ALAD mRNA Expression Is Down-regulated in Ten Solid Tumor Types
To determine the potential role of ALAD in a variety of human cancers, we first verified the expression level of ALAD mRNA using the TCGA database and GEPIA analysis. The results showed that ALAD mRNA expression was significantly down-regulated in breast invasive carcinoma (BRCA), esophageal cancer (ESCA), renal clear cell carcinoma (KIRC), renal papillary cell carcinoma (KIRP), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), thymoma (THYM) and endometrial carcinoma (UCEC). In liver hepatocellular carcinoma (LIHC) and sarcoma (SARC), although the results were not statistically significant, the average expression level of ALAD mRNA in cancer tissues was lower than that in normal tissues (Figure 1).
ALAD mRNA Expression Is Low in BRCA, LUAD and LUSC
By downloading the microarray results related to breast cancer and lung cancer from the NCBI/GEO public database, we found ALAD mRNA expression was obviously down-regulated in cancer tissues compared to adjacent normal tissues in the GSE5364 (ANT=13; T=83), the GES134359 (ANT=12; T=74), the GSE33532 (ANT=20; T=80) and the GSE56044 (ANT=12; T=124) (Figure 2). Moreover, by analysis in UALCAN website, we found the mRNA expression of ALAD was higher in the normal samples (BRCA group n=114; LUAD group n=59; LUSC group n=52) than in the cancer samples (BRCA group n=1097; LUAD group n=515; LUSC group n=503; Figure 3A, 3H, 3O). Further, subgroup analysis showed that ALAD was also significantly down-regulated in different subgroups of BRCA, LUAD, and LUSC, including gender, age, and race subgroups (Figure 3B-D, 3I-K, and 3P-R). Regarding to cancer stages, whether in BRAC, LUAD or in LUSC, we found that ALAD mRNA expression in stage 1-4 was yet lower than that in normal tissues (Figure 3E, 3L, and 3S). In view of lymph node metastasis - N staging, ALAD mRNA expression was also significantly lower in cancer tissues with N0-N3 stages than that in normal tissues (Figure 3F, 3M, and 3T). In addition, ALAD mRNA expression was significantly decreased in BRCA and LUAD tumors with TP53 mutation than that without TP53 mutation. Futhermore, compared with normal tissues, the expression levels of TP53 mutation and without TP53 mutation were lower in BRCA, LUAD and LUSC (Figure 3G, 3N, and 3U). Through all the above analysis, we suggest that ALAD may play a tumor inhibitory role in the progression of breast cancer and lung cancer.
ALAD Protein Expression Is Down-regulated in Ten Solid Tumor Types
The protein expression of ALAD was analyzed using the HPA database in different tumor types. The results showed that ALAD was low expressed and/or absent in breast cancer (91%), renal cancer (91%), lung cancer (89%), endometrial cancer (83%), urothelial cancer (100%), ovarian cancer (91%), gastric cancer (100%), prostate cancer (91%), cervical cancer (100%) and colorectal cancer (83%). However, there are exceptions in liver cancer (42%) and thyroid cancer (50%) whose ALAD protein expression level are not over 50% (Figure 4A-B). These results suggest ALAD may play a tumor inhibitory role in most tumors.
ALAD High Expression Is Associated with Better Overall Survival
To further study the prognostic role of ALAD in different human cancers, KM plotter database was used to evaluate the overall survival time which was calculated according to the high and low expression of ALAD. The results showed that the overall survival time is longer in ALAD high expression group than in ALAD low expression group in BRCA (HR=0.79; p=0.029), ESCA (HR=0.37; p=0.0058), KIRC (HR=0.42; p=5.5e-07), KIRP (HR=0.42; p=0.0041), LIHC (HR=0.51; p=8.8e-05), LUAD (HR=0.67; p=6e-04), LUSC (HR=0.45; p=0.029), SARC (HR=0.56; p=0.0035), THYM (HR=0.12; p=0.016) and UCEC (HR=0.53；p=0.003; Figure 5), and statistical analysis suggested that the p value is less than 0.05 in all the above ten tumors. These results indicate that ALAD may act as a tumor suppressor and may be a valuable biomarker for predicting prognosis of patients with BRCA, ESCA, KIRC, KIRP, LIHC, LUAD, LUSC, SARC, THYM and UCEC.
ALAD Enrichment Analysis of BRCA, LUAD and LUSC
From LinkedOmics website and based on the Spearman test, we screened the differentially expressed genes (DEGs) changed with ALAD expression level in BRCA, LUAD and LUSC (Figure 6A, 6D, 6G). The first 50 positively related genes and the first 50 negatively related genes in different tumors were shown in the form of heat map (Figure 6B-C, 6E-F, 6H-I). Moreover, 561 DEGs were screened by Spearman test more than 0.2 in the BRCA dataset; 1401 DEGs were screened by Spearman test more than 0.4 in LUAD dataset; 1910 DEGs were screened by Spearman test more than 0.4 in LUSC dataset. Among all the above DEGs, 542 DEGs overlap each other (Figure 7A). GO and KEGG enrichment analysis on the DAVID website obtains the relevant data about Biological Process (BP), Cell Composition (CC), Molecular Function (MF) and KEGG pathway. The results are visualized by bioinformatics online tools. For the BP, the negative regulation of transcription from RNA polymerase II promoter, positive regulation of cell proliferation and cell proliferation were the top three relevant pathways (Figure 7B). For the CC, the potential target genes were predominantly enriched in the cytoplasm, cytoso and membrane (Figure 7C). For the MF, the mainly significantly involved items were protein binding (Figure 7D). Furthermore, KEGG analysis showed that cancer pathways, proteoglycans in cancer, Wnt signaling pathways and pathways regulating pluripotency of stem cells were the main pathways related to ALAD regulation (Figure 7E). Collectively, the above results show that ALAD may be involved in many regulation processes in tumors and may be a hopeful therapy target. More studies are needed to further investigate its functions and regulation mechanism.
ALAD Expression Is Negatively Correlated with Cell Cycle and PI3K/AKT/mTOR Signaling Pathway
To more deeply explore the regulation mechanism of ALAD, we conducted GSEA analysis and the results revealed that ALAD expression negatively correlated with the cell cycle characteristics (HALLMARK_G2M_CHECKPOINT) in the publicly available GEO breast cancer database (NCBI/GEO/GES5364, n=341) and lung cancer database (NCBI/GEO/GSE33532, n=100) (Figure 8A). To understand the mechanism of ALAD-mediated cell cycle in breast cancer and lung cancer, we used the same GEO databases to study the relationship between ALAD expression levels and the cancer signaling pathways which closely related to cell cycle regulation, and we found that ALAD expression was negatively correlated with the PI3K/AKT/mTOR pathway (HALLMARK_PI3K_AKT_MTOR_SIGNALING) (Figure 8B). The above results suggest that ALAD may inhibit the cell cycle process and the PI3K/AKT/mTOR signaling pathway and consequently suppress the cancer cells growth and proliferation.