Abstract
Hypoxia and extracellular acidosis are pathophysiological hallmarks of aggressive solid tumors. Regulation of intracellular pH (pHi) is essential for the maintenance of tumor cell metabolism and proliferation in this microenvironment and key proteins involved in pHi 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 pHi 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 pHi 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 pHi. 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.
Highlights
Solid tumors are distinguished from normal tissues by the presence of an acidic and hypoxic extracellular environment arising from exacerbated and predominantly glycolytic tumor cell metabolism
Maintenance of pHi is essential for a wide range of cellular functions and tumor cells have proven to be exceptional at regulating their pHi in the face of extracellular acidosis
The essential nature of stable pHi for cellular function has led to the desire to disrupt pHi regulation in cancer cells for novel therapeutic development
Summary
Solid tumors are distinguished from normal tissues by the presence of an acidic and hypoxic extracellular environment arising from exacerbated and predominantly glycolytic tumor cell metabolism Both hypoxiaregulated and metabolism-related proteins are being pursued as cancer specific targets for therapeutic development (For a recent review refer to [1]). Enthusiasm for the targeting of tumor cell pHi regulation as an www.impactjournals.com/oncotarget anti-cancer strategy was generated from mutated cells exhibiting exclusively glycolysis or respiration in the presence or absence of NHE1 Both in vitro and tumor xenografts using these cells demonstrated the essential nature of pHi regulation via NHE1 for both tumor initiation and growth [5,6,7,8,9]. NHE1 continues to be investigated for its importance in tumor cell progression and in particular cell migration/metastasis and blockade of the H+ secreting strategy in cancer cells remains an attractive therapeutic target [14,15,16,17]
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