Overexpression Of Fatty Acid Synthase Research Articles

SIRT1 histone deacetylase expression is associated with microsatellite instability and CpG island methylator phenotype in colorectal cancer

The class III histone deacetylase SIRT1 (sir2) is important in epigenetic gene silencing. Inhibition of SIRT1 reactivates silenced genes, suggesting a possible therapeutic approach of targeted reversal of aberrantly silenced genes. In addition, SIRT1 may be involved in the well-known link between obesity, cellular energy balance and cancer. However, a comprehensive study of SIRT1 using human cancer tissue with clinical outcome data is currently lacking, and its prognostic significance is uncertain. Using the database of 485 colorectal cancers in two independent prospective cohort studies, we detected SIRT1 overexpression in 180 (37%) tumors by immunohistochemistry. We examined its relationship to the CpG island methylator phenotype (CIMP), related molecular events, clinical features including body mass index, and patient survival. We quantified DNA methylation in eight CIMP-specific promoters (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1) and eight other CpG islands (CHFR, HIC1, IGFBP3, MGMT, MINT1, MINT31, p14, and WRN) by MethyLight. SIRT1 overexpression was associated with CIMP-high (≥6 of 8 methylated CIMP-specific promoters, P=0.002) and microsatellite instability (MSI)-high phenotype (P<0.0001). In both univariate and multivariate analyses, SIRT1 overexpression was significantly associated with the CIMP-high MSI-high phenotype (multivariate odds ratio, 3.20; 95% confidence interval, 1.35–7.59; P=0.008). In addition, mucinous component (P=0.01), high tumor grade (P=0.02), and fatty acid synthase overexpression (P=0.04) were significantly associated with SIRT positivity in multivariate analysis. SIRT1 was not significantly related with age, sex, tumor location, stage, signet ring cells, cyclooxygenase-2 (COX-2), LINE-1 hypomethylation, KRAS, BRAF, BMI, PIK3CA, HDAC, p53, β-catenin, COX-2, or patient prognosis. In conclusion, SIRT1 expression is associated with CIMP-high MSI-high colon cancer, suggesting involvement of SIRT1 in gene silencing in this unique tumor subtype.

P63 Promotes Cell Survival through Fatty Acid Synthase

There is increasing evidence that p63, and specifically ΔNp63, plays a central role in both development and tumorigenesis by promoting epithelial cell survival. However, few studies have addressed the molecular mechanisms through which such important function is exerted. Fatty acid synthase (FASN), a key enzyme that synthesizes long-chain fatty acids and is involved in both embryogenesis and cancer, has been recently proposed as a direct target of p53 family members, including p63 and p73. Here we show that knockdown of either total or ΔN-specific p63 isoforms in squamous cell carcinoma (SCC9) or immortalized prostate epithelial (iPrEC) cells caused a decrease in cell viability by inducing apoptosis without affecting the cell cycle. p63 silencing significantly reduced both the expression and the activity of FASN. Importantly, stable overexpression of either FASN or myristoylated AKT (myr-AKT) was able to partially rescue cells from cell death induced by p63 silencing. FASN induced AKT phosphorylation and a significant reduction in cell viability was observed when FASN-overexpressing SCC9 cells were treated with an AKT inhibitor after p63 knockdown, indicating that AKT plays a major role in FASN-mediated survival. Activated AKT did not cause any alteration in the FASN protein levels but induced its activity, suggesting that the rescue from apoptosis documented in the p63-silenced cells expressing myr-AKT cells may be partially mediated by FASN. Finally, we demonstrated that p63 and FASN expression are positively associated in clinical squamous cell carcinoma samples as well as in the developing prostate. Taken together, our findings demonstrate that FASN is a functionally relevant target of p63 and is required for mediating its pro-survival effects.

High Prevalence of Fatty Acid Synthase Expression in Colorectal Cancers in Middle Eastern Patients and Its Potential Role as a Therapeutic Target

Many human epithelial cancers, particularly those with a poor prognosis, express high levels of fatty acid synthase (FASN), a key metabolic enzyme linked to synthesis of membrane phospholipids in cancer cells. Overexpression of FASN is linked with activation of the phosphatidylinositol-3'-kinase (PI3 K)/AKT pathway. However, the role of FASN in colorectal cancer (CRC) has not been fully elucidated. We investigated the expression of FASN and determined its functional association with the PI3/AKT pathway in CRC. Expression of FASN and its associated targets were studied by immunohistochemistry on 448 CRC tumors in a tissue microarray (TMA) format. Analysis of apoptosis and cell cycle was evaluated in vitro using CRC cell lines by flow cytometry and DNA fragmentation assays. Protein expression was determined by immunohistochemistry and western blotting. In vivo xenograft studies were performed using CRC cell lines and NUDE mice. Correlation of FASN with various clinicopathological parameters on 448 CRC samples was assessed. Activated AKT was found in 294/409 (71.9%) of CRC and was associated with FASN overexpression. FASN expression was observed in 27.1% (109/403) of Middle Eastern CRC. Additionally, FASN expression was significantly more common in tumors characterized by microsatellite instability (MSI) than in those characterized by microsatellite stability (MSS) (P<0.01). Our in vitro data using HCT-15, an MSI CRC cell line, showed a better apoptotic response after inhibition of FASN activity as compared with Colo-320, an MSS CRC cell line. Finally, treatment of HCT-15 cell line xenografts with C-75 resulted in growth inhibition of tumors in NUDE mice via downregulation of FASN and AKT activity. These data identify FASN as a potential biomarker and a novel therapeutic target in distinct molecular subtypes of CRC.

Clinicopathological significance of ubiquitin-specific protease 2a (USP2a), fatty acid synthase (FASN), and ErbB2 expression in oral squamous cell carcinomas

Overexpression of fatty acid synthase (FASN) and ErbB2 has been described in oral squamous cell carcinomas (OSCC). FASN is the key lipogenic enzyme responsible for the endogenous synthesis of fatty acids and its expression can be regulated by ErbB2. The deubiquitinating enzyme (DUB) ubiquitin-specific protease 2a (USP2a) plays a critical role in prostate cancer cell survival by stabilizing the FASN protein. This study investigates whether the gene expression and the immunohistochemical status of FASN, ErbB2, and USP2a correlate with the clinicopathological characteristics of OSCC cases. A strong positive correlation among ErbB2, FASN, and USP2a expression (p=0.001) was observed by qRT-PCR in laser capture microdissected OSCC samples. Perineural infiltration was associated with ErbB2 mRNA expression (p=0.046). The presence of metastatic cervical lymph nodes was associated with FASN (p=0.002), ErbB2 (p=0.001), and USP2a (p=0.006) mRNA levels. ErbB2 staining at the cell membranes was stronger in well-differentiated lesions while a cytoplasmic positivity was found in poorly differentiated tumors. Most of the OSCC (97.06%) that showed a high positivity for FASN were also labeled for ErbB2 at the cell membranes (p=0.001). FASN and ErbB2 positivity was associated with tumor thickness and lymphatic embolization (p=0.006 and p=0.035, p=0.006 and p=0.024 respectively). The membrane expression of ErbB2 as well as FASN and Ki-67 staining were significantly associated with a high risk of recurrence by predicting both disease free survival (log-rank test, p=0.0056, p=0.0011, and p=0.0004, respectively) and overall survival (log-rank test, p=0.0005, p=0.0062, and p=0.0001, respectively). Taken together, the results presented here suggest a molecular connection among FASN, ErbB2, and USP2a in OSCC since their mRNA and protein levels were associated with tumor progression and poor prognosis.

Increased expression of fatty acid synthase in human aberrant crypt foci: Possible target for colorectal cancer prevention

Aberrant crypt foci (ACF), the earliest identified monoclonal lesions in the colon, provide insights into changes that promote and/or accompany the transformation of normal colonic epithelial cells to colorectal cancer. Fatty acid synthase (FAS), the primary enzyme involved in de novo lipogenesis from carbohydrates, is expressed at low levels in most normal human tissues but is elevated in several human neoplasms including colorectal adenomas and carcinomas. To determine if this pathway is altered even earlier in colorectal tumorigenesis, 35 human ACF from 21 patients were evaluated for the immunohistochemical expression of FAS. Sections of colon cancer served as positive controls, and normal colonic mucosa distant from cancer or ACF served as negative controls. FAS expression was increased in 30 (86%) ACF compared with that in adjacent normal colonic mucosa. The expression of FAS in ACF was not related to the degree of dysplasia or to the number of crypts in the ACF. The over expression of FAS in a high proportion of ACF suggests that this enzyme plays an important role very early in colorectal tumorigenesis and may be a target for chemoprevention.

Fatty Acid Synthase: A Metabolic Enzyme and Candidate Oncogene in Prostate Cancer

Overexpression of the fatty acid synthase (FASN) gene has been implicated in prostate carcinogenesis. We sought to directly assess the oncogenic potential of FASN. We used immortalized human prostate epithelial cells (iPrECs), androgen receptor-overexpressing iPrECs (AR-iPrEC), and human prostate adenocarcinoma LNCaP cells that stably overexpressed FASN for cell proliferation assays, soft agar assays, and tests of tumor formation in immunodeficient mice. Transgenic mice expressing FASN in the prostate were generated to assess the effects of FASN on prostate histology. Apoptosis was evaluated by Hoechst 33342 staining and by fluorescence-activated cell sorting in iPrEC-FASN cells treated with stimulators of the intrinsic and extrinsic pathways of apoptosis (ie, camptothecin and anti-Fas antibody, respectively) or with a small interfering RNA (siRNA) targeting FASN. FASN expression was compared with the apoptotic index assessed by the terminal deoxynucleotidyltransferase-mediated UTP end-labeling method in 745 human prostate cancer samples by using the least squares means procedure. All statistical tests were two-sided. Forced expression of FASN in iPrECs, AR-iPrECs, and LNCaP cells increased cell proliferation and soft agar growth. iPrECs that expressed both FASN and androgen receptor (AR) formed invasive adenocarcinomas in immunodeficient mice (12 of 14 mice injected formed tumors vs 0 of 14 mice injected with AR-iPrEC expressing empty vector (P < .001, Fisher exact test); however, iPrECs that expressed only FASN did not. Transgenic expression of FASN in mice resulted in prostate intraepithelial neoplasia, the incidence of which increased from 10% in 8- to 16-week-old mice to 44% in mice aged 7 months or more (P = .0028, Fisher exact test), but not in invasive tumors. In LNCaP cells, siRNA-mediated silencing of FASN resulted in apoptosis. FASN overexpression protected iPrECs from apoptosis induced by camptothecin but did not protect iPrECs from Fas receptor-induced apoptosis. In human prostate cancer specimens, FASN expression was inversely associated with the apoptotic rate (mean percentage of apoptotic cells, lowest vs highest quartile of FASN expression: 2.76 vs 1.34, difference = 1.41, 95% confidence interval = 0.45 to 2.39, Ptrend = .0046). These observations suggest that FASN can act as a prostate cancer oncogene in the presence of AR and that FASN exerts its oncogenic effect by inhibiting the intrinsic pathway of apoptosis.

Fatty acid synthase is over‐expressed in large aberrant crypt foci in rats treated with azoxymethane

Fatty acid synthase (FAS) is over-expressed in many human cancers including colon. High levels of FAS expression have also been observed in a number of lesions that are precursors to invasive colorectal cancer. However, FAS expression in aberrant crypt foci (ACF), the earliest identifiable lesions in colon cancer development, has not been investigated. In this study, we treated Fisher rats with a single dose of the colon carcinogen azoxymethane then evaluated ACF 100 days later. We showed that large ACF (>or=4 crypts/focus) have a significantly higher level of immunohistochemical staining for FAS than either small ACF (<or=3 crypts/focus) or normal crypts. Furthermore, the severity of nuclear atypia in ACF was positively associated with increased expression of FAS. These findings suggest that the genes associated with FAS over-expression are activated early in the stepwise development of colon cancer, though not until the ACF have reached a critical size with a level of nuclear atypia indicative of dysplasia.

Expression of fatty acid synthase as a prognostic marker in triple negative breast cancer.

Abstract Abstract #1082 Background: Fatty acid synthase (FAS), the key enzyme required for fatty acid biosynthesis, is minimally expressed in most normal human tissue. Recent studies have demonstrated that hyperactivity or overexpression of FAS is a common feature in subset of sex steroid-related tumors, including breast carcinoma. Moreover, up-regulation of FAS gene expression is more pronounced in more advanced tumors, and strong expression of FAS seems to be associated with a poor prognosis of breast cancer. We examined the expression of FAS in invasive breast carcinomas to investigate the prognostic value of FAS expression in a large cohort of breast carcinomas with long term follow up data.&amp;#x2028; Methods: Five hundred and ninety six cases of invasive breast carcinoma, retrospectively selected from 1987-2004, were incorporated into tissue microarrays (TMAs). We performed immunohistochemical stains for FAS, ER, progesterone receptor (PR) and Her-2/neu. Triple-negative breast cancers (TNBCs) were defined as those with ER-, PR- and Her-2/neu-negative results. The results of immunohistochemical stains were correlated with other known clinicopathologic parameters and evaluated for an effect on estimating prognosis.&amp;#x2028; Results: The mean age at diagnosis was 47.2 (ranges 20∼85 years) with a mean tumor size of 2.5cm (ranges 0.4∼11cm). The expression of FAS was positive in 437 (73.3%) cases. There was no significant correlation between the expression of FAS and clinicopathologic characteristics of the tumor (age, tumor size, lymph node metastasis and lymphovascular invasion) (p&amp;gt;0.05). FAS expression was inversely correlated with histologic grade (p=0.009) and positively correlated with the expression of ER (p&amp;lt;0.001), PR (p&amp;lt;0.001) and Her-2/neu (p&amp;lt;0.001). One hundred thirty eight of 596 cases (23.1%) were TNBCs. FAS-expressing breast invasive carcinomas had a higher tendency to have triple-negative phenotype when compared to FAS-negative cancers (p&amp;lt;0.001) with a mean follow up of 72.3 months, there was no significant survival difference according to the FAS expression (p&amp;gt;0.05). However, FAS-positive group showed shorter disease free survival (DFS) (p=0.014) and overall survival (OS) (p=0.021) than FAS-negative group in patients with TNBC.&amp;#x2028; Conclusion: This study showed that FAS expression is strongly associated with the expression of hormonal receptors and Her-2/neu in invasive breast carcinomas. Especially FAS could be a potential prognostic marker in TNBCs. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1082.

Activation of the lipogenic phenotype predicts poor prognosis in human hepatocellular carcinoma

Emerging evidence indicates that lipogenic enzymes are induced in hepatocarcinogenesis, but their role liver cancer development and progression remains poorly delineated. In the present investigation, we determined the levels and biologic role of the main lipogenic enzymes in a human hepatocellular carcinoma (HCC) collection. Levels of Fatty Acid Synthase (FASN), Acetyl-CoA Carboxylase (ACACA), ATP citrate lyase (ACLY), and Steroyl-CoA Desaturase (SCD) were all upregulated in HCC compared with corresponding noncancerous livers. Of note, the highest expression of lipogenic enzymes occurred in tumors with poor prognosis, implying their role in tumor aggressiveness. Suppression of FASN, ACACA, ACLY, and SCD by either siRNA or chemical inhibitors strongly reduced the growth of HCC cells in vitro, leading to massive apoptosis, whereas overexpression of FASN, ACACA, ACLY, and SCD induced a significant increase in proliferation and resistance to chemically-induced apoptosis in HCC cells. At the molecular level, inhibition of FASN was paralleled by downregulation of genes involved in cell proliferation and survival, including Akt, epidermal growth factor receptor (EGFR or ERBB1), S-phase kinase-associated protein 2 (SKP2), and NF- kB, and re-expression of putative oncosuppressor genes, including p21, p27, and death associated protein kinase 2 (DAPK2). Furthermore, FASN expression was regulated at post-transcriptional level by the isopeptidase ubiquitin-specific protease-2a (USP2a), whose suppression resulted in FASN downregulation in HCC cells. Taken together, the present data assign a pathogenetic and prognostic role to deregulation of the fatty acid biosynthetic pathway in liver cancer, and envisage the usefulness of FASN inhibitors as therapeutic modality for human HCC.

Overexpression of fatty acid synthase is associated with palmitoylation of Wnt1 and cytoplasmic stabilization of β-catenin in prostate cancer

Fatty acid synthase (FASN), a key metabolic enzyme for liponeogenesis highly expressed in several human cancers, displays oncogenic properties such as resistance to apoptosis and induction of proliferation when overexpressed. To date, no mechanism has been identified to explain the oncogenicity of FASN in prostate cancer. We generated immortalized prostate epithelial cells (iPrECs) overexpressing FASN, and found that 14C-acetate incorporation into palmitate synthesized de novo by FASN was significantly elevated in immunoprecipitated Wnt-1 when compared to isogenic cells not overexpressing FASN. Overexpression of FASN caused membranous and cytoplasmic β-catenin protein accumulation and activation, whereas FASN knockdown by short-hairpin RNA resulted in a reduction in the extent of β-catenin activation. Orthotopic transplantation of iPrECs overexpressing FASN in nude mice resulted in invasive tumors that overexpressed β-catenin. A strong significant association between FASN and cytoplasmic (stabilized) β-catenin immunostaining was found in 862 cases of human prostate cancer after computerized subtraction of the membranous β-catenin signal (P<0.001, Spearman's ρ=0.33). We propose that cytoplasmic stabilization of β-catenin through palmitoylation of Wnt-1 and subsequent activation of the pathway is a potential mechanism of FASN oncogenicity in prostate cancer.

Fatty Acid Synthase and Diffuse Large B Cell Lymphoma

Diffuse Large B-cell Lymphoma (DLBCL) is considered to be the most common type of lymphoma in adults, accounting for 30–40 percent of cases of non-Hodgkin's lymphoma. The incidence of DLBCL is about 40–60% in Saudi Arabian population. The reason of high rate for DLBCL in Saudi Arabian population is not known, however recent studies suggests that differences in molecular signature as compared to western population accounts for this incidence. Although patients with DLBCL are potentially curable with combination chemotherapy, the disease proves fatal in approximately 50% of patients. The cause of most DLBCLs remains unknown; however several studies suggest that dysregulated survival/apoptosis or defective repair pathways have been implicated. Many human cancers, particularly those with a poor prognosis express high levels of fatty acid synthase (FASN), a key metabolic enzyme linked to the synthesis of membrane phospholipids in cancer cells. Over-expression of FASN can be largely attributed to activation of phosphatidylinositol-3′-kinase (PI3K)/AKT pathway. However, the role of FASN in the pathogenesis of DLBCL has not been elucidated. Therefore, in this study, we investigated the role of FASN in a large series (301) of DLBCL patient samples and a panel of DLBCL cell lines. Using immunohistochemistry, FASN was detected in appreciable number of DLBCL tumors and was strongly associated with the expression of p-AKT protein. We next examined the effect of C-75, a synthetic slow binding inhibitor of FASN activity on DLBCL cell lines (SUDHL4, SUDHL5, SUDHL8 and OCI-LY19) in vitro and found that C-75 treatment inhibits growth and induces apoptosis in all 4 DLBCL cell linesused in the study. We show based on in vitro studies employing a variety of experimental tools using different FASN inhibitors, FASN siRNA and AKT siRNA that FASN exert its oncogenic action in DLBCL cells via activated AKT. Our data show that inhibition of FASN leads to de-phosphorylation of p-AKT and it's down stream effectors, FOXO1 and GSK-3. This, in turn leads to activation of the intrinsic apoptotic pathway by, initially causing conformational changes of the Bax protein leading to changes in the mitochondrial membrane potential and release of cytochrome c into cytosole. This causes activation of caspases-9 and -3 and cleavage of PARP. zVAD-fmk, a universal inhibitor of caspases prevents caspase-9 and -3 activation and abrogates apoptosis induced by C-75 treatment. Finally, C-75 treament of DLBCL cell lines causes down-regulation of the inhibitor of apoptosis proteins, XIAP, cIAP1 and Survivin. In summary, data presented here demonstrate a significant correlation between the expression of FASN and active AKT in DLBCL and indicate that inhibition of PI3K/AKT signaling synergize the FAS inhibitors to induce apoptosis in DLBCL cell lines with constitutively active AKT. This may have significant clinical implications. Therefore, FASN has become a promising target for anti cancer drug development.

Cohort Study of Fatty Acid Synthase Expression and Patient Survival in Colon Cancer

Energy balance seems to be important in the pathogenesis of colon cancer. Fatty acid synthase (FASN) is physiologically regulated by energy balance and is often upregulated in colorectal cancer. Nonetheless, the influence of FASN expression on patient outcome is uncertain. Using the database of 647 patients with colon cancer in two independent cohort studies, FASN overexpression was detected in 84 tumors (13%) by immunohistochemistry. Cox proportional hazards models calculated hazard ratios (HRs) of colon cancer-specific and overall mortalities, adjusted for patient characteristics and related tumoral features, including KRAS, BRAF, p53, microsatellite instability and the CpG island methylation phenotype. There were 279 deaths, including 160 colon cancer-specific deaths. FASN overexpression was associated with a significant reduction in colon cancer-specific mortality by both univariate and multivariate analyses (adjusted HR, 0.41; 95% CI, 0.19 to 0.89) and an insignificant trend toward improved overall mortality (adjusted HR, 0.75; 95% CI, 0.50 to 1.13). Notably, the effect of FASN expression on mortality might be different according to body mass index (BMI; P(interaction) = .019); the adjusted HR of overall mortality for FASN overexpression was 0.63 (95% CI, 0.39 to 1.02) among patients with BMI less than 27.5 kg/m(2) and 2.91 (95% CI, 1.19 to 7.12) among those with BMI >or= 27.5 kg/m(2). Moreover, the adverse effect of moderate overweight/obesity on overall survival was limited to FASN-positive tumors (adjusted HR, 4.10; 95% CI, 1.14 to 14.8; BMI >or= 27.5 kg/m(2) v < 27.5 kg/m(2)). CONCLUSION Among nonobese patients with colon cancer, tumoral FASN overexpression is associated with improved survival, whereas among moderately overweight or obese patients (BMI >or= 27.5 kg/m(2)), FASN overexpression may predict a worse outcome.

A new mechanism of drug resistance in breast cancer cells: fatty acid synthase overexpression-mediated palmitate overproduction

Multidrug resistance is a major problem in successful cancer chemotherapy. Various mechanisms of resistance, such as ABC transporter-mediated drug efflux, have been discovered using established model cancer cell lines. While characterizing a drug-resistant breast cancer cell line, MCF7/AdVp3000, we found that fatty acid synthase (FASN) is overexpressed. In this study, we showed that ectopic overexpression of FASN indeed causes drug resistance and that reducing the FASN expression increased the drug sensitivity in breast cancer cell lines MCF7 and MDA-MB-468 but not in the normal mammary epithelial cell line MCF10A1. Use of FASN inhibitor, Orlistat, at low concentrations also sensitized cells with FASN overexpression to anticancer drugs. The FASN-mediated drug resistance appears to be due to a decrease in drug-induced apoptosis from an overproduction of palmitic acid by FASN. Together with previous findings of FASN as a poor prognosis marker for breast cancer patients, our results suggest that FASN overexpression is a new mechanism of drug resistance and may be an ideal target for chemosensitization in breast cancer chemotherapy.

Dietary soy protein inhibits DNA damage and cell survival of colon epithelial cells through attenuated expression of fatty acid synthase

Dietary intake of soy protein decreases tumor incidence in rat models of chemically induced colon cancer. We hypothesized that decreased expression of fatty acid synthase (FASN) underlies, in part, the tumor-preventive effects of soy protein, since FASN overexpression characterizes early tumorigenesis. Here, we show that colonic FASN levels are reduced with dietary intake of soy protein isolate (SPI), compared with a control casein diet, in male Sprague-Dawley rats administered the colon carcinogen azoxymethane. SPI consumption resulted in decreased serum insulin levels and decreased azoxymethane-induced tumor suppressor p53 phosphorylation in colon crypt epithelium. To evaluate potential links between insulin and FASN leading to DNA damage, C2(BBe)1 colon epithelial cells, treated with insulin and/or the carcinogen N-nitroso-N-methylurea (NMU), were evaluated for DNA damage and apoptosis after transfection with control or FASN small interfering RNAs (siRNAs). While the numbers of DNA apurinic/apyrimidinic sites (biomarker of DNA damage) induced by NMU were unaffected by transfection of FASN siRNA, insulin induction of these sites was decreased with FASN knockdown. By contrast, NMU-induced apoptosis of C2(BBe)1, as well as intestinal epithelial cell (IEC)-6, was enhanced by transfected FASN siRNA. Increased FASN expression in IEC-6 cells by addition of liver X receptor agonist T0901317 did not affect apurinic/apyrimidinic site number, but enhanced cell killing by cerulenin, a FASN inhibitor. Moreover, insulin rescued NMU-treated cells from apoptosis in an FASN-dependent manner. Results suggest that dietary SPI, by decreasing circulating insulin levels and colon FASN expression, attenuates insulin-induced DNA damage and FASN-mediated anti-apoptosis during carcinogenesis, resulting in an overall reduced tumorigenic state.

Overexpression of fatty acid synthase gene activates HER1/HER2 tyrosine kinase receptors in human breast epithelial cells

More than 50 years ago, we learned that breast cancer cells (and those of many other types of tumour) endogenously synthesize 95% of fatty acids (FAs) de novo, despite having adequate nutritional lipid supply. Today, we know that breast cancer cells benefit from this phenomenon in terms of enhanced cell proliferation, survival, chemoresistance and metastasis. However, the exact role of the major lipogenic enzyme fatty acid synthase (FASN) as cause, correlate or facilitator of breast cancer remains unidentified. To evaluate a causal effect of FASN-catalysed endogenous FA biosynthesis in the natural history of breast cancer disease, HBL100 cells (an SV40-transformed in vitro model for near-normal gene expression in the breast epithelium), and MCF10A cells (a non-transformed, near diploid, spontaneously immortalized human mammary epithelial cell line) were acutely forced to overexpress the human FASN gene. Following transient transfection with plasmid pCMV6-XL4 carrying full-length human FASN cDNA (gi: NM 004104), HBL100 cells enhanced their endogenous lipid synthesis while acquiring canonical oncogenic properties such as increased size and number of colonies in semisolid (i.e. soft-agar) anchorage-independent cultures. Anchorage-dependent cell proliferation assays in low serum (0.1% foetal bovine serum), MTT-based assessment of cell metabolic status and cell death ELISA-based detection of apoptosis-induced DNA-histone fragmentation, together revealed that sole activation of endogenous FA biosynthesis was sufficient to significantly enhance breast epithelial cell proliferation and survival. When analysing molecular mechanisms by which acute activation of de novo FA biosynthesis triggered a transformed phenotype, HBL100 cells, transiently transfected with pCMV6-XL4/FASN, were found to exhibit a dramatic increase in the number of phosphor-tyrosine (Tyr)-containing proteins, as detected by 4G10 antiphosphor-Tyr monoclonal antibody. Phosphor-Tyr-specific antibodies recognizing the phosphorylation status of either the 1173 Tyr residue of epidermal growth factor receptor (HER1) or the 1248 Tyr residue of HER2, further revealed that FASN-induced Tyr-phosphorylation at approximately 180 kDa region mainly represented that of these key members of the HER (erbB) network, which remained switched-off in mock-transfected HBL100 cells. ELISA and immunoblotting procedures demonstrated that FASN overactivation significantly increased (> 200%) expression levels of epidermal growth factor receptor and HER2 proteins in HBL100 cells. Proteome Profilertrade mark antibody arrays capable of simultaneously detecting relative levels of phosphorylation of 42 phospho-receptor Tyr-kinases (RTKs) confirmed that acute activation of endogenous FA biosynthesis specifically promoted hyper-Tyr-phosphorylation of HER1 and HER2 in MCF10A cells. This FASN-triggered HER1/HER2-breast cancer-like phenotype was specifically inhibitable either by FASN inhibitor C75 or by Tyr-kinase inhibitors (TKIs) gefitinib (Iressa) and lapatinib (Tykerb) but not by chemotherapeutic agents such as cisplatin. Transient overexpression of FASN dramatically increased HBL100 breast epithelial cells' sensitivity to cytotoxic effects of C75, gefitinib and lapatinib (approximately 8, 10 and > 15 times, respectively), while significantly decreasing (approximately 3 times) cisplatin efficacy. Although we cannot definitely establish FASN as a novel oncogene in breast cancer, this study reveals for the first time that exacerbated endogenous FA biosynthesis in non-cancerous epithelial cells is sufficient to induce a cancer-like phenotype functionally dependent on the HER1/HER2 duo. These findings may perhaps radically amend our current perspective of endogenously synthesized fat, as on its own, it appears to actively increase signal-to-noise ratio in the HER1/HER2-driven progression of human breast epithelial cells towards malignancy.

Fatty acid synthase expression in squamous cell carcinoma of the tongue: clinicopathological findings

Overexpression of fatty acid synthase (FAS), the cytosolic enzyme responsible for the conversion of dietary carbohydrates to fatty acids, has been reported in several human malignancies and pointed as a potential prognostic marker for some tumors. This study investigated whether FAS immunohistochemical expression is correlated with the clinicopathological characteristics of oral squamous cell carcinoma (OSCC). The clinical features of 102 patients with OSCC of the tongue treated in a single institution were obtained from the medical records and all histopathological diagnoses were reviewed. The expression of FAS was determined by the standard immunoperoxidase technique in formalin-fixed and paraffin-embedded specimens and correlated with the clinicopathological characteristics of the tumors. Eighty-one cases (79.41%) were positive for FAS. Microscopic characteristics such as histological grade (P < 0.05), lymphatic permeation (P < 0.001), perineural infiltration (P < 0.05), and nodal metastasis (P < 0.02) were associated with FAS status. A significantly lower survival probability for patients with advanced clinical stage (log-rank test, P < 0.001), lymph nodes metastasis (log-rank test, P < 0.001), presence of vascular permeation (log-rank test, P = 0.05), and perineural invasion (log-rank test, P = 0.01) was observed in the studied samples. The expression of FAS in OSCC of the tongue is associated with the microscopic characteristics that determine disease progression and prognosis.

Olanzapine induces SREBP-1-related adipogenesis in 3T3-L1 cells

Olanzapine is a second-generation atypical antipsychotic drug (AAPD). Major side effects of olanzapine are weight gain and development of diabetes mellitus, which are risk factors of cardiovascular diseases. The possible causes of metabolic adverse effects are known as poor satiety and increased food intake due to blockade of receptors such as 5-HT 2C in CNS. In this study, we examine the effect of olanzapine on peripheral adipogenesis using cultured 3T3-L1 cell model. Olanzapine increased triacylglyceride (TG) accumulation during 3T3-L1 preadipocyte differentiation to mature adipocyte phenotype. TG accumulation was accompanied by overexpression of fatty acid synthase and adiponectin that are the downstream genes of sterol regulatory element binding protein-1 (SREBP-1), one of the key transcription factors in lipid homeostasis. We further consisted that mostly SREBP-1 and at a lesser extent peroxisome proliferator-activated receptor gamma (PPAR-γ), but not CCAAT/enhancer binding protein-α (C/EBP-α), were overexpressed and activated in 3T3-L1 adipocytes exposed to olanzapine. Furthermore, we showed that olanzapine enhanced the activity of SRE-1-containing LDLR promoter in transfected 3T3-L1 adipocytes and HepG2 cells. Taken together, olanzapine may cause body weight gain not only through influencing CNS receptors, but also affecting the peripheral adipogenesis regulated by SREBP-1.

Fatty acid synthase overexpression in colorectal cancer is associated with microsatellite instability, independent of CpG island methylator phenotype

Overexpression of fatty acid synthase (FASN), a key enzyme for de novo lipogenesis, is observed in many cancers including colorectal cancer and is associated with poor clinical outcomes. Cellular FASN expression is physiologically upregulated in a state of energy excess. Obesity and excess energy balance have been known to be risk factors for colorectal cancer. High degree of microsatellite instability (MSI-H) is a distinct phenotype in colorectal cancer, associated with CpG island methylator phenotype (CIMP). Previous data suggest that obesity or altered energy balance may potentially modify risks for MSI-H cancers and microsatellite stable (MSS) cancers differently. However, the relationship between MSI and FASN overexpression has not been investigated. Using 976 cases of population-based colorectal cancer samples from 2 large prospective cohort studies, we correlated FASN expression (by immunohistochemistry) with MSI, KRAS and BRAF mutations, p53 expression (by immunohistochemistry), and CIMP status [determined by MethyLight for 8 CIMP-specific gene promoters including CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1]. Marked (2+) FASN overexpression was observed in 110 (11%) of the 976 tumors and was significantly more common in MSI-H tumors (21% [28/135]) than MSI-low (5.6% [4/72], P = .004) and MSS tumors (11% [72/678], P = .001). The association between FASN overexpression and MSI-H persisted even after stratification by CIMP status. In contrast, FASN overexpression was not correlated with CIMP after stratification by MSI status. Fatty acid synthase overexpression was not significantly correlated with sex, tumor location, p53, or KRAS/BRAF status. In conclusion, FASN overexpression in colorectal cancer is associated with MSI-H, independent of CIMP status.

Longitudinal analysis of murine steatohepatitis model induced by chronic exposure to high‐fat diet

Several lines of epidemiological evidence have suggested that non-alcoholic steatohepatitis (NASH) is closely associated with obesity in humans. However, the precise mechanisms of the progression of NASH and its key metabolic abnormalities remain to be elucidated. We found that long-term high-fat diet (HFD) exposure induces NASH, with excess body weight, hyperinsulinemia and hypercholesteremia in mice. Longitudinal analysis of the model showed that steatohepatitis was induced after onset of metabolic abnormalities. In addition, we found that expression of MCP-1 mRNA was induced in the liver before induction of TNFalpha and type I collagen alpha1 mRNAs, and prior to onset of steatohepatitis. We confirmed that hepatic MCP-1 contents were increased in mice fed HFD for 50 weeks, although the precise role of MCP-1 in the development of NASH remains to be addressed. The mouse model was also characterized by moderate reductions in catalase activity and glutathione content, as well as by overexpression of fatty acid synthase, acetyl-CoA carboxylase 1 and FAT/CD36 mRNAs in the liver. The murine NASH model apparently mimics clinical aspects of the condition and provides insight into NASH.

402 POSTER Biological and clinical significance of fatty acid synthase overexpression in cancer

Styrene oxide (SO), ethylene oxide (EO) and gamma-radiation (G) are agents with a well-described metabolism and genotoxicity. EPHX1 and GSTs play an important role in the detoxification of electrophiles and oxidative stress. Enzymes involved in base excision repair (hOGG1, XRCC1), in rejoining single strand breaks (XRCC1) and in repair of cross-links and chromosomal double strand breaks (XRCC3) might have an impact on genotoxicity as well.In this study we assessed the dose-dependent effect of genetic polymorphisms in biotransforming (EPHX (Tyr113/His113 and His139/Arg139), GSTP1 (Ile105/Val105), GSTM1 and GSTT1) and DNA repair enzymes (hOGG1 (Ser326/Cys326), XRCC1 (Arg194/Trp194, Arg280/His280, Arg399/Gln399), XRCC3 (Thr241/Met241)) on the induced genotoxicity.Peripheral blood mononuclear cells from 20 individuals were exposed to 3 doses per agent (+control). Genotoxicity was evaluated by measuring comet tail length (TL) and micronucleus frequencies in binucleated cells (MNCB).Dose-dependent DNA damage was found for all agents and end-points, with the exception of MNCB induced by EO. Repeated measure ANOVA revealed a significant contribution of hOGG1 and XRCC3 genotypes to the inter-individual variability of TL and MNCB in cells exposed to EO and G. Homozygous hOGG1326 wild cells showed significantly lower EO-induced TL than the heterozygous cells. Significantly higher TL and MNCB were found in EO-exposed cells carrying the XRCC3241Met variant and the influence on TL was more pronounced at higher dose. In G-irradiated cells, TL was significantly higher in the hOGG1326 homozygous wild types compared with mutated genotypes. The influence of hOGG1326 on TL was borderline dose-dependent.We conclude that the influence of genetic polymorphisms of enzymes involved in DNA repair on induced genotoxicity depends on exposure dose.