Breast cancer cell-conditioned media inhibit growth and reduce basal and insulin-stimulated glucose uptake by inhibiting Rac1 activation in rat myotubes.

Abstract

Metabolic disorders are common in women with breast cancer, raising mortality and recurrence rates, but their causes remain poorly understood. Given the importance of skeletal muscle metabolism in glucose homeostasis, we investigated the effect of breast cancer cell-conditioned media on insulin-stimulated glucose uptake in muscle. Rat L6 myotubes overexpressing myc-tagged GLUT4 were incubated with 40% conditioned media from tumourigenic MCF7 or BT474, or non-tumourigenic control MCF10A breast cells. Mass-spectrometry-based proteomics was applied to detect molecular rewiring in response to breast cancer in the muscle. Expression of myogenesis and inflammation markers, GLUT4 translocation, [³H]2-deoxyglucose uptake, and intramyocellular insulin signalling were determined. Breast cancer cell-conditioned media induced proteomic changes in pathways linked to sarcomere organisation, actin filament binding and vesicle trafficking. Myogenic differentiation was disrupted, marked by a 50% increase in Mki67 mRNA and trend (P = 0.087) towards reduced myosin heavy chain expression, as shown by immunofluorescence. Additionally, breast cancer cell-conditioned media activated inflammation via nuclear factor-κB and interleukin-6 signalling and reduced myotube width by 70% (P = 0.0524). Myotubes treated with breast cancer cell-conditioned media had a reduced basal and insulin-stimulated GLUT4 translocation and glucose uptake. Insulin signalling via the Rho GTPase Rac1 was reduced by 40%, while absolute Akt-TBC1D4 phosphorylation was unaffected. Conditioned media from MCF7 and BT474 breast cancer cells altered skeletal muscle proteome, induced inflammation, lowered growth markers, reduced glucose uptake, inhibited GLUT4 translocation and blocked insulin-stimulated Rac1 activation. These findings indicate that the rewiring of skeletal muscle could play a role in metabolic dysfunction in patients with breast cancer. KEY POINTS: Metabolic disorders in breast cancer increase mortality and cancer recurrence. Here, we show that incubation with breast cancer cell-conditioned media (CM) alters the proteome in rat skeletal muscle cells. In addition, breast cancer CM activates NF-κB and type 1 interferon pathways, inhibiting muscle growth. Moreover, breast cancer CM inhibits basal and insulin-mediated GLUT4 translocation and glucose uptake, likely by blocking insulin-stimulated Rac1, but not Akt-TBC1D4 activation. These results underscore a potential mechanistic link between breast cancer and metabolic disorders and suggest that skeletal muscle rewiring may play a role.