Amanda L. Davis

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.

Living Shorelines: Coastal Resilience with a Blue Carbon Benefit

Living shorelines are a type of estuarine shoreline erosion control that incorporates native vegetation and preserves native habitats. Because they provide the ecosystem services associated with natural coastal wetlands while also increasing shoreline resilience, living shorelines are part of the natural and hybrid infrastructure approach to coastal resiliency. Marshes created as living shorelines are typically narrow (< 30 m) fringing marshes with sandy substrates that are well flushed by tides. These characteristics distinguish living shorelines from the larger meadow marshes in which most of the current knowledge about created marshes was developed. The value of living shorelines for providing both erosion control and habitat for estuarine organisms has been documented but their capacity for carbon sequestration has not. We measured carbon sequestration rates in living shorelines and sandy transplanted Spartina alterniflora marshes in the Newport River Estuary, North Carolina. The marshes sampled here range in age from 12 to 38 years and represent a continuum of soil development. Carbon sequestration rates ranged from 58 to 283 g C m-2 yr-1 and decreased with marsh age. The pattern of lower sequestration rates in older marshes is hypothesized to be the result of a relative enrichment of labile organic matter in younger sites and illustrates the importance of choosing mature marshes for determination of long-term carbon sequestration potential. The data presented here are within the range of published carbon sequestration rates for S. alterniflora marshes and suggest that wide-scale use of the living shoreline approach to shoreline management may come with a substantial carbon benefit.

The podosome marker protein Tks5 regulates macrophage invasive behavior

Tks5 is a Src substrate and adaptor protein previously recognized for its regulation of cancer cell invasion through modulation of specialized adhesion structures called podosomes/invadopodia. Here we show for the first time that Tks5 localizes to the podosomes of primary macrophages, and that Tks5 protein levels increase concurrently with podosome deposition during the differentiation of monocytes into macrophages. Similar results are reported for model THP‐1 cells, which differentiate into macrophages and form proteolytically active podosomes in response to a PKC signaling agonist (PMA) and with sensitivity to a PKC inhibitor (bisindolylmaleimide). Genetic manipulation of Tks5 expression (silencing and overexpression) in stable THP‐1 cell lines does not independently alter this macrophage differentiation process. Nor do these cells lose the ability to focalize F‐actin and its accessory proteins into podosome‐like structures following PMA treatment. However, Tks5 directly controls podosome‐associated gelatin degradation and invasion through collective changes in adhesion, chemotaxis, and the expression/proteolytic activity of MMP9. The Src family kinase‐dependent phosphorylation of Tks5 is also implicated in the regulation of THP‐1 macrophage invasive behavior. These results therefore define a previously unappreciated function of Tks5 signaling specific to the functional attributes of the macrophage podosome in adhesion, motility, and extracellular matrix‐remodeling.

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.

Combination Treatment with Orlistat-Containing Nanoparticles and Taxanes Is Synergistic and Enhances Microtubule Stability in Taxane-Resistant Prostate Cancer Cells

Taxane-based therapy provides a survival benefit in patients with metastatic prostate cancer, yet the median survival is less than 20 months in this setting due in part to taxane-associated resistance. Innovative strategies are required to overcome chemoresistance for improved patient survival. Here, NanoOrl, a new experimental nanoparticle formulation of the FDA-approved drug, orlistat, was investigated for its cytotoxicity in taxane-resistant prostate cancer utilizing two established taxane-resistant (TxR) cell lines. Orlistat is a weight loss drug that inhibits gastric lipases, but is also a potent inhibitor of fatty acid synthase (FASN), which is overexpressed in many types of cancer. NanoOrl was also investigated for its potential to synergize with taxanes in TxR cell lines. Both orlistat and NanoOrl synergistically inhibited cell viability when combined with paclitaxel, docetaxel, and cabazitaxel in PC3-TxR and DU145-TxR cells, yet these combinations were also additive in parental lines. We observed synergistic levels of apoptosis in TxR cells treated with NanoOrl and docetaxel in combination. Mechanistically, the synergy between orlistat and taxanes was independent of effects on the P-glycoprotein multidrug resistance protein, as determined by an efflux activity assay. On the other hand, immunoblot and immunofluorescence staining with an anti-detyrosinated tubulin antibody demonstrated that enhanced microtubule stability was induced by combined NanoOrl and docetaxel treatment in TxR cells. Furthermore, TxR cells exhibited higher lipid synthesis, as demonstrated by 14C-choline incorporation that was abrogated by NanoOrl. These results provide a strong rationale to assess the translational potential of NanoOrl to overcome taxane resistance. Mol Cancer Ther; 16(9); 1819–30. ©2017 AACR.

SPARC Inhibits Metabolic Plasticity in Ovarian Cancer

The tropism of ovarian cancer (OvCa) to the peritoneal cavity is implicated in widespread dissemination, suboptimal surgery, and poor prognosis. This tropism is influenced by stromal factors that are not only critical for the oncogenic and metastatic cascades, but also in the modulation of cancer cell metabolic plasticity to fulfill their high energy demands. In this respect, we investigated the role of Secreted Protein Acidic and Rich in Cysteine (SPARC) in metabolic plasticity of OvCa. We used a syngeneic model of OvCa in Sparc-deficient and proficient mice to gain comprehensive insight into the paracrine effect of stromal-SPARC in metabolic programming of OvCa in the peritoneal milieu. Metabolomic and transcriptomic profiling of micro-dissected syngeneic peritoneal tumors revealed that the absence of stromal-Sparc led to significant upregulation of the enzymes involved in glycolysis, TCA cycle, and mitochondrial electron transport chain (ETC), and their metabolic intermediates. Absence of stromal-Sparc increased reactive oxygen species and perturbed redox homeostasis. Recombinant SPARC exerted a dose-dependent inhibitory effect on glycolysis, mitochondrial respiration, ATP production and ROS generation. Comparative analysis with human tumors revealed that SPARC-regulated ETC-signature inversely correlated with SPARC transcripts. Targeting mitochondrial ETC by phenformin treatment of tumor-bearing Sparc-deficient and proficient mice mitigated the effect of SPARC-deficiency and significantly reduced tumor burden, ROS, and oxidative tissue damage in syngeneic tumors. In summary, our findings provide novel insights into the role of SPARC in regulating metabolic plasticity and bioenergetics in OvCa, and shines light on its potential therapeutic efficacy.

Trimming the fat in non-small cell lung cancer: a new small molecule inhibitor of acetyl-CoA carboxylase to target fatty acid synthesis

In the October 2016 issue of Nature Medicine, Svensson et al . demonstrate that acetyl-CoA carboxylase 1 (ACC1) is required for non-small cell lung cancer (NSCLC) growth in preclinical models and treatment with an allosteric ACC1 inhibitor, ND-646, suppresses NSCLC through fatty acid synthesis inhibition (1). This study provides answers to questions the cancer metabolism field has asked since the discovery that enzymes within the fatty acid synthesis pathway are overexpressed in cancer (2). Is de novo fatty acid synthesis required for tumors to develop and progress? And can enzymes in the pathway be successfully targeted for therapy?

Abstract A21: ACC1 is required for prostate cancer initiation, but not progression?

The majority of cancers undergo metabolic alterations during initiation and progression of disease. Increased de novo lipogenesis is recognized as metabolic mark of cancer cells. The enzymes responsible for de novo fatty acid synthesis are often overexpressed in cancer, indicating that these enzymes are potential and promising targets for novel therapies. Acetyl CoA Carboxylase 1 (ACC1) is a cytosolic enzyme which catalyzes the rate limiting step of de novo fatty acid synthesis through the ATP and biotin-dependent carboxylation of acetyl-CoA to malonyl-CoA. In doing so, it regulates both fatty acid and acetate metabolism. Both pharmacological inhibition and siRNA knockdown of ACC1 have been demonstrated to deny prostate cancer cells of necessary amounts of fatty acids needs to proliferate, resulting in selective toxicity. Although previous research indicates ACC1 and the entire process of fatty acid metabolism to be promising avenues for novel therapy development, much remains to be understood. To address this, we have generated mice with prostate-specific deletion of exon 22 of the Acetyl CoA Carboxylase 1 (ACCL/L) gene by breeding with Probasin-Cre mice. Loss of ACC1 activity does not affect prostate development as survival, prostate weight, histology, and breeding were similar between ACCL/L; Cre+ and ACCL/L Cre- mice. By breeding ACC+/L mice with the established Pten knockout model of prostate cancer (PtenL/L; Cre+), we have found that homozygous inactivation of ACC1 (ACCL/L; PtenL/L; Cre+) is able to reduce tumor burden in mice at 12 weeks of age when compared to ACC+/+; PtenL/L; Cre+ littermates (average total prostate weight of 90mg versus 120mg respectively, normalized to body weight, p 0.05). In our efforts to detail why ACC1 inactivation dampens early tumor burden but not later cancer progression, we have found that tumors from both 12 and 24 week old ACCL/L; PtenL/L; Cre+ mice show no changes in lipid profile when compared to tumors arising from age-matched ACC+/+; PtenL/L; Cre+ siblings. Interestingly, through metabolic analysis, we have found that different prostate cancer cell lines utilize fatty acids in unique ways. Under conditions that mimic bioenergetic stress, only androgen insensitive DU145 cells, and not LNCaPs, were able to oxidize exogenous fatty acids. Moreover, treatment with the non-isoform specific ACC inhibitor TOFA (5-Tetradecyloxy-2-furoic acid) abolished fatty acid oxidation under such conditions. These data indicate a potential for metabolic rewiring associated with disease progression and/or metabolic stress, and that blockade of ACC1 may not be sufficient to prevent disease progression. The role of ACC1 in acetate metabolism, redox modulation, fatty acid oxidation, and compensation for ACC1 activity by ACC2 are all potential mechanisms by which ACC1-deficient prostate cancer cells are able to survive and proliferate. Ultimately, these data further the notion that tumors rewire their metabolic circuits to meet the demands associated with cancer biology. Citation Format: Amanda L. Davis, Kristen Scott, Wu Jiansheng, Yong Q. Chen, Steven J. Kridel. ACC1 is required for prostate cancer initiation, but not progression? [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 A21.

Abstract 1878: Acetyl coA carboxylase1 and its role in prostate cancer initiation and progression.

The alteration of cellular metabolic processes is a common event in the initiation and progression of several cancer types. One metabolic hallmark of cancer cells is an increase in de novo lipogenesis. The enzymes responsible for de novo fatty acid synthesis are overexpressed in cancer, suggesting that these enzymes may be appropriate targets in efforts to inhibit or treat disease. Specifically, Acetyl CoA Carboxylase 1 (ACC1), the cytosolic enzyme that catalyzes the rate limiting step of de novo fatty acid synthesis through the ATP and biotin-dependent carboxylation of acetyl-CoA to malonyl-CoA, has received attention as a potential target for cancer treatment. Pharmacological inhibition and siRNA mediated knockdown of ACC1 has been shown to selectively kill prostate cancer cells in vitro by denying cells of adequate amounts of fatty acids needed to proliferate and survive. Although ACC1 has been implicated in cancer, its role has not been examined in a spontaneous model of cancer. To address this issue, we first generated mice with prostate-specific deletion of exon 22 of the Acetyl CoA Carboxylase 1 (ACC L/L ) gene by breeding with Probasin-Cre mice. Loss of ACC1 activity did not appear to affect prostate function as survival, prostate weight, histology, and breeding was similar between ACC L/L Cre + and ACC L/L Cre − mice. To determine the role of ACC1 in prostate cancer, the ACC +/L mice were crossed with Pten L/L ; Cre + mice. To our surprise, ACC1 activity was not required for prostate cancer development or progression in vivo, despite its apparent requirement in vitro. Total prostate weight and individual lobe weight did not differ among ACC +/L ; Pten L/L ; Cre + mice, ACC L/L ; Pten L/L ; Cre + mice and ACC1 +/+ ; Pten L/L ; Cre + mice. These early studies suggest that ACC1 activity, and perhaps the fatty acid synthesis pathway, may not be required for prostate cancer development. The role of ACC1 in redox modulation, rescue of impaired de novo fatty acid synthesis by circulating and dietary fatty acid, and the potential compensation for ACC1 activity by ACC2 in prostate cancer are possible explanations for this unexpected phenotype. These results could have significant impact on the development and use of anti-cancer therapeutics that target the fatty acid synthesis pathway. Citation Format: Amanda L. Davis, Kristen Scott, Yong Chen, Steven Kridel. Acetyl coA carboxylase1 and its role in prostate cancer initiation and progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1878. doi:10.1158/1538-7445.AM2013-1878