Na⁺/H⁺ exchanger‐dependent intracellular alkalinization is an early event in malignant transformation and plays an essential role in the development of subsequent transformation‐associated phenotypes

Abstract

In this study we investigate the mechanism of intracellular pH change and its role in malignant transformation using the E7 oncogene of human papillomavirus type 16 (HPV16). Infecting NIH3T3 cells with recombinant retroviruses expressing the HPV16 E7 or a transformation deficient mutant we show that alkalinization is transformation specific. In NIH3T3 cells in which transformation can be turned on and followed by induction of the HPV16 E7 oncogene expression, we demonstrate that cytoplasmic alkalinization is an early event and was driven by stimulation of Na+/H+ exchanger activity via an increase in the affinity of the intracellular NHE-1 proton regulatory site. Annulment of the E7-induced cytoplasmic alkalinization by specific inhibition of the NHE-1, acidification of culture medium, or clamping the pHi to nontransformed levels prevented the development of later transformed phenotypes such as increased growth rate, serum-independent growth, anchorage-independent growth, and glycolytic metabolism. These findings were verified in human keratinocytes (HPKIA), the natural host of HPV. Results from both NIH3T3 and HPKIA cells show that alkalinization acts on pathways that are independent of the E2F-mediated transcriptional activation of cell cycle regulator genes. Moreover, we show that the transformation-dependent increase in proliferation is independent of the concomitant stimulation of glycolysis. Finally, treatment of nude mice with the specific inhibitor of NHE-1, DMA, delayed the development of HPV16-keratinocyte tumors. Our data confirm that activation of the NHE-1 and resulting cellular alkalinization is a key mechanism in oncogenic transformation and is necessary for the development and maintenance of the transformed phenotype.

Reactive oxygen species-mediated regulation of the Na+–H+ exchanger 1 gene expression connects intracellular redox status with cells' sensitivity to death triggers

“…22 Along similar lines, Reshkin et al 31 have recently demonstrated that just as efficient Ras-mediated transformation requires activation of Rac, NHE-1 protein expression was equally implicated in malignant transformation and the development of the transformed phenotype. 32 Taken together, these findings suggest that, in addition to activation of the exchanger leading to alkaline pHi, redox regulation of NHE-1 gene expression could be critical in tumor cell formation. In particular, resistance of tumor cells to death triggers due to a slight increase in intracellular O 2 KÀ could be linked to the regulation of NHE-1 protein expression.…”

Traditional and emerging roles for the SLC9 Na+/H+ exchangers

“…Since these initial observations, numerous studies have examined the role of NHE1 in cancer. NHE1-dependent intracellular alkalinization plays an important role in the development of a transformed phenotype, as inhibition of NHE1 activity prevents this [141]. In breast cancer and leukemic cells, inhibition of NHE1 exerts a protective effect against cancer, inducing apoptosis [142,143].…”

Metabolic reprogramming of the tumor

“…At this level, lactate has a central role in promoting disordered growth by stimulating the production of hyaluronan and the expression of its membrane receptor, CD44, enhancing cell motility and extracellular matrix looseness (Stern et al, 2002). Equally important is the H þ extrusion and the extracellular acidification brought not only by MCT4 (Gallagher et al, 2007) but also by Na þ /H þ exchangers (Reshkin et al, 2000) and other transmembrane channels whose levels are orchestrated in cancer cells (Pouyssegur et al, 2006). This strategy maintains intracellular alkalinization, exerting a positive feedback loop on glycolysis, through activation of 6-phosphofructo-1-kinase (Erecinska et al, 1995), whereas extracellular acidification triggers apoptosis in neighboring cells with normal p53 levels (Williams et al, 1999).…”