RNA interference-mediated silencing of the acetyl-CoA-carboxylase-alpha 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-alpha (ACC-alpha), the enzyme providing the malonyl-CoA substrate. Silencing of the ACC-alpha 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-alpha 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-alpha is a potential target for cancer intervention.

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.

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.

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.

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.

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.