Significance of KDM6A Mutation&nbsp;in Bladder Cancer Immune Escape

doi:10.21203/rs.3.rs-114298/v1

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Significance of KDM6A Mutation in Bladder Cancer Immune Escape

https://doi.org/10.21203/rs.3.rs-114298/v1

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Background: Bladder cancer (BC)is the fourth most prevalent neoplasm in menand is associated with high tumour recurrence rates, leading to major treatment challenges. Lysine-specific demethylase 6A (KDM6A) is frequently mutated in several cancer types; however, its effects on tumour progression and clinical outcome in BCremain unclear. Here, we explored the potential role of KDM6A in regulating the antitumor immune response.

Methods: We mined The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) databases for somatic mutation and clinical data in patients with BC.

Results: We found frequent mutations in 12 genes in both cohorts, including TP53, KDM6A, CSMD3, MUC16, STAG2, PIK3CA, ARID1A, RB1, EP300, ERBB2, ERBB3, and FGFR3. The frequency of KDM6A mutationsin the TCGA and ICGC datasets was 25.97% and 24.27%, respectively. In addition, KDM6A mutation was associated with a lower number oftumour-infiltrating lymphocytes and indicated a state ofimmunetolerance. KDM6A mutation was associated with lower KDM6A expression level compared with that in samples carrying the wild-type gene. Further, survival analysis showed that the prognosis of patients with low KDM6Aexpression was worse than that withhigh KDM6A expression. Using the CIBERSORT algorithm, Tumor Immune Estimation Resource site, and Gene Set Enrichment Analysis, we found that KDM6A mutation downregulated nine signalling pathways that participate in the immune system and attenuated the tumour immune response.

Conclusion: Overall, we conclude that KDM6A mutation is frequent in BC and promotes tumour immune escape, which may serve as a novel biomarker to predict the immune response.

Cancer Biology

KDM6A

bladder cancer

mutation

immune

Bladder cancer (BC) is considered to be the fourth most prevalent neoplasm in men [1]. BC is associated with high rates of tumour recurrence and progression, which represent major challenges in treatment. Lysine-specific demethylase 6A (KDM6A) is a member of the histone H3 lysine 27 demethylase gene family, which is reported to exert pro-tumorigenic effects in some cancer types [2, 3] but is also considered a tumour suppressor in other contexts [4]. Analyses of tumour-associated genes showed frequent loss of KDM6A expression in several cancers, including BC, B-cell lymphoma, lung squamous cell carcinoma, head and neck squamous cell carcinoma, pancreatic adenocarcinoma, and renal papillary cell carcinoma [5-9]. Nevertheless, the molecular mechanism by which KDM6A might suppress tumour progression in BC remains unclear. To provide insight into this mechanism, we conducted data mining of two separate public cohorts of BC patients to explore the frequency of KDM6A mutations and its potential influence on the immune response. This study can help to elucidate the effect of KDM6A on the tumour microenvironment and develop a potential immunotherapy strategy for BC patients.

2.1. Datasets

The somatic gene mutations and clinical data of 412 samples from BC patients in the United States and 101 samples from BC patients in China were downloaded from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) online platforms, respectively.

2.2. Data analysis

All statistical analyses were performed using Stata software 14.0 and GraphPad Prism 8.0 (San Diego, CA, USA); P < 0.05 was defined as statistically significant. Kaplan-Meier survival analysis was conducted to generatesurvival curves, which were evaluated using the log-rank test. Cox regression analyses were performed for assessing the associations of survival with clinical characteristics and KDM6Aexpression levels. The Kruskal-Wallis test was used to analyse the correlation between KDM6A mutation status and overall mutation counts.

CIBERSORT was conducted to evaluate theproportions of 22 tumor-infiltrating lymphocytes (TILs)subsets in 412 samples from BC patients in the United States, and estimatedthe relative abundance of TILs with different KDM6Astatus.

Gene set enrichment analysis (GSEA) was performed using GSEA 4.0. Student’s t-test was used to compare the expression level of KDM6A mRNA according to theKDM6A mutation status. The χ²test was used to evaluate the association between KDM6A mutation status and clinical parameters. In GSEA, P < 0.05 and q < 0.25 were considered statistically significant.

3.1. KDM6A mutation is frequent in BC

We downloaded the data, including follow-up profiles and gene expression levels, of 411 and 103 BC tissues from the TCGA and ICGC databases, respectively. Among these samples, 107/411 (25.97%) and 25/103 (24.27%) harboured a KDM6A mutation (Sup. 1A–C).

Missense and truncating mutations were the main mutation types spanning the entire gene (Sup.2A-B), with the former mutation type being most frequent (Sup. 3A); single nucleotide polymorphism was more common than deletion or insertion (Sup. 3B), and C>T was the most common single nucleotide variant in BC (Sup. 3C). The number of variant bases in each sample was counted and the mutation types are shown in a box plot in different colours in Sup. 3D and 3E.

3.2. KDM6A mutation is associated with higher mutation counts

BC samples with KDM6Amutations had higher overall mutation counts than wild-type samples in the TCGA cohort (210 vs 156.5; p = 0.0383, Kruskal-Wallis equality-of-populations rank test) (Fig. 1).

3.3. KDM6A mutation is associated with reduced KDM6Aexpression levels and poor prognosis in BC

Patients with KDM6A mutations had lower KDM6A expression levels than those with wild-type KDM6A (Fig. 2A). As shown in Sup.4, patients with KDM6A mutationshad a higher metastasis stage than those with wild-type KDM6A. However, no correlation was observed between KDM6A mutation and age, histologic grade, or the tumour-node-metastasis (TNM) stage. Kaplan-Meier analysis showed that BC patients with a high KDM6Aexpression (top 15%) levels hadsignificantly longer overall survival than those with low KDM6A levels (bottom 15%) (Fig. 2B). However, univariate and multivariate Cox regression analyses showed that a low KDM6A level or presence of KDM6A mutation was not an independent prognostic factor.

3.4. KDM6A mutation is significantly correlated with tumour-infiltrating lymphocytes (TILs) in the tumour microenvironment

Tumor Immune Estimation Resource (TIMER) analysis showed that KDM6A mutation was associated with a lower number of TILs. The infiltration levels of macrophages (p = 1.44e-2), CD8+ T cells (p = 5.79e-4), neutrophils (p = 4.40e-3), and resting dendritic cells (p = 3.70e-3) in the KDM6A mutation group were lower than those in the KDM6A wild-type group (Fig.3A). The association between KDM6A mutation and different types of immune cells was significant, including dendritic cells (p = 9.70e-3, cor = –0.1272), CD8⁺ T cells (p = 6.0e-4, cor = –0.1689), macrophages (p = 1.03e-2, cor = –0.1262), and neutrophils (p = 5.1e-3, cor = –0.1375).

To further assess the correlation of KDM6A mutation with TILs in the BC microenvironment, the CIBERSORT algorithm was used. The composition of 22 TILs in each sample varied significantly (Fig. 3B). Moreover, naïve B cells were more enriched in the KDM6A mutation group, whereas M2 macrophages and resting mast cells were more enriched in the KDM6A wild-type group (Fig. 3C). Correlation analysis showed that memory-activated CD4+ T cells were positively correlated with CD8+ T cells, and also had a positive association with the number of M1 macrophages. In turn, resting macrophages were positively correlated with levels of activated natural killer (NK) cells. However, activated NK cells were negatively correlated with activated mast cells (Fig. 3D).

3.5. Enrichment pathway analysis of KDM6A mutation

We further explored the relationship between KDM6A mutation and the immune response. GSEA revealed that the intestinal immune network for IgA production, the chemokine signalling pathway, natural killer cell-mediated cytotoxicity, the B cell receptor signalling pathway, the T cell receptor signalling pathway, the Fc epsilon Ri signalling pathway, Fc gamma R-mediated phagocytosis, primary immunodeficiency, and the Toll-like receptor signal pathway was significantly downregulated in the KDM6A mutation group (Figure 4A–I). These results implied that KDM6A mutation downregulates signalling pathways involved in the immune system in BC.

Dysfunction in demethylation occurs frequently in cancer cells, which can destroy the chromatin configuration and disrupt normal transcriptional processes. KDM6A is a specific H3K27me3 demethylase, and inactivating mutations in KDM6A have been frequently detected in BC [1]. KDM6A was suggested to act as a tumour suppressor in several cancers [10]. Other studies reported abnormal levels of trimethylated H3K27 (H3K27me3) in some cancers, which correlated with a poor prognosis [11-13], suggesting that demethylases are involved in both oncogenesis and tumour progression. Several recent studies identified inactivating mutations of KDM6A in different cancer tissues, along with decreased expression levels of KDM6A in cancer compared with those in normal tissues, further supporting that KDM6A is a tumour suppressor [14-17]. Mutations in or a decreased expression level of KDM6A was also associated with a poor prognosis of female BC patients [18].

Our present analysis of the somatic mutation landscape data of BC samples from the TCGA dataset (412 cases) and ICGC dataset (101 cases) confirmed that KDM6A was frequently mutated in both cohorts, in line with previous studies [6, 19]. Moreover, KDM6A mutation was correlated with higher overall gene mutation counts, but was not associated with clinical prognosis.

Importantly, we identified that KDM6A mutation in BC was negatively associated with signalling pathways involved in the immune response. We observed reduced infiltration of lymphocytes in KDM6A-mutated tissues, including neutrophils, macrophages, and CD8+ T cells, which is consistent with previous reports showing that these immune cells play key roles in the tumour microenvironment and suppress the immune response. A reduced number of CD8+ T cells in the tumour indicated a worse prognosis for patients in a previous study [20]. GSEA of Kyoto Encyclopedia of Genes and Genomes pathways between gene sets of BC samples with mutated and wild-type KDM6A demonstrated that the signalling pathways of cell-adhesion molecules, ECM receptor interaction, and focal adhesion were also suppressed byKDM6A mutation. These findings support the need for further in-depth study of the possible role of KDM6A in regulation of cell adhesion and morphology in BC [21]. KDM6A deficiency has been suggested to activate pathways of chemokines and cytokines, increase M2 macrophage polarization, increase cancer stem cell abundance, and act synergistically with p53 haploinsufficiency [22]. Given the key function of KDM6A in regulating CD4+ T cells[23],concluded that KDM6Alikely regulates multiple immune response genes in autoimmune disease susceptibility.

This study identified that KDM6A was frequently mutated in BC, and KDM6A mutation was correlated with a higher overall gene mutation status. Furthermore, KDM6A mutation was shown to negatively regulate the signalling pathways of the immune system and to suppress antitumor immunity. These findings suggest KDM6A as a latent gene whose mutation could be considered as a predictive biomarker for the immune response, thereby facilitating the development of new strategies for immunotherapy and treatment monitoring in BC patients.

KDM6A: Lysine-specific demethylase 6A; BC: bladder cancer; TCGA: The Cancer Genome Atlas; ICGC: International Cancer Genome Consortium; GSEA: Gene Set Enrichment Analysis; TNM: tumour-node-metastasis; TILs: tumour-infiltrating lymphocytes; TIMER:Tumor Immune Estimation Resource; TP53: tumor protein p53; CSMD3: CUB And Sushi Multiple Domains 3; MUC16: mucin 16; STAG2: Stromal antigen2; PIK3CA: phosphoinositide-3-kinase, catalytic, alpha; ARID1A: AT-rich interactive domain containing protein 1A; RB1: retinoblastoma1; EP300: E1A binding protein p300; ERBB2: erb-b2 receptor tyrosine kinase 2; ERBB3: erb-b2 receptor tyrosine kinase 2; FGFR3: fibroblast growth factor receptor 3; NK cells: natural killer cells.

ACKNOWLEDGEMENTS

Not applicable.

AUTHORS’ CONTRIBUTIONS

Chen X., Lin X.and Zhang Z.conceived of the study, participated in its design and coordinated and drafted the manuscript. Chen X. and Lin X.performed the statistical analyses. Pang G., Deng J., Xie Q.and Zhang Z.gave critical advice and participated in manuscript revising. All authors read and approved the final manuscript.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

The data is publicly available therefore no Ethics approval is required.

FUNDING

None.

AVAILABILITY OF DATA AND MATERIALS

The datasets used during the present study are available from the

corresponding author upon reasonable request.Data were obtained from

The Cancer Genome Atlas (TCGA; https://portal.gdc.cancer.gov), International Cancer Genome Consortium (ICGC; https://dcc.icgc.org/) andTIMER (http://timer.cistrome.org/).

CONFLICT OF INTEREST

None declared.

CONSENT FOR PUBLICATION

Not applicable.

DATAAVAILABLILITYSTATEMENT

The data generated and analysed during the current study are available from the corresponding author on reasonable request. Publicdata and data repositories are referenced within the manuscript.

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You are reading this latest preprint version

Significance of KDM6A Mutation in Bladder Cancer Immune Escape

Status:

Version 1

Abstract

Figures

Introduction

Methods

2.1. Datasets

2.2. Data analysis

Results

3.1. KDM6A mutation is frequent in BC

3.2. KDM6A mutation is associated with higher mutation counts

3.3. KDM6A mutation is associated with reduced KDM6Aexpression levels and poor prognosis in BC

3.4. KDM6A mutation is significantly correlated with tumour-infiltrating lymphocytes (TILs) in the tumour microenvironment

3.5. Enrichment pathway analysis of KDM6A mutation

Discussion

Conclusions

Abbreviations

Declarations

References

Tables

Supplementary Files

Status:

Version 1