Glutamine fuels a vicious cycle of autophagy in the tumor stroma and oxidative mitochondrial metabolism in epithelial cancer cells

Abstract

Glutamine metabolism is crucial for cancer cell growth via the generation of intermediate molecules in the tricarboxylic acid (TCA) cycle, antioxidants and ammonia. The goal of the current study was to evaluate the effects of glutamine on metabolism in the breast cancer tumor microenvironment, with a focus on autophagy and cell death in both epithelial and stromal compartments. For this purpose, MCF7 breast cancer cells were cultured alone or co-cultured with nontransformed fibroblasts in media containing high glutamine and low glucose (glutamine +) or under control conditions, with no glutamine and high glucose (glutamine -). Here, we show that MCF7 cells maintained in co-culture with glutamine display increased mitochondrial mass, as compared with control conditions. Importantly, treatment with the autophagy inhibitor chloroquine abolishes the glutamine-induced augmentation of mitochondrial mass. It is known that loss of caveolin-1 (Cav-1) expression in fibroblasts is associated with increased autophagy and an aggressive tumor microenvironment. Here, we show that Cav-1 downregulation which occurs in fibroblasts maintained in co-culture specifically requires glutamine. Interestingly, glutamine increases the expression of autophagy markers in fibroblasts, but decreases expression of autophagy markers in MCF7 cells, indicating that glutamine regulates the autophagy program in a compartment-specific manner. Functionally, glutamine protects MCF7 cells against apoptosis, via the upregulation of the anti-apoptotic and anti-autophagic protein TIGAR. Also, we show that glutamine cooperates with stromal fibroblasts to confer tamoxifen-resistance in MCF7 cancer cells. Finally, we provide evidence that co-culture with fibroblasts (1) promotes glutamine catabolism, and (2) decreases glutamine synthesis in MCF7 cancer cells. Taken together, our findings suggest that autophagic fibroblasts may serve as a key source of energy-rich glutamine to fuel cancer cell mitochondrial activity, driving a vicious cycle of catabolism in the tumor stroma and anabolic tumor cell expansion.

Expression of glutamine metabolism-related proteins according to molecular subtype of breast cancer

“…Based on the positive correlations between tumoral GLS1-stromal GLS1 (rZ0.191, P!0.001), tumoral GDHstromal GDH (rZ0.106, PZ0.005), and tumoral ASCT2-stromal ASCT2 expression (rZ0.302, P!0.001), we identified a relationship between tumoral and stromal glutamine metabolism. Previous studies have reported that the byproducts of glutaminolysis in cancer cells, such as ammonia, diffuse into the stroma and stimulate autophagy and glutamine synthesis in cancer-associated fibroblasts, which subsequently feeds cancer cells in a vicious cycle (Eng & Abraham 2010, Marino & Kroemer 2010, Pavlides et al 2010, Ko et al 2011, MartinezOutschoorn et al 2011. One previous study utilized co-culturing of MCF-7 breast cancer cells (luminal A type) with fibroblasts and found increased expression of GLS, GDH, and SLC6A14 (glutamine importer) and reduced glutamine neosynthesis when compared with breast cancer single-cell culture without fibroblast, thus demonstrating stroma-tumor glutamine transportation (Ko et al 2011).…”

Cancer metabolism in space and time: Beyond the Warburg effect

“…In deed, re cent work sup ports the hy poth e sis that can cer as so ci ated fi brob lasts (CAFs) un dergo a cata bolic shift to sup ply tu mor cells with ke tone bod ies and glu t a mine [79]. For in stance, Ko et al [80] showed that au tophagy in creased in fi brob lasts grown in the pres ence of MCF7 breast can cer cells. Glu t a mine se creted by au tophagic fi brob lasts in turn fu eled the mi to chon dr ial ac tiv ity of MCF7 can cer cells [79,80].…”

“…For in stance, Ko et al [80] showed that au tophagy in creased in fi brob lasts grown in the pres ence of MCF7 breast can cer cells. Glu t a mine se creted by au tophagic fi brob lasts in turn fu eled the mi to chon dr ial ac tiv ity of MCF7 can cer cells [79,80]. An other study showed that prostate can cer cells can redi rect the me tab o lism of ad ja cent fi brob lasts to wards a gly colytic phe no type.…”

Ethanol exposure induces the cancer-associated fibroblast phenotype and lethal tumor metabolism

“…25 Thus, we explored the possibility that ethanol exposure protects MCF7 cells against autophagy. Figure 3D shows that ethanol treatment decreases the expression of several key autophagy markers, namely BNIP3, LC3, Cathepsin B and, to a lesser extent, Lamp-1, suggesting that ethanol promotes autophagy resistance in epithelial cancer cells.…”