Microenvironmental acidity plays an important role in the response of malignant tumors to a wide variety of drugs and is likely a leading cause of chemotherapeutic failure in cancer treatment. A key factor in this resistance is the “reversed pH” gradient. That is, cancer cells are characterized by both an acidic extracellular pH (pHe) and a normal or alkaline cytoplasmic pH (pHi) [
27,
28]. The alkaline pHi appears to confer resistance to both the hostile acidic milieu and drug cytotoxicity [
29‐
33]. A number of studies have demonstrated that resistance to cisplatin and doxorubicin is associated with an elevation of pHi in multiple tumor cell lines (human epidermoid cancer, human prostate cancer, human ovarian cancer, and myeloma, a series of human lung and breast cancer cell lines) [
33‐
37]. Similarly, cancer cell lines that are evolved to become drug resistant have a more alkaline pHi and a more acidic pH in subcellular organs when compared to the wild-type drug sensitive cells (HL60, K562, CEM, and MCF7) [
38]. Many human spontaneous tumors have similar reversed gradients suggesting a clinical relevance for these studies [
39]. While there are many potential mechanisms of resistance, it is clear that reversed pHe/pHi gradient interferes with the passage of drugs across the lipid bilayer of cells. Many anticancer drugs (such as doxorubicin and mitoxantrone) are weak bases which are neutralized and inactivated by protonation in the acidic microenvironment surrounding the cells or sequestered in intracellular acidic vesicles or endosomes [
40‐
42]. An additional pH-dependent mechanism of drug resistance, recently described for cisplatin, includes both extracellular sequestration and exosomes mediated elimination of the drug from melanoma cells [
43]. Interestingly, other studies have shown that an acidic pH increases the tumor cell exosomes release as well [
44].
3.1 Strategies of tumor cells to survive in an acidic environment
As noted above, cancer cells may use acid as a form of niche engineering in which they actively build an environment that is favorable for their own growth and survival but toxic to competitors and potential predators (such as the immune system). This appears to represent an evolutionary strategy termed “spite” in which an individual evolves a strategy that decreases its own fitness but with the benefit (in this case an acidic environment) that reduces the fitness of other normal and tumor populations and, thus, promotes growth and invasion. A key component of this putative evolutionary sequence is acquisition of adaptive strategies to evade acid-mediated toxicity [
45]. These strategies include a series of proton export mechanisms, which are found both in the lipid bilayer of the external cell membrane and in intracellular compartmental membranes, including vacuolar type ATPase (V-ATPase) and the proton transporters NHE-1, monocarboxylate transporters (MCTs), CAs (mainly CA-IX), adenosine triphosphate synthase, Na(+)/HCO
3(−) co-transporter, and the Cl(−)/HCO
3(−) exchanger. These proton pumps are known to be overexpressed and/or overactivated in cancer cells when compared with their non-transformed counterparts. The availability of several inhibitors specific for these proton extrusion mechanisms has allowed investigation of their role in the maintenance of the reversed proton gradient and consequently in the acquisition of the malignant phenotype.
V-ATPase is an enzyme composed of multiple subunits, ubiquitously present in the membranes of vacuolar systems of animal cells. It is critical in vacuole acidification, thus, playing a crucial role in receptor-mediated endocytosis, intracellular trafficking of late endosomes, the transport of lysosomal enzymes from the Golgi apparatus to lysosomes, and the creation of the microenvironment necessary for proper protein transport, exchange, and secretion [
46,
47]. V-ATPases can also be expressed in the plasma membrane of cancer cells [
48‐
59] probably due to their enhanced exocytotic events and membrane-recycling mechanisms. Messenger RNAs and/or protein expression levels of different V-ATPase subunits have been shown to be increased in several cancer tissues and cell lines (human hepatocellular carcinoma, breast tumors and melanomas, esophageal squamous cancer cells, oral squamous cell carcinoma, human pancreatic carcinoma, and non-small cell lung cancer) compared with normal tissues [
48,
51,
55,
60‐
64]. Moreover, the intensity of V-ATPase expression has been reported to associate to the pathological type and grade, both in non-small cell lung cancer and in pancreatic carcinoma [
48,
55]. V-ATPase overexpression and its localization to the plasma membrane have been associated with the malignant phenotype in terms of invasiveness and metastatic potential and drug resistance [
35,
48‐
50,
61,
62]. Recently, the increased expression of subunit of V-ATPases on the membrane of human melanoma cells deriving from metastatic lesions has been clearly shown [
65] suggesting a role in cancer progression and in the metastatic cascade. These data may provide a new marker of tumor malignancy.
The membrane-bound NHEs represent another class of proteins that can extrude protons in exchange for a cation to maintain intracellular electroneutrality. They are present at the surface of most cells where they have a central role in regulating cellular volume and pH homeostasis. NHE isoform 1 (NHE-1) is the most common isoform of the NHEs family, and it is ubiquitous in all mammalian cells. In normal cells, NHE-1 activity is allosterically increased with decreasing pHi, resulting in rapid activation and subsequent elevation of pHi as a consequence of increased proton extrusion [
66]. An aberrantly elevated NHE-1 activity has been correlated in tumors with pHe/pHi gradient reversal and in turn, associated with tumor origin, local growth, and further progression of the metastatic process [
67,
68]. Molecular mechanisms underlying this tumor associated NHE-1 constitutive activation are only recently becoming evident. NHE-1 regulation occurs through the phosphorylation of key amino acids in the cytosolic domain as well as by its interaction with other intracellular proteins and lipids. Ultimately, NHE-1 regulators alter transport activity by altering its affinity for intracellular H
+ such that it is more active at a more alkaline pHi [
69]. In breast cancer cells, NHE-1 is highly expressed in invadopodia, invasive protrusions capable of proteolytic degradation of the extracellular matrix, where they play an essential role in creating the acidic extracellular microenvironment that facilitates proteases activity [
70,
71]. As yet, large clinical studies examining NHE-1 expression in human tumors are lacking. However, recently NHE genes expression was found to be strongly upregulated in several lung cancer histotypes [
60]. Interestingly, the expression change patterns have been reported to be highly complementary between NHE genes and the V-ATPase genes in different cancer types, suggesting that the NHE antiporters may play a complementary role to that of the V-ATPases [
60].
Monocarboxylate transporters (MCTs) are proton symporters that transport monocarboxylates such as
l-lactate, pyruvate, and the ketone bodies across the plasma membrane. There are four isoforms, MCTs 1–4, which are known to perform this function in mammals, each with distinct substrate and inhibitor affinities. MCTs play essential metabolic roles in most tissues, with their distinct properties, expression profile, and subcellular localization matching the particular metabolic needs of a tissue. They also play a key role in maintaining the pH homeostasis [
72]. MCT1, MCT2, and MCT4 genes have been shown to be upregulated in several cancer histotypes (breast, colon, lung, ovary) with a considerable variation in the MCT isoforms expressed in different tumors [
73,
74]. MCT1, MCT4, and their chaperone CD147 are overexpressed in the plasma membrane of glioblastomas compared with diffuse astrocytomas and non-neoplastic brain [
75]. MCT1 and MCT4 both have elevated activity in human melanoma cells in response to low extracellular pH [
76]. MCT1 has been reported to be upregulated in neuroblastoma cells, and elevated MCT1 mRNA levels have been detected in fresh neuroblastoma biopsy samples, with a positive correlation between expression level and risk of fatal outcome [
77]. Xu et al. [
60] recently reported MCT genes to be upregulated in breast, colon, liver, and two lung (adenocarcinoma, squamous cell carcinoma) cancers, but not in prostate cancer. Interestingly, lactate released as a waste product of glycolytic energy production in hypoxic tumor microenvironment has been demonstrated to constitute a prominent substrate that fuels the oxidative metabolism of tumor cells in oxygenated regions, and MCT1 has been shown to be involved in lactate uptake by a human cervix squamous carcinoma cell line that preferentially utilized lactate for oxidative metabolism [
78].
Carbonic anhydrases (CA) and HCO
3− transporters have also been found to play a role in neutralizing the protons in cancer cells. The membrane-bound CAs catalyze the otherwise slow reaction from CO
2+ H
2O to H
2CO
3, which dissociates into HCO
3
− (bicarbonate) and H
+ in an acidic extracellular environment. The HCO
3
− is then transported across the membrane through an HCO
3
− transporter into the intracellular environment, where it reacts with a H
+ to form CO
2 and H
2O; the CO
2 is freely membrane-permeable and diffuses out of the cell, forming a cycle for removing excess H
+ [
79,
80]. CA isoform 9 is known to be inducible by hypoxia [
81] and, unlike most other CA isoforms, is associated with many tumors [
82,
83]. Very few normal tissues, with the exception of stomach [
84], express significant levels of CA9 so that positive staining for CA9 is considered an established marker of tumor hypoxia and a clinical indicator of aggressive cancers (for example, breast and bone) with poor prognosis [
85‐
87]. In addition to CA9, CA12 and CA14 genes have been recently reported to show upregulation in breast, colon, liver, and two lung (adenocarcinoma, squamous cell carcinoma) cancers (but not in prostate cancer), with two HCO
3
− transporters, NBC2 (SLC4A5) and NBC3 (SLC4A7), also being upregulated in colon, liver, and two lung cancers types analyzed [
60].