RESUMEN
BACKGROUND: Breast cancer clinical outcome relies on its intrinsic molecular subtype and mortality is almost exclusively due to metastasis, whose mechanism remains unclear. We recently revealed the specific contribution of plasma membrane cholesterol to the invasion of malignant MCF10CAIa but not premalignant MCF10AT and normal MCF10A cell lines in 2D, through invadopodia formation and extracellular matrix (ECM) degradation. In the present study, we address the impact of breast cancer subtypes, mutations and aggressiveness on cholesterol implication in breast cancer cell invasion and 3D spheroid invasion and growth. METHODS: We used nine breast cancer cell lines grouped in four subtypes matching breast tumor classification. Four of these cell lines were also used to generate 3D spheroids. These cell lines were compared for cell invasion in 2D and 3D, spheroid growth in 3D, gelatin degradation, cortactin expression, activation and subcellular distribution as well as cell surface cholesterol distribution and lipid droplets. The effect of plasma membrane cholesterol depletion on all these parameters was determined in parallel and systematically compared with the impact of global matrix metalloproteinase (MMP) inhibition. RESULTS: The six invasive cell lines in 2D were sensitive to partial cholesterol depletion, independently of their subtype, aggressiveness or mutation. Nevertheless, the effect was stronger in the three cell lines able to degrade gelatin. 3D spheroid invasion was also reduced after cholesterol depletion in all breast cancer subtypes tested. Notably, targeting cholesterol was more powerful than MMP inhibition in reducing invasion in both 2D and 3D culture models. Moreover, cholesterol depletion in the six invasive cell lines impaired cortactin distribution in the perinuclear region where invadopodia localized. Breast cancer cell line aggressiveness relied on cholesterol-enriched domains at the ECM-free side and intracellular lipid droplets. Furthermore, the three gelatin-degrading cell lines were characterized by increased cholesterol-enriched submicrometric domains at their ECM-contact side. CONCLUSION: Together, our data suggest cell surface cholesterol combined with lipid droplet labeling as a breast cancer cell aggressiveness marker. They also open the way to test other cholesterol-targeting drugs in more complex models to further evaluate whether cholesterol could represent a strategy in breast cancer therapy.
RESUMEN
Energy homeostasis in the central nervous system largely depends on astrocytes, which provide metabolic support and protection to neurons. Astrocytes also ensure the clearance of extracellular glutamate through high-affinity transporters, which indirectly consume ATP. Considering the role of the AMP-activated protein kinase (AMPK) in the control of cell metabolism, we have examined its implication in the adaptation of astrocyte functions in response to a metabolic stress triggered by glucose deprivation. We genetically modified the astrocyte-like C6 cell line to silence AMPK activity by overexpressing a dominant negative mutant of its catalytic subunit. Upon glucose deprivation, we found that C6 cells maintain stable ATP levels and glutamate uptake capacity, highlighting their resilience during metabolic stress. In the same conditions, cells with silenced AMPK activity showed a reduction in motility, metabolic activity, and ATP levels, indicating that their adaptation to stress is compromised. The rate of ATP production remained, however, unchanged by AMPK silencing, suggesting that AMPK mostly influences energy consumption during stress conditions in these cells. Neither AMPK modulation nor prolonged glucose deprivation impaired glutamate uptake. Together, these results indicate that AMPK contributes to the adaptation of astrocyte metabolism triggered by metabolic stress, but not to the regulation of glutamate transport.
