Abstract 4358: Characterization of primary and metastatic pancreatic tumors in pancreatic ductal adenocarcinoma (PDAC)
Authors: Andrés E. Martínez-Muñiz, Sharanya Sivanand, Matthew Vander Heiden
Journal: Cancer Research (2019)

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is the 5th leading cause of cancer death in the U.S. and Europe. It is a very aggressive form of cancer in which patients have a 5-year survival rate lower than 5%. PDAC arises from alterations in the KRAS and TP53 genes. The cell lines used were derived from 2 different mouse models where KRASG12D was combined with a deletion or point mutation of TP53 (TP53-/-, TP53R172H respectively) to drive oncogenic activation of PDAC. The TP53-/- model exhibits rapid pancreatic tumor growth, thus not allowing metastasis to occur, in contrast to the TP53R172H model, where tumors metastasize to the lungs or liver. TP53R172H cells also show a noticeable dependency on pyruvate availability for proliferation in contrast to the opposing model. Other NAD+ replenishing molecules such as duroquinone can have similar effects on proliferation. Cell lines from the primary tumors of both models as well as from the metastasized liver tumors were used for in vitro experimentation. Epithelial-Mesenchymal Transition (EMT) is important for metastasis and cell state can be qualified by measuring E-cadherin expression, which has also been shown to be influenced by pyruvate availability. This project had 2 aims: 1) determine the effect of pyruvate availability on EMT by measuring E-cadherin levels and TWIST1 relative expression and 2) measure the effect that availability of both pyruvate and duroquinone have on cell proliferation. Cells were cultured in 6-well plates. After cell adhesion, they were treated with media containing pyruvate and media lacking pyruvate for comparison. Cells were grown for 24-96 hour periods after which they would be recollected for our designated analyses; western blot, total cell count and qPCR. Measured E-cadherin levels showed that there is higher protein expression in the TP53R172H model. Furthermore, it is shown that E-cadherin levels seem to be regulated by pyruvate in this model. Increase in protein quantity with time also suggests that these cells may be shifting towards an epithelial phenotype. Duroquinone did not rescue proliferation, contrary to the hypothesis. Surprisingly, there was a decrease in proliferation in the TP53-/- model. This could be attributed to experimental errors or loss of pyruvate sensitivity in the cell line. Expression levels were measured for Twist (TWIST1), a transcription factor that serves as a negative regulator for E-cadherin. RNA was isolated from cell lysates and reversed transcribed to measure relative expression of Twist by qPCR. A pronounced effect of pyruvate dependency on Twist expression was observed in both models. This may be meaningful since it could offer a possible explanation on how pyruvate could be influencing EMT in these cells. Citation Format: Andrés E. Martínez-Muñiz, Sharanya Sivanand, Matthew Vander Heiden. Characterization of primary and metastatic pancreatic tumors in pancreatic ductal adenocarcinoma (PDAC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4358.