The Toll-Like Receptor/MyD88/XBP1 Signaling Axis Mediates Skeletal Muscle Wasting during Cancer Cachexia

Abstract

Skeletal muscle wasting causes both morbidity and mortality of cancer patients. Accumulating evidence suggests that the markers of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) pathways are increased in skeletal muscle under multiple catabolic conditions, including cancer. However, the signaling mechanisms and the role of individual arms of the UPR in the regulation of skeletal muscle mass remain largely unknown. In the present study, we demonstrated that gene expression of Toll-like receptors (TLRs) and myeloid differentiation primary response gene 88 (MyD88) was increased in skeletal muscle in a Lewis lung carcinoma (LLC) model of cancer cachexia. Targeted ablation of MyD88 inhibits the loss of skeletal muscle mass and strength in LLC tumor-bearing mice. Inhibition of MyD88 attenuates the LLC-induced activation of the UPR in skeletal muscle of mice. Moreover, muscle-specific deletion of X-box binding protein 1 (XBP1), a major downstream target of IRE1α arm of the UPR, ameliorates muscle wasting in LLC tumor-bearing mice. Our results also demonstrate that overexpression of an active form of XBP1 caused atrophy in cultured myotubes. In contrast, knockdown of XBP1 inhibits myotube atrophy in response to LLC or C26 adenocarcinoma cell conditioned medium. Collectively, our results demonstrate that TLR/MyD88-mediated activation of XBP1 causes skeletal muscle wasting in LLC tumor-bearing mice.

Keywords: MyD88; Toll-like receptors; XBP1; atrophy; cancer cachexia; skeletal muscle; unfolded protein response.

FIG 1

Increased gene expression of TLRs and MyD88 during cancer cachexia. Three-month-old male C57BL/6J mice were injected with saline solution alone or with 2 × 10⁶ Lewis lung carcinoma (LLC) cells in the left flank, and tumor growth was monitored. After 21 days, right-side hind limb muscles were isolated and analyzed by QRT-PCR and Western blotting. (A) Relative mRNA levels of TLR1, TLR2, TLR4, TLR7, TLR8, TLR9, MuRF1, and MAFbx in gastrocnemius (GA) muscle of control (saline solution alone injected) and LLC tumor-bearing mice. n = 4 to 6 in each group. (B and C) Relative mRNA (B) and protein (C) levels of MyD88 in GA muscle of control and LLC tumor-bearing mice. Data represent results of densitometry quantification of bands in immunoblots. n = 3 in each group. C2C12 myotubes were incubated in differentiation medium with or without LLC-conditioned medium (LLC-CM) or C26-CM for 24 h followed by performing QRT-PCR or Western blotting. (D and E) Transcript levels of TLR1, TLR2, TLR4, TLR7, TLR8, TLR9, and MAFbx (D) and MyD88 (E) in control and LLC-CM-treated myotubes. (F) Representative immunoblots and densitometry analysis of MyD88 protein in control and LLC-CM-treated myotubes. (G and H) Relative mRNA levels of TLR1, TLR2, TLR4, TLR7, TLR8, TLR9, and MAFbx (G) and MyD88 (H) in control and C26-CM-treated myotubes assayed by QRT-PCR. n = 3 or 4 in each group. Medians and 25th to 75th percentiles are shown. *, P < 0.05 (values significantly different from corresponding control values). ns, not significant.

FIG 2

Targeted ablation of MyD88 prevents skeletal muscle atrophy in LLC tumor-bearing mice. Three-month-old MyD88^f/f and MyD88^myoKO mice were inoculated with 2 × 10⁶ LLC cells in the left flank and monitored for 21 days. (A and B) Quantification of the maximal (A) and average (B) forelimb strength of control and LLC tumor-bearing MyD88^f/f and MyD88^myoKO mice normalized by body weight. (C and D) Quantification of the maximal (C) and average (D) four-paw strength of control and LLC tumor-bearing MyD88^f/f and MyD88^myoKO mice normalized by body weight. (E) Representative photomicrographs of H&E-stained and antilaminin-stained sections of the TA muscle of control and LLC tumor-bearing MyD88^f/f and MyD88^myoKO mice. Bars, 50 μm. (F and G) Quantification of average cross-sectional area (CSA) (F) and minimal Feret’s diameter (G) of myofibers in TA muscle of control and tumor-bearing MyD88^f/f and MyD88^myoKO mice. (H and I) Quantification of average CSA (H) and minimal Feret’s diameter (I) of myofibers in soleus muscle of control and tumor-bearing MyD88^f/f and MyD88^myoKO mice. (J) Quantification of percentage of loss in myofiber CSA after implantation of LLC tumor in TA and soleus muscle of MyD88^f/f and MyD88^myoKO mice. (K) Average tumor wet weight in MyD88^f/f and MyD88^myoKO mice after 21 days of injection of LLC cells. n = 4 to 7/group. Medians and 25th to 75th percentiles are shown. *, P < 0.05 (values significantly different from those determined for MyD88^f/f mice injected with saline solution alone); &, P < 0.05 values significantly different from those determined for LLC-bearing MyD88^f/f mice; ns, not significant.

FIG 3

Ablation of MyD88 inhibits catabolic pathways in skeletal muscle during cancer cachexia. (A) Representative immunoblots demonstrating levels of MyHC, troponin, tropomyosin, and unrelated protein GAPDH in GA muscle of control (S [saline solution]) and LLC tumor-bearing (L) MyD88^f/f and MyD88^myoKO mice. (B) Results of densitometry quantification of band intensities of MyHC, troponin, and tropomyosin from multiple immunoblots. (C) Relative mRNA levels of MAFbx and MuRF1 in GA muscle of control and LLC tumor-bearing MyD88^f/f and MyD88^myoKO mice. (D) Relative mRNA levels of autophagy genes LC3B and Beclin-1 in GA muscle of control and LLC tumor-bearing MyD88^f/f and MyD88^myoKO mice. (E) Representative immunoblots and quantification of ratio of LC3B-II and LC3B-I in GA muscle of control and LLC tumor-bearing MyD88^f/f and MyD88^myoKO mice. (F) Representative immunoblots demonstrating the levels of phosphorylated and total p65, IκBα, and p38 and unrelated protein GAPDH in GA muscle of control and LLC tumor-bearing mice. (G) Densitometry analysis of ratio of p-p65/p65, p-IκBα/IκBα, and p-p38/p38 from multiple immunoblots. n = 3 or 4 mice/group. Medians and 25th to 75th percentiles are shown. *, P < 0.05 (values significantly different from those determined for MyD88^f/f injected with saline solution alone); &, P < 0.05 (values significantly different from those determined for LLC tumor-bearing MyD88^f/f mice); #, P < 0.05 (values significantly different from those determined for MyD88^myoKO mice injected with saline solution alone); ns, not significant.

FIG 4

Genetic ablation of MyD88 in skeletal muscle inhibits the activation of UPR pathways during cancer cachexia. (A) Immunoblots demonstrating levels of p-eIF2α, total eIF2α, total XBP1 (tXBP1), spliced XBP1 (sXBP1), ATF6, MyD88, and unrelated protein tubulin in the GA muscle of control and LLC tumor-bearing MyD88^f/f and MyD88^myoKO mice. (B) Results of densitometry quantification of bands in immunoblots. (C) Relative mRNA levels of ATF4, CHOP, GRP78, and sXBP1 in GA muscle of control and LLC tumor-bearing MyD88^f/f and MyD88^myoKO mice. n = 3 or 4 in each group. Medians and 25th to 75th percentiles are shown. *, P < 0.05 (values significantly different from those determined for MyD88^f/f injected with saline solution alone); &, P < 0.05 (values significantly different from those determined for LLC tumor-bearing MyD88^f/f mice); ns, not significant.

FIG 5

Targeted deletion of XBP1 inhibits skeletal muscle wasting in LLC tumor-bearing mice. (A) Relative mRNA levels of XBP1, EDEM, and SEC61 in GA muscle of naive XBP1^f/f and XBP1^mKO mice. n = 4 in each group. Three-month-old XBP1^f/f and XBP1^mKO mice were inoculated with 2 × 10⁶ LLC cells in the left flank and monitored for 21 days. (B) Representative photomicrographs of H&E-stained and antilaminin-stained sections of TA muscle of control and LLC tumor-bearing XBP1^f/f and XBP1^mKO mice. Bars, 50 μm. (C and D) Quantification of average myofiber CSA (C) and minimal Feret’s diameter (D) in TA muscle of control and LLC tumor-bearing XBP1^f/f and XBP1^mKO mice. (E and F) Quantification of average myofiber CSA (E) and minimal Feret’s diameter (F) in soleus muscle of control and LLC tumor-bearing XBP1^f/f and XBP1^mKO mice. (G) Quantification of percentage of loss in myofiber CSA in TA and soleus muscle of XBP1^f/f and XBP1^mKO mice after 21 days of inoculation with LLC cells. (H) Quantification of the average tumor wet weight in tumor-bearing XBP1^f/f and XBP1^mKO mice. n = 9 to 12 mice/group. (I to K) Relative mRNA levels of MAFbx and MuRF1 (I), LC3B and Beclin-1 (J), and IL-6 (K) in GA muscle of control and LLC-bearing XBP1^f/f and XBP1^mKO mice. n = 4 mice/group. Medians and 25th to 75th percentiles are shown. *, P < 0.05 (values significantly different from those determined for XBP1^f/f mice injected with saline solution alone); &, P < 0.05 (values significantly different from those determined for LLC tumor-bearing XBP1^f/f mice); #, P < 0.05 (values significantly different from those determined for XBP1^mKO mice injected with saline solution alone); ns, not significant.

FIG 6

Overexpression of sXBP1 causes atrophy in cultured myotubes. C2C12 myotubes were transduced with control adenovirus (Ad.Control) or sXBP1-expressing (Ad.sXBP1) for 24 h. The cultures were incubated in differentiation medium for an additional 48 h. (A) Immunoblots presented here demonstrate the levels of sXBP1 and of an unrelated protein, tubulin, in C2C12 cultures transduced with Ad.Control and Ad.sXBP1. (B) Results of densitometry quantification of band intensity in immunoblots for sXBP1 and tubulin. (C) Representative images of control and sXBP1-overexpressing myotube cultures after staining with DAPI. Bar, 50 μm. (D) Quantification of average myotube diameter in control and sXBP1-overexpressing cultures. (E to G) Relative mRNA levels of MAFbx and MuRF1 (E), LC3B and Beclin-1 (F), and IL-6, TNF-α, and TWEAK (G) in control and sXBP1-overexpressing myotube cultures. (H) Immunoblots demonstrating levels of p-p65, p65, p-p38, and p38 in control and sXBP1-overexpressing cultures. (I) Results of densitometry quantification of band intensities in immunoblots. Ratios of p-p65/p65 and p-p38/p38 are presented here. n = 3 or 4 in each group. Medians and 25th to 75th percentiles are shown. *, P < 0.05 (values significantly different from those determined for cultures transduced with Ad.Control).

FIG 7

Knockdown of XBP1 inhibits atrophy of myotubes in response to tumor-derived factors. C2C12 myotubes were transduced with adenoviral vector expressing a scrambled shRNA (Ad.Control shRNA) or XBP1 shRNA (Ad.XBP1 shRNA) for 24 h. The cells were washed and incubated with or without LLC-CM or C26-CM at a 1:4 ratio for an additional 48 h. (A) Representative images of myotube cultures after staining with DAPI. (B and C) Quantification of average myotube diameters in control and XBP1 knocked-down myotube cultures incubated with normal medium or (B) LLC-CM or (C) C26-CM. (D to F) Relative mRNA levels of MAFbx (D), MuRF1 (E), and sXBP1 (F) in myotube cultures transduced with Ad.Control shRNA or Ad.XBP1 shRNA incubated with or without LLC-CM. n = 4 in each group. C2C12 myotubes were transduced with Ad.Control or Ad.XBP1 shRNA for 24 h followed by treatment with normal medium or LLC-CM for 3 h. (G) Immunoblots demonstrating the levels of phosphorylated and total p65, IκBα, and p38 protein and total LC3B-II/I and CHOP protein in control of XBP1 knocked-down cultures. (H) Results of densitometry quantification of band intensities. Data presented here show ratios of p-p65/p65, p-IκBα/IκBα, p-p38/p38, and LC3B-II/I and total levels of CHOP in control and XBP1 knocked-down myotubes incubated with normal medium or LLC-CM. Medians and 25th to 75th percentiles are shown. *, P < 0.05 (values significantly different from those determined for control cultures transduced with Ad.Control shRNA); &, P < 0.05 (values significantly different from those determined for LLC-CM-treated cultures transduced with Ad.Control shRNA); #, P < 0.05 (values significantly different from those determined for control cultures transduced with Ad.XBP1); ns, not significant.