As solid tumors form, a number of physiological changes occur within the tumor, including low oxygen levels (hypoxia) and an accumulation of lactic acid with concomitant lowered pH levels (lactic acidosis). In order to adapt to and survive in the strong selective pressures imposed by these tumor microenvironment (TME) “stresses”, cancer cells exhibit significant genetic and metabolic changes. While transcriptional responses and signaling events triggered by stresses are well-studied, a systematic genome-wide, forward-genetics screen to identify which genes are critical or restrictive for cell survival in these TME stresses has not been examined. The purpose of this study is to better understand the function of genes that modulate cell survival under TME stresses in order to improve our knowledge of tumor evolution and allow for the development of treatments to specifically target cancer cells under these stresses.

We performed a contextual short-hairpin RNA (shRNA) pooled screen in cancer cells cultured under control, hypoxia or lactic acidosis conditions. Genomic DNA was recovered from each condition to amplify the resulting shRNAs by PCR before quantifying them by microarray. A number of criteria were used to select top candidate hits, including multiple shRNAs targeting a single gene needing to pass stringent statistical criteria. We have successfully validated a number of candidate genes with multiple shRNAs. Importantly, our analysis identified HIF-2α (EPAS1) as critical for cells to survive under hypoxia and this result has been validated.

One “multiple hairpin hit” was ACACA. This gene encodes acetyl-CoA carboxylase (ACC1), which is the enzyme that mediates the rate-limiting step of de novo lipogenesis by converting acetyl-CoA to malonyl-CoA. Validation experiments showed that inhibition of ACC1 by four different shRNAs protected cells from hypoxia-induced apoptosis in several cell lines. This protective affect was specific to hypoxia, since cells were not protected under lactic acidosis or glutamine deprivation. Further interrogation of related genes revealed that the depletion of ATP citrate lyase (ACLY) also prevented hypoxia-induced apoptosis. ACLY acts upstream of ACC1, converting citrate to acetyl-CoA. Metabolomic and transcriptomic analysis revealed that the silencing of ACC1 or ACLY significantly diminished the expected hypoxia and stress responses of these cells. We hypothesize that the metabolic shift resulting from the depletion of ACC1 or ACLY switches cancer cells from a “nutrient rich/proliferative” state to a “nutrient poor/survival” state, allowing cells to better cope with hypoxia. Consistent with this concept, when the gene signatures of ACC1 or ACLY depletion were used to stratify patients in breast cancer datasets, the inactivation of these genes correlated with reduced proliferative and hypoxia pathways and more favorable outcomes. These results may have important implications for the use of metformin and lipogenic inhibitors in the clinic.

Our forward-genetics screen has identified novel genes and biological processes that impact the survival of cancer cells under TME stresses. These findings inform our understanding of the selection of somatic mutations in cancer cells and help reveal potential new therapeutic strategies to selectively target solid human cancers.

This abstract is also presented as Poster B1.

Citation Format: Melissa M. Keenan, Beiyu Liu, Jianli Wu, Derek Cyr, Joseph Lucas, Deborah M. Muoio, So Young Kim, Jen-Tsan Chi. A genome-wide RNAi screen reveals a protective role of decreased lipogenesis under hypoxia. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR09. doi:10.1158/1538-7445.CHTME14-PR09