4986. Dissecting Subcellular Compartmentation of Breast Cancer Glutamine Metabolism
Authors * Denotes Presenting Author
  1. Christopher Hensley *; University of Pennsylvania
  2. Hoon Choi; University of Pennsylvania
  3. Meagan McManus; Children's Hospital of Philadelphia
  4. Hsiaoju Lee; University of Pennsylvania
  5. Austin Pantel; University of Pennsylvania
  6. David Mankoff; University of Pennsylvania
  7. Rong Zhou; University of Pennsylvania
A subset of cancers, including triple negative breast cancer (TNBC), demonstrate glutamine “addiction.” Intracellular glutamine (Gln) can be converted to glutamate (Glu) in the presence of the glutaminase enzyme (GLS), a rate-limiting step for glutamine catabolism. Expression levels of total glutaminase activity have been demonstrated to be correlated to glutamine dependence in breast cancer cell lines in vitro. This dependence on GLS is most prevalent in triple negative breast cancer (TNBC), when compared to estrogen receptor positive (ER+) cell lines. CB-839 (telaglenastat) is an inhibitor of GLS that failed to show the anticipated efficacy in tumor response in clinical trials, suggesting a need for clinical biomarkers. Responding to this need, glutamine PET tracers have been developed to image glutamine metabolism in vivo, including [18F](2S,4R)4-fluoroglutamine ([18F]4F-Gln). We have previously employed a breast cancer cell line mouse flank xenograft model to assess the utility of [18F]4F-Gln. In this model, a TNBC line with high intrinsic GLS activity (HCC1806) is compared to an ER+ line (MCF-7) with lower intrinsic GLS activity. To further efforts toward more predictive PET biomarker development for glutamine “addiction” in breast cancer, we pursued subcellular analysis of glutamine metabolism for a deeper understanding of the critical nodes and thus biomarkers of glutamine “addiction.” Specifically, we hypothesize that the glutaminolysis downstream of glutamine uptake occurs to a greater degree in the mitochondria than the cytosol.

Materials and Methods:
We utilized the methodology of digitonin permeabilization. Digitonin is a detergent that selectively permeabilizes the plasma membrane and leaves the mitochondrial membrane intact and has previously been utilized to assess subcellular enzyme kinetics. Subcellular metabolism in digitonin permeabilized breast cancer cells (HCC1806/TNBC versus MCF-7/ER+) were pursued by measuring glutamate pool sizes.

We have conducted preliminary experiments that support the idea that a 4 - 5 fold concentration gradient exists in TNBC in vitro between the higher mitochondrial glutamate pool compared to the less concentrated cytoplasmic pool, and that this property appears to be not seen in ER+ breast cancer.

The subcellular glutamate pool sizes studies support the mitochondrial glutamate “buffer” hypothesis in TNBC, which we are currently further pursuing.