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Causes of pH Dysregulation in Cancer
Some of the most notable changes in protein expression and activity in cancers are found in proteins that alter the pH environment. These proteins are often membrane transport proteins, and many have attracted interest as drug targets. This section will highlight a variety of proteins that regulate both pHi and pHe, and how they contribute to cancer progression.
MCT1 and MCT4 are two major monocarboxylate-H+ cotransporters associated with cancers17. MCT1 facilitates the export of lactate and protons produced by glycolysis, preventing intracellular acidification under normal conditions. Inhibition and silencing of MCT1 has been demonstrated to drastically decrease pHi, most likely due to inability to excrete excess lactate generated from glycolysis18. The pHi in MCT1 knockouts can be rescued by expression of MCT4, exemplifying the importance and redundancy of these transporters in pH maintenance18. From a mechanistic standpoint, it is not clear whether MCT upregulation is a driver of cancer or a result of cancer, but it is clear that MCTs are drivers of pH change. When expressed in Ras-transformed fibroblasts, MCT4 caused alkaline intracellular pH17. MCTs are also implicated in cell migration, a critical process for metastasis, as they are localized to the leading edge of migrating cells, though their exact role in these processes is unknown19.
Carbonic anhydrases (CAs) are another important class of enzymes commonly upregulated in cancers, and have been found to associate with MCTs in the plasma membrane. Their enzymatic activity catalyzes the conversion of CO2 derived from oxidative phosphorylation into HCO3- in the extracellular matrix, acidifying the pHe. Bicarbonate is then often transported back into the cells, which causes an increase in pHi (see next section). CAIX and CAXII are membrane bound CAs that are commonly overexpressed in aggressive cancers and have been shown to be drivers of pH changes20,21. It seems paradoxical that CAs would be upregulated in cancers, which have decreased oxidative phosphorylation and therefore generate less CO2. In 2015, some insight into this peculiarity was presented by Jamali et al, demonstrating that in breast cancer cells, non-catalytic proton transport activity of the CAIX augments activity of MCT122, showcasing the interplay between different classes of pH regulatory proteins. This process increases pHi, driving glycolysis and proliferation. CAIX has also been suggested to promote migration and invasion23.
When CAIX converts CO2 into HCO3- and H+, HCO3- is driven down its concentration gradient back into the cell, leading to alkalization of the intracellular environment in addition to acidification of the extracellular space. This process is mainly facilitated by Na+/HCO3- cotransporters (NBCs)24. The transport of HCO3- and regulation of NBCs play a role in a variety of cancers22,25. It may also be interesting to consider the role of NBCe1 in the context of a whole tumor. One study showed that NBCe1 is regulated by hypoxia26, which is significant because cells inside a tumor are in a hypoxic environment, while cells on the exterior are more oxygenated. HCO3- produced by more oxygenated cells from higher oxidative phosphorylation could be taken up by glycolytic cells on the interior of the tumor to help increase their pHe. Conversly, lactate from glycolytic cells on the interior of a tumor may be used as a carbon source in oxidative phosphorylation in oxygenated cells on the exterior27. The hypothesis that carbon symbiosis occurs between normoxic and hypoxic regions of tumors has yet to be thoroughly investigated27. This hypothesis would suggest that NBCs are not necessarily drivers of cancer, but are drivers of pH dysregulation after a tumor has already formed.
The ion transporter that is perhaps the most widely accepted as a driver of cancer is the Na+/H+ exchanger NHE1. NHE1 is recognized as a major regulator of cellular pH in an abundance of cancer types28. NHE1 exchanges an extracellular Na+ for an intracellular H+ at the plasma membrane, thus directly decreasing the pHe and increasing the pHi. NHE1 normally helps prevent cytosolic acidification, thereby promoting glycolysis and proliferation and driving cancer28. Furthermore, in migrating cells, NHE1 localizes to pseudopodia and invadopodia13, and also associates with actin and cytoskeletal reorganization kinases28. Taken together, it is clear that NHE1 plays an important role in cancer migration and invasion, though its exact mechanism in these processes is unknown.
Consequences of pH Dysregulation in Cancer
All proteins have an optimal pH range for activity. However, pH sensors are unique in that they change their activity as a direct result of a proton binding to a critical residue. They are usually sensitive within the narrow range of physiological pH fluctuations. Interestingly, 15% of somatic mutations in cancer involve histidine substitutions, which is the amino acid most likely to be implicated in pH sensing and regulation29 due to its near-physiological pKa value of 6.5. Characterizing and determining the nature of these mutations is becoming a new frontier in cancer biology. pH sensors are found in a variety of cancer-related cellular processes, some of which are described below.
NHE1 begs to be mentioned again, as it also functions as a pH sensor in addition to its ion channel activity. NHE1 is highly upregulated by increased intracellular protons, which would occur in glycolytic cancer cells. Though the mechanism of binding and transport of protons is complex and cooperative, it is known that there are four conserved histidine residues in the C-terminal domain whose protonation facilitates binding to a phospholipid, regulating activity of the transporter30.
Perhaps the most significant pH sensors are glycolytic proteins. Glycolysis is regulated via a pH associated negative feedback loop. Glycolysis generates lactic acids and protons, but shuts down if the environment is too acidic to prevent detrimental acidosis of the cell. Two glycolytic enzymes are known pH sensors, lactate dehydrogenase (LDHA) and phosphofructokinase-1 (PFK-1)13,31. The molecular mechanisms for their pH sensitivity were predicted using software called “pHinder”, though their mechanisms have never been experimentally determined32. LDHA is upregulated at higher pHi, consistent with increased glycolysis in cancer33. There exists a plethora of conflicting data for PFK-1, however. While PFK-1 and its activators are upregulated in cancer, so is its glycosylation, which inhibits function34. The precise regulation of PFK-1 in cancer has yet to be determined.
Yet another cellular behavior regulated by pH is actin cytoskeleton remodeling. This is especially important for cancers as cell migration, and therefore metastasis, are impossible without actin remodeling. The enzymes cofilin and talin, which are involved in actin remodeling, are known to increase activity at higher pHi37. Cofilin mediates actin polymerization and membrane protrusion, while talin binds to actin and regulates adhesion. The mechanisms of pH sensing are experimentally characterized38. Both are regulated by protonation states of histidine residues. Talin undergoes a conformational change when deprotonated, and cofilin changes its binding affinity upon deprotonation38. These proteins may be interesting therapeutic targets, considering their mechanisms are well studied and they are so critical for cancer cell migration.
In addition to pH being important for the regulation of ion transporters, glycolysis, and actin remodeling, pH also contributes to cellular signaling. There is a family of GPRCs that are activated by acidic pHe, including GPR4, 65, and 68. Their activation is important for a variety of signaling pathways pertinent to cancer proliferation and metastasis, including MAPK, PIP3K, and cAMP related pathways35. Some cancers even have activating mutations of GPCR. For example, human luteinizing hormone receptor in three case studies of testicular cancer was found to have an activating mutation of Asp578 to a histidine36, yet the effects of pH on this mutant have yet to be explored.
Such pH-sensitive mutations that consistently arise in cancers are of increasing interest in the pH dysregulation in cancer field8. These mutations are often His->Arg or Arg->His mutations39, as these mutations can alter the protein’s ability to modify its structure or function in response to a change in pH. The Arg273His mutation in p53 is one such recurrent mutation. The positive charge on Arg273 is responsible for the protein’s ability to bind to the DNA backbone. When this residue is mutated to a histidine, which can be neutrally charged, especially at higher pH, the protein’s ability to bind DNA decreases drastically.40 While His->Arg or Arg->His mutations are over-represented in cancers, it is currently unclear which of these mutations are drivers of cancer, and which arise later on in cancer progression. An interesting hypothesis is that these mutations are adaptive to the altered pH microenvironments of cancer, and are thus more likely to arise in an environment with increased pH. This modified evolutionary landscape in the tumor microenvironment is currently poorly understood, but has potential to greatly increase our understanding of how mutations arise in cancer and exacerbate the cancerous phenotype.