Design and synthesis of a series of bioavailable fatty acid synthase (FASN) KR domain inhibitors for cancer therapy.

We designed and synthesized a new series of fatty acid synthase (FASN) inhibitors with potential utility for the treatment of cancer. Extensive SAR studies led to highly active FASN inhibitors with good cellular activity and oral bioavailability, exemplified by compound 34. Compound 34 is a potent inhibitor of human FASN (IC50 = 28 nM) that effectively inhibits proliferation of A2780 ovarian cells (IC50 = 13 nM) in lipid-reduced serum (LRS). This cellular activity can be rescued by addition of palmitate, consistent with an on-target effect. Compound 34 is also active in many other cell types, including PC3M (IC50 = 25 nM) and LnCaP-Vancouver prostate cells (IC50 = 66 nM), and is highly bioavailable (F 61%) with good exposure after oral administration. In a pharmacodynamics study in H460 lung xenograft-bearing mice, oral treatment with compound 34 results in elevated tumor levels of malonyl-CoA and decreased tumor levels of palmitate, fully consistent with the desired target engagement.

Selective inhibition of intestinal guanosine 3',5'-cyclic monophosphate signaling by small-molecule protein kinase inhibitors.

The guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase II (cGKII) serine/threonine kinase relays signaling through guanylyl cyclase C (GCC) to control intestinal fluid homeostasis. Here, we report the discovery of small-molecule inhibitors of cGKII. These inhibitors were imidazole-aminopyrimidines, which blocked recombinant human cGKII at submicromolar concentrations but exhibited comparatively little activity toward the phylogenetically related protein kinases cGKI and cAMP-dependent protein kinase (PKA). Whereas aminopyrimidyl motifs are common in protein kinase inhibitors, molecular modeling of these imidazole-aminopyrimidines in the ATP-binding pocket of cGKII indicated an unconventional binding mode that directs their amine substituent into a narrow pocket delineated by hydrophobic residues of the hinge and the αC-helix. Crucially, this set of residues included the Leu-530 gatekeeper, which is not conserved in cGKI and PKA. In intestinal organoids, these compounds blocked cGKII-dependent phosphorylation of the vasodilator-stimulated phosphoprotein (VASP). In mouse small intestinal tissue, cGKII inhibition significantly attenuated the anion secretory response provoked by the GCC-activating bacterial heat-stable toxin (STa), a frequent cause of infectious secretory diarrhea. In contrast, both PKA-dependent VASP phosphorylation and intestinal anion secretion were unaffected by treatment with these compounds, whereas experiments with T84 cells indicated that they weakly inhibit the activity of cAMP-hydrolyzing phosphodiesterases. As these protein kinase inhibitors are the first to display selective inhibition of cGKII, they may expedite research on cGMP signaling and may aid future development of therapeutics for managing diarrheal disease and other pathogenic syndromes that involve cGKII.

Cancer cells differentially activate and thrive on de novo lipid synthesis pathways in a low-lipid environment.

Increased lipogenesis is a hallmark of a wide variety of cancers and is under intense investigation as potential antineoplastic target. Although brisk lipogenesis is observed in the presence of exogenous lipids, evidence is mounting that these lipids may adversely affect the efficacy of inhibitors of lipogenic pathways. Therefore, to fully exploit the therapeutic potential of lipid synthesis inhibitors, a better understanding of the interrelationship between de novo lipid synthesis and exogenous lipids and their respective role in cancer cell proliferation and therapeutic response to lipogenesis inhibitors is of critical importance. Here, we show that the proliferation of various cancer cell lines (PC3M, HepG2, HOP62 and T24) is attenuated when cultured in lipid-reduced conditions in a cell line-dependent manner, with PC3M being the least affected. Interestingly, all cell lines--lipogenic (PC3M, HepG2, HOP62) as well as non-lipogenic (T24)--raised their lipogenic activity in these conditions, albeit to a different degree. Cells that attained the highest lipogenic activity under these conditions were best able to cope with lipid reduction in term of proliferative capacity. Supplementation of the medium with very low density lipoproteins, free fatty acids and cholesterol reversed this activation, indicating that the mere lack of lipids is sufficient to activate de novo lipogenesis in cancer cells. Consequently, cancer cells grown in lipid-reduced conditions became more dependent on de novo lipid synthesis pathways and were more sensitive to inhibitors of lipogenic pathways, like Soraphen A and Simvastatin. Collectively, these data indicate that limitation of access to exogenous lipids, as may occur in intact tumors, activates de novo lipogenesis is cancer cells, helps them to thrive under these conditions and makes them more vulnerable to lipogenesis inhibitors. These observations have important implications for the design of new antineoplastic strategies targeting the cancer cell's lipid metabolism.

Atypical plasma lipid profile in cancer patients: cause or consequence?

The aberrant blood lipoprotein levels in cancer patients are reported to be associated with cancer risk and mortality incidents however, there are several discrepancies in the previous reports. Hence the clinical usefulness of plasma/serum levels in risk stratification of a variety of cancers remains elusive. The present review highlights and compiles findings from different research groups regarding association of plasma lipoprotein levels with the risk of developing various types of cancer. We will discuss some prospective underlying mechanisms for this reported association. In addition to that the potential roles of plasma lipids in promoting carcinogenesis will be conferred.

Lipogenesis and lipolysis: the pathways exploited by the cancer cells to acquire fatty acids.

One of the most important metabolic hallmarks of cancer cells is enhanced lipogenesis. Depending on the tumor type, tumor cells synthesize up to 95% of saturated and mono-unsaturated fatty acids (FA) de novo in spite of sufficient dietary lipid supply. This lipogenic conversion starts early when cells become cancerous and further expands as the tumor cells become more malignant. It is suggested that activation of FA synthesis is required for carcinogenesis and for tumor cell survival. These observations suggest that the enzymes involved in FA synthesis would be rational therapeutic targets for cancer treatment. However, several recent reports have shown that the anti-tumor effects, following inhibition of endogenous FA synthesis in cancer cell lines may be obviated by adding exogenous FAs. Additionally, high intake of dietary fat is reported to be a potential risk factor for development and poor prognosis for certain cancers. Recently it was reported that breast and liposarcoma tumors are equipped for both de novo fatty acid synthesis pathway as well as LPL-mediated extracellular lipolysis. These observations indicate that lipolytically acquired FAs may provide an additional source of FAs for cancer. This review focuses on our current understanding of lipogenic and lipolytic pathways in cancer cell progression.

ATP-citrate lyase: a key player in cancer metabolism.

ATP-citrate lyase (ACLY) is a cytosolic enzyme that catalyzes the generation of acetyl CoA from citrate. Acetyl CoA is a vital building block for the endogenous biosynthesis of fatty acids and cholesterol and is involved in isoprenoid-based protein modifications. Acetyl CoA is also required for acetylation reactions that modify proteins, such as histone acetylation. ACLY is upregulated or activated in several types of cancers, and its inhibition is known to induce proliferation arrest in cancer cells both in vitro and in vivo. The present review highlights current knowledge about the role of ACLY in cancer cells, with special reference to the different pathways that are linked by ACLY.

ATP citrate lyase knockdown induces growth arrest and apoptosis through different cell- and environment-dependent mechanisms.

ATP citrate lyase (ACLY) is a cytosolic enzyme that catalyzes generation of acetyl-CoA, which is a vital building block for fatty acid, cholesterol, and isoprenoid biosynthesis. ACLY is upregulated in several types of cancer, and its inhibition induces proliferation arrest in certain cancer cells. As ACLY is involved in several pathways, its downregulation may affect multiple processes. Here, we have shown that short hairpin RNA-mediated ACLY silencing in cell lines derived from different types of cancers induces proliferation, cell-cycle arrest, and apoptosis. However, this antiproliferative effect of ACLY knockdown was observed only when cells were cultivated under lipid-reduced growth conditions. Proliferation arrest induced by ACLY silencing was partially rescued by supplementing the media with fatty acids and/or cholesterol. This indicates that the ACLY knockdown-mediated growth arrest might be the result of either fatty acid or cholesterol starvation or both. In the absence of ACLY, the cancer cells displayed elevated expression of sterol regulatory element binding protein-regulated downstream genes involved in de novo fatty acid and cholesterol biosynthesis. Furthermore, ACLY suppression resulted in elevated expression of acyl-CoA synthetase short-chain family member 2 (ACSS2), an enzyme that also produces acetyl-CoA using acetate as a substrate. Acetate supplementation partially rescued the cancer cells from ACLY suppression-induced proliferation arrest. We also observed that the absence of ACLY enhanced ACSS2-dependent lipid synthesis. These findings provide new insights into the role of ACLY in cancer cell growth and give critical information about the effects of ACLY silencing on different pathways. This information is crucial in understanding the possible application of ACLY inhibition in cancer therapeutics.

ATP-citrate lyase: a mini-review.

ATP-citrate lyase (ACLY) is a cytosolic enzyme that catalyzes generation of acetyl-CoA from citrate. Acetyl-CoA is a vital building block for the endogenous biosynthesis of fatty acids and cholesterol and is involved in isoprenoid-based protein modifications. Acetyl-CoA is also required for acetylation reactions that modify proteins such as histone acetylation. In the present review some of the known features of ACLY such as tissue distribution, subcellular localization, enzymatic properties, gene regulation and associated physiological conditions are highlighted.

De novo lipogenesis protects cancer cells from free radicals and chemotherapeutics by promoting membrane lipid saturation.

Activation of de novo lipogenesis in cancer cells is increasingly recognized as a hallmark of aggressive cancers and has been implicated in the production of membranes for rapid cell proliferation. In the current report, we provide evidence that this activation has a more profound role. Using a mass spectrometry-based phospholipid analysis approach, we show that clinical tumor tissues that display the lipogenic phenotype show an increase in the degree of lipid saturation compared with nonlipogenic tumors. Reversal of the lipogenic switch in cancer cells by treatment with the lipogenesis inhibitor soraphen A or by targeting lipogenic enzymes with small interfering RNA leads to a marked decrease in saturated and mono-unsaturated phospholipid species and increases the relative degree of polyunsaturation. Because polyunsaturated acyl chains are more susceptible to peroxidation, inhibition of lipogenesis increases the levels of peroxidation end products and renders cells more susceptible to oxidative stress-induced cell death. As saturated lipids pack more densely, modulation of lipogenesis also alters lateral and transversal membrane dynamics as revealed by diffusion of membrane-targeted green fluorescent protein and by the uptake and response to doxorubicin. These data show that shifting lipid acquisition from lipid uptake toward de novo lipogenesis dramatically changes membrane properties and protects cells from both endogenous and exogenous insults. These findings provide important new insights into the role of de novo lipogenesis in cancer cells, and they provide a rationale for the use of lipogenesis inhibitors as antineoplastic agents and as chemotherapeutic sensitizers.

Downregulation of two novel genes in Sl/Sld and W(LacZ)/Wv mouse jejunum.

Interstitial cells of Cajal (ICC) are the so-called pacemaker cells of the gut. W(LacZ)/Wv and Sl/Sld mice lack ICC surrounding the myenteric plexus (MP) in the jejunum. We compared the gene expression profile of wild type (WT) and W(LacZ)/Wv and Sl/Sld mice using suppression subtractive hybridization (SSH), generating a cDNA library of 1303 clones from which 48 unique sequences were differentially expressed with Southern blot. Among them, we identified heme oxygenase2, TROY, and phospholamban in ICC using immunohistochemistry. Using RT-qPCR, c-Kit and two new transcripts Dithp and prenylcysteine oxidase1 were significantly lower expressed in Sl/Sld and W(LacZ)/Wv versus WT. Prenylcysteine oxidase1 appeared cytotoxic for COS-7 cells and was highly expressed in liver while Dithp was mainly expressed in small intestine. The combination of SSH, Southern blot, RT-qPCR, and immunohistochemistry turned out to be a useful approach to identify rarely expressed genes and genes with small differences in expression.

Subtractive hybridization unravels a role for the ion cotransporter NKCC1 in the murine intestinal pacemaker.

In the small intestine, interstitial cells of Cajal (ICC) surrounding the myenteric plexus generate the pacemaking slow waves that are essential for an efficient intestinal transit. The underlying molecular mechanisms of the slow wave are poorly known. Our aim was to identify ICC-specific genes and their function in the mouse jejunum. Suppression subtractive hybridization using two independent ICC-deficient mouse models identified 56 genes putatively downregulated in the muscularis propria compared with wild-type littermates. Differential expression was confirmed by real-time quantitative PCR for the tyrosine kinase receptor KIT, the established marker for ICC, and for the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1). Immunoreactivity for NKCC1 was detected in myenteric ICC but not in the ICC population located at the deep muscular plexus. NKCC1 was also expressed in enteric neurons and mucosal crypts. Bumetanide, an NKCC1 inhibitor, reversibly affected the shape, amplitude, and frequency of the slow waves. Similar alterations were observed in NKCC1 knockout mice. These data support the hypothesis that NKCC1 expressed in myenteric ICC is involved in the mechanism of slow waves in the murine jejunum.

WZsGreen/+: a new green fluorescent protein knock-in mouse model for the study of KIT-expressing cells in gut and cerebellum.

In the small intestine, interstitial cells of Cajal (ICC) surrounding the myenteric plexus generate the pacemaking slow waves that are essential for an efficient intestinal transit. The underlying molecular mechanisms of the slow wave are poorly known. KIT is currently the sole practical marker for ICC. Attempts to purify living ICC have so far largely failed, due to the loss of the KIT epitope during enzymatic dissociation. Aiming to identify and isolate living ICC, we designed a knock-in strategy to express a fluorescent tag in KIT-expressing cells by inserting the sequence of the novel green fluorescent protein ZsGreen into the first exon of the c-Kit gene, creating a null allele called W(ZsGreen). In the gastrointestinal tract of heterozygous W(ZsGreen)/+ mice, tiny ZsGreen fluorescent dots were observed in all KIT-expressing ICC populations, with exception of ICC at the deep muscular plexus in small intestine. During development of the gastrointestinal tract, ZsGreen expression followed KIT expression in a spatiotemporal way. Stellate and basket KIT-expressing cells in the molecular layer of the cerebellum also exhibited ZsGreen dots, whereas no ZsGreen was detected in skin, testis, and bone marrow. ZsGreen dot-containing intestinal cells could be isolated from jejunum and maintained alive in culture for at least 3 days. ZsGreen is a suitable alternative to EGFP in transgenic animals. The novel W(ZsGreen)/+ model reported here appears to be a promising tool for live studies of KIT-expressing cells in the gastrointestinal tract and cerebellum and for the further analysis of pacemaker mechanisms.