MicroRNA-206 as a promising epigenetic approach to modulate tumor-associated macrophages in hepatocellular carcinoma

Abstract

This letter comments on the recently published manuscript by Huang et al in the World Journal of Gastroenterology, which focused on the immunomodulatory effect of Calculus bovis on hepatocellular carcinoma (HCC) tumor microenvironments (TME) by inhibiting M2-tumor-associated macrophage (M2-TAM) polarization via Wnt/β-catenin pathway modulation. Recent research highlights the crucial role of TAMs and their polarization towards the M2 phenotype in promoting HCC progression. Epigenetic regulation, particularly through microRNAs (miR), has emerged as a key factor in modulating immune responses and TAM polarization in the TME, influencing treatment responses and tumor progression. This editorial focuses on miR-206, which has been found to inhibit HCC cell proliferation and migration and promote apoptosis. Moreover, miR-206 enhances anti-tumor immune responses by promoting M1-polarization of Kupffer cells, facilitating CD8+ T cell recruitment and suppressing liver cancer stem cell expansion. However, challenges remain in understanding the precise mechanisms regulating miR-206 and its potential as a therapeutic agent. Targeting epigenetic mechanisms and improving strategies, whether through pharmacological or genetic approaches, offer promising avenues to sensitize tumor cells to chemotherapy. Understanding the intricate interactions between cancer and non-coding RNA regulation opens new avenues for developing targeted therapies, potentially improving HCC prognosis.

Key Words: Epigenetic regulation; Hepatocellular carcinoma; Non-coding RNAs; MicroRNA-206; Tumor-associated macrophages; Tumor microenvironment

Core Tip: Hepatocellular carcinoma is a highly aggressive tumor often associated with chronic liver disease and cirrhosis with limited treatment options for patients ineligible for surgery or transplantation. Thus, there is an urgent need to explore alternative therapeutic options. This letter highlights the modulation of M2-tumor-associated macrophage polarization via Wnt/β-catenin and other pathways, as well as the role of microRNA (miR)-206 in promoting anti-tumor immunity. miR-206 stimulates M1 Kupffer cell polarization, recruits CD8+ T cells, and inhibits the expansion of liver cancer stem cells. Understanding these mechanisms could pave the way for targeted therapies that improve outcomes for patients with hepatocellular carcinoma.

TO THE EDITOR

Huang et al[1] emphasize a fundamental aspect for understanding treatment approaches for hepatocellular carcinoma (HCC). Finding drugs or substances that can modulate the immune response in neoplastic processes is very relevant. HCC is an aggressive tumor that usually occurs in the setting of chronic liver disease and cirrhosis[2]. Although high survival rates are observed after surgical therapy, most patients are not eligible for surgery due to either the extent of the disease or underlying liver dysfunction. Consequently, there is a pressing need to find alternative therapeutic options for patients ineligible for surgery or transplantation. The abovementioned study demonstrates that neoplastic development is facilitated by tumor-associated macrophage (TAM) polarization towards the M2 phenotype, which is associated with Wnt/β-catenin pathway activation[3]. Macrophage polarization into the M2 phenotype is typically driven by cytokines, notably interleukin (IL)-4 and IL-13, which signal through specific receptors to promote this transition. Recent research suggests that certain active compounds may offer protective benefits to HCC patients by significantly regulating the expression of CD206 mRNA in M2-TAMs, leading to increased apoptosis and a more favorable immune response[4]. The response of gastrointestinal tumors, including HCC, appears to be strongly influenced by epigenetic changes and gene regulation, particularly through the action of microRNAs (miR)[5]. Our editorial investigates the role of miR-206 as a modulator of the HCC tumor response. The development of drugs that can modulate epigenetic mechanisms may become a viable therapeutic option in the near future. In this context, we focused on the epigenetic regulation of liver cancer and the potential of certain drugs or chemicals to reverse TAM polarization from the tumor-promoting M2 phenotype to the tumor-suppressing M1 phenotype, with particular emphasis on the role of miR-206.

MIR-206: AN INTERPLAY BETWEEN EPIGENETIC REGULATION AND IMMUNE RESPONSES IN HCC

Overview and outlook

Recently, there has been an increasing focus on the involvement of epigenetic changes, which can regulate various genes at different stages of cancer and, in turn, affect responses to key therapeutic interventions. Given the significant recurrence and mortality rates associated with gastrointestinal malignancies, a deeper understanding of this interplay holds promise for the development of novel therapeutic strategies. In several gastrointestinal tumors, including HCC, epigenetic changes play a role in disease response and prognosis, particularly concerning non-coding RNAs (ncRNAs)[6]. NcRNAs refer to a class of RNA molecules that are not translated into proteins but regulate gene expression at various levels. Among ncRNAs, miRs are small, single-stranded RNA molecules that primarily function in post-transcriptional regulation by targeting messenger RNAs for degradation or inhibiting their translation[7]. Unlike genetic mutations, epigenetic modifications do not change the DNA sequence but involve changes in gene expression regulation. Every hallmark of cancer might be regulated by ncRNAs, which have now emerged as key players in the induction and regulation of the tumor microenvironment (TME). The TME is a dynamic and complex milieu comprising various cell types, extracellular matrix components, and signaling molecules that collectively influence tumor behavior and progression. Among the cellular constituents of the TME, macrophages play a pivotal role in liver cancer progression[8]. These macrophages, referred to as TAMs, are derived from circulating monocytes that infiltrate the tumor site and differentiate in response to TME signals[9].

The complexity of gene expression and protein translation in cancer is increased through the reciprocal regulatory interactions between ncRNAs, TME components, and cancer cells[10]. However, multiple mechanisms of cancer immune suppression in HCC play crucial roles in TME modulation, which remains a significant barrier to immunotherapy. This suggests that the immune response could be precisely regulated by miRNAs to prevent both excessive and inadequate reactions.

TAMs are highly plastic and can adopt either a pro-inflammatory M1 or an anti-inflammatory M2 phenotype depending on the local cytokine milieu and growth factors[11]. M1-TAMs are stimulated by cytokines such as interferon-gamma and tumor necrosis factor-alpha and exhibit anti-tumor properties through the production of pro-inflammatory cytokines and reactive oxygen species that promote tumor cell apoptosis and inhibit proliferation. Conversely, M2-TAMs support tumor growth and metastasis by engaging in immunosuppressive activities and promoting tissue remodeling and angiogenesis through various signaling pathways. M2-TAMs contribute to liver cancer progression through several key pathways, including Nuclear Factor kappa B (NF-κB), IL-6/STAT3, and Wnt/β-catenin[3,12].

These pathways facilitate cancer cell invasiveness and metastatic potential, in addition to survival and proliferation. For instance, the NF-κB pathway is a critical regulator of inflammation and immunity that, when aberrantly activated, can promote tumorigenesis by inducing the expression of genes involved in cell proliferation, survival, and angiogenesis[13]. Additionally, the Wnt/β-catenin pathway is involved in cell proliferation, differentiation, and migration, and it is frequently dysregulated in liver cancer[14,15].

Given the significant role of TAMs in liver cancer, approaches such as inhibiting monocyte recruitment to the tumor site, blocking the signaling pathways that promote M2 polarization, and directly reprogramming M2-TAMs to M1-TAMs are currently under investigation. Specifically, a diverse cellular infiltrate exists in HCC, including resident liver macrophages, such as Kupffer cells (KCs), which protect against HCC by communicating with other immune cells[16]. Traditionally, KCs have been viewed primarily as scavenger cells responsible for clearing particulate materials, pathogens, and cellular debris from the portal circulation. However, their roles extend beyond phagocytosis, encompassing significant cytokine production and immune regulation, distinguishing them from other macrophages[17]. In the context of HCC, KCs undergo a phenotypic shift from a pro-inflammatory M1 state to an anti-inflammatory M2 state, which contributes to tumor progression by creating an immunosuppressive microenvironment. However, how these cells communicate and the mechanisms underlying this process are incompletely understood. Several miRNAs have been identified to play key roles in macrophage polarization toward the M2 phenotype. miR-21 is upregulated in HCC and it promotes M2 polarization by targeting the phosphatidylinositol 3’-kinase/protein kinase B (PI3K/AKT) pathway, which is crucial for cell survival and proliferation[18]. Targeting miR-21 levels reduces the anti-inflammatory and tissue remodeling properties of M2 macrophages, potentially reshaping the immune TME involved in HCC development[19]. Another miRNA implicated in M2 polarization is miR-223, which is downregulated in HCC patients and modulates the NF-κB signaling pathway[20]. By inhibiting NF-κB, miR-223 downregulation promotes the expression of M2-associated markers and functions, thereby facilitating HCC progression[21].

Another nc-RNA involved in liver cancer is miR-122, which can be taken up by non-tumorigenic cells in the liver and other organs from HCC cells. By downregulating targets involved in glucose metabolism and the TME, miR-122 can create a favorable niche for metastatic colonization[22]. Additionally, miR-122 can suppress the expression of genes involved in the cell cycle, promoting an environment conducive to cancer cell survival and proliferation[23].

Similarly, the long nc-RNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) promotes M2 polarization by modulating the expression of oncogenic serine and arginine rich splicing factor 1 (SRSF1), which in turn suppresses apoptosis and autophagy in HCC cells by functioning as an oncogene[24,25]. MALAT1 activates SRSF1 and mTOR via a transcriptional program that triggers Wnt/β-catenin pathways, promoting the expression of M2-associated cytokines. These cytokines modulate the immunosuppressive TME, hence supporting metastasis and tumor proliferation[26,27].

Considering the crucial role of TAMs in the progression of liver cancer, targeting macrophage polarization and the associated signaling pathways offers a promising therapeutic strategy. Some chemicals appear to have immunomodulatory effects on crucial cellular processes. For instance, chloroquine is a potent inhibitor of autophagy, primarily acting by alkalinizing lysosomes and preventing autophagosome-lysosome fusion[28]. Its ability to inhibit autophagy has significant therapeutic implications in cancer therapy, where it can enhance the efficacy of conventional treatments and suppress tumor growth[29]. In the original study[1], flow cytometry analysis demonstrated that treatment with Calculus bovis effectively reduced CD206 mRNA expression in M2-TAMs and appeared to mitigate liver cancer cell proliferation and migration by inhibiting TAM M2 polarization. Strategies to reverse TAM polarization from the tumor-promoting M2 phenotype to the tumor-suppressing M1 phenotype could inhibit tumor growth and improve clinical outcomes, but these approaches are still under investigation.

miR-206 and immunomodulation in HCC

Since their identification, miRNAs have been implicated in various human cancers, including HCC. Among them, miR-206 has garnered attention for its role in inhibiting the proliferation of many cancer types[30,31]. In HCC, one of the major challenges is the immune escape of tumor cells, which allows them to evade detection and destruction by the host immune system. Recent studies have highlighted the role of miR-206 in modulating immune responses within the TME, particularly through the polarization of KCs towards the M1 phenotype[32]. This polarization enhances the hepatic recruitment of cytotoxic T lymphocytes (CTLs), contributing to robust inhibition of HCC progression by modulating protein kinase B/rat sarcoma virus (AKT/RAS) signaling[33,34]. It is now widely recognized that KC polarization contributes to the recruitment of CD8+ T cells, and the failure of these cells to reach tumor sites is a significant factor in immunotherapy resistance[35]. Previous studies have highlighted the role of miR-206 in enhancing anti-tumor immune responses by modulating the communication between macrophages and CD8+ T cells through the activation of the CCL2/CCR2 axis[33,36]. CCL2 is a chemokine that plays a key role in recruiting monocytes, macrophages, and other immune cells to sites of inflammation or tumorigenesis. In the context of cancer, CCL2 is often overexpressed in the TME, where it modulates the immune response and influences tumor progression[37]. CCL2 seems to facilitate hepatic recruitment of CTLs via CCR2[38]. miR-206 expression enhances the production and release of CCL2 and antagonizes the CCL2/CCR2 axis, thereby increasing chemokine availability in the tumor milieu. This activation promotes the migration of CD8+ T cells into the TME and strengthens the interaction between macrophages and CD8+ T cells, facilitating a more effective immune response against HCC[33]. In essence, AKT/RAS signaling activation led to the M2 polarization of KCs, which blocked CD8+ T cells from infiltrating the TME. Conversely, miR-206 facilitated the M1 polarization of KCs and boosted the production of CCL2. This increase in CCL2/CCR2 signaling attracted CD8 suggesting that miR-206 is a potential immunotherapeutic strategy.

Another challenge in HCC treatment is the presence of liver cancer stem cells (CSCs), which are capable of self-renewal and contribute to tumor initiation, progression, and resistance to therapy[39]. Cellular dedifferentiation, a process where differentiated tumor cells revert to a more stem-like state, further exacerbates the problem by promoting CSC expansion and enhancing tumor aggressiveness. A recent study suggested that miR-206 can suppress HCC cell dedifferentiation and liver CSC expansion by targeting the epidermal growth factor receptor (EGFR) signaling pathway[40], a transmembrane receptor tyrosine kinase that activates several downstream signaling cascades, including the PI3K/AKT, MAPK/ERK, and JAK/STAT pathways[41]. EGFR is often overexpressed or hyperactivated in HCC and is implicated in CSC maintenance by promoting their self-renewal and expansion. miR-206 can directly bind to the 3’ untranslated region of EGFR mRNA, leading to its degradation or translational repression[42,43]. By suppressing EGFR, miR-206 inhibits the activation of key downstream pathways such as PI3K/AKT and MAPK/ERK, which reduce the expression of genes involved in self-renewal and CSC maintenance. By downregulating EGFR and its downstream signaling pathways, miR-206 limits the self-renewal and tumorigenic potential of CSCs, making it a promising candidate for therapeutic development in HCC[40].

Finally, the molecular underpinnings of HCC involve the dysregulation of various signaling pathways, including c-MET, which is often upregulated in HCC and associated with tumor cell proliferation, invasion, and resistance to apoptosis. c-MET signaling contributes to resistance against apoptosis-inducing therapies, making it harder to eliminate cancer cells and supports CSC maintenance, which are often resistant to conventional therapies and contribute to tumor relapse[44]. Different studies have highlighted an inverse relationship between c-MET expression and miR-206 in HCC[45]. Specifically, c-MET levels are upregulated in human HCC tissues, while miR-206 expression is significantly downregulated, suggesting that miR-206 may negatively regulate c-MET expression and that restoring miR-206 levels could be a promising therapeutic strategy in HCC[4].

GAPS IN KNOWLEDGE AND FUTURE RESEARCH DIRECTIONS

Despite significant advances in understanding the role of miR-206 and its potential as a therapeutic agent in HCC, several critical gaps in knowledge remain. First, while miR-206 has been shown to modulate signaling pathways involved in HCC progression, the precise mechanisms regulating its expression across different cellular contexts are still unclear. Understanding the upstream regulators of miR-206, such as transcription factors, epigenetic modifications, and the influence of the TME, may reveal additional therapeutic targets and inform strategies to enhance miR-206 expression in HCC. Furthermore, the complex regulatory network involving miR-206 and other ncRNAs in HCC is not fully understood. The heterogeneity of TAMs and KCs in the HCC TME adds another layer of complexity. These cells exhibit significant diversity, with different subpopulations displaying varying degrees of plasticity and functional diversity. Future research should aim to more precisely characterize these subpopulations, investigating how miR-206 influences their polarization and functional states. Considering the multifaceted nature of HCC, combination therapies that simultaneously target multiple pathways are likely to be more effective than single-agent treatments. Future studies should explore the potential of combining miR-206-based therapies with other treatment modalities, such as immune checkpoint inhibitors, targeted therapies (e.g., anti-EGFR or anti-c-MET agents), and conventional chemotherapy. Investigating the synergistic effects of these combinations could lead to more robust and durable responses in HCC patients. Finally, the role of miR-206 in CSC dynamics is another area that requires further investigation. Research should focus on how miR-206 modulate the signaling pathways that maintain CSCs in HCC and whether targeting these pathways can overcome therapeutic resistance and prevent tumor relapse.

CONCLUSION

Although preclinical studies suggest that miR-206 has significant therapeutic potential, the translation of these findings into clinical applications remains challenging. There is a need for research focused on optimizing the delivery mechanisms for miR-206, ensuring its stability and targeting specificity in vivo. The ability of miR-206 to positively impact the prognosis of HCC in patients who are not eligible for surgery or who have multiple comorbidities is increasingly shifting the focus from traditional therapies to epigenetic modulation. Specifically, promoting the polarization of innate immune cells toward a type 1 phenotype appears to slow disease progression, although the precise mechanisms remain underexplored. These findings highlight the potential for developing therapeutic options that directly target ncRNAs, potentially improving the prognosis of typically aggressive cancer.