Key among the genes that encode the helix-loop-helix (HLH) family of transcription factors is the ID, abundant in stem and progenitor cells (1). To date, it is known that ID proteins are encoded by four ID genes in the ID family in vertebrates: ID1-4, all of which encode the corresponding four ID family members (1, 2). These genes are located in different chromosomes and show inconsistent expression profiles and functions (30). Emerging evidence suggests that ID proteins play vital roles in tumorigenesis in several types of malignancies mediated by their ability to regulate cell-cycle, cell differentiation, epithelial-mesenchymal transition, chemoresistance, and immunomodulation (1–3, 5). Yet, the specific roles of the four ID members in OC are obscure. This study evaluated the prognostic value and expression of ID family genes by investigating various large databases. Our study presents the first silico and bioinformatics analysis of the ID family.
ID1 is the most widely characterized component of the HLH transcription factor family (31). Studies show that the molecular functions of ID-1 included induction of cell proliferation, increasing DNA synthesis, and interaction with various oncogenes (32). Aberrant expression of the ID1 protein has not only been detected in multiple types of human cancers, but is also correlated with tumor stages and clinical outcome (33, 34). Furthermore, ectopic expression of ID1 in human cancer cells increases serum-independent cell growth, enhances primary tumor G1/S phase formation and metastatic potential, and protects tumor cells against apoptosis. Conversely, inhibition or inhibition of ID1 in human cancer cells has been shown to suppress cell proliferation, induce cellular senescence, induce G2/M cell-cycle arrest, reduce tumor colony formation or multiplicity, and increase lifespan (35, 36). In OC, Schindl et. al. found that ID1 expression correlates with the malignant potential of OC and is correlated with aggressive behavior, differentiation of tumor cells, and clinical prognosis (12). Several studies have found that increased ID1 may promote cancer cell proliferation and enhance endothelial progenitor cell angiogenesis through regulation or facilitation of EGFR and TGFβ1 expression, and activation NF-κB/MMP-2 and PI3K/Akt signaling pathways in OC cells (6, 37–39). In addition, the study by Li ZD et al demonstrated that apigenin can suppress the expression of ID1, resulting in inhibition of tumorigenesis in human OC A2780 cells (40). Thus, ID1 represents a promising therapeutic target for OC. In our study, the Oncomine and GEPIA datasets indicated that the expression of ID1 was suppressed in human OC. The Kaplan–Meier plotter and PROGgeneV2 analysis revealed a high mRNA expression of ID1, and this was correlated with poor OS in all OC patients. These data reflect the heterogeneity of ID1 expression in mRNA and protein levels, and point to the oncogenic function of ID1.
ID2 belongs to the HLH transcription factor family, which promotes proliferation and invasive growth in multiple solid cancers, e.g., hepatocellular cancer, breast cancer, thyroid cancer, pancreatic cancer, and OC (1, 41). Like ID1, several studies have shown that ID2 promotes the proliferation of human cancer cells by inhibiting cell apoptosis, enhancing cancer stemness of pre-malignant cells, or mediating m6A modifications (42–44). Conversely, reduced ID2 expression increases apoptosis, reduces cell proliferation, and decreases tumor initiation in human cancer cells (16, 45). However, currently there are very few reports on ID2 and OC development in the literature. An earlier study showed the ID2 gene as a candidate for inherited predisposition to breast and ovarian cancer in Jewish women (46). Moreover, the study by Meng et. al. reported that elevated ID2 expression in ERα-positive epithelial tumor cells promoted the invasiveness of cells via a non-canonical pathway independent forming dimers with basic helix-loop-helix factors (47). In this study, ID2 mRNA expression was found to be lower in OC samples than in normal ones, and elevated ID2 expression was strongly related to poor PFS in all patients with OC. Prognostic analysis in patients with OC in different data sets, however, overall effect did not show any significant correlation between ID2 expression and OS. The oncogenic effects, predictive value, and potential molecular targets of ID2 in OC remain to be investigated further.
ID3, associated with HLH transcription factors, has been recognized as a key regulator of cell development, senescence, differentiation, proliferation, stemness, and migration (1, 48). It has been confirmed that ID3 and ID1 can compensate for each other and have similar biological functions (5). Previous studies have demonstrated that aberrant expression of ID3 is associated with advanced tumor stage and poor prognosis in many types of human cancers. In animal experiments, although Id1+/-Id3-/- or Id1-/-Id3+/- mice grow to adulthood, they are unsuitable to implanted tumor xenografts because these mice lack the capacity to recruit hematopoietic precursors and endothelial (48, 49). Furthermore, double knockdown of ID1 and ID3 has been shown to decrease proliferation and anchorage-independent growth, increase apoptosis, and reduce survival in various human cancer cells (16, 17). More importantly, ID3 knockdown improved the survival duration of animals in a seeding model of medulloblastoma. It also compromised the progression of leptomeningeal seeding and the growth of primary tumors (50). Elsewhere, it was recognized that BMP4 signaling is active in ovarian cancer cells where it maintains ID3 expression. This was confirmed by the use of BMP4 blocker Noggin, which decreased endogenous ID3 expression (51). In this study, we also demonstrated that the expression of ID3 in OC tissues was lower than that in normal tissues, and ID3 overexpression was associated with reduced OS and PFS in OC patients. Because ID3 undergoes epigenetic inhibition in multiple cancers, it is therefore thought to be a tumor suppressor.
In comparison with the other ID proteins, ID4 possesses a polyproline domain at its C terminus and a polyalanine domain at its N terminus. Although it harbors the HLH domain, ID4 does not display similar expression and function with D protein (5). Numerous studies have shown that the phenotypic changes and molecular pathways regulated by ID4 are, in general, not like those regulated by ID1, ID2, and ID3. Interestingly, ID4 seems to function as a tumor suppressor in multiple cancers and as a tumor promoter in a small subset of cancers (30, 52, 53). The proposed tumor-suppressing effects of ID4 draw on observations that ID4 undergoes epigenetic silencing in several solid cancers such as esophageal, gastric, pancreatic, colorectal, cholangiocarcinoma and lung cancer. However, ID4 has been reported to be elevated in some small cancers, such as OC, prompting researchers to re-classify it as a tumor promoter (4, 52, 54, 55). Mice deficient in ID4 develop some types of cancers in their lifetime, and the lack of ID4 results in follicular dysplasia and increased atretic follicles due to decreased estrogen biosynthesis (30). It is worth noting that a recent study showed that administration to mice harboring an ovarian tumor with an ID4-specific tumor-penetrating nanocomplex was capable of suppressing the growth of established tumors and significantly improved survival (7). In the current study, unlike ID1, ID2, and ID3, the expression of ID4 was higher in OC tissues than that in normal tissues, and high ID4 expression was significantly correlated with better OS in OC patients, thereby indicating its tumor promoter role in OC.
We also attempted to examine the mechanisms and roles of members of the ID family, we also used the cBioPortal database to explore the mutations in the ID family. The results showed that the genetic alteration rate of the ID family members varied from 3% to 15% for individual genes based on the TCGA provisional dataset, however, there was no significant association between the prognosis of OC with ID gene alteration or without alteration. We then constructed a network of ID family members and 50 of the closest co-expressed genes. The results of the functional analysis indicated that these genes were mainly enriched in tumor-related pathways, including the ID-, c-MYC-, TNF-, and Wnt signaling pathways. In addition, two major highlights of this study were the ID signature and immune infiltration analysis. In the ID signature analysis, the prognostic values of ID signature in patients with OC were evaluated in three datasets based on the SurvExpress platform. The method overcomes the problem single gene with the expression optimal cutoff for prognostic analysis cannot fully reflect the optimal differentiation of survival benefits and performance of potential biomarkers. In the immune infiltration analysis, we explored the correlation between IDs expression with six immune infiltration levels in OC via correlation modules in TIMER. Our results showed that ID expression showed a strong correlation with infiltrating levels of B cells and macrophages, which further confirmed that the biological role of ID may be associated with immune regulation. However, the underlying molecular mechanisms and regulation steps remain largely unexplored.