Ellen De Schrijver

RNA Interference–Mediated Silencing of the Acetyl-CoA-Carboxylase-α Gene Induces Growth Inhibition and Apoptosis of Prostate Cancer Cells

Overexpression of lipogenic enzymes is a common characteristic of many cancers. Thus far, studies aimed at the exploration of lipogenic enzymes as targets for cancer intervention have focused on fatty acid synthase (FAS), the enzyme catalyzing the terminal steps in fatty acid synthesis. Chemical inhibition or RNA interference (RNAi)–mediated knockdown of FAS consistently inhibits the growth and induces death of cancer cells. Accumulation of the FAS substrate malonyl-CoA has been implicated in the mechanism of cytotoxicity of FAS inhibition. Here, using RNAi technology, we have knocked down the expression of acetyl-CoA carboxylase-α (ACC-α), the enzyme providing the malonyl-CoA substrate. Silencing of the ACC-α gene resulted in a similar inhibition of cell proliferation and induction of caspase-mediated apoptosis of highly lipogenic LNCaP prostate cancer cells as observed after FAS RNAi. In nonmalignant cells with low lipogenic activity, no cytotoxic effects of knockdown of ACC-α or FAS were observed. These findings indicate that accumulation of malonyl-CoA is not a prerequisite for cytotoxicity induced by inhibition of tumor-associated lipogenesis and suggest that in addition to FAS, ACC-α is a potential target for cancer intervention.

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.

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.

Mimicry of a cellular low energy status blocks tumor cell anabolism and suppresses the malignant phenotype

Aggressive cancer cells typically show a high rate of energy-consuming anabolic processes driving the synthesis of lipids, proteins, and DNA. Here, we took advantage of the ability of the cell-permeable nucleoside 5-aminoimidazole-4-carboxamide (AICA) riboside to increase the intracellular levels of AICA ribotide, an AMP analogue, mimicking a low energy status of the cell. Treatment of cancer cells with AICA riboside impeded lipogenesis, decreased protein translation, and blocked DNA synthesis. Cells treated with AICA riboside stopped proliferating and lost their invasive properties and their ability to form colonies. When administered in vivo, AICA riboside attenuated the growth of MDA-MB-231 tumors in nude mice. These findings point toward a central tie between energy, anabolism, and cancer and suggest that the cellular energy sensing machinery in cancer cells is an exploitable target for cancer prevention and/or therapy.

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.

Cancer Prevention by Green Tea via EGCG-Mediated Inhibition of Fatty Acid Synthase

Green tea is widely accepted to lower the risk of developing cancer, including hormone-responsive cancers, but the precise mechanism of its cancer-preventive effect is not fully understood. Recently, the green tea component epigallocatechin-3-gallate (EGCG) was demonstrated to inhibit in-vitro enzymatic activity of chicken liver fatty acid synthase (FAS), an enzyme that is frequently overexpressed in many epithelial tumors. Since chemical FAS inhibitors such as cerulenin and C75 are known to inhibit growth and to induce apoptosis of several cancer cell lines in vitro and tumor xenografts in vivo, it was investigated whether EGCG also inhibited FAS activity in cultured prostate cancer (PCA) cells in vivo and how this inhibition affected lipogenesis, cell proliferation, and cell viability. EGCG significantly inhibited FAS activity in PCA cells (with high FAS expression levels). This FAS inhibition was paralleled by decreased lipogenesis, growth inhibition, and apoptosis. In contrast, epicatechin, another closely related catechin that does not influence FAS activity, had no effect on PCA cell proliferation or survival. EGCG also inhibited FAS activity and proliferation of normal fibroblasts (with low FAS expression), but did not induce fibroblast apoptosis. Taken together, it can be concluded that EGCG efficiently inhibits FAS in cultured cells, and specifically induces apoptosis in PCA cells but not in normal fibroblasts, thereby providing interesting perspectives for using EGCG in antineoplastic therapies.

Silencing of the Fatty Acid Synthase Gene by RNA Interference Inhibits Growth and Induces Apoptosis of LNCaP Prostate Cancer Cells

A screening for androgen-regulated genes in prostate cancer (PCA) cells revealed that androgenes, apart from their well known effects on cell survival, proliferation, and differentiation, stimulate the expression of several lipogenic genes, including the gene encoding fatty acid synthase (FAS). FAS, a key enzyme in the biosynthesis of fatty acids, is markedly overexpressed in many epithelial cancers including cancer of the prostate, breast, ovary and endometrium. To gain more insight into the role of FAS in cancer cells and to explore its potential as a novel target for antineoplastic therapy, we have used the potent and highly sequence-specific technique of RNA interference (RNAi) to silence FAS in LNCaP prostate cancer cells. RNAi-mediated down-regulation of FAS expression resulted in a major decrease in the synthesis of triglycerides and of phospholipids partitioning into detergent-resistant membrane microdomains. These effects were accompanied by 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 FAS gene by RNAi significantly inhibited LNCaP cell growth and ultimately resulted in induction of apoptosis. In striking contrast with LNCaP cells, RNAi-mediated inhibition of FAS did not influence viability of nonmalignant fibroblasts. The data presented herein suggest that overexpression of FAS induced by hormones, growth factors, or other mechanisms may play an important role in cancer cell biology, and that RNA interference, particularly targeting lipogenic genes, constitutes a promising tool for the development of new cancer treatments.