A Medicinal Chemistry-Driven Approach Identified the Sterol Isomerase EBP as the Molecular Target of TASIN Colorectal Cancer Toxins.

TASIN (Truncated APC-Selective Inhibitors) compounds are selectively toxic to colorectal cancer cells with APC mutations, although their mechanism of action remains unknown. Here, we found that TASINs inhibit three enzymes in the postsqualene cholesterol biosynthetic pathway including EBP, DHCR7, and DHCR24. Even though all three of these enzymes are required for cholesterol biosynthesis, only inhibition of the most upstream enzyme, EBP, led to cancer cell death via depletion of downstream sterols, an observation that was confirmed by genetic silencing of EBP. Pharmacologic inhibition or genetic silencing of either DHCR7 or DHCR24 had no impact on cell viability. By using photoaffinity probes to generate a relationship between chemical structure and probe competition, we identified compounds that selectively inhibit either EBP or DHCR7. These studies identify EBP, but not downstream enzymes in the cholesterol biosynthetic pathway, as a target in APC mutant colorectal cancer and also have implications for the clinical development of highly selective EBP inhibitors.

Addressing metabolic heterogeneity in clear cell renal cell carcinoma with quantitative Dixon MRI.

BACKGROUND: Dysregulated lipid and glucose metabolism in clear cell renal cell carcinoma (ccRCC) has been implicated in disease progression, and whole tumor tissue-based assessment of these changes is challenged by the tumor heterogeneity. We studied a noninvasive quantitative MRI method that predicts metabolic alterations in the whole tumor. METHODS: We applied Dixon-based MRI for in vivo quantification of lipid accumulation (fat fraction [FF]) in targeted regions of interest of 45 primary ccRCCs and correlated these MRI measures to mass spectrometry-based lipidomics and metabolomics of anatomically colocalized tissue samples isolated from the same tumor after surgery. RESULTS: In vivo tumor FF showed statistically significant (P < 0.0001) positive correlation with histologic fat content (Spearman correlation coefficient, ρ = 0.79), spectrometric triglycerides (ρ = 0.56) and cholesterol (ρ = 0.47); it showed negative correlation with free fatty acids (ρ = -0.44) and phospholipids (ρ = -0.65). We observed both inter- and intratumoral heterogeneity in lipid accumulation within the same tumor grade, whereas most aggressive tumors (International Society of Urological Pathology [ISUP] grade 4) exhibited reduced lipid accumulation. Cellular metabolites in tumors were altered compared with adjacent renal parenchyma. CONCLUSION: Our results support the use of noninvasive quantitative Dixon-based MRI as a biomarker of reprogrammed lipid metabolism in ccRCC, which may serve as a predictor of tumor aggressiveness before surgical intervention. FUNDING: NIH R01CA154475 (YZ, MF, PK, IP), NIH P50CA196516 (IP, JB, RJD, JAC, PK), Welch Foundation I-1832 (JY), and NIH P01HL020948 (JGM).

Fatty Acid Oxidation Mediated by Acyl-CoA Synthetase Long Chain 3 Is Required for Mutant KRAS Lung Tumorigenesis.

KRAS is one of the most commonly mutated oncogenes in human cancer. Mutant KRAS aberrantly regulates metabolic networks. However, the contribution of cellular metabolism to mutant KRAS tumorigenesis is not completely understood. We report that mutant KRAS regulates intracellular fatty acid metabolism through Acyl-coenzyme A (CoA) synthetase long-chain family member 3 (ACSL3), which converts fatty acids into fatty Acyl-CoA esters, the substrates for lipid synthesis and ß-oxidation. ACSL3 suppression is associated with depletion of cellular ATP and causes the death of lung cancer cells. Furthermore, mutant KRAS promotes the cellular uptake, retention, accumulation, and ß-oxidation of fatty acids in lung cancer cells in an ACSL3-dependent manner. Finally, ACSL3 is essential for mutant KRAS lung cancer tumorigenesis in vivo and is highly expressed in human lung cancer. Our data demonstrate that mutant KRAS reprograms lipid homeostasis, establishing a metabolic requirement that could be exploited for therapeutic gain.

Transcription Factor Hepatocyte Nuclear Factor-1ß Regulates Renal Cholesterol Metabolism.

HNF-1ß is a tissue-specific transcription factor that is expressed in the kidney and other epithelial organs. Humans with mutations in HNF-1ß develop kidney cysts, and HNF-1ß regulates the transcription of several cystic disease genes. However, the complete spectrum of HNF-1ß-regulated genes and pathways is not known. Here, using chromatin immunoprecipitation/next generation sequencing and gene expression profiling, we identified 1545 protein-coding genes that are directly regulated by HNF-1ß in murine kidney epithelial cells. Pathway analysis predicted that HNF-1ß regulates cholesterol metabolism. Expression of dominant negative mutant HNF-1ß or kidney-specific inactivation of HNF-1ß decreased the expression of genes that are essential for cholesterol synthesis, including sterol regulatory element binding factor 2 (Srebf2) and 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr). HNF-1ß mutant cells also expressed lower levels of cholesterol biosynthetic intermediates and had a lower rate of cholesterol synthesis than control cells. Additionally, depletion of cholesterol in the culture medium mitigated the inhibitory effects of mutant HNF-1ß on the proteins encoded by Srebf2 and Hmgcr, and HNF-1ß directly controlled the renal epithelial expression of proprotein convertase subtilisin-like kexin type 9, a key regulator of cholesterol uptake. These findings reveal a novel role of HNF-1ß in a transcriptional network that regulates intrarenal cholesterol metabolism.

6-Phosphogluconate dehydrogenase links oxidative PPP, lipogenesis and tumour growth by inhibiting LKB1-AMPK signalling.

The oxidative pentose phosphate pathway (PPP) contributes to tumour growth, but the precise contribution of 6-phosphogluconate dehydrogenase (6PGD), the third enzyme in this pathway, to tumorigenesis remains unclear. We found that suppression of 6PGD decreased lipogenesis and RNA biosynthesis and elevated ROS levels in cancer cells, attenuating cell proliferation and tumour growth. 6PGD-mediated production of ribulose-5-phosphate (Ru-5-P) inhibits AMPK activation by disrupting the active LKB1 complex, thereby activating acetyl-CoA carboxylase 1 and lipogenesis. Ru-5-P and NADPH are thought to be precursors in RNA biosynthesis and lipogenesis, respectively; thus, our findings provide an additional link between the oxidative PPP and lipogenesis through Ru-5-P-dependent inhibition of LKB1-AMPK signalling. Moreover, we identified and developed 6PGD inhibitors, physcion and its derivative S3, that effectively inhibited 6PGD, cancer cell proliferation and tumour growth in nude mice xenografts without obvious toxicity, suggesting that 6PGD could be an anticancer target.

Glutamine oxidation maintains the TCA cycle and cell survival during impaired mitochondrial pyruvate transport.

Alternative modes of metabolism enable cells to resist metabolic stress. Inhibiting these compensatory pathways may produce synthetic lethality. We previously demonstrated that glucose deprivation stimulated a pathway in which acetyl-CoA was formed from glutamine downstream of glutamate dehydrogenase (GDH). Here we show that import of pyruvate into the mitochondria suppresses GDH and glutamine-dependent acetyl-CoA formation. Inhibiting the mitochondrial pyruvate carrier (MPC) activates GDH and reroutes glutamine metabolism to generate both oxaloacetate and acetyl-CoA, enabling persistent tricarboxylic acid (TCA) cycle function. Pharmacological blockade of GDH elicited largely cytostatic effects in culture, but these effects became cytotoxic when combined with MPC inhibition. Concomitant administration of MPC and GDH inhibitors significantly impaired tumor growth compared to either inhibitor used as a single agent. Together, the data define a mechanism to induce glutaminolysis and uncover a survival pathway engaged during compromised supply of pyruvate to the mitochondria.

C-terminus of apolipoprotein A-I removes phospholipids from a triolein/phospholipids/water interface, but the N-terminus does not: a possible mechanism for nascent HDL assembly.

Apolipoprotein A-I (ApoA-I) is the principle protein component of HDL, also known as "good cholesterol," which is an inverse marker for cardiovascular disease. The N-terminal 44 amino acids of ApoA-I (N44) are predicted to be responsible for stabilization of soluble ApoA-I, whereas the C-terminal 46 amino acids (C46) are predicted to initiate lipid binding and oligomerization. In this work, we apply what we believe to be a novel application of drop tensiometry to study the adsorption and desorption of N44 and C46 at a triolein/POPC/water (TO/POPC/W) interface. The amount of peptide that adsorbed to the surface was dependent on the surface concentration of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and pressure (Π) before adsorption. At a TO/POPC/W interface, the exclusion pressure (Π(EX)) of C46 was 25.8 mN/m, and was 19.3 mN/m for N44. Once adsorbed, both peptides formed a homogeneous surface with POPC but were progressively ejected from the surface by compression. During a compression, C46 removed POPC from the surface whereas N44 did not. Repeated compressions caused C46 to deplete entirely the surface of phospholipid. If full-length ApoA-I could also remove phospholipid, this could provide a mechanism for the transfer of surface components of chylomicrons and very low density lipoprotein to high density lipoprotein with the assistance of phospholipid transfer protein.

The adsorption of biological peptides and proteins at the oil/water interface. A potentially important but largely unexplored field.

This review focuses on some new techniques to study the behavior of peptides and proteins bound to oil droplets. We will show how model peptides e.g., amphipathic alpha helices (AalphaH) and amphipathic beta strand (AbetaS) and some apolipoproteins adsorb to triacylglycerol (TAG) droplets and how they behave once adsorbed to the interface. While most of the studies described involve peptides and proteins at an oil/water interface, studies can also be carried out when the surface has been partially covered with phospholipids. This work is important because it examines biophysical changes that take place at lipid droplet interfaces and how this may relate to the metabolism of lipoproteins and lipid droplets.