Rethinking the Combination of Proton Exchanger Inhibitors in Cancer Therapy.

1. Introduction

Tumor cells often grow in a hypoxic microenvironment where there is low nutrient supply, and they upregulate glycolysis to sustain their high proliferation rate [ 1 , 2 ]. Growing evidence suggests that cancer cells take up much more glucose than normal cells, and mainly process it through aerobic glycolysis, the so-called “Warburg effect” [ 3 , 4 ]. This phenomenon leads to the conversion of one molecule of glucose into two molecules of lactic acid and 2 H + to produce 2 ATP, compared to the 36 ATP produced by oxidative metabolism [ 1 , 2 ]. Thus, tumor cells implement glycolysis, promoting an abnormally high rate of glucose utilization, which in turn, leads to the accumulation of lactic acid and to the production of a large amount of H + associated with proton efflux and extracellular pH reduction [ 5 ]. Associated with high glycolysis rate, high levels of carbonic dioxide produced during mitochondrial respiration of oxygenated cancer cells may also contribute to a substantial release of H + into the tumor environment [ 6 , 7 , 8 , 9 , 10 ]. The complete oxidation of one glucose to carbonic dioxide yields 6 HCO 3 − and 6 H + , leading to three times greater production of H + than when glucose is converted to lactate, significantly accounting for tumor extracellular acidosis [ 6 , 7 , 8 , 9 , 10 ]. Uncontrolled growth, lactic and carbonic acid production, low blood and nutrient supply, contribute to the generation of a tumor microenvironment that is extremely toxic for either normal or more differentiated cells, and therefore, progressively selects cells able to survive in these adverse conditions. It is therefore conceivable that malignant cancer cells survive in this hostile microenvironment, thanks to the upregulation of the expression and activity of several proton extrusion mechanisms [ 11 ], which release protons and lactate into extracellular environment, avoiding the acidification of the cytosol. Among proton flux regulators are vacuolar H + -ATPases (V-ATPases), Na + /H + exchanger (NHE), monocarboxylate transporters (MCTs), carbonic anhydrase IX (CA-IX) [ 11 , 12 , 13 , 14 ], and Na + /HCO 3 co-transporters (NBC) [ 15 ] ( Figure 1 ).

Two of the most studied proton flux regulators are vacuolar H + -ATPases (V-ATPase) [ 16 , 17 , 18 ] and carbonic anhydrase (CA) IX/XII [ 19 , 20 , 21 , 22 ]. The aberrant activity of these proton extruders creates a reversed pH gradient across the plasma membrane that is considered a hallmark of malignancy: extracellular acidity and alkaline conditions in the organelle-free cytosol, while normal cells show a neutral pH extracellularly, with a weakly acidic organelle-free cytosolic pH [ 23 , 24 ]. For this reason, the tumor pH gradient is called “reversed pH gradient” [ 5 , 16 , 25 , 26 , 27 , 28 ]. More precisely, the pH of tumor microenvironments have been shown to range between 6.0 and 6.8, with median values around 6.5, and the level of acidity was related with tumor malignancy [ 29 , 30 , 31 , 32 ]. Direct measurements of both intratumoral pO 2 and pH revealed either a spatial heterogeneity between hypoxia and acidosis gradients [ 10 , 33 , 34 ], meaning that the areas of hypoxia and acidosis in tumors may not overlap in mouse tumor models, and a lack of correlation between CA-IX expression and various hypoxia markers [ 35 , 36 , 37 ]. In many tumors, chronic exposure to acidic pH has been reported to promote invasiveness, metastatic behavior, and resistance to cytotoxic agents [ 38 , 39 , 40 , 41 , 42 ]. All in all, tumor extracellular acidity is considered a crucial phenotype of malignant tumors, subjecting cancer cells to a sort of selective pressure, that independently from the tumor histotype, leads to the development of cells able to survive in such a hostile microenvironment. Notably, normal cells at pH ranging from very acidic to weakly acidic die or are entirely blocked in their functions [ 43 , 44 ]. For this reason, the acidic pH of solid tumors has been proposed as a therapeutic target and a drug delivery system for selective anticancer treatments [ 25 , 32 , 45 ]. Indeed, inhibition of these pH regulation systems has been reported to lead to potent antitumor effects [ 13 ]. Therefore, approaches aimed at inhibiting the proteins involved in pH regulation are now under exploitation for the design of novel promising alternative therapeutic anticancer strategies. NHE inhibitors were the first studied as interesting pharmacological agents for interfering with tumor hypoxia/acidosis [ 12 ]. Considering their significant toxicity and the lack of isoform-selectivity for other proteins involved in these processes (such as the MCTs) [ 46 , 47 ], the attention has been focused on the V-ATPases and CAs. Indeed, we have already reported that a specific inhibition of H + release through proton pump inhibitors (PPIs) was able to induce acidification of the tumor cell cytosol [ 48 ], and acidic vesicle retention within tumor cells, with consequent increase in the antitumor activity of chemotherapeutic drugs [ 49 ], and significant antitumor effects [ 31 , 32 , 48 , 50 ]. Moreover, growing evidence in the literature is supporting the evidence that inhibition of CAs has potent antiproliferative and antimetastatic action [ 22 , 51 ]. A recent study has shown for the first time that the combination of proton pump and CA inhibitors (PPIs and CAIs) leads to a more efficient antitumor effect as compared to single treatments, representing the first attempt aimed at targeting, in a unique treatment, two important mechanisms involved in tumor acidification (i.e., tumor acidity and hypoxia) [ 52 ]. Herein, we will review the fields of the proton pump and CA inhibitors as promising agents in the management of solid tumors. This review will also attempt to emphasize the importance of using a cocktail of proton exchangers inhibitors as a new and innovative therapeutic strategy against cancer.

2. The pH Regulators Vacuolar H + -ATPases and Carbonic Anhydrase IX/XII

Tumor microenvironments are characterized by hypoxia, low blood supply, and acidity, which favor the generation of a microenvironment that is hostile and highly toxic for normal or more differentiated cells, inducing progressively, the selection against a more aggressive phenotype. Therefore, to avoid intracellular accumulation of toxic molecules, tumor cells upregulate the expression and activity of proton exchangers that maintain a relative neutral or even alkaline intracellular pH, through pumping protons into the extracellular environment or within the lumen of some membrane-bound organelles. Proton pump exchangers and CAs have been reported to be largely responsible for this hypoxic and acidic microenvironment [ 17 , 53 , 54 ].

Vacuolar-type ATPase (V-ATPase) is a ubiquitous proton pump shuttling protons from the cytoplasm towards intracellular organelles, and from inside to outside the cell plasma membrane [ 17 , 27 , 55 , 56 ]. It is a complex multi-subunit protein, composed of a transmembrane subunit, named V0 complex, devoted to proton transfer and a cytoplasmic portion, named V1 complex, that provides the necessary energy for proton translocation [ 55 ]. Its expression and activity are upregulated in many cancer cells. Augmented expression of V-ATPase is considered to be a well-designed compensatory mechanism that confers survival and growth advantages to cancer cells [ 57 , 58 , 59 , 60 , 61 ]. In tumor cells, the extrusion of protons by V-ATPases causes intracellular alkalinization and extracellular acidification, which are important mechanisms favoring the increased activation of extracellular metalloproteinases, thus contributing to tumor cell survival and growth, motility, invasion, metastasis, resistance to apoptosis, and multidrug resistance [ 62 , 63 , 64 , 65 , 66 , 67 , 68 ]. The V-ATPases are also expressed in vacuolar membranes (i.e., lysosomes), where they are involved in the transport of H + into the lumen of intracellular organelles. Lysosomal acidification by V-ATPases has been reported to be under the control of lactate dehydrogenase B (LDHB), and to be facilitated by a physical interaction between LDHB and V-ATPase at the lysosomal surface [ 69 ]. Data obtained by Lu and collaborators [ 70 ] strongly demonstrated that inhibition of the V-ATPase has an antineoplastic activity [ 45 ]. In fact, the inhibition of V-ATPase function via knockdown of ATP6L expression using siRNA suppresses cancer metastasis by decreased proton extrusion, and downregulated protease activity [ 70 ]. Thus, this proton pump may be considered a suitable target for the development of novel anticancer strategy.

Carbonic anhydrases (CAs) are cellular pH regulators with a key role in the maintenance of pH homeostasis in cancer cells, thus representing suitable targets for anticancer therapies. CAs are a family of metalloenzymes that catalyze the reversible hydration of carbonic dioxide to bicarbonate and protons [ 22 , 71 , 72 ]. They are present in several tissues such us the gastrointestinal tract, the reproductive tract, the nervous system, kidney, lungs, skin, and eyes [ 20 , 22 , 73 , 74 , 75 ]. CAs are involved in respiration and acid–base equilibrium, electrolyte secretion, bone resorption, calcification, ureagenesis, gluconeogenesis, and lipogenesis [ 20 , 72 ]. Several isoforms are known, which are subdivided according to their location: membrane-bound/transmembrane, cytosolic, mitochondrial and secreted [ 21 , 22 , 72 ]. CA-IX and CA-XII are the two major tumor-related CA isoforms [ 76 , 77 ]. The two enzymes are transmembrane, multi-domain proteins formed of a short intra-cytosolic tail, a transmembrane short domain, an extracellular catalytic domain and a proteoglycan (PG)-like domain. CA-IX and CA-XII contribute significantly to the acidification of tumor microenvironment together with lactic acid production. Indeed, their inhibition has been reported to revert this phenomenon [ 19 ]. These two CA isoforms are predominantly found in hypoxic tumors with restricted expression in normal tissue, where they seem to be in their catalytically inactive state. Thus, their inhibition showed an anticancer effect with less side effects compared to other anticancer drugs [ 72 , 78 ]. For this reason, CA-IX and CA-XII have been considered attractive targets for cancer therapy, being validated recently as antitumor/antimetastatic targets [ 22 , 79 ].

Taking into account all these considerations, V-ATPases and tumor-associated CAs have been considered good candidates and ideal targets for the design of novel and innovative anticancer therapy, which interfere with tumor microenvironmental acidification, thus gaining a renewal interest in the last decade for oncologists.

3. V-ATPase Inhibitors

A bulk of evidence correlated hyperfunction of V-ATPases with tumor migration and invasion, multidrug resistance and metastatic processes. Consequently, inhibition of V-ATPase has become an attractive and promising strategy to counteract the tumor hypoxic and acidic microenvironment and to develop novel drugs for the benefit of cancer patients. Different anti-VATPases compounds have then been assessed as potential anticancer agents. Several studies are currently ongoing in order to better investigate in vitro and in vivo, in both preclinical and clinical settings, the binding properties and the mode of inhibition of V-ATPase inhibitors.

Among the V-ATPase inhibitors, the first identified and most frequently used are the natural compounds of microbial origin, bafilomycins, and concanamycins (belonging to plecomacrolide antibiotics) [ 80 , 81 ]. They are lipophilic compounds that have been reported to inhibit growth and to induce apoptosis in human cancer cells [ 59 , 82 , 83 , 84 , 85 , 86 , 87 ]. Other molecules capable of inhibiting V-ATPases via different mechanisms of action, such as benzolactone enamides, archazolid, and indolyls, were later discovered [ 88 , 89 , 90 , 91 , 92 ]. These compound, like bafilomycin A1 or concanamycin, have also investigated as anticancer agents [ 93 , 94 ]. Unfortunately, many reports evidenced high cytotoxicity of these ATPase inhibitors for normal cells, probably because V-ATPase is ubiquitously expressed and active in all types of cells, strongly hampering their potential clinical applications [ 16 , 45 , 55 ].

Our group focused the attention on another class of promising V-ATPase inhibitors, the family of proton pump inhibitors (PPIs), that include omeprazole, esomeprazole, lansoprazole, pantoprazole, and rabeprazole [ 95 ]. They are currently used as antiacid drugs against peptic diseases, including gastroesophageal reflux disease, peptic ulcers or functional dyspepsia [ 96 ]. These compounds are weak bases that need an acidic milieu in order to be transformed into the active molecule. Indeed, after protonation in the acidic spaces of the stomach, PPIs irreversibly bind to the cysteine residues of proton pump, dramatically inhibiting proton translocation and acidification of the extracellular environment [ 97 ] ( Figure 2 ).

The specific targets of PPIs are H-ATPases contained within the lumen of gastric parietal cells, and through a lesser activity, they inhibit the activity of V-ATPases, thus blocking proton transport across membranes [ 12 , 13 , 45 , 95 ]. Some reports also refer to that PPIs could be useful in blocking ATPase activity in tumor cells. These agents did not show relevant systemic toxicity for normal cells, even in prolonged treatments and at very high dosages [ 98 ]. Therefore, PPIs have represented an attractive possibility as V-ATPases inhibitors, as they require an acidic environment to be activated, such as that found in the tumor microenvironment, which provides the possibility of tumor specific selectivity, and thus, of targeted anticancer strategy [ 56 ]. Therefore, several in vitro and in vivo studies have been carried out on PPIs, in both preclinical and clinical settings, for the design of novel anticancer therapy which target specifically tumor acidity.

3.1. PPIs as Therapeutic Agents

The first evidence supporting the use of PPIs in cancer was provided in 2004 when our group demonstrated that pre-treatment with PPIs (such as omeprazole, esomeprazole, or pantoprazole) in vitro and in vivo (i) reverted chemoresistance of different human tumor cell lines to cisplatin, 5-fluorouracile and doxorubicin; and (ii) increased the sensitivity of drug-sensitive cells to anticancer agents [ 49 ]. These effects were mediated by the intracellular retention of chemotherapeutic agents into the lysosomal organelles, associated with a “normalization” of the pH gradients of the tumor cells [ 49 ]. Additional studies investigated the response of B-cell lymphoma cell lines and acute lymphoblastic leukemia (ALL) bone marrow blasts, to omeprazole and esomeprazole. PPIs were able to induce a cytotoxic effect against both B-cell tumors and pre-B acute lymphoblastic leukemia cells obtained from patients with acute lymphoblastic leukemia (ALL). The cytotoxic effect was exerted through (i) activation of reactive oxygen species (ROS); (ii) perturbation of lysosomal membranes; (iii) alkalinization of acidic vesicles; (iv) acidification of the cytosol; (v) caspase-independent cell death [ 48 ]. PPIs were also able to significantly delay human tumor growth in SCID mice engrafted with B cell lymphomas [ 48 ]. Similarly, PPIs exerted a strong tumor inhibitory action against different human melanoma cell lines as well as in tumor-bearing mice [ 31 ]. Treatments with PPIs in different pH conditions, induced a marked cytotoxicity that was exerted through a caspase-dependent mechanism strongly influenced by low pH. In vivo studies in SCID mice engrafted with human melanoma pointed out that the tumor growth delay induced by PPIs was consistent with a clear reduction of pH gradients in tumor tissue [ 31 ]. We further demonstrated either in vitro and in vivo that pre-treatment with PPIs were able to improve the cytotoxic activity of suboptimal doses of the chemotherapeutic agent paclitaxel against human melanoma cells [ 32 ]. Then, we focused on identifying the most effective and promising agent within the PPI family, and discovered that lansoprazole was the most effective single agent against tumor cells [ 50 ]. Based on this finding, we investigated the efficacy of lansoprazole against multiple myeloma cell lines, showing that this drug was capable of inducing a strong tumor inhibitory action against this troublesome neoplasm, leading to a caspase-independent cell death [ 99 ]. Lastly, we showed that acidity represents a potent mechanism of tumor immune escape, and that PPIs increase the immune reaction against tumors [ 25 , 44 , 100 ]. Beside the direct toxic effects, PPIs were also able to inhibit mTOR signaling and other metabolic pathways in gastric carcinoma cell line, and to potentiate the antitumor effectiveness of Adriamycin in mice harboring gastric carcinoma xenografts [ 101 , 102 , 103 , 104 , 105 , 106 ]. In gastric cancer models, PPIs induced cancer stem cell depletion which passed through inhibition of proliferation, sphere formation, and 5-fluorouracil chemoresistance [ 107 ]. PPIs were also tested on breast cancers. Different studies demonstrated that pre-treatments with lansoprazole were able to potentiate the cytotoxic effect of doxorubicin, with no significant effect on non-neoplastic breast epithelial cells [ 108 , 109 , 110 , 111 , 112 , 113 , 114 ]. In esophageal cancer cell lines, esomeprazole significantly reduced cell viability, adhesion, and migration, as well as enhanced the cytotoxic effects of cisplatin and 5-fluorouracil [ 115 ]. Moreover, in ovarian carcinomas, omeprazole pre-treatments have been reported to induce a synergistic effect with paclitaxel on tumor growth in orthotopic and patient-derived xenograft mouse models [ 116 ]. Comparable results have been obtained by other groups that have been focused their investigations on hepatocellular, pancreatic, prostatic, and brain tumors [ 85 , 117 , 118 , 119 , 120 , 121 , 122 ].

Therefore, these results provided the proof of concept that PPIs may be considered, not only chemosensitizer agents, but also a new class of antineoplastic drugs, and described the background for a series of clinical studies aimed at supporting the use of PPIs as chemosensitizers.

3.2. PPIs in Clinical Trials

Up to now, two main clinical trials in humans investigated the effect of combined application of PPIs and chemotherapy treatment in cancer. The studies were performed in either osteosarcomas or metastatic breast cancer patient (MBC) [ 123 , 124 ]. The results showed that pre-treatment with PPIs increased the effectiveness of neoadjuvant chemotherapy in osteosarcomas patients, increasing the overall response rate [ 123 ]. Confirmation came with the second trial that enrolled women with metastatic breast cancer [ 124 ]. This phase II clinical trial aimed at evaluating whether esomeprazole improves efficacy of docetaxel and cisplatin treatment in metastatic breast cancer. The results showed that high dose PPIs proved beneficial and improved chemotherapy efficacy [ 124 ]. Indeed, patients that underwent esomeprazole treatment experienced the highest response rates and the longest survivals.

Another phase I clinical trial assessed the effectiveness and safety of the combined use of pantoprazole and doxorubicin in patients with advanced solid tumors, identifying the dose of 240 mg as the baseline for future phase II studies in patients with castration-resistant prostate cancer [ 125 ]. Moreover, a study on head and neck tumor patients confirmed that treatments with PPIs increase the overall survival of patients [ 126 ].

A more recent study on three patients affected with gastrointestinal cancer, refractory to standard chemotherapy, reported that high-dose of the PPI rabeprazole were able to sensitize human metastatic colorectal cancer to metronomic capecitabine, leading to a good quality of life with acceptable side effects [ 127 ]. This combined approach (rabeprazole with capecitabine) is currently under investigation in an approved clinical II trial, proving to be beneficial for the patients [ 128 ].

Lastly, two studies in companion animals with spontaneous tumors have been performed in order to explore the effect of PPIs treatment in combination with chemotherapy. Interestingly, PPIs were able to reverse chemoresistance in refractory tumors, both hematopoietic (lymphoma) or solid (melanoma and squamous cell carcinoma) [ 129 ]. Moreover, PPIs increased the efficacy of metronomic chemotherapy, independently from the tumor histotype or the animal species affected by cancer [ 130 ].

All these studies provided the first clinical evidence that PPIs pretreatment could be easily included into the standard protocols in clinical oncology with a clear benefit for patients having the less favorable prognostic factors. Indeed, pretreatment with PPIs, by inhibiting proton pumps, induced a decrease of the protonation of extracellular tumor environment, in turn allowing the chemotherapeutics to be fully effective, improving the effectiveness of either chemical and biological drugs against cancer. Thus, tumor alkalinization could improve the outcome of patients by counteracting tumor chemoresistance.

4. Inhibitors of Carbonic Anhydrase IX/XII

In recent years, the involvement of CA-IX/XII in generating the peculiar pH gradients of tumor cells is becoming more and more evident [ 12 ]. In fact, inhibition of the enzyme catalytic activity with fluorescent sulfonamides has been reported to be able to revert this phenomenon [ 19 ]. Moreover, accumulating experimental evidence recognizes that disruption of CA-IX by gene knockdown or inhibition of its catalytic activity with small molecules and/or antibodies strongly correlates with both extracellular and intracellular pH, tumor growth [ 78 , 131 , 132 , 133 ], tumor cell migration/invasion [ 134 , 135 , 136 ], chemo- or radiotherapy resistance [ 137 , 138 ].

As a consequence, different pharmacological inhibitors that specifically target the tumor-associated isoforms CA-IX and -XII were developed and tested for their antitumor activity during the last years [ 20 ].

4.1. CA-IX/XII Inhibitors as Therapeutic Agents

Many sulfonamide, sulfamate, sulfamide, and coumarine CA inhibitors were reported to efficiently target CA-IX [ 139 , 140 , 141 ]. The compounds specifically designed for targeting CA-IX were: (i) fluorescent sulfonamides, used for imaging purposes [ 19 , 142 ]; (ii) positively or negatively-charged compounds that inhibit selectively extracellular CAs [ 74 ]; (iii) nanoparticles coated with CAs [ 143 ]; (iv) monoclonal antibodies, among which M75 is a highly specific anti-CAIX mAb targeting the PG domain of CA-IX, discovered by Pastorekova’s group [ 144 , 145 , 146 ].

The most studied and important CA-IX/XII inhibitors are the sulfonamides, that exert their potent CA inhibitory properties by binding to the catalytic site of Cas, blocking then, its function [ 147 , 148 ]. Several inhibitors of the sulfonamide type have been identified for both CA-IX [ 76 ] and CA-XII [ 77 ]. Unfortunately, most of these compounds did not show specificity for the inhibition of the tumor-associated isoforms IX and XII versus the remaining CAs. Therefore, different sulfonamide derivates, more selective inhibitors of the tumor-associated CAs (CA-IX and XII) were designed and developed for targeting these agents, mainly through structure-based drug design approaches [ 149 ]. With the discovery of the X-ray crystal structure of CA-IX by the De Simone’s group in 2009 [ 150 ], the drug-design studies of CA inhibitors targeting isoform IX/XII were highly favored, and highly isoform-selective inhibitors were identified [ 139 , 140 , 141 ]. These compounds have been shown, over the past years, to be very promising in anticancer therapies. For instance, a fluorescent sulfonamide with high affinity for CA-IX was generated. This compound was used to determine the role of CA-IX in tumor acidification and for imaging purposes [ 19 ]. Indeed, Dubois et al. showed that fluorescent sulfonamides accumulate selectively in the hypoxic regions of xenograft animals with transplanted hypoxic colorectal cancers [ 142 , 151 ]. The in vivo proof of concept that sulfonamide CA-IX inhibitors may show antitumor effects has been first published by Neri’s group [ 152 ]. Similar studies from different laboratories on diverse models and cancer types demonstrated that sulfonamide CA-IX/XII inhibitors have a profound effect in inhibiting the growth of primary tumors, and great in vivo anti-metastatic effects [ 79 , 152 , 153 , 154 , 155 , 156 ]. These inhibitors were also able to induce depletion of cancer stem cells in models of breast cancer metastasis, leading, in turn, to diminished tumor growth and metastasis, and showing a promising efficacy for the recurrence of some cancers [ 157 ]. Supuran and coworkers developed a sulfonamide-derived compound, SLC-0111 (also known as WBI-5111), that strongly and specifically inhibits CA-IX, in vitro and in vivo [ 79 , 154 ]. This compound inhibits tumor growth and metastasis formation alone or in combination with antineoplastic drug, and decreased the cancer stem cell population in breast cancer models [ 79 , 154 , 158 ]. It has now completed the phase I clinical trials and is scheduled for phase II trials later this year [ 22 , 156 ].

Coumarins are another class of CA-IX and XII inhibitors. These natural compounds, together with the highly isoforms-selective CA-Is, are selective against the tumor-associated isoforms IX and XII at nanomolar concentrations, being ineffective against the broadly expressed isoforms CA-I and CA-II [ 153 , 159 ]. One of these derivatives, a glycosyl coumarin, was recently shown to inhibit the growth of primary tumors and the formation of metastasis in the highly aggressive 4T1 syngeneic mouse metastatic breast cancer [ 153 ]. These inhibitors are still under investigation in pre-clinical studies [ 160 ].

Monoclonal antibodies may represent another venue for the selective CA-IX and XII inhibition [ 144 , 161 ]. M75 is a highly specific anti-CAIX monoclonal antibody targeting the proteoglycan domain of CA-IX, discovered by Pastorekova’s group [ 78 ] and widely used in immunohistochemical and Western blot studies. WX-G250 (known also as girentuximab), the first CA-IX inhibitor to enter clinical trials [ 162 ], is another chimeric monoclonal antibody that is actually in phase III clinical trials, as an adjuvant therapy for the treatment of non-metastasized renal cell carcinoma. CA-XII specific monoclonal antibodies have also been generated and characterized. The first and most significant anti-CAXII monoclonal antibody (6A10) was created by Battke and coworkers [ 144 ]. The antibody binds to the catalytic domain of CA-XII, inhibiting its activity at nanomolar concentrations. 6A10 was shown to successfully inhibiting the growth of tumor spheroids in vitro and efficiently decreased tumor growth in vivo [ 144 , 163 ].

4.2. CA-IX Inhibitors in Clinical Trials

In the past few years, the tumor-associated cell surface CA isoform, CA-IX, has been validated as new anticancer drug target for the treatment and imaging of cancers expressing this enzyme, and some therapeutic strategies against CA-IX have already entered in clinical studies.

Serum levels of CA-IX have been explored as a potential biomarker for the treatment response in patients with metastatic renal cell cancer (mRCC) in a pilot human trial [ 164 ]. The study collected blood samples from 91 patients with mRCC and 32 healthy individuals, and associated serum CA-IX levels with the occurrence of tumor progression (stage, tumor grade, tumor size, recurrence, and metastasis), suggesting that CA-IX may be a valuable diagnostic and prognostic tool in RCC [ 164 ]. Serum CA-IX levels have also been suggested as a potential biomarker to predict the outcome for patients with mRCC [ 165 ]. Correlation between CA-IX level of expression and recurrence, survival, and clinical response to therapy was also observed in patients with high-risk, nonmetastatic renal cell carcinoma [ 166 ] and laryngeal cancer [ 167 ].

A selective small molecule, an ureido-substituted benzenesulfonamide derivative, SLC-0111, entered, in 2014, a phase I clinical trial for the treatment of advanced, metastatic solid tumors to evaluate the safety, tolerability, and pharmacokinetics of this compound ( NCT02215850 ). Although the study was completed in March 2016, the results have not yet been published.

A novel small molecule radiotracer, 18F-VM4-037, that binds to CA-IX in clinically localized kidney tumors, has been submitted to a phase II clinical trial sponsored by National Cancer Institute (NCI) ( NCT01712685 ). The main objective of the study was to test the safety and effectiveness of 18F-VM4-037 during imaging studies of kidney cancer. The study enrolled 12 patients with renal cell carcinoma, and demonstrated that it is a well-tolerated agent which allows imaging of CA-IX expression both in primary tumors and metastases, highlighting 18F-VM4-037 as a useful drug in the evaluation of metastatic ccRCC lesions [ 168 ].

Regarding antibodies, the monoclonal antibody G250 was the first monoclonal antibody anti-CAIX introduced in a phase I/II clinical trials in combination with interferon-alpha-2a in metastatic renal cell carcinoma patients (mRCC), who are at a high risk of recurrence after resection of the primary tumor [ 162 ]. The study on 31 patients with mRCC treated with both drugs demonstrated that the treatment was safe, well tolerated, and led to clinical disease stabilization [ 162 ]. This antibody also progressed to a phase III clinical trial as an adjuvant therapy for the treatment of patients with metastatic renal cell carcinoma ( NCT00087022 ). This study had proposed to investigate the efficacy and safety of adjuvant G250. The adjuvant treatment demonstrated a safe and well tolerated profile. However, G250 had no clinical benefit for the patients [ 169 ]. Antibodies were also proposed as imaging agents for CA-IX positive tumors, originally by Neri’s group [ 152 ]. Therefore, radiolabelled chimeric G250 was also tested as a valuable imaging agent for the diagnosis of patients with renal cell carcinoma. Zirconium-89- labeled girentuximab has been developed and entered in a phase II/III clinical trial ( NCT02883153 ) to study the impact of the Zirconium-89-girentuximab in clinical management. Unfortunately, even if the study is completed, the results have not been published yet. Lastly, radioimmunotherapy with Lutetium 177–labeled girentuximab are also well tolerated in metastatic ccRCC patients [ 170 ].

5. Cocktail of Proton Exchanger Inhibitors as a Novel Therapeutic Approach against Cancer

Triggered from such encouraging results, we explored the hypothesis that PPIs could increase the effectiveness of CA-IX inhibitors against very malignant human melanoma cells, fully expressing the enzyme [ 52 ]. To this purpose, human melanoma cells have been treated with potent CA-IX inhibitors, the sulfamates S4, and p -nitrophenyl derivative FC9-399A (selective ureido-sulfamate derivatives), in combination with lansoprazole [ 52 ]. First of all, we observed that treatment with these CA-IX inhibitors induced a slight but significant inhibition of melanoma cell growth, which was strongly impaired under acidic conditions, typical of malignant cell growth [ 52 ]. We postulated that the impairment of CA-IX inhibitor activity was probably due to their neutralization by protonation outside the cells induced by acidity. When we compared the combination treatment at suboptimal doses (lansoprazole followed by one of the two CA-IX inhibitors), we observed a more efficient and significantly increased tumor cell growth inhibition, and a straightforward cytotoxic effect against metastatic melanoma cells, compared to each single agent [ 52 ]. We also demonstrated that the effect of combined treatments was not due to changes in the CA-IX protein expression, but rather probably to induction of an inhibition in CA-IX activity [ 52 ]. The pre-treatment of human melanoma cells with lansoprazole significantly improved the antitumor effect of CA-IX inhibitors, probably because lansoprazole, fully active in acidic conditions, induced an alkalinization of the tumor extracellular environment, in turn leading to stronger and the greatest activation and effectiveness of CA-IX inhibitors. Therefore, our results provided the first evidence that combinations of lansoprazole with two different CA-IX inhibitors were more effective than single treatments in inhibiting cell proliferation and inducing cell death in human melanoma cells. These results were supported by another recent study where we combined the proton pump inhibitor lansoprazole with the inhibitor of the reverse transcriptase, Efavirenz, in order to target in a unique treatment two new oncotypes, i.e., proton pumps and reverse transcriptase [ 171 ]. Again, the results clearly showed that pre-treatment of human melanoma cells with the proton pump inhibitor lansoprazole significantly improved the Efavirenz antitumor effect [ 171 ]. These two recent works of ours highlighted, for the first time, that the combination of proton pump and CA-IX/reverse transcriptase inhibitors possess a more efficient antitumor action compared to single treatments. Actually, they represent the first attempt aimed at combining either different proton exchangers inhibitors or a proton exchanger with the reverse transcriptase inhibitor in a unique antitumor approach, with conceivably more effectiveness and less toxicity.

Altogether, the results of these two studies clearly supported the hypothesis that treatment aimed at buffering the acidic tumor microenvironment, through the use of PPIs, that contrary to other drugs are activated in acidic conditions, may improve the effectiveness of CA-IX and reverse transcriptase inhibitors, significantly improving their antitumor effects. Thus, they open the way to novel and alternative antitumor strategies that are more specific, and probably less toxic for tumor patients. Of course, clinical trials obtained with lansoprazole followed by CA-IX or reverse transcriptase inhibitors are needed in order to further support the use of combination therapies that these studies were proposing.

6. Conclusions

Despite the great efforts of the scientific community in finding proper treatments for cancer, the responsiveness of human tumors to chemotherapy has not changed in the last decades, and resistance or refractoriness to chemotherapeutic drugs has become a key problem in the therapy of tumor patients and still remains unsolved. Therefore, novel antitumor strategies, which are more specific and probably less toxic, have become an urgent medical need. During the last decades, tumor metabolism and microenvironmental acidity are increasingly considered important determinants of tumor progression and drug resistance. Therefore, the mechanisms controlling the acidic pH of solid tumors have been proposed as selective and specific therapeutic targets in setting up novel antitumor strategies. Proton exchangers, whose expression and activity are upregulated by hypoxia and acidity, have gained attention in the last years, due to their crucial function in determining the acidification of the tumor microenvironment. In particular, V-ATPases and CA-IX represented interesting targets for the development of novel approaches in anticancer therapy. For these reasons, several V-ATPases and CA-IX inhibitors have been tested for their antitumor activity. Investigations reported that several inhibitors of the tumor-associated carbonic anhydrase isoform IX, and of V-ATPases, had a clear antineoplastic action, and may be useful to be combined and tested for developing novel antitumor therapies. We tested combinations of lansoprazole, targeting proton pumps, followed by CA-IX inhibitors, and observed that combined treatments, while inducing the alkalinization of tumor microenvironment, led to an increased effectiveness of CA-IX inhibitors against very malignant human melanoma cells [ 52 ]. The same effect was observed combining lansoprazole with the inhibitor of the reverse transcriptase, another important hallmark of cancers [ 171 ]. These results clearly supported the hypothesis that an approach aimed at targeting tumor extracellular acidity, combining two or more proton exchanger inhibitors with different antitumor actions, may open the way to the development of innovative and alternative antitumor strategies that are more specific, effective, and hopefully less toxic for tumor patients. Of course, data from clinical trials obtained through combination of PPIs with CA-IX inhibitors are needed, in order to further support the use of proton pump and CA-IX inhibitor combination therapies, and to translate these results to the patients. Finally, the results of these studies give support to new investigations aimed at the setting up of either hybrids or combined molecules containing both proton pump and CA-IX inhibitors.

Acknowledgments

This study was supported by a grant from the Italian Ministry of Health.

Author Contributions

Elisabetta Iessi conceptualized and designed the structure of the article, and wrote the article. Mariantonia Logozzi and Rossella Di Raimo assisted in literature search. Davide Mizzoni prepared figures presented in this review. Claudiu T. Supuran edited and supervised the manuscript. Stefano Fais edited and supervised the manuscript draft and figures. All authors were involved in the critical reading and editing of the review. All authors have read and approved the final version of the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

References

1. Gatenby R.A. Gillies R.J. Why do cancers have high aerobic glycolysis? Nat. Rev. Cancer 2004 4 891 899 10.1038/nrc1478 15516961

2. Vander Heiden M.G. Cantley L.C. Thompson C.B. Understanding the Warburg effect: The metabolic requirements of cell proliferation Science 2009 324 1029 1033 10.1126/science.1160809 19460998 PMC2849637

3. Warburg O. On the origin of cancer cells Science 1956 123 309 314 10.1126/science.123.3191.309 13298683

4. Cairns R.A. Harris I.S. Mak T.W. Regulation of cancer cell metabolism Nat. Rev. Cancer 2011 11 85 95 10.1038/nrc2981 21258394

5. Tredan O. Galmarini C.M. Patel K. Tannock I.F. Drug resistance and the solid tumor microenvironment J. Natl. Cancer Inst. 2007 99 1441 1454 10.1093/jnci/djm135 17895480

6. Helmlinger G. Sckell A. Dellian M. Forbes N.S. Jain R.K. Acid production in glycolysis-impaired tumors provides new insights into tumor metabolism Clin. Cancer Res. 2002 8 1284 1291 11948144

7. Newell K. Franchi A. Pouyssegur J. Tannock I. Studies with glycolysis-deficient cells suggest that production of lactic acid is not the only cause of tumor acidity Proc. Natl. Acad. Sci. USA 1993 90 1127 1131 10.1073/pnas.90.3.1127 8430084 PMC45824

8. Yamagata M. Hasuda K. Stamato T. Tannock I.F. The contribution of lactic acid to acidification of tumours: Studies of variant cells lacking lactate dehydrogenase Br. J. Cancer 1998 77 1726 1731 10.1038/bjc.1998.289 9667639 PMC2150353

9. Mookerjee S.A. Goncalves R.L. Gerencser A.A. Nicholls D.G. Brand M.D. The contributions of respiration and glycolysis to extracellular acid production Biochim. Biophys. Acta 2015 1847 171 181 10.1016/j.bbabio.2014.10.005 25449966

10. Corbet C. Feron O. Tumour acidosis: From the passenger to the driver’s seat Nat. Rev. Cancer 2017 17 577 593 10.1038/nrc.2017.77 28912578

11. Izumi H. Torigoe T. Ishiguchu H. Uramoto H. Yoshida Y. Tanabe M. Ise T. Murakami T. Yoshida T. Nomoto M. Cellular pH regulators: Potentially promising molecular targets for cancer chemotherapy Cancer Treat. Rev. 2003 29 541 549 10.1016/S0305-7372(03)00106-3 14585264

12. Parks S.K. Chiche J. Pouysségur J. Disrupting proton dynamics and energy metabolism for cancer therapy Nat. Rev. Cancer 2013 13 611 623 10.1038/nrc3579 23969692

13. Spugnini E.P. Sonveaux P. Stock C. Perez-Sayans M. De Milito A. Avnet S. Garcìa A.G. Harguindey S. Fais S. Proton channels and exchangers in cancer Biochim. Biophys. Acta 2015 1848 2715 2726 10.1016/j.bbamem.2014.10.015 25449995

14. Spugnini E. Fais S. Proton pump inhibition and cancer therapeutics: A specific tumor targeting or it is a phenomenon secondary to a systemic buffering? Semin. Cancer Biol. 2017 43 111 118 10.1016/j.semcancer.2017.01.003 28088584

15. Gorbatenko A. Olesen C.W. Boedtkjer E. Pedersen S.F. Regulation and roles of bicarbonate transporters in cancer Front. Physiol. 2014 5 130 10.3389/fphys.2014.00130 24795638 PMC3997025

16. De Milito A. Fais S. Proton pump inhibitors may reduce tumour resistance Expert Opin. Pharmacother. 2005 6 1049 1054 10.1517/14656566.6.7.1049 15957961

17. Nishi T. Forgac M. The vacuolar (H + )-ATPases-nature’s most versatile proton pumps Nat. Rev. Mol. Cell Biol. 2002 3 94 103 10.1038/nrm729 11836511

18. Martinez-Zaguilan R. Lynch R.M. Martinez G.M. Gillies R.J. Vacuolar-type H(+)-ATPases are functionally expressed in plasma membranes of human tumor cells Am. J. Physiol. 1993 265 C1015 C1029 8238296 10.1152/ajpcell.1993.265.4.C1015

19. Svastová E. Hulíková A. Rafajová M. Zat’ovicová M. Gibadulinová A. Casini A. Cecchi A. Scozzafava A. Supuran C.T. Pastorek J. Hypoxia activates the capacity of tumor-associated carbonic anhydrase IX to acidify extracellular pH FEBS Lett. 2004 577 439 445 10.1016/j.febslet.2004.10.043 15556624

20. Supuran C.T. Carbonic anhydrases: Novel therapeutic applications for inhibitors and activators Nat. Rev. Drug Discov. 2008 7 168 181 10.1038/nrd2467 18167490

21. Supuran C.T. Carbonic anhydrase inhibition/activation: Trip of a scientist around the world in the search of novel chemotypes and drug targets Curr. Pharm. Des. 2010 16 3233 3245 10.2174/138161210793429797 20819070

22. Supuran C.T. Carbonic Anhydrase Inhibition and the Management of Hypoxic Tumors Metabolites 2017 7 48 10.3390/metabo7030048 28926956 PMC5618333

23. Huber V. De Milito A. Harguindey S. Reshkin S.J. Wahl M.L. Rauch C. Chiesi A. Pouysségur J. Gatenby R.A. Rivoltini L. Proton dynamics in cancer J. Transl. Med. 2010 8 57 10.1186/1479-5876-8-57 20550689 PMC2905351

24. Webb B.A. Chimenti M. Jacobson M.P. Barber D.L. Dysregulated pH: A perfect storm for cancer progression Nat. Rev. Cancer 2011 11 671 677 10.1038/nrc3110 21833026

25. Fais S. Venturi G. Gatenby B. Microenvironmental acidosis in carcinogenesis and metastases: New strategies in prevention and therapy Cancer Metastasis Rev. 2014 33 1095 1108 10.1007/s10555-014-9531-3 25376898 PMC4244550

26. Barar J. Omidi Y. Dysregulated pH in tumor microenvironment checkmatescancer therapy Bioimpacts 2013 3 149 162 24455478 10.5681/bi.2013.036 PMC3892734

27. Daniel C. Bell C. Burton C. Harguindey S. Reshkin S.J. Rauch C. The role of proton dynamics in the development and maintenance of multidrug resistance in cancer Biochim. Biophys. Acta 2013 1832 606 617 10.1016/j.bbadis.2013.01.020 23376112

28. Gillies R.J. Raghunand N. Karczmar G.S. Bhujwalla Z.M. MRI of the tumor microenvironment J. Magn. Reson. Imaging 2002 16 430 450 10.1002/jmri.10181 12353258

29. Gallagher F.A. Kettunen M.I. Day S.E. Hu D.E. Ardenkjaer-Larsen J.H. Zandt Ri. Jensen P.R. Karlsson M. Golman K. Lerche M.H. Magnetic resonance imaging of pH in vivo using hyperpolarized 13 C-labelled bicarbonate Nature 2008 453 940 943 10.1038/nature07017 18509335

30. Van Sluis R. Bhujwalla Z.M. Raghunand N. Ballesteros P. Alvarez J. Cerdán S. Galons J.P. Gillies R.J. In vivo imaging of extracellular pH using 1H MRSI Magn. Reson. Med. 1999 41 743 750 10.1002/(SICI)1522-2594(199904)41:4<743::AID-MRM13>3.0.CO;2-Z 10332850

31. De Milito A. Canese R. Marino M.L. Borghi M. Iero M. Villa A. Venturi G. Lozupone F. Iessi E. Logozzi M. pH-dependent antitumor activity of proton pump inhibitors against human melanoma is mediated by inhibition of tumor acidity Int. J. Cancer 2010 127 207 219 10.1002/ijc.25009 19876915

32. Azzarito T. Venturi G. Cesolini A. Fais S. Lansoprazole induces sensitivity to suboptimal doses of paclitaxel in human melanoma Cancer Lett. 2015 356 697 703 10.1016/j.canlet.2014.10.017 25449440

33. Helmlinger G. Yuan F. Dellian M. Jain R.K. Interstitial pH and pO 2 gradients in solid tumors in vivo: High-resolution measurements reveal a lack of correlation Nat. Med. 1997 3 177 182 10.1038/nm0297-177 9018236

34. Vaupel P.W. Frinak S. Bicher H.I. Heterogeneous oxygen partial pressure and pH distribution in C3H mouse mammary adenocarcinoma Cancer Res. 1981 41 2008 2013 7214369

35. Bittner M.I. Wiedenmann N. Bucher S. Hentschel M. Mix M. Rücker G. Weber W.A. Meyer P.T. Werner M. Grosu A.L. Analysis of relation between hypoxia PET imaging and tissue-based biomarkers during head and neck radiochemotherapy Acta Oncol. 2016 55 1299 1304 10.1080/0284186X.2016.1219046 27593107

36. Le Q.T. Kong C. Lavori P.W. O’byrne K. Erler J.T. Huang X. Chen Y. Cao H. Tibshirani R. Denko N. Expression and prognostic significance of a panel of tissue hypoxia markers in head-and-neck squamous cell carcinomas Int. J. Radiat. Oncol. Biol. Phys. 2007 69 167 175 10.1016/j.ijrobp.2007.01.071 17707270

37. Rademakers S.E. Lok J. van der Kogel A.J. Bussink J. Kaanders J.H. Metabolic markers in relation to hypoxia; staining patterns and colocalization of pimonidazole, HIF-1α, CAIX, LDH-5, GLUT-1, MCT1 and MCT4 BMC Cancer 2011 11 167 10.1186/1471-2407-11-167 21569415 PMC3115911

38. Lee A.H. Tannock I.F. Heterogeneity of intracellular pH and of mechanisms that regulate intracellular pH in populations of cultured cells Cancer Res. 1998 58 1901 1908 9581831

39. Martinez-Zaguilan R. Seftor E.A. Seftor R.E. Chu Y.W. Gillies R.J. Hendrix M.J. Acidic pH enhances the invasive behavior of human melanoma cells Clin. Exp. Metastasis 1996 14 176 186 10.1007/BF00121214 8605731

40. Rofstad E.K. Mathiesen B. Kindem K. Galappathi K. Acidic extracellular pH promotes experimental metastasis of human melanoma cells in athymic nude mice Cancer Res. 2006 66 6699 6707 10.1158/0008-5472.CAN-06-0983 16818644

41. Wachsberger P.R. Landry J. Storck C. Davis K. O’Hara M.D. Owen C.S. Leeper D.B. Coss R.A. Mammalian cells adapted to growth at pH 6.7 have elevated HSP27 levels and are resistant to cisplatin Int. J. Hyperth. 1997 13 251 255 10.3109/02656739709023533 9222809

42. Raghunand N. Mahoney B. van Sluis R. Baggett B. Gillies R.J. Acute metabolic alkalosis enhances response of C3H mouse mammary tumors to the weak base mitoxantrone Neoplasia 2001 3 227 235 10.1038/sj.neo.7900151 11494116 PMC1505597

43. Lugini L. Matarrese P. Tinari A. Lozupone F. Federici C. Iessi E. Gentile M. Luciani F. Parmiani G. Rivoltini L. Cannibalism of live lymphocytes by human metastatic but not primary melanoma cells Cancer Res. 2006 66 3629 3638 10.1158/0008-5472.CAN-05-3204 16585188

44. Calcinotto A. Filipazzi P. Grioni M. Iero M. De Milito A. Ricupito A. Cova A. Canese R. Jachetti E. Rossetti M. Modulation of microenvironment acidity reverses anergy in human and murine tumor-infiltrating T lymphocytes Cancer Res. 2012 72 2746 2756 10.1158/0008-5472.CAN-11-1272 22593198

45. Fais S. De Milito A. You H. Qin W. Targeting vacuolar H + -ATPases as a new strategy against cancer Cancer Res. 2007 67 10627 10630 10.1158/0008-5472.CAN-07-1805 18006801

46. Pettersen E.O. Ebbesen P. Gieling R.G. Williams K.J. Dubois L. Lambin P. Ward C. Meehan J. Kunkler I.H. Langdon S.P. Targeting tumour hypoxia to prevent cancer metastasis. From biology, biosensing and technology to drug development: The METOXIA consortium J. Enzyme Inhib. Med. Chem. 2015 30 689 721 10.3109/14756366.2014.966704 25347767

47. Perez-Sayans M. Garcia-Garcia A. Scozzafava A. Supuran C.T. Inhibition of V-ATPase and carbonic anhydrases as interference strategy with tumor acidification processes Curr. Pharm. Des. 2012 18 1407 1413 10.2174/138161212799504876 22360559

48. De Milito A. Iessi E. Logozzi M. Lozupone F. Spada M. Marino M.L. Federici C. Perdicchio M. Matarrese P. Lugini L. Proton pump inhibitors induce apoptosis of human B-cell tumors through a caspase-independent mechanism involving reactive oxygen species Cancer. Res. 2007 67 5408 5417 10.1158/0008-5472.CAN-06-4095 17545622

49. Luciani F. Spada M. De Milito A. Molinari A. Rivoltini L. Montinaro A. Marra M. Lugini L. Logozzi M. Lozupone F. Effect of proton pump inhibitor pretreatment on resistance of solid tumors to cytotoxic drugs J. Natl. Cancer Inst. 2004 96 1702 1713 10.1093/jnci/djh305 15547183

50. Lugini L. Federici C. Borghi M. Azzarito T. Marino M.L. Cesolini A. Spugnini E.P. Fais S. Proton pump inhibitors while belonging to the same family of generic drugs show different anti-tumor effect J. Enzyme Inhib. Med. Chem. 2016 31 538 545 10.3109/14756366.2015.1046062 26018420

51. Thiry A. Supuran C.T. Masereel J.M. Dogne´ J.M. Recent developments of carbonic anhydrase inhibitors as potential anticancer drugs J. Med. Chem. 2008 51 3051 3056 10.1021/jm701526d 18348513

52. Federici C. Lugini L. Marino M.L. Carta F. Iessi E. Azzarito T. Supuran C.T. Fais S. Lansoprazole and carbonic anhydrase IX inhibitors sinergize against human melanoma cells J. Enzyme Inhib. Med. Chem. 2016 31 119 125 10.1080/14756366.2016.1177525 27142956

53. Reshkin S.J. Cardone R.A. Harguindey S. Na + –H + exchanger, pH regulation and cancer Recent Pat Anticancer Drug Discov. 2013 8 85 99 10.2174/1574892811308010085 22738122

54. Swayampakula M. McDonald P.C. Vallejo M. Coyaud E. Chafe S.C. Westerback A. Venkateswaran G. Shankar J. Gao G. Laurent E.M.N. The interactome of metabolic enzyme carbonic anhydrase IX reveals novel roles in tumor cell migration and invadopodia/MMP14-mediated invasion Oncogene 2017 36 6244 6261 10.1038/onc.2017.219 28692057 PMC5684442

55. Forgac M. Vacuolar ATPases: Rotary proton pumps in physiology and pathophysiology Nat. Rev. Mol. Cell Biol. 2007 8 917 929 10.1038/nrm2272 17912264

56. Spugnini E.P. Citro G. Fais S. Proton pump inhibitors as anti vacuolar-ATPases drugs: A novel anticancer strategy J. Exp. Clin. Cancer Res. 2010 29 44 10.1186/1756-9966-29-44 20459683 PMC2876100

57. Von Schwarzenberg K. Wiedmann R.M. Oak P. Schulz S. Zischka H. Wanner G. Efferth T. Trauner D. Vollmar A.M. Mode of cell death induction by pharmacological vacuolar H + -ATPase (V-ATPase) inhibition J. Biol. Chem. 2013 288 1385 1396 10.1074/jbc.M112.412007 23168408 PMC3543021

58. Schempp C.M. von Schwarzenberg K. Schreiner L. Kubisch R. Muller R. Wagner E. Vollmar A.M. V-ATPase inhibition regulates anoikis resistance and metastasis of cancer cells Mol. Cancer Ther. 2014 13 926 937 10.1158/1535-7163.MCT-13-0484 24482380

59. Sennoune S.R. Bakunts K. Martinez G.M. Chua-Tuan J.L. Kebir Y. Attaya M.N. Martínez-Zaguilán R. Vacuolar H + -ATPase in human breast cancer cells with distinct metastatic potential: Distribution and functional activity Am. J. Physiol. Cell Physiol. 2004 286 C1443 C1452 10.1152/ajpcell.00407.2003 14761893

60. Martinez-Zaguilan R. Raghunand N. Lynch R.M. Bellamy W. Martinez G.M. Rojas B. Smith D. Dalton W.S. Gillies R.J. pH and drug resistance. I. functional expression of plasmalemmal V-type H + -ATPase in drug-resistant human breast carcinoma cell lines Biochem. Pharmacol. 1999 57 1037 1046 10.1016/S0006-2952(99)00022-2 10796074

61. Raghunand N. Martinez-Zaguilan R. Wright S.H. Gillies R.J. pH and drug resistance. II. Turnover of acidic vesicles and resistance to weakly basic chemotherapeutic drugs Biochem. Pharmacol. 1999 57 1047 1058 10.1016/S0006-2952(99)00021-0 10796075

62. Von Schwarzenberg K. Lajtos T. Simon L. Muller R. Vereb G. Vollmar A.M. V-ATPase inhibition overcomes trastuzumab resistance in breast cancer Mol. Oncol. 2014 8 9 19 10.1016/j.molonc.2013.08.011 24055142 PMC5528506

63. Perez-Sayans M. Somoza-Martin J.M. Barros-Angueira F. Diz P.G. Rey J.M. Garcia-Garcia A. Multidrug resistance in oral squamous cell carcinoma: The role of vacuolar ATPases Cancer Lett. 2010 295 135 143 10.1016/j.canlet.2010.03.019 20418015

64. Huang L. Lu Q. Han Y. Li Z. Zhang Z. Li X. ABCG2/V-ATPase was associated with the drug resistance and tumor metastasis of esophageal squamous cancer cells Diagn. Pathol. 2012 7 180 10.1186/1746-1596-7-180 23244569 PMC3542252

65. Xu J. Xie R. Liu X. Wen G. Jin H. Yu Z. Jiang Y. Zhao Z. Yang Y. Ji B. Expression and functional role of vacuolar H + -ATPase in human hepatocellular carcinoma Carcinogenesis 2012 33 2432 2440 10.1093/carcin/bgs277 22962303

66. Chung C. Mader C.C. Schmitz J.C. Atladottir J. Fitchev P. Cornwell M.L. Koleske A.J. Crawford S.E. Gorelick F. The vacuolar-ATPase modulates matrix metalloproteinase isoforms in human pancreatic cancer Lab. Investig. 2011 91 732 743 10.1038/labinvest.2011.8 21339745 PMC3084324

67. Lu Q. Lu S. Huang L. Wang T. Wan Y. Zhou C.X. Zhang C. Zhang Z. Li X. The expression of V-ATPase is associated with drug resistance and pathology of non-small-cell lung cancer Diagn. Pathol. 2013 8 145 10.1186/1746-1596-8-145 23984887 PMC3846167

68. Perut F. Avnet S. Fotia C. Baglio S.R. Salerno M. Hosogi S. Kusuzaki K. Baldini N. V-ATPase as an effective therapeutic target for sarcomas Exp. Cell Res. 2014 320 21 32 10.1016/j.yexcr.2013.10.011 24416789

69. Brisson L. Bański P. Sboarina M. Dethier C. Danhier P. Fontenille M.J. Van Hée V.F. Vazeille T. Tardy M. Falces J. Lactate Dehydrogenase B Controls Lysosome Activity and Autophagy in Cancer Cancer Cell 2016 30 418 431 10.1016/j.ccell.2016.08.005 27622334

70. Lu X. Qin W. Li J. Tan N. Pan D. Zhang H. Xie L. Yao G. Shu H. Yao M. The growth and metastasis of human hepatocellular carcinoma xenografts are inhibited by small interfering RNA targeting to the subunit ATP6L of proton pump Cancer Res. 2005 65 6843 6849 10.1158/0008-5472.CAN-04-3822 16061667

71. Ekinci D. Cavdar H. Durdagi S. Oktay T. Murat Ş. Claudiu T.S. Structure-activity relationships for the interaction of 5,10-dihydroindeno[1,2- b ]indole derivatives with human and bovine carbonic anhydrase isoforms I, II, III, IV and VI Eur. J. Med. Chem. 2012 49 68 73 10.1016/j.ejmech.2011.12.022 22245047

72. Supuran C.T. Inhibition of carbonic anhydrase IX as a novel anticancer mechanism World J. Clin. Oncol. 2012 3 98 103 10.5306/wjco.v3.i7.98 22787577 PMC3394083

73. Said H.M. Supuran C.T. Hageman C. Staab A. Polat B. Katzer A. Scozzafava A. Anacker J. Flentje M. Vordermark D. Modulation of carbonic anhydrase 9 (CA9) in human brain cancer Curr. Pharm. Des. 2010 16 3288 3299 10.2174/138161210793429788 20819065

74. Neri D. Supuran C.T. Interfering with pH regulation in tumours as a therapeutic strategy Nat. Rev. Drug Discov. 2011 10 767 777 10.1038/nrd3554 21921921

75. Pastorekova S. Parkkila S. Pastorek J. Supuran C.T. Carbonic anhydrases: Current state of the art, therapeutic applications and future prospects J. Enzyme Inhib. Med. Chem. 2004 19 199 229 10.1080/14756360410001689540 15499993

76. Vullo D. Franchi M. Gallori E. Pastorek J. Scozzafava A. Pastorekova S. Supuran C.T. Carbonic anhydrase inhibitors: Inhibition of the tumor-associated isozyme IX with aromatic and heterocyclic sulfonamides Bioorg. Med. Chem. Lett. 2003 13 1005 1009 10.1016/S0960-894X(03)00091-X 12643899

77. Vullo D. Innocenti A. Nishimori I. Pastorek J. Scozzafava A. Pastorekova S. Supuran C.T. Carbonic anhydrase inhibitors. Inhibition of the transmembrane isozyme XII with sulfonamides—A new target for the design of antitumor and antiglaucoma drugs? Bioorg. Med. Chem. Lett. 2005 15 963 969 10.1016/j.bmcl.2004.12.053 15686894

78. Zatovicova M. Jelenska L. Hulikova A. Csaderova L. Ditte Z. Ditte P. Goliasova T. Pastorek J. Pastorekova S. Carbonic anhydrase IX as an anticancer therapy target: Preclinical evaluation of internalizing monoclonal antibody directed to catalytic domain Curr. Pharm. Des. 2010 16 3255 3263 10.2174/138161210793429832 20819068

79. Lou Y. McDonald P.C. Oloumi A. Chia S. Ostlund C. Ahmadi A. Kyle A. Auf dem Keller U. Leung S. Huntsman D. Targeting tumor hypoxia: Suppression of breast tumor growth and metastasis by novel carbonic anhydrase IX inhibitors Cancer Res. 2011 71 3364 3376 10.1158/0008-5472.CAN-10-4261 21415165

80. Bowman E.J. Siebers A. Altendorf K. Bafilomycins: A class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells Proc. Natl. Acad. Sci. USA 1988 85 7972 7976 10.1073/pnas.85.21.7972 2973058 PMC282335

81. Dröse S. Bindseil K.U. Bowman E.J. Siebers A. Zeeck A. Altendorf K. Inhibitory effect of modified bafilomycins and concanamycins on P- and V-type adenosinetriphosphatases Biochemistry 1993 32 3902 3906 10.1021/bi00066a008 8385991

82. McSheehy P.M. Troy H. Kelland L.R. Judson I.R. Leach M.O. Griffiths J.R. Increased tumour extracellular pH induced by Bafilomycin A1 inhibits tumour growth and mitosis in vivo and alters 5-fluorouracil pharmacokinetics Eur. J. Cancer 2003 39 532 540 10.1016/S0959-8049(02)00671-8 12751386

83. Lim J.H. Park J.W. Kim M.S. Park S.K. Johnson R.S. Chun Y.S. Bafilomycin induces the p21-mediated growth inhibition of cancer cells under hypoxic conditions by expressing hypoxia-inducible factor-1alpha Mol. Pharmacol. 2006 70 1856 1865 10.1124/mol.106.028076 16940187

84. Ohta T. Arakawa H. Futagami F. Fushida S. Kitagawa H. Kayahara M. Nagakawa T. Miwa K. Kurashima K. Numata M. Bafilomycin A1 induces apoptosis in the human pancreatic cancer cell line Capan-1 J. Pathol. 1998 185 324 330 10.1002/(SICI)1096-9896(199807)185:3<324::AID-PATH72>3.0.CO;2-9 9771488

85. Morimura T. Fujita K. Akita M. Nagashima M. Satomi A. The proton pump inhibitor inhibits cell growth and induces apoptosis in human hepatoblastoma Pediatr. Surg. Int. 2008 24 1087 1094 10.1007/s00383-008-2229-2 18712525

86. Nakashima S. Hiraku Y. Tada-Oikawa S. Hishita T. Gabazza E.C. Tamaki S. Imoto I. Adachi Y. Kawanishi S. Vacuolar H + -ATPase inhibitor induces apoptosis via lysosomal dysfunction in the human gastric cancer cell line MKN-1 J. Biochem. 2003 134 359 364 10.1093/jb/mvg153 14561721

87. Hishita T. Tada-Oikawa S. Tohyama K. Miura Y. Nishihara T. Tohyama Y. Yoshida Y. Uchiyama T. Kawanishi S. Caspase-3 activation by lysosomal enzymes in cytochrome c -independent apoptosis in myelodysplastic syndrome-derived cell line P39 Cancer Res. 2001 61 2878 2884 11306462

88. Erickson K.L. Beutler J.A. Cardellina J.H. II Boyd M.R. Salicylihalamides A and B, novel cytotoxic macrolides from the marine sponge Haliclona sp. J. Org. Chem. 1997 62 8188 8192 10.1021/jo971556g 11671930

89. Sasse F. Steinmetz H. Hofle G. Reichenbach H. Archazolids, new cytotoxic macrolactones from Archangium gephyra (Myxobacteria). Production, isolation, physico-chemical and biological properties J. Antibiot. Tokyo 2003 56 520 525 10.7164/antibiotics.56.520 12931860

90. Gagliardi S. Nadler G. Consolandi E. Parini C. Morvan M. Legave M.N. Belfiore P. Zocchetti A. Clarke G.D. James I. 4-Pentadienamides: Novel and selective inhibitors of the vacuolar H + -ATPase of osteoclasts with bone antiresorptive activity J. Med. Chem. 1998 41 1568 1573 10.1021/jm9800144 9572882

91. Nadler G. Morvan M. Delimoge I. Belfiore P. Zocchetti A. James I. Zembryki D. Lee-Rycakzewski E. Parini C. Consolandi E. (2Z,4E)-5-(5,6-dichloro-2-indolyl)-2-methoxy-N-(1,2,2,6,6-pentamethylpiperidin-4-yl)-2,4-pentadienamide, a novel, potent and selective inhibitor of the osteoclast V-ATPase Bioorg. Med. Chem. Lett. 1998 8 3621 3626 10.1016/S0960-894X(98)00660-X 9934482

92. Huss M. Wieczorek H. Inhibitors of V-ATPases: Old and new players J. Exp. Biol. 2009 212 341 346 10.1242/jeb.024067 19151208

93. Lebreton S. Jaunbergs J. Roth M.G. Ferguson D.A. De Brabander J.K. Evaluating the potential of Vacuolar ATPase inhibitors as anticancer agents and multigramsynthesis of the potent salicylihalamide analog saliphenylhalamide Bioorg. Med. Chem. Lett. 2008 18 5879 5883 10.1016/j.bmcl.2008.07.003 18657422 PMC2593418

94. Wiedmann R.M. von Schwarzenberg K. Palamidessi A. Schreiner L. Kubisch R. Liebl J. Schempp C. Trauner D. Vereb G. Zahler S. The V-ATPase-inhibitor archazolid abrogates tumor metastasis via inhibition of endocytic activation of the rho-GTPase Rac1 Cancer Res. 2012 72 5976 5987 10.1158/0008-5472.CAN-12-1772 22986742

95. Mullin J.M. Gabello M. Murray L.J. Farrell C.P. Bellows J. Wolov K.R. Kearney K.R. Rudolph D. Thornton J.J. Proton pump inhibitors: Actions and reactions Drug Discov. Today 2009 14 647 660 10.1016/j.drudis.2009.03.014 19443264

96. Schwartz M.D. Dyspepsia, peptic ulcer disease, and esophageal reflux disease West. J. Med. 2002 176 98 103 11897730 PMC1071675

97. Ward R.M. Kearns G.L. Proton pump inhibitors in pediatrics: Mechanism of action, pharmacokinetics, pharmacogenetics, and pharmacodynamics Paediatr. Drugs 2013 15 119 131 10.1007/s40272-013-0012-x 23512128 PMC3616221

98. Han Y.M. Hahm K.B. Park J.M. Hong S.P. Kim E.H. Paradoxically augmented anti-tumorigenic action of proton pump inhibitor and Gastrinin APCMin/+ intestinal polyposis model Neoplasia 2014 16 73 83 10.1593/neo.131510 24563620 PMC3924549

99. Canitano A. Iessi E. Spugnini E.P. Federici C. Fais S. Proton pump inhibitors induce a caspase-independent antitumor effect against human multiple myeloma Cancer Lett. 2016 376 278 283 10.1016/j.canlet.2016.04.015 27084522

100. Bellone M. Calcinotto A. Filipazzi P. De Milito A. Fais S. Rivoltini L. The acidity of the tumor microenvironment is a mechanism of immune escape that can be overcome by proton pump inhibitors Oncoimmunology 2013 2 e22058 10.4161/onci.22058 23483769 PMC3583905

101. Chen M. Huang S.L. Zhang X.Q. Zhang B. Zhu H. Yang V.W. Zou X.P. Reversal effects of pantoprazole on multidrug resistance in human gastric adenocarcinoma cells by down-regulating the V-ATPases/mTOR/HIF-1α/P-gp and MRP1 signaling pathway in vitro and in vivo J. Cell. Biochem. 2012 113 2474 2487 10.1002/jcb.24122 22396185 PMC3762681

102. Yeo M. Kim D.K. Kim Y.B. Oh T.Y. Lee J.E. Cho S.W. Kim H.C. Hahm K.B. Selective induction of apoptosis with proton pump inhibitor in gastric cancer cells Clin. Cancer Res. 2004 10 8687 8696 10.1158/1078-0432.CCR-04-1065 15623654

103. Yeo M. Kim D.K. Park H.J. Cho S.W. Cheong J.Y. Lee K.J. Blockage of intracellular proton extrusion with proton extrusions with proton pump inhibitor induces apoptosis in gastric cancer Cancer Sci. 2008 99 185 10.1111/j.1349-7006.2007.00642.x 18224727

104. Gu M. Zhang Y. Zhou X. Ma H. Yao H. Ji F. Rabeprazole exhibits antiproliferative effects on human gastric cancer cell lines Oncol. Lett. 2014 8 1739 1744 10.3892/ol.2014.2354 25202402 PMC4156221

105. Huang S. Chen M. Ding X. Zhang X. Zou X. Proton pump inhibitor selectively suppresses proliferation and restores the chemosensitivity of gastric cancer cells by inhibiting STAT3 signaling pathway Int. Immunopharmacol. 2013 17 585 592 10.1016/j.intimp.2013.07.021 23973653

106. Shen Y. Chen M. Huang S. Zou X. Pantoprazole inhibits human gastric adenocarcinoma SGC-7901 cells by downregulating the expression of pyruvate kinase M2 Oncol. Lett. 2016 11 717 722 10.3892/ol.2015.3912 26870273 PMC4727057

107. Feng S. Zheng Z. Feng L. Yang L. Chen Z. Lin Y. Gao Y. Chen Y. Proton pump inhibitor pantoprazole inhibits the proliferation, self-renewal and chemoresistance of gastric cancer stem cells via the EMT/β-catenin pathways Oncol. Rep. 2016 36 3207 3214 10.3892/or.2016.5154 27748935

108. Patel K.J. Lee C. Tan Q. Tannock I.F. Use of the proton pump inhibitor pantoprazole to modify the distribution and activity of doxorubicin: A potential strategy to improve the therapy of solid tumors Clin. Cancer Res. 2013 19 6766 6776 10.1158/1078-0432.CCR-13-0128 24141627

109. Yu M. Lee C. Wang M. Tannock I.F. Influence of the proton pump inhibitor lansoprazole on distribution and activity of doxorubicin in solid tumors Cancer Sci. 2015 106 1438 1447 10.1111/cas.12756 26212113 PMC4638015

110. Fan S. Niu Y. Tan N. Wu Z. Wang Y. You H. Ke R. Song J. Shen Q. Wang W. LASS2 enhances chemosensitivity of breast cancer by counteracting acidic tumor microenvironment through inhibiting activity of V-ATPase proton pump Oncogene 2013 32 1682 1690 10.1038/onc.2012.183 22580606

111. Glunde K. Guggino S.E. Solaiyappan M. Pathak A.P. Ichikawa Y. Bhujwalla Z.M. Extracellular acidification alters lysosomal trafficking in human breast cancer cells Neoplasia 2003 5 533 545 10.1016/S1476-5586(03)80037-4 14965446 PMC1502575

112. Jin U.H. Lee S.O. Pfent C. Safe S. The aryl hydrocarbon receptor ligand omeprazole inhibits breast cancer cell invasion and metastasis BMC Cancer 2014 14 498 10.1186/1471-2407-14-498 25011475 PMC4226953

113. Robey I.F. Martin N.K. Bicarbonate and dichloroacetate: Evaluating pH altering therapies in a mouse model for metastatic breast cancer BMC Cancer 2011 11 235 10.1186/1471-2407-11-235 21663677 PMC3125283

114. Udelnow A. Kreyes A. Ellinger S. Landfester K. Walther P. Klapperstueck T. Wohlrab J. Goh W. Sleptsova-Freidrich I. Petrovic N. Use of proton pump inhibitors as adjunct treatment for triple-negative breast cancers. An introductory study J. Pharm. Pharm. Sci. 2014 17 439 446 25224353 10.18433/j34608

115. Lindner K. Borchardt C. Schopp M. Burgers A. Stock C. Hussey D.J. Haier J. Hummel R. Proton pump inhibitors (PPIs) impact on tumour cell survival, metastatic potential and chemotherapy resistance, and affect expression of resistance-relevant miRNAs in esophageal cancer J. Exp. Clin. Cancer Res. 2014 33 73 10.1186/s13046-014-0073-x 25175076 PMC4431491

116. Lee Y.Y. Jeon H.K. Hong J.E. Cho Y.J. Ryu J.Y. Choi J.J. Lee S.H. Yoon G. Kim W.Y. Do I.G. Proton pump inhibitors enhance the effects of cytotoxic agentsin chemoresistant epithelial ovarian carcinoma Oncotarget 2015 6 35040 35050 26418900 10.18632/oncotarget.5319 PMC4741507

117. Song J. Ge Z. Yang X. Luo Q. Wang C. You H. Ge T. Deng Y. Lin H. Cui Y. Hepatic stellate cells activated by acidic tumor microenvironment promote the metastasis of hepatocellular carcinoma via osteopontin Cancer Lett. 2015 356 713 720 10.1016/j.canlet.2014.10.021 25449435

118. Henne-Bruns D. Knippschild U. Würl P. Omeprazole inhibits proliferation and modulates autophagy in pancreatic cancer cells PLoS ONE 2011 6 e20143 21629657 10.1371/journal.pone.0020143 PMC3101238

119. Jin U.H. Kim S.B. Safe S. Omeprazole inhibits pancreatic cancer cell invasion through a nongenomic aryl hydrocarbon receptor pathway Chem. Res. Toxicol. 2015 28 907 918 10.1021/tx5005198 25826687 PMC4948974

120. Ihling A. Ihling C.H. Sinz A. Gekle M. Acidosis-induced changes in proteome patterns of the prostate cancer-derived tumor cell line AT-1 J. Proteome Res. 2015 14 3996 4004 10.1021/acs.jproteome.5b00503 26214752

121. Harris R.J. Cloughesy T.F. Liau L.M. Prins R.M. Antonios J.P. Li D. Yong W.H. Pope W.B. Lai A. Nghiemphu P.L. pH-weighted molecular imaging of gliomas using amine chemical exchange saturation transfer MRI Neuro Oncol. 2015 17 1514 1524 10.1093/neuonc/nov106 26113557 PMC4648305

122. Coman D. Huang Y. Rao J.U. De Feyter H.M. Rothman D.L. Juchem C. Hyder F. Imaging the intratumoral-peritumoral extracellular pH gradient of gliomas NMR Biomed. 2016 29 309 319 10.1002/nbm.3466 26752688 PMC4769673

123. Ferrari S. Perut F. Fagioli F. Brach Del Prever A. Meazza C. Parafioriti A. Picci P. Gambarotti M. Avnet S. Baldini N. Proton pump inhibitor chemosensitization in human osteosarcoma: From the bench to the patients’ bed J. Transl. Med. 2013 11 268 10.1186/1479-5876-11-268 24156349 PMC3815282

124. Wang B.Y. Zhang J. Wang J.L. Sun S. Wang Z.H. Wang L.P. Zhang Q.L. Lv F.F. Cao E.Y. Shao Z.M. Intermittent high dose proton pump inhibitor enhances the antitumor effects of chemotherapy in metastatic breast cancer J. Exp. Clin. Cancer Res. 2015 34 85 10.1186/s13046-015-0194-x 26297142 PMC4546346

125. Brana I. Ocana A. Chen E.X. Razak A.R. Haines C. Lee C. Douglas S. Wang L. Siu L.L. Tannock I.F. A phase I trial of pantoprazole in combination with doxorubicin in patients with advanced solid tumors: Evaluation of pharmacokinetics of both drugs and tissue penetration of doxorubicin Investig. New Drugs 2014 32 1269 1277 10.1007/s10637-014-0159-5 25213162

126. Papagerakis S. Bellile E. Peterson L.A. Pliakas M. Balaskas K. Selman S. Hanauer D. Taylor J.M. Duffy S. Wolf G. Proton pump inhibitors and histamine 2 blockers are associated with improved overall survival in patients with head and neck squamous carcinoma Cancer Prev. Res. 2014 7 1258 1269 10.1158/1940-6207.CAPR-14-0002 25468899 PMC4372797

127. Falcone R. Roberto M. D’Antonio C. Romiti A. Milano A. Onesti C.E. Marchetti P. Fais S. High-doses of proton pumps inhibitors in refractory gastro-intestinal cancer: A case series and the state of art Dig. Liver Dis. 2016 48 1503 1505 10.1016/j.dld.2016.08.126 27651344

128. Marchetti P. Milano A. D’Antonio C. Romiti A. Falcone R. Roberto M. Fais S. Association between proton pump inhibitors and metronomic capecitabine as salvage treatment for patients with advanced gastrointestinal tumors: A randomized phase II trial Clin. Colorectal Cancer 2016 15 377 380 10.1016/j.clcc.2016.06.005 27435761

129. Spugnini E.P. Baldi A. Buglioni S. Carocci F. de Bazzichini G.M. Betti G. Pantaleo I. Menicagli F. Citro G. Fais S. Lansoprazole as a rescue agent in chemoresistant tumors: A phase I/II study in companion animals with spontaneously occurring tumors J. Transl. Med. 2011 9 221 10.1186/1479-5876-9-221 22204495 PMC3264547

130. Spugnini E.P. Buglioni S. Carocci F. Francesco M. Vincenzi B. Fanciulli M. Fais S. High dose lansoprazole combined with metronomic chemotherapy: A phase I/II study in companion animals with spontaneously occurring tumors J. Transl. Med. 2014 12 225 10.1186/s12967-014-0225-y 25143012 PMC4145230

131. Chiche J. Ilc K. Laferriere J. Trottier E. Dayan F. Mazure N.M. Brahimi-Horn M.C. Pouyssegur J. Hypoxia-inducible carbonic anhydrase IX and XII promote tumor cell growth by counteracting acidosis through the regulation of the intracellular pH Cancer Res. 2009 69 358 368 10.1158/0008-5472.CAN-08-2470 19118021

132. Gondi G. Mysliwietz J. Hulikova A. Jen J.P. Swietach P. Kremmer E. Zeidler R. Antitumor efficacy of a monoclonal antibody that inhibits the activity of cancer-associated carbonic anhydrase XII Cancer Res. 2013 73 6494 6503 10.1158/0008-5472.CAN-13-1110 24030978

133. Chiche J. Brahimi-Horn M.C. Pouyssegur J. Tumour hypoxia induces a metabolic shift causing acidosis: A common feature in cancer J. Cell. Mol. Med. 2010 14 771 794 10.1111/j.1582-4934.2009.00994.x 20015196 PMC3823111

134. Pastorek J. Pastorekova S. Hypoxia-induced carbonic anhydrase IX as a target for cancer therapy: From biology to clinical use Semin. Cancer Biol. 2015 31 52 64 10.1016/j.semcancer.2014.08.002 25117006

135. Svastova E. Witarski W. Csaderova L. Kosik I. Skvarkova L. Hulikova A. Zatovicova M. Barathova M. Kopacek J. Pastorek J. Carbonic anhydrase IX interacts with bicarbonate transporters in lamellipodia and increases cell migration via its catalytic domain J. Biol. Chem. 2012 287 3392 3402 10.1074/jbc.M111.286062 22170054 PMC3270993

136. Parks S.K. Cormerais Y. Marchiq I. Pouyssegur J. Hypoxia optimises tumour growth by controlling nutrient import and acidic metabolite export Mol. Asp. Med. 2016 47 3 14 10.1016/j.mam.2015.12.001 26724171

137. Doyen J. Parks S.K. Marcie S. Pouyssegur J. Chiche J. Knock-down of hypoxia-induced carbonic anhydrases IX and XII radiosensitizes tumor cells by increasing intracellular acidosis Front. Oncol. 2012 2 199 10.3389/fonc.2012.00199 23316475 PMC3539669

138. Dubois L. Peeters S. Lieuwes N.G. Geusens N. Thiry A. Wigfield S. Carta F. McIntyre A. Scozzafava A. Dogné J.M. Specific inhibition of carbonic anhydrase IX activity enhances the in vivo therapeutic effect of tumor irradiation Radiother. Oncol. 2011 99 424 431 10.1016/j.radonc.2011.05.045 21676479

139. Supuran C.T. How many carbonic anhydrase inhibition mechanisms exist? J. Enzyme Inhib. Med. Chem. 2016 31 345 360 10.3109/14756366.2015.1122001 26619898

140. Supuran C.T. Advances in structure-based drug discovery of carbonic anhydrase inhibitors Expert Opin. Drug Discov. 2017 12 61 88 10.1080/17460441.2017.1253677 27783541

141. Supuran C.T. Structure-based drug discovery of carbonic anhydrase inhibitors J. Enzyme Inhib. Med. Chem. 2012 27 759 772 10.3109/14756366.2012.672983 22468747

142. Dubois L. Lieuwes N.G. Maresca A. Thiry A. Supuran C.T. Scozzafava A. Wouters B.G. Lambin P. Imaging of CA IX with fluorescent labelled sulfonamides distinguishes hypoxic and (re)-oxygenated cells in a xenograft tumour model Radiother. Oncol. 2009 92 423 428 10.1016/j.radonc.2009.06.019 19616332

143. Stiti M. Cecchi A. Rami M. Abdaoui M. Barragan-Montero V. Scozzafava A. Guari Y. Winum J.Y. Supuran C.T. Carbonic anhydrase inhibitor coated gold nanoparticles selectively inhibit the tumor-associated isoform IX over the cytosolic isozymes I and II J. Am. Chem. Soc. 2008 130 16130 16131 10.1021/ja805558k 18989963

144. Battke C. Kremmer E. Mysliwietz J. Gondi G. Dumitru C. Brandau S. Lang S. Vullo D. Supuran C. Zeidler R. Generation and characterization of the first inhibitory antibody targeting tumour-associated carbonic anhydrase XII Cancer Immunol. Immunother. 2011 60 649 658 10.1007/s00262-011-0980-z 21298264 PMC11028429

145. Hoeben B.A. Kaanders J.H. Franssen G.M. Troost E.G. Rijken P.F. Oosterwijk E. van Dongen G.A. Oyen W.J. Boerman O.C. Bussink J. PET of hypoxia with 89 Zr-labeled cG250-F(ab′) 2 in head and neck tumors J. Nucl. Med. 2010 51 1076 1083 10.2967/jnumed.109.073189 20554724

146. Perez-Sayans M. Suarez-Penaranda J.M. Pilar G.D. Supuran C.T. Pastorekova S. Barros-Angueira F. Gándara-Rey J.M. García-García A. Expression of CA-IX is associated with advanced stage tumors and poor survival in oral squamous cell carcinoma patients J. Oral Pathol. Med. 2012 41 667 674 10.1111/j.1600-0714.2012.01147.x 22486898

147. Winum J.Y. Vullo D. Casini A. Montero J.L. Scozzafava A. Supuran C.T. Carbonic anhydrase inhibitors: Inhibition of transmembrane, tumor-associated isozyme IX, and cytosolic isozymes I and II with aliphatic sulfamates J. Med. Chem. 2003 46 5471 5477 10.1021/jm030911u 14640555

148. Winum J.Y. Vullo D. Casini A. Montero J.L. Scozzafava A. Supuran C.T. Carbonic anhydrase inhibitors. Inhibition of cytosolic isozymes I and II and transmembrane, tumor-associated isozyme IX with sulfamates including EMATE also acting as steroid sulfatase inhibitors J. Med. Chem. 2003 46 2197 2204 10.1021/jm021124k 12747791

149. Guler O.O. De Simone G. Supuran C.T. Drug design studies of the novel antitumor targets carbonic anhydrase IX and XII Curr. Med. Chem. 2010 17 1516 1526 10.2174/092986710790979999 20166929

150. Alterio V. Hilvo M. Di Fiore A. Supuran C.T. Pan P. Parkkila S. Scaloni A. Pastorek J. Pastorekova S. Pedone C. Crystal structure of the catalytic domain of the tumor-associated human carbonic anhydrase IX Proc. Natl. Acad. Sci. USA 2009 106 16233 16238 10.1073/pnas.0908301106 19805286 PMC2752527

151. Dubois L. Douma K. Supuran C.T. Chiu R.K. van Zandvoort M.A. Pastoreková S. Scozzafava A. Wouters B.G. Lambin P. Imaging the hypoxia surrogate marker CA IX requires expression and catalytic activity for binding fluorescent sulfonamide inhibitors Radiother. Oncol. 2007 83 367 373 10.1016/j.radonc.2007.04.018 17502120

152. Ahlskog J.K. Dumelin C.E. Trüssel S. Mårlind J. Neri D. In vivo targeting of tumor-associated carbonic anhydrases using acetazolamide derivatives Bioorg. Med. Chem. Lett. 2009 19 4851 4856 10.1016/j.bmcl.2009.06.022 19615903

153. McDonald P.C. Winum J.Y. Supuran C.T. Dedhar S. Recent developments in targeting carbonic anhydrase IX for cancer therapeutics Oncotarget 2012 3 84 97 10.18632/oncotarget.422 22289741 PMC3292895

154. Pacchiano F. Carta F. McDonald P.C. Lou Y. Vullo D. Scozzafava A. Dedhar S. Supuran C.T. Ureido-substituted benzene sulfonamides potently inhibit carbonic anhydrase IX and show antimetastatic activity in a model of breast cancer metastasis J. Med. Chem. 2011 54 1896 1902 10.1021/jm101541x 21361354

155. Gieling R.G. Babur M. Mamnani L. Burrows N. Telfer B.A. Carta F. Winum J.Y. Scozzafava A. Supuran C.T. Williams K.J. Antimetastatic effect of sulfamate carbonic anhydrase IX inhibitors in breast carcinoma xenografts J. Med. Chem. 2012 55 5591 5600 10.1021/jm300529u 22621623

156. Supuran C.T. Winum J.Y. Carbonic anhydrase IX inhibitors in cancer therapy: An update Future Med. Chem. 2015 7 1407 1414 10.4155/fmc.15.71 26230880

157. Lock F.E. McDonald P.C. Lou Y. Serrano I. Chafe S.C. Ostlund C. Aparicio S. Winum J.Y. Supuran C.T. Dedhar S. Targeting carbonic anhydrase IX depletes breast cancer stem cells within the hypoxic niche Oncogene 2013 32 5210 5219 10.1038/onc.2012.550 23208505

158. Boyd N.H. Walker K. Fried J. Hackney J.R. McDonald P.C. Benavides G.A. Spina R. Audia A. Scott S.E. Libby C.J. Addition of carbonic anhydrase 9 inhibitor SLC-0111 to temozolomide treatment delays glioblastoma growth in vivo JCI Insight 2017 2 10.1172/jci.insight.92928 29263302 PMC5752277

159. Maresca A. Scozzafava A. Supuran C.T. 7,8-disubstituted- but not 6,7-disubstituted coumarins selectively inhibit the transmembrane, tumor-associated carbonic anhydrase isoforms IX and XII over the cytosolic ones I and II in the low nanomolar/subnanomolar range Bioorg. Med. Chem. Lett. 2010 20 7255 7258 10.1016/j.bmcl.2010.10.094 21067924

160. Touisni N. Maresca A. McDonald P.C. Lou Y. Scozzafava A. Dedhar S. Winum J.Y. Supuran C.T. Glycosyl coumarin carbonic anhydrase IX and XII inhibitors strongly attenuate the growth of primary breast tumors J. Med. Chem. 2011 54 8271 8277 10.1021/jm200983e 22077347

161. Stillebroer A.B. Mulders P.F. Boerman O.C. Oyen W.J. Oosterwijk E. Carbonic anhydrase IX in renal cell carcinoma: Implications for prognosis, diagnosis, and therapy Eur. Urol. 2010 58 75 83 10.1016/j.eururo.2010.03.015 20359812

162. Siebels M. Rohrmann K. Oberneder R. Stahler M. Haseke N. Beck J. Hofmann R. Kindler M. Kloepfer P. Stief C. A clinical phase I/II trial with the monoclonal antibody cG250 (RENCAREX ® ) and interferon-alpha-2a in metastatic renal cell carcinoma patients World J. Urol. 2011 29 121 126 10.1007/s00345-010-0570-2 20512580

163. Mishra C.B. Kumari S. Angeli A. Monti S.M. Buonanno M. Prakash A. Tiwari M. Supuran C.T. Design, synthesis and biological evaluation of N-(5-methyl-isoxazol-3-yl/1,3,4-thiadiazol-2-yl)-4-(3-substitutedphenylureido) benzenesulfonamides as human carbonic anhydrase isoenzymes I, II, VII and XII inhibitors J. Enzyme Inhib. Med. Chem. 2016 31 174 179 10.1080/14756366.2016.1197221 27314170

164. Li G. Feng G. Gentil-Perret A. Genin C. Tostain J. Serum carbonic anhydrase 9 level is associated with postoperative recurrence of conventional renal cell cancer J. Urol. 2008 180 510 514 10.1016/j.juro.2008.04.024 18550116

165. Gigante M. Li G. Ferlay C. Perol D. Blanc E. Paul S. Zhao A. Tostain J. Escudier B. Negrier S. Prognostic value of serum CA9 in patients with metastatic clear cell renal cell carcinoma under targeted therapy Anticancer Res. 2012 32 5447 5451 23225450

166. Chamie K. Klöpfer P. Bevan P. Störkel S. Said J. Fall B. Belldegrun A.S. Pantuck A.J. Carbonic anhydrase-IX score is a novel biomarker that predicts recurrence and survival for high-risk, nonmetastatic renal cell carcinoma: Data from the phase III ARISER clinical trial Urol. Oncol. 2015 33 e25 e33 10.1016/j.urolonc.2015.02.013 25823535

167. Rademakers S.E. Hoogsteen I.J. Rijken P.F. Oosterwijk E. Terhaard C.H. Doornaert P.A. Langendijk J.A. van den Ende P. Takes R. De Bree R. Pattern of CAIX expression is prognostic for outcome and predicts response to ARCON in patients with laryngeal cancer treated in a phase III randomized trial Radiother. Oncol. 2013 108 517 522 10.1016/j.radonc.2013.04.022 23719582

168. Turkbey B. Lindenberg M.L. Adler S. Kurdziel K.A. McKinney Y.L. Weaver J. Vocke C.D. Anver M. Bratslavsky G. Eclarinal P. PET/CT imaging of renal cell carcinoma with 18 F-VM4-037: A phase II pilot study Abdom. Radiol. 2016 41 109 118 10.1007/s00261-015-0599-1 26830617 PMC7788639

169. Belldegrun A.S. Chamie K. Kloepfer P. Fall B. Bevan P. Störkel S. ARISER: A randomized double blind phase III study to evaluate adjuvant cG250 treatment versus placebo in patients with high-risk ccRCC—Results and implications for adjuvant clinical trials J. Clin. Oncol. 2013 31 4507

170. Stillebroer A.B. Boerman O.C. Desar I.M. Boers-Sonderen M.J. van Herpen C.M. Langenhuijsen J.F. Smith-Jones P.M. Oosterwijk E. Oyen W.J. Mulders P.F. Phase 1 radioimmunotherapy study with lutetium 177-labeled anti-carbonic anhydrase IX monoclonal antibody girentuximab in patients with advanced renal cell carcinoma Eur. Urol. 2013 64 478 485 10.1016/j.eururo.2012.08.024 22980441

171. Lugini L. Sciamanna I. Federici C. Iessi E. Spugnini E.P. Fais S. Antitumor effect of combination of the inhibitors of two new oncotargets: Proton pumps and reverse transcriptase Oncotarget 2017 8 4147 4155 10.18632/oncotarget.13792 27926505 PMC5354819