**2. Development of the tumor acidic microenvironment**

The acidic microenvironment is mainly produced by tumor energy metabolism and hypoxia in solid tumors. Stephen Paget proposed the "seed and soil" hypothesis, stating that metastasizing cancer cells "seed" only in certain especially hospitable tissues, akin to seeding in "fertile soil" [5]. Since then, subsequent studies have revealed several classes of metastasis causes, including the tumor acidic microenvironment. In the 1920s, Otto Warburg first described a phenomenon that cancer cells displayed an altered metabolism, attaining energy through glycolysis at disproportionately high rates even under hypoxic conditions (**Figure 1**) [6]. Glucose is converted into lactic acid through glycolysis. Nevertheless, energy can still be obtained through the glutaminolysis pathway instead of glycolysis. From both pathways, high amounts of lactic acid are generated and subsequently discharged into the extracellular space between cancer cells [7, 8]. Some key transporters and enzymes, including ras, src, p53, glut, etc., are regulated in cancer cells to ensure that the impact of expanded H+ is removed. These changes enhance the rate of glucose uptake to support the rapid proliferation of tumor cells [4].

Hypoxia-inducible factors cannot be effectively degraded by enzymes under hypoxic conditions that are mainly caused by the rapid proliferation of tumor cells, resulting in stable and sustained expression in tumor cells [9]. The proliferation, invasion, migration, and energy metabolism of tumor cells are affected by these changes.

### **2.1 Energy metabolism**

Normal cells consume oxygen for energy production. Liver cancer aerobic glycolysis, otherwise known as the Warburg effect, to produce energy [10]. The end products of aerobic glycolysis contribute to the establishment of the acidic microenvironment.

**Figure 1.** *Energy metabolism in acidic microenvironment.*

### *Mechanisms of Hepatocarcinogenesis Development in an Acidic Microenvironment DOI: http://dx.doi.org/10.5772/intechopen.108559*

In the acidic microenvironment, liver cancer cells regulate aerobic glycolysis by activating AMP-activated protein kinase (AMPK), PI3K/activity, and other related pathways, resulting in the increased expression of related proteins and enzymes, including glucose transporter, hexokinase, fructose-6-phosphate kinase, pyruvate kinase, and so on (**Figure 2**) [11]. As a carrier responsible for glucose transportation across the cell membrane, glucose transporter (GLUT) promotes higher glucose uptake in liver cancer cells [12]. Hexokinase, fructose-6-phosphate kinase, and pyruvate kinase are key enzymes of glycolysis. Relevant studies have shown that inhibition of the glucose transporter, hexokinase, and pyruvate kinase can attenuate the glucose metabolism of liver cancer cells, affecting the occurrence and development of liver cancer [13–16]. Upregulation of the activation ratio of phosphofructokinase [17] facilitates the adaptation of liver cancer cells to the microenvironment and accelerates the proliferation and growth of liver cancer cells. The energy needed for the proliferation of liver cancer cells is provided by these changes, and the same with the material basis required for establishment of the acidic microenvironment. By adopting a pattern of energy metabolism that is different from normal cells, the internal and external microenvironment of liver cancer cells is changed, improving their survival advantage. From this perspective, inhibiting the activity of glycolysis-related proteases in liver cancer cells may be an effective way to treat liver cancer.

#### **2.2 Hypoxia**

Hypoxia is a key feature of liver cancer, in which hypoxia-inducible factors (HIFs) play a key role [18, 19]. As the most studied hypoxia factor in liver cancer, the adaptive response of cells to the hypoxic microenvironment can be mediated by HIF-1, which is composed of HIF-1α and HIF-1β, as heterodimers that are expressed stably only in an anoxic environment but degraded rapidly in a normoxic environment [20, 21]. A variety of genes (such as Ras, C-MYC, p53, AMPK, etc.) and signaling pathways (such as PKB/Akt, PI3K, mTOR, etc.) can be transcriptionally regulated by HIF-1 and regulate the energy production of cancer cells to maintain their proliferation and survival [22]. It has been reported [23–26] that lactic acid production from of liver cancer cells can be amplified by HIF-1, which results in altering the activity of enzymes associated with glycolysis that lead to microenvironment acidification of liver cancer tissues. This inhibition can affect the growth and proliferation of liver cancer by regulating the energy metabolism. In addition, it has also been reported that liver cancer cells and tissues can upregulate the expression of HIF-1 and vascular endothelial growth factor (VEGF) during hypoxia [27–29]. VEGF is central for neovascularization of expanding tumor nodules.

#### **3. Maintenance of tumor acidic microenvironment**

The production of lactic acid causes the tumor microenvironment to be low in pH. Tumor cells excrete H+ and acid metabolites into the extracellular environment to avoid intracellular acidosis (**Figure 3**). To achieve this goal, tumor cells use transporters that mainly include vacuolar-H+ ATPase (V-ATPase), Na+ /H+ exchanger (NHE), monocarboxylic acid transporters (MCTs), bicarbonate transporters, and hydrochloric acid transporters to expel intracellular H+ . In addition, the carbonic anhydrases (CAs) can be used to regulate pH by catalyzing the reversible hydration of CO2 to form bicarbonate and a proton in aqueous solutions. The reduction of extracellular pH is sensed by other complex mechanisms that include G-protein-coupled receptors, T-cell death-related gene 8 (TDAG8), acid-sensitive ion channels (ASICs), and the transient receptor potential channels, vanillin subfamily 1(TRPV1), to regulate the tumor microenvironment [30]. We can understand the occurrence and development of liver cancer and provide favorable conditions for targeted therapy of liver cancer by studying these transporters and complex pH-sensing molecular mechanisms in liver cancer.

### **3.1 Study on the mechanism of intracellular pH regulation of liver cancer in an acidic microenvironment**

#### *3.1.1 Vacuolar H+ -ATPase (V-ATPase)*

Vacuolar H<sup>+</sup> -adenosine triphosphatase (V-ATPase) is ubiquitously expressed in eukaryotic cells [31], being situated not only in the membranes of many organelles but also in the plasma membrane [32]. Studies have demonstrated that V-ATPase is functionally expressed in some human tumor cell lines and plays an important role in the regulation of tumor acidic microenvironment [33–35]. V-ATPase has multiple subunits, and the C subunit of V-ATPase, ATP6L, is the most thoroughly studied. Xu et al. [36] showed that the expression of ATP6L, the C subunit of V-ATPase, was elevated on the plasma membrane of liver cancer cells. As it indiscriminately inhibits *Mechanisms of Hepatocarcinogenesis Development in an Acidic Microenvironment DOI: http://dx.doi.org/10.5772/intechopen.108559*

**Figure 3.** *Molecular mechanism of transport in acidic microenvironment.*

V-ATPase in both mammals and non-mammals, palomycin was used to inhibit V-ATPase, thereby reducing the acid load and pH of liver cancer cells. Further, in an orthotopic xenograft model, the growth of liver cancer cells was delayed [37]. Tang et al. [38] showed that the protein expressed by LASS2 can bind to ATP6L and can inhibit the transmembrane transport of H+ by V-ATPase proton pump. In this case, the mitochondrial apoptosis pathway is activated by increasing the concentration of hydrogen ions in the cell to induce apoptosis and inhibit the growth of tumor cells. These results imply that the expression of V-ATPase is increased in liver cancer cells and involved in the regulation of intracellular pH, while the growth of liver cancer cells can be effectively delayed by its corresponding inhibition.

#### *3.1.2 Na+ /H+ exchangers (NHEs)*

Na+ /H+ exchangers (NHEs) are a family of membrane proteins that contribute to exchanging one intracellular proton for one extracellular sodium. The family of NHEs consists of nine known members, NHE1-9. Each isoform represents a different gene product that has unique tissue expression, membrane localization, physiological effects, pathological regulation, and sensitivity to drug inhibitors [39]. NHE1 was the first to be discovered and is often referred to as the "housekeeping" isoform of the NHE family [40]. The NHE protein is activated by increased intracellular H<sup>+</sup> , can achieve a one-to-one exchange between intracellular H<sup>+</sup> and extracellular Na<sup>+</sup> , and excess Na+ can be regulated by Na<sup>+</sup> /K+ -ATPase in cells, which is a key ingredient in preventing cellular acidosis [40]. Enhanced glycolysis increases the amount of H+ in cancer cells, by which the NHE protein is effectively activated. Yang et al. [41] showed that the expression of NHE1 was increased in liver cancer and in cells and closely related to tumor size, venous invasion, and tumor stage. Kim et al. [42] showed that

curcumin combined with GR was used to inhibit NHE1 expression in liver cancer cells characterized by low pH. In addition, Li et al. [43] showed that the growth and metastasis of liver cancer cells can be inhibited by Ginsenoside (Rg3), which blocks the EGF-EGFR-ERK1/2-HIF pathway by decreasing the expression of NHE1. As these results imply that NHE1 is involved in regulating intracellular pH and is upregulated in liver cancer cells, inhibition of it can effectively delay tumor cell growth and metastasis. Further study of NHE1 may effectively inhibit the progression of liver cancer. However, the role of other subtypes of NHEs in liver cancer is unclear and needs to be further studied.

## *3.1.3 Monocarboxylic acid transporters (MCTs)*

MCTs belong to the SLC16 gene family and consist of 14 members, in which MCT1, MCT2, and MCT4 can act as proton transporters to participate in the transport of pyruvate and lactate [44]. In the process of glycolysis a lot of lactic acid is produced by tumor cells that require large amounts of monocarboxylic acid transporters to pump these acids out of the cell to regulate pH and maintain homeostasis in the tumor cell environment, which prevents cell apoptosis caused by lactic acid accumulation [45]. The distribution of MCTs in tissues is determined by the physiological requirements of lactate metabolism (**Figure 4**). In general, because it has a high affinity for lactic acid, MCT1 and MCT4, which are expressed in most tissues, are mainly responsible for the transport of lactic acid inside cells. However, glucose metabolism determines the level of MCT2 expression, where its affinity for pyruvate is much higher than other MCTs molecules, and intracellular pyruvate transport is mainly completed by it. In some literature reports, MCT1 and MCT4 are highly expressed in liver cancer, and the high expression of it was significantly correlated with the malignant phenotype and prognosis of the tumor, but MCT2 expression is low in hepatocellular carcinoma [46, 47]. Chen et al. found that the expression of MCT4 was positively correlated with the expression of GLUT1 and speculated that

**Figure 4.** *Related mechanism of MCT in acidic microenvironment.*

#### *Mechanisms of Hepatocarcinogenesis Development in an Acidic Microenvironment DOI: http://dx.doi.org/10.5772/intechopen.108559*

there was a positive feedback loop between the growth of HCC and the upregulation of MCT4/GLUT1 [48]. As an antitumor drug, lonidamine effectively inhibits MCTs, and it can impair the proliferation, metastasis, and invasion of liver cancer cells by reducing glycolysis [49]. These results imply that the specific molecular mechanism of MCTs involvement in the development of liver cancer may be related to MCTs mediating the shuttle of lactate and pyruvate in and out of liver cancer cells, which prevents lactic acid from accumulating in the liver cancer microenvironment and pyruvate from being transported extracellular that inhibits the proliferation and survival of HCC cells. However, the specific molecular pathways and related mechanisms remain unclear and need to be further studied.

#### *3.1.4 Carbonic anhydrases (Cas)*

Tumor tissues are often exposed to low oxygen that can activate CAs by which H2O and CO2 are reversibly converted to HCO3 so that the chemical reaction can maintain the normal pH of the cell. CAs can be divided into four categories according to their distribution location (cell and subcellular) [50], namely cytoplasmic type (CAI, II, III, VII, XIII), mitochondrial type (cava, VB), secretory type (CAVI), and membrane-related type (CAIX, XII, XIV, XV). Among them, CAII and CAXII are the most studied in liver cancer. Xing et al. [51] showed that the expression of carbonic anhydrase II (CAII) was significantly upregulated in liver cancer compared with serum CAII concentrations in the normal population and among patients with non-recurrent liver cancer. Further investigating the molecular mechanisms involved suggests that CaII increases the migration and invasion of liver cancer cells by activating the epithelial-mesenchymal transition (EMT) pathway. Finkelmeier et al. [52] showed that the serum CAXII level was significantly increased in patients with advanced liver cancer (BCLC and ALBI scores). Zeng et al. [53] showed that the high expression of CAXII was associated with poor prognosis in patients with liver cancer. UDA et al. [54] showed that the changes in intracellular pH can be caused by knocking out CAII, which inhibits the progression of liver cancer. Han et al. [55] showed that the proliferation of liver cancer can be inhibited by tiliroside by blocking CAXII. These findings suggest that CAXII may also be a prognostic indicator of poor prognosis in patients with liver cancer. The molecular mechanisms of CAII and CAXII affecting the progression of liver cancer need to be further explored, and the specific roles of other subtypes of CAs in the progression of liver cancer need to be further determined. However, according to current studies, CAII and CAXII may be potential treatment targets sites for liver cancer.

## **3.2 Study on the mechanism of extracellular pH regulation of liver cancer in an acidic microenvironment**

#### *3.2.1 Acid sensing ion channels (ASICs)*

Changes in extracellular pH can be sensed by acid-sensitive ion channels (ASICs). Six ASICs subunits are encoded by four genes have been cloned, which comprise ASIC1a, 1B, 2A, 2B, 3, and 4 [56]. A large number of crucial biological functions, such as inflammation, ischemia, and tissue acidification of tumors, are represented in ASICs. ASIC1a is of particular interest as one of its six subunits that play an important physiological and pathological role from mediating Ca2+influx [57]. Our team has been working on ASIC1a for many years since it is involved in the proliferation,

invasion, and metastasis of liver cancer. When liver cancer tissues were in a pH 6.5 microenvironment, the expression of ASIC1a in liver cancer tissues was significantly higher than that in adjacent non-tumor tissues in previous studies [3]. ASIC1a protects against phosphorylation and ubiquitination of β-catenin and promotes β-catenin nuclear aggregation to stimulate the proliferation of HCC cells. ASIC1a can promote invasion and migration of liver cancer as demonstrated by cell scratch and trans-well assay data [58]. Downstream differentially expressed genes of ASIC1a were mainly concentrated in the transcription factor AP-1 in the MAPK-related signaling pathways [59]. These findings suggest that intracellular Ca2+ concentrations and changes in downstream AP-1 expression can be increased by ASIC1a to affect the migration and invasion of liver cancer. Thus, ASIC1a is an effective target for the treatment of liver cancer, and a precise study of the relevant mechanisms may provide a new diagnostic method or target.
