Tubulin alpha-1b chain was identified as a prognosis and immune biomarker in pan-cancer combing with experimental validation in breast cancer

Abstract

The α-tubulin subtype, Tubulin α-1b chain (TUBA1B), has been shown to influence immune cell infiltration, cancer growth, and survival across various malignancies. However, a comprehensive study has not yet been undertaken examining the immunological and predictive effects of TUBA1B in a pan-carcinoma context. Using data from TCGA, GEO, and other databases, we analyzed TUBA1B expression across various carcinoma types using transcriptional profiling, prognostic implications, genetic and epigenetic alterations, methylation patterns, and immunological significance. To validate our findings, we conducted Western blot analysis to assess TUBA1B protein levels in matched breast cancer tissue samples and performed CCK-8 proliferation assay, flow cytometry, transwell invasion, and migration assays to comprehensively examine the functional impact of TUBA1B on breast cancer cells. Our pan-cancer analysis found TUBA1B upregulation across most tumor types, with varying expression patterns in distinct immune and molecular subtypes. High TUBA1B expression was an independent risk factor and associated with poor prognoses in several cancers, including BRCA, KICH, LGG, LUAD, and MESO. TUBA1B also demonstrates moderate to high diagnostic accuracy in most tumor types. Increased m6A methylation levels were observed in the TUBA1B gene, while its promoter region displayed low methylation levels. TUBA1B's expression impacted some cancers by elevating tumor mutation burden, microsatellite instability, neoantigen formation, immune cell infiltration, and the modulation of immune checkpoints. Functional enrichment analysis highlights TUBA1B's involvement in important cellular processes such as the cell cycle, p53 signaling, cell senescence, programmed cell death, and the regulation of immune-related pathways. Moreover, our study reveals higher TUBA1B protein expression in breast cancer tissues compared to adjacent tissues. In vitro experiments confirm that TUBA1B deletion reduces breast cancer cell proliferation, invasion, and migration while increasing apoptosis. In conclusion, our study suggests that TUBA1B could potentially serve as a diagnostic marker for predicting cancer immunological profiles and survival outcomes and shed light on the expression and role of TUBA1B in breast cancer, providing a solid foundation for considering it as a promising therapeutic target for breast cancer patient treatment.

Keywords: Methylation; Pan-cancer; Prognosis; TUBA1B; Tumor immunity.

Figure 1

TUBA1B expression variations across 33 cancer types. (A) Comparison of TUBA1B mRNA expression between TCGA tumor samples and corresponding normal tissues. (B) Differences in TUBA1B mRNA expression between tumor and normal tissues, integrating data from TCGA and GTEx datasets. (C) TUBA1B mRNA expression in TCGA tumor samples compared to paired normal tissues. Analysis of TUBA1B expression differences was conducted using GEO datasets for specific cancers: (D) BRCA (GSE42568), (E) CESC (GSE66791), (F) COAD (GSE20916), (G) HNSC (GSE66791), (H) KIRC (GSE105261), (I) LIHC (GSE121248), (J) PAAD (GSE15471), and (K) STAD (GSE54129). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

Correlation between TUBA1B expression and tumor stages in different cancers, including ACC (A), LIHC (B), LUAD (C), and COAD (D). (E) Relationships between immune subtypes and TUBA1B expression across various TCGA tumors, encompassing BLCA, BRCA, COAD, ESAD, HNSC, KIRP, LGG, LIHC, LUAD, LUSC, OV, PAAD, PCPG, PRAD, SARC, STAD, THCA, and UCEC. (F) Associations between molecular subtypes and TUBA1B expression in diverse TCGA tumors, comprising BRCA, COAD, ESCA, HNSC, KIRP, LGG, LUSC, OV, PCPG, PRAD, STAD, and UCEC.

Figure 3

Survival analysis of TUBA1B in various GEO datasets from the PrognoScan database.

Figure 4

Association between TUBA1B expression and prognosis in cancer patients. (A) Association between TUBA1B expression and overall survival (os) in cancer patients. (B) Association between TUBA1B expression and disease-specific survival (DSS) in cancer patients. (C) Association between TUBA1B expression and progression-free survival (PFS) in cancer patients. (D) The Venn diagram shows the intersection of OS, DSS, and PFS for different cancers. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

Receiver operating characteristic (ROC) curve for TUBA1B expression in pan-cancer. (A) TUBA1B expression in cancers with good diagnostic value (AUC > 0.9), including CESC, CHOL, COAD, GBM, LAML, OV, PAAD, STAD, and TGCT. (B) TUBA1B expression in cancers with some diagnostic value (AUC > 0.9), including ACC, BLCA, BRCA, DLBC, ESCA, HNSC, KICH, KIRC, KIRP, LGG, LIHC, LUSC, READ, SKCM, THYM, UCEC, and UCS.

Figure 6

Nomograms and calibration curves predicting patient OS in four cancers. Nomograms for BRCA (A), KICH (B), LGG (C), and LUAD (D). Calibration curves for BRCA (E), KICH (F), LGG (G), and LUAD (H). The horizontal and vertical coordinates represent the model-predicted and observed survival probability, respectively. A closer alignment with the ideal line indicates better model performance.

Figure 7

Mutated features of TUBA1B in various tumors. (A) Overview of TUBA1B expression alterations in different tumors. (B) Frequency distribution of mutation types. (C) Mutation sites in the TUBA1B amino acid sequences. (D) Visualization of select TUBA1B mutations on the 3D protein structure. (E) Association between CNV in TUBA1B and cancer patient prognosis.

Figure 8

Epigenetic methylation analysis of TUBA1B. (A) Relationship between TUBA1B mRNA expression and m6A methylation regulators in various cancers. (B) Differential promoter methylation levels of TUBA1B in normal tissues and tumors based on UALCAN data (β values).

Figure 9

Association of TUBA1B expression with TMB, MSI, NEO, TME, and immune checkpoints in 33 cancer types. (A) Correlation between TUBA1B expression and TMB, MSI, and NEO in 33 cancers. (B) Correlation between TUBA1B expression and StromalScore, ImmuneScore, and ESTIMATEScore in 33 cancers. (C) Correlation between TUBA1B expression and immune checkpoint expression in 33 cancers. (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 10

Relationships between immune cell infiltration levels and TUBA1B expression in pan-cancer. (A) Correlation between TUBA1B expression and immune infiltration using the ssGSEA algorithm. (B) Correlation analysis of TUBA1B expression with immune infiltration of CAF cells based on the Timer2.0 database. Scatter plots include ACC, KICH, KIRP, MESO, PAAD, THCA, THYM, and UVM. (*p < 0.05, **p < 0.01).

Figure 11

TUBA1B-related genes, interacting proteins, and functional enrichment analysis. (A) Protein–protein interaction (PPI) network for TUBA1B. (B) Venn diagram showing the intersection of TUBA1B-binding and interacting genes after selection. (C) and (D) depict the correlation between TUBA1B and six associated genes in 33 cancers. GO analysis includes biological processes (E), cellular components (F), molecular functions (G), and KEGG pathways (H).

Figure 12

GSEA Functional Enrichment Analysis of TUBA1B in Eight Cancers. In ACC (A), BLCA (B), BRCA (C), LGG (D), LGG (E), LIHC (F), LUAD (G), MESO (H), and SARC (I), the first 10 pathways are positively correlated with TUBA1B expression.

Figure 13

Expression levels of TUBA1B in paired breast cancer tissues, knockdown of TUBA1B inhibiting proliferation, migration, and invasion of breast cancer cells, and promoting apoptosis. (A, B) TUBA1B protein expression in breast cancer and adjacent tissues. Original blots are presented in Fig. S9. (C) Analysis of knockdown efficiency of TUBA1B transfected with siRNA-359, siRNA-535, and siRNA-1314 in MDA-MB-231 cell lines. (D) Knockdown efficiency of TUBA1B transfected with siRNA-359 in MDA-MB-468 cell line. (E) Detection of TUBA1B protein expression in MDA-MB-231 cell line transfected with siRNA-359 and siRNA-535. (F) Expression of TUBA1B protein in MDA-MB-231 cell line transfected with siRNA-1314. (G) Expression of TUBA1B protein in MDA-MB-468 cell line transfected with siRNA-359. (H, I) Knockdown of TUBA1B inhibited the proliferation of breast cancer cell lines. (J, K) Knockdown of TUBA1B promoted apoptosis of breast cancer cell lines. (L, M) Knockdown of TUBA1B inhibited the invasion of breast cancer cell lines. (N, O) Knockdown of TUBA1B inhibited the metastasis of breast cancer cell lines. (**p < 0.01, ***p < 0.001).