Aging-associated skeletal muscle defects in HER2/Neu transgenic mammary tumor model

Abstract

Background: Loss of skeletal muscle volume and resulting in functional limitations are poor prognostic markers in breast cancer patients. Several molecular defects in skeletal muscle including reduced MyoD levels and increased protein turn over due to enhanced proteosomal activity have been suggested as causes of skeletal muscle loss in cancer patients. However, it is unknown whether molecular defects in skeletal muscle are dependent on tumor etiology.

Methods: We characterized functional and molecular defects of skeletal muscle in MMTV-Neu (Neu+) mice (n= 6-12), an animal model that represents HER2+ human breast cancer, and compared the results with well-characterized luminal B breast cancer model MMTV-PyMT (PyMT+). Functional studies such as grip strength, rotarod performance, and ex vivo muscle contraction were performed to measure the effects of cancer on skeletal muscle. Expression of muscle-enriched genes and microRNAs as well as circulating cytokines/chemokines were measured. Since NF-κB pathway plays a significant role in skeletal muscle defects, the ability of NF-κB inhibitor dimethylaminoparthenolide (DMAPT) to reverse skeletal muscle defects was examined.

Results: Neu+ mice showed skeletal muscle defects similar to accelerated aging. Compared to age and sex-matched wild type mice, Neu+ tumor-bearing mice had lower grip strength (202±6.9 vs. 179±6.8 g grip force, p=0.0069) and impaired rotarod performance (108±12.1 vs. 30±3.9 seconds, P<0.0001), which was consistent with reduced muscle contractibility (p<0.0001). Skeletal muscle of Neu+ mice (n=6) contained lower levels of CD82+ (16.2±2.9 vs 9.0±1.6) and CD54+ (3.8±0.5 vs 2.4±0.4) muscle stem and progenitor cells (p<0.05), suggesting impaired capacity of muscle regeneration, which was accompanied by decreased MyoD, p53 and miR-486 expression in muscles (p<0.05). Unlike PyMT+ mice, which showed skeletal muscle mitochondrial defects including reduced mitochondria levels and Pgc1β, Neu+ mice displayed accelerated aging-associated changes including muscle fiber shrinkage and increased extracellular matrix deposition. Circulating "aging factor" and cachexia and fibromyalgia-associated chemokine Ccl11 was elevated in Neu+ mice (1439.56±514 vs. 1950±345 pg/ml, p<0.05). Treatment of Neu+ mice with DMAPT significantly restored grip strength (205±6 g force), rotarod performance (74±8.5 seconds), reversed molecular alterations associated with skeletal muscle aging, reduced circulating Ccl11 (1083.26 ±478 pg/ml), and improved animal survival.

Conclusions: These results suggest that breast cancer subtype has a specific impact on the type of molecular and structure changes in skeletal muscle, which needs to be taken into consideration while designing therapies to reduce breast cancer-induced skeletal muscle loss and functional limitations.

Keywords: NF-κB; breast cancer; cytokines/chemokines; functional limitations; skeletal muscle.

Figure 1:

Functional limitations in mammary tumor-bearing mice. A. Reduced grip strength force in mammary tumor-bearing Neu+ mice was restored upon oral administration of DMAPT (n=8–12; p=0.0069). B. Reduced rotarod performance in mammary tumor-bearing Neu+ mice was reversed by DMAPT treatment (n=8–12, p<0.0001). * indicates significance between wild type and tumor-bearing groups. # indicates significance between vehicle-treated tumor-bearing group and DMAPT-treated tumor-bearing group. C. Skeletal muscle of tumor-bearing Neu+ mice displayed lower contraction force compared to age and sex-matched controls (p<0.0001). D. Tumors in PyMT+ mice similarly affected skeletal muscle contraction force (p<0.0001).

Figure 2:

Altered levels of CD82+ and CD54+ stem/progenitor cells in the skeletal muscle of tumor-bearing mice. A. Skeletal muscle of Neu+ mice contained lower number of CD82+ (p=0.0407) and CD54+ cells (top panel, p=0.0431). Circles denote CD82+/CD54+ double positive cells. B. Skeletal muscle composition in PyMT+ mice is different from that of Neu+ mice as there were higher numbers of CD82+ cells (p=0.0416) without any changes in CD54+ cells (bottom panel).

Figure 3:

The effect of Neu+ tumor on gene expression in the skeletal muscle. A. Expression pattern of skeletal muscle-enriched genes in wild type, Neu+, and Neu+ DMAPT-treated mice (n=8–12). B. MyoD and p53 protein levels in the skeletal muscle of indicated mice (four per group). Same blots were stained with Ponceau Staining to demonstrate equal loading. C. Quantitative analyses of western blots using the Image J software (p=0.0452). D. mRNA levels of p53 (p=0.0311), Mdm2 and Hey1 (0.0472) in the skeletal muscle of Neu+ mice. * indicates significance between wild type group and tumor-bearing groups. # indicates significance between vehicle-treated tumor-bearing group and DMAPT-treated tumor-bearing group. E. p53 protein levels were lower in the skeletal muscle of PyMT+ mice, which could be reversed by DMAPT treatment (p=0.0092). F. Depletion of p53 protein in the purified skeletal muscle cells of Neu+ mice (p<0.0001). G. Conditioned medium (CM) from Neu+ (p=0.0409), but not PyMT+, tumor-derived cell line downregulated p53 in differentiated but not undifferentiated C2C12 myoblast cell line.

Figure 4:

Tumor-induced changes in miRNAs in circulation and in skeletal muscles. A. Circulating miR-486 was lower in Neu+ mice, which was reversed by DMAPT treatment (n=8–12, p=0.0049). B. Skeletal muscle miR-486 was lower in Neu+ mice (p=0.022), which was partially reversed by DMAPT treatment (n=8–12). * indicates significance between wild type group and tumor-bearing groups. # indicates significance between vehicle-treated tumor-bearing group and DMAPT-treated tumor-bearing group.

Figure 5:

Circulating cytokines/chemokines in Neu+ mice. Cytokine/chemokine levels in plasma of age-matched animals of three groups were assessed using multiplex ELISA kit (n=6 per group). In case of no detectable cytokine/chemokine, a value of zero was given. Only data with at least three animals per group with detectable expression were included. Data were graphed in four panels based on cytokine/chemokine levels. * indicates significance between wild type group and tumor-bearing group (p<0.05). # indicates significance between vehicle-treated tumor-bearing group and DMAPT-treated tumor-bearing group (p<0.05).

Figure 6:

Morphological changes in the skeletal muscles of Neu+ mice. A. Mammary tumors led to significant shrinkage of muscle fibers in Neu+ mice compared to wild type mice. DMAPT treatment reversed muscle shrinkage (top row). Arrows indicate shrunk muscle cells (n=6, p=0.0033). B. Mitochondria content in muscles was measured through IHC of Cox IV. No significant changes between groups (n=8; middle row). C. ECM in the skeletal muscles of Neu+ tumor-bearing mice was higher compared to wild type mice, which was reversed by DMAPT treatment (n=8; bottom row, p<0.0001). * indicates significance between wild type group and tumor-bearing groups. # indicates significance between vehicle-treated tumor-bearing group and DMAPT-treated tumor-bearing group.