Assessing Na+/H+ exchange and cell effector functionality in metastatic breast cancer

Abstract

Metastasis is the leading cause of mortality in patients with breast cancer. In triple-negative breast cancer, high recurrence rates, increased invasive capacity of cells, and their aggressive ability to metastasize at secondary sites dictate patient survival. The Na+/H+ exchanger isoform 1 (NHE1) plays a critical role in controlling the metastatic potential of these cells. Its activity results in an elevation of intracellular pH and in extracellular acidification, a key step in the establishment of the tumor microenvironment. Here, we describe assays for characterization of Na+/H+ exchanger activity and its related downstream physiological effects on triple-negative breast cancer cells. Na+/H+ exchanger activity can be routinely and rapidly measured in live cells with a fluorometric assay that assesses changes in intracellular pH. Characterization of downstream cell effector function as a result of Na+/H+ exchanger activation can be evaluated by measuring directed cell migration and invasion. Cell migration is assessed with wound-healing assays, where a gap is introduced in a confluent monolayer of cells and the rate of gap closure is measured over time. Cell invasion is assessed in the short-term by transwell invasion assays that track cell movement through an extracellular matrix. Long-term invasiveness, growth and proliferation can be assessed with 3-D invasion assays using transwell inserts fitted with specialized scaffolds optimized for 3-D cell culture. Taken together these assays provide powerful tools for testing the effects of altering Na+/H+ exchanger activity with chemical inhibition on the metastatic capacity of breast cancer cells.

Towards an Integral Therapeutic Protocol for Breast Cancer Based upon the New H+-Centered Anticancer Paradigm of the Late Post-Warburg Era

“…Most importantly, a series of publications in this line have shown that the H + extrusion in BC cells, especially in triple-negative breast cancer (TNBC), is mainly mediated by NHE1 overexpression and H + -extrusion, with NHE1 being the real actor that induces a high pHi-mediated carcinogenic effect on breast cells [ 44 ]. Furthermore, H + extrusion also is the main etiological mediator in the transition from ductal carcinoma in situ to invasive BC, where even a precancerous lesion already shows a higher than normal proton export rate [ 44 , 45 , 46 ] ( Figure 2 ). Most recently, the intricate relationships of tumor pHi and pHe with ion channels and changes in membrane potential have been widely discussed and reviewed [ 47 ].…”

“…Most recently, however, other channels, transporters and even certain aquaporins (AQP), mainly AQP1 and AP3, as well as Cl − channels with Ca 2+ influx, have also been shown to be important in the initiation and progression of TNBC [ 49 , 50 ]. All these fundamental data clarify why and how NHE1, and its main consequences, H + extrusion and an elevated pHi, are involved in BC etiology, invasion, and the metastatic process, as well as in multiple drug resistance (MDR) [ 43 , 44 , 45 , 46 , 51 , 52 ]. While a pathologically elevated pHi should be considered to be the main responsible factor of BC-promoting activity, it secondarily induces a highly pathological and damaging extracellular/intratumoral TME with the final result of inducing CPR.…”

A New and Integral Approach to the Etiopathogenesis and Treatment of Breast Cancer based upon its Hydrogen Ion Dynamics

“…Importantly, H+ extrusion alone has been implicated in the transition and progression from precancerous ductal carcinoma in situ to invasive BC. Of note, the precancerous lesion already shows a higher than normal proton export rate [36,38,102]. Indeed, the invasive BC cells show a higher pHi and a higher production and exportation of H + into the TME than noninvasive cells [36,97,105].…”

“…extrusion has a carcinogenic effect by itself on breast cells [36]. The same alteration also plays a fundamental role in the metastatic process and in multiple drug resistance (MDR) [37,38,102,103]. In these studies, NHE1 hyperactivity and/or a high pHi act as an early and decisive driver in BC carcinogenesis and also in most, if not all, other human malignancies [33,104].…”

“…To this already extensive list, PPs should be added, as well so many different natures and origins are known to be carcinogenic, the up-regulation of any of of them or several ones in a synergistic combination with other stimulators in the same direction, indicates that the pHi/pHe abnormalities so induced may exert their effect through the same acid-base intracellular (IC)/extracellular (EC) deregulated dynamics, which suggests the existence of a universality of phenomenon involved in human carcinogenesis and etiopathogenesis, BC being no exception to this rule [25]. 4.9) NHE1-related genetics of breast cancer.The Na+/H+ exchanger isoform 1 (NHE1) is nowadays recognized as one of the most important factors involved in BC etiology and etiopathogenesis[34,[36][37][38]102]. NHE1 has been found to be produced from the APNH gene located on chromosome 1p35-36, whose deletion has been blamed to be involved in the development of different tumors, BC among them[162].…”