Regulation of pH by Carbonic Anhydrase 9 Mediates Survival of Pancreatic Cancer Cells With Activated KRAS in Response to Hypoxia

Abstract

See Covering the Cover synopsis on 589. BACKGROUND & AIMS: Most pancreatic ductal adenocarcinomas (PDACs) express an activated form of KRAS, become hypoxic and dysplastic, and are refractory to chemo and radiation therapies. To survive in the hypoxic environment, PDAC cells upregulate enzymes and transporters involved in pH regulation, including the extracellular facing carbonic anhydrase 9 (CA9). We evaluated the effect of blocking CA9, in combination with administration of gemcitabine, in mouse models of pancreatic cancer. METHODS: We knocked down expression of KRAS in human (PK-8 and PK-1) PDAC cells with small hairpin RNAs. Human and mouse (Kras G12D /Pdx1-Cre/ Tp53/Rosa YFP) PDAC cells were incubated with inhibitors of MEK (trametinib) or extracellular signal-regulated kinase (ERK), and some cells were cultured under hypoxic conditions. We measured levels and stability of the hypoxia-inducible factor 1 subunit alpha (HIF1A), endothelial PAS domain 1 protein (EPAS1, also called HIF2A), CA9, solute carrier family 16 member 4 (SLC16A4, also called MCT4), and SLC2A1 (also called GLUT1) by immunoblot analyses. We analyzed intracellular pH (pHi) and extracellular metabolic flux. We knocked down expression of CA9 in PDAC cells, or inhibited CA9 with SLC-0111, incubated them with gemcitabine, and assessed pHi, metabolic flux, and cytotoxicity under normoxic and hypoxic conditions. Cells were also injected into either immunecompromised or immune-competent mice and growth of xenograft tumors was assessed. Tumor fragments derived from patients with PDAC were surgically ligated to the pancreas of mice and the growth of tumors was assessed. We performed tissue microarray analyses of 205 human PDAC samples to measure levels of CA9 and associated expression of genes that regulate hypoxia with outcomes of patients using the Cancer Genome Atlas database. RESULTS: Under hypoxic conditions, PDAC cells had increased levels of HIF1A and HIF2A, upregulated expression of CA9, and activated glycolysis. Knockdown of KRAS in PDAC cells, or incubation with trametinib, reduced the

Actively Targeted Nanodelivery of Echinomycin Induces Autophagy-Mediated Death in Chemoresistant Pancreatic Cancer In Vivo

“…Therefore, relative levels of autophagy observed in S2VP10 and S2CP9 models agree with behavior of clinical pancreatic cancer [44,45] and suggest that autophagy increases with cancer progression and poor outcomes. It is noted that both S2VP10 and S2CP9 are more tolerant of harsh microenvironmental conditions, including hypoxia ( Figure S4) and acidic extracellular pH [46,47], which promotes chronic autophagy, metastasis [48], and resistance to apoptosis-inducing treatments at greater rates than MiaPaCa-2 and Panc 1 [49,50], both in vitro and in vivo.…”

CA IX Stabilizes Intracellular pH to Maintain Metabolic Reprogramming and Proliferation in Hypoxia

“…Previous reports have shown that suppression or inhibition of CA IX disrupts pH control in hypoxia and reduces cellular proliferation and/or tumor growth ( 20 – 22 ). In order to investigate the mechanism of reduced proliferation in the context of our observation that CA IX suppression decreases the glycolytic flux, we tested several compounds that modify different aspects of glucose metabolism to see if they could restore the growth of CA IX deficient cells in hypoxia.…”

Identification of a metabolic signature genes predicting overall survival for Head and neck squamous cell carcinoma

“…They are involved in various biological processes, including bone resorption, respiration, calci cation, and acid-base balance. Study found that pancreatic ductal adenocarcinomas (PDACs) cells that express activated KRAS increase expression of CA9, via stabilization of hypoxia-inducible factor 1 subunit alpha (HIF1A) and HIF2A, to regulate pH and glycolysis 25 . Similarly, CA9 is an independent prognostic factor for OSCC patients and therefore a potential therapeutic target 26 .…”