Inhibition of Stat3 activation suppresses caspase-3 and the ubiquitin-proteasome system, leading to preservation of muscle mass in cancer cachexia

Abstract

Cachexia occurs in patients with advanced cancers. Despite the adverse clinical impact of cancer-induced muscle wasting, pathways causing cachexia are controversial, and clinically reliable therapies are not available. A trigger of muscle protein loss is the Jak/Stat pathway, and indeed, we found that conditioned medium from C26 colon carcinoma (C26) or Lewis lung carcinoma cells activates Stat3 (p-Stat3) in C2C12 myotubes. We identified two proteolytic pathways that are activated in muscle by p-Stat3; one is activation of caspase-3, and the other is p-Stat3 to myostatin, MAFbx/Atrogin-1, and MuRF-1 via CAAT/enhancer-binding protein δ (C/EBPδ). Using sequential deletions of the caspase-3 promoter and CHIP assays, we determined that Stat3 activation increases caspase-3 expression in C2C12 cells. Caspase-3 expression and proteolytic activity were stimulated by p-Stat3 in muscles of tumor-bearing mice. In mice with cachexia caused by Lewis lung carcinoma or C26 tumors, knock-out of p-Stat3 in muscle or with a small chemical inhibitor of p-Stat3 suppressed muscle mass losses, improved protein synthesis and degradation in muscle, and increased body weight and grip strength. Activation of p-Stat3 stimulates a pathway from C/EBPδ to myostatin and expression of MAFbx/Atrogin-1 and increases the ubiquitin-proteasome system. Indeed, C/EBPδ KO decreases the expression of MAFbx/Atrogin-1 and myostatin, while increasing muscle mass and grip strength. In conclusion, cancer stimulates p-Stat3 in muscle, activating protein loss by stimulating caspase-3, myostatin, and the ubiquitin-proteasome system. These results could lead to novel strategies for preventing cancer-induced muscle wasting.

Keywords: Cancer Biology; Cancer Cachexia; Caspase; Caspase-3; Muscle Atrophy; STAT3; Ubiquitin.

FIGURE 1.

Conditioned medium from C26 cancer cells activates p-Stat3 in C2C12 myotubes, a model of skeletal muscle. A, representative Western blots for p-Stat3 and Stat3 exposed to the conditioned medium of C26 for different times. B, representative Western blots for p-Stat3 or Stat3 with or without C188-9 in conditioned medium for 15 min. C, the average sizes of C2C12 myotubes following a 72-h incubation in C26-conditioned medium with or without C188-9. Error bars indicate means ± S.E. D, representative Western blots of caspase-3 and cleaved caspase-3 of C2C12 incubated in C26-conditioned medium. E, representative Western blots of pro-caspase-3 in C2C12 myotubes treated with C26-conditioned medium with or without C188-9. F, representative Western blots of C/EBPδ and myostatin in C2C12 myotubes treated with C26-conditioned medium with or without C188-9. CTRL, control.

FIGURE 2.

p-Stat3 stimulates caspase-3 transcription, augmenting cancer-induced muscle loss. A, representative Western blot of caspase-3 and cleaved (activated) caspase-3 in muscles of mice bearing C26 or LLC tumors. CTRL, control. B, representative Western blots demonstrating increased caspase-3 activity in muscles of mice bearing C26 or LLC tumors. Activity was assessed from the cleavage of actomyosin, which produces a 14-kDa actin fragment in muscle found in cancer and other catabolic conditions. C, the three putative Stat3 binding sites in the caspase-3 promoter. D, a CHIP assay revealed that p-Stat3 binds to the caspase-3 promoter in C2C12 myotubes that had been treated with conditioned medium from C26 cells. E, C2C12 myotubes were infected with an adenovirus expressing GFP or Stat3. After 24 h, cells expressing Stat3 were stimulated by adding IL-6. Results of the CHIP assay revealed binding of p-Stat3 to the caspase-3 promoter. M indicates molecular marker. F, immunoprecipitated DNA was obtained as in panel E and subjected to RT-PCR analysis. The -fold change of Stat3 or p-Stat3 that is associated with DNA when compared with the value obtained with anti-IgG is shown (*, p < 0.05 versus GFP expressing cells). G, C2C12 cells were transfected with plasmids expressing different deletions of the caspase-3 promoter plus a plasmid that expresses constitutively active Stat3. Cells were then treated with or without IL-6 for 6 h, and luciferase activity was measured to assess caspase-3 promoter activity. (Results are mean ± S.E.; *, p < 0.05 versus results obtained in cells cultured in serum-free (SF) medium.) H, representative Western blots demonstrating caspase-3 proteolytic activity as an increase in the 14-kDa actin fragment in muscles of mice bearing tumor or being treated with or without C188-9.

FIGURE 3.

Muscle-specific Stat3 KO in mice suppresses LLC-induced loss of muscle mass. Mice with muscle-specific Stat3 KO (KO) or control mice (Stat3^flox/flox (F/F)) were used; 10 mice in each group were injected with LLC cells. A–G, after 18 days, we assessed: body weight (A); weights of gastrocnemius (Gast.) muscles (B); differences in cross-sections of tibialis anterior muscles that were immunostained with anti-laminin (C); the average sizes of myofibers (D); weights of tumors (E); muscle grip (mean values ± S.E.) (F); and representative Western blots of Stat3, C/EBPδ, and myostatin in gastrocnemius muscles of Stat3 KO mice versus control, floxed Stat3 (F/F mice) that had been treated with and without LLC tumors (G). CTRL, control. Densities of these Western blots were corrected for GAPDH and quantified (bar graphs).

FIGURE 4.

Elimination of C/EBPδ in mice suppresses LLC-induced cachexia. A–F, C/EBPδ KO and control mice were injected with LLC cells. Eighteen days later, we measured: body weight (A); weights of gastrocnemius (Gast.) (B); cross-sections of tibialis anterior muscles following immunostaining with anti-laminin (C); average myofiber sizes (D); rates of muscle protein degradation (E); and muscle grip strength (F). CTRL, control. G, representative Western blots of myostatin in muscles of C/EBPδ KO or control mice inoculated with or without LLC (upper panel) are shown. The -fold changes in myostatin expression when compared with results in control mice are shown (lower panel, quantification of myostatin levels corrected for GAPDH). H, representative Western blots of 14-kDa actin fragment in muscles of wild type or C/EBPδ KO mice bearing tumors. The -fold changes in 14 kDa to total actin are shown (lower panel,* p < 0.05 versus non-tumor mice). Results are reported as mean ± S.E. NS, not significant.

FIGURE 5.

Blocking Stat3 activation with C188-9, a Stat3 inhibitor, suppresses cancer cachexia. CD2F1 mice bearing C26 tumors for 5 days were subsequently treated with C188-9 or D5W twice daily for 14 days. CD2F1 mice without C26 tumors served as controls (CTRL). Twelve mice were in each group. A, upper panel, representative Western blots of different proteins from lysates of gastrocnemius muscles (Gast.). Lower panel, quantification was corrected for GAPDH levels. B–G, we also measured: body weights (B); muscle mass (C); TA, tibialis anterior; the distribution of myofiber sizes in tibialis anterior muscles of the three groups of mice (D).; muscle grip strength (E); the rate of protein synthesis (F); and the rate of protein degradation in soleus and extensor digitorum longus (EDL) muscles (G). Results are mean ± S.E.

FIGURE 6.

Activation of C/EBPδ induces proteolysis in the UPS in cancer cachexia. A, C2C12 myotubes were treated with conditioned medium from C26 cells with or without C188-9 for 72 h. A representative Western blot shows a decrease in myosin heavy chain protein that was blocked by C188-9. B, C2C12 myotubes were incubated in conditioned medium from C26 cells for 24 h with or without C188-9. Levels of mRNAs of MAFbx/Atrogin-1 and MuRF-1 are shown. C and D, CD2F1 mice bearing C26 tumors were treated with C188-9 for 2 weeks. mRNAs of MAFbx/Atrogin-1 and MuRF-1 and representative Western blots of MAFbx/Atrogin-1 in gastrocnemius muscle are shown. CTRL, control. E, LLC tumors were injected into mice with muscle-specific KO of Stat3 or Stat3^flox/flox (F/F). After 18 days, representative Western blots from muscle show an increase in the MAFbx/Atrogin-1 protein corrected for GAPDH (mean ± S.E.). F, LLC cells were injected into C/EBPδ KO mice, and 14 days later, a representative Western blot from muscle shows the MAFbx/Atrogin-1 protein. Results corrected for GAPDH are presented as mean ± S.E. G, MAFbx/Atrogin-1 promoter activity was increased in cells that overexpress C/EBPδ. H, MuRF-1 promoter activity was increased in cells that overexpress C/EBPδ. I, proteasomes isolated from muscles of mice with or without tumors were used to measure the proteasome activity using the fluorogenic peptide, LLVY-AMC, as a substrate. Results are mean ± S.E.

FIGURE 7.

Cancer activates Stat3 in muscle to stimulate loss of muscle mass via two signaling pathways. In one pathway, p-Stat3 stimulates caspase-3 transcription and activity. In a second pathway, p-Stat3 stimulates C/EBPδ expression and activity, which increases myostatin and MAFbx/Atrogin-1 and MuRF-1. Both pathways result in protein losses in muscle.