Pyridine derivatives as anticancer lead compounds with Fatty Acid Synthase as the target: An in silico-guided in vitro study.

For the past few decades, structure-based drug discovery (SBDD) has become an inevitable technique in the drug development process for screening hit compounds against therapeutic targets. Here, we have successfully used the SBDD approach viz. virtual high-throughput screening to identify potential inhibitors against the Ketoacyl synthase (KS) domain of Fatty acid synthase (FASN). Overexpression of FASN, and subsequent enhancement of de novo lipogenesis is a key survival strategy of cancer cells. Hence, targeting lipid metabolism using FASN inhibitors has been considered as a promising method to induce metabolic stress, thereby posing a survival disadvantage to cancer cells. In the present study, we have successfully identified eight FASN inhibitors from Asinex Elite database by implementing in silico tools. Five of the hit compounds share a common ring structure, which enables characteristic binding interactions with FASN-KS. Among them, in vitro validation showed that SFA 22637550 possesses significant FASN inhibitory activity and antiproliferative effect in human cancer cells of various origins. The maximum sensitivity was exhibited towards HepG2 hepatocellular carcinoma cells (IC50 = 28 µM). The mode of cell death was found to be apoptosis with a significant increase in SubG0 population without affecting any other phases of the cell cycle. The current study puts forward an excellent core structure for the development of potent FASN inhibitors for successfully targeting cancer cell metabolism, thereby causing selective cell death.

Ginseng metabolite protopanaxadiol interferes with lipid metabolism and induces endoplasmic reticulum stress and p53 activation to promote cancer cell death.

Protopanaxadiol (PPD), a ginseng metabolite generated by the gut bacteria, was shown to induce colorectal cancer cell death and enhance the anticancer effect of chemotherapeutic agent 5-FU. However, the mechanism by which PPD promotes cancer cell death is not clear. In this manuscript, we showed that PPD activated p53 and endoplasmic reticulum (ER) stress and induced expression of BH3-only proteins Puma and Noxa to promote cell death. Induction of Puma by PPD was p53-dependent, whereas induction of Noxa was p53-independent. On the other hand, PPD also induced prosurvival mechanisms including autophagy and expression of Bcl2 family apoptosis regulator Mcl-1. Inhibition of autophagy or knockdown of Mcl-1 significantly enhanced PPD-induced cell death. Interestingly, PPD inhibited expression of genes involved in fatty acid and cholesterol biosynthesis and induced synergistic cancer cell death with fatty acid synthase inhibitor cerulenin. As PPD-induced ER stress was not significantly affected by inhibition of new protein synthesis, we suggest PPD may induce ER stress directly through causing lipid disequilibrium.

FASN-TGF-ß1-PD-L1 axis contributes to the development of resistance to NK cell cytotoxicity of cisplatin-resistant lung cancer cells.

Cisplatin remains the most effective therapy for non-small cell lung cancer (NSCLC). We previously have found cisplatin-resistant lung cancer cells (A549CisR and H157CisR) were more resistant to natural killer (NK) cell-mediated cytotoxicity than parental cells. We also discovered that fatty acid synthase (FASN) levels in cisplatin-resistant cells were significantly higher than in parental cells. To reveal whether a link exists between the up-regulated FASN levels and higher resistance to NK cell cytotoxicity, we performed inhibition studies using a FASN inhibitor and applied the FASN knockdown approach. In both approaches, we found that the FASN inhibition/knockdown significantly increased the susceptibility of cisplatin-resistant cells to NK cell cytotoxicity. We further found such decreased susceptibility was associated with an increased programmed death receptor ligand (PD-L1) level in cisplatin-resistant cells. In mechanisms studies, TGF-ß1 was found to be the FASN downstream signaling molecule that was responsible for modulating the PD-L1 levels in cisplatin-resistant cells. Accordingly, TGF-ß1 inhibition resulted in significantly increased susceptibility of cisplatin-resistant cells to NK cell cytotoxicity. We suggest that the inhibition of FASN-TGFß1-PD-L1 axis may improve the efficacy of immunotherapy in treating cisplatin-resistant lung cancer.

The Role of Fatty Acid Synthase in the Vascular Smooth Muscle Cell to Foam Cell Transition.

Vascular smooth muscle cells (VSMCs), in their contractile and differentiated state, are fundamental for maintaining vascular function. Upon exposure to cholesterol (CHO), VSMCs undergo dedifferentiation, adopting characteristics of foam cells-lipid-laden, macrophage-like cells pivotal in atherosclerotic plaque formation. CHO uptake by VSMCs leads to two primary pathways: ABCA1-mediated efflux or storage in lipid droplets as cholesterol esters (CEs). CE formation, involving the condensation of free CHO and fatty acids, is catalyzed by sterol O-acyltransferase 1 (SOAT1). The necessary fatty acids are synthesized by the lipogenic enzyme fatty acid synthase (FASN), which we found to be upregulated in atherosclerotic human coronary arteries. This observation led us to hypothesize that FASN-mediated fatty acid biosynthesis is crucial in the transformation of VSMCs into foam cells. Our study reveals that CHO treatment upregulates FASN in human aortic SMCs, concurrent with increased expression of CD68 and upregulation of KLF4, markers associated with the foam cell transition. Crucially, downregulation of FASN inhibits the CHO-induced upregulation of CD68 and KLF4 in VSMCs. Additionally, FASN-deficient VSMCs exhibit hindered lipid accumulation and an impaired transition to the foam cell phenotype following CHO exposure, while the addition of the fatty acid palmitate, the main FASN product, exacerbates this transition. FASN-deficient cells also show decreased SOAT1 expression and elevated ABCA1. Notably, similar effects are observed in KLF4-deficient cells. Our findings demonstrate that FASN plays an essential role in the CHO-induced upregulation of KLF4 and the VSMC to foam cell transition and suggest that targeting FASN could be a novel therapeutic strategy to regulate VSMC phenotypic modulation.

Inhibition of fatty acid synthase suppresses U-2 OS cell invasion and migration via downregulating the activity of HER2/PI3K/AKT signaling pathway in vitro.

FASN plays an important role in the malignant phenotype of various tumors. Our previous studies show that inhibition FASN could induce apoptosis and inhibit proliferation in human osteosarcoma (OS) cell in vivo and vitro. The aim in this study was to investigate the effect of inhibition FASN on the activity of HER2/PI3K/AKT axis and invasion and migration of OS cell. The expression of FASN, HER2 and p-HER2(Y1248) proteins was detected by immunohistochemistry in OS tissues from 24 patients with pulmonary metastatic disease, and the relationship between FASN and p-HER2 as well as HER2 was investigated. The results showed that there was a positive correlation between FASN and HER2 as well as p-HER2 protein expression. The U-2 OS cells were transfected with either the FASN specific RNAi plasmid or the negative control RNAi plasmid. FASN mRNA was measured by RT-PCR. Western blot assays was performed to examine the protein expression of FASN, HER2, p-HER2(Y1248), PI3K, Akt and p-Akt (Ser473). Migration and invasion of cells were investigated by wound healing and transwell invasion assays. The results showed that the activity of HER2/PI3K/AKT signaling pathway was suppressed by inhibiting FASN. Meanwhile, the U-2OS cells migration and invasion were also impaired by inhibiting the activity of FASN/HER2/PI3K/AKT. Our results indicated that inhibition of FASN suppresses OS cell invasion and migration via down-regulation of the "HER2/PI3K/AKT" axis in vitro. FASN blocker may be a new therapeutic strategy in OS management.

In-silico investigations on the anticancer activity of selected 2-aryloxazoline derivatives against breast cancer.

As the in-silico study has become an important tool to search for new drugs in the concurrent era with towering acceptance and accuracy, it has been employed in our research to unearth effective cancer drugs. Breast cancer has accounted for the most serious diseases for both men and women. Although few research outputs have been obtained on breast cancer, these are not an adequate amount to ascertain new drugs. Due to this gap, virtual screening, in-silico study, and computational techniques have been used to provide the ability to design and select anticancer compounds with desirable drug-like properties of breast cancer protein, which is commonly known as fatty acid synthase. A total of nine derivatives of 2-aryloxazoline compounds were chosen, and In-silico was studied to evaluate as a potential anticancer agent with the comparison of seven Food and Drug Administration(FDA) approved breast cancer drugs. These compounds were subjected to computational studies for quantum calculations, ADME and Lipinski analysis, as well as molecular docking and MD simulations against a variety of therapeutic targets involved in cell proliferation of fatty acid synthase (PDB ID:3TJM, 3ERT, 4OAR, 2J6M). An in-silico docking study reveals that ligands Hit-4, Hit-6, and Hit-8 had the highest docking scores at -10.3 kcal/mol, -10.3 kcal/mol, and -10.2 kcal/mol towards the protein of fatty acid synthase. The ligands had docking scores better than the standard anti-breast cancer drug gefitinib (-5.3 kcal/mole). Our findings demonstrate how crucial it is for pharmaceutical researchers to develop novel drugs for the treatment of breast cancer.Communicated by Ramaswamy H. Sarma.

Prognostic and clinicopathological significance of fatty acid synthase in breast cancer: A systematic review and meta-analysis.

Background: Aberrant expression of fatty acid synthase (FASN) was demonstrated in various tumors including breast cancer. A meta-analysis was conducted to investigate the role of FASN in breast cancer development and its potential prognostic significance. Methods: The Web of Science, PubMed, Embase, and Cochrane Library databases were searched to identify studies that evaluated the relationship between FASN expression and overall survival (OS), relapse-free survival (RFS), and disease-free survival (DFS) of breast cancer patients. To analyze the clinicopathological and prognostic values of FASN expression in breast cancer, pooled hazard ratios (HRs), odds ratios (ORs), and 95% confidence intervals (CIs) were clustered based on random-effects models. To confirm whether the findings were stable and impartial, a sensitivity analysis was performed, and publication bias was estimated. Data were analyzed using Engauge Digitizer version 5.4 and Stata version 15.0. Results: Five studies involving 855 participants were included. Patients with higher FASN expression did not have a shorter survival period compared to those with lower FASN expression (summary HR: OS, 0.73 [95% CI, 0.41-1.32; P=0.300]; DFS/RFS, 1.65 [95% CI, 0.61-4.43; P=0.323]). However, increased FASN expression was correlated with large tumor size (OR, 2.04; 95% CI, 1.04-4.00; P=0.038), higher human epidermal growth factor receptor 2 (HER2) positivity (OR, 1.53; 95% CI, 1.05-2.23; P=0.028). No significant associations were observed between FASN expression and histological grade (OR, 0.92; 95% CI, 0.41-2.04; P=0.832), Tumor Node Metastasis (TNM) stage (OR, 1.11; 95% CI, 0.49-2.53; P=0.795), nodal metastasis (OR, 1.42; 95% CI, 0.84-2.38; P=0.183), Ki-67 labelling index (OR, 0.64; 95% CI, 0.15-2.63; P=0.533), estrogen receptor (ER) status (OR, 0.90; 95% CI, 0.61-1.32; P=0.586), or progesterone receptor (PR) status (OR, 0.67; 95% CI, 0.29-1.56; P=0.354). Conclusion: FASN is associated with HER2 expression and may contribute to tumor growth, but it has no significant impact on the overall prognosis of breast cancer.

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

Background: 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. Methods: 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. Results: 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. Conclusions: 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.

Proteomic and miRNA profiling of radon-induced skin damage in mice: FASN regulated by miRNAs.

Radon is a naturally occurring radioactive gas and considered as a serious carcinogen to humans. Continuous radioactive decay of this gas emits high-energy alpha particles. Long-term radon exposure induces oxidative stress and inflammatory response, which results in chronic lung diseases. However, biological effects after radon exposure in other organs have been rarely reported. As the outermost organ of the human body, the skin suffers from environmental damage to agents such as air pollution. Epidemiological studies indicated that areas with high level of radon had a high incidence of skin cancer. However, whether radon exposure induces skin damage has not been reported yet. In this study, we established a radon-exposed mouse model and found that radon exposure affected the structure of skin tissues, which was manifested by inflammatory cell infiltration and skin atrophy. Using proteomic approach, we found 45 preferentially expressed proteins in 60 Working Level Months (WLM) group and 314 preferentially expressed proteins in 120 WLM group from radon-exposed skin tissues. Through microRNA (miRNA) sequencing profiling analysis, 57 dysregulated miRNAs were screened between the control and radon-treated mouse skin. By integrating the dysregulated proteins and miRNAs, radon-induced fatty acid synthase (FASN) was investigated in greater detail. Results showed that FASN was regulated by miR-206-3p and miR-378a-3p and involved in the pathogenesis of radon-induced skin damage. Overexpression of FASN inhibited the proliferation, and induced in WS1 cells. Our present findings illustrate the molecular change during radon-induced skin damage and the potential role of FASN during this process.

Visualization and quantification of de novo lipogenesis using a FASN-2A-GLuc mouse model.

Background: De novo lipogenesis (DNL) is a dynamic process that converts excess carbohydrates into fatty acids to maintain cellular homeostasis. Dysregulation of DNL is associated with diverse obesity-related diseases and many tumor types. Therefore, monitoring DNL in real-time with high sensitivity should be highly beneficial when screening therapeutic agents for their potential use as obesity treatments. Methods: A sequence coding for Gaussia luciferase (GLuc) preceded by a 2A peptide was inserted into the murine fatty acid synthase (FASN) genetic locus by homologous recombination to generate FASN-2A-GLuc mice. The luciferase mouse model was evaluated in conditions of physical and pharmacological stimuli by in vivo and ex vivo imaging. Results: The distribution of bioluminescence signals in different organs was identical to the FASN expression: high in white fat, brown fat, and the lungs. In addition, the bioluminescence signals accurately recapitulated the dynamic change of FASN in response to fasting and refeeding conditions. Moreover, with this murine reporter model, we also discovered that fatostatin, a synthetic inhibitor of sterol regulatory element-binding proteins, effectively inhibited DNL in multiple organs, especially in adipose tissues under a high-carbohydrate diet. Conclusions: Our FASN-2A-GLuc reporter mouse model proved to be a sensitive visualization tool for monitoring both systemic and organ-specific DNL in real time.

Characterization of Total RNA, CD44, FASN, and PTEN mRNAs from Extracellular Vesicles as Biomarkers in Gastric Cancer Patients.

In-depth characterization has introduced new molecular subtypes of gastric cancer (GC). To identify these, new approaches and techniques are required. Liquid biopsies are trendsetting and provide an easy and feasible method to identify and to monitor GC patients. In a prospective cohort of 87 GC patients, extracellular vesicles (EVs) were isolated from 250 µL of plasma. The total RNA was isolated with TRIZOL. The total RNA amount and the relative mRNA levels of CD44, PTEN, and FASN were measured by qRT-PCR. The isolation of EVs and their contained mRNA was possible in all 87 samples investigated. The relative mRNA levels of PTEN were higher in patients already treated by chemotherapy than in chemo-naïve patients. In patients who had undergone neoadjuvant chemotherapy followed by gastrectomy, a decrease in the total RNA amount was observed after neoadjuvant chemotherapy and gastrectomy, while FASN and CD44 mRNA levels decreased only after gastrectomy. The amount of RNA and the relative mRNA levels of FASN and CD44 in EVs were affected more significantly by chemotherapy and gastrectomy than by chemotherapy alone. Therefore, they are a potential biomarker for monitoring treatment response. Future analyses are needed to identify GC-specific key RNAs in EVs, which could be used for the diagnosis of gastric cancer patients in order to determine their molecular subtype and to accompany the therapeutic response.

Serum fatty acid synthase levels and n-3 fatty acid intake in patients with breast cancer.

PURPOSE: Fatty acid synthase (FASN) is a key enzyme in fatty acid biosynthesis that is usually over-expressed in patients with breast cancer, but its relationship with the patient's dietary habit is not clear. A higher intake of n-3 polyunsaturated fatty acids is related to reduced breast carcinogenesis in vitro and in vivo. The aim of this study was to clinically investigate the association between serum FASN levels and fatty acid intake in women newly diagnosed with breast cancer. METHODS: In a case-control cross-sectional study, with 18 breast cancer patients and 29 controls, we evaluated nutritional status, dietary intake, and serum FASN levels. Statistical analyses were carried out with parametric and non-parametric tests, according to the sample's normality distribution. RESULTS: The mean age of breast cancer group (n = 18) and control group (n = 29) was 46.8 ± 9.7 y and 44.4. ± 8.6 y, respectively. Mean serum concentration of FASN in breast cancer group was significantly higher (132.51 ± 95.05 ng/mL) than in control group (36.88 ± 20.87 ng/mL) (p < 0.0001). Among breast cancer group, serum FASN levels of premenopausal women were significantly higher than those of postmenopausal women (p = 0.026). There was no significant difference between the early and late disease stages in regard to serum FASN levels in breast cancer group. Mean nutrient intake was similar and n-3 docosahexaenoic acid intake was low in both groups. We observed no association regarding fatty acid intake and serum FASN levels. CONCLUSION: These data suggest that dietary n-3 fatty acid has no association with serum FASN levels among newly diagnosed breast cancer patients.

Repurposing Combination Therapy of Voacamine With Vincristine for Downregulation of Hypoxia-Inducible Factor-1α/Fatty Acid Synthase Co-axis and Prolyl Hydroxylase-2 Activation in ER+ Mammary Neoplasia.

The current study investigated the role of combination therapy with voacamine and vincristine in preventing mammary gland carcinoma through prolyl hydroxylase-2 activation. Prolyl hydroxylase-2 activation leads to the downregulation of hypoxia-inducible factor-1α and fatty acid synthase. Overexpression of hypoxia-inducible factor-1α and fatty acid synthase has been previously reported in solid tumors of the mammary gland. After screening a battery of natural compounds similar to vincristine, voacamine was selected as a possible prolyl hydroxylase-2 activator, and its activity was evaluated using a 7,12-dimethylbenz[a]anthracene-induced rat model. The combination therapy was evaluated for cardiac toxicity using a hemodynamic profile. Angiogenic markers were evaluated by carmine staining. Monotherapy and combination therapy were also evaluated for liver and kidney toxicity using hematoxylin and eosin staining. The antioxidant potential was delineated using oxidative stress markers. The serum metabolomic profile was studied using NMR spectroscopy, and the disruption of fatty acids was evaluated using gas chromatography. Western blotting of proteins involved in hypoxic pathways was performed to decipher the action of therapy at the molecular level. Immunoblotting analysis validated that combination therapy has potential toss with prolyl hydroxylase-2 activity and thus initiates proteolytic degradation of hypoxia-inducible factor-1α and its consequent effects. Combination therapy also stimulated programmed cell death (apoptosis) in rapidly dividing cancer cells. The present study explored the role of voacamine inactivation of prolyl hydroxylase-2, which can decrease the overexpression of hypoxia-inducible factor-1α and fatty acid synthase in mammary gland carcinoma cells.

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.

Fatty acid synthase is a prognostic marker and associated with immune infiltrating in gastric cancers precision medicine.

Aim: Fatty acid synthase (FASN), a key enzyme for de novo synthesis of fatty acids, has been identified as an oncogene in some tumor types; however, the function of FASN in gastric cancer (GC) is poorly elucidated. Method: Integrative bioinformatics analyses were performed to unveil the role of FASN in tumor progression and cancer-associated immunology of GC. Result:FASN was overexpressed in the GC tissues and correlated with an inferior survival outcome, and largely contributed to the carcinogenesis of GC. Moreover, FASN expression was closely associated with the immune-infiltrating levels of CD8+ T, CD4+ T, neutrophils, macrophages and dendritic cells. Conclusion:FASN was closely associated with GC and may be involved in the tumorigenesis and cancer-immune interactions, and could be a promising prognostic and therapeutic biomarker in GC.

Orlistat as a FASN inhibitor and multitargeted agent for cancer therapy.

INTRODUCTION: Cancer cells have increased glycolysis and glutaminolysis. Their third feature is increased de novo lipogenesis. As such, fatty acid (FA) synthesis enzymes are over-expressed in cancer and their depletion causes antitumor effects. As fatty acid synthase (FASN) plays a pivotal role in this process, it is an attractive target for cancer therapy. AREAS COVERED: This is a review of the lipogenic phenotype of cancer and how this phenomenon can be exploited for cancer therapy using inhibitors of FASN, with particular emphasis on orlistat as a repurposing drug. EXPERT OPINION: Disease stabilization only has been observed with a highly selective FASN inhibitor used as a single agent in clinical trials. It is too early to say whether the absence of tumor responses other than stabilization results because even full inhibition of FASN is not enough to elicit antitumor responses. The FASN inhibitor orlistat is a 'dirty' drug with target-off actions upon at least seven targets with a proven role in tumor biology. The development of orlistat formulations suited for its intravenous administration is a step ahead to shed light on the concept that drug promiscuity can or not be a virtue.

Multi-level suppression of receptor-PI3K-mTORC1 by fatty acid synthase inhibitors is crucial for their efficacy against ovarian cancer cells.

Receptor-PI3K-mTORC1 signaling and fatty acid synthase (FASN)-regulated lipid biosynthesis harbor numerous drug targets and are molecularly connected. We hypothesize that unraveling the mechanisms of pathway cross-talk will be useful for designing novel co-targeting strategies for ovarian cancer (OC). The impact of receptor-PI3K-mTORC1 onto FASN is already well-characterized. However, reverse actions-from FASN towards receptor-PI3K-mTORC1-are still elusive. We show that FASN-blockade impairs receptor-PI3K-mTORC1 signaling at multiple levels. Thin-layer chromatography and MALDI-MS/MS reveals that FASN-inhibitors (C75, G28UCM) augment polyunsaturated fatty acids and diminish signaling lipids diacylglycerol (DAG) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) in OC cells (SKOV3, OVCAR-3, A2780, HOC-7). Western blotting and micropatterning demonstrate that FASN-blockers impair phosphorylation/expression of EGF-receptor/ERBB/HER and decrease GRB2-EGF-receptor recruitment leading to PI3K-AKT suppression. FASN-inhibitors activate stress response-genes HIF-1α-REDD1 (RTP801/DIG2/DDIT4) and AMPKα causing mTORC1- and S6-repression. We conclude that FASN-inhibitor-mediated blockade of receptor-PI3K-mTORC1 occurs due to a number of distinct but cooperating processes. Moreover, decrease of PI3K-mTORC1 abolishes cross-repression of MEK-ERK causing ERK activation. Consequently, the MEK-inhibitor selumetinib/AZD6244, in contrast to the PI3K/mTOR-inhibitor dactolisib/NVP-BEZ235, increases growth inhibition when given together with a FASN-blocker. We are the first to provide deep insight on how FASN-inhibition blocks ERBB-PI3K-mTORC1 activity at multiple molecular levels. Moreover, our data encourage therapeutic approaches using FASN-antagonists together with MEK-ERK-inhibitors.

BNIP3 induction by hypoxia stimulates FASN-dependent free fatty acid production enhancing therapeutic potential of umbilical cord blood-derived human mesenchymal stem cells.

Mitophagy under hypoxia is an important factor for maintaining and regulating stem cell functions. We previously demonstrated that fatty acid synthase (FASN) induced by hypoxia is a critical lipid metabolic factor determining the therapeutic efficacy of umbilical cord blood-derived human mesenchymal stem cells (UCB-hMSCs). Therefore, we investigated the mechanism of a major mitophagy regulator controlling lipid metabolism and therapeutic potential of UCB-hMSCs. This study revealed that Bcl2/adenovirus E1B 19kDa protein-interacting protein 3 (BNIP3)-dependent mitophagy is important for reducing mitochondrial reactive oxygen species accumulation, anti-apoptosis, and migration under hypoxia. And, BNIP3 expression was regulated by CREB binding protein-mediated transcriptional actions of HIF-1α and FOXO3. Silencing of BNIP3 suppressed free fatty acid (FFA) synthesis regulated by SREBP1/FASN pathway, which is involved in UCB-hMSC apoptosis via caspases cleavage and migration via cofilin-1-mediated F-actin reorganization in hypoxia. Moreover, reduced mouse skin wound-healing capacity of UCB-hMSC with hypoxia pretreatment by BNIP3 silencing was recovered by palmitic acid. Collectively, our findings suggest that BNIP3-mediated mitophagy under hypoxia leads to FASN-induced FFA synthesis, which is critical for therapeutic potential of UCB-hMSCs with hypoxia pretreatment.

A FASN-TGF-ß1-FASN regulatory loop contributes to high EMT/metastatic potential of cisplatin-resistant non-small cell lung cancer.

Cisplatin-resistant A549CisR and H157CisR cell lines were developed by treating parental A549 (A549P) and H157 (H157P) cells. These cisplatin-resistant cells showed slight growth retardation, but exhibited higher epithelial-mesenchymal transition (EMT) and increased metastatic potential compared to parental cells. We observed a highly up-regulated fatty acid synthase (FASN) level in A549CisR and H157CisR cells compared to parental cells and the up-regulation of FASN was also detected in A549P and H157P cells after short time treatment with cisplatin, suggesting that the high level of FASN in cisplatin-resistant cells may be from the accumulated cellular responses during cisplatin-resistance developmental process. We next investigated whether the inhibition of FASN by using a specific FASN inhibitor, cerulenin, can influence growth and EMT/metastatic potential of A549CisR and H157CisR cells. There was slight growth inhibition, but significantly reduced EMT/metastatic potential in cisplatin-resistant cells upon inhibitor treatment. The in vitro result was further investigated in orthotopic xenograft mouse models established with luciferase-tagged H157P and H157CisR cells. Mice were injected with cerulenin or vehicle after tumors were developed. No significant tumor regression was detected at the end of cerulenin treatment, but IHC staining showed higher expression of EMT/metastasis markers in H157CisR cell-derived tumors than H157P cell-derived tumors, and showed dramatic reduction of these markers in tumor tissues of cerulenin-treated mice, confirming the in vitro results. In mechanism dissection studies, we revealed the existence of the FASN-TGF-ß1-FASN positive loop in A549CisR and H157CisR cells, but not in parental cells, which is believed to augment the FASN function in cisplatin-resistant cells.

Fatty acid synthase is a primary target of MiR-15a and MiR-16-1 in breast cancer.

Fatty acid synthase (FASN) is upregulated in breast cancer and correlates with poor prognosis. FASN contributes to mammary oncogenesis and serves as a bona fide target in cancer therapies. MicroRNAs inhibit gene expression through blocking mRNA translation or promoting mRNA degradation by targeting their 3'-UTRs. We identified four microRNAs in two microRNA clusters miR-15a-16-1 and miR-497-195 that share a common seed sequence to target the 3'-UTR of the FASN mRNA. In reporter assays, both of these microRNA clusters inhibited the expression of a reporter construct containing the FASN 3'-UTR. However, only ectopic miR-15a-16-1, but not miR-497-195, markedly reduced the levels of endogenous FASN in breast cancer cells. Both miR-15a and miR-16-1 contributes to inhibiting FASN expression and breast cancer cell proliferation. Consistently, a sponge construct consisting of eight repeats of the FASN 3'-UTR region targeted by these microRNAs could markedly increase endogenous FASN levels in mammary cells. When FASN expression was restored by ectopic expression in breast cancer cells, retarded cell proliferation caused by miR-15a-16-1 was partially rescued. In conclusion, we demonstrated that FASN expression is primarily downregulated by miR-15a and miR-16-1 in mammary cells and FASN is one of the major targets of these two tumor suppressive microRNAs.