Fatty Acid Synthase Knockdown Research Articles

Abstract 1796: Role of FASN in pancreatic neuroendocrine cancer cells survival

Abstract Introduction. Pancreatic neuroendocrine tumors (pNETs) are among the most frequently occurring neuroendocrine neoplasms. Up-regulation of fatty acid synthase (FASN) has been reported in many cancers, but the role of lipid metabolism in pNETs remains unclear. The purpose of this study was to determine whether FASN inhibition can be used as a target for pNET treatment. Methods. Immunohistochemistry and western blot analysis were used to determine protein levels of FASN in pNET tissues and cell lines. Expression of FASN was evaluated in pancreatic neuroendocrine primary and metastatic tumors and scored by a pathologist. Neutral lipid droplet levels in pNET cell lines were determined with BODIPY 493/503 staining. Clonogenic and SRB assays were used to evaluate cytotoxic effects of FASN inhibition by TVB-3664 in QGP-1 and BON pancreatic NET cell lines. Western blot analysis was used to determine levels of cleaved PARP and cyclin D1 after FASN inhibition. FASN knockdown was used to determine the effects of FASN downregulation on proliferation and lipid synthesis. Results. FASN expression was determined at the maximum scores of 6 and 5 in 98% of patient samples. BON, QGP-1 and NT-3 cell lines were stained with BODIPY 493/503; the highest levels of neutral lipid droplets were observed in the BON cell line. BON and QGP-1 cells were treated with TVB-3664 in either complete media with fatty acids (FA+) or complete media prepared with fatty acid depleted FBS (FA-). Treatment with TVB-3664 resulted in significant inhibition of BON and QGP-1 cell proliferation and cyclin D1 expression in both FA+ and FA- conditions. Depletion of extracellular FA enhanced the effect of TVB-3664 therapy on NET cells, especially in the BON cell line. FASN inhibition with TVB-3634 suppressed pNET cell proliferation and colony formation. Conclusions. Our study identified high protein levels of FASN in pNET patient samples and cell lines. Moreover, we demonstrated variability in the response of pNET cells to FASN inhibition. QGP cell viability was significantly decreased with FASN inhibition even in the presence of extracellular lipids. FASN inhibition in BON cells had only a minimal effect on cell viability and required depletion of extracellular lipids to achieve a decrease in cell viability and cyclin D1 expression. These data demonstrate that de novo lipid synthesis is involved in the pathogenesis and progression of pNETs. Importantly, our findings show that some pNETs are highly sensitive to FASN inhibition, suggesting that FASN inhibition may be considered as a treatment option in selected pNETs. Citation Format: Benjamin M. Evers, Zeta Chow, Yekaterina Zaytseva, Eun Y. Lee, Courtney M. Townsend, Tianyan Gao, B. Mark Evers, Piotr G. Rychahou. Role of FASN in pancreatic neuroendocrine cancer cells survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1796.

FASN promotes gallbladder cancer progression and reduces cancer cell sensitivity to gemcitabine through PI3K/AKT signaling.

Lipid metabolism plays an important role in the growth and development of tumors. However, the role of lipid metabolism in gallbladder cancer (GBC) has not been clearly clarified. Here, we demonstrated that fatty acid synthase (FASN), a key enzyme in de novo fatty acid biosynthesis, had upregulated expression in GBC samples both at protein and mRNA levels. Analysis of clinical data indicated the association between elevated FASN expression and poorer histology grades. Furthermore, FASN activity impairment through FASN knockdown or treatment with orlistat resulted in the inhibition of cell proliferation and migration, as well as increased sensitivity to gemcitabine. Both FASN knockdown and orlistat treatment induced cell apoptosis. Mechanistically, impairment of FASN activity suppressed the activation of the PI3K/AKT signaling pathway, which led to increased cell apoptosis and sensitivity to gemcitabine. These findings were also validated through nude mouse xenograft models, thus highlighting the potential of targeting FASN as a clinical treatment strategy. Collectively, the present study underscores the crucial role of FASN in the progression of gallbladder cancer via the PI3K/AKT pathway.

Overexpression of fatty acid synthase attenuates bleomycin induced lung fibrosis by restoring mitochondrial dysfunction in mice

Proper lipid metabolism is crucial to maintain alveolar epithelial cell (AEC) function, and excessive AEC death plays a role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). The mRNA expression of fatty acid synthase (FASN), a key enzyme in the production of palmitate and other fatty acids, is downregulated in the lungs of IPF patients. However, the precise role of FASN in IPF and its mechanism of action remain unclear. In this study, we showed that FASN expression is significantly reduced in the lungs of IPF patients and bleomycin (BLM)-treated mice. Overexpression of FASN significantly inhibited BLM-induced AEC death, which was significantly potentiated by FASN knockdown. Moreover, FASN overexpression reduced BLM-induced loss of mitochondrial membrane potential and the production of mitochondrial reactive oxygen species (ROS). Oleic acid, a fatty acid component increased by FASN overexpression, inhibited BLM-induced cell death in primary murine AECs and rescue BLM induced mouse lung injury/fibrosis. FASN transgenic mice exposed to BLM exhibited attenuated lung inflammation and collagen deposition compared to controls. Our findings suggest that defects in FASN production may be associated with the pathogenesis of IPF, especially mitochondrial dysfunction, and augmentation of FASN in the lung may have therapeutic potential in preventing lung fibrosis.

Abstract 414: Targeting lipid metabolism to improve efficacy of braf-targeted therapy in colorectal cancer

Abstract Background: Aberrant lipid metabolism is associated with poor prognosis in colorectal cancer (CRC). Fatty acid synthase (FASN), a key enzyme of lipogenesis, is overexpressed and a therapeutic target in CRC. Overexpression of CD36, a fatty acid translocase, plays a pro-tumorigenic role in CRC. BRAFV600E is the mutation occurring in about 10-15% of CRC cases. BRAF-targeted therapy is effective, but quickly developed resistance is an issue. Our preliminary data show that development of resistance to BRAF-targeted therapy is associated with an increase in expression of FASN, CD36, accumulation of triglycerides (TGs), and mitochondrial respiration. Therefore, our central hypothesis is that inhibition of lipid metabolism will sensitize CRC cells to BRAF inhibitors and overcome acquired resistance. Methods: We established HT29 cells and primary PT130 and PT2449pt cells resistant to PLX8394, a novel BRAF inhibitor. IC50 curves, PrestoBlue viability, CytoSelect™ 24-Well Cell Invasion, and Triglyceride Assays, Seahorse XF analysis, western blot, and confocal microscopy were used to evaluate differences between parental and resistant cells. RNA-seq and lipid analysis were used to evaluate changes in gene expression and lipids levels. Combination of PLX8394 and TVB3664 (FASN inhibitor) was tested on cell viability in parental and resistant cells. Results: PLX8394 resistant cells have a higher IC50, an increased proliferation, and invasion compared to parental cells. An increase in invasion of resistant cells is associated with a decrease in E-cadherin. RNA sequencing shows a significant increase in FASN expression. Western blot on resistant cells confirms upregulation of FASN, CD36, and other lipogenic markers. Consistently, resistant cells have an increase in levels of triglycerides as compared to parental cells. Seahorse XF Cell Mito Stress Test shows that resistant cells forgo the Warburg effect and instead rely more heavily on oxidative phosphorylation. To further support our hypothesis, FASN shRNA knockdown of FASN in parental HT29 cells is more susceptible to PLX8394 treatment compared to control. We also observed the synergetic effect of PLX8394 and TVB3664 treatment on cell viability in parental cells, but not in resistant cells. Conclusion: Our study demonstrates that resistance to BRAF inhibitors is associated with a significant increase in proliferation, metastasis, and upregulation of lipid metabolism. We show that combination of FASN and BRAF inhibitors has a combinational effect on inhibiting cell viability in parental but not resistant cells, suggesting that the addition of FASN inhibitor to the standard regimen for BRAFV600E mutation positive patients can improve efficacy of these therapies. Additional screening of lipid metabolism-targeted therapies in combination with standard BRAF regimens are needed to develop novel and more efficacious strategies for CRC patients with BRAF mutations. Citation Format: Mariah E. Geisen, Dang he, Chi Wang, Jill M. Kolesar, Yekaterina Zaytseva. Targeting lipid metabolism to improve efficacy of braf-targeted therapy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 414.

Anlotinib suppresses lung adenocarcinoma growth via inhibiting FASN-mediated lipid metabolism.

Anlotinib, a vascular endothelial growth factor receptor (VEGFR) inhibitor, has been widely used in advanced lung cancer patients, but the intrinsic mechanism of cancer cell elimination is not fully disclosed. In this study, we reported that anlotinib suppressed lung adenocarcinoma (LUAD) growth through inhibiting fatty acid synthase (FASN)-mediated lipid metabolism. To investigate the underlying mechanisms of anlotinib, an A549 cell line-derived xenograft model was constructed and a proteomics technique was employed to screen potential markers. Gas chromatography-mass spectrometry (GC-MS) profiling of medium-long chain fatty acid and neutral lipid droplet fluorescence staining were employed to detect lipid metabolism in cancer cells. Subsequently, the effects of anlotinib on FASN expression were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot. Short hairpin RNA (shRNA) knockdown of FASN was used to assess the role of FASN in the antitumor effect of anlotinib. A patient-derived xenograft (PDX) model was established to validate the efficacy of anlotinib in the patient and IHC staining of FASN was examined. Our data revealed that anlotinib significantly decreased the expression of proteins related to lipid metabolism. GC-MS profiling of medium-long chain fatty acid and neutral lipid droplet fluorescence staining validated that anlotinib could disturb the fatty acid metabolism in cancer cells, especially de novo lipogenesis. Mechanically, the messenger RNA (mRNA) and protein of FASN were down-regulated by anlotinib in A549 cells and FASN knockdown could diminish the antitumor effect of anlotinib in vitro. Remarkable tumor shrinkage by anlotinib was further shown in a patient with multiple-line treatment failure, and FASN reduction was evidenced in the corresponding patient-derived xenograft (PDX) model. Anlotinib could inhibit the growth of LUAD through FASN-mediated lipid metabolism. Our findings provide new insights into the antitumor mechanism of anlotinib in lung adenocarcinoma.

Fatty acid synthase (FASN) inhibits the cervical squamous cell carcinoma (CESC) progression through the Akt/mTOR signaling pathway

BackgroundCervical cancer is a malignant tumor that affects females and remains the cause of the highest morbidity and mortality among women worldwide. Currently, gene-targeted therapy is a novel treatment option for clinicians. Furthermore, fatty acid synthase (FASN) plays a therapeutic role in various cancers. Nonetheless, the mechanism of action of this enzyme in cervical squamous cell carcinoma and cervical duct adenocarcinoma (CESC) has not yet been reported. MethodsRNA (ribonucleic acid) sequencing data and clinical information were obtained from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx). The expression levels of FASN were obtained from Gene Expression Profiling Interactive Analysis 2 (GEPIA2) and Human Protein Atlas (HPA). Univariate and multivariate Cox regression analyses were utilized to assess independent prognostic factors associated with survival. A nomogram and receiver operating characteristic curve (ROC) were employed to evaluate survival and predictive power. In vitro experiments and real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) were conducted to identify cell interference efficiency. MTS, monoclonal formation, and EDU assays were used to determine cell viability. Wound healing and invasion assays (transwell assay) were used to evaluate cell migration and invasion. Finally, Hoechst 33342, propidium iodide (PI) staining and Annexin V-FITC staining were used to assess apoptosis and the cell cycle, while western blotting was utilized to determine the protein expression levels. ResultsFASN was aberrantly expressed in various cancers, including CESC, where it was highly expressed. Kaplan-Meier, univariate, multivariate Cox regression analyses and ROC curve indicated that FASN is a potential key indicator of survival prognosis among CESC patients and demonstrated good predictive ability and efficacy. Complementary in vitro experiments confirmed that FASN is an important target for CESC therapy. ConclusionThe current study validated the biological and clinical significance of FASN in CESC prognosis, suggesting that FASN knockdown may exert antitumor activity against cervical cancer through the Akt/mTOR signaling pathway.

Cryptotanshinone suppresses tumorigenesis by inhibiting lipogenesis and promoting reactive oxygen species production in KRAS‑activated pancreatic cancer cells.

Pyruvate dehydrogenase kinase 4 (PDK4) is an important regulator of energy metabolism. Previously, knockdown of PDK4 by specific small interfering RNAs (siRNAs) have been shown to suppress the expression of Kirsten rat sarcoma viral oncogene homolog (KRAS) and the growth of lung and colorectal cancer cells, indicating that PDK4 is an attractive target of cancer therapy by altering energy metabolism. The authors previously reported that a novel small molecule, cryptotanshinone (CPT), which inhibits PDK4 activity, suppresses the in vitro three-dimensional (3D)-spheroid formation and in vivo tumorigenesis of KRAS-activated human pancreatic and colorectal cancer cells. The present study investigated the molecular mechanism of CPT-induced tumor suppression via alteration of glutamine and lipid metabolism in human pancreatic and colon cancer cell lines with mutant and wild-type KRAS. The antitumor effect of CPT was more pronounced in the cancer cells containing mutant KRAS compared with those containing wild-type KRAS. CPT treatment decreased glutamine and lipid metabolism, affected redox regulation and increased reactive oxygen species (ROS) production in the pancreatic cancer cell line MIAPaCa-2 containing mutant KRAS. Suppression of activated KRAS by specific siRNAs decreased 3D-spheroid formation, the expression of acetyl-CoA carboxylase 1 and fatty acid synthase (FASN) and lipid synthesis. The suppression also reduced glutathione-SH/glutathione disulfide and increased the production of ROS. Knockdown of FASN suppressed lipid synthesis in MIAPaCa-2 cells, partially promoted ROS production and mildly suppressed 3D-spheroid formation. These results indicated that CPT reduced tumorigenesis by inhibiting lipid metabolism and promoting ROS production in a mutant KRAS-dependent manner. This PDK4 inhibitor could serve as a novel therapeutic drug for KRAS-driven intractable cancers via alteration of cell metabolism.

Berberine suppresses the migration and invasion of colon cancer cells by inhibition of lipogenesis through modulation of promyelocytic leukemia zinc finger-mediated sterol-regulatory element binding proteins cleavage-activating protein ubiquitination

This study was aimed to explore whether and how berberine suppresses colon cancer cell metastasis via lipid modulation. Lipid accumulation was measured by an oil red O staining kit. The expression of proteins and message RNA was detected by Western blot and quantitative real-time PCR. The interaction of sterol-regulatory element-binding proteins cleavage-activating protein (SCAP) with promyelocytic leukaemia zinc finger (PLZF) was confirmed by co-immunoprecipitation assay. Expressions of fatty acid synthase (FASN) and PLZF were knocked down by specific small interfering RNA. Berberine inhibited the migration and invasion of HCT-8, HCT-116 and HT-29 cells. Moreover, it was observed that berberine decreased lipid droplet accumulation. FASN knockdown abolished the inhibitory effects of berberine on cell migration and invasion. Further investigation revealed that berberine induced the ubiquitination degradation of SCAP. And PLZF interacted with SCAP and promoted its ubiquitination, which was inhibited by berberine treatment. Silence of PLZF impaired the effects of berberine on SCAP ubiquitination and lipogenesis. Berberine suppressed lipogenesis via promotion of PLZF-mediated SCAP ubiquitination, thereby inhibiting colon cancer cell metastasis.

FASN promotes lymph node metastasis in cervical cancer via cholesterol reprogramming and lymphangiogenesis

Cervical cancer (CC) patients with lymph node metastasis (LNM) have a poor prognosis. Clarification of the detailed mechanisms underlying LNM may provide potential clinical therapeutic targets for CC patients with LNM. However, the molecular mechanism of LNM in CC is unclear. In the present study, we demonstrated that fatty acid synthase (FASN), one of the key enzymes in lipid metabolism, had upregulated expression in the CC samples and was correlated with LNM. Moreover, multivariate Cox proportional hazards analysis identified FASN as an independent prognostic factor of CC patients. Furthermore, gain-of-function and loss-of-function approaches showed that FASN promoted CC cell migration, invasion, and lymphangiogenesis. Mechanistically, on the one hand, FASN could regulate cholesterol reprogramming and then activate the lipid raft-related c-Src/AKT/FAK signaling pathway, leading to enhanced cell migration and invasion. On the other hand, FASN induced lymphangiogenesis by secreting PDGF-AA/IGFBP3. More importantly, knockdown of FASN with FASN shRNA or the inhibitors C75 and Cerulenin dramatically diminished LNM in vivo, suggesting that FASN plays an essential role in LNM of CC and the clinical application potential of FASN inhibitors. Taken together, our findings uncover a novel molecular mechanism in LNM of CC and identify FASN as a novel prognostic factor and potential therapeutic target for LNM in CC.

α-Linolenic acid induces apoptosis, inhibits the invasion and metastasis, and arrests cell cycle in human breast cancer cells by inhibiting fatty acid synthase

• Discover α-linolenic acid (ALA) as a novel fatty acid synthase (FASN) inhibitor. • ALA binds to the TE domain of FASN. • ALA obviously reduces the viability of human breast cancer cells, while palmitic acid shows no effect. • ALA induces breast cancer cell apoptosis, inhibits the invasion and metastasis, and arrests cell cycle in breast cancer cells. Fatty acid synthase (FASN) is an enzyme that synthesizes endogenous fatty acids. FASN overexpressed in various cancers, which indicates the involvement of FASN in cancer progression. α-Linolenic acid (ALA) is an omega-3 fatty acid with many biological activities, including anti-cancer effects. The aim of the present study was to investigate the inhibitory effect of ALA on fatty acid synthesis pathway and breast cancer cells apoptosis. We found FASN expression decreased significantly in ALA treated breast cancer cells. Compared with palmitic acid (PA), ALA reduced cell viability in a dose-dependent manner. ALA showed a higher affinity with the TE domain than PA. ALA induced breast cancer cells apoptosis, which effects were similar with the knockdown of FASN. In addition, ALA inhibited the invasion and metastasis, and arrested cell cycle in breast cancer cells. We propose a hypothesis that ALA could contribute to the treatment of human breast cancer by inhibiting FASN.

Sulforaphanes: disruptors of phagophores and autolysosomes

Sulforaphane and its metabolites (SFNs) cause apoptosis in cancers and could be potential anti-cancer drugs. We focused on investigating the underlying mechanisms through which SFNs inhibit cancers. First, SFNs cause microtubule disruption by phosphorylated MAPK1/ERK2-MAPK3/ERK1-mediated activation of 26S proteasome leading to a microtubule-associated protein degradation and microtubule depolymerization. Second, SFNs cause the accumulation of autophagosomes and mitophagosomes via blocking their fusion with lysosomes. These changes might be involved in multiple signaling pathways. High-performance liquid chromatography-tandem mass spectrometry showed that SFN regulates the expression of lipoproteins; highly expressed FASN (fatty acid synthase) correlates with cancer malignancy and poor prognosis. More, SFN lowers the expressions of FASN, ACACA (acetyl-CoA carboxylase alpha), and ACLY (ATP citrate lyase) by activating the 26S proteasome; SFN inhibits the interactions of TUBA/α-tubulin with FASN, ACACA or ACLY; SFN also reduces the production of intracellular fatty acids; knockdown of FASN increases mitochondrial abnormality and apoptosis. Moreover, SFN decreases the expressions of mitophagy-associated proteins BNIP3L/NIX and BNIP3 and the interaction between BNIP3L/NIX and LC3-II/-I and upregulates mitochondria-associated LC3-II/-I. Therefore, SFN might cause apoptosis via inhibiting the microtubule-mediated lipoprotein activity and the fusion of lysosomes with autophagosomes and mitophagosomes.

Suppressing fatty acid synthase by type I interferon and chemical inhibitors as a broad spectrum anti-viral strategy against SARS-CoV-2

SARS-CoV-2 is an emerging viral pathogen and a major global public health challenge since December of 2019, with limited effective treatments throughout the pandemic. As part of the innate immune response to viral infection, type I interferons (IFN-I) trigger a signaling cascade that culminates in the activation of hundreds of genes, known as interferon stimulated genes (ISGs), that collectively foster an antiviral state. We report here the identification of a group of type I interferon suppressed genes, including fatty acid synthase (FASN), which are involved in lipid metabolism. Overexpression of FASN or the addition of its downstream product, palmitate, increased viral infection while knockout or knockdown of FASN reduced infection. More importantly, pharmacological inhibitors of FASN effectively blocked infections with a broad range of viruses, including SARS-CoV-2 and its variants of concern. Thus, our studies not only suggest that downregulation of metabolic genes may present an antiviral strategy by type I interferon, but they also introduce the potential for FASN inhibitors to have a therapeutic application in combating emerging infectious diseases such as COVID-19.

TAMI-65. FASN-MEDIATED LIPID SYNTHESIS HAMPERS ANTI-CANCER IMMUNITY OF GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most aggressive type of primary brain tumor in adults. Radiation therapy (RT) is an essential modality for GBM treatment and is recognized to stimulate anti-cancer immunity by at least generating type I interferon (IFN-I) responses. However, RT also exacerbates potent immune inhibitory mechanisms that facilitate immune evasion. Notably, increased lipid synthesis by the fatty acid synthase (FASN) is an emerging mechanism that can account for the deceiving treatment efficacy and immune escape of GBM. Therefore, we hypothesize that FASN-mediated lipid synthesis represents an innate immune evasion mechanism of irradiated GBM. Supporting this hypothesis, we observed that 10 gray (Gy) irradiation of murine GBM cell lines, GL261 and CT2A, upregulates FASN protein expression and increase cellular lipid content determined by BODIPY staining and electronic microscopy. Interestingly, this effect was abrogated when GBM cells were incubated with an inhibitor of FASN. Next, to ask whether FASN was impairing RT-induced IFN-I, GL261 and CT2A cells were engineered to express an inducible shRNA silencing FASN (GBMshFASN) or its non-silencing control (GBMshNS). Irradiation of GBMshNS cells enhanced the secretion of IFN-beta and CXCL10, but this effect was more pronounced when FASN was blocked. Finally, GBMshNS and GBMshFASN cells were orthotopically implanted in mice on day 0. On day 10, selective irradiation (10Gy) was performed to the tumor. Tumor growth and immune contexture were evaluated on day 17. Magnetic resonance imaging revealed that FASN knockdown reduces tumor growth independently from RT. However, in situ immunofluorescence of GBMshFASN tumors showed increased infiltration of CD8+ T cells and CD11c+ cells only in irradiated mice bearing GBMshFASN tumors. Altogether, our data suggest that RT rewires the energy supply of GBM by promoting FASN-mediated lipid synthesis to foster immune evasion. Targeting FASN is a promising strategy to promote anti-cancer immunity and sensitize irradiated GBM to immunotherapies.

905 FASN prevents immunogenicity of irradiated glioblastoma by inhibiting ER stress

BackgroundGlioblastoma (GBM) is the most aggressive and incurable adult brain tumor. Radiation therapy (RT) is an essential modality for GBM treatment and is recognized to stimulate anti-tumor immunity by inducing immunogenic cell death (ICD) subsequent to endoplasmic reticulum (ER) stress. However, RT also exacerbates potent immunosuppressive mechanisms that facilitate immune evasion. Notably, increased de novo lipid synthesis by the fatty acid synthase (FASN) is emerging as a mechanism of therapy resistance and immune escape. Here, we hypothesize that RT induces FASN to promote GBM survival and evade immune recognition by inhibiting ER stress and ICD.MethodsTo determine if lipid synthesis is altered in response to RT, we first assessed FASN expression by western blot (WB) and lipid accumulation by BODIPY staining in murine (CT2A and GL261) and human (U118) GBM cell lines. Next, FASN expression was blocked in CT2A cells using CRISPR-Cas9 or an inducible shRNA directed against Fasn to evaluate ICD and ER stress markers by ELISA, WB, and electron microscopy. Finally, CT2AshFASN cells or its non-silencing control (CT2AshNS) were orthotopically implanted and FASN knockdown was induced by feeding the mice with doxycycline. The immune contexture was determined by in situ immunofluorescence (n=3/group). Remaining mice were followed for survival (n=7/group).ResultsWe found that in vitro irradiation of GBM cells induces lipid accumulation in a dose-dependent fashion; an effect that is magnified over time lasting at least 6/7 days. Consistent with these findings, FASN expression was upregulated in irradiated GBM cells. Confirming the role of FASN, RT-induced accumulation of lipids was reverted when GBM cells were incubated with a FASN inhibitor. Next, we found that FASN ablation in CT2A cells induces mitochondria disruption and was sufficient to increase the expression of the ER stress makers BIP and CHOP. Along similar lines, shFASN enhances the secretion of the ICD markers HMGB1, IFN-beta and CXCL10 in irradiated CT2A cells. In vivo, CT2AshFASN tumors presented increased infiltration of CD11c+ cells and CD8+ T cells, consistent with prolonged mice survival (56 days vs. 28 days for CT2AshNS). Importantly, 43% of CT2AshFASN-bearing mice remained tumor-free for more than 70 days, while none of the CT2AshNS-bearing mice survived.ConclusionsAltogether, our data suggest that FASN-mediated lipid synthesis is an important mechanism to prevent ER stress, ICD, and anti-tumor immune responses in GBM. While much work remains to be done, our data propose FASN as a novel therapeutic target to overcome immunosuppression and sensitize GBM to immunotherapies.

Sulforaphane downregulated fatty acid synthase and inhibited microtubule-mediated mitophagy leading to apoptosis

We previously demonstrated that sulforaphane (SFN) inhibited autophagy leading to apoptosis in human non-small cell lung cancer (NSCLC) cells, but the underlying subcellular mechanisms were unknown. Hereby, high-performance liquid chromatography-tandem mass spectrometry uncovered that SFN regulated the production of lipoproteins, and microtubule- and autophagy-associated proteins. Further, highly expressed fatty acid synthase (FASN) contributed to cancer malignancy and poor prognosis. Results showed that SFN depolymerized microtubules, downregulated FASN, and decreased its binding to α-tubulin; SFN downregulated FASN, acetyl CoA carboxylase (ACACA), and ATP citrate lyase (ACLY) via activating proteasomes and downregulating transcriptional factor SREBP1; SFN inhibited the interactions among α-tubulin and FASN, ACACA, and ACLY; SFN decreased the amount of intracellular fatty acid (FA) and mitochondrial phospholipids; and knockdown of FASN decreased mitochondrial membrane potential (ΔΨm) and increased reactive oxygen species, mitochondrial abnormality, and apoptosis. Further, SFN downregulated mitophagy-associated proteins Bnip3 and NIX, and upregulated mitochondrial LC3 II/I. Transmission electron microscopy showed mitochondrial abnormality and accumulation of mitophagosomes in response to SFN. Combined with mitophagy inducer CCCP or autophagosome–lysosome fusion inhibitor Bafilomycin A1, we found that SFN inhibited mitophagosome–lysosome fusion leading to mitophagosome accumulation. SFN reduced the interaction between NIX and LC3 II/I, and reversed CCCP-caused FA increase. Furthermore, knockdown of α-tubulin downregulated NIX and BNIP3 production, and upregulated LC3 II/I. Besides, SFN reduced the interaction and colocalization between α-tubulin and NIX. Thus, SFN might cause apoptosis via inhibiting microtubule-mediated mitophagy. These results might give us a new insight into the mechanisms of SFN-caused apoptosis in the subcellular level.

Targeting Fatty Acid Synthase Modulates Metabolic Pathways and Inhibits Cholangiocarcinoma Cell Progression.

An aberrant regulation of lipid metabolism is involved in the pathogenesis and progression of cancer. Up-regulation of lipid biosynthesis enzymes, including acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN) and HMG-CoA reductase (HMGCR), has been reported in many cancers. Therefore, elucidating lipid metabolism changes in cancer is essential for the development of novel therapeutic targets for various human cancers. The current study aimed to identify the abnormal expression of lipid-metabolizing enzymes in cholangiocarcinoma (CCA) and to evaluate whether they can be used as the targets for CCA treatment. Our study demonstrated that a high expression of FASN was significantly correlated with the advanced stage in CCA patients. In addition, survival analysis showed that high expression of FASN and HMGCR was correlated with shorter survival of CCA patients. Furthermore, FASN knockdown inhibited the growth, migration and invasion in CCA cell lines, KKU055 and KKU213, as well as induced cell cycle arrest and apoptosis in the CCA cell lines. In addition, metabolomics study further revealed that purine metabolism was the most relevant pathway involved in FASN knockdown. Adenosine diphosphate (ADP), glutamine and guanine levels significantly increased in KKU213 cells while guanine and xanthine levels remarkably increased in KKU055 cells showing a marked difference between the control and FASN knockdown groups. These findings provide new insights into the mechanisms associated with FASN knockdown in CCA cell lines and suggest that targeting FASN may serve as a novel CCA therapeutic strategy.

Fatty Acid Synthase Is Involved in Classical Swine Fever Virus Replication by Interaction with NS4B.

Classical swine fever virus (CSFV), a member of the genus Pestivirus of the family Flaviviridae, relies on host machinery to complete its life cycle. Previous studies have shown a close connection between virus infection and fatty acid biosynthesis, mainly regulated by fatty acid synthase (FASN). However, the molecular action of how FASN participates in CSFV replication remains to be elucidated. In this study, two chemical inhibitors of the fatty acid synthesis pathway [5-(tetradecyloxy)-2-furoic acid (TOFA) and tetrahydro-4-methylene-2R-octyl-5-oxo-3S-furancarboxylic acid (C75)] significantly impaired the late stage of viral propagation, suggesting CSFV replication required fatty acid synthesis. We next found that CSFV infection stimulated the expression of FASN, whereas knockdown of FASN inhibited CSFV replication. Furthermore, confocal microscopy showed that FASN participated in the formation of replication complex (RC), which was associated with the endoplasmic reticulum (ER). Interestingly, CSFV NS4B interacted with FASN and promoted overexpression of FASN, which is regulated by functional Rab18. Moreover, we found that FASN regulated the formation of lipid droplets (LDs) upon CSFV infection, promoting virus proliferation. Taken together, our work provides mechanistic insight into the role of FASN in the viral life of CSFV, and it highlights the potential antiviral target for the development of therapeutics against pestiviruses. IMPORTANCE Classical swine fever, caused by classical swine fever virus (CSFV), is one of the notifiable diseases by the World Organization for Animal Health (OIE) and causes significant financial losses to the pig industry globally. CSFV, like other (+)-strand RNA viruses, requires lipid and sterol biosynthesis for efficient replication. However, the role of lipid metabolism in CSFV replication remains unknown. Here, we found that fatty acid synthase (FASN) was involved in viral propagation. Moreover, FASN is recruited to CSFV replication sites in the endoplasmic reticulum (ER) and interacts with NS4B to regulate CSFV replication that requires Rab18. Furthermore, we speculated that lipid droplet (LD) biosynthesis, indirectly regulated by FASN, ultimately promotes CSFV replication. Our results highlight a critical role for de novo fatty acid synthesis in CSFV infection, which might help control this devastating virus.

The Pharmacological or Genetic Blockade of Endogenous De Novo Fatty Acid Synthesis Does Not Increase the Uptake of Exogenous Lipids in Ovarian Cancer Cells.

Ovarian cancer(OC) is a serious threat to women worldwide. Peritoneal dissemination, ascites and omental metastasis are typical features for disease progression, which occurs in a micro-environment that is rich in high-energy lipids. OC cells require high amounts of lipids for survival and growth. Not only do they import lipids from the host, they also produce lipids de novo. Inhibitors of fatty acid(FA) synthase(FASN) – the rate-limiting enzyme of endogenous FA synthesis that is overexpressed in OC – induce growth-arrest and apoptosis, rendering them promising candidates for cancer drug development. However, cancer researchers have long hypothesized that the lipid deficiency caused by FASN inhibition can be circumvented by increasing the uptake of exogenous lipids from the host, which would confer resistance to FASN inhibitors. In contrast to a very recent report in colorectal cancer, we demonstrate in OC cells (A2780, OVCAR3, SKOV3) that neither FASN inhibitors (G28UCM, Fasnall) nor FASN-specific siRNAs can stimulate a relief pathway leading to enhanced uptake of extrinsic FAs or low density lipoproteins (LDLs). Instead, we observed that the growth-arrest due to FASN inhibition or FASN knock-down was associated with significant dose- and time-dependent reduction in the uptake of fluorescently labeled FAs and LDLs. Western blotting showed that the expression of the FA receptor CD36, the LDL receptor(LDLR) and the lipid transport proteins fatty acid binding proteins 1–9 (FABP1–9) was not affected by the treatment. Next, we compared experimental blockade of endogenous lipid production with physiologic depletion of exogenous lipids. Lipid-free media, similar to FASN inhibitors, caused growth-arrest. Although lipid-depleted cells have diminished amounts of CD36, LDLR and FABPs, they can still activate a restorative pathway that causes enhanced import of fluorophore-labeled FAs and LDLs. Overall, our data show that OC cells are strictly lipid-depend and exquisitely sensitive to FASN inhibitors, providing a strong rationale for developing anti-FASN strategies for clinical use against OC.

Abstract PR-007: Targeting FASN improves type I interferon responses in irradiated glioblastoma

Abstract Glioblastoma (GBM) is a devastating primary brain cancer with a median survival of 11-15 months. Radiotherapy (RT), the standard of care for GBM, increases adjuvanticity of cancer cells, by at least generating cancer cell intrinsic type I interferon (IFN-I) responses to jumpstart antitumor immunity. However, RT can also exacerbate potent immune inhibitory mechanisms to facilitate immune evasion. Among which, metabolic reprogramming of irradiated GBM represents an emerging mechanism of immune resistance. Notably, increased de novo lipid synthesis by the fatty acid synthase (FASN) is a hallmark of GBM that is emerging as a mechanisms of therapy resistance and immune escape. Because FASN was found to impair IFN-I in antiviral immunity, we hypothesize that FASN is preventing RT-induced IFN-I responses to promote GBM survival and evade immune recognition. To test this hypothesis, we first defined RT-induced metabolic changes by seahorse assay and metabolomics in the GL261 murine GBM model. We observed an increase in mitochondrial respiration, glycolysis and in lipid metabolism-related pathways in 10 gray (Gy) irradiated GL261 cells. Consistent with this last observation, we found upregulation of FASN expression by western blot and intracellular neutral lipid accumulation by BODIPY staining and transmission electron microscopy (TEM) in irradiated GL261 cells (10 Gy). Confirming the role of FASN, RT-induced accumulation of neutral lipids was reverted when GL261 cells were incubated with a pharmacologic FASN inhibitor. Next, to ask whether FASN was impairing IFN-I in irradiated GBM, GL261 cells were engineered to express an inducible shRNA silencing FASN (GL261shFASN) or its non-silencing control (GL261shNS). As expected, irradiation of GL261shNS cells enhanced the secretion of IFN-I cytokines. Strikingly, we found that FASN knockdown spontaneously induced IFN-beta and CXCL10 secretion, and this effect was even more pronounced when cells were irradiated. Finally, GL261shNS and GL261shFASN cells were orthotopically implanted in mice and IFN-I signaling was blocked using an antibody against the interferon alpha/beta receptor (IFNAR). FASN knockdown alone was sufficient to increase the median survival of mice bearing GL261shFASN tumors by 51 days vs. 35 days for GL261shNS tumors. Moreover, 33% of mice bearing GL261 tumors unable to express FASN remained tumor free for up to 100 days, while none of the GL261shNS-tumor bearing animal survived. This significant survival prolongation of mice bearing GL261shFASN tumor was completely abrogated when IFNAR was blocked. Overall, our findings suggest that FASN-mediated lipogenesis prevents RT-induced cancer cell intrinsic IFN-I to promote GBM survival. Consequently, it is possible that FASN acts as an immuno-metabolic checkpoint capable to regulate the immune system upon metabolic cues generated by RT. Citation Format: Mara De Martino, Camille Daviaud, Claire Vanpouille-Box. Targeting FASN improves type I interferon responses in irradiated glioblastoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PR-007.

Blockade of fatty acid signalling inhibits lipopolysaccharide-induced macrophage recruitment and progression of apical periodontitis.

To examine the role of palmitic acid in lipopolysaccharide (LPS)-stimulated chemotaxis of macrophages and the potential contribution of saturated fatty acid in signalling during the pathogenesis of apical periodontitis. J774, a mouse macrophage cell line, was used in the experiments. After treatment with LPS, proteolytic maturation of sterol regulatory element-binding protein-1c (SREBP-1c) and expression of fatty acid synthase (FASN) were examined by Western analysis. Levels of palmitic acid were measured by reverse phase-high performance liquid chromatography-mass spectrometry. Knockdown of SREBP-1c and FASN was accomplished by small interfering RNA technology. Secretion of CC-chemokine ligand 2 (CCL2) and cellular chemotaxis were assessed by enzyme-linked immunosorbent assay and transwell migration assay, respectively. Sulfo-N-succinimidyl oleate (SSO) treatment was used to inhibit fatty acid signalling in vitro and also in a rat model of apical periodontitis. All data were first subjected to Levene's test. In vitro data were then analysed using ANOVA followed by Tukey's multiple comparison test. Data from animal experiments were analysed by independent t-tests. The significant level was set at 0.05. LPS stimulated proteolytic maturation of SREBP-1c and FASN expression in macrophages and significantly enhanced palmitic acid synthesis (P<0.05). Knockdown of SREBP-1c attenuated LPS-enhanced FASN expression. Knockdown of FASN significantly suppressed LPS-enhanced palmitic acid synthesis (P<0.05). LPS and exogenous palmitic acid significantly enhanced CCL2 secretion and macrophage chemotaxis (all P<0.05). Inhibition of FASN expression significantly alleviated LPS-augmented CCL2 secretion (P<0.05). SSO significantly suppressed CCL2 secretion and macrophage chemotaxis augmented by LPS and palmitic acid (all P<0.05). In a rat model of induced apical periodontitis, SSO treatment significantly attenuated progression of apical periodontitis and macrophage recruitment (all P<0.05). LPS/SREBP-1c/FASN/palmitic acid signalling contributed to tissue destruction caused by bacterial infection. Modulation of lipid metabolism and signalling may be helpful for the management of apical periodontitis.