According to the latest cancer statistics, breast cancer (BRCA) surpassed lung cancer as the leading cancer in the world, with 2.26 million new cases reported in 2020. BRCA is thus a serious threat to human health (Sung et al., 2021). Owing to the development of medical technology and genomics, malignant tumor has been gradually revealed to be caused by genetic mutation. The diagnosis and treatment of cancer have evolved from empirical medicine and evidence-based medicine to precision medicine (Liu et al., 2019). Despite receiving highly effective and comprehensive treatment, some patients develop drug resistance or recurrence and metastasis, which markedly affect their prognosis and survival. The molecular characteristic of BRCA determine the therapeutic response and prognosis (Dai et al., 2015). The innovation of genetic testing technology has paved the way for identifying new cancer biomarkers and therapeutic targets.

Ribosomes are important organelles responsible for human protein synthesis; however, their assembly and maturation are highly complex. Based on accumulating evidence, abnormalities in ribosomal biosynthesis can affect tumorigenesis (Pelletier et al., 2018). In addition to ribosomal RNA (rRNA) and ribosomal protein, more than 300 ribosomal assembly factors perform important regulatory function in ribosomal biosynthesis (de la Cruz et al., 2018). It is reported that ribosome export 7 (IRix7), ribosome export/assembly 1 (Rea1) and diazaborine resistance gene 1 (Drg1), which belong to the ATPase associated with diverse activities (AAA-ATPase) family, are associated with the formation of the large 60S subunit (Kressler et al., 2012).

MDN1, also known as Midasin AAA-ATPase 1, KIAA0301, and Real1, is a protein coding gene. MDN1 provides energy for the efficient removal of specific assembly factors from pre-60S particles after they have fulfilled their function (Prattes et al., 2019). Only a few studies have reported that the MDN1 mutation exists in carcinomas and may be associated with tumor oncogenesis and prognosis. For example, Liu et al. successfully identified two distinct mutation signature clusters (MSCs) featured by dominant somatic copy number alterations and massive mutations, respectively. MSC-1 displayed higher immunogenicity and immune infiltration, and among them, MDN1 was identified as a novel driver gene that could effectively distinguish different subtypes (Zaoqu Liu et al., 2021). Besides, Lobl et al. conducted a comprehensive synthesis and analysis of cutaneous squamous cell carcinoma (SCC) sequencing studies with individual clinical data. Based on their findings, MDN1 was found to be one of the mutations in more than 25% of SCCs, which indicates a potential applicability in targeted therapy. (Lobl et al., 2021). However, the relationship between MDN1 mutation and BRCA has not been reported to date. Therefore, the diagnostic and prognostic value of MDN1 mutation in BRCA require further assessment.

In this study, we aimed to analyze the mutation signature of MDN1 in BRCA based on TCGA and the influence of MDN1 mutation on TMB and immune infiltrating cells. MDN1 mutation-related biological pathways involved in BRCA were also detected using gene set enrichment analysis (GSEA), and a multivariate logistic regression based on the MDN1 mutation and clinicopathological parameters was established as a nomogram to predict BRCA prognosis.

BRCA is the most common malignant tumor and the second leading cause of death due to cancer in women, thereby posing a serious threat to human health (Sung et al., 2021). The role of somatic mutations in cancer development and progression has been confirmed with the innovation of genetic testing technology, which brings new hope for identifying novel cancer markers and therapeutic targets. To our knowledge, the function and potential prognostic impact of MDN1 mutation on BRCA have not been investigated.

The protein encoded by the MDN1 gene is one of the largest proteins in the body with a relative molecular mass of 632,000 and containing 5,596 amino acid residues. It is a member of the AAA-ATPases family, which plays an important role in pre-60S ribosome maturation. Abnormalities in ribosome biosynthesis, including ribosomal RNA instability, protein mutations, and assembly factor abnormities, might induce tumorigenesis and progression. As a result, these factors have potential applicability as therapeutic targets for a variety of tumors, such as colorectal cancer (Tsoi et al., 2017), cervical cancer (Zhou et al., 2016), and thyroid cancer (Guimaraes and Zavolan, 2016). As an important protein enzyme affecting the process of ribosome synthesis, MDN1 has been found to be mutated in a few cancers, such as colorectal cancer (Zaoqu Liu et al., 2021) and SCC of the skin (Lobl et al., 2021), and may act as a driver gene to promote the occurrence and development of cancer. In BRCA, MDN1 has a high mutation rate of 2.77%. However, the dysfunction of MDN1, especially its mutation, has rarely been studied in BRCA. Our result provided a foundation for the research of MDN1 mutation in BRCA for the first time.

First, we comprehensively analyzed the mutational signature of MDN1 in BRCA, in which missense mutation constitutes the main mutation type. Besides, we observed that the CNV levels of several genes were changed significantly between the MDN1 MUT and MDN1 WT groups. In addition, based on survival analysis, patients in the MDN1 MUT group were found to have a worse prognosis. Accordingly, we sought to further explore the underlying mechanisms.

Somatic mutations might affect the expression of downstream genes, leading to alterations in the transcriptional network that affect different phenotypes and functions. To determine the effect of MDN1 mutations on patients with BRCA, we performed transcriptome analysis and found that 910 genes were significantly upregulated and 77 genes were significantly downregulated in the MDN1 MUT group in TCGA-BRCA dataset. We respectively selected the top 25 upregulated and downregulated genes to plot a heatmap.

Proline dehydrogenase (PRODH) overexpression was identified in the MDN1 MUT group relative to the WT group. PRODH can catalyze the first step of proline catabolism and has diverse functional roles in regulating pathophysiological processes, including apoptosis, autophagy, cell senescence, and tumorigenesis. In many types of cancer, PRODH acts as oncogene to promote cancer occurrence and progression (Yating Liu et al., 2021). Elia et al. found that the upregulation of PRODH supported 3D growth and metastasis, generated ATP, and acted as a drug target in MCF10A BRCA cells (Elia et al., 2017). These findings confirmed the potential role of PRODH expression as a prognostic marker for BRCA. However, the relationship between MDN1 and PRODH remains to be evaluated. Another upregulated gene, PHLDA2, belongs to the pleckstrin homology like domain, family A (PHLDA) gene family and encodes protein that can inhibit AKT signaling through phosphoinositol binding competition. Mangone et al. found that breast tumor samples showed higher levels of PHLDA2 transcripts (p < 0.0001) than normal tissue samples. Besides, PHLDA2 could act as a prognostic marker and predict a response to endocrine therapy in BRCA (R Mangone et al., 2020). Moon et al. also concluded that the expression of PHLDA was high in triple negative BRCA cells (Moon et al., 2015).

Some members of the ABC family are downregulated in MDN1 mutation patients, such as ABCG2, ABCB5, ABCA9, and ABCA10. These proteins are alternatively referred to as BRCA resistance proteins and function as a xenobiotic transporter, which may play a major role in multi-drug resistance (Hlavac et al., 2020). Similarly, SNPH is downregulated, which suppresses metastasis by inhibiting the movement of mitochondria in cancer cells (Seo et al., 2018). These results indicated that the MDN1 mutation may influence BRCA drug resistance and induce tumor progression.

Concerning the DEGs, GSEA revealed samples with MDN1 mutation mainly enriched in “retinol metabolism, drug metabolism cytochrome P450, glucuronidation, miscellaneous transport and binding events pathways.” Among them, glucuronidation is the major pathway in phase II metabolism and accounts for approximately 35% of drug conjugation (Wells et al., 2004). Glucuronidation conjugation utilizes UDP-glucuronosyltransferases to catalyze a wide range of diverse endogenous and xenobiotic compounds. Zhou et al. found that UGT contributes to estrogen elimination, and its glucuronidation capacity influences the estrogen signaling pathway and pathogenesis of BRCA (Zhou et al., 2017). Similarly, the cytochrome P450 (P450) enzymes constitute the predominant enzyme system involved in human drug metabolism (Tracy et al., 2016), which plays a multi-faceted role in contributing to carcinogenesis, tumor growth, invasion and metastasis, especially in BRCA (Luo et al., 2021).

Whether MDN1 mutations promote BRCA development by affecting the enrichment of drug metabolism-related pathways is unclear. Further in-depth analysis using a drug-gene interaction database revealed that multiple drug classes correlated with MDN1 gene mutation, especially with the druggable genome. Oncogenic signaling pathways indicated that RTK-Ras pathway, NOTCH pathway, and WNT pathway were significantly enriched in patients with BRCA, suggesting that patients with MDN1 mutation had potential targets for prevention and therapy.

Immunotherapy based on immune checkpoint inhibitors has significantly improved the survival prognosis of patients with malignancies; however, BRCA is mostly considered to be a weakly immunogenic tumor type, which limits the effectiveness of immunotherapy. As a result, identifying patients that can benefit from immunotherapy and discovering reliable biomarkers are important in devising novel treatment strategies. (Savas et al., 2016). We found that patients in the MDN1 MUT groups had significantly increased levels of TMB compared to those in the MDN1 WT group. TMB, which generally refers to the number of nonsynonymous mutations per megabase pair (Mb) of somatic cells within a specific genomic region, has been shown to predict the effectiveness of immunotherapy for a variety of tumors (Chalmers et al., 2017). Patients with MDN1 mutations had potentially higher therapeutic benefit with immunotherapy. The analysis of immune components in the tumor microenvironment could provide insight into tumorigenesis and progression. For example, in the BRCA tumor microenvironment, tumor infiltrating lymphocyte (TIL) can respond to the treatment effect and prognosis of multiple BRCA subtypes and serve as a potential biomarker (Denkert et al., 2018).

We then analyzed the relationship between MDN1 gene mutation and immune cell infiltration in the BRCA microenvironment. Although some immune cells showed differences in different subgroups, none of the 22 immune cells showed significant differences. We further analyzed the expression of HLA family genes in the MDN1 MUT group versus the MDN1 WT group. MHC class I genes, such as HLA-A, HLA-B, and HLA-C, were not statistically different in the mutant group versus the wild group; however, significant differences were found in the expression of MHC class II genes, such as HLA-DQA2. HLA-DQA2 was found to play a central role in the peptide loading of MHC class II molecules by aiding the release of the CLIP molecule from the peptide binding site, which influences the MHC class II receptor activity (Lenormand et al., 2012). In conclusion, MDN1 mutation could increase TMB levels and promote MHC-II activity, thereby potentially enhancing the immunotherapeutic effects.

To further explore the impact of MDN1 gene mutation on the prognosis of TCGA-BRCA patients, prognostic models were established using univariate and multivariate Cox regression analysis in combination with the clinicopathological characteristics of patients. As a result, age, TNM stage, and MDN1 mutation-type were identified as risk factors for patients with BRCA (HR > 1). Based on the results, a nomogram was developed to predict the 1-, 3-, and 5- survival probabilities of patients with BRCA. Further, a ROC curve was used to determine the predictive validity of the nomogram to prospective outcomes, which revealed AUC values of 0.794, 0.679, and 0.635 for 1-year, 3-years, and 5-years OS, respectively.

Although our approach improved the understanding of the relationship between MDN1 mutation and BRCA, this study has some limitations. First, all data were obtained using online databases, although similar sample setting from TCGA database can be found in many studies (Wang et al., 2020; Zhang and Zhang, 2020; <u>Li et al., 2021), only one database was adopted as data source may cause sample bias. Second, despite one tumor “TCGA-AC-A23H” had several more mutations relative to the rest of the samples in the MDN1-MUT cohort. The current limited number of sample cases may also cause bias in the results if the individual mutation is excluded. In addition, other similar studies have included the entire sample and did not perform sample exclusion (Zhang and Zhang, 2020; Guo et al., 2021). Third, even several of the recently published high-scoring articles were analyzed based exclusively on the TCGA or/and GEO public databases, and all of these studies have implications for future clinical research (Hong et al., 2020; Yi et al., 2020; Guo et al., 2021). The functional pathway analysis of MDN1 mutations needs to be verified in vitro and in vivo. Besides, further experimental verifications are necessary to elucidate the detailed and direct mechanisms of the correlation between MDN1 mutation and BRCA.

In conclusion, through the analysis of somatic mutation data in TCGA database, we found that MDN1 was a high frequency mutation gene in BRCA. Further, DEGs and pathway enrichment analysis revealed that MDN1 mutation affected multiple biological functions, including drug metabolism-related genes. MDN1 mutation was closely correlated with high TMB and MHC class II receptor activity, suggesting that it might be a potential therapeutic target and could contribute to the development of immunotherapy for BRCA. Furthermore, MDN1 mutation was an unfavorable prognostic biomarker and revealed the potential mechanism of MDN1 mutation on BRCA.