Introduction
Ovarian cancer is one of the most common gynecologic malignant tumors, with a relatively high mortality rate. The statistics showed that 5-year survival rate of ovarian cancer patients was less than 44% [
1]. The first-line treatment for ovarian cancer remains tumor cell reduction surgery combined with post-operative chemotherapy. However, the recurrence rate of ovarian cancer is as high as 70%. Studies have shown that tumor microenvironments (TME) participated in regulating the immune response and inflammatory response of cancer through various mechanisms [
2‐
4]. Therefore, immunotherapy targeting TME has attracted extensive attentions.
TME refers to the tumor microenvironment which surrounds and nourishes the tumor cells, including blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, various signaling molecules and extracellular matrix. Macrophages are one of the most important immune cells in TME, which mainly work as recognition, phagocytosis and degradation of foreign bodies, bacteria and dead cells, etc. In addition, macrophages can also play a role in presenting antigens to T cells to initiate adaptive immune response, which means macrophages not only participate in innate immunity, but also adaptive immunity. Macrophages can be divided into M1 and M2 polarization phenotypes, tumor-associated macrophages (TAMs) are a type of macrophages, which status is similar to M2 macrophages [
5]. Here, we aimed to explore the role of m6A methylation enzymes ALKBH5 and IGF2BP2 in regulating macrophage polarization in ovarian cancer microenvironment.
M6A methylation is one of the most common RNA modifications, and participates in all stages of RNA life cycle, including RNA transcription, translation and degradation [
4]. In fact, m6A methylation also participates in cancer progression. ALKBH5 is one of the most important and classical demethylases. In many cancers, ALKBH5
functions as
an oncogene. In GBM, ALKBH5 worked as an oncogene and influences the self-renewal and proliferation of cancer stem cells [
6]. In ovarian cancer, ALKBH5 was up-regulated to enhance the stability of BCL-2, thus inhibiting the autophagy, and promoting the invasion and proliferation of cancer cells [
7]. In addition, ALKBH5-HOXA10 loop could promote the cisplatin resistance in epithelial ovarian cancer through demethylating JAK2 [
8]. As an important m6A recognition protein, IGF2BP2 mainly acts to promote the stability and translation of mRNA. In ovarian cancer, the knockdown of circ-0001756 could suppress IGF2BP2 mediated RAB5A expression, thus inhibiting malignant progression of ovarian cancer [
9]. As for the role that IGF2BP2 plays in immune response, study showed that IGF2BP2 could affect the immune-related biological pathways in oral squamous cell carcinoma, thus leading to the worse prognosis for cancer [
10]. However, few studies have focused on the role of ALKBH5 and IGF2BP2 in regulating macrophage polarization in ovarian cancer.
Compared to the M0 macrophages, we first identified that ALKBH5 and IGF2BP2 were up-regulated in M2 macrophages. To deepen our understanding the role of ALKBH5 and IGF2BP2 play in ovarian cancer immune microenvironment, we used various databases to explore their relationships with immune cells, not only at tissues level, but also at single-cell level. Finally, we validated the role of ALKBH5 plays in macrophage polarization through RT-PCR. Taken together, our results could potentially represent the roles of ALKBH5 and IGF2BP2 played in macrophages in ovarian cancer, which might work as the potential immunotherapy biomarkers.
Discussion
Ovarian cancer is always characterized by the late stage once found, with high recurrence rate and mortality. It is known as the “silent killer” for women. Studies have shown that more than 25% of ovarian cancer patients represented chemoresistance at the first relapse [
21]. So it is important to find new approaches for ovarian cancer therapy. M6A methylation is the most common nucleotide modification in mRNA, about 1/4 mRNA molecules contain at least one m6A modification site [
22]. M6A modification is involved in various processes of mRNA metabolism [
23‐
25]. Recent studies have shown that m6A could affect a variety of cellular biological processes and played an important role in cell fate determination, lipid metabolism and immunity [
26‐
28]. ALKBH5 works as an important demethylase, while IGF2BP2 works as the m6A recognition protein. However, its role in ovarian cancer needs further study. We first found that ALKBH5 and IGF2BP2 were up-regulated in M2 macrophages, and both showed significantly correlated with immune cells in ovarian cancer, especially macrophages. Importantly, we found that the closely correlation between ALKBH5 and IGF2BP2 with immune cells due to its mRNA expression levels, rather than their CNV status. Furthermore, we investigated the prognostic role of ALKBH5 and IGF2BP2 in ovarian cancer, which both showed the overexpression of ALKBH5 and IGF2BP2 represented the worse prognosis for ovarian cancer.
The mechanisms that can regulate the macrophages polarization has been investigated for years. However, the studies focused on investigating the role that m6A methylation-related enzymes that played in cancer immune microenvironment were very limited. According to our analysis, we found that ALKBH5 and IGF2BP2 were significantly correlated with the immune response in ovarian cancer. ALKBH5 is a classical m6A demethylase that primarily functions to remove m6A modification to promote mRNA nuclear processing and export. In ovarian cancer, high expression of TLR4 could activate NF-κB signaling pathway, thus up-regulated ALKBH5 to influence the m6A methylation of NANOG, which further promoted carcinogenesis in ovarian cancer [
29]. In epithelial ovarian cancer, ALKBH5-HOXA10 loop regulated the methylation of JAK2, which could activate JAK2-STAT3 signaling, thus promoting chemoresistance of cancer cells [
8]. Moreover, IGF2BP2 worked as an important m6A recognition protein, which mainly acted to promote the stability and translation of mRNA. In ovarian cancer, the study showed that circITGB6 can directly interact with IGF2BP2 and FGF9, thus stabilized FGF9 and promoted M2 polarization in ovarian cancer [
30]. In fact, research on whether the expression of ALKBH5 and IGF2BP2 can regulate the polarization of macrophages were very limited. Most of the studies correlating m6A methylation enzymes and macrophages function have focused on studying the role of METTL3 in macrophages. In myeloid cells, the ablation of METTL3 could promote tumor metastasis, which was due to the deficiency of METTL3 increased the M1 macrophages and regulatory T cells infiltration [
31]. In breast cancer, METTL14 and ZC3H13 were down-regulated, indicating a positive correlation between the expression of METTL14 and ZC3H13 positively correlated with various kinds of immune cells, including macrophages, T cells and DCs [
32]. In this study, we found that among all the immune cells, ALKBH5 and IGF2BP2 showed the closest correlation with macrophages in ovarian cancer. However, we found that the expression level of IGF2BP2 was much lower than ALKBH5 in ovarian cancer immune microenvironment.
As one of the most important immune cells in cancer, macrophages have a relatively high expression of immune cells, which can not only regulate the nonspecific immunity, but also influence the specific immunity. In nonspecific immunity, macrophages can kill and clear pathogens through phagocytosis and mediate inflammatory response. In specific immunity, macrophages mainly focus on immune regulation and antigen presentation. Studies have shown that repolarization of macrophages from M2 to M1 could enhance the macrophages’ ability to
kill cancer cells, including ovarian cancer cells [
33‐
35]. Thus, we investigated the correlation between ALKBH5 and IGF2BP2 with the M2 macrophage markers IL-10 and MRC1, while ALKBH5 showed positively correlation, however, no obvious correlation was observed with IGF2BP2.
Single-cell sequencing has been gaining momentum in recent years. The single-cell sequencing technology involves sequencing and analyzing the genome, transcriptome and epigenome at the single-cell level [
36]. Traditional sequencing is carried out on the basis of multicellularity. In fact, what we get is the average value of signals in a cluster of cells, which
eliminates the information of cell heterogeneity. Single-cell sequencing technology can detect the heterogeneity information that can’t be obtained by hybrid sample sequencing, so it effective in addressing this
issue. Thus, we analyzed the ALKBH5 and IGF2BP2 expression in immune cells through single-level analysis. The results showed that ALKBH5 is widely expressed in various cells, with the highest up-regulation observed in macrophages not only in ovarian cancer, but also in ovarian tissues. However, the expression of IGF2BP2 was relatively low in macrophages in ovarian cancer microenvironment. Thus, we chose ALKBH5 for further study. Here, we chose THP-1 cell line induced by PMA as the macrophage model, which is widely acknowledged macrophage lineage in many researches. Most of the studies investigating the role of macrophages in human use THP-1 cell line and induced it into macrophage by PMA, which could imitate the status of macrophage in vivo, including in ovarian cancer [
34,
37‐
39]. To validate whether ALKBH5 could promote the M2 polarization of macrophages in vitro, macrophages transfected with ALKBH5 that could overexpress or inhibit its expression were used to evaluate the polarization markers of macrophages through RT-PCR. The results showed that overexpression of ALKBH5 could promote high expression of M2 marker IL-10, ARG-1 and low expression of M1 marker TNF-α, and inhibition of ALKBH5 could promote high expression of M1 marker CD86 with low expression of M2 marker CD163 and CCL22. In fact, many genes and proteins can regulate the polarization of macrophages. However, the function of enzymes that can regulate the m6A methylation in controlling macrophage polarization has not been widely investigated yet. Based on the GO and KEGG analysis, results showed that the biological process of ALKBH5 in ovarian cancer was mostly correlated with immune response, including T cell receptor signaling pathway, inflammatory response, immune response and antimicrobial humoral immune response mediated by antimicrobial peptide. These processes were confirmed as the main processes in immune response in cancer metabolism. What’s more, in KEGG analysis, we
identified pathways that related to ALKBH5 were Th17 cell differentiation, intestinal immune network for IgA production, NOD-like receptor signaling pathway and NF-κB signaling pathway. Those pathways also showed significantly correlation with immune response. Thus, we speculated that ALKBH5 might participate in regulating the immune response in ovarian cancer microenvironment.
Conclusion
In conclusion, our study demonstrated that ALKBH5 and IGF2BP2 were significantly up-regulated in M2 macrophages, which not only showed closely correlation with macrophage expression in ovarian cancer, but also correlated with the prognosis of ovarian cancer. In single-cell analysis, we found that ALKBH5 was mainly expressed in macrophages in ovarian cancer. Finally, we verified that ALKBH5 could regulate M2 macrophage polarization in vitro study. Thus, targeting ALKBH5 in macrophages might be a promising target for regulating the immune microenvironment in ovarian cancer, which could further influence the prognosis of ovarian cancer patients.
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