Inhibition of M2 tumor-associated macrophages polarization by modulating the Wnt/β-catenin pathway as a possible liver cancer therapy method

Abstract

The problem of liver cancer is becoming increasingly important due to the epidemic of metabolic diseases and persistent high alcohol consumption. This determines great attention to the development and improvement of methods for early diagnosis and treatment of liver cancer. Huang et al presented a study in the World Journal of Gastroenterology, in which they showed that the use of the traditional Chinese medicine Calculus bovis (CB) can suppress tumor growth in mice by inhibiting M2 tumor-associated macrophages (TAM) through modulating the activity of the Wnt/β-catenin pathway. The interaction of CB components with the Wnt/β-catenin pathway, M2 TAM polarization, and tumor dynamics were studied using network pharmacology, transcriptomics, and molecular docking. It is now generally accepted that the polarization of TAM and the differentiation of the functions of M1 and M2 phagocytes are of great importance for the progression of neoplasms. It is assumed that M2 TAM promote proliferation and migration of tumor cells. Attempts to medicinally influence the Wnt/β-catenin pathway in order to modulate phagocyte polarization now belong to one of the most promising areas of immunotherapy of oncological diseases. Undoubtedly, the work of the Chinese authors deserves attention and further development.

Key Words: Liver cancer; Treatment; Calculus bovis; Tumor-associated macrophages; M2 tumor; Macrophage polarization; Wnt/β-catenin pathway

Core Tip: The editorial article is devoted to the possibility of drug inhibiting M2 tumor-associated macrophages polarization by modulating the Wnt/β-catenin pathway for suppressing liver cancer. The article by Chinese authors presented in the World Journal of Gastroenterology draws attention to this issue, in their work they showed that the use of a traditional Chinese medicine-Calculus bovis allows suppressing liver cancer growth in mice through this mechanism. This study is distinguished by its extremely promising and strategically new aim and a very impressive methodological level of research.

TO THE EDITOR

Two million people die every year worldwide from complications of liver cirrhosis and hepatocellular carcinoma (HCC)[1]. The problem of liver cancer is becoming increasingly important due to the epidemic of obesity, diabetes mellitus, metabolic dysfunction–associated steatotic liver disease and persistent high alcohol consumption[1-3]. This leads to great attention to the development and improvement of methods for early diagnosis and treatment of HCC[4,5]. An important place among new approaches to the treatment of liver cancer is occupied by immunotherapy[6]. In this connection the work of Chinese authors Huang et al[7], devoted to the study of the possibility of inhibiting the polarization of M2 tumor-associated macrophages (TAM) by modulating the Wnt/β-catenin pathway for the suppression of liver cancer, seems very relevant and modern[7].

Huang et al[7] carried out an experimental study in which liver cancer mice were divided into a control group and groups treated with different doses of Calculus bovis (CB). Clinical dynamics were assessed by determining the size, weight, and histological analysis of tumors. The interaction of CB components with the Wnt/β-catenin pathway, M2 TAM polarization, and tumor dynamics were analyzed using network pharmacology, transcriptomics, and molecular docking. Network pharmacology is a method for discovering new drug targets and molecular mechanisms by combining computational analysis with in vivo and in vitro experiments and integrating a large volume of information. This area of research is based on systems biology, genomics, transcyptomics, proteomics and other disciplines[8]. The methods used in network pharmacology for traditional Chinese medicine research include network-based disease gene prediction, drug targets, drug function prediction to specific diseases, network construction of Chinese herbal medicine, and construction and analysis of drug-gene-disease network[9]. Molecular docking allows one to predict the preferred orientation of one molecule relative to another when a ligand and target are bound to each other to form a stable complex[10]. Transcriptomics studies the complete set of transcripts (RNA molecules) synthesized in a cell or organism under certain conditions. The Chinese authors conducted a transcriptome analysis to study the effects of CB and identified significant changes in 820 genes. The positive aspect of network pharmacology is that it is suitable for studying multi-component drugs, which include traditional Chinese medicine. It is generally accepted that network pharmacology has several limitations when used alone: The inability to avoid false positive results, the inability to fully assess the effects and toxicity profile of a multicomponent drug, and the difficulty in establishing a therapeutic dose[11].

The work by Huang et al[7] is complicated by the descriptive nature of the methods scattered throughout the article. It is not clear which component of CB has a significant effect on the Wnt/β-catenin pathway functioning. It can be assumed that the composition of the CB is not standard, which may become an obstacle to the widespread use of the method. At the same time, molecular docking results from the study by Huang et al[7] showed that bilirubin and bile acid-like compounds such as glycocholic acid, taurodeoxycholic acid, glycohyodeoxycholic acid, hyodeoxycholic acid, and 7-ketolithocholic acid exhibited binding energies ≤ -6.5 kcal/mol with Wnt5B, β-catenin, and Axin2 proteins. Which the authors considered as an indicator of strong affinity between the active ingredients and their target proteins. The results of transcriptome analysis showed changes in genes associated with M2 polarization[7]. Thus, CB components, by binding Wnt5B, β-catenin and Axin2, block the Wnt/β-catenin pathway, thereby inhibiting M2 polarization of TAM. Finally, the authors did not focus on the idea of which particular component of CB allows tumor growth suppression in experimental animals by inhibiting M2 TAM macrophages through modulating the activity of the Wnt/β-catenin pathway. The goal of further research can be considered to be the specification of the answer to this question.

CB is dried gallstones of domesticated cows, contains about 43 chemical components, including bile pigments, bile acids, cholesterols, amino acids, microelements, and has been used in traditional Chinese medicine for about two thousand years to treat diseases of the nervous system, cardiovascular system, respiratory system, digestive system, and affects the immune system[12]. Previous studies have shown the effectiveness of CB for the treatment of hepatic cholestasis by regulating inflammation, oxidative stress, apoptosis, and bile acid metabolism[13,14]. Since the 18^th century, CB has been used either as part of a combination therapy (Xihuang Pill) or as monotherapy to treat breast cancer, stomach cancer, and liver cancer[15,16].

Macrophages are important components of both the innate and adaptive immune systems, and contribute to the destruction of pathogens and the regulation of homeostasis in the organism. Macrophages can polarize, acquiring different phenotypes. Despite the complexity of the problem, it is customary to distinguish the proinflammatory phenotype M1 and the anti-inflammatory phenotype M2[17]. Macrophages located inside the tumor are called TAM[18]. A number of studies have shown that TAM M2 promotes the proliferation and migration of tumor cells[18-21], while M1 macrophages have an antitumor effect[18,20]. It should be noted that some authors call not to evaluate the polarization of macrophages according to the principle of division into “black” and “white”, pointing to the functional diversity of subtypes of macrophages M1 and M2[19].

The involvement of macrophages in the pathogenesis and development of HCC is of key importance. Zhang et al[22] found that M2 macrophages increased the proliferation, migration and invasion of HCC cells through a process dependent on fatty acid oxidation. Specifically, IL-1β instigated the pro-migratory effect of M2 cells, and fatty acid oxidation was responsible for the upregulated secretion of IL-1β, which depended on reactive oxygen species and NLRP3 inflammasome[22]. In M2 polarization of macrophages and during the process of monocyte to macrophage differentiation, upregulation of Wnt is observed. When Wnt silencing occurs in macrophages, the anti-tumor activity is observed. However, increase in expression level of Wnt can result in upregulation of c-Myc in mediating M2 polarization of macrophages. Such function of Wnt on macrophages can significantly increase growth and invasion of HCC cells. Increase in M2 polarization of macrophages can be obtained by ZIP9 and this transcription factor decreases M1 polarization of macrophages in increasing HCC malignancy[23]. Autophagy studies have shown that this process promotes tumor progression through M2 macrophage polarization and TAM accumulation in the tumor microenvironment[24].

The Wnt signaling pathway is one of the intracellular signaling pathways in animals, it is evolutionarily conserved and is necessary for embryonic development and tissue homeostasis[25]. This pathway is activated by Wnt ligands, which are secreted proteins. Currently, 19 Wnt ligands have been described[26]. The Wnt signaling pathway can be non-canonical and canonical[27]. Non-canonical Wnt pathway is β-catenin-independent, such as regulating cell polarity and migration[28], as well as calcium metabolism[29]. The canonical Wnt pathway (Wnt/β-catenin pathway) involves the nuclear translocation of β-catenin and activation of target genes via T cell factor (TCF)/lymphoid enhancer-binding factor (LEF). These two pathways form a network of mutual regulation. Wnt signaling itself is inherently complex due to the large number of Wnt proteins, which in turn have multiple receptors, resulting in a huge number of possible ligand-receptor interactions[30]. The Wnt/β-catenin pathway comprises four segments: The extracellular signal (mediated by Wnt ligands), membrane segment (mainly includes the Wnt receptors Frizzled and LRP5/6), cytoplasmic segment (mainly contains β-catenin, DVL, glycogen synthase kinase-3β, AXIN, APC, and casein kinase I), and nuclear segment (β-catenin, which translocate to the nucleus, TCF/LEF, and β-catenin downstream target genes). A simplified view of the Wnt/β-catenin pathway is as follows. In the absence of Wnt signaling, β-catenin is degraded. When Wnt ligand acts on cell, it results in a decrease in the rate of degradation of β-catenin, which accumulates in the cytoplasm, enters the nucleus, where it ultimately activates transcription of target genes via TCF/LEF[27].

The Wnt/β-catenin pathway is an important regulator that controls growth, metabolic zonation, and regeneration of the liver when it is damaged[31]. Through this pathway, tissue integrity is restored after acute liver injury. Under normal physiological conditions, the Wnt/β-catenin signaling pathway is carefully regulated. However, aberrant activation of this pathway and its downstream target genes can occur due to mutations in key pathway components, epigenetic modifications, and other causes[32]. Therefore, the Wnt/β-catenin pathway is one of the most crucial mediators of carcinogenesis[33,34]. Mutations in the CTNNB1 gene encoding β-catenin in Exon3 have been shown to be the most common activation mechanism of this pathway and are present in approximately 20%-40% of patients with HCC[35].

Complex relationships between the Wnt/β-catenin pathway and macrophages have been established. Macrophages are both a source and recipient of Wnt signals. Normally, Wnt/β-catenin signaling is involved in the differentiation of myeloblasts into monocytes, as well as in the transition of monocytes into macrophages. Wnt signals regulate macrophage functions such as phagocytosis, adhesion and migration[36]. In oncological diseases, tumor cells actively release Wnt ligands, which activate the Wnt/β-catenin pathway in the cells of their microenvironment, including TAM. Wnt ligands derived from HCC tumor cells can activate canonical Wnt/β-catenin signaling of macrophages and then induce their polarization to M2 TAMs, which promotes tumor growth, migration, and metastasis[37]. In this regard, the effect on the function of the Wnt/β-catenin pathway and the production of M2 TAM is a promising target for the therapy of oncological diseases. Currently, a number of substances that affect the WNT/β-catenin pathway are in preclinical trials and in phases 1-2 of clinical trials[33,38]. But the number of studies devoted to the pharmacological effect on the polarization of macrophages through the Wnt/β-catenin pathway is not numerous. A study by Chinese authors showed that andrographolide affects the Wnt/β-catenin pathway, which significantly suppresses M2 polarization and stimulates M1 polarization of macrophages in breast cancer[39]. In an experimental study, it was demonstrated that Oudemansiella raphanipes limited the polarization of M1 macrophages into M2 macrophages through the suppression of Wnt/β-catenin signaling in tumor tissues, thereby inhibiting the growth and metastasis of breast cancer in mice[40]. In a model of pulmonary fibrosis, Icariside II was found to have an antifibrotic effect by inhibiting the profibrotic polarization of macrophages into the M2 phenotype through modulation of the Wnt/β-catenin signaling pathway[41]. In another study, the role of Wnt2b-mediated signaling activation for macrophage polarization in the HCC microenvironment was investigated. The authors proposed a TLR9 agonist, CpG ODN, as an inhibitor of the Wnt2b signaling for HCC therapy[42].

CONCLUSION

The work by Huang et al[7], presented in World Journal of Gastroenterology, is distinguished by an extremely promising and strategically new target and a very impressive methodological level of research. Currently, the world is experiencing a boom in the development of drug effects on the Wnt/β-catenin pathway function to influence the M2 polarization of TAMs and suppression of liver cancer. The decision to choose the traditional Chinese medicine CB as a therapeutic agent looks interesting. The authors will likely have a large and complex work ahead of them to establish which specific CB components modulate the function of the Wnt/β-catenin pathway. At this stage, demonstrating this possibility seems essential. Undoubtedly, the direction of the work of the Chinese authors deserves attention and further development.