Indiana University-Purdue University Indianapolis (IUPUI) / Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer with a dismal prognosis. Targeted therapies for breast cancer with expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) are currently available; however, due to the lack of ER, PR, and HER2 in TNBC, targeted therapies are limited. While surgery and traditional chemotherapy remain the standard of care, development of a new treatment strategy for TNBC is needed to improve clinical outcomes. Fatty acid synthase (FASN) has been implicated as a metabolic oncogene and has given cancer cells a survival advantage by increasing NHEJ repair. Recently, it has been shown that FDA-approved proton pump inhibitors, used for the treatment of acid related digestive diseases, have antitumor effects. Here, I show that a metabolite of lansoprazole, 5-hydroxy lansoprazole sulfide, has increased potency over parent compound lansoprazole. 5-hydroxy lansoprazole sulfide inhibits FASN, leading to a decrease in PARP and NHEJ DNA repair activity in TNBC. Ultimately, this leads to an increase in DNA damage and cell death via apoptosis. These findings suggest that 5-hydroxy lansoprazole sulfide, as a metabolite of lansoprazole, may have better activity in suppressing TNBC cells and that 5-hydroxy lansoprazole sulfide may be developed as a therapeutic for TNBC treatment. Furthermore, due to the role of FASN in increasing NHEJ repair, we hypothesized that FASN played a role in resistance to CC-115, a dual mTOR/DNA-PK inhibitor currently in clinical trials, by increasing DNA-PK activity. However, it was found that ABCG2, an ATP-binding cassette transporter, and not FASN, has a role in CC-115 resistance. ABCG2 effluxes CC-115 from cancer cells, increasing resistance to treatment. Inhibition of ABCG2 by FTC or PZ39C8 led to accumulation of CC-115 within cells and sensitization to treatment. Therefore, ABCG2 status should be assessed to stratify patients into treatment groups, increasing the efficacy of CC-115 treatment. / 2020-02-21

Indiana University-Purdue University Indianapolis (IUPUI) / The overexpression of the multi-domain enzyme fatty acid synthase (FASN) has long been associated with poor clinical prognosis and treatment outcome in various cancers. Previous research in the Zhang lab has determined a role for FASN in mediating increases in non-homologous end-joining (NHEJ) DNA double-strand break repair activity allowing for increased cancer cell survival, and this mechanism was found to involve inhibition of NF-kB/p65. The mechanism responsible for the regulation of NF-kB/p65 by FASN in cancer cells, however, remains unknown. To this end, I was able to determine that FASN negatively regulates both the expression and activity of NF-kB/p65 in breast cancer cells, and that this effect was likely mediated by the 16-carbon saturated fatty acid palmitate, the end product of FASN catalytic activity. Specifically, FASN was found to negatively regulate p65 expression by disrupting its protein stability as a result of an increase in poly-ubiquitination of p65 protein and subsequent proteasomal degradation. Further, I found that the phosphorylation site Thr254 of p65 is involved in the regulation of p65 protein stability by FASN, in that mutation of this residue resulted in a disruption in p65 stability. Finally, I was able to determine that FASN likely inhibits the ability of the peptidyl-prolyl cis/trans isomerase Pin1 to assist in maintaining p65 stability, in that both siRNA knockdown and pharmacological inhibition of Pin1 resulted in a reduction of p65 expression in FASN shRNA knockdown cells. The determination of this signaling mechanism serves to expand our understanding of the role of FASN in breast cancer cells and has the potential to assist in uncovering more effective ways to target the oncogenic FASN pathway to kill breast tumor cells and to overcome resistance to drug treatment.

L’acide gras synthase, codé par le gène FASN, est une protéine multi-enzyme homodimérique responsable de la lipogenèse de novo à partir de l’acétyl-CoA et du malonyl- CoA. La finalité de cette lipogenèse est la production de l’acide palmitique, un acide gras simple, précurseur des acides gras à très longues chaînes. L’acide palmitique est impliqué dans plusieurs processus biologiques, dont la palmitoylation qui permet d’ancrer diverses protéines à la membrane cellulaire sous-tendant, entre autres, la transmission synaptique. Le rôle de l’acide gras synthase dans le développement embryonnaire est bien établi. En effet, il est exprimé de manière ubiquitaire dans l’embryon, principalement dans les tissus en pleine croissance et soumis à un remodelage, participant ainsi activement au développement cérébral. Par conséquent, des mutations du gène FASN ont été associées à plusieurs maladies, incluant divers types de cancers, les maladies cardiovasculaires, mais également, plus récemment, à certaines maladies du neurodéveloppement, incluant les troubles du spectre de l’autisme. Des données récentes des laboratoires Rossignol et Campeau, au CHU Ste-Justine, suggèrent un lien entre des mutations récessives ou de novo du gène FASN et des formes précoces d’épilepsie avec atteinte cognitive (encéphalopathies épileptogènes). Nous postulons que les mutations du gène FASN modifient la synthèse de l’acide palmitique et perturbent le développement des réseaux neuronaux, en altérant la migration, le développement morphologique, l’excitabilité et/ou la fonction synaptique de populations neuronales spécifiques, résultant en une hyperexcitabilité neuronale et à l’épilepsie. Pour explorer cette hypothèse, nous avons recueilli les informations cliniques de dix patients porteurs de mutations du gène FASN dans le cadre d’études génomiques en cours au CHU Ste- Justine et à travers le monde. Nous avons également généré un nouveau modèle murin de la maladie, exprimant une mutation retrouvée chez un membre de notre cohorte clinique, que nous avons caractérisé sur les plans histochimique et électrophysiologique. Nos données suggèrent que les mutations du gène FASN induisent chez l’humain un phénotype clinique de retard global du développement évoluant vers une déficience intellectuelle, s’accompagnant d’un éventail de signes neurologiques (déficit moteur, spasticité, réflexes ostéotendineux vifs, hypotonie et ataxie) et d’un risque accru d’épilepsie. De plus, notre modèle de souris knock-in Fasn.S154N révèle la fonction critique de ce gène dans le développement embryonnaire puisqu’une mutation homozygote entraîne une mortalité in utero. Par ailleurs, les souris porteuses de mutations hétérozygotes survivent et présentent un phénotype clinique rappelant celui observé chez les patients, incluant un comportement anxieux, une activité épileptique interictale à l’électroencéphalogramme ainsi qu’un abaissement du seuil convulsif lors d’une exposition au pentylenetetrazole (PTZ). Nous discutons certains mécanismes sous-jacents contribuant potentiellement au développement de l’épilepsie dans cette maladie, incluant une altération de l’activité de l’acide gras synthase au niveau du cortex préfrontal et de l’amygdale, une palmitoylation aberrante des protéines synaptiques, une plus grande vulnérabilité des cellules granulaires du gyrus denté, un dysfonctionnement des cellules souches neurales, une neurogénèse insuffisante, ainsi qu’une altération de la myélinisation et de la croissance axonale impactant la migration des interneurones. Ces mécanismes sont prédits pour altérer l’excitabilité neuronale et la transmission synaptique, perturbant la fonction des circuits. Des études subséquentes permettront d’élucider lesquels de ces divers mécanismes contribuent au phénotype clinique dans notre nouveau modèle murin de la maladie. / Fatty Acid Synthase is a large protein complex encoded by the FASN gene, which is responsible for de novo lipogenesis from acetyl-CoA and malonyl-CoA in the presence of NADPH. The endpoint of this process is the production of palmitic acid. The roles of fatty acid synthase in embryonic development are well established: it is ubiquitously expressed in early embryos, particularly in tissues undergoing active proliferation, outgrowth, and remodelling, and it is thus essential for normal brain development and neuronal function. Consequently, FASN gene mutations have been associated with several neurodevelopmental conditions, including autism spectrum disorders (ASD). Recently, the laboratories of Drs. E. Rossignol and P. Campeau at the CHU Ste-Justine (Université de Montréal), with their international collaborators, have identified 10 patients with neurodevelopmental disorders (i.e., developmental delay, intellectual disability and/or epilepsy) carrying recessive or de novo mutations in the FASN gene, supporting a critical role of FASN in regulating neuronal circuit development and function. However, the mechanisms by which mutations in the FASN gene result in epilepsy are unknown. We postulate that FASN mutations alter palmitic acid synthesis and disrupt neuronal network development, resulting in network hyperexcitability and epilepsy. In this study, we expand the phenotypic description of patients carrying FASN mutations, while generating a novel mouse model carrying a patient-derived FASN mutation to explore the underlying cellular and network mechanisms. Our data reveal that FASN mutations, in humans, generate neurodevelopmental disorders characterized by epilepsy, global developmental delay (GDD), intellectual disability (ID), and a broad range of neurological signs (motor deficit, spasticity, hyperreflexia, hypotony, and ataxia). In our knock-in FasnS154N mouse model, homozygous mutations resulted in prenatal lethality. In contrast, heterozygous mutations caused a clinical phenotype reminiscent of the patient phenotype, with anxiety-like behaviors, spontaneous interictal spikes on electroencephalograms (EEG), and a tendency to a reduced PTZ-induced seizure threshold. We discuss the potential underlying mechanisms, including an altered FAS activity within the prefrontal cortex and the amygdala, aberrant palmitoylation of postsynaptic density proteins, the vulnerability of dentate gyrus granules cell, altered neural stem cells activity and neurogenesis, improper axonal growth and myelination, resulting in altered neuronal excitability and synaptic function, aberrant network activities and epilepsy. These mechanisms will be explored in subsequent studies using our novel animal model.

Conjugated linoleic acid (CLA) exhibits multiple biological and molecular activities that have made it the subject of considerable nutrition-related research. Numerous studies support broad acting anti-tumor effects including anti-inflammatory, anti-proliferation, and pro-apoptosis in a variety of model systems. CLA’s ability to influence multiple tumor promoting pathways, without toxicity, may prove valuable in the chemoprevention of breast cancer. The overall objective of this dissertation research was to investigate the potential of CLA in the chemoprevention of breast cancer in a subgroup of women at risk of developing estrogen receptor (ER) negative disease. Overexpression of either the ERBB2 oncogene or the epidermal growth factor receptor (EGFR) is a common event in ER negative breast cancer. To respond to this association, the stated research objective was pursued in relevant model systems. The primary hypothesis was that CLA would downregulate the ERBB2 receptor in vitro and inhibit mammary tumorigenesis in vivo. The t10c12 CLA isomer significantly reduced ERBB2 protein expression in the ERBB2 overexpressing cell line SKBr3. This was accompanied by a decrease in NFκB nuclear localization, cyclooxygenase-2 (COX2)-derived prostaglandin (PG) E2 production, increased apoptosis, and inhibition of proliferation. In contrast to the in vitro data, however 1% dietary CLA had pro-tumor effects in the PyV-mT transgenic mouse model, Mammary gland whole mounts indicated a significant loss of adipose in the CLA-treated group compared to controls that was confirmed by the downregulation of adipocyte-specific genes including PPARγ and adiponectin. CLA’s effect on the adipose was supported by decreases in fatty acid synthase at the protein and mRNA level. cDNA microarray revealed significant downregulation of cytoskeletal and adhesion-related genes in the CLA-treated group. These data suggest CLA’s combined effect on the adipose and epithelial architecture may have promoted tumor growth in this model While the large body of evidence supporting an anti-tumor effect of CLA can not be discounted, the studies herein demonstrate the complexity of its action that may not be captured in simple model systems. Reports of adverse effects of mixed isomers or the t10c12 purified isomer lend caution to supplementation that is supported by our in vivo data.

Submitted by Claudia Rocha (claudia.rocha@udesc.br) on 2018-03-09T14:47:58Z No. of bitstreams: 1 PGCA16MA201.pdf: 1020205 bytes, checksum: d956db0f093e7d366bba8bbcc7198546 (MD5) / Made available in DSpace on 2018-03-09T14:47:58Z (GMT). No. of bitstreams: 1 PGCA16MA201.pdf: 1020205 bytes, checksum: d956db0f093e7d366bba8bbcc7198546 (MD5) Previous issue date: 2016-07-13 / UNIEDU / FUMDES / The biosynthesis of lipids is dependent of a coordinated action of several lipogenic enzymes, such as acetyl-CoA carboxylase alpha (ACC-α) and fatty acid synthase (FASN), in different tissues. This study evaluated the gene expression of acetyl-CoA carboxylase alpha (ACC-α) transcripts tissue-specific expressed from promoters I, II and III (PI, PII and PIII), fatty acid synthase (FASN), leptin and sterol regulatory element binding protein 1 (SREBP1) in mammary gland and adipose tissues of lactating ewes at three different stages of lactation. Eight crossbred Lacaune/Texel ewes weighing 62 ± 1.8 kg (mean ± SE), with mean parity number of 3.1 ± 0.23 and producing 0.94 ± 0.12 kg milk/d were used in three different stages of lactation (days in milk-DIM): a) Early, (15 DIM), b) Mid, (70 DIM) and; c) Late (120 DIM). Biopsies were taken after the morning milking, the total RNA was extracted, complementary DNA (cDNA) synthesized and qRT-PCR analysis carried out. The gene expression data were analyzed using the MIXED procedure of SAS using the geometric mean of two housekeeping genes, ribosomal protein S18 (RPS18) and beta actin (β-ACT), as a covariate. In mammary tissue, PII transcripts were reduced 82, 93 and 59% between early and mid, early and late and mid and late stages of lactation. PIII transcripts were decreased 73, 89 and 59% between early and mid, early and late and mid and late, respectively. Gene expression of FASN was reduced respectively, 54 and 83% in mid and late stages compared to early lactation. In mammary tissue SREBP1 was decreased 33 and 42% between early and late and mid to late lactation. In adipose tissue, the expression of PI transcripts was increased more than 30 fold between early and mid lactation. There is an adaptation for fatty acid synthesis in adipose and mammary tissues to meet the demands of lactation is associated with changes in gene expression of the transcription factor and codifying genes involved in fat synthesis in both tissues / A biossíntese de lipídeos é dependente de uma ação coordenada de várias enzimas lipogênicas, como a acetil-CoA carboxilase alfa (ACC-α) e a ácido graxo sintase (FASN) em diferentes tecidos. Este estudo mensurou a expressão gênica dos transcritos tecido-específicos oriundos de diferentes regiões promotoras I, II e III (PI, PII e PIII) do gene da ACC-α e transcritos da FASN, leptina e da SREBP1 na glândula mamária e no tecido adiposo de ovelhas lactantes em três diferentes estágios de lactação. Oito ovelhas mestiças Lacaune/Texel pesando 62 ± 1,8 kg (média ± SE), com média de parições de 3,1 ± 0,23 e produzindo 0,94 ± 0,12 kg leite/d foram utilizadas em três diferentes estágios de lactação (Dias em lactação - DEL): a) Inicial (15 DEL), b) Intermediário (70 DEL) e c) Final (120 DEL). As biópsias da glândula mamária e do tecido adiposo foram coletadas após a ordenha da manhã, o RNA total foi extraído, o DNA complementar (cDNA) foi sintetizado e análises de qRT-PCR foram realizadas. A expressão gênica foi analisada utilizando o procedimento MIXED do SAS usando a média geométrica de dois housekeeping genes, proteína ribossomal S18 (RPS18) e beta-actina (β-ACT), como covariável. No tecido mamário os transcritos do PII reduziram 82% entre os períodos inicial e intermediário, 93% inicial e final e 59% entre o período intermediário e final. Os transcritos do PIII decresceram respectivamente 73, 89 e 59% entre os períodos inicial e intermediário, inicial e final e intermediário e final. A expressão gênica da FASN na glândula mamária reduziu respectivamente 54 e 83% quando o período inicial foi comparado com os períodos intermédiario e final. No tecido mamário a expressão da SREBP1 decresceu 33 e 42% entre o período inicial e final e entre o intermediário e final. No tecido adiposo a expressão do transcrito PI aumentou mais de 30 vezes entre os períodos inicial e intermediário. Há uma adaptação para a síntese de ácidos graxos nos tecido adiposo e mamário a fim de suprir as demandas fisiológicas da lactação, associada à mudanças na expressão gênica dos fatores de transcrição e genes envolvidos na síntese de lipídeos em ambos os tecidos

Global studies show the effect of polymorphism of selected genes on dairy production and fatty acid spectrum. The aim of the diploma thesis was genotyping of candidate FASN locus with a focus on milk yield and fatty acid spectrum depending on genotype. The thesis describes the characteristics of cow's milk, milk fat and fatty acids in milk fat. Furthermore, the thesis deals with the genome of cattle and the potential influence of polymorphism of candidate genes affecting fatty acids of milk fat. Genotypes for FASN were determined by the PCR-RFLP method, the milk yield of selected dairy cattle was statistically evaluated according to the milk production indices for 1st lactation in individual breeds, and the determination of fatty acids was performed by spectrophotometry followed by statistical evaluation. The resulting genotypes in selected breeds were only two, the GG genotype with a higher relative frequency than the genotype AG. There is no statistically significant difference between FASN genotypes depending on the milk yield and the fatty acids spectrum.

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.

Adiponectin is a protein secreted by adipose tissue which possesses insulin-sensitivity and anti-atherosclerotic properties. In this work, we will study if high molecular weight adiponectin is a key molecule in the association between energy metabolism, thyroid axis and bone metabolism in children. To this end, we studied a population of 234 asymptomatic Caucasian children. A sample of blood was also collected to quantify HMW adiponectin, thyroid hormones, bone markers, vitamin D and sFASN. We also obtained 6 biopsies of visceral adipose tissue. The results derived from this study show a positive association of high molecular weight adiponectin with free T4 and soluble FASN and a negative association with carboxylated OC. And provide further evidence of the important role of HMW adiponectin as a biomarker of the common regulation between adipose tissue, the thyroid axis and bone metabolism in healthy children. / L'adiponectina és una proteïna secretada pel teixit adipós que té propietats insulinosensibilitzants i anti-arterioscleròtiques. L’objectiu d’aquest estudi és estudiar a l’adiponectina d’alt pes molecular com a molècula clau en la interacció entre el metabolisme energètic, l’eix tiroïdal i el metabolisme ossi. Per a tal fi, s’ha dissenyat un estudi clínic descriptiu transversal en una mostra de nens sans. A tots ells se'ls va quantificar l’adiponectina d'APM, les hormones tiroïdals, marcadors ossis, la vitamina D i FASN soluble. Addicionalment, es van obtenir 6 biòpsies de teixit adipós visceral.Els resultats derivats d’aquest estudi mostren una associació positiva de l’adiponectina d’alt pes molecular amb la T4 lliure i la FASN soluble i negativa amb la OC carboxilada. I aporten noves evidències de l'important paper de l'adiponectina d'APM com a marcador biològic de la regulació comuna entre el teixit adipòs, l’eix tiroïdal i el metabolisme ossi en edat pediàtrica.

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