Sterol Regulatory Element-binding Protein Pathway Research Articles

A High-Throughput Screening Platform Identifies FDA-Approved Drugs That Inhibit SREBP Pathway Activation.

Sterol regulatory element-binding protein (SREBP) transcription factors are central regulators of lipid homeostasis and are essential for lipid metabolic reprogramming that supports tumor growth in multiple cancers. SREBP pathway inhibitors have been identified, but bioavailable compounds are lacking. To address this need, we designed a novel approach for screening a collection of 4,474 FDA-approved drugs. SREBPs are conditionally essential and required under low lipid conditions. Leveraging this property, we screened for drugs that inhibited pancreatic cancer cell growth in lipid-poor, but not lipid-rich, medium. The primary screen identified 83 drugs that inhibited cell growth in a lipid-dependent manner. Secondary assays examining SREBP target gene expression, SREBP proteolytic cleavage, and effects on human breast cancer cells identified 13 FDA-approved drugs that inhibit SREBP pathway activation. Taken together, we demonstrated that our screening approach can identify SREBP inhibitors from a small library of compounds. This high-throughput screening platform enables screening of large compound collections to discover novel small molecule SREBP inhibitors.

Identification of Potential New Genes Related to the SREBP Pathway in Xanthophyllomyces dendrorhous.

The sterol regulatory element-binding protein (SREBP) pathway is an integral cellular mechanism that regulates lipid homeostasis, in which transcriptional activator SREBPs regulate the expression of various genes. In the carotenogenic yeast Xanthophyllomyces dendrorhous, Sre1 (the yeast SREBP homolog) regulates lipid biosynthesis and carotenogenesis, among other processes. Despite the characterization of several components of the SREBP pathway across various eukaryotes, the specific elements of this pathway in X. dendrorhous remain largely unknown. This study aimed to explore the potential regulatory mechanisms of the SREBP pathway in X. dendrorhous using the strain CBS.cyp61- as a model, which is known to have Sre1 in its active state under standard culture conditions, resulting in a carotenoid-overproducing phenotype. This strain was subjected to random mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (NTG), followed by a screening methodology that focused on identifying mutants with altered Sre1 activation phenotypes. Single-nucleotide polymorphism (SNP) analysis of 20 selected mutants detected 5439 single-nucleotide variants (SNVs), narrowing them down to 1327 SNPs of interest after a series of filters. Classification based on SNP impact identified 116 candidate genes, including 49 genes with high impact and 68 genes with deleterious moderate-impact mutations. BLAST, InterProScan, and gene ontology enrichment analyses highlighted 25 genes as potential participants in regulating Sre1 in X. dendrorhous. The key findings of this study include the identification of genes potentially encoding proteins involved in protein import/export to the nucleus, sterol biosynthesis, the ubiquitin-proteasome system, protein regulatory activities such as deacetylases, a subset of kinases and proteases, as well as transcription factors that could be influential in SREBP regulation. These findings are expected to significantly contribute to the current understanding of the intricate regulation of the transcription factor Sre1 in X. dendrorhous, providing valuable groundwork for future research and potential biotechnological applications.

Dominant missense variants in SREBF2 are associated with complex dermatological, neurological, and skeletal abnormalities

PurposeWe identified 2 individuals with de novo variants in SREBF2 that disrupt a conserved site 1 protease (S1P) cleavage motif required for processing SREBP2 into its mature transcription factor. These individuals exhibit complex phenotypic manifestations that partially overlap with sterol regulatory element binding proteins (SREBP) pathway-related disease phenotypes, but SREBF2-related disease has not been previously reported. Thus, we set out to assess the effects of SREBF2 variants on SREBP pathway activation. MethodsWe undertook ultrastructure and gene expression analyses using fibroblasts from an affected individual and utilized a fly model of lipid droplet (LD) formation to investigate the consequences of SREBF2 variants on SREBP pathway function. ResultsWe observed reduced LD formation, endoplasmic reticulum expansion, accumulation of aberrant lysosomes, and deficits in SREBP2 target gene expression in fibroblasts from an affected individual, indicating that the SREBF2 variant inhibits SREBP pathway activation. Using our fly model, we discovered that SREBF2 variants fail to induce LD production and act in a dominant-negative manner, which can be rescued by overexpression of S1P. ConclusionTaken together, these data reveal a mechanism by which SREBF2 pathogenic variants that disrupt the S1P cleavage motif cause disease via dominant-negative antagonism of S1P, limiting the cleavage of S1P targets, including SREBP1 and SREBP2.

SREBP signaling is important for pathogenic B cell responses in allergy.

Abstract Severe IgE antibody-mediated allergic conditions impacts millions worldwide, significantly influencing both the survival and quality of life of those affected. Our previous research indicated the critical role of sterol regulatory element-binding protein (SREBP) signaling in mediating lipid homeostasis during B cell activation and the formation of germinal centers following immunization and viral infection. However, it remained unclear whether this pathway is also involved in the pathogenic B cell responses triggered by chronic allergen sensitization. Our latest findings show that ablation of SREBP signaling in B cells doesn't affect overall IgE levels in the serum. However, during chronic sensitization to house dust mites (HDM), SREBP signaling was essential for allergen-triggered germinal center responses and pathogenic antibody production. These results suggest the potential of targeting the B cell SREBP pathway as a treatment approach for allergies.

Cholesterol metabolism: physiological versus pathological aspects in intracerebral hemorrhage.

Cholesterol is an important component of plasma membranes and participates in many basic life functions, such as the maintenance of cell membrane stability, the synthesis of steroid hormones, and myelination. Cholesterol plays a key role in the establishment and maintenance of the central nervous system. The brain contains 20% of the whole body's cholesterol, 80% of which is located within myelin. A huge number of processes (e.g., the sterol regulatory element-binding protein pathway and liver X receptor pathway) participate in the regulation of cholesterol metabolism in the brain via mechanisms that include cholesterol biosynthesis, intracellular transport, and efflux. Certain brain injuries or diseases involving crosstalk among the processes above can affect normal cholesterol metabolism to induce detrimental consequences. Therefore, we hypothesized that cholesterol-related molecules and pathways can serve as therapeutic targets for central nervous system diseases. Intracerebral hemorrhage is the most severe hemorrhagic stroke subtype, with high mortality and morbidity. Historical cholesterol levels are associated with the risk of intracerebral hemorrhage. Moreover, secondary pathological changes after intracerebral hemorrhage are associated with cholesterol metabolism dysregulation, such as neuroinflammation, demyelination, and multiple types of programmed cell death. Intracellular cholesterol accumulation in the brain has been found after intracerebral hemorrhage. In this paper, we review normal cholesterol metabolism in the central nervous system, the mechanisms known to participate in the disturbance of cholesterol metabolism after intracerebral hemorrhage, and the links between cholesterol metabolism and cell death. We also review several possible and constructive therapeutic targets identified based on cholesterol metabolism to provide cholesterol-based perspectives and a reference for those interested in the treatment of intracerebral hemorrhage.

Effects of Fermented Extracts of Wuniuzao Dark Loose Tea on Hepatic Sterol Regulatory Element-Binding Protein Pathway and Gut Microbiota Disorder in Obese Mice

BackgroundArtificially fermented dark loose tea is a type of novel dark tea prepared via fermentation by Eurotium cristatum. The effects of artificially fermented dark loose tea on lipid metabolism are still unclear. ObjectivesThis study aimed to explore if artificially fermented dark loose tea has the same effects as naturally fermented dark loose tea in regulating hepatic lipid metabolism. MethodsThirty-six 8-wk-old male C57BL/6 mice were randomly divided into 6 treatment groups, including normal control (NC), high-fat diet (HFD), positive control (PC), Wuniuzao dark raw tea (WDT), Wuniuzao naturally fermented dark loose tea (NFLT), and Wuniuzao artificially fermented dark loose tea (AFLT) groups. The HFD, PC, WDT, NFLT, and AFLT groups were fed a HFD. The PC group was supplemented with atorvastatin (10 mg/kg). The WDT group was supplemented with WDT (300 mg/kg), the NFLT group with NFLT (300 mg/kg), and the AFLT group with AFLT (300 mg/kg). ResultsThe study compared the effect of WDT, NFLT, and AFLT on liver steatosis and gut microbiota disorder in obese mice. All 3 tea extracts reduced body weight, glucose tolerance, and serum lipid concentrations. Via sterol-regulatory element binding protein (SREBP)-mediated lipid metabolism, all 3 tea extracts alleviated hepatic steatosis in mice with obesity. Furthermore, NFLT and AFLT intervened in the abundance of Firmicutes, Bacteroidetes, Clostridia, Muribaculaceae, and Lachnospiraceae. ConclusionIn mice with obesity induced by a HFD, WDT, NFLT, and AFLT may improve hepatic steatosis through an SREBP-mediated lipid metabolism. Moreover, NFLT and AFLT improved the composition of gut microbiota.

Inhibition of the SREBP pathway prevents SARS-CoV-2 replication and inflammasome activation.

SARS-CoV-2 induces major cellular lipid rearrangements, exploiting the host's metabolic pathways to replicate. Sterol regulatory element binding proteins (SREBPs) are a family of transcription factors that control lipid metabolism. SREBP1 is associated with the regulation of fatty acids, whereas SREBP2 controls cholesterol metabolism, and both isoforms are associated with lipid droplet (LD) biogenesis. Here, we evaluated the effect of SREBP in a SARS-CoV-2-infected lung epithelial cell line (Calu-3). We showed that SARS-CoV-2 infection induced the activation of SREBP1 and SREBP2 and LD accumulation. Genetic knockdown of both SREBPs and pharmacological inhibition with the dual SREBP activation inhibitor fatostatin promote the inhibition of SARS-CoV-2 replication, cell death, and LD formation in Calu-3 cells. In addition, we demonstrated that SARS-CoV-2 induced inflammasome-dependent cell death by pyroptosis and release of IL-1β and IL-18, with activation of caspase-1, cleavage of gasdermin D1, was also reduced by SREBP inhibition. Collectively, our findings help to elucidate that SREBPs are crucial host factors required for viral replication and pathogenesis. These results indicate that SREBP is a host target for the development of antiviral strategies.

Multiomics Analysis of IMiD/CELMoD Resistant Multiple Myeloma Models Uncovers Novel and Targetable Vulnerabilities in the SREBP Lipid Synthesis Pathway

Background Acquired resistance to immunomodulatory drugs (IMiDs)/cereblon (CRBN) E3 ligase modulators (CELMoDs) is a major challenge in multiple myeloma (MM) treatment. These drugs bind to the CRBN component of the CRL4CRBN E3 ubiquitin ligase and designate a new set of substrates for degradation via the proteasome. Generation of resistance is frequently associated with decreased CRBN expression and this is due to genetic alteration in ~1/3rd of patients. Alternative drivers of the low CRBN state, and other mechanisms of resistance, need to be elucidated. To tackle this complex problem, MM models with acquired IMiD/CELMoD resistance were generated and multiomics analysis performed. Methods IMiD/CELMoD resistant human MM cell lines were generated by treating MM1s and H929 cells with lenalidomide (Len), pomalidomide (Pom) or iberdomide (Iber) at ~10x GI50 concentration for ~12 weeks until resistance was achieved. Resistant cell lines and controls were characterised by whole exome sequencing (WES), RNA-Seq and proteomics. A genome-wide loss-of-function CRISPR screen (Brunello library) was carried out in Iber-resistant MM1s; gene effect scores were calculated with the Chronos model and compared to parental MM1s using data from the Broad Institute DepMap portal. Pathway analysis was performed using g:Profiler. The Membrane Bound Transcription Factor Peptidase, Site 1 (MBTPS1) inhibitor PF-429242 was used to inhibit activation of the Sterol Regulatory-Element Binding Protein (SREBP) pathway and the effect on cell viability measured using CellTiter-Blue®. The Multiple Myeloma Research Foundation CoMMpass database was used to explore the correlation between mRNA expression of SREBP pathway genes in newly diagnosed patients and progression-free survival (PFS). Results All models were resistant to the IMiD/CELMoD with which they were generated (to 20-100x the GI50 concentration) and exhibited cross-resistance to other IMiDs/CELMoDs. Functional assays showed that well-characterised effects of IMiD/CELMoD treatment, e.g. degradation of Ikaros/Aiolos, were abrogated. WES showed Pom-resistant MM1S and Pom- and Iber-resistant H929 cells had acquired mutations in CRBN predicted to have a high impact on function. Len-resistant H929 cells had new copy number loss at the CRBN locus but Len- and Iber-resistant MM1s cells had no genetic changes in CRBN. CRBN protein expression was reduced in all resistant lines compared to control (log2 fold changes (FCs) by proteomics ranging from -0.25 to -1.95, adj p <0.05). Together these models display diverse resistance mechanisms, reflecting the clinical picture. Proteomic analysis of the resistant lines identified key changes in the SREBP pathway. The proteomes of the resistant lines were heterogeneous and the only pathway with common significant enrichment was SREBP/fatty acid metabolism (on analysis performed per cell line of proteins with significantly decreased expression). Stearoyl-CoA Desaturase (SCD), a key effector of the SREBP pathway, was one of only 5 proteins with significantly altered expression in all 6 cell lines compared to control. SCD had log2FCs ranging from -0.37 to -1.50 (adj p <0.05). Other key pathway members are shown in Figure 1. A genome-wide CRISPR screen using Iber-resistant MM1s cells identified potential new dependencies (gene effect score <-1 in resistant cells and >-0.5 in parental cells) in 47 genes including SCD and MBTPS1. MBTPS1 is critical for activation of the SREBP pathway and demonstrated one of the largest changes in gene effect (-1.4 vs -0.3). The activity of PF-429242, an inhibitor of MBTPS1, was explored in the resistant lines. A significant difference in GI50 between Iber-resistant H929 cells and control was found (1uM and >10uM respectively) with greater activity in the resistant cells. The same pattern was observed with the other resistant H929 cell lines, but not the resistant MM1s. Other pathway inhibitors are being explored. The expression of genes encoding SREBP pathway components was explored in the CoMMpass dataset. High SCD or MBTPS1 mRNA expression was associated with significantly worse PFS (logrank p<0.01), providing a further rationale for targeting the pathway. Conclusions Models representing multiple different IMiD/CELMoD resistance mechanisms have unified alterations in the SREBP pathway, highlighting a role in resistance biology and potential novel and targetable vulnerabilities. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract B011: Identifying druggable lipid synthesis targets as novel therapeutics for pancreatic cancer

Abstract Lipid metabolic reprogramming is a hallmark of cancer that contributes to tumorigenesis by supporting energy metabolism and the synthesis of signaling molecules, cellular membranes, and protein modifications. Cells modulate lipid metabolic genes through the regulation of sterol regulatory element-binding proteins (SREBPs), which serve as transcriptional activators of genes responsible for fatty acid and cholesterol synthesis and uptake. Despite efforts demonstrating that the SREBP pathway is an attractive chemotherapeutic target, a potent bioavailable inhibitor of the pathway has not yet been discovered. Here, we identify druggable SREBP target genes as potential targets in pancreatic ductal adenocarcinoma (PDAC). Results: To discover candidate genes, we constructed a CRISPR knockout library containing 148 known SREBP target genes. We used a patient-derived PDAC cell line to conduct the knockout screens in vivo (n = 7 tumors) by injecting library-infected cells into the pancreas of nude mice. After 4 weeks of growth, mouse PDAC tumors showed a statistically significant depletion of guide RNAs targeting 8 genes within the mevalonate pathway, which is responsible for synthesizing cholesterol and isoprenoids such as farnesyl-diphosphate and geranyl-diphosphate. One of the most significant hits was HMG-CoA reductase (HMGCR), which catalyzes the formation of mevalonate. We chose to investigate statins, which are a well-characterized class of FDA-approved drugs that inhibit HMGCR. Using flow cytometry, we stained for annexin-V as a marker for apoptosis and found minimal cell death induced by Fluvastatin alone. Given that inhibiting HMGCR would disrupt lipid homeostasis and in turn activate the SREBP pathway, we hypothesized that a combination therapy with an SREBP inhibitor, TMDP, would potentiate statin-induced cell death. We observed minimal cell death with TMDP alone, but striking synergy between Fluvastatin and TMDP, showing a five-fold increase in annexin-V staining after 48 hours. Mevalonate and geranylgeranyl-diphosphate both fully rescued this cell death to basal levels. Conclusions: In vivo CRISPR screening is a powerful tool for the identification of novel therapeutic targets for cancer. We found that the mevalonate pathway plays a significant role in pancreatic tumor growth in vivo and that existing FDA-approved drugs can be used in combination with an SREBP inhibitor to dramatically enhance cell killing in vitro. Citation Format: Casie S. Kubota, Chiaki T. Ishida, Stephanie Myers, Peter J. Espenshade. Identifying druggable lipid synthesis targets as novel therapeutics for pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B011.

Effects of copper exposure on lipid metabolism and SREBP pathway in the Chinese mitten crab Eriocheir sinensis

Copper (Cu) is not only a common metal pollutant in the aquatic environment but also an essential trace element for aquatic organisms such as the Chinese mitten crab (Eriocheir sinensis). Cu is known to regulate lipid metabolism yet exert toxic effects if ingested in excess. However, the molecular regulatory roles of Cu in the lipid metabolism of crabs remains unclear. Thus, this study investigated the potential regulatory mechanism of Cu onto lipid metabolism of E. sinensis following acute Cu exposure. Crabs were exposed to environmental concentration of Cu (50 μg/L) for 96 h, and the expression of sterol regulatory element binding protein (SREBP) was knocked down by RNA interference (RNAi) to test its effect on Cu exposure. The results showed that RNAi significantly attenuated the Cu exposure-induced increase in lipid synthesis and triglycerides (TG) hydrolysis, while significantly inhibited the Cu exposure-induced decrease in fatty acid β-oxidation, suggesting that SREBP is involved in Cu-induced lipid metabolism. Subsequent analyses of the transcriptome results further revealed potential responsive genes of SREBP that were linked to lipid metabolism and immune regulation. Moreover, Cu may affect lipid metabolism through the TOR-SREBP pathway in E. sinensis. This work provides a reference for exploring the effects of Cu on lipid metabolism disorders in crustaceans.

The SREBP-dependent regulation of cyclin D1 coordinates cell proliferation and lipid synthesis.

The sterol regulatory-element binding protein (SREBP) family of transcription factors regulates cholesterol, fatty acid, and triglyceride synthesis and metabolism. However, they are also targeted by the ubiquitin ligase Fbw7, a major tumor suppressor, suggesting that they could regulate cell growth. Indeed, enhanced lipid synthesis is a hallmark of many human tumors. Thus, the SREBP pathway has recently emerged as a potential target for cancer therapy. We have previously demonstrated that one of these transcription factors, SREBP1, is stabilized and remains associated with target promoters during mitosis, suggesting that the expression of these target genes could be important as cells enter G1 and transcription is restored. Activation of cyclin D-cdk4/6 complexes is critical for the phosphorylation and inactivation of the retinoblastoma protein (Rb) family of transcriptional repressors and progression through the G1 phase of the cell cycle. Importantly, the cyclin D-cdk4/6-Rb regulatory axis is frequently dysregulated in human cancer. In the current manuscript, we demonstrate that SREBP1 activates the expression of cyclin D1, a coactivator of cdk4 and cdk6, by binding to an E-box in the cyclin D1 promoter. Consequently, inactivation of SREBP1 in human liver and breast cancer cell lines reduces the expression of cyclin D1 and attenuates Rb phosphorylation. Rb phosphorylation in these cells can be rescued by restoring cyclin D1 expression. On the other hand, expression of active SREBP1 induced the expression of cyclin D1 and increased the phosphorylation of Rb in a manner dependent on cyclin D1 and cdk4/6 activity. Inactivation of SREBP1 resulted in reduced expression of cyclin D1, attenuated phosphorylation of Rb, and reduced proliferation. Inactivation of SREBP1 also reduced the insulin-dependent regulation of the cyclin D1 gene. At the same time, SREBP1 is known to play an important role in supporting lipid synthesis in cancer cells. Thus, we propose that the SREBP1-dependent regulation of cyclin D1 coordinates cell proliferation with the enhanced lipid synthesis required to support cell growth.

Rotavirus exploits SREBP pathway for hyper lipid biogenesis during replication.

Species A rotavirus (RVA) is one of the pathogens causing severe acute gastroenteritis in young children and animals worldwide. RVA replicates and assembles its immature particle within electron dense compartments known as viroplasm. Despite the importance of lipid droplet (LD) formation in the RVA viroplasm, the upstream molecules modulating LD formation have remained elusive. Here, we demonstrate that RVA infection reprogrammes sterol regulatory element binding proteins (SREBPs)-dependent lipogenic pathways in virus-infected cells. Interestingly, silencing of SREBPs significantly reduced RVA protein synthesis, genome replication and progeny virus production. Moreover, knockout of SREBP-1c gene conferred resistance to RVA-induced diarrhoea, reduction of RVA replication, and mitigation of small intestinal pathology in mice. This study identifies SREBPs-mediated lipogenic reprogramming in RVA-infected host cells for facilitating virus replication and SREBPs as a potential target for developing therapeutics against RVA infection.

SREBP and central nervous system disorders: genetic overlaps revealed by in silico analysis.

The central nervous system (CNS) is enriched in lipids; despite this, studies exploring the functional roles of lipids in the brain are still limited. Sterol regulatory element binding protein (SREBP) signaling is a transcriptomic pathway that predominantly participates in the maintenance of lipid homeostasis; however, its involvement in the CNS dysfunction is not well-established. In this study, we aimed to characterize and pinpoint specific genes of the SREBP pathway which may be implicated in neurodegenerative, neurological, and neuropsychiatric diseases. In silico bioinformatic analysis was performed using the open-source databases DisGeNET and MSigDB. Protein-protein interaction data were visualized and analyzed using STRING, after which GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses were conducted via DAVID (Database for Annotation, Visualization and Integrated Discovery). Several common genes were identified between the SREBP pathway and CNS disorders. In GO enrichment analysis, the most enriched biological processes included lipid, cholesterol, and steroid biosynthetic processes; the most enriched molecular functions were transcription factor-related; and the most enriched subcellular compartments revealed that the genes involved in CNS disorders were mainly associated with the enzyme complexes of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN). In KEGG enrichment analysis, the most enriched pathway was the AMP-activated protein kinase (AMPK) signaling pathway, and the top-ranked genes significantly enriched under this pathway were ACACA, ACACB, FASN, HMGCR, MTOR, PPARGC1A, PRKAA1, SCD, SIRT1, and SREBF1. The findings of this study strengthen the evidence linking the involvement of lipid homeostasis in CNS functions. We suggest herein the roles of downstream ACC and FASN enzymes and upstream AMPK signaling in the SREBP pathway as mechanisms underlying neurodegenerative, neurological, and neuropsychiatric CNS disorders.

Monensin supplementation downregulated the expression signature of genes involved in cholesterol synthesis in the ruminal epithelium and adipose tissue of lambs

To understand the metabolic mechanisms regulating lipid metabolism by monensin, Afshari male lambs (n = 16) with 41.0 ± 2.4kg body weight (BW, mean ± SD) at approximately 180days of age were randomly assigned equally to two dietary treatments. After a 21-day pre-adaptation period, all animals in two groups continued to receive the basal diet, but one group received no monensin supplementation (control) while the other group received 30mg/day of monensin per animal. Individual BW was recorded weekly to determine the average daily body weight gain (ADG). At the end of the 56-day experimental period, lambs were weighed and slaughtered. Monensin supplementation did not affect BW, ADG, and rumen fermentation characteristics. However, monensin significantly downregulated the sterol regulatory element-binding protein (SREBP)-2 gene expression in all sample tissues (p < 0.05). Also, monensin downregulated expressions of SREBP-1c and peroxisome proliferator-activated receptor (PPAR)-γ in back fat tissues. Monensin increased the expression of 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS)-2, but it decreased the mRNA abundance of HMGCS-1 in the rumen epithelial tissues (p < 0.05). Our data suggest that monensin downregulates cholesterol synthesis via inhibition of HMGCS-1 and impairment of the SREBP pathway, probably due to a crosstalk among different tissues to control energy metabolism.

Abstract PR-009: Targeting the sterol regulatory element-binding protein pathway in pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive tumor with limited diagnostic and therapeutic options. Due to its proliferative nature and thick, desmoplastic stroma, PDAC tumor cells are challenged with meeting a high demand for lipids in a hypoxic, lipid-poor environment. Cancer cells respond to lipid conditions through sterol regulatory element-binding proteins (SREBPs). SREBPs are master transcriptional regulators of lipid homeostasis and require SREBP cleavage activating protein (SCAP) during signaling. While the SREBP pathway has been implicated in several cancers, its role in PDAC has not been examined. In this study, we assess the requirement of this pathway using both in vitro and in vivo model systems. Furthermore, we target the SREBP pathway using a novel combination therapy of two FDA-approved compounds: dipyridamole and statins. Methods: We acquired four patient-derived pancreatic adenocarcinoma cell lines containing mutations in both Kras and p53 and knocked out SCAP in each cell line followed by gene-based rescue. Functional growth assays were performed in both lipid-poor and lipid-rich media conditions. Subcutaneous and pancreatic orthotopic xenografts were performed in nude mice using these same cell lines. A previously established mouse line, LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1 Cre (KPC) on a C57Bl/6 background, was utilized as a PDAC model. KPC mice lacking Scap in one or both alleles (Sfl/+ or Sfl/fl) were generated. Cohort survival and histologic analysis were performed for all mice, and results plotted on a Kaplan-Meier survival curve. In each patient cell line, the following drugs were applied individually and in combination: Dipyridamole (DP), tetramethyl-DP (TMDP), Fluvastatin, and Simvastatin. Cell viability post-treatment was assessed, and synergy calculated for each combination using SynergyFinder. Results: In lipid-poor conditions, SCAP knockout cells showed significantly reduced growth when compared to wild type or SCAP rescued cells. In tumor xenograft models, SCAP knockout cells exhibited reduced tumor growth and tumor volume when compared to wild-type cells. In the genetically engineered mouse models, KPC mice exhibited a median survival time of 289 days, while KPCSfl/+ mice show a significantly increased median survival time of 425 days. Additionally, KPCSfl/fl mice did not develop invasive PDAC. Cells treated individually with DP, TMDP and both statins exhibit lipid-dependent growth defects in all cell lines tested. In combination, DP with either statin as well as TMDP with either statin demonstrate synergy in lipid-poor conditions. Conclusions: Our results demonstrate that loss of SCAP in PDAC tumor cells alters the growth capability both in vitro and using mouse xenograft models. Additionally, heterozygous loss of Scap in the KPC mouse model significantly increased survival. Finally, dipyridamole works in synergy with statins to alter growth of PDAC tumor cells. These findings suggest that targeting the SREBP pathway has significant therapeutic potential in PDAC. Citation Format: Stephanie Myers, Meredith McGuire, Wei Shao, Chine Liu, Theodore Ewachiw, Zeshaan Rasheed, William Matsui, Toni Sepalla, Richard Burkhart, Peter Espenshade. Targeting the sterol regulatory element-binding protein pathway in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-009.

ERAD components Derlin-1 and Derlin-2 are essential for postnatal brain development and motor function

SummaryDerlin family members (Derlins) are primarily known as components of the endoplasmic reticulum-associated degradation pathway that eliminates misfolded proteins. Here we report a function of Derlins in the brain development. Deletion of Derlin-1 or Derlin-2 in the central nervous system of mice impaired postnatal brain development, particularly of the cerebellum and striatum, and induced motor control deficits. Derlin-1 or Derlin-2 deficiency reduced neurite outgrowth in vitro and in vivo and surprisingly also inhibited sterol regulatory element binding protein 2 (SREBP-2)-mediated brain cholesterol biosynthesis. In addition, reduced neurite outgrowth due to Derlin-1 deficiency was rescued by SREBP-2 pathway activation. Overall, our findings demonstrate that Derlins sustain brain cholesterol biosynthesis, which is essential for appropriate postnatal brain development and function.

Structural basis for sterol sensing by Scap and Insig.

The sterol regulatory element-binding protein (SREBP) pathway monitors the cellular cholesterol level through sterol-regulated association between the SREBP cleavage-activating protein (Scap) and the insulin-induced gene (Insig). Despite structural determination of the Scap and Insig-2 complex bound to 25-hydroxycholesterol, the luminal domains of Scap remain unresolved. In this study, combining cryogenic electron microscopy (cryo-EM) analysis and artificial intelligence-facilitated structural prediction, we report the structure of the human Scap/Insig-2 complex purified in digitonin. The luminal domain loop 1 and a co-folded segment in loop 7 of Scap resemble those of the luminal/extracellular domain in NPC1 and related proteins, providing clues to the cholesterol-regulated interaction of loop 1 and loop 7. An additional luminal interface is observed between Scap and Insig. We also show that Scap(D428A), which inhibits SREBP activation even under sterol depletion, exhibits an identical conformation with the wild-type protein when complexed with Insig-2, and its constitutive suppression of the SREBP pathway may also involve a later step in protein trafficking.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Apigenin Ameliorates Insulin Resistance and Lipid Accumulation by Endoplasmic Reticulum Stress and SREBP-1c/SREBP-2 Pathway in Palmitate-Induced HepG2 Cells and High-Fat Diet-Fed Mice.

Insulin resistance (IR) is the common basis of diabetes and cardiovascular diseases, and its development is closely associated with lipid metabolism disorder. Flavonoids have definite chemical defense effects, including anti-inflammatory effects, anticancer effects, and antimutation effects. However, the function and mechanism of apigenin (AP, a kind of flavonoid) in IR are still unclear. In our study, intracellular fat accumulation model cells and high-fat diet (HFD)-fed model mice were established using palmitate (PA) and HFD. Mechanistically, we first demonstrated that AP could notably downregulate sterol regulatory element-binding protein 1c (SREBP-1c), sterol regulatory element-binding protein 2 (SREBP-2), fatty acid synthase, stearyl-CoA desaturase 1, and 3-hydroxy-3-methyl-glutaryl-CoA reductase in PA-induced hyperlipidemic cells and mice. Functionally, we verified that AP could markedly reduce lipid accumulation in PA-induced hyperlipidemic cells and decrease the body weight, visceral fat weight, IR, and lipid accumulation in HFD-induced hyperlipidemic mice. Besides, we showed that PA could significantly downregulate endoplasmic reticulum stress (ERS)-related proteins and inhibit ERS. Furthermore, we proved that AP could reduce blood lipids by inhibiting ERS in PA-induced hyperlipidemic cells. Meanwhile, 4-phenyl butyric acid (also called ERS alleviator), like AP, could significantly reduce blood lipids and alleviate IR in HFD-fed model mice. Therefore, we concluded that AP could substantially improve the disorder of lipid metabolism, and its mechanism might be related to the decrease of SREBP-1c, SREBP-2, and downstream genes, the inhibition of ERS, and the reduction of blood lipids and IR. SIGNIFICANCE STATEMENT: Apigenin, a nontoxic and naturally sourced flavonoid, has antihyperlipidemic properties in mice and hepatocyte. This study highlights a new mechanism of apigenin and proposes that these hypolipidemic effects are associated with the mitigation of endoplasmic reticulum stress and insulin resistance in diet-induced obesity. This study might provide translational insight into the prevention and treatment of apigenin in hyperlipidemia-related diseases.

Role of the Sterol Regulatory Element Binding Protein Pathway in Tumorigenesis.

Metabolic changes are a major feature of tumors, including various metabolic forms, such as energy, lipid, and amino acid metabolism. Sterol regulatory element binding proteins (SREBPs) are important modules in regulating lipid metabolism and play an essential role in metabolic diseases. In the previous decades, the regulatory range of SREBPs has been markedly expanded. It was found that SREBPs also played a critical role in tumor development. SREBPs are involved in energy supply, lipid supply, immune environment and inflammatory environment shaping in tumor cells, and as a protective umbrella to support the malignant proliferation of tumor cells. Natural medicine and traditional Chinese medicine, as an important part of drug therapy, demonstrates the multifaceted effects of SREBPs regulation. This review summarizes the core processes in the involvement of SREBPs in tumors and provides a comprehensive understanding of the pathways through which natural drugs target the SREBP pathway and regulate tumor progression.