Genetic disruption of the pHi-regulating proteins Na+/H+ exchanger 1 (SLC9A1) and carbonic anhydrase 9 severely reduces growth of colon cancer cells

Abstract

Hypoxia and extracellular acidosis are pathophysiological hallmarks of aggressive solid tumors. Regulation of intracellular pH (pH_i) is essential for the maintenance of tumor cell metabolism and proliferation in this microenvironment and key proteins involved in pH_i regulation are of interest for therapeutic development. Carbonic anhydrase 9 (CA9) is one of the most robustly regulated proteins by the hypoxia inducible factor (HIF) and contributes to pH_i regulation. Here, we have investigated for the first time, the role of CA9 via complete genomic knockout (ko) and compared its impact on tumor cell physiology with the essential pH_i regulator Na⁺/H⁺ exchanger 1 (NHE1). Initially, we established NHE1-ko LS174 cells with inducible CA9 knockdown. While increased sensitivity to acidosis for cell survival in 2-dimensions was not observed, clonogenic proliferation and 3-dimensional spheroid growth in particular were greatly reduced. To avoid potential confounding variables with use of tetracycline-inducible CA9 knockdown, we established CA9-ko and NHE1/CA9-dko cells. NHE1-ko abolished recovery from NH₄Cl pre-pulse cellular acid loading while both NHE1 and CA9 knockout reduced resting pH_i. NHE1-ko significantly reduced tumor cell proliferation both in normoxia and hypoxia while CA9-ko dramatically reduced growth in hypoxic conditions. Tumor xenografts revealed substantial reductions in tumor growth for both NHE1-ko and CA9-ko. A notable induction of CA12 occurred in NHE1/CA9-dko tumors indicating a potential means to compensate for loss of pH regulating proteins to maintain growth. Overall, these genomic knockout results strengthen the pursuit of targeting tumor cell pH regulation as an effective anti-cancer strategy.