Calcineurin Homologous Protein Expression Regulates Na+/H+ Exchanger 1 Dependent Tumor Survival
Authors: Cottle Wayne Taylor , Wallert Mark Anthony , Provost Joseph John 
Published: November 2023

Calcineurin Homologous Proteins 2 (CHP2) is a cofactor which regulates the Sodium Hydrogen Exchanger Isoform 1 (NHE1), a transmembrane proton pump vital in maintaining pH homeostasis. During nascent tumor development, cancer cells experience an acidic and serum deprived microenvironment as a result of anaerobic metabolism and poor diffusion kinetics. To survive, cells must engage regulatory proteins like NHE1 to prevent acidosis. CHP1 is an isoform required for basal NHE1 activity, and CHP2 is thought to compete for, and augment this role by an unknown mechanism. CHP2 binds to a common CHP site but is the only isoform shown to be uniquely expressed in malignant tissues. Maintenance of steady state intracellular pH (pHi) in serum starved human lung fibroblasts is NHE1 and CHP2 dependent. Real time PCR found a reduced NHE1 expression was concomitant with a decrease in pHi of 0.19 (s=0.02, n=12) and CHP2 expression knockdown yielded an intracellular pH decrease of 0.23 (s=0.02, n=12). These data suggest that both NHE1 and CHP2 play a significant role in pHi homeostasis regulation and that cells experiencing serum deprivation are more susceptible to acidification when CHP2 expression is reduced. Though proton transport is clearly effected by CHP2 expression, it's effect on the kinetics of proton transport is still unclear. To determine the effect of NHE1 and CHP2 on proton transport kinetics we are measuring the rate of pHi change during recovery from an ammonium chloride acid load. Preliminary results show the ΔpHi in wildtype lung fibroblasts to be 0.31 pH/min (n=32) while NHE1 KD and CHP2 KD both show a rate of 0.16 pH/min (n=28, n=32). Tests conducted in serum deprivation and hypoxic conditions, mimicking the nascent tumor environment, will determine the effect of the tumor microenvironment on proton transport kinetics. To uncover the mechanism driving transport kinetics, we are also studying NHE1, CHP1, and CHP2 expression. Western blot analysis of cultured cells and human tissues reveals an increase in CHP2 expression in nearly each lung tumor cell line and tissue examined. To quantitate this change in expression, real‐time PCR (RT‐PCR) was used to measured RNA expression of NHE1, CHP1, and CHP2 in control and serum‐deprived conditions. Serum deprivation in H1299 lung cells has no significant effect on gene expression of NHE1 (n=3) and decreased other CHP isoform expression by 0.4 fold (CHP1 [n=3]) but does increase CHP2 expression. This supports western blot analysis suggesting that neither the transcriptional nor peptide level regulation of NHE1 and CHP1 are responsible for the ability of tumor cells to maintain pHi homeostasis during serum deprivation. CHP and NHE expression in paired diseased and adjacent normal tissue is also examined. Understanding the expression levels of NHE1, CHP1, and CHP2 along with their cellular effects will help characterize the mechanism governing nascent tumor survival and will aid in the development of treatment modalities for non‐small cell lung cancer.

Support or Funding Information

This work was funded by the Beckman Scholars Program.