Muscle wasting and impaired myogenesis in tumor bearing mice are prevented by ERK inhibition

Abstract

Background: The onset of cachexia is a frequent feature in cancer patients. Prominent characteristic of this syndrome is the loss of body and muscle weight, this latter being mainly supported by increased protein breakdown rates. While the signaling pathways dependent on IGF-1 or myostatin were causally involved in muscle atrophy, the role of the Mitogen-Activated-Protein-Kinases is still largely debated. The present study investigated this point on mice bearing the C26 colon adenocarcinoma.

Methodology/principal findings: C26-bearing mice display a marked loss of body weight and muscle mass, this latter associated with increased phosphorylated (p)-ERK. Administration of the ERK inhibitor PD98059 to tumor bearers attenuates muscle depletion and weakness, while restoring normal atrogin-1 expression. In C26 hosts, muscle wasting is also associated with increased Pax7 expression and reduced myogenin levels. Such pattern, suggestive of impaired myogenesis, is reversed by PD98059. Increased p-ERK and reduced myosin heavy chain content can be observed in TNFα-treated C2C12 myotubes, while decreased myogenin and MyoD levels occur in differentiating myoblasts exposed to the cytokine. All these changes are prevented by PD98059.

Conclusions/significance: These results demonstrate that ERK is involved in the pathogenesis of muscle wasting in cancer cachexia and could thus be proposed as a therapeutic target.

Figure 1. ERK is activated in the skeletal muscle of TB animals.

Levels of phosphorylated MAPKs in the GSN. Panel A: control (C; n = 6) and AH-130-bearing rats (n = 8). Panel B: control (C, n = 8) and C26-bearing mice (n = 8). Levels of phosphorylation were normalized by total protein content. Data (means ± SD) expressed as percentages of controls. Significance of the differences: *p<0.05 vs C.

Figure 2. PD98059 counteracts the onset of cachexia in C26-bearing mice.

(A) Body weight, expressed as percent changes respect to initial body weight (i.b.w., C = 19.14±1.68 g; C26 = 19.14±0.69 g; PD = 18.57±2.37 g; C26 PD = 17.56±0.89 g), (B) muscle weight, (C) voluntary strength (grasping test), (D) atrogin-1 and p-c-Jun protein expression in the GSN of controls (n = 6) and C26 hosts (n = 8) either untreated or administered PD (1 mg/kg; see Materials and methods). Densitometric quantifications were normalized according to tubulin levels. Data (means ± SD) expressed as percentages of controls. Significance of the differences: *p<0.05 vs C; **p<0.01 vs C; ***p<0.001 vs C; $ p<0.05 vs C26; $$ p<0.01 vs C26.

Figure 3. ERK inhibition prevents TNFα-induced alterations in C2C12 myotube cultures.

C2C12 myotubes (5 days differentiation) treated for 48 h with 100 ng/ml TNFα, in the presence or in the absence of PD (20μM). (A) MyHC immunostaining (red: MyHC; blue: nuclei). (B) Protein expression levels of p-ERK, MyHC, p-Akt and atrogin-1, the latter two evaluated at both 6 and 48 h (see Li et al., 2005). Densitometric quantifications were normalized according to tubulin levels. Data (means ± SD; n = 3) expressed as percentages of controls. Significance of the differences: *p<0.05 vs C; $ p<0.05 vs TNFα.

Figure 4. Follistatin overexpression prevents TNFα-induced MyHC loss without interfering with ERK activation.

C2C12 myotubes (5 days differentiation), transfected or not with myc-follistatin (FST) and treated or not with 100 ng/ml TNFα for 48 h. (A) Phase contrast microscopy showing the hypertrophy induced by FST in cells treated or not with TNFα. (B) Western blots for MyHC, p-ERK and p-Akt protein levels and (C) corresponding densitometric analysis normalized by tubulin. Data (means ± SD; n = 3) expressed as percentage of controls. Significance of the differences: *p<0.05 vs C; $ p<0.05 vs TNFα.

Figure 5. ERK inhibition restores the myogenic potential in TB mice.

(A) Immunostaining of Pax7, laminin and caveolin1 (Cav1) in tibialis transverse sections of controls and TB mice. (B) Pax7, MyoD and myogenin protein expression in the GSN of controls (n = 6) and TB mice (n = 8) either untreated or administered PD (1 mg/kg) and (C) corresponding densitometric analysis normalized by tubulin. Data (means ± SD) are expressed as percentages of controls. Significance of the differences: **p<0.01 vs C; $ p<0.05 vs C26; $$ p<0.01 vs C26.

Figure 6. ERK inhibition promotes myogenic differentiation of C2C12 cells.

Subconfluent C2C12 cells switched to DM and simultaneously treated for 48 h with 100 ng/ml TNFα in the presence or in the absence of PD (20μM). (A) MyHC immunostaining. (B) Protein expression levels of p-ERK, MyHC, p-Akt, MyoD, myogenin and Pax7. Densitometric quantifications were normalized according to tubulin levels. Data (means ± SD; n = 3) expressed as percentages of controls. Significance of the differences: *p<0.05 vs C; **p<0.01 vs C; $ p<0.05 vs TNFα; $$ p<0.01 vs TNFα.