WNT/β-catenin-M2 macrophage interplay as a target for therapy against hepatocellular carcinoma: Role of Calculus bovis

Abstract

Liver cancer, and in particular hepatocellular carcinoma (HCC) is a disease of rising prevalence and incidence. To date, definitive treatment options include either surgical excision or ablation of the affected area. With increasing research on several pathways that could be involved in the progression of HCC, new elements within these pathways emerge as potential targets for novel therapies. The WNT/β-catenin pathway favors the presence of M2 tumor-associated macrophages which in turn promote tumor growth and metastasis. The inhibition of this pathway is considered a good candidate for such targeted therapeutic interventions. Interestingly, as Huang et al show in their recently published article, Calculus bovis which is used in traditional Chinese medicine can exert an inhibitory effect on the β-catenin pathway and become a potential candidate for targeted pharmacotherapy against liver cancer.

Key Words: Hepatocellular carcinoma; Calculus bovis; WNT/β-catenin pathway; Tumor-associated macrophages

Core Tip: Activation of the WNT/β-catenin pathway plays an important role in hepatocellular carcinoma (HCC) development and the modulation of the tumor microenvironment through M2 macrophage production. Calculus bovis (CB), an herb used in traditional Chinese medicine, exhibits liver anti-tumor properties: Inhibition of the WNT/β-catenin pathway and subsequent decrease in M2 tumor-associated macrophages polarization. In all, the inhibition of WNT/β-catenin pathway emerges as a therapeutic target while the use of CB surfaces as potential medium to achieve this kind of modulation. CB, therefore, appears to be a lucrative candidate that can aid in the pharmacotherapy of HCC.

TO THE EDITOR

Liver cancer is the sixth most common cancer worldwide, with hepatocellular carcinoma (HCC) accounting for 75%-85% of cases[1,2]. We read with great interest in the article published by Huang et al[3] in the recent issue of the World Journal of Gastroenterology, about the effect of Calculus bovis (CB) on HCC. To date, available treatment modalities consist of pharmacotherapy as well as more invasive interventions, including surgical resection[4]. The increased frequency and poor prognosis of HCC has fueled, and shifted research towards the “manipulation” of tumor microenvironment and the reduction of tumor progression and metastasis through therapeutic targeting of relevant molecular and cellular pathways[4,5].

A well-studied cellular pathway is that of WNT/β-catenin. The term WNT is a fusion of the name of a segment-polarity gene in Drosophila, wingless, and that of the vertebrate homolog, integrated or int-1. The WNT/β-catenin pathway is a modal mechanism that regulates cell proliferation and differentiation. WNT protein binds to the frizzled (FZD) receptor on the cell surface, thereby activating the intracellular LRP5/6 complex which in turn inhibits β-catenin phosphorylation, thus inducing its stabilization[2]. The stable form of β-catenin is transported into the cell nucleus, where it triggers transcription of target genes.

Based on available data, there are two patterns of WNT pathway activation: a. a canonical WNT/β-catenin pathway, which plays a role in the regulation of tissue growth and homeostasis; b. a non-canonical WNT/β-catenin pathway associated with calcium channel activation, also involved in processes related to cell proliferation, cell migration and embryonic development[2]. Inhibition of the WNT/β-catenin pathway, on the other hand, has been shown to be regulated by extracellular proteins that antagonize the binding site, thus preventing further activation[6]. The role of the WNT/FZD as a key player in the pathogenesis of HCC is supported by the fact that activation of β-catenin is present in 20%-35% of cases[7]. In addition, the β-catenin encoding CTNNB1 gene is one of the most frequently mutated genes in cases of HCC[1,6,8].

Tumor-associated macrophages (TAMs) are a component of the HCC-related tumor microenvironment, often associated with poor prognosis[8], and depending on their differentiation, exert either anti-tumor or tumorigenic properties[6], including neo-angiogenesis and tumor metastasis[9]. These TAMs are macrophages that differentiate either through the classical pathway (M1), or through alternative cytokines and growth factors modulation (M2)[10].

M1 macrophages exhibit anti-inflammatory and anti-tumor activity by activating Th1 cells and producing pro-inflammatory cytokines [interleukin-6 (IL-6)], tumor necrosis factor alpha (TNF-α), and oxygen-free radicals. M2 macrophages grow under the influence of IL-4/IL-13, IL-10, and TGF-β, induce a Th2 immune response, exhibit precancerous activity and predominate in most tumors, favoring their progression[11].

Among several formulations used in traditional medicine worldwide, some exhibit rather intriguing anti-tumor properties[12]. CB, a major herbal component of traditional Chinese medicine, is such an example, as it exerts several anti-tumor effects on different neoplasms, including HCC[13]. The recently published study by Huang et al[3] shed light into the molecular pathways through which CB successfully suppresses HCC progression. This is, to our knowledge, the first attempt to use CB as a “stand-alone” monotherapy, rather than as part of a multi-substance regimen, while “decoding” and determining its molecular composition and bioactive constituents[3]. Interestingly, CB has been found to consist of 22 components, 11 of which are bioactive. Through in vivo experimentation, it has been shown that CB exerts immunomodulatory effects, subsequently leading to the inhibition of tumor growth. This inhibitory role is evidenced through a series of advanced in vivo and in vitro assays, network pharmacology, transcriptomics and molecular docking, which ultimately helped researchers confirm the hypothesis that CB can suppress liver cancer growth through modulation of the WNT/β-catenin pathway and M2 TAMs polarization. CB’s interplay during the initial steps of the WNT/β-catenin pathway, results in a reduced expression of the CCL22 gene which in turn inhibits M2 polarization while suppressing TNF-α and IL-6 production, and ultimately tumor growth. This anti-tumor effect is amplified by CB’s additional ability to promote the apoptosis of M2 macrophages. The aforementioned WNT/β-catenin-mediated actions of CB can be reversed with the use of a WNT agonist[3]. Based on recent reports, however, the potential of CB to “block” the Wnt /β-catenin pathway can be hindered by the presence of β-catenin gene mutations, found in 25%-30% of HCC patients. Although CB can also promote β catenin degradation by optimizing the activity of the β catenin destruction complex, such mutations can lead to the production of a more stable β catenin molecule. This modified β catenin may elude CB-facilitated degradation, enter the nucleus and exert uncontrolled transcriptional activity[14,15].

As tumor growth in HCC is a complex phenomenon, resulting from activation of numerous interconnected molecular pathways, ongoing research attempts to “map” these pathways and their constituents while shedding light into their actions and interactions[4,5]. This process also facilitates the identification of novel candidates for a more targeted, yet effective, pharmacotherapy that can “manipulate” inherent molecular pathways involved in tumor genesis and growth, through activation or inhibition of key molecules and cells within the tumor microenvironment. The study by Huang et al[3] confirms the tumor microenvironment’s key-role in HCC progression, and places it under the spotlight as a target for novel therapeutic approaches. Encouraged by recent data[12,13] showing that the addition of CB to established anti-cancer drugs may exert synergistic antineoplastic effects, the authors take a step back and examine CB’s composition and its individual antitumor properties. Based on their findings, CB’s anti-liver tumor potential is the result of its ability to inhibit the WNT/β-catenin pathway, regulate cytokines and decrease the polarization of M2 TAMs[3].

In all, the inhibition of WNT/β-catenin pathway emerges as a target for therapy against HCC, while the use of CB surfaces as potential medium or instruments to achieve this kind of modulation. Further research will hopefully consolidate these findings and clarify whether one or more of CB’s bioactive components contribute to its therapeutic effect, so they can be used in an effective and safe manner.