Inhibition of ER stress and unfolding protein response pathways causes skeletal muscle wasting during cancer cachexia

Abstract

Cachexia is a devastating syndrome that causes morbidity and mortality in a large number of patients with cancer. However, the mechanisms of cancer cachexia remain poorly understood. Accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes stress. The ER responds to this stress through activating certain pathways commonly known as the unfolding protein response (UPR). The main function of UPR is to restore homeostasis, but excessive or prolonged activation of UPR can lead to pathologic conditions. In this study, we examined the role of ER stress and UPR in regulation of skeletal muscle mass in naïve conditions and during cancer cachexia. Our results demonstrate that multiple markers of ER stress are highly activated in skeletal muscle of Lewis lung carcinoma (LLC) and Apc(Min/+) mouse models of cancer cachexia. Treatment of mice with 4-phenylbutyrate (4-PBA), a chemical chaperon and a potent inhibitor of ER stress, significantly reduced skeletal muscle strength and mass in both control and LLC-bearing mice. Blocking the UPR also increased the proportion of fast-type fibers in soleus muscle of both control and LLC-bearing mice. Inhibition of UPR reduced the activity of Akt/mTOR pathway and increased the expression of the components of the ubiquitin-proteasome system and autophagy in LLC-bearing mice. Moreover, we found that the inhibition of UPR causes severe atrophy in cultured myotubes. Our study provides initial evidence that ER stress and UPR pathways are essential for maintaining skeletal muscle mass and strength and for protection against cancer cachexia.-Bohnert, K. R., Gallot, Y. S., Sato, S., Xiong, G., Hindi, S. M., Kumar, A. Inhibition of ER stress and unfolding protein response pathways causes skeletal muscle wasting during cancer cachexia.

Keywords: Lewis lung carcinoma; PERK; XBP-1; autophagy; mTOR.

Figure 1.

Activation of ER stress in mouse models of cancer cachexia. LLC cells were implanted in the left flank of wild-type mice, and tumor growth was monitored. Right hindlimb muscles were isolated and used to measure the levels of various markers of ER stress. A) Relative mRNA levels of PERK, IRE1α, XBP-1, ATF6, GRP78, GRP94, GADD34, and DR5 in GA muscle of control and LLC-bearing mice. B) Western blot analysis of levels of phosphorylated and total eIF2α and total CHOP and of total GADD34 protein in GA muscle of control and LLC-bearing mice. C) Densitometry quantification of phosphorylated vs. total eIF2α ratio and CHOP in GA muscle of control and LLC-bearing mice. D) Spliced (s)XBP-1 and unspliced (u)XBP-1 levels in control and LLC-bearing mice measured by semiquantitative RT-PCR assay using primers that detected both sXBP-1 and uXBP-1. Black vertical lines in gel images indicate that intervening lanes have been spliced out. E) Relative mRNA levels of PERK, IRE1α, XBP-1, ATF6, GRP78, GRP94, GADD34, and DR5 in GA muscle of 4-mo-old control and Apc^Min/+ mice measured by qRT-PCR. Error bars represent sd (n = 4/group); unpaired Student’s t test. *P < 0.05, vs. control mice.

Figure 2.

Activation of UPR markers in cultured myotubes treated with LLC-CM. Primary myotubes prepared from WT mice were treated with LLC-CM in a 1:4 ratio for 24 h and processed for a qRT-PCR assay or Western blot analysis. A) Relative mRNA levels of PERK, ATF6, GRP94, GADD34, CHOP, ATF4, and DR5 in control and LLC-CM-treated myotubes. B) Immunoblots demonstrate the levels of phosphorylated and total eIF2α and ATF6, CHOP, and GADD34 protein in control and LLC-CM-treated myotubes. C) Densitometry quantification of phosphorylated vs. total p-eIF2α and total ATF6 and CHOP in control and LLC-CM-treated myotubes (n = 3/group). Error bars represent sd; unpaired Student’s t test. *P < 0.05, vs. control cultures.

Figure 3.

ER stress inhibitor 4-PBA causes loss of muscle strength and mass in naïve conditions and in LLC-bearing mice. C57BL6 12-wk-old mice were inoculated with 2 × 10⁶ LLC cells in the left flank. They were also treated daily with 4-PBA (100 mg/kg body weight, i.p.). After 18 d, muscle strength, body weight, and wet muscle mass were measured. A) Average body weight of mice in each group. B, C) Maximum (B) and average (C) forelimb grip strength of mice in each group. D, E) Maximum (D) and average (E) total 4-paw grip strength of mice in each group. F–H) Average wet weight of isolated soleus (F), TA (G), and GA (H) muscle in each group. I) Wet weight of tumor after 18 d of inoculation with LLC in vehicle alone or 4-PBA-treated mice (n = 5/group). Error bars represent sd; paired Student’s t test. *P < 0.05, vs. control mice; ^#P < 0.05, vs. LLC-inoculated mice.

Figure 4.

Inhibition of ER stress by 4-PBA exacerbates skeletal muscle atrophy in control and LLC-bearing mice. A) Representative photomicrographs of H&E-stained sections of mouse TA muscle. Scale bar, 10 µm. B) Quantification of average fiber CSA of TA muscle in control and LLC-bearing mice, with or without 4-PBA treatment. C) Representative photomicrographs of H&E-stained sections of soleus muscle. Scale bar, 10 µm. D) Quantification of average fiber CSA of soleus muscle in control and LLC-bearing mice, with or without 4-PBA treatment. E) GA muscle of vehicle, 4-PBA, LLC, and LLC+4-PBA mice were processed for Western blot analysis to detect levels of specific muscle proteins. Representative immunoblots demonstrate the levels of MyHC, troponin, tropomyosin, sarcomeric α-actin, and unrelated protein α-tubulin. F) Densitometry quantification of MyHC. G) Representative immunoblots demonstrate that the levels of phosphorylated and total eIF2α, total IRE1, total sXBP-1, and ATF6 were reduced by treatment with 4-PBA, confirming suppression of ER stress. H) Transcript levels of ATF4 in GA muscle of vehicle, 4-PBA alone, LLC alone, and LLC+4-PBA groups measured by performing qRT-PCR assay. Black vertical lines in immunoblots indicate that intervening lanes have been spliced out (n = 5/group). Error bars represent sd; paired Student’s t test. *P < 0.05, vs. mice treated with vehicle alone; ^#P < 0.05 vs. LLC-inoculated mice.

Figure 5.

Blocking ER stress increases the proportion of fast-type fibers in normal and LLC-bearing mice. Soleus muscle sections prepared from vehicle, 4-PBA, LLC, and LLC+4-PBA mice were subjected to triple immunostaining against MyHC I, IIA, and IIB protein. A) Representative photomicrographs of triple-stained sections of soleus muscle. Scale bar, 50 µm. B) Quantification of the percentage of each fiber type in different groups. C) Average CSA of type I and IIa fibers in soleus muscle of mice in each group (n = 5 in each group). Error bars represent sd; paired Student’s t test. *P < 0.05, vs. control mice; ^#P < 0.05, vs. LLC-inoculated mice.

Figure 6.

The effect of 4-PBA treatment on the activation of ubiquitin proteasome system and autophagy. GA muscle of vehicle, 4-PBA, LLC, and LLC+4-PBA mice were processed for Western blot and qRT-PCR analyses. A) Representative immunoblots demonstrate the levels of ubiquitinated proteins in GA muscle of mice in each group. B) Relative mRNA levels of MAFBx, MuRF1, MUSA1, and TRAF6 in GA muscle measured by performing qRT-PCR assay. C) Representative immunoblots demonstrates the levels of LC3B-I and -II, Beclin1, p62, and the unrelated protein GAPDH in GA muscle of mice. D) Densitometry quantification of the ratio of LC3B-II vs. LC3B-I and total levels of Beclin1, and p62. Black vertical lines in immunoblots indicate that intervening lanes have been spliced out (n = 4/group). Error bars represent sd; paired Student’s t test. *P < 0.05, vs. control mice; ^#P < 0.05, vs. LLC inoculated mice.

Figure 7.

Effects of 4-PBA on activation of Akt/mTOR and AMPK signaling pathways in skeletal muscle of normal and LLC-bearing mice. GA muscle of vehicle-, 4-PBA-, LLC-, and LLC+4-PBA-treated mice were processed by Western blot analysis. A) Representative immunoblots demonstrate the levels of phosphorylated and total Akt, mTOR, p70S6 kinase (p70S6K), rpS6, and the unrelated protein α-tubulin. Black vertical lines indicate that intervening lanes have been spliced out. B) Quantification of phosphorylated and total Akt, mTOR, p70S6K, and rpS6 levels after normalizing with α-tubulin in GA muscle of vehicle-, 4-PBA-, LLC-, and LLC+4-PBA-mice. C) Fold change in enzymatic activity of AMPK in GA muscle of vehicle-, 4-PBA-, LLC-, and LLC+4-PBA-treated mice measured with a commercially available kit (n = 4/group). Error bars represent sd; paired Student’s t test. *P < 0.05, vs. control mice; ^#P < 0.05, vs. LLC-inoculated mice.

Figure 8.

Inhibition of ER stress causes atrophy in cultured primary myotubes. Differentiated myotubes were treated with vehicle alone, 4-PBA (5 mM), LLC-CM (1:4 ratio), or LLC-CM+4-PBA for 24 h. A) Representative photomicrographs of myotubes in each group taken at 24 h. Scale bar, 10 µm. B) Average myotube diameter in each group. C) Immunoblots demonstrate the levels of phosphorylated and total eIF2α, sXBP-1, and tXBP-1 in cultured myotubes after treatment with 4-PBA (5 mM) for 24 h. D) Relative mRNA levels of the E3 ubiquitin ligases MuRF1, MAFBx, MUSA1, and TRAF6 and the autophagy marker LC3B and Beclin1 in vehicle, 4-PBA, LLC-CM, or LLC-CM+4-PBA-treated myotubes measured after 12 and 24 h. E) Representative immunoblot (top) from the Sunset assay demonstrating relative amounts of newly synthesized protein in each condition. Equal loading of protein in each lane was confirmed by staining nitrocellulose membrane with Ponceau S dye (bottom). F) Immunoblots demonstrate the levels of phosphorylated and total Akt, mTOR, and rpS6 after 12 h in the indicated groups (n = 3/group). Error bars represent sd; paired Student’s t test *P < 0.05, vs. control cultures; ^#P < 0.05, vs. LLC-CM-treated myotube cultures at corresponding time points.