RNA interference-mediated silencing of the fatty acid synthase gene attenuates growth and induces morphological changes and apoptosis of LNCaP prostate cancer cells.

Fatty acid synthase (FASE), a key enzyme in the biosynthesis of fatty acids, is markedly overexpressed in many human epithelial cancers, rendering it an interesting target for antineoplastic therapy. Here, using the potent and highly sequence-specific mechanism of RNA interference (RNAi), we have silenced the expression of FASE in lymph node carcinoma of the prostate (LNCaP) cells. RNAi-mediated down-regulation of FASE expression resulted in a major decrease in the synthesis of triglycerides and phospholipids and induced marked morphological changes, including a reduction in cell volume, a loss of cell-cell contacts, and the formation of spider-like extrusions. Furthermore, silencing of the FASE gene by RNAi significantly inhibited LNCaP cell growth and ultimately resulted in induction of apoptosis. Importantly and in striking contrast with LNCaP cells, RNAi-mediated inhibition of FASE did not influence growth rate or viability of nonmalignant cultured skin fibroblasts. These data indicate that RNAi opens new avenues toward the study of the role of FASE overexpression in tumor cells and provides an interesting and selective alternative to chemical FASE inhibitors in the development of antineoplastic therapy.

Role of the phosphatidylinositol 3'-kinase/PTEN/Akt kinase pathway in the overexpression of fatty acid synthase in LNCaP prostate cancer cells.

One of the most common molecular changes in cancer cells is the overexpression of fatty acid synthase (FAS), a key metabolic enzyme catalyzing the terminal steps in the synthesis of long chain saturated fatty acids. As part of our efforts to elucidate the mechanisms responsible for FAS overexpression, we have addressed the question whether overexpression of FAS may be linked to the frequently observed inactivation of PTEN and subsequent activation of the phosphatidylinositol 3'-kinase (PI3k) pathway. Using LNCaP prostate cancer cells as an experimental paradigm of FAS-overexpressing PTEN-null cancer cells, we demonstrate that LY294002, an inhibitor of the PI3k pathway causes a dramatic decrease in FAS protein expression. Smaller but still substantial effects are seen at the FAS mRNA level and at the level of transcriptional activity of FAS promoter-reporter constructs. Consistent with these findings, reintroduction of PTEN results in decreased levels of FAS expression in a manner that is dependent on its lipid phosphatase activity. In support of a role for Akt/protein kinase B as a downstream effector, cotransfection of constitutively active Akt1/protein kinase B alpha abrogates the inhibitory effects of PTEN expression and restores FAS promoter activity. Taken together, these results demonstrate that inactivation of PTEN and subsequent activation of the PI3k/Akt kinase pathway may play an important role in the overexpression of the FAS protein in cancer cells.

Androgens stimulate coordinated lipogenic gene expression in normal target tissues in vivo.

In prostate cancer cell lines in culture androgens cause a marked and coordinated upregulation of the expression of several lipogenic genes. Here, using castrated male Wistar rats as an experimental paradigm, we investigated whether coordinated androgen stimulation of lipogenic gene expression represents a more general physiological regulation in non-cancerous androgen-responsive cells as well. In typical target tissues for androgen action such as the ventral prostate and the lacrimal gland, androgen deprivation resulted in a marked reduction in the mRNA and protein levels of genes involved in fatty acid (fatty acid synthase and acetyl-CoA-carboxylase) and cholesterol synthesis (HMG-CoA-reductase and farnesyl diphosphate synthase). Readministration of testosterone immediately following orchidectomy restored the expression of all four genes. Substitution of testosterone by the non-aromatizable androgen dihydrotestosterone gave rise to comparable changes in the mRNA and protein levels of the lipogenic genes under investigation, confirming the involvement of the androgen receptor in the observed effects. In support of the coordinate nature of this regulation, androgen-induced upregulation of lipogenic gene expression is accompanied by an increase in the nuclear content of SREBP, a key lipogenic transcription factor. Taken together, these findings provide evidence for a coordinate regulation of lipogenic gene expression not only in prostate cancer cell lines in culture but also in non-cancerous androgen-responsive tissues in vivo.

Androgens, lipogenesis and prostate cancer.

Both experimental and epidemiological data indicate that androgens are among the main factors controlling the development, maintenance and progression of prostate cancer. Identifying the genes that are regulated by androgens represents a major step towards the elucidation of the mechanisms underlying the impact of androgens on prostate cancer cell biology and is an attractive approach to find novel targets for prostate cancer therapy. Among the genes that have been identified thus far, several genes encode lipogenic enzymes. Studies aimed at the elucidation of the mechanisms underlying androgen regulation of lipogenic genes revealed that androgens coordinately stimulate the expression of these genes through interference with the molecular mechanism controlling activation of sterol regulatory element-binding proteins (SREBPs), lipogenic transcription factors governing cellular lipid homeostasis. The resulting increase in lipogenesis serves the synthesis of key membrane components (phospholipids, cholesterol) and is a major hallmark of cancer cells. Pharmacologic inhibition of lipogenesis or RNA-interference-mediated down-regulation of key lipogenic genes induces apoptosis in cancer cell lines and reduces tumor growth in xenograft models. While increased lipogenesis is already found in the earliest stages of cancer development (PIN) and initially is androgen-responsive it persists or re-emerges with the development of androgen-independent cancer, indicating that lipogenesis is a fundamental aspect of prostate cancer cell biology and is a potential target for chemoprevention and for antineoplastic therapy in advanced prostate cancer.

Coactivation of an endogenous progesterone receptor by TIF2 in COS-7 cells.

Transfection experiments, a powerful tool to study the function of steroid hormone receptors and their coregulators, are often performed in COS-7 cells, because of high transfection efficiencies and expression levels. Here we report on the presence in COS-7 cells of an endogenous steroid hormone receptor, which is highly responsive to progesterone and the synthetic steroids R1881 and ORG2058, but not to 5 alpha-DHT. A 10-fold excess of the progesterone antagonist RU486 abolishes the stimulation by progesterone, while cotransfection with the coactivator TIF2 increases its activity 6- to 7-fold. A comparison of the ligand specificity with transfected androgen or progesterone receptors indicates that the endogenous receptor is a progesterone receptor. Its presence is confirmed by steroid-binding experiments, RT-PCR and Northern blot analysis. Consequently, progesterone receptor function may be studied conveniently in COS-7 cells without cotransfection of receptor, but the endogenous receptor may interfere in studies of ligand specificity and coactivation of cotransfected receptors.

Epigallocatechin-3-gallate is a potent natural inhibitor of fatty acid synthase in intact cells and selectively induces apoptosis in prostate cancer cells.

Chemical inhibitors of fatty acid synthase (FAS) inhibit growth and induce apoptosis in several cancer cell lines in vitro and in tumor xenografts in vivo. Recently the green tea component epigallocatechin-3-gallate (EGCG) was shown to act as a natural inhibitor of FAS in chicken liver extracts. Here we investigated whether EGCG inhibits FAS activity in cultured prostate cancer cells and how this inhibition affects endogenous lipid synthesis, cell proliferation and cell viability. The high levels of FAS activity in LNCaP cells were dose-dependently inhibited by EGCG and this inhibition was paralleled by decreased endogenous lipid synthesis, inhibition of cell growth and induction of apoptosis. In contrast, epicatechin (EC), another closely related green tea polyphenolic compound, which does not inhibit FAS, had no effect on LNCaP cell growth or viability. Treatment of nonmalignant cells with low levels of FAS activity (fibroblasts) with EGCG led to a decrease in growth rate but not to induction of apoptosis. These data indicate that EGCG inhibits FAS activity as efficiently as presently known synthetic inhibitors and selectively causes apoptosis in LNCaP cells but not in nontumoral fibroblasts. These findings establish EGCG as a potent natural inhibitor of FAS in intact cells and strengthen the molecular basis for the use of EGCG as a chemopreventive and therapeutic antineoplastic agent.

Overexpression of fatty acid synthase is an early and common event in the development of prostate cancer.

The expression of fatty acid synthase (FAS), a key lipogenic enzyme and potential target for antineoplastic therapy, was analyzed in 87 frozen needle biopsies of prostate cancer using a highly sensitive immunohistochemical detection technique (Envision). In comparison to normal or benign, hyperplastic glandular structures, which were all negative for FAS staining, immunohistochemical signal was evident in 24/25 low grade prostatic epithelial neoplasia (PIN) lesions, in 26/26 high grade PIN lesions and in 82/87 invasive carcinomas. Staining intensity tended to increase from low grade to high grade PIN to invasive carcinoma. Cancers with a high FAS expression had an overall high proliferative index. No correlation was found between FAS expression and lipid accumulation. These findings indicate that increased FAS expression is one of the earliest and most common events in the development of prostate cancer, suggesting that FAS may be used as a general prostate cancer marker and that antineoplastic therapy based on FAS inhibition may be an option for chemoprevention or curative treatment for nearly all prostate cancers.

Identification of an androgen response element in intron 8 of the sterol regulatory element-binding protein cleavage-activating protein gene allowing direct regulation by the androgen receptor.

Sterol regulatory element-binding proteins (SREBPs) are transcription regulators that play a pivotal role in intracellular lipid homeostasis. They are synthesized as inactive precursor proteins in the endoplasmic reticulum, where they are retained by SREBP cleavage-activating protein (SCAP), a sterol sensing protein that in turn is linked to a retention protein complex. Low intracellular sterol concentrations weaken the interaction of SCAP with its retention proteins and allow translocation of the SREBP.SCAP complex to the Golgi compartment where SREBP is proteolytically cleaved and activated. Previous studies on the mechanisms by which androgens provoke a coordinated activation of lipogenic pathways in prostate cancer cells have suggested an alternative pathway of activation in which androgens increase the expression of SCAP and favor translocation of the SREBP.SCAP complex to the Golgi apparatus by disturbing the balance between SCAP and its retention proteins. Here we show that the SCAP gene contains an androgen-responsive region located in intron 8. This region interacts directly with the androgen receptor and confers androgen responsiveness to promoter-reporter constructs transfected in LNCaP cells. It contains a noncanonical androgen response element GGAAGAaaaTGTACC that interacts not only with the androgen receptor but also with the glucocorticoid receptor and that also confers glucocorticoid responsiveness. The identification of a steroid response element in intron 8 of the SCAP gene further supports the contention that SCAP is a direct target for steroid hormone action.

A Sertoli cell-selective knockout of the androgen receptor causes spermatogenic arrest in meiosis.

Androgens control spermatogenesis, but germ cells themselves do not express a functional androgen receptor (AR). Androgen regulation is thought to be mediated by Sertoli and peritubular myoid cells, but their relative roles and the mechanisms involved remain largely unknown. Using Cre/loxP technology, we have generated mice with a ubiquitous knockout of the AR as well as mice with a selective AR knockout in Sertoli cells (SC) only. Mice with a floxed exon 2 of the AR gene were crossed with mice expressing Cre recombinase ubiquitously or selectively in SC (under control of the anti-Müllerian hormone gene promoter). AR knockout males displayed a complete androgen insensitivity phenotype. Testes were located abdominally, and germ cell development was severely disrupted. In contrast, SC AR knockout males showed normal testis descent and development of the male urogenital tract. Expression of the homeobox gene Pem, which is androgen-regulated in SC, was severely decreased. Testis weight was reduced to 28% of that in WT littermates. Stereological analysis indicated that the number of SC was unchanged, whereas numbers of spermatocytes, round spermatids, and elongated spermatids were reduced to 64%, 3%, and 0% respectively of WT. These changes were associated with increased germ cell apoptosis and grossly reduced expression of genes specific for late spermatocyte or spermatid development. It is concluded that cell-autonomous action of the AR in SC is an absolute requirement for androgen maintenance of complete spermatogenesis, and that spermatocyte/spermatid development/survival critically depends on androgens.

Fatty acid synthase drives the synthesis of phospholipids partitioning into detergent-resistant membrane microdomains.

Fatty acid synthase (FAS) is a key metabolic enzyme catalyzing the synthesis of long-chain saturated fatty acids. It plays a central role in the production of surfactant in fetal lungs, in the supply of fatty components of milk, and in the conversion and storage of energy in liver and adipose tissue. Remarkably high levels of FAS expression are found in the majority of human epithelial cancers. As the role of FAS in cancer cells remains largely unknown, we have initiated studies to assess the fate of newly synthesized lipids in cancer cells and have estimated the contribution of FAS to the synthesis of specific lipid classes by treating the cells with small interfering RNAs targeting FAS. Here, we show that in cancer cells FAS plays a major role in the synthesis of phospholipids partitioning into detergent-resistant membrane microdomains. These are raft-aggregates implicated in key cellular processes including signal transduction, intracellular trafficking, cell polarization, and cell migration. These findings reveal a novel role for FAS, provide important new insights into the otherwise poorly understood mechanisms underlying the control of lipid composition of membrane microdomains, and point to a link between FAS overexpression and dysregulation of membrane composition and functioning in tumor cells.