by: Elizabeth Kolar
To start, let me introduce myself as I am a new writer for HBN! I am a Ph.D. candidate in the Biochemistry, Cell, and Molecular Biology graduate program at The Johns Hopkins University School of Medicine. I work on the fourth floor of the Kennedy Krieger Institute for Paul Watkins, M.D., Ph.D. As a lab, we study the role of fatty acid metabolism and the associated enzymes in different neurological diseases. My thesis research focuses on one enzyme that belongs to the acyl-CoA synthetase family of enyzmes known as ACSVL3.
Acyl-CoA synthetases (ACSs) are a family of proteins that “activate” fatty acids that have been synthesized de novo by the cell or those that have been brought into the cell. This occurs by adding a Coenzyme A via a thioester bond. After this step, the fatty acid can be metabolized in a number of different pathways. These activated fatty acids can be incorporated into phospholipids, used in beta-oxidation to create energy, or be used for post-translational modifications of proteins, to name a few. The human genome encodes for 26 different ACS proteins, and these can be characterized by their specificity for fatty acid chain length. Acyl-CoA synthetase family members that have the ability to activate or transport very long chain fatty acids are known as the ACSVL family (acyl-CoA synthetase for very long chain fatty acids), in which ACSVL3, my enzyme of interest, is characterized.
Our lab discovered ACSVL3 to be highly expressed in a malignant glioma cell line known as the U87 cell line. It was confirmed to be medically important by performing immunohistochemistry on a tissue array of 79 tumors, of varying degrees of malignancy using an antibody raised against ACSVL3. ACSVL3 was detected in all tissues, and when compared to normal tissues, the amount of staining in the malignant tissue was very obvious. This prompted the lab’s interest in this particular ACS’s role in malignant brain cancer. We use the U87 cell line as our model system, with plans to do some animal modeling in the future.
We have created an ACSVL3 knockout (KO) cell line using zinc-finger nuclease gene editing technology. This KO has a large, in-frame deletion in a critical part of the DNA/protein sequence that is necessary to confer enzymatic activity. From a morphological point of view, these cells grow very slowly when compared to the original U87 cell line, the cells appear much larger, and rarely form subcutaneous tumors when injected right below the skin of mice with a compromised immune system, which allows the tumors to grow without the mice rejecting the foreign tissue. The original U87 cells form subcutaneous tumors 100% of the time. These KO cells are also deficient in acyl-CoA synthetase activity when looking at activation of the long-chain fatty acid 16:0 (palmitic acid) and the very long chain fatty acid, C24:0 (lignoceric acid).
The evidence that makes our focus on this particular ACS interesting is that cells that lack the enzyme seem to have aberrant signaling from the receptor tyrosine kinase c-Met (Pei et al., 2009). When the receptor’s ligand, hepatocyte growth factor (HGF), is bound, c-Met signals through the Akt/Protein Kinase B pathway, and can trigger multiple pathways involved in cell growth and proliferation, glucose and lipid metabolism, and cell migration, which are all characteristics important for tumor malignancy. By adding exogenous HGF to the media, the KO cells showed a lack of response, while activation of c-Met and Akt (denoted by phosphorylation of both) were increased in the U87 cells. In a separate experiment using a glioma line that had very low levels of ACSVL3, the addition of HGF increased Akt activation and led to a robust expression of ACSLV3. This suggests that ACSVL3 is an important enzyme in the transformation of cells to a more oncogenic phenotype, as well as potentially necessary to maintain phospholipid membrane requirements necessary for Akt stability, since Akt activation occurs at the membrane.
There was some evidence prior to my joining the lab that ACSVL3 levels affected cholesterol metabolism. I struggled for a few years trying to repeat the results that suggested a decrease in ACSVL3 led to a decrease in total cholesterol and cholesterol synthesis. Cholesterol is thought to reside in the plasma membrane in high concentrations in what are known as lipid rafts. These microdomains are thought to act as platforms for signaling through receptor tyrosine kinases, such as c-Met. What started out as a simple experiment to see if lipid rafts in the KO cell line were any different, led to a line of experiments I had not really planned, but what has now become the bulk of my thesis. We had hypothesized that with a decrease in either fatty acid activation that we saw with the synthetase assay using C24:0 and slightly decreased fatty acid synthesis, we would see a decrease in lipid rafts in the KO cells. Using a fluorescently tagged Cholera toxin B subunit protein, we looked at the lipid rafts in our U87 cells and the ACSVL3 KO cells. The tagged protein binds to a ganglioside found to be enriched in these lipid rafts, GM1. What we found was surprising – more GM1 was found on the surface of the KO cells when compared to the U87 line! Because of this result, I am focusing on the synthesis of ceramide, a lipid precursor to gangliosides and other sphingolipids, as well as ganglioside levels in these cells, and hopefully I will be able to dissect what role ACSVL3 plays in the synthesis and metabolism of these different lipids.
We cannot rule out that other ACSs contribute to a tumor cell’s malignancy, but I believe our lab has some very good evidence that ACSLV3 seems to be an important enzyme. The Watkins’ lab has shown that ACSVL3 is also up-regulated in prostate cancer and lung cancer. As of right now, there are no known inhibitors specifically to ACSVL3. This is a very active area of research in my lab right now, and we are hoping to find a promising one soon.
Ever since high school, I have wanted to do cancer research, so this feels like dream comes true! I’ve learned so much working in this lab, but more importantly, it has shown me how much there is still to learn about cancer biology as well as science in general. Thank you for letting me share my experiences here, and I look forward to sharing many other thoughts and reading contributions from other writers in the network!
*Pei et al. Acyl-CoA Synthetase VL3 knockdown inhibits human glioma cell proliferation and tumorigenicity. Cancer Research. 2009; 69(24); 9175-82.