Cancer is a multifactorial disease influenced by a variety of factors, such as intracellular metabolism, immune evasion, apoptosis and inflammation [33]. A fascinating concept known as the 'tubulin code'[34] has emerged, highlighting the significance of molecular patterns formed by combinations of tubulin isotypes and PTMs. TUBB6, as a member of the β-tubulin gene family, can undergo multiple PTMs and is engaged in microtubule formation. Perturbation of TUBB6 expression can lead to the development of several cancers, including colorectal cancer, gastric cancer, breast cancer, glioblastoma and bladder cancer. Furthermore, abnormal TUBB6 expression has been associated with the prognosis and survival outcomes of these tumors [12, 14, 15, 17, 18, 35]. We used various databases, such as TCGA, GTEx, UALCAN, and cBioPortal, to comprehensively elucidate the expression of TUBB6 in 33 tumors and its correlation with prognosis, immune infiltration, gene mutation, DNA and RNA methylation, and drug sensitivity profiles. Our aim was to gain comprehensive insights into the multifaceted role of TUBB6 in the development of various tumors and to elucidate potential regulatory pathways associated with its activity. Given the limited number of studies on TUBB6, we conducted a systematic pancancer analysis to gain a comprehensive understanding of its role. First, the expression of TUBB6 was evaluated using the TCGA database, GTEX database and HPA database. The results showed that TUBB6 was markedly overexpressed in 13 types of cancer and notably underexpressed in 13 other types of cancer. Moreover, we identified the top three tissues with the highest TUBB6 expression in normal tissues, namely, the esophagus, blood vessel, and fallopian tube. Additionally, when comparing TUBB6 protein expression levels between cancer tissues and normal tissues, we found higher expression in LGG, LIHC, and TGCT, while lower expression was observed in COAD, KICH, and OV. These findings indicated that TUBB6 plays an essential role in different cancers, but its exact mechanisms remain poorly understood. Interestingly, in several tumors, TUBB6 showed higher expression in advanced clinical stages (higher N and T stages). However, in BLCA, COAD, LUAD, and STAD, TUBB6 expression was lower than that in normal tissues. Subsequently, prognostic analysis indicated that increased TUBB6 expression was linked to poorer OS, PFI, and DSS in ACC, KIRC, LGG, STAD and UVM. Thus, we chose COAD to verify our bioinformatics result. We transfected CRC cell lines HCT116 and HCT8 with TUBB6 overexpression vector or it negative control vector to investigate the function of TUBB6 in CRC cell lines. And the results revealed overexpression of TUBB6 significantly inhibited cell proliferation, and cycle. These controversial findings raise questions about the precise role of TUBB6 in tumor initiation or progression. By reviewing the literature, similar situations have been encountered by other researchers, underscoring the need for further exploration of TUBB6's potential functions due to the limited available research [36, 37].
Our findings also pointed to an important role of TUBB6 in the TME, which plays an influential role in affecting tumor growth, metastasis and response to treatment resistance and targeted therapy [38]. Considering the vital role of the TME in cancer, we investigated the relationship between TUBB6 expression and the TME across cancers. The results obtained through the ssGSEA and MCP-counter algorithms revealed a positive correlation between TUBB6 expression and the levels of immune cells and stromal cells across various cancers, with notable associations observed for NK cells, CD4+ T cells, CD8+ T cells, macrophages, and fibroblasts. One possible explanation for these findings lies in the role of CAFs in modulating immunotherapy resistance by separating immune cells from the tumor mass and releasing them to the tumor margin, while remodeled ECM constructed by the interaction of CAFs and cancer cells acts as a physical barrier to the infiltration of tumor-killing immune cells and the delivery of anticancer drugs to solid tumors [39, 40]. Interestingly, high TUBB6 expression was associated with worse prognosis, suggesting that TUBB6 may influence cancer development and prognosis by shaping the TME. Furthermore, based on ESTIMATE scores, we found that TUBB6 expression was positively correlated with stromal and immune scores in 23 cancers and negatively correlated in 4 cancers. This showed that TUBB6 may interact with tumor cells and immune cells within tumors, either promoting or antagonizing tumorigenesis. This discovery provides a novel perspective for the development of effective therapies. Currently, as immunotherapies, particularly immune checkpoint inhibitors (ICIs), have become central to cancer treatment, the levels of immune checkpoints are crucial indicators of tumor progression and can yield significant clinical benefits for patients [41–43]. Remarkably, our analysis demonstrated that TUBB6 expression positively correlates with several common immune checkpoints, including TGFB1, PD-L1, PD-1, CTLA4, HAVCR2, CD70, CD276, C10orf54, ICAM1, IL1B and LAG3, indicating that TUBB6 holds promise as a potent biomarker for predicting the response to immune checkpoint blockade therapy. These results highlight that TUBB6 expression is closely associated with immune cell infiltration, influences the prognosis of tumor patients, and could be a potential biomarker for immunotherapy response. Nevertheless, little is known about the role of TUBB6 in the human immune system. The role of TUBB6 in the TME remains a research gap worthy of further investigation.
TMB and MSI are classical biomarkers for predicting the response to immunotherapy, guiding the application of tumor immunotherapies. High TMB and MSI show a better response to immunotherapy [44, 45]. In our research, we made noteworthy observations regarding the correlation between TUBB6 expression and TMB and MSI in various tumors. We found significant correlations between TUBB6 expression and TMB in 13 different tumors, as well as with MSI in 6 tumors. Interestingly, TUBB6 expression in BRCA was positively correlated with TMB and MSI, suggesting that it may indicate a beneficial response to immunotherapy. Conversely, in ESCA, PAAD and STAD, TUBB6 expression displayed a negative correlation with TMB and MSI. This suggests that in these specific cancer types, high TUBB6 expression may not necessarily translate to a greater survival benefit after immunotherapy.
DNA methylation is a prevalent epigenetic event that holds significant importance in regulating gene expression, maintaining genomic stability, and contributing to tumorigenesis [46]. Dysregulation of DNA methylation promotes cellular oncogenesis by silencing tumor suppressor genes (TSGs) [47]. Using the UALCAN tool, we found that the DNA methylation levels of TUBB6 were upregulated in the majority of cancer types, suggesting potential implications for its role in tumorigenesis. Furthermore, RNA methylation has emerged as a critical factor in cancer cell proliferation, metastasis, and the immune response. RNA methylation-related proteins have emerged as promising targets for cancer therapy [48]. In addition, we discovered a correlation between TUBB6 expression and RNA methylation-regulated genes in a multitude of cancers. Thus, these findings suggest that TUBB6 may be involved in promoting tumorigenesis through its influence on DNA or RNA methylation processes.
We conducted a comprehensive pancancer functional analysis of TUBB6 using CancerSEA and GSEA. Through single-cell functional analysis, we identified a positive association between TUBB6 and the migration and invasion of several tumors, suggesting its potential involvement in tumor aggressiveness. Intriguingly, GSEA of TUBB6-related genes showed its participation in various crucial biological pathways across different cancers. These pathways included drug metabolism, cytokine–cytokine receptor interaction, ECM receptor interaction, hematopoietic cell lineage, inflammatory response, hypoxia, epithelial-mesenchymal transition, apoptosis, angiogenesis, oxidative phosphorylation and JAK/STAT, TGF-beta and KRAS signaling pathways. However, it is essential to emphasize that the functions attributed to TUBB6 based on these analyses require further validation in future research. In addition, the correlation between TUBB6 mRNA and anticancer drug sensitivity was examined through the CellMiner database, and the expression of TUBB6 was negatively correlated with the sensitivity to six drugs, including tamoxifen, vinorelbine, eribulin mesilate, PX-316, volasertib and barasertib. These drugs have the potential to prevent cancer progression. Therefore, we hypothesize that TUBB6 may play a role in the chemotherapy response and may be associated with chemotherapy resistance.
Indeed, while our study provided valuable insights into the role of TUBB6 in multiple cancers through comprehensive bioinformatics analyses, it is essential to acknowledge its limitations. The primary limitations is the depth of our study regarding TUBB6's role in cancer. While our pancancer analysis elucidated TUBB6's association with various aspects of tumorigenesis, such as gene expression, prognosis, immune infiltration, DNA and RNA methylation, and drug sensitivity, as well as validated in vitro experiments on COAD, the underlying molecular mechanisms remain unclear. Further research is necessary to delve deeper into the specific molecular pathways and signaling cascades through which TUBB6 exerts its effects in different cancer types. Understanding these mechanisms could lead to valuable insights and potentially identify TUBB6 as a therapeutic target for cancer treatment.