Molecular machineries of pH dysregulation in tumor microenvironment: potential targets for cancer therapy

Introduction

Solid tumors, as a class of complex diseases, are characterized by their unique hallmarks, including: anomalous initiation and development through altered genotype and phenotype, self-organization and adaptation, collective specific behavior with molecular networks and irregular pattern formation and dynamism within tumor microenvironment (TME). Such traits result in an uncontrolled process of cell growth, division, metastasis and progression.

The cancer cells, in comparison with normal cells, exhibit distinctive physiopathology, including: (a) autonomous mechanisms of cell growth, (b) divergence from the factors involved in growth inhibition, (c) evasion from anoikis, immunosurveillance and apoptosis, (d) evolutionary regulation of growth, (e) invasiveness and metastatic colonization. These characteristics are manifested through genetic and epigenetic changes.^1,2 The initiation of cancer is deemed to be linked with the hypoxia and irregular metabolism in energetic pathway, in particular glucose, resulting in production of acidic byproducts. Such dysregulation of pH within the cancer cells and TME, in comparison with the normal cells, seems to be one of the main causes for the metastasis, drug resistance, and the recurrence of the disease after the course(s) of treatment. The normal cells have intracellular pH (pH_i) of ~ 7.2, but in cancer cells, it is about 7.4. Further, in the normal cells extracellular pH (pH_e) is approximately 7.4, while in cancer cells it reduces to 6.7-7.1.³ Therefore, any changes in the activity and/or expression of membrane ion pumps and proton transfers may lead to a lower pH_e and higher pH_i. All these occur because the cancer cells can significantly increase the functional expression of several key molecular machineries, including: Na⁺–H⁺ exchanger (NHE1), H⁺-ATPases, carbonic anhydrase IX (CA IX) and carbonic anhydrase XII (CA XII).^1,4-7

Based on scientific praxis, a number of theories have been coined to explain the unique behavior of cancer cells. Some of studies have shown that the hypoxia, Warburg effect and aberrant metabolism play central roles in the pH dysregulation and also progression and metastasis of cancer cells.^8-10 In 1880s, Steven Paget articulated the "seed and soil" notion. Based on this theory, environment (soil) is necessary for the tumor cells (seed).^11,12 In this concept, however, there still remain some key questions. How exactly cancer cells can escape from the immune system and apoptosis? What are the main causes of their resistance to chemotherapy? In fact, answering all these questions can improve the missing link(s) between the actual mechanisms of cancer progression and pharmacotherapy strategies. The right answers to these questions may be entombed within the TME as a crucial part of the cancer puzzle. In this review, we will discuss the TME and its role in cancer development and the factors involved in the pH dysregulation. Further, the relationship between the pH dysregulation and some key molecular machineries such as NHE1, H⁺-ATPases, CA9 and CA12 are articulated in terms of their association with invasion, metastasis, and drug resistance. At the end of each section, we will focus on some genetic changes that can be occurred in the TME on the molecular machineries such as NHE1, H⁺-ATPases, CA9 and CA12.

Tumor microenvironment

Intricate interacting network between the cancerous and non-cancerous cells often lead to formation of a permissive milieu called tumor microenvironment. From pathological perspective, the TME include blood vessels, bone marrow-derived inflammatory cell, signaling molecules, extracellular matrix (ECM) and lymphocytes.¹³ As shown in Fig. 1, the cells involved with TME are recognized by various cell-specific molecular markers often expressed on the cell surface.¹⁴

Fig. 1. Schematic representation of tumor microenvironment. NK-cell: natural killer cell, CAF: cancer associated-fibroblast.

× Schematic representation of tumor microenvironment. NK-cell: natural killer cell, CAF: cancer associated-fibroblast.

Fig. 1.

Based upon the Warburg effect, tumor cells tend to be anaerobic glycolytic pathway even in the presence of oxygen, and hence produce lactate. This is important in the acidic TME, as well as contribution to the homeostasis and the immune defense system. From a holistic point of view, lactate production may contribute in several functions, including: (a) evasion of cancer cells from immunosurveillance of the immune system, (b) alteration of the metabolism processes in the stromal cells function such as T cells, (c) increase of the inflammation mediated IL-17 production by T cells and macrophages, (d) inhibition of the activity of dendritic cells (DCs), (e) enhanced mobility of tumor cells, (f) prevention of the monocyte migration and cytokine extrication, and (g) initiation of angiogenesis and tumor vascularization by induction of factors such as IL-8, NF-κB and VEGF/VEGF-A via HIF-1.¹⁵

Na⁺/H⁺ exchanger 1 (NHE1)

The pH change in the cancerous cells, in comparison with the normal tissue, seems to be common in most solid tumors. In fact, they show some similar phenotypic properties in terms of pH dysregulation, which includes some common key metabolic changes. Such alterations seem (i) to promote acid-producing pathways, (ii) to activate the oncogenic signals, (iii) to induce and develop the hypoxia, and (iv) to alter the functional expression of some key molecular transporters involved in the regulation of pH. Despite these changes, most of studies have focused on the clinical importance of proton transporters, in particular NHE1 and V-ATPase. Factors that promote the cellular pH or enhance the functional expression of NHE1 leading to an increased pH_i and carcinogenicity include viruses (e.g., human papilloma virus), persistent hypoxia and HIF, oncogenes and viral proteins, p53 defect, growth factors, hormones, and chemicals carcinogenic and genomic products.⁷⁷

Structure and function of NHE1

Changes in the functional expression of plasma membrane ionic pumps and also transmitters which facilitates the distribution of H⁺ in the extracellular fluid may result in stabilized increased the pH_i and decreases the pH_e. One of the plasma membrane ionic pumps with enhanced functional expression is NHE1. The human NHE1 gene is a member of a gene family that is also called solute carrier 9 (SLC9). The NHE gene family is pH regulator of the cytoplasm and intracellular organelles. There are 9 different genes locus (NHE1-9 or /SLC9A1-9) in the human genome for the NHE gene family while some related pseudogenes have also been identified in human genome. These genes encode proteins that have 25 to 75% similarity with each other.⁷⁸ The human NHE1 (Fig. 2) is an integral membrane protein and encoded by SLC9A1 gene, which is composed of 815 amino acids, and comprises two domains:

Fig. 2. Three-dimensional representation of the modeled human NHE1. The membrane-spanning protein, NHE1, was modeled by means of Chimera software. Subcellular localization of different domains of the protein is predicted via Uniprot database.

× Three-dimensional representation of the modeled human NHE1. The membrane-spanning protein, NHE1, was modeled by means of Chimera software. Subcellular localization of different domains of the protein is predicted via Uniprot database.

Fig. 2.

a hydrophobic N-terminal membrane domain which contains amino acids 1 to 500 and show a secondary structure with 12 transmembrane spans, to which the NHE1 transport activity is attributed, and

a domain embracing 501 to 815 amino acids constituting the cytoplasmic domain (C-terminus tail) that is fundamental for the NHE1 regulation.

The N-terminal domain of NHE1 exchanges intracellular H⁺ with extracellular Na⁺. This molecular exchanger protects cells from the acidification and regulates pH_i.⁷⁹ Moreover, human NHE1 is involved in some important biological and physiological activities of cells, including: regulation of intracellular pH, acid-base electrolyte control, homeostasis, adhesion, migration and proliferation.⁷⁸ Studies have shown that early stage transformation by oncogene(s) in normal cells occurs along with NHE1 activity, and then the cytosolic alkalinization happens in consistent correlation with the occurrence of glycolysis. In addition, it has been shown that such alkalinization stimulates a set of transformation markers such as an increase in the growth rate, substrate-independent growth, and growth factor(s)-independent growth.

The intracellular pH determines that how cells obtain their energy, in which an alkalinized pH_i stimulates an anaerobic glycolysis, while an acidic pH provokes an oxidative phosphorylation process. Such molecular storms caused by the activation of NHE1 decrease with its inhibition leading to suppression of tumor progression, in large part by acidifying the intracellular fluid of cancer cells.⁷⁷

Further, it should be noted that the tissue distribution and intracellular position of NHEs varry markedly. Recent studies have identified a variety of plasma membrane NHEs and several organelle-based isoforms with similar functions in mammalian physiology. NHE1 exists abundantly in various types of cells, which is found as an extended member. Although it is placed on the cell surface, its functional presences at different domains in various cell types reflects its diverse functions. For example, NHE1 in fibroblasts is localized at the boundary of lamellipodia, the basolateral membrane of epithelia, and the disks and t-tubules of cardiac.^80-82 It is believed that NHE1 performs two important functions. First, it is the chief alkalinizing mechanism in various types of cells to protect cells against the harmful effects of increased acidity. If the production of acidic metabolites increases, the H⁺-derived from the related metabolic pathways via different secretory routes are intensified and remain unchecked. Hence, to maintain the acid-base balance in the cytoplasm, it plays a central role along with bicarbonate-transporting systems such as, Na⁺-HCO3- co‏-transporters, Na⁺-dependent HCO3-/Cl- exchangers and Cl-/HCO3-exchangers. Second, it provides a channel for the traverse of Na⁺, which is paired with Cl^¯ and absorbs H₂O necessary to maintain the cell volume at a steady state level. As a result, the cytoplasmic contents is modulated by a sharp rise in extracellular osmolality. In many specialized secretive cell types, for example the acinar cells of the parotid and sublingual glands, the activation of NHE1 is also necessary for the secretion of derived fluids. Change in the activity of NHEs is related to the pathogenesis of several diseases, such as hypertension, congenital secretive diarrhea, diabetes, and tissue harm affected by ischemia/reperfusion.⁷⁸

The phosphorylation alteration of basic amino acids in C-terminal and the reaction with intracellular lipids and proteins can regulate the NHE1 activities (Fig. 3).⁸³ Transport activity of NHE1 is changed by regulators through altering affinity to intracellular H⁺, in which it is activated in alkaline pH_i.⁸⁴

Fig. 3. The main residues at the human NHE1 3D structure. A) Phenylalanine (F) 161 has a main role for covering the channel pore. B) Serine (S) 703 can be modified to phosphoserine, in this form it is ready for binding to 14-3-3 adaptor protein. C) The site of interaction between NHE1 and calcineurin homologous protein II (CHP2). D) The actin-binding site between ERM (ezrin, radixin and moesin) proteins and NHE1.

× The main residues at the human NHE1 3D structure. A) Phenylalanine (F) 161 has a main role for covering the channel pore. B) Serine (S) 703 can be modified to phosphoserine, in this form it is ready for binding to 14-3-3 adaptor protein. C) The site of interaction between NHE1 and calcineurin homologous protein II (CHP2). D) The actin-binding site between ERM (ezrin, radixin and moesin) proteins and NHE1.

Fig. 3.

The approximate binding sites of regulatory proteins and cofactors which bind to NHE1 in breast cancer cells are shown in Table 1. A number of protein kinases are involved in the regulation of NHE1, protein kinase p160ROCK facilitates the assembly of actin stress fibers through RhoA which leads to the activation of fibroblastic NHE1.⁸⁵ Angiotensin II stimulates the NHE1 through ERK phosphorylation, which depends on p38MAPK. Therefore, the phosphorylation of NHE1 at residues T⁷¹⁸, S⁷²³, S⁷²⁶, and S⁷²⁹ seem to regulate the apoptosis as reported previously for the pro-B cells.⁸⁶ Protein kinase B (Akt) plays a key role in phosphorylation of S⁶⁴⁸, and inhibits the activity of NHE1 in myocardial cells, perhaps by interfering with CaM binding. In other cell types, although Akt phosphorylation leads to the activation of NHE1, and plays a key role in the survival, growth, and perhaps metastasis. In the phosphorylation of NHE, some other protein kinases are involved through various mechanisms such as Ca²⁺/calmodulin-dependent kinase (CaMKII) and Nck-interacting kinase (NIK) mechanisms.⁴

Studies have shown that the activity of NHE1 in the breast cancer leads to acidification of extracellular microenvironment. This process facilitates the degradation ECM by protease and increases the invasion and metastasis.⁴ Fibroblast cells with mutated NHE1 show lower pH_i as compared to the NHE1 of wild type cells. In such circumstances, the expression of enzymes related to the glycogenesis reduces 3-4 times, which include fructose1,6bisphosphatase, galactokinase and phosphorylase kinase. Besides, the concentration of lactate dehydrogenase (LDH) is reduced, which is an enzyme crucial for the Warburg effect.¹ There are several key inhibitors and protein kinases that justify the activity of NHE1. These regulators can be considered as interesting molecular target for pharmacotherapy of cancer, (e.g., ErbB2).⁹⁶ The NHE1 has a critical role in chronic and acute myeloid leukemia differentiation. The simultaneous pharmacological inhibition of P38 MAPK and NHE1 under hypoxia significantly suppresses the expression of C/EBP. These results show the hypoxia-induced K562 differentiation by inhibiting NHE1, in large part due to upregulation of C/EBPα through‏ p38 MAPK signaling pathway. This suggests that the inhibition of NHE1 in hypoxic microenvironment could be considered as possible treatment for the leukaemic disease.⁹⁷

The genetic variation and diversity

The NHEs have been detected generally in organisms from prokaryotes to eukaryotes such as plants, fungi and animals. In prokaryotes like the Escherichia coli, there are two types of NHEs (NhaA and NhaB) that are employed in H⁺ gradient produced by H⁺-ATPase to export Na⁺ or Li⁺. In terms of stoichiometry, the NhaA and NhaB transported 1Na⁺:2H⁺ and 2Na⁺:3H⁺, respectively. Various eukaryotes encoding different types of NHEs, for example, Arabidopsis thaliana contains at least several NHE proteins so-called AtSOS1 or AtNHX1–6 and the nematode Caenorhabditis elegans encodes nine NHE-like proteins such as CeNHX1–9.⁷⁸

Wakabayashi et al identified mutations of Tyr⁴⁵⁴or Arg⁴⁵⁸ in transmembrane domain 11 (TM11) that disrupt the plasma membrane domain expression of NHE1 and possibly play a role in protein folding. Their studies showed that mutation of Arg⁴⁴⁰ in intracellular loop 5 (IL5) could reduce the sensitivity of NHE1 to pH_i sensitivity, even though, mutation in Gly amino acid in Gly-rich area could increase its in TM11 domain.¹⁰⁰ Genomic analyses of NHE1 display mutation at Phe¹⁶² amino acid of human NHE1 that extremely decrease the affinity of M4 domain to Na⁺. In return, the mutations of Glu³⁵⁰ and Gly³⁵⁶ in TM9 considerably reduce the catalytic efficiency of the transporter without affecting other subsections (Fig. 4).⁷⁸ Genetic change in human NHE1 is related to hypertension, which is clearly related to NHE1‏ phosphorylation that increases the activation of MAPK pathway. It is suggested that the change in protein kinase pathway possibility results in cation homeostasis, hence leading to hypertension.¹⁰¹

Fig. 4. NHE1 sequence analysis in terms of probability variant(s). A and B) Some of main natural variants, C) Polymorphism at the position 682 lead to change from asparagine (N) to lysine (K). D) The 261-length domain that is missed in NHE1 isoform 2.

× NHE1 sequence analysis in terms of probability variant(s). A and B) Some of main natural variants, C) Polymorphism at the position 682 lead to change from asparagine (N) to lysine (K). D) The 261-length domain that is missed in NHE1 isoform 2.

Fig. 4.

Vacuolar H⁺ ATPases (V-ATPases)

The V-ATPases are ATP-dependent H⁺ pumps that show a great variation in the cell membrane. The proteins are composed of several subunits (up to 14), a transmembrane domain is called V0 complex and V1 complex embedded in the cytoplasm. The V-ATPase functions includ pH homeostasis, contribution in endocytosis, participation in activation of proteases and invasion of tumor cells. Furthermore, it is involved in several cellular activities such as angiogenesis, autophagy and also interplay with mTOR for amino acids sensing.^98,102

Carbonic anhydrases

The aberrant metabolism of cancer cells may lead to the production of proton and carbon dioxide (CO₂), which can be converted into carbonic acids by the carbonic anhydrases (CAs) - a process that occurs due to high consumption of glucose. Many proteins are involved in the metabolism of glucose such as glucose transporters and pH regulatory proteins. Of these, carbonic anhydrase family plays an important role, especially CAIX and CAXII. It should be noted that little change (even 0.1 pH units) in the intracellular and extracellular pH could disrupt the molecular functions of solid tumor cells, including: ATP synthesis, enzyme functionalities, migration, invasiveness and metastasis. Also, different isoforms of extracellular proteins (e.g., fibronectin and tenascin) might be generated by alternative splicing that may not occur in the normal cells.¹³⁰

The carbonic anhydrases are zinc metalloproteinase enzymes which can be found among all organisms, including in microbes, fungi, plants, and animals.¹³⁰ Five groups of carbonic anhydrases (α, β, γ, δ, and ζ) have evolved independently, among which alpha-carbonic anhydrases were found in the human. There are 16 isoforms of alpha-carbonic anhydrase in primates, 15 isoforms of which are common and also shared in human. They are classified based on their location in the cell, catalytic activity and response to several groups of inhibitors, as follows: (i) some are placed in the cell membrane (CAI, CAII, CAIII, CAVII and CAXIII), (ii) several are attached to the membrane (CAIV, CAIX, CAXII and CAXIV), (iii) two of them are found in the mitochondria (CAVA and CAVB) and (iv) secretory isoforms (CAVI), three of them are catalytic isoforms, so-called CA-related proteins (CARPs) which include CAVIII, CAX and CAXI.^130-132

The carbonic anhydrases perform a vital reaction in cells, that is: “CO₂⁺ + H₂O↔HCO₃- + H⁺”. Also, CA isoforms are involved in many process and functions in mammals. Among the most important ones, we can point out the acid-base balance, release of electrolytes, bone resorption, calcification, lipogenesis, gluconeogenesis and ureagenesis in human. Further, this enzyme appears to contribute in photosynthesis and carbon dioxide fixation reaction in bacteria and plants.^132,133 In human, CAs are present in a very wide range of tissues. In fact, they are present almost in most tissues, including: digestive, nervous and genital systems, renal, lungs, skin and others organs.¹³³ In 1992, Pastoreková et al showed that CAs are associated with cancer. Thus, it became clear that CAIX and CAXII increase simultaneously in many tumor tissues and normal tissue could be related to the cancer development and progression.¹³⁴

Sulphonamides, sulphamates and sulphamides are the main compounds for inhibition of carbonic anhydrases, which are able to bind to zinc ion and active site of the enzyme resulting in their inactivation.135 It has been over 50 years that sulphonamides have been used as diuretics and antiglaucoma medicine. Now, this component is proposed as anticonvulsant, anti-obesity, anticancer and anti-pain agent.¹³⁶ Various compounds have been designed for the inhibition of carbonic anhydrases, and some of them are mentioned in the following context:

Fluorescent sulphonamides are used for imaging purposes and CAs are involved in tumor acidity.¹³⁷

Addition of positive and negative charges to sulphonamide and sulphamate enable them more likely to cross the cell membrane and influence the intracellular CAs such as CAIX and CAXII.¹³⁰

Acetazolamide inhibits all CA isoforms with a high capability. To date, new acetazolamide derivatives have been produced with much better and efficient functional properties than the parent drug acetazolamide.¹³⁰

Sugar-containing sulfonamides-sulfamates-sulfamides are extremely hydrophilic compounds that cannot easily cross the cell membrane. Thus, they influence the extracellular CAs, including CAIX and CAXII.¹³⁵

Coumarins, thiocoumarins and polyamines are new chemotypes that have CAs inhibitory properties. These inhibitors bind to CAs active site and do not have interaction with zinc ions of this enzyme.¹³⁸

Protein tyrosine kinase (PTK) inhibitors (e.g., imatinib and nilotinib) are also the CAs inhibitors, however, they are not as effective as sulphonamides and coumarins.^139,140

Monoclonal antibodies such as M75 and WX-G250 have been used.^141,142

CA inhibitors coated gold nanoparticles have also been used.¹⁴³

Structure and function of CAIX in tumor cells

CAIX seems to be more complex than the other isoforms of CAs. Unlike other isoforms that contain a polypeptide chain with just one domain, CAIX is a multidomain protein (Fig. 5). The CAIX isoform is comprised of (i) a short intracytosolic sector with an unknown role, (ii) a small transmembrane section, (iii) an extracellular catalytic domain, (iv) a proteoglycan like domain that exclusively belongs to CAIX - not seen in other isoforms - and is involved in cell adhesion processes, and (v) a stocky peptide signal.

Fig. 5. Subcellular localization, transmembrane topology and sequence processing of carbonic anhydrase IX (CAIX). A) The residues from 436 to 459 that is localized in cytoplasm. B) The residues from 38 to 414 that known as extracellular domain. C) Transmembrane segment of the CA9 between amino acids 415 – 435. D) The N-terminal signal peptide at the first 37 residues are denoted as signal peptide.

× Subcellular localization, transmembrane topology and sequence processing of carbonic anhydrase IX (CAIX). A) The residues from 436 to 459 that is localized in cytoplasm. B) The residues from 38 to 414 that known as extracellular domain. C) Transmembrane segment of the CA9 between amino acids 415 – 435. D) The N-terminal signal peptide at the first 37 residues are denoted as signal peptide.

Fig. 5.

CAIX may be seen in the form of a dimmer, in which they may be connected to each other through disulfide bonds. Among CAs protein families, CAIX has a high catalytic activity. The catalytic domain of this protein contains zinc (Zn²⁺) ion as well as three histidine amino acids and a water molecule (Fig. 6).¹³¹

Fig. 6. Functional sites, disulfide bond and post-translational modifications of the carbonic anhydrase IX. A) Zinc binding to the histidine 226, 228 and 251 require a catalytically activity for the CA9 protein. B) Histidine 200 act as a proton acceptor that is crucial for the enzyme catalysis activity. C) The positions of cysteine 156 and 336 that are participated in formation of disulfide bond. D and E) the O- and N-linked glycosylation relating to threonine 115 and asparagine 346.

× Functional sites, disulfide bond and post-translational modifications of the carbonic anhydrase IX. A) Zinc binding to the histidine 226, 228 and 251 require a catalytically activity for the CA9 protein. B) Histidine 200 act as a proton acceptor that is crucial for the enzyme catalysis activity. C) The positions of cysteine 156 and 336 that are participated in formation of disulfide bond. D and E) the O- and N-linked glycosylation relating to threonine 115 and asparagine 346.

Fig. 6.

CAIX is highly expressed in a variety of different types of cancers, whose expression is regulated by the transcription factor HIF-1 and is highly inducted in the hypoxic condition, and also associate with the cellular responses to radiotherapy and chemotherapy.¹³² The transcription factor HIF-1 consists of two subunits, i.e., alpha (α) and beta (β). In normoxia, the unit α is connected to pVHL (von Hippel–Lindau tumor suppressor protein), and therefore prevents the assembly of two subunits of α and β. However, under hypoxia, pVHL is separated from the unit α and leads to the connection of two α and β subunits to each other, at which point an active form of HIF-1 is created. Such phenomena result in the functional expression of the hypoxia responsive elements (HREs) such as glucose transporters (GLUT-1 and GLUT-3), vascular endothelial growth factor (VEGF) and CAIX.^56,134,144 Of these, CAIX contributes to the acidification of TME. It may also lead to the phenotype of metastasis. Moreover, this isoform is involved in the pH regulation and cellular connection in association with ion exchanger and Na⁺-HCO3- co-transporters.^145-147 Studies have shown that CAIX expression is under the control of promoter methylation in the kidney cells, which is an epigenetic mechanism.¹⁴⁶

Reports have shown that inactivation of CAIX gene with short hairpin RNA or inhibition of catalytic domain activity by drug could lead to a decrease in xenograft tumor size. Further, CAXII mRNA levels are upregulated in some cancer cells such as LS174Tr and HT29. Overall, suppression of both CAIX and CAXII appears to be the reason for 85% reduction in tumor growth. Intriguingly, CAIX expression in cancer cells could be a compensatory response to reduce the damaging effects of acidosis and hypoxia.^148-150 The use of CAIX inhibitors reduces proliferation of cancer cells and increases apoptosis in hypoxic conditions, sensitizing cancer cells to chemotherapy and radiotherapy.¹⁵¹ The membrane transporters, pH regulation machineries (e.g., CAIX) and hypoxia in cancer cells are mostly located in invadopodia areas (i.e., membranous actin-rich protrusios of the cells). Using the import mechanism of HCO₃- and export mechanism of H⁺, lactate and CO₂ are able to facilitate the cell movement.¹⁵² Special conditions of solid tumors (i.e., low pH_e, hypoxia and its molecular responses) allow cancer cells to show stem cell-like phenotype.^153,154 In esophageal cancer model, it was found that the expression of CAIX and some other genes (i.e., Sox-2, Nanog and Lin28) are related to an increase in stem cell-like phenotype.¹⁵⁵ Studies about breast cancer have shown that the expression of CAIX with stem cell-related factors is increased markedly.^156,157 The expression of CAIX is related to the expression of CD44, which is a stem cell marker. Besides, CAIX contributes to the activation of EMT phenomenon in epithelial cancer cells.^155,158 It has been shown that the overexpression CAIX is involved in the formation of focal adhesion in the cells.¹⁵⁹ It associate with β-catenin and the separation of E-cadherin from cytoskeletal - a key process for the separation of cell-cell junction.¹⁶⁰ Further, the activations of PI3K/Akt and FAK/PI3K/mTOR/p70S6K pathways seem to be in association with CAIX.^161,162

Table 1. Summarizing NHE1 regulation by proteins, cofactors, and protein kinases
Regulators	Mechanism of action	Biding site(s)	Ref.
Calmodulin (CaM)	Binding to a high- and low-affinity, blocks an auto-inhibitory site, consequently activating NHE1	From AAs 636 to 700	79
Calcineurin homologous protein (CHP)	(i) CHP1: NHE1 activity and its stabilization and localization to the plasma membrane (ii) CHP2: overexpressed in tumor cells; it is protective against serum deprivation–induced cell death by enhancing pHi and promoted proliferation of tumor cells (iii) CHP3: promote maturation and cell surface stability	518 to 537	87-89,90
phosphatidylinositol 4,5‏- biphosphate (PIP2)	Depletion of PIP2 results in ATP-dependent inhibition of NHE1	513, 520, 556 and 564	91
Carbonic anhydrase II (CAII)	Catalyzing the production of HCO3- and H+ from the hydration of CO2	790 to 802	92
Actin-binding ERM	Proper localization of NHE1 to the plasma membrane and maintaining cell shape	552 and 560	93
Heat shock protein 70 (Hsp70)	Binding to NHE1 and possible play a role in protein folding	Unknown site	94
14–3-3 adaptor protein	Binding to NHE1 when it is phosphorylated at S703. Thereby NHE1 has activate by protecting S703 from dephosphorylation	703	95
Several protein kinases	NHE1 stimulation and activation		85

Present and future prospects

Solid tumors display complex hallmarks. They are able to form a permissive microenvironment and show adaptation and evolution ed through functional expression of a number of different molecular machineries. Such traits make the treatment of solid tumors very hard. Although acid-base balance in cells is one of the main parameters to cellular homeostasis, unfortunately, this balance is disturbed in solid tumors‏. This is a unique characteristic of solid tumors, which seems to be initiated by hypoxia and aberrant metabolism of glucose resulting in production of acidic byproducts in cancerous cells. Such phenomena give rise to functional expression of array of functional proteins including transcription factors, enzymes and transporters. These biological elements are considered as potential targets, inhibition of which may provide effective pharmacotherapy in combination with chemotherapy and/or immunotherapy. Use of the proton transporter inhibitors as a new treatment for cancer were proposed by several research groups. Pouysségur group showed that use of these this inhibitor in combination with anti-angiogenic inhibitors could effectively abolish the solid tumors.⁷⁷ One advantage of this treatment modality seems to be the induction of less toxicity in comparison with the traditional chemotherapy strategies. In fact, the main purpose of this method was based on acid-base balance, employing the forces involved in controlling pH dysregulation in cancer cells and tissue, resulting in regression of tumor growth, prohibition of invasion and suppression of metastasis. This has been shown in many different human solid tumors. This could be beneficial because such treatment may lead to the inhibition of metastasis process and‏ neutralization of drug resistance, resulting in much more sensitization of cancer cells to chemotherapy, radiotherapy and immunotherapy. Cancer cells in different tissues show distinctive behaviors because they have different genetic pattern and mutations or expression . For instance, lower metastatic cells in breast cancer often use NHEs and HCO₃-- and H⁺- based transporting system to transfer proton, whereas extremely metastatic cells employ plasma membrane V-ATPases.¹⁶³ This issue is also true about‏ CAs, for example, CAII mRNA level was shown to be decreased in the lung and colon cancers, while the level of CAIX mRNA was reported to be extremely increased in basal and triple-negative breast cancers. Further, CAXII mRNA levels was shown to be substantially enhanced in all types of breast cancer. In accordance with these, CAIX and CAXII mRNA levels were reported to be increased in squamous cell carcinoma of the lung, but not in the lung adenocarcinomas.¹⁶⁴ A variety of mechanisms are involved in pH-dependent cancer cells' behaviors such as proliferation and survival, metabolic adaptation, metastatic and invasion. Detection of the molecular basis of this phenomenon is very important in understanding of how pH dysregulation can influence the progression and metastasis of solid tumors. It is now well-known that an increased pHi is in favor of cancer cells proliferation, and it also increases the cell survival through inhibition of apoptosis. In such circumstances, cancer cells proliferation seems to be somewhat independent from the influence of growth factors. Further, cancer cells need high energy and nucleotid biosynthesis for growth and proliferation in comparison with normal cells. As a result, metabolic program changes in cancer cells, which is known as the Warburg effect or ‘aerobic’ glycolysis. In solid tumors, cancerous cells are able to modulate/remodel the ECM through pH dysregulation resulting in metastasis. The pH_i increase seems to necessary for the migration of cells, while the pH_e decrease appears to interfere with the degradation of ECM that favors further invasion of cancer. Researchers who attempt to discover pH dysregulation in solid tumors have focused on proton transport, while other ions may be involved in the cancer, including HCO₃-. However, many fundamental questions remain unanswered. We need to address how cancer cells get capability to escape from the immune system surveillance within TME? What are the holistic roles of solute transporters? How the vesicular enzymes behave in lower pH_e? Is pH dysregulation involved in anoikis? How single cancer cells communicate with neighboring cells during metastasis? How different types of cells communicate with TME? And, many other questions.

We have previously capitalized on development of immunotherapies and various multifunctional nanomedicines to combat different types of solid tumors.^165-182 However, it seems that ultimate therapy of solid tumors might need use of robust synthetic lethality through combination therapy via targeting different key molecules involved in the dysregulation of pH, formation of TME, tumor vascularization, and even tumor metabolism. he main aim of this review was to bring about the importance of molecular machineries related to the pH dysregulation within TME and that the use of relevant inhibitors against key regulators of pH might suppress tumor progression, metastasis and further invasion. It can be suggested that targeting the key elements involved in formation of TME and pH along with other conventional therapies can sensitize cancer cells to combinational therapy and hence reduce drug resistances. This may improve the survival rate of cancer patients. It should be also noted that the cancerous cells in different part of solid tumors may present distinct molecular patterns in terms of pH dysregulation, and hence some proof-of-concept conforming studies on epigenetic/genetic and proteomic/metabolomic aspects may help us to design much more effective treatment modalities to improve the currently used chemotherapy and immunotherapy.

Acknowledgment

The authors are very grateful for the financial support (grant No: RCPN-94012) provided by the Research Center for Pharmaceutical Nanotechnology (RCPN), BioMedicine Instiute, Tabriz University of Medical Sciences.

Conflict of interest

Authors declare no conflict of interest in this study.

Ethical approval

Not applicable.

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