Extracellular acidity, a - Springer Link

Cancer Metastasis Rev (2014) 33:823–832 DOI 10.1007/s10555-014-9506-4

CLINICAL

Extracellular acidity, a “reappreciated” trait of tumor environment driving malignancy: perspectives in diagnosis and therapy Silvia Peppicelli & Francesca Bianchini & Lido Calorini

Published online: 2 July 2014 # Springer Science+Business Media New York 2014

Abstract Tumors are ecosystems which develop from stem cells endowed with unlimited self-renewal capability and genetic instability, under the effects of mutagenesis and natural selection imposed by environmental changes. Abnormal vascularization, reduced lymphatic network, uncontrolled cell growth frequently associated with hypoxia, and extracellular accumulation of glucose metabolites even in the presence of an adequate oxygen level are all factors contributing to reduce pH in the extracellular space of tumors. Evidence is accumulating that acidity is associated with a poor prognosis and participates actively to tumor progression. This review addresses some of the most experimental evidences providing that acidity of tumor environment facilitates local invasiveness and metastatic dissemination, independently from hypoxia, with which acidity is often but not always associated. Clinical investigations have also shown that tumors with acidic environment are associated with resistance to chemotherapy and radiation-induced apoptosis, suppression of cytotoxic lymphocytes, and natural killer cells tumoricidal activity. Therefore, new technologies for functional and molecular imaging as well as strategies directed to target low extracellular pH and low pH-adapted tumor cells might represent important issues in oncology.

Keywords Tumor metabolism . “Warburg effect” . Extracellular acidity . Invasiveness . Metastatic dissemination . Stemness . Molecular imaging . Novel therapeutic strategies

S. Peppicelli : F. Bianchini : L. Calorini (*) Department of Experimental and Clinical Biomedical Sciences, Section of Experimental Pathology and Oncology, University of Florence, Viale G.B. Morgagni, 50, 50134 Florence, Italy e-mail: [email protected]

1 Introduction Tumor cells accumulate increasingly genetic and epigenetic alterations, leading them to acquire all the characteristics of malignant phenotype. However, in concert with this “genetic instability,” a key role in favoring changes in tumor cells is played by local host factors [1, 2], among which, particular attention has been devoted to the interactions that tumor cells establish with various host cells that reside in or are attracted into tumor environment. The bidirectional interaction between tumor cells and host cells is recognized as crucial for the decision whether tumor cells progress toward metastatic dissemination or remain dormant [3–7]. Changes expressed by host cells during tumor development might be also related to their location inside tumor mass. Indeed, most tumors show heterogeneous areas often characterized by low oxygen tension (hypoxia), elevated interstitial fluid pressure, and high lactate and H+ concentration. These changes are largely caused by a combination of poor tissue perfusion due to abnormal tumor vasculature, uncontrolled proliferation, and altered energy metabolism [8]. In addition, the uncontrolled growth of tumor cells compresses the intra-tumor lymphatic vessels. Consequently, there are no functional lymphatic vessels inside solid tumors, whereas functional lymphatic vessels are present only in peri-tumoral tissues [9, 10]. Both, the high permeability of tumor blood vessels and the lack of functional lymphatics are key contributors to the development of an interstitial hypertension in neoplastic tissues [11]. As a result, the hydrostatic and colloid osmotic pressures become almost equal between intravascular and extravascular spaces, compromising the delivery of nutrients as well as therapeutic agents. The consequent hallmarks of tumor environment are hypoxia, elevated interstitial fluid pressure, low glucose, and high lactate leading to a low extracellular pH (pHe). In this review, we will discuss on tumor cell metabolism and evidence that acidity of tumor extracellular space

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represents a direct contributor to the process of tumor progression and normalization of pHe emerges as a new supportive strategy for tumor therapy.

2 Tumor cell metabolism and acidosis of extracellular space of tumors Most of tumor cells use glycolysis to produce more than 50 % of energy requested, even when there is enough O2 to support mitochondrial function. In most mammalian cells, which rely on mitochondrial oxidative phosphorylation to generate the energy needed for cellular processes, glycolysis is inhibited by the presence of oxygen, which allows mitochondria to oxidize pyruvate to CO2 and H2O. This inhibition is termed the “Pasteur effect,” after Louis Pasteur, who first demonstrated that glucose flux was reduced by the presence of oxygen [12]. In contrast to normal cells, cancer cells, even in the presence of sufficient oxygen to support mitochondrial respiration, use glycolysis, the so-called “Warburg effect” [13, 14]. This phenomenon was first reported by Warburg in the 1920s, leading to hypothesis that cancer results from impaired mitochondrial metabolism. Although the “Warburg hypothesis” has proven incorrect, an increased conversion of glucose to lactic acid and H+ in tumors has been constantly demonstrated. Dr. A. Braunstein, late as in 1921, observed that in those diabetic patients who developed a cancer, glucose secretion in the urine disappeared [15]. “Aerobic glycolysis” leads to the conversion of one molecule of glucose into 2 molecules of lactic acid and 2 H+ to produce 2 ATP compared to the 36 ATP produced with aerobic metabolism [13, 14]. Although ATP production by glycolysis is more rapid than by oxidative phosphorylation, it is far less efficient in terms of ATP generated per unit of glucose consumed; therefore, tumor cells implement an abnormally high rate of glucose uptake through an enhanced expression of HIF-1α-dependent glucose transporters (GLUT) [16]. The diffusion coefficient for glucose is also larger than for oxygen, thus high rate of glucose capture is facilitated. A number of oncogenes and tumor suppressor genes are involved in the metabolic switch to glycolysis, such as PI3K (phophatidylinositol-3 kinase)/Akt/mTOR (mechanistic target of rapamycin), HIF-1α, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), and c-Myc [17, 18]. In tumor cells, Akt suppresses β-oxidation of fatty acids, but increases de novo lipid synthesis. Akt also activates mTOR, a key regulator of cell proliferation integrating signaling from insulin, amino acid availability, cellular energy status, and oxygen levels. In cancer cells, mTOR induces aerobic glycolysis by upregulating key glycolytic enzymes, especially through its downstream effectors HIF-1α. HIF-1α is stabilized under normoxic conditions by the products of glycolysis, lactate, and pyruvate [19]. Consequently, HIF-1α regulates an elevated number of genes involved in glycolysis, lactate

Cancer Metastasis Rev (2014) 33:823–832

production, and lactate/proton extrusion, including iron metabolism, angiogenesis, and metastasis. Such genes include glucose transporters (GLUT1 and 3) [20], glycolytic enzymes (hexokinase I and II, phosphofructokinase-1, aldolase-A and C, phosphoglycerate kinase 1, enolase-a), the pyruvate dehydrogenase kinase 1 (PDK1, that inactivates the enzyme responsible for conversion of pyruvate to acetyl-CoA [21] and leads to an enhanced flux of glycolysis), lactate production (LDHA, encoding lactate dehydrogenase A, which converts pyruvate to lactate [22]), and lactate extrusion (MTC-4, the monocarboxylate transporter-4). Lactate production in cancer cells is also facilitated by the increased expression of PK type M2 (PKM2) produced by alternative splicing of the PKM gene. PKM2 catalyzes the rate-limiting step of glycolysis, controlling the conversion of phosphoenolpyruvate (PEP) to pyruvate, and thus ATP generation. By slowing the passage of metabolites through glycolysis, PKM2 promotes the shuttling of these substrates through the pentose phosphate pathway (PPP) and other alternative pathways, so that large quantities of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and other macromolecules needed to support the rapid cell division are produced [23]. So lactate, the end product of glycolysis, is produced in large excess in tumors and might constitute an alternative metabolic fuel for those cancer cells characterized by a high energetic demand [24–26]. This is a phenomenon that has been well characterized in non-tumor tissues, including in skeletal muscle, brain, and liver [27, 28]. Furthermore, Sonveaux et al. demonstrated the existence of a “metabolic symbiosis” between hypoxic and aerobic cancer cells, in which lactate produced by hypoxic cells is taken up by aerobic cells, which use it as their principal substrate for oxidative phosphorylation [24]. This is the socalled “Reverse Warburg effect” also described studying the metabolic symbiosis between cancer-associated fibroblasts (CAFs) and cancer cells. Following activation, CAFs shift their metabolism toward glycolysis and generate lactate which cancer cells can use for tricarboxylic acid cycle (TCA), growth, and survival [29, 30]. Cells derived from tumors typically maintain their metabolic phenotypes in culture under normoxic conditions, indicating that aerobic glycolysis is constitutively upregulated through stable genetic or epigenetic changes [13] and it could be a crucial component of the malignant phenotype. A possible explanation for the switch to aerobic glycolysis is that proliferating tumor cells have important metabolic requirements beyond ATP, and some glucose must be diverted to macromolecular precursors such as acetyl-CoA for fatty acids, glycolytic intermediates for nonessential amino acids, and ribose for nucleotides. A high glycolytic rate has additional advantages, including it avoids the production of ROS through respiration and may evade apoptotic signaling which is linked to mitochondrial function. The increased glucose uptake is useful for diagnostic purposes, as in the case of monitoring uptake of glucose analog


tracer 2-(18F)-fluoro-2-deoxy-D-glucose (18FdG) by positronemission tomography (PET). The glucose analog tracer used in PET imaging of oncology patients has unequivocally shown that numerous primary and metastatic human cancers show significantly increased glucose uptake. The constitutive upregulation of glycolysis requires an additional adaptation in order to maintain an intracellular pH compatible with a high proliferation rate. Short-term and longterm mechanisms exist [31]. The short-term mechanisms consisting of rapid buffering responses such as physiochemical buffering of the acids, metabolic consumption of nonvolatile acid or transfer of acid from the cytosol to the organelles, act quickly but fail to maintain the intracellular environment at neutral pH for a long time. The mechanisms for long-term pHi regulation are represented by multiple and redundant families of H+ transporters, which release protons and lactate into extracellular environment, such as Na+/H+ exchanger 1 (NHE1), carbonic anhydrases (CAIX, CAXII, and CAII), Cl−/HCO3− transporters (Na+/HCO3− co-transporters, NBCs), anion exchange protein 1 (AE1), proton-linked monocarboxylate transporters (MCT1 and 4), and vacuolar H+-ATPases (V-ATPases) [32]. Thus, the acidic pHe of tumors is not solely due to poor perfusion but instead is an integral property of cancer cells themselves, although we cannot exclude that low lymphatic removing activity and diffusion coefficient, consistent with a reduced flux relative to proton production rate, participate in falling pHe. Tumor acidification is now considered a critical hallmark of tumor environment [33].

3 Cancer acidosis drives local aggressiveness and distant colonization of tumor cells Clinical investigations have shown that tumors with acidic environment are associated with poorer prognosis and an enhanced metastatic incidence [34], increased mutation rate [35], resistance to chemotherapy [36] and radiotherapy [37]. Tumor acidity also confers multiple advantages to the aggressiveness of tumor cells affecting several properties involved in metastatic dissemination [38]. Frequently, a minimum in pHe has been observed near tumor periphery, where tumor cells are invading normal tissues [39]. Hypoxia also stimulates invasiveness of tumor cells [40], thus we could expect that low extracellular pH and hypoxia always co-localize within tumor regions, and instead there is often a lack of spatial correlation among these parameters [41]. Potential explanations of this lack of correlation could be due to the enhanced glucose uptake for glycolytic ATP generation in conditions of high oxygen tension, or to the possibility that some tumor vessels carrying hypoxic blood are unable to deliver adequate quantity of oxygen to the cells but are able to carry away the waste products (e.g., lactic acid). This latter explanation is consistent with anemia of tumor-bearing patients, a key effector in

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worsening tumor oxygenation. Anemia in tumor-bearing patients is referred to as “anemia of chronic disease.” One essential characteristic of migrating tumor cells is their polarization along the direction of movement [42]. Recent studies indicate that the intracellular pH may serve locally as a regulator of cell polarization and migration because it modulates cytoskeletal dynamics directly by affecting the actinbinding proteins cofilin [43], profilin [44], twinfilin [45], villin [46], and talin [47] and indirectly by enhancing the activity of the low-molecular-weight GTPase CDC42 [48]. In addition to the de novo assembly of actin filaments, migration requires the remodeling of cell-substrate adhesion, a process that requires an increased pHi and a decreased pHe [49]. Human melanoma cells migrate faster at a pHe 7 compared with a pHe 7.4 because low pH regulates the dynamics of extracellular matrix (ECM)-integrin attachments directly altering integrin conformation and affecting integrin-ligand binding. Through computational molecular dynamics simulations, Paradise et al. found that acidic extracellular pH promotes opening of the αVβ3 headpiece, indicating that acidic pH can thereby facilitate integrin activation [50]. A recent aspect of tumor cell invasiveness includes the invadosome microdomains. Invadosomes are proteolytic actin-rich protrusion structures involved in cancer-associated matrix remodeling and whose formation, maturation, and function are highly stimulated by the reversed pH gradient of tumors [51]. Also hypoxic conditions improve invadosome formation, with a relative increase in invasiveness of cancer cells, through a NHE1-dependent activity [52]. Two studies demonstrated that glycolytic enzymes are enriched in the invadosome structures [53], providing new insights on the link between cell metabolism and the resulting effects on pHe and ECM degradation at the invadosome site. The ATP produced locally could be used for energy-demanding processes involved in cytoskeleton remodeling, secretion/ endocytic cycles and even for the functioning of pH regulators. It could be postulated that invadosome represents a critical microdomain enriched in metabolic enzymes, proton producers and proteases, able to promote and sustain ECM degradation and subsequent tumor cell invasion. It has been reported that an acidic pHe-stimulated invadosome structures enhance invasion of tumor cells facilitating the redistribution of active cathepsin B, a lysosomal aspartic proteinase with acidic pH optima, to the surface of malignant cells [54, 55]. Acid-activated cathepsins L also participate to amplify proteinase cascade through activation of urokinase-type plasminogen activator (uPA) [56] that converts plasminogen to plasmin, which degrades various components of the ECM such as fibronectin, laminin, proteoglycan, and collagen [57], and also activates latent collagenase and growth factors [58]. Concomitant expression of uPA and uPA receptor (uPAR) by tumor cells results in a high invasive capability [59]. Additionally, acid-activated cathepsin B cleaves secreted latent metallo-

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proteases (MMPs) into active enzymes. MMPs are a family of structurally related zinc-dependent endopeptidases, collectively capable of degrading all components of ECM [60]. MMPs have long been associated with invasiveness, angiogenesis, and tumor cell dissemination [61–63] due to their capacity to help tumor cells to cross structural barriers, including basement membranes, to migrate into the blood, and to extravasate and colonize in distant host tissues. Kato et al. have shown that, in mouse metastatic B16 melanoma cells, expression of MMP-9 and in vitro invasiveness are induced by acidic pHe (pHe 5.4–6.5) [64]. Another component of basement membrane to be degraded by tumor cells to disseminate are the heparan sulphate chains. Toyoshima and Nakajima have reported that heparanase has an optimal pH of 4.2, but a significant heparanase activity persists at pH 6.0–6.5, suggesting that the acidic environment of tumors may activate the degrading properties of tumor heparanases [65]. Dissemination of tumor cells was either favored by angiogenesis and lymphangiogenesis through the preeminent activity of specific growth factors, VEGF-A and C, respectively. It was demonstrated that a transient exposure to acidosis stimulates a NF-κB-dependent upregulation of VEGF-A in human tumor cells [66, 67]. Angiogenesis is favored also by acidinduced IL-8 [68]. Our laboratory showed that acidity stimulates VEGF-C expression and secretion in melanoma cells through NF-κB transcription factor. We also demonstrated that esomeprazole, a proton pump inhibitor (PPI) activated by a low pH, inhibits VEGF-C expression in acidic melanoma cells downregulating NF-κB [69]. VEGF-C, in addition to the well known role of VEGF-A in metastasis, might represent a new causal link between lymphangiogenesis and distant metastasis development [70]. A program which contributes to malignancy of tumor cells is the epithelial-to-mesenchymal transition (EMT), able to orchestrate series of events in which cell-cell and cell-ECM interactions are altered. During EMT, epithelial cells are released from the surrounding tissue and alter their polarity, the cytoskeleton is reorganized to confer the ability to move through a three-dimensional ECM, and a new transcriptional program with multiple biochemical changes are induced to assume and maintain a mesenchymal cell phenotype [71]. These include enhanced migratory capacity, elevated resistance to apoptosis, and greatly increased production of ECM components [72, 73]. We found that a reduced pH may promote in melanoma cells some aspects of EMT, such as several mesenchymal markers, resistance to the proapoptotic agents, MMP release and invasiveness through Matrigel. Acidicexposed tumor cells were also able to help invasiveness and in vivo organ colonization of nonacidic tumor cells, indicating a new cooperation between EMT-acidic cells and the nonacidic counterparts [74]. Extracellular acidity has a robust protective effect against apoptosis induced by multiple cytotoxic metabolic stresses, as low pH activates MAPK signaling in a


variety of cell type [75, 76], inducing suppression of apoptosis. Kumar et al. [77] found that acidic preconditioning suppresses apoptosis increasing expression of Bcl-xl in ischemic endothelial cells. In addition, Ryder et al. examined the role of the Bcl-2 family in apoptosis showing the promotion of high anti-apoptotic Bcl-2 family member by acidosis, probably mediated by the acid-sensing G protein-coupled receptor (the GPR65), via a MEK/ERK pathway [78]. An additional survival mechanism used by tumor cells exposed to low pH is the autophagy [79], an evolutionarily conserved catabolic process through which cytoplasmic proteins and organelles are self-digested, maintaining cellular metabolism through recycling of the degraded components [80]. EMT was proved to be associated with stemness of tumor cells [81], although some other studies indicate an inverse correlation between these two processes [82]. Acidic condition, independent of restricted oxygen, promotes the expression of stem cell markers, self-renewal, and tumor growth in glioma cells. Glioma stem cells (GSC) exert paracrine effects on tumor growth through elaboration of angiogenic factors, and low pH conditions augment this expression associated with induction of HIF-2α, a GSC-specific regulator. Induction of HIF-2α and other GSC markers by acidic stress can be reverted by elevating pH in vitro, suggesting that raising pHe may be beneficial for targeting the GSC phenotype [83]. There is increasing evidence that cancers can escape immune destruction by suppressing the anticancer immune response through maintaining a relatively low pH in their environment. The acquired properties enable cancer cells to suppress the anticancer activity of immune cells secreting chemokines to enhance recruitment of T regulatory cells to the tumor site or producing immunosuppressive cytokines [84]. The acidic environment has been shown to impair natural killer (NK) cells via restriction of IFNγ, IL-10 and TGFβ (Fig. 1) [85], to inhibit lymphocyte proliferation and dendritic cell maturation [86] and to lead to loss of T cell function of tumor-infiltrating lymphocytes. The T cell function could be completely restored by buffering the pH at physiological values [87]. Extracellular lactic acid generated by cancer metabolism has been identified as primary cause responsible for the pH-dependent T cell function-suppressive effect and new results suggest that tumor lactic acidosis suppresses cytotoxic T lymphocyte functions via inhibition of p38 and JNK/ c-Jun activation [88]. Ohashi et al. found that lactic acid secreted by tumor cells promotes tumor development by inhibiting the immune response by increasing arginase-1 (ARG1) expression in tumor-associated macrophages and inducing IL-23/IL-17 secretion [89]. Elevated lactate levels correlate with poorer prognosis and poor disease-free survival in several epithelial cancers, such as cervical, head and neck, non-small cell lung, and breast cancers [90]. In addition to the contribution to the immunologic escape, lactate mediates cell mobility [91], stimulates VEGF production by endothelial


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Fig. 1 Holistic framework of acidity on tumor cells

cells [92], NF-κB activity and IL-8 expression [93], and upregulates genes associated with aerobic metabolism, including factors involved in TCA cycle and electron transport [94]. Acidosis can also influence the resistance to anticancer chemotherapy. A principal mechanism leading to a multidrug-resistant (MDR) phenotype of tumor cells is represented by the ability of acidic pHe to increase the pump activity of the P-glycoprotein, a drug efflux transporter, presumably as a result of lowered intracellular calcium levels and inhibition of PKC [95]. Acidosis may also interfere in drug distribution [96] and efficacy favoring reduction in cycling cell fraction [97] and selection for apoptosis-resistant phenotypes [98]. The acidic pHe in tumors traps weakly basic drugs, thereby hindering influx of these drugs into the cells. In particular, it was reported that uptake of doxorubicin, vinblastine, and mitoxantrone is reduced in an acidic environment. Acidity also reduces sensitivity of tumor cells to radiation therapy [37, 99, 100]. Indeed, an acidic environment induces p53 expression [101], suppresses radiation-induced apoptosis [102, 103], and prolongs radiation-induced G2/M arrest, resulting in an increased DNA damage repair during the prolonged G2 arrest [104].

4 Potential diagnostic and therapeutic implications of cancer acidosis Perspectives in diagnosis Imaging is important for diagnosis, staging, and monitoring of response to treatment in patients with cancer. Newer imaging methods, termed functional and molecular imaging, can provide information about the physiology, biochemistry, protein expression, and genetics of tissues. The low extracellular pH of tumors by itself may function as a marker of most solid tumors and target of imaging

technology. Indeed, several advances were made in the in vivo measurement of tumor pH by pH-sensitive PET radiotracers, magnetic resonance spectroscopy (MRS), and imaging (MRI) [105]. PET scanning is based on the detection of tracer compounds conjugated with a positron emitter isotope, and its main advantage is the high-resolution 3D images of radiotracer distribution specific to a particular biochemical pathway. The tracer used most often in PET scanning is [18F]-FDG [106]. Recently, a low pH-dependent peptide has been developed, which selectively targets acidic tissues. The pH-low insertion peptide (pHLIP) is a water-soluble membrane peptide of 37 amino acids that inserts and folds across a cellular membrane lipid bilayer in response to low pH but not at a normal physiologic pH [107–109]. This pHLIP was used to detect tissue acidity and to diagnose primary tumors and metastatic lesions. Vavere et al. extended this method to investigate the acidic environment in prostate tumors using 64Cu-conjugated pHLIP (64Cu-DOTA-pHLIP) [110]. This construct accumulates in LNCaP and PC-3 tumors, with higher uptake and retention, that were correlated with differences in the bulk of pHe, measured by MRS using 31P chemical shift of pHe marker 3-aminopropyl phosphonate (3-APP). MRS analyses are based on a difference in chemical shifts between pH-dependent and -independent resonances. Several isotopes have been evaluated to determine tissue pH with MRS. 31P-MRS also provides a robust technique for simultaneously measuring pHi from the chemical shift of endogenous inorganic phosphate and pHe from the chemical shift of exogenous indicators, such as 3-APP [105]. Measurement was improved using 1H-MRS with the pH-sensitive H2 resonance of 2-imidazole-1-yl-3-ethoxycarbonyl propionic acid. Although these studies showed that tumor pHe was heterogeneous, they are still limited in spatial and temporal resolution [105]. Provent et al. (2007) have evaluated in vivo

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pHe in C6 glioma in rat brain using 1H MRS with the new probe 2(imidazol-1-yl)succinic acid (ISUCA). Voxel-byvoxel analysis showed that, both before and during glucose infusion, distribution of lactate and extracellular acidity were very different [39]. The authors suggest that proton influx and efflux cause pHe to be acidic at sites remote from lactate production. An alternative approach using MRI relies on perturbing the relaxivity of water via pH-dependent relaxation agents. A small molecule approach to measuring pH has been developed by Aime et al. [111] and Zhang et al. [112], who have synthesized gadolinium-based agents whose relaxivity is pH-dependent. In the case of the tetraphosphonate, gadolinium-DOTA-4AmP52, the hydrogen-bonding network created by phosphonate sidearm protonation provides a catalytic pathway for hydrogen exchange. For quantification, this approach requires accurate knowledge of the agent concentration in each voxel. Raghunand solved that problem using sequential injection of two gadolinium agents, one of which was pH-insensitive. This method has been applied to imaging pH in the kidneys and in rat brain gliomas [113]. Perspectives in therapy Robey et al. (2009) have shown that chronic use of oral bicarbonate as a cancer intervention selectively increased the pHe of tumors and reduced the formation of spontaneous metastases in mouse models of metastatic breast cancer and the rate of lymph node involvement [114]. Computer simulation used to verify the ability of sodium bicarbonate to increase pHe of tumors in vivo also indicates that the normalization of tumor acidity reduces invasiveness of tumor cells without altering the pH of blood or normal tissues [115]. Other studies testing the efficacy of the 2imidazole-1-yl-3-ethoxycarbonylpropionic acid (IEPA) demonstrated that it can reduce spontaneous and experimental metastases with the same efficacy of bicarbonate [116]. As an alternative strategy, a number of researchers have explored inhibition of membrane ion pumps involved in the maintenance of an alkaline internal pH by extruding protons or importing bicarbonate ions. Proton pump inhibition tents to decrease intracellular pH and the consequent intracellular acidification can have an impact on cancer cell behavior, for example suppressing the efficiency of glycolysis [117], and exerting antiproliferative and proapoptotic effects [118–120]. NHE is crucial in pH regulation and is expressed in every cell type. Of the nine members of the NHE family, NHE1 has received the most attention in the context of tumor celltargeted therapy [32]. Both the acidic pHe and the constitutively active NHE1 play a key role in driving proteasemediated digestion and remodeling of the ECM and the turning on of invasive phenotypes of the cells, scavenging normal tissue, and increasing motility through the formation of invasive structures such as leading-edge pseudopodia and invadopodia [121]. Amiloride was the first NHE inhibitor developed and it was shown to decrease VEGF-A production,


uPA and MMP activities, all of which essential for the metastatic process [122, 123]. Moreover, EIPA (5-(N-ethyl-Nisopropyl)amiloride) was recently demonstrated to compromise the growth of KRAS-transformed tumor xenografts, an effect that was attributed to inhibition of nutrient uptake by macropinocytosis [124]. Cariporide, another NHE1 inhibitor, was found to lower the intracellular pH of cancer cells [125], affect tumor angiogenesis, and inhibit tumor growth [126, 127]. Proton extrusion is also achieved by V-ATPase which influences various biological properties of tumor cells [128]. Several studies have shown that proton pump inhibitors (PPIs) such as esomeprazole, omeprazole, and pantoprazole, activated by acidic pHe of tumor environment, have an antineoplastic activity toward human hematopoietic and solid tumors [129]. PPI pretreatment induces a marked reduction of drug efflux and sensitizes tumor cells to the effects of cisplatin, 5fluorouracil, and vinblastine with an IC50 value reduction up to 2 logs, disclosing the possibility to combine cytotoxic drugs with the well-tolerated PPI [130]. PPI also acquire a particular interest in view of their capacity to inhibit HIF-1α [118] and VEGF-C release by melanoma cells [69]. Moreover, the inhibition of MCT-1 was found to be particularly effective against malignant melanoma [131]. Finally, there are inhibitors of the CAIX which are currently undergoing clinical trials for several cancers [132].

5 Concluding remarks A large variety of chemotherapeutic drugs are being used to treat cancer; however, due to problems of delivery and penetration, many compounds hold limited efficacy. In addition, tumor cells are a rapidly changing target leading to selection and overgrowth of drug-resistant subpopulations. Acidosis of most solid growing tumors induces an aggressive and chemo- and radio-resistant phenotype, and in some circumstances, an EMT program and stemness. As a consequence, acidic-adapted tumor cells are likely able to survive to endogenous and exogenous stressors and represent a hidden reserve of tumor cells from which a tumor could relapse. Thus, intratumoral acidosis of solid tumors acquires a prognostic value and therapeutic manipulation of acidosis of tumor environment and/or targeting acidic-adapted tumor cells might represent a new strategy for a successful supportive treatment of cancer. Acknowledgments We acknowledge the “Istituto Toscano Tumori” and Ente Cassa di Risparmio di Firenze for their financial support. Conflict of interest The authors declare that they have no conflict of interest.


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