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The causes and consequences of metabolic reprogramming during human breast cancer metastasis remain largely unknown. We performed metabolomics in the human triple-negative breast cancer cell line MDA-MB-231 (231-Parental) and its metastatic subclones that display tissue-tropism towards the brain (831-BrM), the bone (1833-BoM), and the lung (4175-LM). Using molecular and metabolic flux analysis, we uncovered that the mitochondrial serine and one-carbon (1C) unit pathway is upregulated in the metastatic subclones. Inhibition of the first rate-limiting enzyme of the pathway, serine hydroxymethyltransferase (SHMT2), potently suppresses proliferation of metastatic subclones in culture and impairs growth of lung metastatic subclones at both primary and metastatic sites in mice. In addition to breast cancer, a few other cancer types, such as adrenocortical carcinoma (ACC) and kidney chromophobe cell carcinoma (KICH), also display increased SHMT2 expression during disease progression. Moreover, we found that metastatic cells are exquisitely adapted to low serine conditions by synthesizing serine de novo from glucose to generate 1C units, and blocking serine catabolism abrogates metastatic growth under serine deprivation. How do metastatic breast cancer cells acquire new metabolic properties such as de novo serine synthesis? We observed that 231-Parental cells grown in media lacking serine for up to ten passages exhibited metastatic cell-like metabolic properties. Subsequent RNA-sequencing revealed dramatic global gene expression changes during serine starvation in more primary tumor-like cells. Several pathways involved in metastasis such as TNFα signaling, mTOR1 signaling, and Ras signaling were modulated upon serine starvation. These data suggest that the availability of the nonessential amino acid serine may play a key regulatory role in breast cancer progression and metastasis, and highlight the potential for metabolite supplementation to block or halt these transformations. Citation Format: Albert Mao Li, Gregory S. Ducker, Yang Li, Jose A. Seoane, Yiren Xiao, Stavros Melemenidis, Yiren Zhou, Ling Liu, Sakari Vanharanta, Edward E. Graves, Erinn B. Rankin, Christina Curtis, Joan Massague, Joshua D. Rabinowitz, Craig B. Thompson, Jiangbin Ye. Reprogramming of serine metabolism during breast cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5713.

The Warburg effect is a metabolic hallmark of all cancer cells, characterized by increased glucose uptake and glycolysis for lactate generation. The generation and excretion of lactate would appear be a waste of carbon backbone and energy that is needed for proliferation. It was proposed by Warburg that the cause and consequence of the Warburg effect were the injury of respiration and cell dedifferentiation, respectively. One common factor that damages mitochondrial respiration is hypoxia, which is a metabolic stress that blocks cell differentiation and promotes cancer progression. The underlying mechanism by which this occurs is poorly understood, and no effective therapeutic strategy has been developed to overcome this resistance to differentiation. Using a neuroblastoma (NB) differentiation model, we have discovered that hypoxia represses the differentiation induced by retinoic acid (RA) as demonstrated by loss of neuron differentiation markers and changes in cell morphology, associated with reduction of global histone acetylation, that are caused by the induction of pyruvate dehydrogenase kinases (PDKs). PDKs phosphorylate pyruvate dehydrogenase (PDH), thereby blocking pyruvate entry into the TCA cycle, reducing acetyl-CoA generation, and promoting the Warburg effect. Genetic and pharmaceutical inhibition of PDK restores histone acetylation and NB cell differentiation morphology. Acetate supplementation restores histone acetylation, along with differentiation markers expression and neuron differentiation. In addition, ATAC-Seq analysis demonstrated that hypoxia treatment significantly reduces chromatin accessibility at RAR/RXR binding sites, which can be restored by acetate supplementation. These findings suggest that (1) combining RA and acetate supplementation represents a potentially effective therapeutic strategy for neuroblastoma treatment; (2) diverting pyruvate away from acetyl-CoA generation is a key mechanism by which the Warburg effect blocks cell differentiation. Citation Format: Jiangbin Ye, Yang Li, Joshua J. Gruber, Ulrike M. Litzenburger, Yiren Zhou, Yu R. Miao, Edward L. LaGory, Albert M. Li, Zhen Hu, Lori S. Hart, John M. Maris, Howard Y. Chang, Amato J. Giaccia. Deciphering Warburg effect: hypoxia inhibits tumor cell differentiation through reducing acetyl-CoA generation and chromatin accessibility [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5708.

Neuroblastoma is the most common extracranial solid tumor in childhood. Retinoic acid (RA) leads to cell cycle arrest and induces cell differentiation in neuroblastoma cells, thus it has been used to treat neuroblastoma as differentiating agent for decades. However, therapy resistance often occurs and the overall survival remains low. Hypoxia, a common metabolic stress existing in tumor microenvironment, increases tumor resistance to RA by promoting dedifferentiation of neuroblastoma cells. The aim of this study was to elucidate the mechanisms underlying hypoxia-mediated therapy resistance and develop therapeutic approach to improve the response to differentiation therapy. By RNA-Seq analysis, we identified a group of neuron-specific differentiation markers that induced by RA treatment, while the induction was diminished under hypoxia. Associated with the silencing of differentiation markers expression, a significant loss of histone acetylation of H3K9, H3K27 and total H3 was observed under hypoxia. Metabolomics analysis indicated hypoxia inhibited mitochondrial oxidative phosphorylation to decrease intracellular acetyl-CoA and citrate, which provides acetyl-CoA for histone acetyltransferase. Importantly, acetate supplementation restored cellular acetyl-CoA level, histone acetylation, and the expression of differentiation markers under hypoxia. Differentiation morphology was also restored by acetate supplementation under hypoxia. In conclusion, our finding suggested hypoxia contributed to the resistance to RA-induced differentiation by reprogramming cellular metabolism and inducing histone hypoacetylation. Acetate supplementation is an efficacious therapeutic approach to improve the sensitivity of hypoxic tumor to RA-based differentiation therapy. Citation Format: Yang Li, Yiren Zhou, John M. Maris, Amato J. Giaccia, Jiangbin Ye. Acetate supplementation eliminates hypoxia mediated resistance to differentiation therapy in neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3798.