Catabolism of Extracellular Protein by Pancreatic Cancer Cells

Abstract

All cells require amino acids to support protein synthesis and cell growth. Until recently, mammalian cells were thought to depend on monomeric amino acids in the environment. I showed that pancreatic tumor cells can use extracellular protein as a source of amino acids. These cells take up intact protein via macropinocytosis and catabolize it in lysosomes. This process – “protein eating” – enables cultured pancreatic cancer cells to grow in amino acid-deficient environments. In this thesis, I present my work on protein eating.

To show that protein eating is capable of fueling growth, I cultured murine pan- creatic cancer cells in medium lacking leucine (an essential amino acid) and supple- mented with a physiological concentration of serum albumin. Many cells cells died in this medium, but some survived and grew to confluence. I passaged these survivors for months, and they gradually adapted to growth fueled by protein eating. This proved that protein eating is a viable form of amino acid uptake.

I developed isotope tracer-based methods to quantitatively measure protein eating. Cells are grown in medium with stable isotope-labeled amino acids and unlabeled serum protein. Mass spectrometry enables distinction of amino acids taken up as monomers (labeled) from amino acids taken up as intact protein and catabolized (unlabeled).

I conducted genome-wide screens to systematically identify genes essential for growth fueled by protein eating. The most essential gene was GCN2, which suppresses translation initiation in cells starved for amino acids. I discovered that loss of GCN2 impairs catabolism in amino acid-deprived cells. I propose that GCN2 supports catabolism by directing amino acids emerging from lysosomes into newly synthesized proteins that increase the catabolic capacity of the cell – for example, the lysosomal hydrolase cathepsin L.

Advances in our understanding of protein eating may lead to the development of better therapies for pancreatic cancer patients. The importance of protein eating as an amino acid supply route for cells in healthy tissues remains unexplored.