Cervical cancer risk continues to be significantly increased by human papillomavirus (HPV) infection[23]. The way tumor patients are managed has undergone a significant paradigm shift thanks to tumor immunotherapy. Despite the fact that treatments for CC that target the programmed cell death protein 1 (PD-1) pathway have been licensed, a sizable portion of patients show innate resistance[3].
Immune checkpoints are involved in tumor immunosuppression and are supposed to be ideal modulation targets for tumor immunotherapy. The sialic acid-binding immunoglobulin-like lectins (Siglecs), novel kinds of immune checkpoints involved in tumor immunosuppression, may be new targets, or biomarkers or prognostic factors of immunotherapy[24, 25]. A member of the CD33-related sialic acid-binding immunoglobulin-like lectin family is SIGLEC9 (CD329). Its cytoplasmic tail comprises immunoreceptor tyrosine-based inhibitory (ITIM) and ITIM-like motifs, like the majority of other immunoinhibitory Siglec family members[9, 26]. The traditional mechanism of inhibitory SIGLEC9 signaling would involve the recruitment of inhibitory tyrosine phosphatases Src homology region 2 domain-containing phosphatase (SHP) SHP1 and/or SHP2 and the phosphorylation of tyrosine residues in the ITIM domain by SRC kinase upon binding to their sialic acid ligand[27]. Blockade of Siglec-9, an emerging immune checkpoint treatment target, seems to strengthen immunity against cancer[28]. The CD33-related Siglec-7 and Siglec-9 are of particular interest in the context of tumor immunotherapy, as a variety of human cancers express ligands for both of these Siglecs[29]. Some articles have highlighted a significant role for SIGLEC9 in controlling immune suppression and provides new potential targets for cancer immunotherapy in PDAC[26] and in breast cancer[30]. However, the underlying mechanism of SIGLEC9 in cervical cancer remains unclear.
In this study, we comprehensively analyzed the expression pattern of SIGLEC9 in TMN Plot databases. We found that SIGLEC9 was highly expressed in cancer and metastasis groups. In addition, SIGLEC9 expression was significantly upregulated in tumor M stage. Then, we investigated the protein levels of SIGLEC9 in 40 cervical cancer patients and 30 myoma of uterus patients who screen negative for cervical cancer and undergo total hysterectomy from the First Affiliated Hospital of Xinjiang Medical University. The IHC and West blotting results indicated that SIGLEC9 is highly expressed in cervical cancer patients. What’s more, high SIGLEC9 expression presented to have a higher tumor stage in cervical cancer patients. And there was a correlation between SIGLEC9 and MUC16(CA125). And some researches show that MUC16-Siglec-9 binding likely mediates inhibition of anti-tumor immune responses[31, 32]. These results showed that SIGLEC9 expression was positively correlated with a malignant biologic process, indicating that SIGLEC9 might play important roles in the progression of CC.
Then, we investigated the underlying mechanisms of SIGLEC9 in CC, and our results showed that SIGLEC9 might regulate the tumor microenvironment (TME) in CC. TME is a dynamic condition in such a way that different immune cells interplay with cancer cells[33]. TME has different inflammatory mediators, extracellular matrix, and signaling molecules to induce tumor progression and therapy resistance[33]. Our results revealed that tumor-related immune cells, such as macrophage M2 and dendritic cell (DC), were positively correlated with SIGLEC9 expression in CC by CIBERSORT algorithm. Then, we found that SIGLEC9 was highly positively correlated with the number of Treg by used the TISIDB database. And the results of immunohistochemical staining indicated that a positive correlation between the SIGLEC9 and Treg. A study indicates that SIGLEC9 and its ligands play an important role in limiting human mast cell activation in vitro[34]. Monocytes and macrophages also express SIGLEC9. Classical, human myeloid-derived classical DCs (cDCs) are expressing various Siglecs including Siglec-9[24].
To further determine the biological functions about SIGLEC9, the results of GO, KEGG and GSEA indicated that this functional module may play an important role in Interactions between cells. Furthermore, SIGLEC9 expression was also positively correlated with immune checkpoints, including LAG3, HAVCR2, CD86, and PD1. Thus, SIGLEC9 was considered to exacerbate the cervical cancer by suppressing the anti-tumor immune response.
As an important member of the immune tumor microenvironment, macrophages play an important therapeutic role in CC[35, 36]. In the results of our single-cell sequencing analysis, compared with normal samples, we found that SIGLEC9 expresses highly in macrophages in tumor samples. Furthermore, we found that the SIGLEC9 gene is related to the ability of macrophages to process antigens and the proliferation of macrophages. In the future, we will demonstrate the regulatory role of SIGLEC9 in macrophages through further experiments. Thus, SIGLEC9 was considered to exacerbate the CC by suppressing the anti-tumor immune response. Some articles indicated that SIGLEC9 was mainly expressed on tumor-associated macrophages (TAMs)[18, 30].
To further analyze the function of SIGLEC9, we collected some genes directly interacting with SIGLEC9 from the protein interaction network database, and jointly constructed an interaction network module dominated by SIGLEC9 genes. From the interaction network diagram, we can see that MUC1 and LGALS3BP directly interact with SIGLEC9. Examples of glycoproteins in cancer that display an aberrantly sialylated glycan landscape capable of binding to Siglec-9 include mucin 1 (MUC1) and lectin galactoside-binding soluble 3 binding protein (LGALS3BP)[18, 37]. Beatson et al continued to show that SIGLEC9 expressing myeloid cells can bind strongly to MUC1-ST, suggesting boosted interaction involving SIGLEC9, sialic acid, and MUC1 protein, this underscored a cryptic protein–protein interaction between the MUC1 protein backbone and Siglec-9 receptors, as alternative epitopes for SIGLEC9 antibodies[19, 30]. However, the mechanism of the MUC1-ST/ SIGLEC9 axis action in cervical cancer has not been investigated. Hence, we analyzed the expression pattern of MUC1 in CC with GEO and GEPIA databases. And these results indicated that MUC1 was highly expressed in cancer groups. In addition, MUC1 expression was significantly upregulated in tumor stage.
Specific sialyltransferases (ST) that transfer sialic acids to acceptor carbohydrate residues in a linkage-dependent way are involved in the manufacture of particular siglec ligands. TCGA database was used to analyze the expression of 20 STs in CESC (ST3GAL5 was not found in CESC database), the results indicated that ST3GAL1 and ST6GAL1 were highly expressed in cervical cancer. Meanwhile, we used GEO databases to validation. Defective mucus proteins and congenital inflammatory bowel disease (IBD) are caused by reduced sialylation, according to glycoproteomic profiling and biochemical investigation of ST6 mutations found in patients[38]. And the results of immunohistochemical staining indicated that MUC1 and ST3GAL1 were high expression in cervical cancer. Lastly, we used the area under the curve (AUC) of the ROC curve to analyze the diagnostic role of SIGLEC9, MUC1, ST3GAL-I and ST6GAL-1 in cervical cancer.
In conclusion, Treg and macrophages are crucial to SIGLEC9's activity. SIGLEC9 may regulate the TME in CC to worsen the condition. To further decrease the anti-tumor immune response in CC, SIGLEC9 may upregulate the expression of immune checkpoint genes. Finally, by attaching to MUC1, which has been sialylated by the sialyltransferases in cervical cancer tumor cells, SIGLEC9 may support cervical cancer cells. These findings suggested that high SIGLEC9 expression may serve as a poor prognosis signal for cervical cancer patients and that SIGLEC9 may one day act as a therapeutic target for the disease.