Tumor metabolism, cancer cell transporters, and microenvironmental resistance

Abstract

Cancer cells reprogram their metabolic machineries to enter into permanent glycolytic pathways. The full reason for such reprogramming takes place is unclear. However, this metabolic switch is not made in vain for the lactate that is generated and exported outside cells is reused by other cells. This results in the generation of a pH gradient between the low extracellular pH that is acidic (pHe) and the higher cytosolic alkaline or near neutral pH (pHi) environments that are tightly regulated by the overexpression of several pumps and ion channels (e.g. NHE-1, MCT-1, V-ATPase, CA9, and CA12). The generation of this unique pH gradient serves as a determining factor in defining “tumor fitness”. Tumor fitness is the capacity of the tumor to invade and metastasize due to its ability to reduce the efficiency of the immune system and confer resistance to chemotherapy. In this article, we highlight the importance of tumor microenvironment in mediating the failure of chemotherapeutic agents.

• Cancer
• metabolism
• pH
• proton transporters

Introduction

In contrast to normal eukaryotic cells that generate energy by the breakdown of pyruvate, cancer cells generate energy by the nonoxidative breakdown of glucose with tumor cells displaying glycolytic rates up to 200 times higher than normal tissues. In the early 1920s, a technician at Otto Warburg’s lab spilled NaHCO₃ by accident and created an opportunity for Otto Warburg to observe that those cancer cells became highly fermentative. This has led to the development of the Warburg hypothesis that states that the driver for tumor development is a mitochondrial insult that leads to insufficient cellular respiration, explaining why even in the presence of adequate oxygen tumor cells preferentially turn to fermentation of lactic acid for an energy supply “Warburg Effect”1. While in yeast the fermentation results in the formation of ethanol, in eukaryotic cells glycolysis results in the formation of lactate. Cancer cells are unique in the way by which they switch to anaerobic glycolysis in the presence of a plentiful supply of oxygen1,2. Glycolysis occurs through the glycolysis pathway or the pentose phosphate pathway (PPP; Figure 1).

Figure 1. A model that describes the cytoplasmic utilization of glucose through either glycolysis or PPPs.

Glycolysis is an energy-rich process in that it requires two molecules of ATP to produce four ATP molecules. In contrast, PPP requires less energy as it requires only one ATP to produce four ATP molecules. Therefore, PPP has a lower metabolic cost in comparison to glycolysis. Furthermore, PPP maintains cellular redox status by providing a reduced form of glutathione which removes reactive oxygen species (ROS). Although the generation of ROS is an indication of cell death or induction of gene variation (mutation), ROS are also signaling molecules that are useful in the manipulation of cellular migration3–6. The generation of lactic acid is very helpful in allowing the progression of metastasis.

Warburg effect

Warburg effect is a cellular behavior rather than a disease that has been exploited clinically as diagnostic tool with Positron Emission Tomography (PET) scan7–9 to detect a tumor and to some extent for therapeutic purposes10–12. Since the 1920s the mitochondrion–cancer correlation plays a critical part of tumor biology. Otto Warburg and recent scientists have considered cancer as mitochondrial injury13–15. In particular, several studies have shown a decrease in mitochondrion number within cells16–19. Decreasing the efficiency of Krebs’ cycle pushes tumor cells to utilize the glucose in the cytoplasm either through glycolysis or the PPP20–22. In this context, it is suggested that the Warburg effect reveals that some cancer undergoes a loss of mitochondrial activity (i.e. loss of endosymbiotic relationship between mitochondrion and the cell), and so most probably are returning to a primitive state (primitive microorganisms)1. Diminishing the mitochondrial activity has evolutionarily advantaged such as keeping cancer cell survival through inhibition of apoptosis23–25. Mitochondrial dynamic (fission/fusion processes) perturbations have been detected in cancer26–28.

Causes of metabolic transformation

Numerous studies have identified the changes that occur leading to the metabolic transformation of tumor cells. In 2000, Reshkin et al. found that the first step in carcinogenesis was the development of cytoplasmic alkalinity due to Sodium-Hydrogen Exchanger-1 (NHE1) overexpression following human papilloma virus (HPV) transfection29. Later in 2007, Gatenby suggested that the Warburg effect is an adaptive strategy used by tumor cells to enable them to survive during periods of intermittent hypoxia29,30.

Sodium–hydrogen exchanger1 (NHE1)

The sodium-hydrogen antiporter is a housekeeping protein found in the plasma membrane31. It is an integral membrane protein having isoforms (NHE-1–9)32,33. These isoforms are expressed differently in tissues and localized differently sub-cellularly (Table 1). The physiological role of NHE is to maintain the pH of the cell and its organelles. Also, NHE1 acts as an anchor for actin filaments to control the integrity of the cortical cytoskeleton34. This occurs through a previously unrecognized structural link between NHE1 and the actin-binding proteins ezrin, radixin, and moesin (ERM)34. Therefore, it plays a role in the regulation of the cytoskeleton. NHE1 is the most extensively studied of the four isoforms. It has been implicated in pain35, several diseases, e.g. myocardial hypertrophy36 and cancer. In 2000, Reshkin et al. observed that NHE1 stimulation by HPV is a core event in malignant transformation and inhibition of NHE1 delayed such transformation29. NHE1 plays a critical role in tumor progression and invasion37. NHE-1 could be considered as a key regulator in tumor cell migration due to its role in the formation of lamellipodium, which are needed for migration38 and formation of invadopodium that degrades the extracellular matrix39. NHE-1 may also confer resistance to cancer chemotherapy either physiologically or biochemically. Physiologically NHE1 confers resistance to chemotherapy by inhibiting the diffusion of weakly basic drugs due to extracellular acidification, or by increasing the solubility of weakly acidic drugs when reaching cytoplasm and so preventing weakly acidic drugs from reaching their target. This type of resistance is known as ion trapping40–42. Biochemically, cancer cells develop antiacidifying mechanisms such as hyperactivity of the group of membrane-bound proton extrusion transporters, inactivation of Bcl-2, Bcl-xI, and/or a pH-dependent destabilization of p5343. These concerted dynamic changes work as an anti-chemotherapeutic shield involved in multiple drug resistance (MDR) and in the development of newly resistant subpopulations of tumor cells43. The final therapeutic aim is to target the selective acid–base disruption of cancer cell metabolism based on the H⁺-dependent thermodynamic advantages that malignant cells possess for their evolutionary survival as compared to their normal counterparts. Currently, NHE1 inhibitors such as amiloride and cariporide are being investigated as potential novel chemotherapeutic agents43,44. NHE1 inhibitors could be used directly due to their tumoricidal activity or they may function as chemosensitizers as in adjuvant therapy45.

Table 1. The level of expression of NHE isoforms in different tissues.

NHE isoform	Subcellular localization	Tissue distribution
NHE1	Plasma membrane	Ubiquitous protein found in most of tissues136
NHE2	Plasma membrane	Na+-absorptive epithelium of jejunum, ileum, and ascending and descending colon137
NHE3	Plasma membrane	Colon and small intestine and proximal tubule of138,139
NHE4	Plasma membrane	Stomach and inner medulla of the kidney139,140
NHE5	Plasma membrane	Nervous system
NHE6	Endosomes, plasma membrane141	Nervous system142
NHE7	Trans-Golgi network, endosomes and plasma membrane143	Putamen, occipital lobe of the brain, skeletal muscle and secretory tissues e.g. prostate, stomach, pancreas, pituitary gland, mammary gland, thyroid gland, salivary gland, and adrenal gland. Also, it found in liver small intestine and colon144
NHE8	Trans-Golgi network, endosomes	Gastric mucosa145
NHE9	Late recycling endosome 146	Nervous system142

Monocarboxylate transporter 1 (MCT-1)

Monocarboxylate transporters (MCTs) belong to the SLC16 gene family and are composed of 14 members46,47. MCTs handle lactate and pyruvate transport across the plasma membrane48,49. MCT is a symporter that transports H⁺and L-lactate or pyruvate bidirectionally48. Lactate is extruded continuously from transformed cells due to a switch from the Pasteur effect to the Warburg effect1. The presence of lactate extracellularly preserves a favorable habitat for malignant cells and an unfavorable environment for normal cells, so it offers and evolutionary advantage to tumor cells50. Tumor cells are highly adaptable, and it has been noted that individual tumors are composed of cells of different metabolic phenotypes. As the outer edge of the tumor cells may be in contact with blood vessels and thus have adequate oxygen and nutrients to allow glycolysis and oxidative phosphorylation to occur. In contrast, those cells in the hypoxic center of the tumor will utilize this switch to anaerobic glycolysis as dictated by their microenvironment. The lactate produced by oxidative tumor cells on the outer edge of the tumor is also thought to be an important signaling molecule, also known as “lactormone” (Figure 2)51 and, therefore, the presence of lactate transporters is very useful in cancer growth52,53. MCT-1 is expressed on the plasma membrane of the cell54. According to the endosymbiotic theory, mitochondria originated from bacteria that were engulfed by the plasma membrane55–57. Therefore, it is not surprising that MCT-1 is localized at both the mitochondrial and peroxisomal membranes (while evolution peroxisomes originated from mitochondria)58,59. One of the great evolutionary advantages is that MCT-1 plays a critical role in maintaining the redox status of the cell through inter-organelle lactate shuttling within the cytoplasm of the cell60. For example, NADH is regenerated from NAD⁺in the cytoplasm by lactate that has been generated from pyruvate inside a peroxisome and is subsequently translocated into the cytoplasm via MCT60. The presence of this shuttle is crucial to maintaining beta-oxidation of free fatty acids. Upregulation of this oxidation process has been correlated with cancer and its progression61. MCT1 also interacts with CD147/basigin and promotes cellular migration62,63. Moreover, MCT1 overexpression is correlated with metastasis64. MCT1 has been used as a prognostic factor65, and it is thought to contribute to tumor recurrence (relapse)66 and may mediate chemotherapy resistance too67. MCT1 is thought to be as a selective target in cancer68,69.

Figure 2. A tumor colony consists of a heterogeneous group of cell population that clusters around the blood vessel.

Box 1. Lactate Paradox in Cancer:

On the one hand, lactate supports tumor growth, cell migration, and metastasis147–150. One of the suggested mechanism is its interaction with NF-kappaB151,152. On the other hand, lactate is associated with spontaneous regression of cancer when exogenously administrated153. The presence of pyruvate also offers defense strategy against ROS-induced apoptosis154 and so prevents chemotherapy-induced apoptosis. So, overexpression of MCTs is a successful adaptive strategy against stress-induced cellular death.

Vacuolar ATPase (V-ATPase)

In 1981, Anraku and Ohsumi discovered Vacuolar–ATPase (V-ATPase) at the membrane of Saccharomyces cerevisiae70. V-ATPase is an evolutionarily conserved transmembrane enzyme that might originate from archaebacteria71. It consists of two major domains with each domain being composed of several subunits72. V-ATPase translocates protons (H⁺) to maintain the intracellular cytosol and organelles at optimal pH73. Organelles that contain V-ATPases are clathrin-coated vesicles, the Golgi complex, lysosomes, synaptic vesicles, and chromaffin granules. Examples of plasma membranes that contain V-ATPases are kidney proximal and distal tubule cells, the mitochondria-rich cells of the epididymis, macrophages, and osteoclasts74. V-ATPase is overexpressed in cancer. It works to increase extracellular acidity and maintains cytoplasmic alkalinity too75. It has been shown that metastatic phenotypes have higher expression of V-ATPase compared to NHE176. Overexpression of V-ATPase is associated with a drug-resistant phenotype77,78. Weakly basic drugs such as doxorubicin are ionized extracellularly. Moreover, the small amount of weakly basic drugs that can enter the cells becomes trapped within acidic vesicles75 (Figure 3). Inhibition of V-ATPase serves to re-sensitize tumor cells to such weakly basic drugs79–81. V-ATPase inhibitors include proton-pump inhibitor (e.g. lansoprazole), bafilomycin, concanamycin, the benzolactoneenamides salicylihalamides, and lobotamides, and, more recently, the macrolide lactams chondropsin and poecillastrin76,80,82–84.

Figure 3. Different pH zones induced by V-ATPase. BOH (unionized, lipid soluble) OH⁻ + B⁺ (ionized, lipid insoluble).

Carbonic anhydrases

Carbonic anhydrases 9 (CAIX)

Carbonic anhydrases (CAs) are a large family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide. They participate in a variety of biological processes, including respiration, calcification, acid–base balance, bone resorption, and the formation of aqueous humor, cerebrospinal fluid, saliva, and gastric acid.

Carbonic anhydrase 9 (CA9/CAIX) is an enzyme that in human is encoded by the CA9 gene85. It has also been linked to malignant transformation and hypoxia in various cancers86,87. CA9 also supports tumor growth and survival under normoxia too88. CA9 is a cancer-associated transmembrane enzyme, involving in pH regulation and ion transport89. The CA9 expression is driven by hypoxia90, and it is overexpressed preferentially at the edge of the tumor90 while other did observe it at the core of tumor (necrotic region)91. CA9 is correlated with tumor cell invasion/migration90,92; it is associated with poor prognosis of the tumor93,94. Also, CA9 is associated with resistance to chemotherapy95–99.

Carbonic anhydrase 12 (CAXI)

Carbonic anhydrase 12 is an enzyme that in humans is encoded by the CA12 gene100. CA12 is associated with poor prognosis101 also responsible for tumor invasion102 and associated with poor prognosis103 while other data suggested CA12 is related to good prognosis104,105. These data collectively reveal that CA12 overexpressed preferentially at the edge of the tumor (distally from necrotic regions). Our data demonstrate that CA9 is correlated with P-gp overexpression (unpublished DATA), and hence, it is correlated with cancer chemotherapy resistance.

Carbonic anhydrase inhibition to alter tumor pH gradient becomes a promising strategy for treating cancer89,106. Carbonic anhydrase inhibitors are divided into four groups: inorganic anions, sulfonamide-based compounds (sulphonamides, sulfanilamides, sulphamates, and their derivatives), phenols, and coumarins105. A sulfonamide CA9 inhibitor (SLC-0111) is presently in Phase I clinical trials107. It has been shown that simultaneous inhibition of both CA9 and CA12 has a greater impact than inhibition of each individually or even inhibition of HIF-α105. Indisulam is an example of nonselective CAs inhibitor where it inhibits both CA9 and CA12108.

Impact of these channels in creating the tumor microenvironment

The tumor microenvironment consists of blood vessels109, immune cells110,111, fibroblasts112,113 other cells, signaling molecules114–116, and the extracellular matrix (ECM)117. In tumorigenesis, all of these components are working together in this unusual acidic medium50. Although some studies suggest that these ion channels mediate carcinogenesis by creating a hostile and sanctuary environment29,39,118, other suggests that these ion channels are an adaptation to the intolerable acidity created within cancer cells119. Nevertheless, the presence of this microenvironment has evolutionary advantages by reducing the stress e.g. defeating chemotherapy i.e. chemotherapy resistance.

Resistance

The prevailing medical dogma of cancer chemotherapy resistance is the overexpression of P-gp (MDR)120–124 which is a very narrow perspective. Cancer chemotherapy resistance is a multifactorial phenomenon that can occur at multiple levels (local, regional, and systemic)125. The tumor microenvironment is considered at a regional level that alters efficacy of cancer chemotherapy by:

1. Tumor vascularization: The tumor is like a fetus that grows outside the uterus. The placenta provides the fetus with nutrients and eliminates wastes; tumor vascularization serves as the “tumor placenta” supporting tumor growth by supplying nutrients126 and removing waste metabolites127. However, tumor vascularization differs from the physiology of the placenta in that tumor vascularization occurs as a response to hypoxia that may be regarded as detrimental to tumor progression. Inhibition of tumor angiogenesis would lead to the selective survival of hypoxic cancer cells, and it is these hypoxic cells at the center of tumor masses that are the most difficult to target with chemotherapeutic drugs and thus the larger the number of hypoxic tumor cells the worse the prognosis. Limiting the vascularization that occurs within tumors also restricts the perfusion of the cytotoxic agents as these are unable to perfuse the hypoxic central region of the tumor and can only penetrate those cells at the periphery of the tumor near to a blood supply at128,129.

2. Physicochemical properties of drugs: The tumor microenvironment alters the physicochemical properties of proton transporter130,131. Extrusion of protons into the extracellular milieu results in the generation of an acidic pH1,50. This extracellular acidity leads to protonation of weakly basic drugs as doxorubicin, decreasing their partitioning inside cells42 (Figure 4). Thus, the tumor microenvironment creates another equilibrium as far as drug uptake is involved. Finally, it was also suggested that membrane stiffness increases due to the cytosolic alkaline pH reducing the ability of any chemotherapeutic agent to enter cancer cells130–132. Finally, the changes in membrane stiffness increase membrane recycling augmenting drug trapping inside acidified compartments133–135.

Figure 4. Le Chatelier’s principle, tumor acidity, shift the reaction to the right direction, and so the probability of drug will be in ionized form that decreases its partitioning.

Conclusion

Malignant transformation is a highly organized process accompanied by (i) reprogramming of the cellular metabolism that creates a unique pH gradient and (ii) the overexpression of several proton transporters and ion channels (e.g. MCTs, NHE1, V-ATPase, CA9, and CA12) that maintain this pH gradient as well as promote cancer cell survival. This acidic tumor microenvironment suppresses the growth of normal cells, supports cancer cell growth and migration, and blunts the response of the immune system. The acidic tumor environment also mediates microenvironmental resistance by either affecting drug permeability or altering the physicochemical properties of the chemotherapeutic agent at the site of the tumor. The cell transporters and ion channels represent the core of this complex and coordinated system. Therefore, the targeting of these transporters and ion channels will represent a new class of potential anticancer treatments and combination strategies that contributes to the war against cancer by controlling the progression of the disease, improving the tumor prognosis, improving the quality of life as well as increasing the survival rate of the patients, and finally decreasing the medicines cost.

Acknowledgements

The author thank Professors Salvador Harguindey, Stephan Joel Reshkin, Cyril Rauch, and Megan Walsh for the excellent discussion on the current topic.

Declaration of interest

The author reports no conflicts of interest. The author alone is responsible for the content and writing of this article.