Evaluation of Hypoxia Induced Regulation of Nucleoside and Amino Acid Membrane Transporters in Breast Cancer

• Author / Creator

  Krys, Daniel

• Positron Emission Tomography (PET) utilizes radioactively tagged molecules to identify cancerous tissue. In most cases, membrane transporters control entry of these molecules into human cancer cells. Hypoxia-inducible factor (HIF)-1 is the master homeostatic regulator during hypoxia in human cancer cells. Rapid cell proliferation, as seen in cancer, isolates regions of tumors from blood vessels. Oxygen diffusion becomes too distant, which induces hypoxia and activates the HIF-1 pathway and induces changes within those cells. Regulation of hypoxic responses and induction of malignant reprogramming is maintained by HIF-1. HIF-1 controls expression of hexose, nucleoside, and amino acid transporters and for this reason is partly responsible for increased proliferation and metabolism seen in cancer cells. Upregulated expression of multiple target genes such as membrane transporters like Glucose transporter 1 (GLUT1), the primary glucose transporter is necessary to facilitate the Warburg Effect. Recently we have also demonstrated regulation of GLUT5, the cell’s primary fructose transporter, under hypoxia in breast cancer (BC). Currently, [18F]-fluorodeoxyglucose ([18F]FDG), a radiotracer transported into cells by GLUT1, is the “gold” standard and the only approved radiotracer for PET imaging of BC. [18F]FDG uptake, is not consistent across BC subtypes therefore other radiotracers must be explored. We studied protein levels and functionality of human equilibrative nucleoside transporters 1 and 2, and amino acid transporters LAT1, xc-, ASCT1, and ASCT2 under normoxic and hypoxic conditions using western blot experiments, flow cytometry, and/or confocal microscopy. Cellular uptake experiments were performed with 3'-deoxy-3'-L-[18F]fluorothymidine ([18F]FLT) for hENT1, 3,4-dihydroxy-6-(18F)-fluoro-L-phenylalanine ([18F]FDOPA) for LAT1, and (4S)-4-(3-18F-fluoropropyl)-L-glutamate ([18F]FPSG) for xc-, in estrogen receptor positive (ER(+)) MCF7 and MDA-MB231 triple-negative BC (TNBC) cells.
  In vitro [18F]FLT uptake was higher in MDA-MB231 than in MCF7: 242 ± 9 % vs. 147 ± 18 % radioactivity/mg protein after 60 minutes under normoxia. Immunoblot analysis revealed higher hENT1 levels in MDA-MB231, and higher thymidine kinase 1 (TK1) in MCF7. Data indicated that [18F]FLT uptake and hENT1 levels were not influenced significantly by hypoxia. In vitro inhibition experiments suggest involvement of both hENT1 and hENT2 [18F]FLT uptake into MDA-MB231. In vivo PET imaging revealed comparable tumor uptake in MCF7 and MDA-MB231 tumors: SUV60min 0.96±0.05 vs. 0.89±0.08 (n=3). LAT1 expression was higher in ER(+) MCF7 versus MDA-MB231 cells. [18F]FDOPA cell uptakes revealed greater uptake in ER(+) MCF7 cells (467 ± 98 %) compared to TNBC MDA-MB231 cells (105 ± 54 %) under normoxic conditions at 30 min. ASCT1 and ASCT2 did not exhibit any differentiating protein levels in the BC cell lines. In MDA-MB231, system xc- displayed high protein levels and high in vitro and in vivo uptakes with [18F]FSPG. Hypoxia significantly increased [18F]FDOPA uptake in MCF7 cells at 15 and 30 mins, and in MDA-MB231 cells at 30 and 60 mins. [18F]FSPG uptake was increased in MDA-MB231 cells under hypoxia at 30 and 60 mins: 95 ± 34 % vs. 40 ± 9.8 % and 164 ± 37 % vs. 127 ± 33 %. In vivo PET imaging revealed greater tumor uptake of [18F]FDOPA in MCF7 tumor models and [18F]FSPG in MDA-MB231 tumor models.
  Our study demonstrates that hypoxia has significant effects on amino acid transport as tested with [18F]FDOPA and [18F]FSPG in BC. Overall, there is increased transport of [18F]FLT and of [18F]FPSG in MDA-MB231 cells, while [18F]FDOPA is increased in MCF7 cells. Higher hENT1 expression in MDA-MB231 seems to drive nucleoside transport, while higher TK1 expression in MCF7 seems to be responsible for [18F]FLT retention in ER(+) tumors. We see minimal hypoxic regulation of protein levels of these transporters in both cell lines, but do see high activity of xc- in MDA-MB231 and LAT1 in MCF7.

• Subjects / Keywords

  • Nucleoside Membrane Transporters
  • Hypoxia
  • Cancer
  • Amino Acid Membrane Transporters
  • Positron Emission Tomography
  • Breast Cancer

• Graduation date

  Spring 2020

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-qmhg-wv89

• License

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