Research in the Bode Lab
Amino Acid Transporters as Potential Therapeutic Targets in Hepatocellular Cancer
Nutrient transporter proteins in the plasma membrane deliver the extracellular metabolic fuels that allow all cells to function, grow and thrive. Because of their gate-keeping role in nutrient delivery, transporter proteins represent a major regulatory step in cellular growth dynamics. Not only do the nutrients delivered to the cell provide metabolic energy sources and biochemical building blocks, but they also regulate (via excess or deficit) intracellular signaling and gene expression.
My laboratory studies the role and regulation of amino acid transporters in human cancer cell growth and survival signaling. Specifically, we focus on two amino acid transporters: ASCT2 (encoded by the SLC1A5 gene) and LAT1 (encoded by the SLC7A5 gene). A bioinformatics study from our laboratory showed that expression of both transporters is coordinately enhanced in a broad spectrum of human cancers compared to corresponding normal tissue. In normal liver, expression of ASCT2 and LAT1 is nearly undetectable, but is robustly increased upon cancerous transformation, making these transporters potential targets for directed therapies in hepatocellular carcinoma (HCC) – a cancer with limited treatment options, a poor prognosis and high mortality rates. Both ASCT2 and LAT1 are amino acid exchangers and have been shown to physically associate in a plasma membrane-localized complex with monocarboxylic acid (e.g. lactate and pyruvate) transporters (MCT) in human cancer cells. This collection of transporters (ASCT2, LAT1, MCT1/2) and associated chaperone proteins (CD98, CD147) has been termed the Metabolic Activation - related Complex ((MArC); Xu and Hemler, Mol Cell Proteomics 4:1061-1071, 2005). Work from our laboratory demonstrated an essential role for ASCT2 expression in human liver cancer growth and survival, as its targeted silencing led to rapid growth cessation and programmed cell death, independent of its role in amino acid delivery. Thus, nutrient transporters not only deliver nutrients, but also appear to play other roles in cellular signal transduction by mechanisms that are currently unknown.
Individual cancers display a dizzying array of disparate oncogenic signatures, usually a combination of loss of tumor suppressor functions and activation of cellular oncogenes. Despite this inherent complexity, most cancers display common physiological aberrations relative to normal tissue. Among these are the acquisition of cell-autonomous (external growth factor-independent) nutrient transporter expression and trafficking and enhanced aerobic glycolysis (the Warburg Effect), which is the basis for PET-imaging in cancer detection using 18F-deoxyglucose. These physiological deviations are in turn linked to unchecked signaling via the Akt kinase / mammalian Target-Of-Rapamycin (mTOR) axis. Significantly, mTOR kinase activity is highly stimulated by amino acids, particularly by essential amino acids in humans. Most mTOR-stimulating amino acids are preferred substrate of LAT1. Thus, in cancers a strong relationship exists between glycolytic metabolism, Akt/mTOR signaling and amino acid delivery. Recent work from our laboratory demonstrated that ASCT2 silencing inhibits mTOR signaling in human liver cancer cells. Based on those studies, our current hypothesis is that enhanced expression of ASCT2 and LAT1 in cancer is necessary to drive growth via stimulated mTOR signaling, and by extension, glycolytic metabolism. This hypothesis is bolstered by the observation that ASCT2 and LAT1 are physically associated with MCT1/2 in the MArC, linking amino acid transport to glycolytic end-product (lactate) transport.
Our laboratory currently uses a panel of several human liver cancer cell lines that have been characterized by gene expression profiling (microarray analysis), and represent a broad spectrum of clinically-relevant HCC phenotypes. Using these cell lines as the experimental model system, the overarching goal of our research is to define roles for ASCT2 and LAT1 in hepatocellular cancer, and to target these transporters as potential therapies for this recalcitrant malignancy.
Our research is currently supported by Grant # 2R15CA 108519-02 from the National Cancer Institute.
Specifically, ongoing research projects in the lab examine:
- The consequence of ASCT2 or LAT1 silencing on glycolysis, growth and mTOR signaling in human HCC cell lines.
- The effects of ASCT2 or LAT1 overexpression on growth, metabolism and stress responses in liver cells.
- The effect of tumor microenvironmental stresses on ASCT2 and LAT1 expression.
- The regulation of ASCT2 and LAT1 expression in cells grown as “mini tumors” in three dimensional culture (spheroids); reciprocally, the impact of transporter expression on spheroid formation.
- The role of ASCT2 and LAT1 expression in human vs. rodent liver cell transformation
Research opportunities: The study of amino acid transporters in cancer biology in my laboratory currently involves a broad spectrum of techniques in cell culture and molecular biology:
Gene Expression Analysis:
- RNA isolation, agarose gel electrophoresis, real-time quantitative RT-PCR, northern blotting*
- Protein extraction and purification, Antibody-based assays: western blotting, immunostaining, immunoprecipitation
- Bioinformatic analysis (computer-based) of transporter gene expression in normal and cancerous tissue, and across species
- Western blot analysis and Enzymatic assays
Targeted Gene Manipulation:
- Post-transcriptional gene silencing (RNAi)
- Molecular cloning (PCR, plasmid vector construction, analysis and purification)
- Transfection of cells with plasmid DNA
Cell Biology, Physiology, Metabolism:
- Cell growth quantification, cell cycle analysis
- Spheroid growth and analysis
- Use of biochemical inhibitors of transport and metabolism
- Radiotracer nutrient uptake*
- Enymatic/ colormetric assays
Each of these techniques is utilized alone or in combination to address specific questions in cancer biology. Undergraduate research opportunities can involve any of these techniques, except those that involve the use of radioactivity(*), depending on the current problems that we are addressing in my research program.
Professor Barrie P. Bode
Phone: (815) 753-1753
Office: MO 350,
Lab: MO 412
Dept. of Biological Sciences
Northern Illinois University
DeKalb, IL 60115