In this paper we show that forced overexpression of a metabolic enzyme—FASN—together with expression of the AR in prostate epithelial cells facilitates the development of invasive cancers when these cells are injected orthotopically into mice and results in age-dependent PIN when expressed as a transgene in the murine prostate. We also show that FASN exerts its oncogenic effects at least in part by protecting prostate epithelial cells from apoptosis in transgenic mice and in a large series of human prostate cancers.
Prostate cancer is a leading cause of male cancer-related death, second only to lung cancer, and represents about 10% of all cancer deaths among men in the United States (45
). Dietary and lifestyle factors appear to be important risk factors for prostate cancer mortality; specifically, higher body mass index and adult weight gain are associated with an increased risk of dying from prostate cancer (20
). Dietary intervention or regulation of metabolic pathways may therefore potentially affect prostate cancer incidence and, perhaps, tumor aggressiveness.
FASN catalyzes the synthesis of palmitate from the condensation of malonyl-CoA and acetyl-CoA and plays an important role in energy homeostasis by converting excess carbon intake into fatty acids for storage. It has been shown that FASN expression is markedly increased in several human malignancies, notably breast and prostate cancer, and its overexpression in tumor tissues from patients with colon, breast, and prostate carcinomas as well as melanoma and gastrointestinal stromal tumors has been associated with a poor prognosis (3
). In addition, one-fourth of human prostate cancers have genomic amplification of FASN
). Despite these clinical association studies, to our knowledge, there has been no bona fide demonstration of FASN
oncogenicity in cultured cells or in vivo. To this end, we have demonstrated that forced overexpression of FASN
in immortalized prostate epithelial cells results in transformation of immortalized epithelial cells and in tumor formation in vivo. More importantly, when expressed as a transgene in mice, FASN
was associated with the development of prostate epithelial hyperplasia and intraepithelial neoplasia. Taken together with the previous evidence of FASN overexpression and/or of FASN
gene amplification in prostate cancer, these data provide the first evidence to our knowledge that the mere overexpression of a metabolic enzyme can result in neoplastic transformation of epithelial cells.
The biochemical and metabolic basis, as well as the biological consequences, of FASN overexpression are not well understood. The antiproliferative and proapoptotic effects of FASN inhibition have been shown in several systems (40
). In addition, functional interference, mostly by RNA interference, of enzymes that precede FASN in the fatty acid synthetic pathway, such as ATP citrate lyase and acetyl-CoA carboxylase, has been shown to result in G1
arrest and/or induction of apoptosis (49
). The reported induction of programmed cell death through FASN blockade in other studies led us to investigate inhibition of apoptosis as the predominant mechanism of oncogenic action of FASN in this study. We showed that FASN
overexpression protected cells from apoptosis via stabilization of mitochondrial membrane potential but had no effect on the extrinsic apoptotic cascade that involves Fas and Fas ligand. Importantly, we showed that castration-induced apoptosis was statistically significantly inhibited in FASN
-transgenic mice. Furthermore, the increase in ROS that resulted from treatment of LNCaP cells with FASN siRNA was reversed by supplying palmitate, the principal enzymatic product of FASN, to the culture media, suggesting that the products of de novo FASN synthesis, such as palmitate, may alter mitochondrial membranes and protect LNCaP cells from diffusion of ROS to outside the mitochondria. Phospholipids, the end product of nearly 85% of all lipids synthesized de novo by FASN in tumor cells, have been observed to end up in lipid rafts in plasma membranes (52
). Therefore, general alterations in the lipid compositions of the cellular and mitochondrial membranes may confer a selective growth advantage to cells that display increased FASN activity by inhibition of apoptosis. We previously reported (53
) that human prostate adenocarcinomas with the lowest expression of USP2a, an isopeptidase that stabilizes and prolongs the half-life of FASN (16
), also had the lowest expression of FASN and were specifically and statistically significantly associated with a cell death gene set when tested against 440 predetermined gene sets by a gene-set enrichment analysis (16
). These data together with the reciprocal correlation between low FASN expression and high apoptosis that we report here further support an inverse association between FASN levels and proapoptotic gene expression.
The transition from an androgen-dependent status to androgen independence is the key feature of lethal prostate cancers. Hormone resistance occurs as a result of a diminished apoptotic response to castration, resulting in androgen-independent disease. In addition, anticancer therapeutic regimens, including chemotherapy, radiation therapy, and immunotherapy also trigger tumor cell death through the induction of apoptosis. We showed that overexpression of FASN protein in immortalized or transformed prostate epithelial cells, which in clinical samples occurs predominantly in castration-resistant disease metastatic to bone (7
), results in resistance to apoptosis induced by chemotherapeutic regimens. Importantly, we also showed that a strong and inverse relationship exists between FASN expression and apoptosis measured by DNA fragmentation assays in a large cohort of prostate cancer patients, as was previously suggested in more limited series of patients (54
The experimental evidence we provide on the oncogenic role of FASN in the prostate suggests that pharmacological targeting of FASN might represent an effective treatment for prostate cancer. The recent description of the crystal structure of a mammalian FASN revealed an important opportunity for the discovery of new FASN-targeting drugs (56
). FASN inhibitors, such as C75 (α-methylene-γ-butyrolactone), the mycotoxin cerulenin, and their derivatives induce apoptosis in several types of cancer cells including prostate, breast, sarcoma, and melanoma and decrease the size of prostate cancer xenografts and autochthonous tumors that overexpress FASN (57
). Orlistat, an antiobesity drug approved by the Food and Drug Administration, induces apoptosis in FASN-overexpressing prostate tumors by inhibiting the thioesterase domain of FASN (15
). More recent discoveries include the novel drug GSK837149A (58
) and new beta-lactone inhibitors (59
), which target the β-ketoacyl reductase and the thioesterase domains of FASN, respectively. These advances, taken together with our data on FASN oncogenicity, suggest that pharmacological targeting of FASN may be a possible and effective means of preventing prostatic tumorigenesis as well as tumor maintenance in prostate cancers that overexpress this enzyme.
The data we show provide only a preliminary and limited explanation for the undoubtedly complex function of FASN and for the consequences of the induction of the lipogenic phenotype in prostate epithelial transformation. Specifically, the antiapoptotic activity of FASN reported here represents perhaps just one of many potential mechanisms of FASN-mediated oncogenicity. For example, increased production of fatty acids, particularly palmitate, in prostate tumor cells may result in the activation through palmitoylation of various cell signaling–related proteins such as such as, RAS, mitogen-activated protein kinases, and Wnt . Our study was limited to the antiapoptotic function of FASN. Another limitation of our study that deserves further investigation is that the models we used did not address the role played by lipid-modifying enzymes such as desaturases on FASN enzymatic products, specifically palmitate. These modifications may play an important role in alterations in cellular and mitochondrial membranes, and in turn would affect signaling and apoptotic response in cells that overexpress FASN. Finally, overexpression of FASN
as a transgene did not result in invasive adenocarcinomas. Development of age-dependent PIN but not invasive cancer in our FASN
-transgenic mouse model indicates that although increased expression of FASN is able to transform prostate cells, it is not sufficient to induce invasive prostate cancer. FASN
has indeed been found to be amplified in prostatic adenocarcinomas (13
) and overexpression of FASN protein is associated with higher stage and grade (7
), suggesting that it plays a role in prostatic tumorigenesis. However, further studies are required to precisely identify the potential additional mechanisms of action through which FASN overexpression confers oncogenicity in the prostate.
In summary, we provide evidence that FASN
, which encodes a metabolic enzyme and is overexpressed in many human tumors, is a bona fide oncogene in prostate cancer that exerts its oncogenic effect, at least in part, by inhibiting the intrinsic pathway of apoptosis. The identification of FASN-overexpressing tumors, which can be achieved in vivo by monitoring 11
C acetate uptake (60
), may thus provide prognostic and biologically relevant information in patients with advanced prostate cancer. Furthermore, we speculate that a high-fat diet and obesity may also represent a permissive background for the development of a lipogenic phenotype leading to neoplastic transformation in the prostate cancer as they do in breast cancer (10
), although data to support this possibility are lacking. More importantly, however, we suggest that FASN-overexpressing tumors may be resistant to chemotherapeutic agents that induce apoptosis and that inhibition of the FASN
gene or protein should be considered as a therapeutic strategy in prostate cancer.