Nianli Sang

Abstract Glutamine is a critical nutrient for proliferating cells and most tumor cells. Glutamine addiction as one of the dramatic tumor cell adaptive metabolisms is characterized by increased glutaminolysis. To better understand how tumor cells respond to glutamine deficiency, we cultured Hep3B cells in glutamine free media supplemented with ammonia, and isolated survival clones (MM01). These clones are capable of perpetual survival in glutamine free media with ammonia, but assume a slow growing phenotype, which represents the model adapted to long-term glutamine insufficiency. Comparing MM01 and Hep3B by microarray-based genome-wide gene expression profiling, we identified functional activation of Smad2/3 in MM01 cells, suggesting that long-term glutamine insufficiency activates a TGF-β signaling pathway. We have validated that glutamine insufficiency triggers Samd2 phosphorylation, which in turn, stimulates the expression of p15INK4B, providing a link to the slow growing phenotype. To elucidate how glutamine insufficiency triggers TGF-β signaling, we examined the effects of glutamine insufficiency on the expression levels of TGF-β family ligands, and found that Inhibin-βE, a newly discovered inhibin subunit isoform in the TGF-β superfamily, is up-regulated at both mRNA and protein levels in MM01. Luciferase assays demonstrate that glutamine insufficiency enhances the activity of Inhibin-βE promoter. Furthermore, we show that overexpressing Inhibin-βE is sufficient to induce Smad2/3 activation and growth inhibition in Hep3B cells. Taken together, our data suggest that Inhibin-βE likely plays a crucial role in cell adaptation to metabolic stress, facilitating cancer cell survival by slowing down biosynthesis and proliferation. A better understanding of the novel Inhibin-βE-triggered TGF-β signaling pathway may provide potential new therapeutic targets for cancer treatment. Citation Format: Dan He, Chengqian Yin, Shuo Qie, Shuyang Chen, Nianli Sang. A novel TGF-β signaling pathway promotes a slow growing phenotype of cancer cells in response to glutamine insufficiency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3562. doi:10.1158/1538-7445.AM2017-3562