Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with poor treatment options. PDAC progression involves a series of driver mutations in genes such as KRAS, TP53, SMAD4, CDKN2A. PDAC is characterized by a desmoplastic, acidic and hypoxic tumor microenvironment, which is suggested to contribute to epithelial-mesenchymal transition (EMT), metabolic changes, and drug resistance. However, the molecular mechanisms of how chronic acidosis and driver mutations impact tumorigenesis remain essentially unknown. The overall aim of this PhD thesis was to investigate the effects of chronic acidosis and driver mutations on pH regulation, 3D growth, drug resistance, invasiveness, stemness and aggressiveness of pancreatic cancer cells.

In Paper I, we identified a cancer-associated response of three different cancer models to chronic acidosis, which correlated to overall survival and tumor-specific expression changes in pancreatic cancer patients. We identified that during chronic acidosis, cancer cells underwent a profound metabolic shift and extracellular matrix (ECM) remodeling, and exhibited altered cell cycle regulation and DNA damage response. In Paper II we proposed a classification of pancreatic cancer subtypes according to their metabolic preferences, highlighted the impact of driver mutations on cancer metabolism and summarized metabolismtargeted therapeutic options for treatment of pancreatic cancer. In Paper III, we demonstrated that acid adaptation of pancreatic cancer cells with wild-type (WT) p53 and their subsequent return to physiological extracellular pH (pHe) increased 3D growth, invasive outgrowth and adhesion-independent colony formation. A similar pattern of increased aggressive traits was observed for 3D spheroid- and organotypic cultures, the latter grown in ECM matrices such as matrigel and/or collagen I to mimic the ECM of early and late stages of cancer progression. Acidosis-driven aggressive characteristics were further exacerbated by p53 loss in specific ECMs. In addition, acid adaptation promoted upregulation of Akt-, TGFβ-signaling pathways and upregulation of the Na+/H+ exchanger NHE1 (SLC9A1), frequently implicated in cancers. Akt-signaling was equally important for growth of both WT p53 and p53-null pancreatic cancer cells, whereas TGFβ-signaling facilitated 3D growth only in WT p53 cells. In Paper IV, we reported that chronic acidosis altered transcriptional activity, increased viability and promoted gemcitabine- and erlotinib resistance in murine pancreatic cancer organoids. Notably, drug resistance was most prominent in acid adapted WT p53 organoids returned to physiological pH. Our transcriptional data revealed that acid adaptation upregulated cytidine deaminase (Cda) and ribonucleotide reductase regulatory subunit 2 (Rrm2), and their inhibition partially restored gemcitabine resistance, the remainder of the resistance likely reflecting an acidadaptation-induced increase in proliferation. In Paper V, we established that pH regulation capactiy of organoids is increased by p53 loss and decreased in acid adapted organoids cultured at acidic pHe. Moreover, despite increased baseline DNA damage, p53 loss protected organoids from olaparib-induced loss of cell viability, especially when cultured in acidic pHe. We suggest that increased capacity for net acid extrusion could favor tumorigenicity of p53deficient pancreatic cancer organoids. Finally, in Paper VI, we investigated whether chronic acidosis can promote stemness properties in human pancreatic cancer cells. RNA-seq data showed a distinct cluster of upregulated genes related to cancer stemness, confirmed by qPCR and Western blot analysis. Acid adaptation increased the fraction of CD44+/CD24cancer stem cells, ALDH activity, β-catenin signaling, and pancreatosphere formation capacity. Finally, acid adapted cancer cells gave rise to more aggressive PDAC development and liver metastases after orthotopic transplantation in NOD/SCID mice.

Collectively, the work of this PhD thesis demonstrates that chronic acid adaptation and p53 knockout increase pH regulation capacity, 3D growth, drug resistance, invasiveness and stem cell-like phenotype in pancreatic cancer cells, compared to respective controls. These findings extend the current understanding of how chronic acidosis and driver mutations favor tumor progression, of potential relevance for development of new acidosis-targeted therapeutics.