Malonyl-CoA decarboxylase inhibition is selectively cytotoxic to human breast cancer cells.

Fatty acid synthase (FAS) inhibition initiates selective apoptosis of cancer cells both in vivo and in vitro, which may involve malonyl-CoA metabolism. These findings have led to the exploration of malonyl-CoA decarboxylase (MCD) as a potential novel target for cancer treatment. MCD regulates the levels of cellular malonyl-CoA through the decarboxylation of malonyl-CoA to acetyl-CoA. Malonyl-CoA is both a substrate for FAS and an inhibitor of fatty acid oxidation acting as a metabolic switch between anabolic fatty acid synthesis and catabolic fatty acid oxidation. We now report that the treatment of human breast cancer (MCF7) cells with MCD small interference RNA (siRNA) reduces MCD expression and activity, reduces adenosine triphosphate levels, and is cytotoxic to MCF7 cells, but not to human fibroblasts. In addition, we synthesized a small-molecule inhibitor of MCD, 5-{(Morpholine-4-carbonyl)-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-phenyl]-amino}-pentanoic acid methyl ester (MPA). Similar to MCD siRNA, MPA inhibits MCD activity in MCF7 cells, increases cellular malonyl-CoA levels and is cytotoxic to a number of human breast cancer cell lines in vitro. Taken together, these data indicate that MCD-induced cytotoxicity is likely mediated through malonyl-CoA metabolism. These findings support the hypothesis that MCD is a potential therapeutic target for cancer therapy.

AMP-activated protein kinase and human cancer: cancer metabolism revisited.

AMP-activated protein kinase (AMPK) and its upstream kinase, LKB1, act to both monitor and restore cellular energy in response to energy depletion. Studied extensively in liver and skeletal muscle, AMPK is phosphorylated and activated by LKB1 in response to increasing AMP/ATP ratios, which occur in a variety of settings including hypoxia, nutrient starvation and redox imbalance. Interest in the roles of both AMPK and LKB1 in cancer has grown substantially, following the identification of LKB1 as the tumor suppressor gene mutated in the Peutz-Jegher familial cancer syndrome. Patients with the Peutz-Jegher syndrome harbor a single inactive LKB1 gene, and acquisition of a second inactivating lesion (loss of heterozygosity) leads to the development of the cancer in a variety of organs. Thus, the loss of AMPK activation is hypothesized to promote the development of malignancy. Conversely, pharmacological AMPK activation has recently been shown to be cytotoxic to many established human cancer cell lines in vitro and in human cancer xenograft and mouse cancer allografts. Previously, changes in cell metabolism that accompanied the malignant phenotype have largely been considered a consequence of cellular transformation. Now, AMPK and energy metabolism are linked to the development and maintenance of the malignant phenotype. These findings have led to renewed interest in AMPK and cancer cell metabolism in general as potential targets for cancer therapy.

Fatty acid synthase inhibition in human breast cancer cells leads to malonyl-CoA-induced inhibition of fatty acid oxidation and cytotoxicity.

Inhibition of fatty acid synthase (FAS) induces apoptosis in human breast cancer cells in vitro and in vivo without toxicity to proliferating normal cells. We have previously shown that FAS inhibition causes a rapid increase in malonyl-CoA levels identifying malonyl-CoA as a potential trigger of apoptosis. In this study we further investigated the role of malonyl-CoA during FAS inhibition. We have found that: [i] inhibition of FAS with cerulenin causes carnitine palmitoyltransferase-1 (CPT-1) inhibition and fatty acid oxidation inhibition in MCF-7 human breast cancer cells likely mediated by elevation of malonyl-CoA; [ii] cerulenin cytotoxicity is due to the nonphysiological state of increased malonyl-CoA, decreased fatty acid oxidation, and decreased fatty acid synthesis; and [iii] the cytotoxic effect of cerulenin can be mimicked by simultaneous inhibition of CPT-1, with etomoxir, and fatty acid synthesis with TOFA, an acetyl-CoA carboxylase (ACC) inhibitor. This study identifies CPT-1 and ACC as two new potential targets for cancer chemotherapy.

Fatty acid synthase (FAS) expression in human breast cancer cell culture supernatants and in breast cancer patients.

Fatty acid synthase (FAS) is selectively expressed in certain human cancers, including carcinoma of the breast, prostate, colon, ovary, and endometrium, compared to normal human tissues and therefore is a putative tumor marker. In this study, we found FAS concentrations were elevated in cell culture supernatants during cell growth in two human breast cancer cell lines but not other cancer cell lines. A quantitative enzyme-linked immunosorbent assay and Western blot analysis were employed in this study. In addition, serum FAS levels were significantly higher in breast cancer patients with different clinical stages (Stage II: 0.59+/-0.09 units/l, Stage III: 0.79+/-0.13 units/l, and Stage IV: 1.39+/-0.35 units/l) compared with healthy subjects (0.27+/-0.02 units/l, P<0.05). Taken together, our data suggest that FAS expression may be a useful tumor marker for breast cancer and play a role in assessing cancer virulence.

Increased fatty acid synthase is a therapeutic target in mesothelioma.

Many common human cancer tissues express high levels of fatty acid synthase (FAS), the primary enzyme for the synthesis of fatty acids, and the differential expression of FAS between normal and neoplastic tissues has led to the consideration of FAS as a target for anticancer therapy. To investigate the potential of targeting FAS for the treatment of pleural mesothelioma, we first determined whether FAS is overexpressed in human mesothelioma. By immunohistochemistry, we found 22 of 30 human mesothelioma tissue samples tested to express significantly increased levels of FAS compared with normal tissues, including mesothelium. To further explore FAS as a therapeutic target in mesothelioma, we established a nude mouse xenograft model for human mesothelioma using the H-Meso cell line. The i.p. xenografts of this cell line have high levels of FAS expression and fatty acid synthesis pathway activity and grow along mesothelial surfaces in a manner similar to the growth pattern of human mesothelioma. Growth of these tumor xenografts was essentially abolished in mice treated with weekly i.p. injections of C75, a synthetic, small molecule inhibitor of FAS, at levels that resulted in no significant systemic toxicity except for reversible weight loss. These results suggest that FAS may be an effective target for pharmacological therapy in a high proportion of human mesotheliomas.

Two-site ELISA for the quantitative determination of fatty acid synthase.

Fatty acid synthase (FAS) is an enzyme which plays a central role in the de novo biosynthesis of fatty acids. FAS is selectively expressed in certain human cancers and therefore is a putative tumor marker. We developed an enzyme-linked immunosorbent assay (ELISA) for measuring FAS, and investigated its expression and clinical features. In this two-site sandwich ELISA, a polyclonal antibody was used as a capture on Nunc MaxiSorp ELISA/EIA modules and a monoclonal antibody labeled with biotin was used as a signal antibody. The assay was linear with no cross-reactivity with other tumor markers. The within- and between-run CVs were <10%, and the detection limit was 0.15 arbitrary Units/l. Recoveries were 92.4-105.1%. FAS was stable in buffer at 4 degrees C for more than 10 days and stable at 37 degrees C for 2 days. In human serum, FAS levels were significantly higher in patients with breast (1.01+/-0.71 Units/l, mean+/-S.D.), prostate (0.79+/-0.76 Units/l), colon (0.89+/-0.49 Units/l), and ovarian (0.84+/-0.9 Units/l) cancers compared to normal subjects (0.27+/-0.09 Units/l, P<0.01). This assay is sensitive, accurate, and precise and can distinguish between patients with various types of cancer and normal subjects.

Reduced food intake and body weight in mice treated with fatty acid synthase inhibitors.

With the escalation of obesity-related disease, there is great interest in defining the mechanisms that control appetite and body weight. We have identified a link between anabolic energy metabolism and appetite control. Both systemic and intracerebroventricular treatment of mice with fatty acid synthase (FAS) inhibitors (cerulenin and a synthetic compound C75) led to inhibition of feeding and dramatic weight loss. C75 inhibited expression of the prophagic signal neuropeptide Y in the hypothalamus and acted in a leptin-independent manner that appears to be mediated by malonyl-coenzyme A. Thus, FAS may represent an important link in feeding regulation and may be a potential therapeutic target.

Fatty-acid synthase and human cancer: new perspectives on its role in tumor biology.

This review documents the changing perspectives on the function of fatty-acid synthase and fatty-acid synthesis in human tumor biology. With the recent discovery that human cancer cells express high levels of fatty-acid synthase and undergo significant endogenous fatty-acid synthesis, our understanding of the role of fatty acids in tumor biology is expanding. Once considered largely an anabolic-energy-storage pathway, fatty-acid synthesis is now associated with clinically aggressive tumor behavior and tumor-cell growth and survival and has become a novel target pathway for chemotherapy development. These findings will ultimately enhance our understanding of fatty acids in tumor biology and may provide new diagnostic and therapeutic moieties for patient care.

Synthesis and antitumor activity of an inhibitor of fatty acid synthase.

Compared to normal human tissues, many common human cancers, including carcinoma of the colon, prostate, ovary, breast, and endometrium, express high levels of fatty acid synthase (FAS, EC ), the primary enzyme responsible for the synthesis of fatty acids. This differential expression of FAS between normal tissues and cancer has led to the notion that FAS is a target for anticancer drug development. Recent studies with C75, an inhibitor of fatty acid synthesis, have shown significant antitumor activity with concomitant inhibition of fatty acid synthesis in tumor tissue and normal liver. Importantly, histopathological analysis of normal tissues after C75 treatment showed no adverse effects on proliferating cellular compartments, such as bone marrow, gastrointestinal tract, skin, or lymphoid tissues. In this study, we describe the de novo synthesis of C75 based on the known mechanism of action of cerulenin and the theoretical reaction intermediates of the beta-ketoacyl synthase moiety of FAS. In addition, we demonstrate that C75 is a synthetic, chemically stable inhibitor of FAS. C75 inhibits purified mammalian FAS with characteristics of a slow-binding inhibitor and also inhibits fatty acid synthesis in human cancer cells. Treatment of human breast cancer cells with [5-(3)H]C75 demonstrates that C75 reacts preferentially with FAS in whole cells. Therefore, we have shown that the primary mechanism of the antitumor activity of C75 is likely mediated through its interaction with, and inhibition of, FAS. This development will enable the in vivo study of FAS inhibition in human cancer and other metabolic diseases.

Malonyl-coenzyme-A is a potential mediator of cytotoxicity induced by fatty-acid synthase inhibition in human breast cancer cells and xenografts.

A biologically aggressive subset of human breast cancers and other malignancies is characterized by elevated fatty-acid synthase (FAS) enzyme expression, elevated fatty acid (FA) synthesis, and selective sensitivity to pharmacological inhibition of FAS activity by cerulenin or the novel compound C75. In this study, inhibition of FA synthesis at the physiologically regulated step of carboxylation of acetyl-CoA to malonyl-CoA by 5-(tetradecyloxy)-2-furoic acid (TOFA) was not cytotoxic to breast cancer cells in clonogenic assays. FAS inhibitors induced a rapid increase in intracellular malonyl-CoA to several fold above control levels, whereas TOFA reduced intracellular malonyl-CoA by 60%. Simultaneous exposure of breast cancer cells to TOFA and an FAS inhibitor resulted in significantly reduced cytotoxicity and apoptosis. Subcutaneous xenografts of MCF7 breast cancer cells in nude mice treated with C75 showed FA synthesis inhibition, apoptosis, and inhibition of tumor growth to less than 1/8 of control volumes, without comparable toxicity in normal tissues. The data suggest that differences in intermediary metabolism render tumor cells susceptible to toxic fluxes in malonyl-CoA, both in vitro and in vivo.

Antimycobacterial activity of cerulenin and its effects on lipid biosynthesis.

Cerulenin is a potent inhibitor of fatty acid synthase (FAS) in a variety of prokaryotic and eukaryotic cells. Using a standardized mycobacterial susceptibility test, we have observed that cerulenin inhibits the growth of several species of mycobacteria, including tuberculous species such as Mycobacterium tuberculosis (H37Rv and clinical isolates) and Mycobacterium bovis BCG (hereafter called BCG), as well as several non-tuberculous species: Mycobacterium smegmatis, the Mycobacterium avium-intracellulare complex (MAC), Mycobacterium kansasii and others. All species and strains tested, including multi-drug resistant isolates of M. tuberculosis, were susceptible to cerulenin with MICs ranging from 1.5 to 12.5 mg/L. Two-dimensional thin-layer chromatography revealed different inhibition patterns of lipid synthesis between tuberculous and non-tuberculous mycobacteria. Cerulenin treatment resulted in a relative increase in phospholipids and mycolic acids in MAC and M. smegmatis, whereas in cerulenin-treated BCG, phospholipids and mycolic acids diminished relative to controls. In addition, long-chain extractable lipids (intermediate in polarity), triglycerides and glycopeptidolipids decreased with cerulenin treatment in all three species of mycobacteria tested. Qualitative changes in several of these lipid classes indicate inhibition in the synthesis of intermediate and long-chain fatty acids. Our results suggest that cerulenin's primary effect may be inhibition of intermediate and long-chain lipid synthesis, with little effect on the synthesis of other lipid classes. In addition, the BCG-specific reduction in phospholipids and mycolic acids suggests the presence of a unique cerulenin-sensitive FAS system in tuberculous mycobacteria. Since pathogenic mycobacteria produce novel long-chain fatty acids, inhibition of fatty acid synthesis offers a potential target for the development of antimycobacterial drugs.

Insulin depletion leads to adipose-specific cell death in obese but not lean mice.

Mutation of the obese gene produces obesity, hyperinsulinemia, and compensatory "overexpression" of the defective gene. As insulin activates obese gene expression, it seemed possible that hyperinsulinemia might be responsible for overexpression of the gene. To address this question we rapidly neutralized circulating insulin by injection of an insulin antibody. Unexpectedly, insulin depletion in obese (ob/ob or db/db) mice caused massive adipose RNA degradation confirmed by histological analysis to result from adipocyte cell death by a largely necrotic mechanism. This effect was not observed in lean littermates and was completely corrected by coadministration of insulin. Comparison of multiple tissues demonstrated that the effect was restricted to adipose tissue. Insulin depletion in obese mice by administration of streptozotocin also led to cell death, but this death was less extensive and appeared to be apoptotic in mechanism. Thus insulin may promote the survival side of the physiological balance between adipocyte survival and death.

Fatty acid synthase expression in endometrial carcinoma: correlation with cell proliferation and hormone receptors.

BACKGROUND: Fatty acid synthase (FAS), a biosynthetic enzyme, normally functions in the liver to convert dietary carbohydrate to fat, but it is minimally expressed in most other normal adult tissues. FAS is expressed at markedly elevated levels in subsets of human breast, ovarian, and prostate carcinomas that are associated with poor prognoses. During the menstrual cycle, the expression of FAS in the human endometrium is closely linked to the expression of the proliferation antigen Ki-67, estrogen receptor (ER), and progesterone receptor (PR). METHODS: This study reports the expression patterns of these antigens in 35 endometrial carcinomas as determined by immunohistochemical analysis. RESULTS: All cases demonstrated a close direct correlation between FAS and Ki-67 expression. Average FAS expression levels were correlated with tumor grade. Twenty-five carcinomas that were positive for ER and PR showed close correlation in expression of FAS, Ki-67, and hormone receptors. Individual tumors displayed varying degrees of heterogeneity of expression. A few well-differentiated carcinomas showed very low expression of all four antigens, similar to the antigenic profile of secretory endometrium. Nine high grade carcinomas that were negative for ER and PR also showed close correlation in expression of FAS and Ki-67 with uniformly high expression. CONCLUSIONS: These data suggest the following hypothesis: In hormone-dependent endometrial cells, FAS expression is part of the estrogen-driven cellular response that leads to proliferation; however, its linkage to proliferation is such that FAS expression is maintained in proliferating cells in endometrial carcinomas that acquire hormone independence. The use of these four antibodies as a panel may increase the diagnostic utility of ER and PR immunohistochemistry for tumor classification and prediction of the responsiveness of tumors to hormonal therapy.

Novel nuclear phosphoprotein pp32 is highly expressed in intermediate- and high-grade prostate cancer.

BACKGROUND: pp32 is a differentiation-regulated nuclear phosphoprotein that is highly expressed in many cancers, but is restricted to self-renewing and long-lived normal cell populations. During murine embryogenesis, pp32 is expressed in primitive cell populations, diminishing as tissues terminally differentiate. Functionally, pp32 confers resistance to programmed cell death and, paradoxically, inhibits transformation mediated in vitro by a broad range of oncogenes, suggesting that pp32 is a multifunctional molecule with potentially complex activities in cancer. METHODS: We studied pp32 expression in prostatic adenocarcinomas and benign prostatic hyperplasia by in situ hybridization. RESULTS: In benign prostatic tissues, moderate pp32 expression occurs only in the basal cells. This study found elevated pp32 expression in 98% (54/55) of prostatic adenocarcinomas of Gleason score > or = 5 (P < 0.0001). CONCLUSIONS: These results suggest that pp32 may be diagnostically useful and may contribute mechanistically to prostate tumor development. In comparison to other molecular alterations, increased pp32 expression is one of the most frequent events in primary prostate cancer.

Obese gene expression at in vivo levels by fat pads derived from s.c. implanted 3T3-F442A preadipocytes.

3T3-F442A preadipocytes implanted s.c. into athymic mice develop into fat pads that are indistinguishable from normal adipose tissue. Implanted preadipocytes harboring a beta-galactosidase transgene gave rise to fat pads in which almost all adipocytes expressed beta-galactosidase. This finding proved that the implanted 3T3-F442A preadipocytes, rather than endogenous preadipose cells, gave rise to the newly developed "adipose tissue." 3T3-F442A preadipocytes, when differentiated into adipocytes in cell culture, express the obese gene at an unexpectedly low level, i.e.,

Elevated expression of fatty acid synthase and fatty acid synthetic activity in colorectal neoplasia.

Expression of the primary enzyme catalyzing the synthesis of fatty acids, ie, fatty acid synthase (FAS), and ex vivo fatty acid synthetic activity were examined in colorectal epithelium and neoplasms, including the relationship to tumor progression and prognosis. Immunohistochemistry for FAS showed only faint staining of native colorectal mucosa, but increased expression was found in all sporadic adenomas (n = 18), adenomas associated with familial adenomatous polyposis (n = 7), hyperplastic polyps (n = 3), dysplasias arising in ulcerative colitis (n = 17), and colorectal carcinomas (n = 130) including 11 with contiguous adenomas. The intensity of staining was strong in 53% of carcinomas, intermediate in 38%, and weak in 9%. Activity of the fatty acid synthetic pathway measured by labeling of six surgical specimens with [U-14C]acetate was 2- to 7-fold higher in colorectal carcinomas than adjacent native mucosa (P = 0.006) and 6- to 16-fold higher than serosal fat (P = 0.01). Activity correlated with immunohistochemical expression (Spearman's rank correlation coefficient = 0.85; P < 0.001). There was no statistically significant association between patient survival and FAS staining intensity of carcinomas. Our study shows that FAS is expressed in all colorectal neoplasms and there is a concomitant increase in fatty acid synthesis. FAS may therefore represent a potential therapeutic target.

Expression of fatty acid synthase is closely linked to proliferation and stromal decidualization in cycling endometrium.

Estrogen-driven proliferative phase growth is the most rapid physiological proliferative process that occurs in the adult. The tissue growth that occurs during this phase of the menstrual cycle requires incorporation of a substantial quantity of fatty acid into the structural lipids of cell membranes. Fatty acid synthase (FAS) is the major biosynthetic enzyme required for de novo synthesis of fatty acids. In this immunohistochemical study, we have observed that human endometrium displays distinct patterns of FAS expression in the proliferative and secretory phases of the normal menstrual cycle. Proliferative endometrial glands and stroma show high FAS expression that closely correlates with expression of Ki-67, estrogen and progesterone receptors, supporting the view that FAS expression plays a role in cellular proliferation in response to estrogen. FAS expression declines during early to midsecretory phase, then reappears in decidualized stromal cells in late secretory phase as well as in the decidua of pregnancy. The second wave of FAS expression correlates with progesterone-receptor localization in the decidual cells, a finding suggesting a second induction of FAS expression in the endometrium, associated with differentiation, that may be regulated by progesterone.

Enzymes of the fatty acid synthesis pathway are highly expressed in in situ breast carcinoma.

Expression of high levels of fatty acid synthase (FAS), an important enzyme in fatty acid synthesis, has been identified in a wide variety of human carcinomas. In breast and prostate carcinoma, FAS expression appears to be associated with aggressive disease. Recent biochemical studies have demonstrated that FAS expression in cancer cells connotes activation of the entire fatty acid synthesis pathway leading to the production of palmitic acid. Here, we explore the immunohistochemical expression of FAS and human acetyl-CoA carboxylase (HACC), the rate-limiting enzyme in fatty acid synthesis, in breast cancer progression from histologically normal breast through the development of in situ duct and lobular carcinoma to infiltrating carcinoma. Both FAS and the Mr 275,000 isoform of HACC are expressed in a small subset of cells in normal breast lobules and terminal ducts. Upon development of either in situ duct or lobular carcinoma, FAS and both isoforms of HACC are expressed at higher levels and in a majority of the cells. These findings suggest that expression of the enzymes of fatty acid synthesis are frequently altered early in the progression of human breast carcinoma.

Structure of pp32, an acidic nuclear protein which inhibits oncogene-induced formation of transformed foci.

pp32 is a nuclear protein found highly expressed in normal tissues in those cells capable of self-renewal and in neoplastic cells. We report the cloning of cDNAs encoding human and murine pp32. The clones encode a 28.6-kDa protein; approximately two-thirds of the N-terminal predicts an amphipathic alpha helix containing two possible nuclear localization signals and a potential leucine zipper motif. The C-terminal third is exceptionally acidic, comprised of approximately 70% aspartic and glutamic acid residues; the predicted pI of human pp32 is 3.81. Human and murine pp32 cDNAs are 88% identical; the predicted proteins are 89% identical and 95% similar. Although the structure of pp32 is suggestive of a transcription factor, pp32 did not significantly modulate transcription of a reporter construct when fused to the Gal4 DNA-binding domain. In contrast, in cotransfection experiments, pp32 inhibited the ability of a broad assortment of oncogene pairs to transform rat embryo fibroblasts, including ras + myc, ras + jun, ras + E1a, ras + mutant p53, and E6 + E7. In related experiments, pp32 inhibited the ability of Rat 1a-myc cells to grow in soft agar, whereas it failed to affect ras-induced focus formation in NIH3T3 cells. These results suggest that pp32 may play a key role in self-renewing cell populations where it may act in the nucleus to limit their sensitivity to transformation.

pp32 overexpression induces nuclear pleomorphism in rat prostatic carcinoma cells.

Nuclear pleomorphism is an important diagnostic factor in tumour pathology. Traditionally, nuclear pleomorphism is evaluated qualitatively or semiquantitatively, often as a component of tumour grade; the molecular basis of nuclear pleomorphism, however, remains unclear. In this study, we investigated the quantitative effects on nuclear morphology of overexpressing pp32, a recently described nuclear phosphoprotein highly expressed in self-renewing and neoplastic cell populations. Assessment of Feulgen-stained transfected and control lines of AT3.1, a rat prostatic carcinoma cell line, using a computerized Cellular Image Analysis System (BD CAS-200) showed that stable overexpression of human pp32 in AT3.1 cells is accompanied by marked increases in the coefficient of variation of nuclear shape, nuclear size and chromatin textures but not in DNA content. In contrast, stable transfection with control vector, with ras, or with bcl-2 failed to affect nuclear morphology. Cell cycle analysis further showed that pp32-related increases in variation of nuclear structure manifested principally in G1. These studies suggest that pp32 plays a role either directly or indirectly in the control of nuclear shape of G1 cells.