FLAD1 mRNA Expression Level in Pan-Cancer
FLAD1 expression is higher in bladder, brain, breast, cervical, colorectal, gastric, kidney, liver, lung, and ovarian cancers and leukemia, lymphoma, and myeloma (Fig. 1A). We found no cancer in which FLAD1 expression in tumor tissue was lower than in the normal tissue. Detailed information on FLAD1 mRNA expression in pan-cancer is summarized in Table S1. FLAD1 mRNA expression level was in the top 1% in BRCA.
The TIMER database was used to analyze single gene expression in pan-cancer in relation to FLAD1. The differential expression of FLAD1 between cancer and normal tissues is shown in Fig. 1B. BRCA had higher FLAD1 expression than the other cancer types.
FLAD1 Prognostic Value in Pan-Cancer
We used PrognoScan and Kaplan-Meier Plotter for a double validation exploring the relationship between FLAD1 expression and prognosis in pan-cancer. High FLAD1 expression influenced survival in seven cancers (lung, breast, skin, colorectal, head and neck, bladder, and blood) in the PrognoScan database. Increased FLAD1 expression had a protective role in colorectal and head and neck cancer, while it was negatively associated with survival prognosis in the other cancer types (breast, lung, skin, brain, and bladder). However, the Kaplan-Meier Plotter showed that prognosis was not associated with FLAD1 expression in bladder, colorectal, lung, and head and neck cancer. Data on skin and blood cancers was absent from this database. Detailed information on these cancer cohorts and FLAD1 expression in PrognoScan and Kaplan-Meier Plotter is presented in Table S2 and Figures S2 and S3. Using FLAD1 median expression level as a standard, analysis of the BRCA cohorts in PrognoScan and Kaplan-Meier Plotter showed that increased FLAD1 expression was associated with poor OS, RFS, and disease metastasis-free survival (DMFS), which presented in Fig. 2.
Increased FLAD1 expression is Correlated with poor BRCA Prognosis
After finding that high FLAD1 expression was associated with poor BRCA prognosis, we performed a subgroup analysis of FLAD1 expression and survival using the UALCAN database. The data were stratified by clinical characteristics, including sample type, sex, age, race, major molecular classification, nodal metastasis status, and clinical stage (Fig. 2A-G). Subgroup analysis showed that sex did not influence FLAD1 expression in tumor tissue (Fig. 2C). However, FLAD1 expression was higher in the 21–40-year-old group than in the other age groups analyzed (Fig. 2B). The FLAD1 expression in African-Americans was higher than in Caucasians (Fig. 2D). The luminal BRCA subtype had lower FLAD1 expression than other subtypes (Fig. 2E), and BRCA patients with lymph node metastases had significantly higher FLAD1 expression than normal tissues, suggesting that FLAD1 might affect BRCA patients by increasing lymph node metastasis (Fig. 2F).
We also performed a survival analysis based on different pathological characteristics in the Kaplan-Meier Plotter (Table S3). The results showed that triple-negative BRCA (P = 0.0348) had poorer RFS, and luminal A-type BRCA had poor RFS and OS (P = 0.0015 and P = 0.0044, respectively) when FLAD1 expression was high. In BRCA patients negative for lymph node metastasis, high FLAD1 expression was also associated with a poor OS, indicating that FLAD1 expression level detection could improve the prognostic accuracy in lymph node-negative patients.
FLAD1 Co-expression Networks in BRCA
We analyzed the FLAD1 co-expression model in the BRCA cohort in the LinkedOmics database to explore FLAD1 biological significance. FLAD1 was significantly correlated with 9 733 genes (black dots in Fig. 4A), of which 4,883 were negatively correlated 9 and 4 850 were positively correlated (red). Heat maps of the top 50 genes positively and negatively correlated with FLAD1 are presented in Figs. 4B and 4C, respectively. Information on the co-expressed genes is detailed in Table S4.
The KEGG pathway analysis results showed that the co-expressed genes were enriched in the proteasome, RNA degradation, aminoacyl-tRNA biosynthesis, RNA transport, and more (Fig. 4D). GO-BP analysis showed that enrichment was mainly in the tRNA metabolic process, mitochondrial gene expression, telomere organization, deoxyribonucleotide metabolic process, and others (Fig. 4E). These results indicated that FLAD1 might have an extensive effect on the genome replication, transcription, and repair systems.
We further analyzed the FLAD1 network regulators in BRCA, including kinases, miRNA, and transcription factors enrichment (Table 1). We found no significant kinase enrichment among genes co-expressed with FLAD1 (Table S5). Among the miRNAs co-expressed with FLAD1, AAGTCA, miR-422B, miR-422A; AAGCACA, miR218; CTCTAGA, miR526C, miR518F, miR526A; TCCCCAC, miR491; ATCTTGC, miR31 are significant miRNAs associated with FLAD1 and negatively expressed in BRCA (Table S6). The serum response factor (SRF) transcription factor family, including V$SRF_C, V$SRF_Q4, V$SRF_Q5_01, and V$SRF_Q6, was the most highly enriched among the genes co-expressed with FLAD1 (Table S7).
FLAD1 Expression is Associated with MMR in BRCA
We analyzed DNA methylation and repair system status to explore the relationship between FLAD1 expression and DNA genome activity. DNMTs are essential for genome integrity in humans. Promoter hypermethylation inhibits gene expression, while methylation of the gene body contributes to its expression 10, 11. Meanwhile, gene body DNA methylation in cancer tissues occurs more frequently than in the promoters 12, 13. The levels of DNMT1 (R = 0.21, P < 0.001), DNMT2 (R = 0.08, P = 0.013), DNMT3A (R = 0.32, P < 0.001), and DNMT3B (R = 0.33, P < 0.001) were all positively correlated with FLAD1 expression in BRCA (Fig. 5B).
Expression of the four MMR genes, MLH1, MSH2, MSH6, and PMS2, and epithelial cell adhesion molecule (EpCAM), a transmembrane glycoprotein, is crucial for cell migration and metastasis. The relationship between FLAD1 expression and the mutation status of the MMR genes was analyzed by the Sangerbox online tools. The results showed that higher mutation rates of MLH1 (P < 0.05), MSH2 (P < 0.0001), MSH6 (P < 0.0001), and EpCAM (P < 0.0001) were significantly correlated with FLAD1 expression in BRCA (Fig. 5A).
FLAD1 Expression is Associated with Immune Infiltration in BRCA
The expression status of MMR and DNMTs was reported to be correlated with tumor-infiltrating lymphocytes 3, 14, 15. TIL is an independent predictor that can guide survival and improve treatment in various cancers 16, 17. Our results showed that FLAD1 expression was negatively correlated with immune cell infiltration in many cancer types (Fig. 6A). Furthermore, high FLAD1 expression was positively associated with BRCA tumor infiltration purity (R = 0.131, P < 0.001), and negatively associated with infiltration by CD8 + T cells (R = -0.264, P < 0.001), neutrophils (R = -0.128, P < 0.001), macrophages (R = -0.255, P < 0.001), and DCs (R = -0.136, P < 0.001; Fig. 6B). No association was found between FLAD1 expression and the infiltrating levels of B cells and CD4 + T cells. These results suggest that FLAD1 might affect survival by influencing the immune infiltration into BRCA.
Immune Markers Correlation with FLAD1 Expression in BRCA
The results, adjusted by tumor purity in the TIMER database, indicated a strong correlation between FLAD1 expression and immune markers present on macrophages, monocytes, and NK cells in BRCA (Table 2). The NOS2 and TLR4 of M1 macrophages, MRC1 and PPARG of M2 macrophages, PARK2 and CSF1R of monocytes, CD7 and KIR2DL4 of NK cells, and CD1C, HLA-DPA1, and ICAM1 of DCs were specifically correlated with FLAD1 expression in BRCA (P < 0.001; Figure S3). Therefore, we used the GEPIA database to analyze these markers and their correlation with FLAD1 expression in BRCA tumor tissues. The results indicated that FLAD1 expression was mainly associated with macrophage infiltration into tumor tissue (Table 2). Furthermore, PPARG, a protective factor, was negatively correlated with FLAD1 expression. Treg gene marker FOXP3 and Exhausted T cell marker LAG3 were significantly and positively correlated with FLAD1 expression.