Daniel Ackerman

Hypoxia, lipids, and cancer: surviving the harsh tumor microenvironment

Solid tumors typically develop hostile microenvironments characterized by irregular vascularization and poor oxygen (O2) and nutrient supply. Whereas normal cells modulate anabolic and catabolic pathways in response to changes in nutrient availability, cancer cells exhibit unregulated growth even under nutrient scarcity. Recent studies have demonstrated that constitutive activation of growth-promoting pathways results in dependence on unsaturated fatty acids for survival under O2 deprivation. In cancer cells, this dependence represents a critical metabolic vulnerability that could be exploited therapeutically. Here we review how this dependence on unsaturated lipids is affected by the microenvironmental conditions faced by cancer cells.

Dysregulated mTORC1 renders cells critically dependent on desaturated lipids for survival under tumor-like stress

Solid tumors exhibit heterogeneous microenvironments, often characterized by limiting concentrations of oxygen (O2), glucose, and other nutrients. How oncogenic mutations alter stress response pathways, metabolism, and cell survival in the face of these challenges is incompletely understood. Here we report that constitutive mammalian target of rapamycin complex 1 (mTORC1) activity renders hypoxic cells dependent on exogenous desaturated lipids, as levels of de novo synthesized unsaturated fatty acids are reduced under low O2. Specifically, we demonstrate that hypoxic Tsc2(-/-) (tuberous sclerosis complex 2(-/-)) cells deprived of serum lipids exhibit a magnified unfolded protein response (UPR) but fail to appropriately expand their endoplasmic reticulum (ER), leading to inositol-requiring protein-1 (IRE1)-dependent cell death that can be reversed by the addition of unsaturated lipids. UPR activation and apoptosis were also detected in Tsc2-deficient kidney tumors. Importantly, we observed this phenotype in multiple human cancer cell lines and suggest that cells committed to unregulated growth within ischemic tumor microenvironments are unable to balance lipid and protein synthesis due to a critical limitation in desaturated lipids.

Imaging Cancer Metabolism: Underlying Biology and Emerging Strategies

Dysregulated cellular metabolism is a characteristic feature of malignancy that has been exploited for both imaging and targeted therapy. With regard to imaging, deranged glucose metabolism has been leveraged using 18F-FDG PET. Metabolic imaging with 18F-FDG, however, probes only the early steps of glycolysis; the complexities of metabolism beyond these early steps in this single pathway are not directly captured. New imaging technologies—both PET with novel radiotracers and MR-based methods—provide unique opportunities to investigate other aspects of cellular metabolism and expand the metabolic imaging armamentarium. This review will discuss the underlying biology of metabolic dysregulation in cancer, focusing on glucose, glutamine, and acetate metabolism. Novel imaging strategies will be discussed within this biologic framework, highlighting particular strengths and limitations of each technique. Emphasis is placed on the role that combining modalities will play in enabling multiparametric imaging to fully characterize tumor biology to better inform treatment.

Triglycerides Promote Lipid Homeostasis during Hypoxic Stress by Balancing Fatty Acid Saturation

Summary Lipid droplets, which store triglycerides and cholesterol esters, are a prominent feature of clear cell renal cell carcinoma (ccRCC). Although their presence in ccRCC is critical for sustained tumorigenesis, their contribution to lipid homeostasis and tumor cell viability is incompletely understood. Here we show that disrupting triglyceride synthesis compromises the growth of both ccRCC tumors and ccRCC cells exposed to tumor-like conditions. Functionally, hypoxia leads to increased fatty acid saturation through inhibition of the oxygen-dependent stearoyl-CoA desaturase (SCD) enzyme. Triglycerides counter a toxic buildup of saturated lipids, primarily by releasing the unsaturated fatty acid oleate (the principal product of SCD activity) from lipid droplets into phospholipid pools. Disrupting this process derails lipid homeostasis, causing overproduction of toxic saturated ceramides and acyl-carnitines as well as activation of the NF-κB transcription factor. Our work demonstrates that triglycerides promote homeostasis by “buffering” specific fatty acids.

Abstract B33: Assessing the role of DGAT activity on lipid homeostasis and cancer cell survival

Studies on the effects of modulating the lipid composition of cells have found that increases in saturated fatty acid levels can lead to ER stress, activation of the unfolded protein response (UPR) and cell death. These effects may occur through saturation of the phospholipid pool and subsequent deterioration of ER membrane function. Changes in the composition of the ER membrane can be sensed by the two UPR stress sensors IRE1 and PERK and lead to activation of UPR targets. Rapidly proliferating cancer cells frequently exhibit elevated ER stress due to their increased protein translation rate. In addition, the tumor microenvironment is frequently hypoxic and a lack of oxygen can inhibit the activity of the fatty acid desaturase SCD1. This leads to saturation of the fatty acid pool and renders the ER less able to cope with high levels of protein synthesis, making the maintenance of adequate lipid homeostasis of paramount importance to cancer cells within hypoxic tumor domains. Tumor cells adapt to these conditions through a variety of mechanisms, including the uptake of exogenous unsaturated lipid. Both hypoxia and the presence of certain oncogenic drivers have been found to increase lipid uptake, which was found to protect cancer cells against inhibition of SCD1. Since fatty acids can be converted to triglycerides and stored within lipid droplets, we asked whether cancer cells also cope with saturation of their fatty acid pool by storing excess saturated fatty acids as triglycerides. Clear cell renal cell carcinoma (ccRCC) tumors are very fatty and lipidomic analyses have found high levels of triglycerides in ccRCC compared to normal kidney tissue. We inhibited triglyceride synthesis in A498 ccRCC cells by shRNA –mediated knockdown of DGAT enzymes, which catalyze the final step in the synthesis of triglycerides from fatty acids. We tested whether loss of DGAT activity causes sensitivity to tumor-like stresses and investigated its effect on xenograft tumor growth. Remarkably, while loss of DGAT activity has little effect on cancer cell proliferation under oxygen- and nutrient-rich conditions, loss of DGAT activity has large effects on xenograft tumor growth. We also find that loss of DGAT activity is often accompanied by increases in markers of ER stress. We are currently assessing the lipid composition of these tumors and determining what aspects of the tumor microenvironment sensitize cells to loss of DGAT activity. Citation Format: Daniel Ackerman, Bo Qiu, Hong Xie, Jurre Kamphorst, M. Celeste Simon. Assessing the role of DGAT activity on lipid homeostasis and cancer cell survival. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B33.

Abstract PR04: HIF-2α dependent lipid storage promotes endoplasmic reticulum homeostasis in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most common form of renal cancer, defined pathologically by abundant intracellular lipid droplets (LDs) that impart the clear cell phenotype, and molecularly by constitutive activation of the hypoxia inducible factors (HIFs). The primary aim of this study was to identify the mechanisms driving enhanced neutral lipid storage and the function of this phenotype in ccRCC. Our work demonstrates that HIF-2α promotes neutral lipid storage in ccRCC through up-regulation of the LD coat protein PLIN2. Expression profiling in multiple cohorts of primary ccRCC and normal kidney samples revealed that PLIN2 was overexpressed in all stages of disease and correlated with constitutive activation of HIF-2α, but not HIF-1α. Using ccRCC cell lines, 3D tumor spheroids, and tumor xenografts, we showed that HIF-2α dependent PLIN2 expression was required for neutral lipid storage and tumor cell viability. We demonstrated, for the first time, that PLIN2 dependent lipid storage promoted ccRCC tumor growth by maintaining integrity of the endoplasmic reticulum (ER), an organelle that is functionally and physically associated with LDs. Recent work indicates that cellular transformation commits tumors to growth programs that strain ER homeostasis, including elevated protein and lipid synthesis. Such ER stress is exacerbated by conditions of oxygen and nutrient deprivation characteristic of solid tumor microenvironments, which further disrupt cellular protein and lipid homeostasis. Consistent with this theme, we found that the enhanced requirement for PLIN2 in ccRCC was due to heightened ER stress downstream of cellular transformation, including elevated protein synthesis and growth within nutrient and oxygen limited tumor microenvironments. In these settings, PLIN2 depletion elicited irremediable ER stress and triggered a cytotoxic unfolded protein response. Remarkably, suppression of protein synthesis via cycloheximide or pharmacologic inhibitors of mTORC1, which is activated in >80% of ccRCC, prevented ER stress and cell death in PLIN2 depleted cells. On the other hand, inhibition of lipid synthesis was selectively toxic to PLIN2 depleted cells, consistent with an adaptive function of lipid synthesis downstream of ER stress. Our results suggest that PLIN2 is not merely a marker of lipid accumulation in ccRCC. Instead, it promotes lipid storage and maintains integrity of the ER, a vital hub for tumor cell protein and lipid metabolism. Consistent with this model, HIF-2α/PLIN2 dependent lipid storage also protected ccRCC cells against pharmacological ER stress inducing agents, including the proteasome inhibitor Bortezomib (Velcade). These findings reveal a novel function of the well documented clear cell phenotype in ccRCC and identify ER stress as a targetable vulnerability created by HIF-2α/PLIN2 suppression in this common renal malignancy. Citation Format: Bo Qiu, Daniel Ackerman, Danielle J. Sanchez, Bo Li, Joshua D. Ochocki, Alison Grazioli, Ekaterina Bobrovnikova-Marjon, J. Alan Diehl, Brian Keith, M. Celeste Simon. HIF-2α dependent lipid storage promotes endoplasmic reticulum homeostasis in clear cell renal cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr PR04.

Abstract IA16: Dysregulated mTORC1 renders cells critically dependent on desaturated lipids for survival under tumor-like stress

Solid tumors exhibit heterogeneous microenvironments, often characterized by limiting concentrations of oxygen (O2), glucose, and other nutrients. How oncogenic mutations alter stress response pathways, metabolism, and cell survival in the face of these challenges is incompletely understood. Here we report that constitutive mTORC1 activity renders hypoxic cells dependent on exogenous desaturated lipids, as levels of de novo synthesized unsaturated fatty acids are reduced under low O2. Specifically, we demonstrate that hypoxic Tsc2-/- cells deprived of serum lipids exhibit a magnified UPR response, but fail to appropriately expand their ER, leading to IRE1-dependent cell death that can be reversed by the addition of unsaturated lipids. UPR activation and apoptosis were also detected in Tsc2-deficient kidney tumors. Importantly, we observe this phenotype in multiple human cancer lines and suggest that cells committed to unregulated growth within ischemic tumor microenvironments are unable to balance lipid and protein synthesis due to a critical limitation in desaturated lipids. Citation Format: Regina Young, Daniel Ackerman, Zachary Quinn, Anthony Mancuso, Michaela Gruber, Liping Liu, Dionysios Giannoukos, Ekaterina Bobovnikova-Marjon, J. Alan Diehl, Brian Keith, M. Celeste Simon. Dysregulated mTORC1 renders cells critically dependent on desaturated lipids for survival under tumor-like stress. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr IA16.