Su Chii Kong:
pH Regulatory Transporters in Pancreatic Ductal Adenocarcinoma

Date: 31-08-2015    Supervisor: Stine Falsig Pedersen

The abnormal features of hypoxia and altered metabolisms in solid tumours lead to an increased glycolysis that is uncoupled from oxidative phosphorylation in the TCA cycle. Tumoural cells often exhibit dysregulated expressions and activities of various membrane pH regulatory transporters to cope with the elevated acid production from this glycolysis, as well as from cellular ATP hydrolysis, sequentially creating a favourable intracellular pH and hostile acidic tumour microenvironment, fortify the tumour cells with highly invasive, metastatic and drug resistant phenotype. In current work, we study the expressions and functional roles of these pHregulating transporter in pancreatic ductal adenocarcinoma (PDAC), one of the deadliest human malignancies with an overall 5-year survival rate of only 6%.

Herein we focus on two pH-regulating transporter families, monocarboxylate transporters (MCTs) and V-ATPases. MCT isoforms 1 to -4 are the only proton-coupled isoforms transporting monocarboxylates such as L-lactate. We show that MCT1 and MCT4 are robustly expressed in all PDAC cell lines studied. These transporters were found localized on the plasma membrane of PDAC cells, colocalizing with MCT chaperone protein basigin. Lactate influx capacity was reduced upon siRNA-mediated silencing and pharmacological inhibition of MCT1 and/or MCT4. PDAC cell migration was not significantly affected by MCT1 inhibition with AR-C155858 and MCT1 silencing, yet was inhibited by the general MCT inhibitor 4-CIN. The migration of the fastest moving PDAC cells detected, BxPC-3, was also reduced after MCT4 knockdown. Both MCT inhibition with AR-C155858 and 4-CIN as well as MCT1 and MCT4 silencing reduce the PDAC cell invasiveness in the Boyden chamber assay. In addition, the silencing of MCT1 and/or MCT4 reduced the invasiveness of BxPC-3 cells in the spheroid outgrowth assays. We also observed that the silencing of MCT1 and MCT4 inhibited the proliferation of the studied PDAC cells, where MCT4 silencing exerts a bigger effect than MCT1 silencing.

V-ATPases are multi-subunit proton pumps whose involvement in the generation of tumour microenvironmental acidosis has recently emerged and have been implicated in the promotion of tumour aggressive phenotypes. In present study, substantial up-regulation of subunit a3 was detected in a panel of PDAC cells tested as compared to non-cancerous control HPDE. Silencing of subunit a3 inhibited the invasiveness in two PDAC cell lines studied yet increased their 2D cell motility on a tumour ECM-mimicking substratum. V-ATPases were also found to be associated with several cancer-related signaling pathways. V-ATPase inhibition with concanamycin A and a3 silencing up-regulated the HIF-1α and p21 protein level. Concanamycin A treatment in PDAC cells provoked an increase in p62, indicative of reduced autophagic flux; whereas silencing of a3 subunit did not. The underlying mechanisms leading to these observations remain to be elucidated. Finally, in BxPC-3 cells, cell proliferation was found to be decreased while apoptosis was increased with concanamycin A treatment, indicative of V-ATPases being involved in PDAC cell survival mechanisms as well.

Comprehending pH regulation in tumour cells might provide insights in preventing tumourigenesis by pH disruptions. Data presented in this thesis corroborate the roles of pH-regulating transporter proteins in PDAC progression and pave the way for future studies in search of specific therapeutic targets for this malignancy.