Cancer affects microRNA expression, release, and function in cardiac and skeletal muscle

Abstract

Circulating microRNAs (miRNA) are emerging as important biomarkers of various diseases, including cancer. Intriguingly, circulating levels of several miRNAs are lower in patients with cancer compared with healthy individuals. In this study, we tested the hypothesis that a circulating miRNA might serve as a surrogate of the effects of cancer on miRNA expression or release in distant organs. Here we report that circulating levels of the muscle-enriched miR486 is lower in patients with breast cancer compared with healthy individuals and that this difference is replicated faithfully in MMTV-PyMT and MMTV-Her2 transgenic mouse models of breast cancer. In tumor-bearing mice, levels of miR486 were relatively reduced in muscle, where there was elevated expression of the miR486 target genes PTEN and FOXO1A and dampened signaling through the PI3K/AKT pathway. Skeletal muscle expressed lower levels of the transcription factor MyoD, which controls miR486 expression. Conditioned media (CM) obtained from MMTV-PyMT and MMTV-Her2/Neu tumor cells cultured in vitro were sufficient to elicit reduced levels of miR486 and increased PTEN and FOXO1A expression in C2C12 murine myoblasts. Cytokine analysis implicated tumor necrosis factor α (TNFα) and four additional cytokines as mediators of miR486 expression in CM-treated cells. Because miR486 is a potent modulator of PI3K/AKT signaling and the muscle-enriched transcription factor network in cardiac/skeletal muscle, our findings implicated TNFα-dependent miRNA circuitry in muscle differentiation and survival pathways in cancer.

Figure 1. The sera of transgenic mice with mammary tumors display distinct miRNA profile

A) Principle Component Analysis (PCA) of miRNAs in sera of control, MMTV-Her2/Neu, and MMTV-PyMT mice without normalization. B) PCA of miRNAs in sera of control, MMTV-Her2/Neu, and MMTV-PyMT mice after normalization with miR-202. C) qRT-PCR analysis confirmed down-regulation of miR-486 in sera of transgenic animals with mammary tumors. D) Analysis of circulating miR-486 levels in healthy and metastatic patients using miRNA preparations after spiking sera with C. elegans miR-39 to correct for technical variability. E) miR-486 levels in the normal mammary gland (n, 7–8), MMTV-Her2/Neu (n=6) and MMTV-PyMT-derived mammary tumors (n=10).

Figure 2. Cancer cell-derived soluble factors reduce miR-486 expression in muscle and increase miR-486 target proteins

A) Heart of both MMTV-Her2/Neu and MMTV-PyMT and skeletal muscle of MMTV-Her2/Neu mice with mammary tumors expressed lower levels of miR-486 compared with healthy controls (N=6). B) CM from MMTV-Her2/Neu and MMTV-PyMT tumor-derived cell lines reduced miR-486 expression in C2C12 cells. CM pre-heated at 80 C partially lost the ability to reduce miR-486 expression. Asterisks and dollar sign indicate statistically significant differences. C) CM from MMTV-Her2/Neu and MMTV-PyMT tumor-derived cell lines reduced Ank1 mRNA. D) CM from MMTV-Her2/Neu and MMTV-PyMT tumor-derived cell lines up-regulated miR-486 target proteins in C2C12 cells.

Figure 3. TNFα mediates miR-486 repression

A) Cytokine array identified GM-CSF, CCL2, IFNγ, IL-1α, and TNFα as common cytokines secreted by both MMTV-Her2/Neu and MMTV-PyMT tumor-derived cell lines. B) The effects of CCL2, IFNγ, IL-1α, and TNFα on miR-486 expression in C2C12 cells. C) The effects of CCL2, IFNγ, IL-1α, and TNFα on Ank1 expression in C2C12 cells. D) Neutralizing antibody against TNFα partially reversed the effects of CM from MMTV-Her2/Neu and MMTV-PyMT tumor-derived cell lines on miR-486 expression in C2C12 cells. E) Neutralizing antibody against TNFα reduced the effects of CM from MMTV-Her2/Neu and MMTV-PyMT tumor-derived cell lines on Ank1 expression in C2C12 cells. F) CM from MMTV-Her2/Neu and MMTV-PyMT tumor-derived cell lines contained TNFα. G) Sera of MMTV-Her2/Neu and MMTV-PyMT mice contained higher levels of TNFα compared with control mice.

Figure 4. CM from tumor cell lines decrease miR-486 expression in differentiated C2C12 cells

C2C12 cells were allowed to undergo differentiation by replacing FBS with horse serum. Phase contrast images of myotubes under control or CM-treated conditions are shown. The levels of miR-486 with or without CM treatment are also shown.

Figure 5. Heart and muscle of transgenic mice display defective PI3K/AKT pathway compared with control mice

A) pAKT, PTEN, MyoD, and pp38 levels in the heart of mice. Quantitative differences are shown at the bottom. Extracts from four mice in each group were used. B) pAKT, PTEN, MyoD, and pp38 levels in the muscle of mice. C) Model depicting the effects of cancer on signaling events in heart and muscle leading to lower miR-486 in circulation.