Abstract LB-302: Mutant KRAS causes diverse metabolic reprogramming and subsequent autophagy early in the pancreatic carcinogenesis
Authors: Suzuki Tatsunori , Otsuka Motoyuki , Koike Kazuhiko 
Published: September 2020

Abstract
Background: Pancreatic cancer is a lethal disease and is profoundly resistant to all forms of therapy. In recent years, understanding metabolic reprogramming in cancer has attracted interests as novel therapeutic targets. In addition to the well-established role of the altered glucose metabolism in cancer, recent studies have shown the altered amino acid metabolism, especially glutamine, in malignant cells. Because the initiation of most pancreatic carcinoma is triggered by KRAS gene mutation, we examined the state of metabolic reprogramming in KRAS gene mutation in human pancreatic ductal epithelial cells to clarify the metabolic reprogramming at the early stages during the pancreatic carcinogenesis. Methods: To investigate the specific pathway by which mutant KRAS exhibits metabolic alterations in human pancreatic epithelial cells, we introduced KRAS G12V mutation using CRISPR/Cas9 system in hTERT-HPNE cells, an immortalized human pancreatic non-cancerous epithelial cells. Comprehensive metabolome analyses were performed and further studies were done based on the results. Results: We confirmed the precise single nucleotide substitution by droplet digital PCR and by western blotting using mutant-specific antibodies. Mutant KRAS promoted cellular proliferation in a glucose- and glutamine-dependent manner. To clarify the metabolic alterations, CE mass spectrometry (CE/MS) metabolomic analyses were performed to characterize intracellular metabolites comprehensively. These analyses revealed that mutant KRAS caused the decreases in almost all amino acids, especially asparagine. Evaluation of the degree of protein translation by incorporation of puromycin, a tyrosyl-tRNA mimetic, showed that mutant KRAS caused enhanced protein synthesis. By quantitative RT-PCR, we also confirmed that various amino acid transporters including SLC1A5, SLC7A11/SLC3A2 was significantly upregulated by mutant KRAS, suggesting that KRAS mutant cells take in amino acids from extracellular space and actively consume them. Consistent to the previous reports, autophagy was activated, which was considered as a compensatory mechanism for the decreased amino acids levels. The lysosomal inhibitor, chloroquine, significantly inhibited the proliferation of the cells with mutant KRAS, suggesting that autophagy is required to promote oncogenic KRAS-driven tumorigenesis and to maintain tis metabolic reprogramming state. Conclusions: These results suggest that metabolic reprogramming and the subsequent autophagic activity are the key biological changes at the early stages of carcinogenetic changes in the cells harboring KRAS gene mutation and could be molecular targets to develop preventive methods against pancreatic carcinogenesis.
Citation Format: Tatsunori Suzuki, Motoyuki Otsuka, Kazuhiko Koike. Mutant KRAS causes diverse metabolic reprogramming and subsequent autophagy early in the pancreatic carcinogenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-302.