Prognostic Potential of MAGEH1 and Its Correlation with Immune infiltrates in Gastric Cancer


 Background: MAGEH1 is a critical gene for regulatory T cell function. However, correlations of MAGEH1 to prognosis and tumor-infiltrating cells in different cancers remain unclear. This study was to determine the prognostic impact of MAGEH1 as well as evaluate MAGEH1 expression and immune infiltrate relevance in gastric cancer.Methods: Oncomine, TIMER2.0, Kaplan-Meier Potter, PrognoScan, Gene Expression Profiling Interactive Analysis (GEPIA), and the Human Protein Atlas database were utilized to analyze the differential MAGEH1 expression and its prognostic value. The relationship between MAGEH1 and gene markers of immune infiltration was confirmed by TIMER2.0 and GEPIA.Results: The prognostic effect of MAGEH1 varied across cancers. Particularly in gastric cancer, highly expressed MAGEH1 showed a significant association with poor prognosis in multiple databases whereas was linked to a better prognosis in patients with adjuvant 5-fluorouracil therapy. MAGEH1 expression positively correlated with infiltrating levels of B cells, CD8+ T cells, CD4+ T cells, neutrophils, macrophages, and dendritic cells in stomach adenocarcinoma (STAD). Furthermore, MAGEH1 expression showed a strong link with markers for monocytes, macrophages, neutrophils and Tregs in STAD tissues.Conclusions: MAGEH1 can serve as a prognostic biomarker in gastric cancer and is related to immune infiltrates.


MAGEH1 Expression-prognosis Association
We next investigated MAGEH1 and oncologic outcome relevance in order to verify its clinical signi cance. Figure S3 provided an overview of the MAGEH1 expression-prognosis association in PrognoScan. Prognosis of 8 cancers dramatically associated with MAGEH1 in PrognoScan was screened with Cox-P < 0.05 as a threshold, which including bladder cancer, acute myeloid leukemia, Glioma, breast cancer, CRC, eye cancer, LUAD, and liposarcoma ( Figure S3). Multiple cohorts of CRC (GSE17536, GSE14333, GSE17537) and breast cancer (GSE12276, GSE1378, GSE1379, GSE9893, GSE2034) showed that MAGEH1 was a markedly favorable prognostic marker. Results infer that MAGEH1 highly expressed was an independent risk factor for the adverse outcome of CRC and breast cancer patients.
The prognostic value of MAGEH1 in diverse neoplasms was further validated by Gene Expression Pro ling Interactive Analysis (GEPIA) ( Figure S1) and Human Protein Atlas database (HPA) analysis, which based on RNA-seq datasets from TCGA. Highly expressed MAGEH1 was bene cial to prognosis for OS and disease-free survival (DFS) in LGG (Brain Lower Grade Glioma), OV (ovarian serous cystadenocarcinoma) and PAAD (Pancreatic adenocarcinoma) ( Figure S1.ac-ad, am-an, ao-ap), OS in LUAD and DFS in SKCM (Skin Cutaneous Melanoma) and THCA ( Figure S1ag

The Discrepancy Expression of MAGEH1 in a layered STAD Population
We validated the relationship between MAGEH1 and clinicopathological factors in GC in Kaplan-Meier Plotter for exploring its relevance and potential mechanisms. As shown in Fig. 3 The discrepancy between ACC, STAD and LGG in Associations of MAGEH1 Expression and Immune In ltration According to the importance of tumor-in ltrating lymphocytes [23,24]and MAGEH1 in neoplasm prognosis, we explored whether MAGEH1 would be conducive to recruiting more immune cells into TME, thus a icting the oncologic outcome. We carried out TIMER2.0 to assess the relationship between MAGEH1 and immunoin ltration in 39 neoplasms. The results displayed MAGEH1 expression remarkably linked to tumor purity of 16 types of cancer. Moreover, the levels of in ltration of B cells, CD8 + T cells, CD4 + T cells, dendritic cells (DCs), macrophages, and neutrophils (19, 19, 11, 24, 29, and 28 cancer types, respectively) had an association with MAGEH1 expression through TIMER algorithms ( Figure S2).
The data of TIMER2.0, GEPIA and HPA are all mainly from TCGA database. Therefore, based on the above prognostic consequences in GEPIA and HPA, we nally selected LGG and ACC (Adrenocortical carcinoma) with different associations between prognosis and MAGEH1 expression than STAD as the control for comparison. Interestingly, we found that the relevance between increased expression of MAGEH1 and immune in ltration after adjusting for tumor purity was dramatically distinct in STAD and LGG. A positive correlation was found betweenMAGEH1 expression and the levels of in ltration of Therefore, we next investigated the relationship between MAGEH1 and immunomarker genes, which characterized immune cells among STAD, ACC and LGG. We found that after adjustment for tumor purity, not statistically signi cant correlations existed in ACC. However, in STAD and LGG, MAGEH1 expression was markedly related to most markers of multiple innate and acquired immune cells ( Table 1). As expected, most immune markers had positive coe cients in STAD negatively correlated in LGG, but the two focused on different types of immune cells. Hence, it could conceivably be hypothesized that the expression pro le of MAGEH1 in STAD and LGG was associated with separate in ltrating patterns of TME, which can explain the noticeable variations in the associations across MAGEH1 and patient survival in the two types of cancers. Speci cally, A strong link with MAGEH1 expression was observed in genetic markers of macrophages (monocytes, tumor-related macrophages (TAMs) and M2 macrophages), neutrophils and Treg cells than others in STAD (Table 1), while LGG was mainly associated with antigen-presenting DCs.  Table 1), which was also corroborated by GEPIA later (Table 2). Together, these results reveal that MAGEH1 may be crucial in immunologic escape and immune tolerance in TME in STAD. Normal, correlation analysis in normal tissue of stomach adenocarcinoma in TCGA. *P < 0.01; **P < 0.001; ***P < 0.0001.

Discussion
Although MAGEH1 is rarely examined, it is known to be upregulated in activated Treg and CD8 + T cells. Recent investigates have stressed the importance of MAGEH1 in tumorigenesis and cancer development [17-19, 21, 25]. In this study, variations in MAGEH1 expression levels appeared to associate with prognosis in different cancer types. MAGEH1 highly expressed correlated with a favorable prognosis in LUAD and PRAD and worsened the prognosis in STAD and BRCA. Unexpectedly, the increased expression of MAGEH1 was bene cial to the outcome of GC patients with adjuvant therapy, pointing out the MAGEH1's prediction for tumor e cacy. Furthermore, we found that STAD and LGG have different patterns for relevance between immune in ltration and MAGEH1. Therefore, this work provides insights into MAGEH1 as a biomarker for prognosis and therapeutic effect of GC from the tumor immunology viewpoint, which contributes to future mechanism research and the development of immunotherapy.
Here we pro led the difference of MAGEH1 expression and the visualization of prognostic landscape in various tumors utilizing data from Oncomine, PrognoScan and TCGA. Compare to normal tissues, the mRNA levels of MAGEH1 varied among different tumors, and most of them were downregulated. For example, decreased expression of MAGREH1 in liver cancer tissues corroborates the previous nding [18] (Fig. 1a and Table S1.). This might be due to certain factors leading to gene silencing, such as transformation-dependent DNA hypermethylation, which is common in cancer cells [26]. However, further investigates are needed to determine the speci c relationship between MAGEH1 and methylation. In multiple databases, high MAGEH1 expression dramatically linked to better survival of LUAD and PAAD, and worse prognosis of STAD and BRCA, even though there are remarkable variations among different databases due to diversities in sample collection methods, processing standards, as well as samples themselves (Fig. 2, Table S1, Figure S1, 3). Unlike De Simone [21], who argued highly expressed MAGEH1 predicted a poor 5-year survival of NSCLC patients after adjusting for T cell density, we found LUAD patients with elevated MAGEH1 was remarked related to positive prognosis ( Fig. 2 and Figure   S1). The different range of tumor types included may account for this contradiction, such as MAGEH1 had a correlation with LUAD prognosis in lung cancer, rather than LUSC ( Figure S1). Moreover, various clinical trial endpoints ( Figure S1), as well as treatment methods in the study population may lead to diversity in results. Therefore, further research is required, including more patients and more detailed and improved designs. In addition, MAGEH1 was an adverse prognostic factor for GC in stage N1, M0, intestinal and diffuse by Lauren classi cation, as well as surgery only (Fig. 3).
Taken together, MAGEH1 may be a promising prognostic marker for gastric, lung, breast, and prostate cancers.
Interestingly, in the treatment of GC, the elevated expression of MAGEH1 in patients with adjuvant 5-FU was substantially correlated with a favorable prognosis, which differed from whose surgery alone. We speculate MAGEH1 can reverse 5-FU resistance of GC. Over-expression of MAGEH1 has been reported to overcome melanoma resistance to current therapies by interacting with the juxtamembrane region of p75 neurotrophic receptor [29].
Disordered apoptosis is linked to cancer treatment resistance [27]. While 5-FU interferes with DNA replication by inhibiting thymidylate synthase, and consequently, apoptosis and cell cycle arrest [28], and its effect is limited by the drug resistance of GC. Therefore, it is reasonable to consider that the anti-apoptotic effect of MAGEH1 can partly overcome the tumor resistance to 5-FU and thus improve the prognosis of patients. Up to now, there is little published data on the relationship between MAGEH1 and GC. Additional research is needed to validate this hypothesis, which may help to better de ne patients who are clinically appropriate to receive adjuvant chemotherapy. This result points to MAGEH1 as a predictive factor responding to postsurgical chemotherapy, rather than being only a prognostic indicator.
Another nding was the association of MAGEH1 expression with varying degrees of immune in ltrates in TME, especially in STAD. TME contains tumor cells and other cells, with tumor-in ltrating lymphocytes accounts for a large proportion. Accumulation of TAMs and Tregs in the TME is a major element in immune evasion and immunosuppression, thus promoting tumor growth and invasion. Moreover, a variety of immunoin ltrating cells contained in the TME, also participate in cancer development [30]. MAGEH1 expression was negatively correlated to tumor purity in STAD, contrary to the correlation in LGG, which denotes it is relatively enriched in tumor cells in STAD. Therefore, the assessment regarding the MAGEH1-immune in ltration relationship adjusted the purity of the tumor to obviate the confounding effects by tumor purity [31]. We found that MAGEH1 expression optimistically associated with the 6 immune in ltrating cells in STAD, while most negatively correlated in LGG, as expected. This also re ects the heterogeneity of tumors and various roles of MAGEH1 in different tumors.
Meanwhile, the MAGEH1 and immune-related markers relevance also demonstrate MAGEH1 in the regulation of tumor immunity in STAD. Our results revealed a strong link between MAGEH1 and neutrophils. We know that neutrophils regulate many malignance-related behaviors of cancer cells, such as migration and invasion, and may accelerate GC progression by inhibiting T cell function [32]. Under certain assumptions, we can be construed that MAGEH1 may partially inhibit T cell function in the regulation of neutrophils, but not in the way of PD-1 dependence. Interestingly, MAGEH1 was positively correlated with immune markers regarding TAM, monocytes, macrophages, and Treg cells. Although we cannot account for this phenomenon by describing a cell-speci c mechanism, clues may be found from ex-pressed markers concerning TAM. The most critical pathways for TAM recruitment and proliferation are the CSF-1 /CSR-1 signal and the CCL2/CCR2 axis [33], which are crucial for macrophage survival and M1 to M2 transformation. What's more, some drugs inhibiting TAM recruitment have achieved de nite clinical e cacy [33]. The strong correlation between CSF-1, CCL2, M2, and TAM-related immune markers and MAGEH1 reveals the potential regulatory effect of MAGEH1 on the recruitment of monocytes/macrophages, Tregs and M2 polarization. Tumor-in ltrating Tregs can inhibit anti-cancer immunity, thus hindering immunosurveillance and hampering effective antitumor immune responses, sequentially accelerating tumor progression in a broad range of cancer types including GC [34].
FOXP3 and TGF-β are essential for Tregs to regulate effector T cells' function [35,36]. Based on the strong correlation between FOXP3, TGF-β and MAGEH1, presumably, MAGEH1 is a potential marker for the tumor immunization of Tregs, in uencing the immune response of effector T cells, thereby promoting tumor development. Although the results showed that there was positive relevance between MAGEH1 and CD8 + T cell (Fig. 4, Table 1 Recent work about MAGEH1 may provide some explanations for the potential mechanisms. MAGEH1 may act as E3 ubiquitin ligase, which affects the function or survival of Treg in cancer tissues and accelerates tumor growth and metastasis [37,38]. Increased expression of MAGEH1 has been reported in numerous tumors' Treg cells, such as breast cancer, CRC, and malignant cell lung cancer [21,37]. Indeed, Tregs have been emphasized in accelerating tumorigenesis and progression by speci cally inhibiting tumor-reactive T cells [34]. Besides, altered subcellular localization of MAGEH1 may affect its function. MAGEH1 is mainly localized to the nucleus in normal cells but is predominantly detected in the cytoplasm of tumor cells [39].
The subcellular localization of proteins is vital for their physiological function [40]. MAGEH1 may transfer from nucleus to cytoplasm, afterwards the different domains and subcellular location of MAGEH1 affect its subcellular functions, such as regulating cell cycle and promoting apoptosis [15,16,25,29].
However, despite the integration of information from multiple databases to analyze the expression and the impact of MAGEH1 on the oncologic outcome, the mechanisms of MAGEH1 have not been fully elucidated in cell and animal studies. Moreover, potential epigenetic mechanisms of MAGEH1 are yet to be explored concerning prognosis. Besides, due to some con icting ndings from different databases, we still can't make a blanket statement about MAGEH1.

Materials And Methods
Genetic differential analysis The discrepancy of MAGEH1 expression in various tumor tissues versus corresponding normal tissues was examined in the Oncomine database (https://www.Oncomi ne.org/resource/login.html) [41]. And statistical analysis of the difference was used by the student's T-test. The threshold was set at: P-value: 0.001, fold change: 1.5, gene rank: ALL. Then the differential expression of MAGEH1 gene was ascertained in the TCGA database via the Gene_DE part in TIMER2.0 (http://timer.comp-genomics.org/) [42] and the results were shown with boxplots.

Estimation of immune in ltration
We applied Gene and Survival Module in TIMER2.0 (http://timer.comp-genom ics.org/) to calculate the relationships between MAGEH1 expression and 6 types of tumor-in ltrating cells (B cells, CD4 + T cells, CD8 + T cells, macrophages, neutrophils, and dendritic cells), in STAD, LGG and ACC. The connection with genetic markers of tumor-in ltrating cells conditioned on tumor purity was computed in Gene_Corr module. We further veri ed associations in GEPIA (http://gepia.cancer-pku.cn/index.html); under other default settings, spearman's correlation analysis was selected to analyze the tumor and normal tissue datasets.

Statistical analysis
The statistically signi cant difference of various expression levels of MAGEH1 in tumor versus normal groups was calculated by T-test. Kaplan-Meier curves were utilized to compare the prognostic differences, which displayed as P or Cox P-values determined by the log-rank test and HR with 95% CI.
Relation of gene expression was tested with Spearman's correlation. The level of signi cance was de ned by a P-value < 0.05, if not especially noted. Declarations