off-target effects (9, 46). By contrast, the data presented herein introduce rAAV6:Smad7 as a novel approach for preferentially attenuating ActRIIB signaling in striated muscle (12, 15) with little risk of producing the off-target effects. Further development of this approach could include cis elements (promoters, silencers, and microRNA-binding sites) capable of restricting SMAD7 production to striated muscle, because this could enhance the long-term applicability and potential safety. Clinical effectiveness will be contingent upon widespread transduction of striated musculature in adult humans. Although others have demonstrated the feasibility of administering AAV vectors systemically and regionally (for example, to limb muscles) via vascular infusion in large animal models, ongoing refinement of vector designs and administration protocols will support the clinical translation of these therapeutic strategies. Advancing this concept toward clinical trials will also require studies to establish a minimum effective dose and complete toxicological profiling in nonhuman primate models (9).

In terms of limitations, this study only examined the effects of rAAV6:Smad7 in mouse models. Although these models have been used previously (6), evaluating efficacy in other even more clinically relevant models remains a challenge not only for this study but also for the development of prospective cachexia therapeutics in general (47). Because of ethical considerations, our study did not test whether rAAV6:Smad7 was sufficient to extend survival. Also, the intervention did not prevent the loss of fat mass. However, preservation of muscle mass without protection of fat mass in tumor-bearing mice via administration of ActRIIB ligand traps has been reported to increase survival (6), which supports the importance, and prospects, of interventions that preserve muscle mass and strength.

Beyond cancer cachexia, muscle wasting occurs with many disease states, including chronic kidney disease, chronic obstructive pulmonary disease, and heart failure, and is also the defining feature of age-related sarcopenia. Circulating levels of myostatin and/or activin are often elevated under these conditions. Attenuating ActRIIB signaling in striated muscle with rAAV6:Smad7 may therefore prove useful in treating many conditions in addition to cancer cachexia.

MATERIALS AND METHODS

Study design

The aim of this study was to investigate whether a genetic intervention targeting ActRIIB signaling within striated muscle could prevent muscle wasting associated with cancer cachexia. We evaluated Smad7 gene delivery to musculature, because SMAD7 prevents SMAD2/3 signaling subsequent to ActRIIB activation. A recombinant AAV-based vector comprising the serotype-6 capsid and a Smad7 expression cassette was administered to mice via intramuscular or intravenous delivery. This vector configuration was chosen on the basis of the capacity of rAAV6 vectors to achieve sustained transduction of mammalian (rodent, canine, and nonhuman primate) striated muscle via local and systemic administration. rAAV6 vectors carrying a geneless construct were used as controls in all experiments. This intervention was evaluated in established models of muscle wasting and cancer cachexia, including mice implanted with C-26 carcinoma tumors and mice that develop tumors and cachexia due to inhibin knockout (Inha−/−) (6, 24, 39, 48, 49). Primary outcomes of muscle mass and force-producing capacity were assessed as clinically relevant markers of cachexia. These data were supported by secondary measures of muscle fiber morphology and ex vivo protein synthesis, as well as assays for protein signaling and gene expression to examine underlying mechanisms of action (Supplementary Materials and Methods). Time points of analyses were preselected on the basis of the onset and progression of cachexia in the different models and are presented in relation to specific observations reported. Ethical considerations regarding tumor burden also limited the duration of study and treatment for mice developing or receiving tumors. Data are presented inclusive of outliers. Age-matched mice were randomly assigned to experimental cohorts. Samples were deidentified for biochemical, histological, and functional analyses. Biological replicates for cohorts are indicated by n values in the figure legends. All experiments with mice and viral vectors were conducted in accordance with the relevant codes of practice for the care and use of animals for scientific purposes set by the U.S. National Institutes of Health and the National Health and Medical Council of Australia.

Cachexia animal models

Implantation of C-26–derived tumor tissue was carried out using CD2F1 or BALB/c mice. Cells or tissue was implanted into the flank as described previously (48, 49). Briefly, cells were passaged before implantation and reconstituted in 10% Dulbecco’s modified Eagle’s medium, whereas tumor pieces were thawed from liquid nitrogen. Cells were injected subcutaneously, or 1-mm3 tumor pieces were implanted using a trocar needle through a small incision in the skin overlying the flank. The mice were analyzed within 28 days of tumor implantation. Fat, lean, and total body masses of inhibin-α–null mice were analyzed using quantitative magnetic resonance (Echo Medical Systems).

Gene delivery in vivo

For local vector delivery, mice were anesthetized deeply with isoflurane or a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg; intraperitoneal injection) (VM Supplies). Individual vectors were administered at doses of 1 to 10 × 109 vector genomes (vg, defined by optimization pilot studies) in 30 μl of Hanks’ balanced salt solution (HBSS) directly into the anterior compartment of the hindlimb, which is occupied by the TA and extensor digitorum longus muscles. Control injections of the contralateral limb used a vector lacking a functional gene (rAAV6:MCS) (16).

For systemic delivery studies, 3 × 1012 to 5 × 1012 vg of rAAV6:Smad7 or control vector were administered in HBSS to a total volume of 200 μl via the tail vein. For rapamycin experiments, mice received rapamycin (2 mg/kg per day) (Calbiochem, Merck Millipore) or vehicle as a daily intraperitoneal injection commencing 3 hours before rAAV6:Smad7 injection and continuing for 14 days inclusive. Rapamycin was dissolved overnight in a solution containing 0.2% carboxymethylcellulose sodium salt (Sigma) and 0.25% polysorbate 80 (Sigma) in water, as described previously (50). For tissue harvest, mice were humanely sacrificed via a cervical dislocation, and the muscles were rapidly excised and weighed before subsequent processing.

Western blotting

TA muscles were homogenized in radioimmunoprecipitation assay–based lysis buffer (Merck Millipore) with EDTA-free protease and phosphatase inhibitor cocktails (cOmplete, Roche). Lysis was followed by centrifugation at 13,000g for 10 min at 4°C, and samples were denatured for 5 min at 95°C. Protein concentration was determined using a protein assay kit (Pierce, Thermo Scientific). Protein fractions were subsequently separated by SDS–polyacrylamide gel electrophoresis using precast 4 to 12% Bis-Tris gels (Life Technologies), blotted onto nitrocellulose membranes (Bio-Rad), and incubated with the appropriate antibody overnight. All antibodies used were obtained from Cell Signaling, except for antibodies against SMAD7 (Imgenex), phosphorylated SMAD3 (Epitomics), and glyceraldehyde phosphate dehydrogenase (Santa Cruz Biotechnology). Quantifications of labeled Western blots were performed using ImageJ pixel analysis (National Institutes of Health), and data were normalized to a control. Densitometric analyses of Western blots are presented as band density normalized to the loading control and are representative of at least three independent samples.

Assessment of muscle function

As described previously (16, 51), the contractile properties of the TA muscles of mice were assessed in situ by delivering a series of electrical stimuli to the tibial motor nerve via percutaneous electrodes and recording tension generated during contraction via a force transducer attached to the distal tendon with surgical silk suture. Mice were anesthetized with sodium pentobarbitone (Nembutal; 60 mg/kg; Sigma-Aldrich) via intraperitoneal injection before testing and were humanely sacrificed via cardiac excision at the conclusion of evaluation while still anesthetized deeply. At the conclusion of the protocol, muscles were rapidly excised, dissected free of tendon and connective tissue, and weighed.

Statistical analysis

Sample sizes were initially estimated on the basis of analyzing differences in morphology (that is, muscle mass and muscle fiber size), previous in-house characterization of the C-26 and rAAV6:Mstn and rAAV6:Act A models, and published characterizations of Inha−/− mice. Statistical differences were assessed across multiple conditions using one-way or two-way ANOVA tests, with the Student-Newman-Keuls post hoc test used for comparisons between the group means. Comparisons between two conditions used the two-tailed Student’s t test. Differences between groups were reported as statistically significant for values of P < 0.05.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/8/348/348ra98/DC1

Materials and Methods

Fig. S1. rAAV6:Smad7 administration induces skeletal muscle hypertrophy.

Fig. S2. rAAV6:Smad7 administration regulates muscle mass independently of Akt and mTOR signaling.

Fig. S3. Systemic rAAV6:Smad7 administration ameliorates muscle atrophy in a mouse model of cancer cachexia.

Fig. S4. Specificity of systemic rAAV6:Smad7 administration in mice bearing C-26 tumors.

REFERENCES AND NOTES

  1. Acknowledgments: We thank D. Coletti (University of Rome, Italy; Université Pierre et Marie Curie, France) for advice on the C-26 model of cachexia. We thank A. Chee and T. Naim (The University of Melbourne) for technical assistance with the assessment of the contractile properties and fiber-type composition of mouse muscles. Funding: This work was supported by grant funding (526648 and 566820) from the National Health and Medical Research Council (NHMRC, Australia). J.R.M. is supported by a Senior Research Fellowship (1078985) from NHMRC. K.T.M., C.A.H., and P.G. are supported by Career Development Fellowships (1023178, 1013533, and 1046782, respectively) from NHMRC. B.D.R. is supported by the National Science Foundation (NSF, USA; IOS1147275) and the Muscular Dystrophy Association (USA). The Baker IDI Heart and Diabetes Institute is supported in part by the Operational Infrastructure Support Program of the Victorian Government, Australia. Author contributions: C.E.W., K.T.M., C.A.H., G.S.L., and P.G. designed the research. C.E.W. performed most of the experimental work and the statistical analyses; K.T.M. coordinated the assessment of muscle function; B.C.B. analyzed cardiac attributes; H.Q. manufactured the viral vectors; P.V.S. performed the protein synthesis assays; Y.L., C.B., A.H., R.E.T., J.L.C., and K.L.W. assisted with the collection, processing, and analyses of tissue samples for Western blotting, RT-PCR, and histology; J.R.M. analyzed cardiac attributes; and P.G. performed small animal procedures and assisted with sample collection and processing. K.L.L., B.D.R., J.R.M., C.A.H., G.S.L., and P.G. contributed reagents, analytical tools, and technical advice. C.E.W., K.T.M., B.C.B., P.V.S., J.L.C., A.H., J.R.M., C.A.H., and P.G. analyzed the data. C.E.W. B.D.R., J.R.M., C.A.H., G.S.L., and P.G. prepared the manuscript. All authors had the opportunity to comment on the manuscript. Competing interests: Components of research described herein were made the subject of a provisional patent application (“Smad7 Gene Delivery as a Therapeutic to Treat and Prevent Muscle Wasting”) jointly filed by Washington State University and The Baker IDI Heart and Diabetes Institute. Data and materials availability: C-26 tumor fragments were a gift of D. Coletti. Myostatin-null mice were a gift of S. J. Lee (Johns Hopkins University). Inhibin-null mice were a gift of M. M. Matzuk (Baylor College of Medicine). Plasmids for AAV vector production are available via a material transfer agreement.
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