Frances B. Wheeler

Inhibition of Fatty Acid Synthase Induces Endoplasmic Reticulum Stress in Tumor Cells

Fatty acid synthase (FAS), the cellular enzyme that synthesizes palmitate, is expressed at high levels in tumor cells and is vital for their survival. Through the synthesis of palmitate, FAS primarily drives the synthesis of phospholipids in tumor cells. In this study, we tested the hypothesis that the FAS inhibitors induce endoplasmic reticulum (ER) stress in tumor cells. Treatment of tumor cells with FAS inhibitors induces robust PERK-dependent phosphorylation of the translation initiation factor eIF2alpha and concomitant inhibition of protein synthesis. PERK-deficient transformed mouse embryonic fibroblasts and HT-29 colon carcinoma cells that express a dominant negative PERK (DeltaC-PERK) are hypersensitive to FAS inhibitor-induced cell death. Pharmacologic inhibition of FAS also induces the processing of X-box binding protein-1, indicating that the IRE1 arm of the ER stress response is activated when FAS is inhibited. Induction of ER stress is further confirmed by the increased expression of the ER stress-regulated genes CHOP, ATF4, and GRP78. FAS inhibitor-induced ER stress is activated prior to the detection of caspase 3 and PARP cleavage, primary indicators of cell death, whereas orlistat-induced cell death is rescued by coincubation with the global translation inhibitor cycloheximide. Lastly, FAS inhibitors cooperate with the ER stress inducer thapsigargin to enhance tumor cell killing. These results provide the first evidence that FAS inhibitors induce ER stress and establish an important mechanistic link between FAS activity and ER function.

1-11C-Acetate as a PET Radiopharmaceutical for Imaging Fatty Acid Synthase Expression in Prostate Cancer

Although it is accepted that the metabolic fate of 1-11C-acetate is different in tumors than in myocardial tissue because of different clearance patterns, the exact pathway has not been fully elucidated. For decades, fatty acid synthesis has been quantified in vitro by the incubation of cells with 14C-acetate. Fatty acid synthase (FAS) has been found to be overexpressed in prostate carcinomas, as well as other cancers, and it is possible that imaging with 1-11C-acetate could be a marker for its expression. Methods: In vitro and in vivo uptake experiments in prostate tumor models with 1-11C-acetate were performed both with and without blocking of fatty acid synthesis with either C75, an inhibitor of FAS, or 5-(tetradecyloxy)-2-furoic acid (TOFA), an inhibitor of acetyl-CoA carboxylase (ACC). FAS levels were measured by Western blot and immunohistochemical techniques for comparison. Results: In vitro studies in 3 different prostate tumor models (PC-3, LNCaP, and 22Rv1) demonstrated blocking of 1-11C-acetate accumulation after treatment with both C75 and TOFA. This was further shown in vivo in PC-3 and LNCaP tumor-bearing mice after a single treatment with C75. A positive correlation between 1-11C-acetate uptake into the solid tumors and FAS expression levels was found. Conclusion: Extensive involvement of the fatty acid synthesis pathway in 1-11C-acetate uptake in prostate tumors was confirmed, leading to a possible marker for FAS expression in vivo by noninvasive PET.

Differential regulation of calcitonin gene-related peptide and substance P in cultured neonatal rat vagal sensory neurons ☆

Nodose (inferior vagal sensory) ganglia were removed from neonatal rats, enzymatically dispersed using neutral protease, and maintained on previously dispersed rat atriacytes. After 7-10 days in culture, calcitonin gene-related peptide (CGRP) was present in 1-3 times the molar amount of substance P (SP). The content of SP was doubled by the addition of nerve growth factor (NGF) whereas CGRP was significantly less increased by 50% or less. The addition of forskolin increased SP and CGRP levels in cultures with or without NGF by 60-80 percent. Phorbol ester (PMA) did not alter SP content but significantly raised CGRP content by 40% in NGF supplemented cultures (P less than 0.001). Corticosterone, 0.01-0.1 microM, reduced SP content by 30% independently of NGF but had no effect on CGRP. These studies demonstrate that SP in vagal sensory neurons is more sensitive than CGRP to the effects of NGF or corticosterone. Both peptides are up-regulated by presumed increases in intracellular cyclic AMP, while CGRP (or CGRP neurons) may be independently regulated by protein kinase C.

Metabolic Regulation of Invadopodia and Invasion by Acetyl-CoA Carboxylase 1 and De novo Lipogenesis

Invadopodia are membrane protrusions that facilitate matrix degradation and cellular invasion. Although lipids have been implicated in several aspects of invadopodia formation, the contributions of de novo fatty acid synthesis and lipogenesis have not been defined. Inhibition of acetyl-CoA carboxylase 1 (ACC1), the committed step of fatty acid synthesis, reduced invadopodia formation in Src-transformed 3T3 (3T3-Src) cells, and also decreased the ability to degrade gelatin. Inhibition of fatty acid synthesis through AMP-activated kinase (AMPK) activation and ACC phosphorylation also decreased invadopodia incidence. The addition of exogenous 16∶0 and 18∶1 fatty acid, products of de novo fatty acid synthesis, restored invadopodia and gelatin degradation to cells with decreased ACC1 activity. Pharmacological inhibition of ACC also altered the phospholipid profile of 3T3-Src cells, with the majority of changes occurring in the phosphatidylcholine (PC) species. Exogenous supplementation with the most abundant PC species, 34∶1 PC, restored invadopodia incidence, the ability to degrade gelatin and the ability to invade through matrigel to cells deficient in ACC1 activity. On the other hand, 30∶0 PC did not restore invadopodia and 36∶2 PC only restored invadopodia incidence and gelatin degradation, but not cellular invasion through matrigel. Pharmacological inhibition of ACC also reduced the ability of MDA-MB-231 breast, Snb19 glioblastoma, and PC-3 prostate cancer cells to invade through matrigel. Invasion of PC-3 cells through matrigel was also restored by 34∶1 PC supplementation. Collectively, the data elucidate the novel metabolic regulation of invadopodia and the invasive process by de novo fatty acid synthesis and lipogenesis.

Disruption of crosstalk between the fatty acid synthesis and proteasome pathways enhances unfolded protein response signaling and cell death

Fatty acid synthase (FASN) is the terminal enzyme responsible for fatty acid synthesis and is up-regulated in tumors of various origins to facilitate their growth and progression. Because of several reports linking the FASN and proteasome pathways, we asked whether FASN inhibitors could combine with bortezomib, the Food and Drug Administration-approved proteasome inhibitor, to amplify cell death. Indeed, bortezomib treatment augmented suboptimal FASN inhibitor concentrations to reduce clonogenic survival, which was paralleled by an increase in apoptotic markers. Interestingly, FASN inhibitors induced accumulation of ubiquinated proteins and enhanced the effects of bortezomib treatment. In turn, bortezomib increased fatty acid synthesis, suggesting crosstalk between the pathways. We hypothesized that cell death resulting from crosstalk perturbation was mediated by increased unfolded protein response (UPR) signaling. Indeed, disruption of crosstalk activated and saturated the adaptation arm of UPR signaling, including eIF2α phosphorylation, activating transcription factor 4 expression, and X-box-binding protein 1 splicing. Furthermore, although single agents did not activate the alarm phase of the UPR, crosstalk interruption resulted in activated c-Jun NH2-terminal kinase and C/EBP homologous protein-dependent cell death. Combined, the data support the concept that the UPR balance between adaptive to stress signaling can be exploited to mediate increased cell death and suggests novel applications of FASN inhibitors for clinical use. [Mol Cancer Ther 2008;7(12):3816–24]

Development of a Self-Assembled Nanoparticle Formulation of Orlistat, Nano-ORL, with Increased Cytotoxicity against Human Tumor Cell Lines

Fatty acid synthase (FASN), the enzyme that catalyzes de novo synthesis of fatty acids, is expressed in many cancer types. Its potential as a therapeutic target is well recognized, but inhibitors of FASN have not yet been approved for cancer therapy. Orlistat (ORL), an FDA-approved lipase inhibitor, is also an effective inhibitor of FASN. However, ORL is extremely hydrophobic and has low systemic uptake after oral administration. Thus, new strategies are required to formulate ORL for cancer treatment as a FASN inhibitor. Here, we report the development of a nanoparticle (NP) formulation of ORL using amphiphilic bioconjugates that are derived from hyaluronic acid (HA), termed Nano-ORL. The NPs were loaded with up to 20 wt % weight of ORL at greater than 95% efficiency. The direct inhibition of the human recombinant thioesterase domain of FASN by ORL extracted from Nano-ORL was similar to that of stock ORL. Nano-ORL demonstrated a similar ability to inhibit cellular FASN activity when compared to free ORL, as demonstrated by analysis of (14)C-acetate incorporation into lipids. Nano-ORL treatment also disrupted mitochondrial function similarly to ORL by reducing adenosine triphosphate turnover in MDA-MB-231 and LNCaP cells. Nano-ORL demonstrated increased potency compared to ORL toward prostate and breast cancer cells. Nano-ORL decreased viability of human prostate and breast cancer cell lines to 55 and 57%, respectively, while free ORL decreased viability to 71 and 79% in the same cell lines. Moreover, Nano-ORL retained cytotoxic activity after a 24 h preincubation in aqueous conditions. Preincubation of ORL dramatically reduced the efficacy of ORL as indicated by high cell viability (>85%) in both breast and prostate cell lines. These data demonstrate that NP formulation of ORL using HA-derived polymers retains similar levels of FASN, lipid synthesis, and ATP turnover inhibition while significantly improving the cytotoxic activity against cancer cell lines.

Abstract 42: Biological significance of the lipogenic enzyme acetyl-CoA carboxylase in the control of metastatic properties of murine and human transformed cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC In recent years, it has been determined that the fatty acid synthesis pathway is upregulated in several types of cancers of epithelial origin at both transcriptional and translational levels. Unlike normal cells, cancer cells rely on this pathway to provide the fatty acid precursors needed for synthesizing lipids that preferentially segregate into the liquid ordered microdomains of the plasma membrane. Acetyl-CoA carboxylase (ACC) is the ATP dependent and rate limiting step of fatty acid synthesis and catalyzes the carboxylation of acetyl-CoA to malonyl-CoA. Interestingly, overexpression of this enzyme increases with severity of breast cancer patient samples and cell lines, indicating a possible role for ACC in the metastatic process. Preliminary findings from our lab indicate that ACC is important for the proteolytic invasion and migration of transformed cells independently of cell death. Moreover, ACC is critical for formation of F-actin rich membrane structures known as podosomes, which have been implicated in the metastatic process in recent years. Restoration of podosomes in cells with ACC inhibition was achieved with the addition of the soluble fatty acid, C18:1 (oleate). In contrast, inhibition of the fatty acid synthetic enzyme, fatty acid synthase (FASN), or the Δ9 desaturase, stearoyl-CoA desaturase 1 (SCD-1), does not affect podosome formation. However, inhibition of FASN resulted in attenuated proteolytic invasion and migration of transformed cells with a phenotype similar to cells with ACC inhibition although not quite to the same degree. In addition to regulation at transcriptional and translational levels, ACC activity is also regulated by phosphorylation/dephosphorylation by AMP activated protein kinase (AMPK). Decreased ACC phosphorylation at serine 79 (S79) has been shown to correlate with increased disease severity and reduced disease free survival of lung adenocarcinoma patients. This indicates that the phosphorylation status of ACC may be an important mechanism that cancer cells manipulate to successfully achieve metastasis. Therefore, induction of phosphorylation, and inactivation of ACC, either by activation of AMPK or by direct mutation of S79 may be sufficient to reduce invasion and migration of cancer cells. Taken together, investigating ACC phosphorylation status and analyzing the resulting phenotype will advance the mechanistic understanding of how cancer cells metastasize and possibly provide new therapeutic avenues to explore in the treatment of disseminated disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 42.

Abstract A33: CD38 and Nampt regulate tumor cell metabolism through modulation of NAD+

In order to maintain high rates of proliferation, tumor cells must alter their metabolic machinery in favor of increased macromolecule synthesis and energy production. A key factor in tumor cell metabolism is Nicotinamide Adenine Dinucleotide, NAD+. NAD+ is used as a cofactor for catabolic reactions (NAD(H)), or after conversion to NADP(H), for anabolic reactions. In addition, NAD+ is utilized as part of the catalytic mechanism for several classes of enzymes including the sirtuins, PARPs, and CD38. Given the juxtaposition of NAD+ to cell metabolism and other critical cellular process we hypothesize a balance between synthesis and consumption of NAD+ is a primary metabolic determinant in cancer. In prostate cancer, the primary pathway by which NAD+ is synthesized is via the salvage pathway, which recycles nicotinamide into Nicotinamide Mononucleotide (NMN) via the rate-limiting enzyme Nicotinamide Phosphoribosyltransferase (Nampt). We determined the metabolic consequences of Nampt blockade in prostate cancer and glioblastoma (GBM) cell lines using the pharmacological inhibitor FK866. As expected, treatment with FK866 decreases the levels of NAD+/NADP+ and the NAD+ precursor nicotinamide. In relation to glycolysis, there were increases in metabolites upstream of glyceraldehyde-3-phosphate dehydrogenase, which requires NAD+ as a cofactor. Specifically, glucose is increased 1.6 fold in PC3 cells (p≤0.05), glucose-6-phosphate is decreased 1.7 fold SNB19s (p≤0.05), and both lines have decreased fructose-1-phosphate levels between 6 and11 fold (p≤0.05), respectively. To compliment these changes we measured the effects of Nampt inhibition on glycolysis in real time using a Seahorse XF extracellular flux analyzer. Nampt inhibition lowers basal glycolysis 40% (p Citation Format: Jeffrey P. Chmielewski, Frances Wheeler, Scott Cramer, Shi Lihong, Joseph Sirintrapun, Steven J. Kridel. CD38 and Nampt regulate tumor cell metabolism through modulation of NAD+. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A33.