Toll-like receptor 4 mediates Lewis lung carcinoma-induced muscle wasting via coordinate activation of protein degradation pathways

Abstract

Cancer-induced cachexia, characterized by muscle wasting, is a lethal metabolic syndrome with undefined etiology. Current consensus is that multiple factors contribute to cancer-induced muscle wasting, and therefore therapy requires combinational strategies. Here, we show that Toll-like receptor 4 (TLR4) mediates cancer-induced muscle wasting by directly activating muscle catabolism as well as stimulating an innate immune response in mice bearing Lewis lung carcinoma (LLC), and targeting TLR4 alone effectively abrogate muscle wasting. Utilizing specific siRNAs we observed that LLC cell-conditioned medium (LCM)-treated C2C12 myotubes underwent a rapid catabolic response in a TLR4-dependent manner, including activation of the p38 MAPK-C/EBPβ signaling pathway as well as the ubiquitin-proteasome and autophagy-lysosome pathways, resulting in myotube atrophy. Utilizing a reporter cell-line it was confirmed that LCM activated TLR4. These results suggest that LLC-released cachexins directly activate muscle catabolism via activating TLR4 on muscle cells independent of immune responses. Critically, LLC tumor-bearing TLR4^-/- mice were spared from muscle wasting due to a blockade in muscle catabolic pathways. Further, tumor-induced elevation of circulating TNFα and interleukin-6 (IL-6) was abolished in TLR4^-/- mice. These data suggest that TLR4 is a central mediator and therapeutic target of cancer-induced muscle wasting.

Figure 1

LLC cell-conditioned medium activates myotube catabolism through TLR4. C2C12 myoblasts were transfected with indicated siRNAs. After incubation in differentiation medium for 96 h, myotubes were treated with Lewis lung carcinoma cell-conditioned medium (LCM) or control medium. Activation of NF-κB (p65), p38 MAPK and C/EBPβ was evaluated by Western blot analysis of cell lysate in 1 h (A). Levels of UBR2, atrogin1 and LC3-II were evaluated by Western blot analysis of cell lysate in 8 h (B). Autophagy flux increase was demonstrated by measuring LC3-II levels in myotubes pre-treated with 20 μM of lysosome inhibitor chloroquine (CQ, C). Levels of myosin heave chain (MHC) were evaluated by Western blot analysis of cell lysate in 72 h (D). TLR2 and TLR4 knockdown was monitored at each of the time points. Myotubes treated for 72 h were immunofluorescence-stained for MHC and their diameter was measured (E). Data was analyzed by ANOVA. *Denotes a difference (P < 0.05).

Figure 2

LLC cell-conditioned medium activates TLR4 in reporter cells. TLR4 reporter cell line HEK-Blue hTLR4 (InvivoGen) was treated with LCM or control medium for 24 h and TLR4 activation was measured as the enzymatic activity of secreted embryonic alkaline phosphatase (SEAP) in arbitrary unit (a.u.). Data was analyzed by Student t test. *Denotes a difference (P < 0.05).

Figure 3

TLR4^−/− mice are resistant to LLC tumor-induced muscle wasting. Wild-type C57BL/6 and TLR4^−/− mice (7-week old male) were inoculated with LLC cells. Muscle wasting was evaluated in 21 days by measuring tumor mass, body weight change (excluding tumor weight), grip strength, tyrosine release from EDL, muscle weight (TA and EDL) (A) as well as muscle fiber cross-sectional area (B). Bar = 100 μm. Data was analyzed by ANOVA (A) or Chi-square analysis (B). *Denotes a difference (P < 0.05).

Figure 4

LLC tumor-induced muscle catabolism is dependent on TLR4. (A) TLR4 is required for the activation of catabolic pathways in muscle of LLC-bearing mice. TA muscle collected from mice described in Fig. 3 was analyzed by Western blotting for markers of TLR4-mediated signaling and muscle catabolism. Data was analyzed by ANOVA. *Denotes a difference (P < 0.05). (B) Autophagosome formation is activated in cachectic muscle of LLC-bearing mice in a TLR4-dependent manner. The TA of wild type C57BL/6 or TLR4^−/− mice that had been implanted with LLC cells (controlled by PBS injection) were transfected with GFP-LC3 or GFP-expressing plasmid on day 14. In 7 days, autophagosome formation was evaluated by fluorescence microscopy examination of frozen sections of TA. Bars represent 50 μM.

Figure 5

TLR4 is required for LLC tumor-induced elevation of circulating catabolic cytokines in mice. Sera collected from mice described in Fig. 3 on day 21 of LLC tumor implant were analyzed for catabolic cytokines TNFα, IL-6 and IL-1β by multiplex immunoassay (Bio-Rad). Data was analyzed by ANOVA. *Denotes a difference (P < 0.05).