ATP citrate lyase inhibition can suppress tumor cell growth

Submitted by Daniel Rosen

Georgia Hatzivassiliou1, Fangping Zhao1, Daniel E. Bauer1, Charalambos Andreadis2, Anthony N. Shaw3, Dashyant Dhanak4, Sunil R. Hingorani1, 2, David A. Tuveson1, 2 and Craig B. Thompson1, Corresponding Author Contact Information, E-mail The Corresponding Author

1Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104 2Department of Medicine, Abramson Cancer Center, University of Pennsylvania, Philadelphia, Pennsylvania 19104 3Department of Medicinal Chemistry, Metabolic and Viral Diseases Center of Excellence for Drug Discovery, GlaxoSmith-Kline, Collegeville, Pennsylvania 19426 4Department of Medicinal Chemistry, Musculoskeletal, Microbial and Proliferative Diseases Center of Excellence for Drug Discovery, GlaxoSmith-Kline, Collegeville, Pennsylvania 19426


Received 11 April 2005;

revised 9 September 2005;

accepted 28 September 2005.

Published: October 17, 2005.

Available online 17 October 2005.



Summary:

Human cancers are often detected by 18-F-2-deoxyglucose positron emission tomography (PET) because the cancers tend to exhibit high levels of aerobic glycolyis which can be detected by the PET scan. Although the increased levels of aerobic metabolism fulfill a need for increased ATP production, the majority of the ATP derives from glycolytic activity. The pyruvate end-product enters a modified Citric Acid/Krebs cycle and is shunted to producing citrate and then acetyl-CoA. The acetyl-CoA is a necessary building block for de-novo synthesis of fatty acids, which cancers cells do almost exclusively regardless of external supplies. Cancer cells engage in upregulated levels of fatty acid synthesis to (1) support membrane production and (2) permit post-translational modification of proteins (i.e. acetylation) specifically lipid modified signaling molecules. Both functions are essential for cancer growth. ATP Citrate Lyase (ACL) is an enzyme which converts Citrate to cytosolic acetyl-coA and it is also coordinately regulated with other lipgenic enzymes. For this potential to link glucose and lipid metabolism, ACL was studied and its inhibition was determined to suppress proliferation and promote differentiation in glucose dependent cancers.

Endogenous ACL levels in human lung adenocarcinoma cell line A549 were knocked down with siRNA oligonucleotides. As a control the siACL knockdowns were compared to siLUC (luciferase) knockdowns. In the first experiment a Western Blot was run comparing the two siRNA transfections production of ACL, which was compared to Actin (Figure 1). The results show decreased levels of ACL in the knockdowns which increases with time and there is no change in the controls. This is also quantified in figure 1B. Data in Figure 1 also shows a difference in lipid synthesis when tagged with D-[6-14C] glucose (for the glucose dependent lipid synthesis) for the siACL vs. siLUC lines and no change for acetate tagging (glucose independent lipid synthesis). Viability only decreased for the siACL line at extended time periods.

As shown in Figure 4. the in vivo effect of short hairpin knockdown strains of A549 cells resulted in reduced tumor weight and differentiation into glandular structures when injected in vivo into nude mice. The differentiated phenotype, being unexpected, was confirmed with a replicated experiment using K562 chronic myelogenous leukemia cell line. The pharmoacologic inhibitor of ACL, SB-204990 (not stated but presumably a Glaxo-smith-kline (GSK) propriety compound) was tested on a IL-3 dependent cell lines. The IL-3 stimulates glycolysis and cell proliferation which replicates a tumor cell profile. The SB-204990 compound induced delayed cell cycle entry (arrest in G1 phase at 15 microMolar and complete inhibition at higher doses). The compound was then tested against three tumor forming cell lines in vitro and in vivo via xenographs in nude mice. Two of the cell lines (A549 and PC3) exhibited sensitivity to SB-204990 treatment while the third line SKOV3 did not. The difference is indicated to be a result of the SKOV3 lower dependence and uptake of glucose.

Significance:

Because many human cancers display a higher than average glucose dependency, fueling growth and malignancy, an inhibitor of the utilization of that glucose makes an attractive target in anti-cancer therapy. Cancer cells require the extra glucose in order to generate de novo lipids specifically signaling molecules and membrane. One relatively upstream enzyme involved in this lipogenesis is ACL which when downregulated was shown in vitro and in vivo to have negative effects on cancer growth and also promoted differentiation—another indication of cancer retreat. Although several methods of downregulation including siRNA and pharmaceutical compounds produced the desired effect, it was only significant in cancer lines representative of cells which require an abnormally high amount of exogenous glucose and had a minor effect on cancer lines with lower glucose to lipid production ratios. Additionally, a separate glucose-independent pathway can rescue lipogenesis function in several cell lines. Despite these setbacks, ACL inhibitors remain attractive anti-cancer therapeutics due to upstream regulation of other lipogenic factors, selective targeting of cancer tissues and anti-neoplastic properties.

From the perspective of BioE 115 this paper employs three of the available cell lines, Western Blotting, and BrdU staining.