TUBB4A is a Novel Prognostic Biomarker and Therapeutic Target For Melanoma


 Background: Melanoma is one of the most malignant skin carcinomas with high metastatic potential. Increasing evidences have demonstrated that β-tubulin 4A (TUBB4A) play key role in development and progression of several types of human cancer. However, the potential function of TUBB4A in cutaneous melanoma remains to be determined. Methods: We first performed differential expression analysis between skin melanoma tissues and normal tissues from GEO and TCGA datasets, and then ran survival analysis to identify prognostic-related key genes. We went further to conduct the verification of in vitro biochemical experiments to explore the functional roles of key gene TUBB4A. Two small molecule inhibitors of TUBB4A, Dihydroartemisinin (DHA) and Nocodazole, were used to examined the effect on apoptosis and cell cycle progression of melanoma cells.Results: We found that TUBB4A is markedly correlated to the overall survival of cutaneous melanoma patients. The co-expressed genes with TUBB4A are enriched in the melanoma-related pathways and function. Then, the experimental results showed that knockdown of TUBB4A inhibit the proliferation and migration of A375 and B16-F10 melanoma cells. Moreover, two small molecular agents targeting TUBB4A, Dihydroartemisinin and Nonocodazole, dpromote the apoptosis of melanoma cells and made the tumor cells significantly blocked in G2/M stage. Conclusion: We suppose that TUBB4A may be a potential prognostic marker and therapeutic target of melanoma.

of carcinogenesis, and overexpression of β-tubulins is associated to the progression, prognosis and resistance to chemotherapeutic agents in different cancers [10]. For example, it is reported that βIII and βIV-tubulins are highly expressed in pancreatic tumors, compared to normal pancreas tissue [11]. Haider et al. also reported that the aberrant expression of the β-tubulins is commonly observed in metastatic cancer cells [12].
TUBB4A is a member of the β-tubulin family and encodes β-tubulin 4A. β-tubulin is involved in several intracellular processes like mitosis, motility and transport [13]. Previous studies have shown that GLUT1 and its binding partner TUBB4 as potentially druggable targets in glioblastoma multiforme. Silencing TUBB4 reduces glioblastoma stem cell tumorsphere formation, self-renewal and proliferation in vitro [14]. The down-regulation of βIVa-tubulin in lung cancer cells increases their sensitivity to tubulin-binding agents, such as vinorelbine, vincristine and paclitaxel [15]. Ross et al. explored the impact of hypoxia on the proteome, and found that TUBB4A differential expression primarily involved in structural and binding processes of the prostate cancer [16]. Atjanasuppat et al. also reported that ERK signaling mediates upregulation of βIVa-tubulin gene and confers paclitaxel resistance in H460 oating lung cancer cells [17]. However, the potential function of TUBB4A has not been reported in melanoma to date.
In this study, we conducted differential expression analysis on two large SKCM cohorts, and found that TUBB4A is signi cantly up-regulated in SKCM tissues compared to normal tissues. Signi cant correlation between TUBB4A expression and patient overall survival was observed. Moreover, our in vitro experiments showed that inhibition of TUBB4A reduce the migration and proliferation of melanoma cells. Two drugs reported to target TUBB4A in DrugBank, Dihydroartemisinin and Nocodazole, were evaluated by ow cytometry whether they promote the apoptosis and block cell cycle of SKCM cells. The assay results showed that both drugs signi cantly induced cell apoptosis and G2/M cell cycle arrest in the group received treatment of Dihydroartemisinin and Nocodazole. In summary, we would draw the conclusion that TUBB4A may be a potential prognostic marker and therapeutic target of melanoma.

Gene expressions and differential expression analysis
The gene expression pro les of SKCMs were downloaded from GEO (https://www.ncbi.nlm.nih.gov/geo/) and TCGA (http://cancergenome.nih.gov/) database [18]. For GEO, we selected human melanoma datasets according to the criteria: sample size is more than 10, both tumor and nontumor samples are included. As a result, three GEO datasets GSE46517, GSE15605 and GSE3189 are selected, and the combined dataset include 207 tumor samples and 31 normal samples. Details of the three datasets were listed in Table 1. The differential expression analysis by integration of TCGA and GTEx datasets, and identi cation of the differentially expressed genes (DEGs) between melanoma and normal samples was conducted as described in our previous study [19].
The Limma R package was used to conduct differential expression analysis [20]. The genes with P-value < 0.05 and absolute fold change > 2 were considered as differentially expressed genes (DEGs). The R packages ggplot2 was used to draw the volcano plots of the DEGs. Venn diagram analyses were conducted to obtain the overlapping DEGs among the three datasets mentioned above.
Enrichment, survival and co-expression analysis Enrichment analysis on Gene Ontology (GO) functional annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were performed on the set of overlapping DEGs using R package clusterPro ler [21]. All three type GO functional annotations, including biological process (BP), cellular composition (CC), and molecular function (MF), were covered.
Survival analysis was conducted on 458 SKCM patients with RNA-seq expression pro les and clinical information obtained from TCGA. The patients were divided into high and low groups by TUBB4A median expression levels. The prognostic effect of the expression of TUBB4A on overall survival (OS) and disease-free survival (DFS) were estimated using the Kaplan-Meier method and survival curves were evaluated using the log-rank test.
Co-expressed genes of TUBB4A were clustered and demonstrated by the heat map generated by LinkedOmics [22]. The top 50 positive and negative correlated genes with TUBB4A were identi ed by Pearson correlations (P-value<0.05).

Cell lines and cell culture
The commercially available melanoma cell lines A375 and B16-F10 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). A375 and B16-F10 cells were cultured in Dulbecco's modi ed Eagle's medium (DMEM) containing 10% FBS at 37 °C in a humidi ed incubator under 5% CO 2 condition.

siRNA transfection
The speci c small interfering RNA was performed using custom-made siRNA targeting the TUBB4A mRNA region (siTUBB4A sense: GGAGGUUAUCAGUGACGAATT, siTUBB4A antisense: UUCGUCACUGAUAACCUCCTT) and negative control (siNC sense: UUCUUCGAACGUGUCACGUTT, antisense: ACGUGACACGUUCGGAGAATT) (GenePharma, China). Cells were cultured in a 6-well plate and transfected with lip3000 (Invitrogen, Carlsbad, CA, USA) following the instructions of the manufacturer. After 48 h of transfection, the cells were used for subsequent experiments.

Cell proliferation assay
For CCK8 proliferation assay, A375 and B16-F10 cells were transfected as previously described. Then, 800 cells were resuspended in 100 µL DMEM supplemented with 10% FBS and then added to a 96 well plate. After 24, 48, 72 and 96 hours, cell proliferation was investigated using CCK8 (Dojindo Molecular Technologies, USA) according to the manufacturer's instruction.
Wound healing and transwell assay Cells were seeded in a 6-well plate and cultured for a certain time to reach approximately 100% con uence. The sterile pipette tip was used to scratch a linear wound and serum free DMEM was added for further culturing. Wound healing images were captured using an inverted microscope. For transwell assay, appropriated melanoma cells were seeded into the upper well (Corning, USA). The lower chamber was lled with 700µL of DMEM containing 20% FBS. Transwell chambers were placed in an incubator (37°C , 5% CO 2 ) for 12 h. Cells in the upper chamber were xed with 4% paraformaldehyde for 15 min, stained with 0.1% crystal violet for 15 min and counted under an inverted microscope.
Drug treatments and ow cytometric analysis TUBB4A inhibitors were retrieved from DrugBank [23]. Two small molecule agents, Dihydroartemisinin (DHA) and Nocodazole, are reported to bind TUBB4A protein. Cells seeded in a 6-well plate were treated with Dihydroartemisinin (20 and 40μM) for 48 h and Nocodazole (0.075 and 0.15μM) for 24 h separately [24,25]. Annexin V-FITC/PI kit (Becton Dickinson, USA) were used to measure the apoptosis of melanoma cells. All operations were carried out strictly according to the manufacturer instruction. In brief, cells were harvested and washed with phosphate buffer saline (PBS), and resuspended in 1×binding buffer. Next, cells were stained with 5μL FITC-Annexin and 5 PI for 15 min under dark conditions. The apoptosis of cells was measured by FACS Calibur ow cytometer (BD Biosciences, USA). Early apoptosis and late apoptosis were summed and the total apoptosis rate was calculated.

Statistical analysis
Statistical analyses were performed using SPSS 23.0 software (IBM, Chicago, IL, USA) and GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA). Student's t test was used to analyze the differences between two groups. One-way analysis of variance (ANOVA) was used for the comparison among three or more groups. A P-value less than 0.05 was considered as statistical signi cance.

Results
Dysregulated genes closely related to melanoma Differential expression analysis on were conducted on three GEO datasets independently, and the volcano plots of DEGs (P-value<0.05, absolute fold change>2) were displayed in Fig. 1a-c. Subsequently, DEGs common in three SKCM datasets are ltered out and illustrated in Venn diagram. As shown in Fig. 1d-f, there are 580 overlapping DEGs in all three datasets, in which 165 genes were upregulated and 393 genes were downregulated. Moreover, overexpression of TUBB4A was found in melanoma tissues compared with normal tissues in all three datasets ( Fig. 1h-j).
To uncover the role of these differentially expressed genes in the pathogenesis of melanoma, GO and KEGG enrichment analysis was carried out. As shown in Fig. 2a-c, they are signi cantly enriched in skin epidermis-related biological processes, such as skin and epidermis development, epidermal cell and keratinocyte differentiation, and extracellular structure organization. As for cellular component, these It has been reported that melanin is partially attributed to the secretion of -melanocyte stimulating hormone ( -MSH) induced by keratinocytes, while ultraviolet (UV) induction of -MSH in skin is directly regulated by p53 [26]. Key signal molecule p53 activated in response to DNA damage, tra cked in melanoma and thyroid cancer cells and was associated with cell proliferation and progression. Moreover, it also mediates cell-cycle arrest, apoptosis, and senescence [27].
TUBB4A is over-expressed and correlated to prognosis in melanoma We conducted differential expression analysis by integration of TCGA and GTEx datasets. The differential expression analysis revealed 1,485 genes that are differentially expressed between tumor and normal samples. Among them, 514 genes are up-regulated and 972 genes are down-regulated, as shown in Supplementary Fig. S1. Since dysregulated genes are often main cancerogenic factors, we performed survival analysis on dysregulated genes independently. The Kaplan-Meier analysis results showed that ve genes are signi cantly related to the both overall survival and disease-free survival of cancer patients. They are TUBB4A, PSEN2, SLC45A2, QPRT and TRPV2. Table 2 listed the details of the ve genes.
We have conducted extensive literature search to verify the genes associated to melanoma survival rate. SLC45A2 is involved in melanosome maturation and pigmentation and already reported to be associated with risk of cutaneous malignant melanoma [28,29]. The upregulation of PSEN2 is associated with human melanoma aggressiveness and poor prognosis, and it is also the target of MYC [30]. According to previous study [31], TRPV2 exhibited ectopic distribution both in melanocytes and melanoma cells. Moreover, activation of TRPV2 could lead to the decline of cell viability for melanoma A2058 and A375 cells [31].
Most importantly, TUBB4A is the gene with the most statistical signi cant in the prediction of patient survival status. The Kaplan-Meier survival curve showed that patients with high TUBB4A expression had signi cantly shorter overall survival and disease-free survival than those with low expression (Fig. 3a, b).
Then, we preformed co-expression analysis of TUBB4A in SKCM cases through LinkedOmics in the TCGA database. As shown in Fig. 3c, d, fty signi cantly co-expressed genes were demonstrated by heatmap, showing that they were positively or negatively correlated with TUBB4A.

TUBB4A knockdown signi cantly inhibits SKCM cell proliferation
To further explore the role of TUBB4A in SKCM, small interfering RNAs (siRNAs) to silence TUBB4A gene was transfected in A375 and B16-F10 cells. We ensure the transfection resulted in su cient knockdown of TUBB4A expression. The downregulation at RNA level was evident at 48h past transfection for both cell lines (Fig. 4a, b). Cell viability was determined using the CCK8 assay based on absorbance at 450 nm after 24, 48, 72 and 96 h. Both A375 and B16-F10 cells with TUBB4A knockdown by siRNA showed reduced proliferation compared to the control group (si-NC, Fig. 4c, d). These results indicate that the higher expression of TUBB4A is closely associated to the proliferation of melanoma cells.
TUBB4A knockdown reduce SKCM cell migration in vitro SKCM cell migration capacity was detected by wound healing assay. A375 and B16-F10 cells of which TUBB4A is knocked down by siRNA showed a larger open wound area compared with the control group after 24h and 48 h (Fig. 5a, c). The difference in open wound area after 24h and 48h was quanti ed by calculating the percentage of change in the open wound area (Fig. 5b, d, p<0.05). In addition, transwell assay is also used to evaluate its impact on migration capacity. Results demonstrated that TUBB4A knockdown caused a strong reduction in cell migration in A375 (t test, p<0.001) and B16-F10 (t test, p<0.05) cells, compared to the control group (Fig. 6a-d). These ndings indicate that TUBB4A functions as a driver factor in increasing melanoma cell motility.

TUBB4A inhibitors signi cantly induce apoptosis of melanoma cells
We went further to search for small molecule drugs that target TUBB4A in the DrugBank database. There are ve small molecule agents potentially target to TUBB4A. We selected two drugs, Dihydroartemisinin (DHA) and Nocodazole, which have been approved or already in experimental stage. Dihydroartemisinin is an artemisinin derivative and antimalarial agent used in the treatment of uncomplicated Plasmodium falciparum infections, and it has been also reported to bind to TUBB4A protein [32]. Nocodazole is a 16-membered macrolide that mimics the biological effects of taxol and functions as inhibitor of microtubule function. Nocodazole has also been reported to inhibit the activity of TUBB4A [33].
Next, we went to test whether treatment with these two drugs separately can induce apoptosis of melanoma cell. As A375 is human melanoma cell line, we conducted assays on A375 cell line. The A375 cells were treated with DHA (20μM and 40μM) for 48h and Nocodazole (0.075μM and 0.15μM) for 24h separately. As shown in Fig. 7a, c, microscopy observations revealed that the exposure to DHA and Nocodazole triggered fragmentation in the cancer cells. The A375 cell apoptosis induced by these two agents was con rmed by staining with Annexin V-FITC/PI and subsequent analysis by ow cytometry. As shown in Fig. 7a-d, the proportion of apoptotic cells (quadrant Q2 An+/PI+ -late apoptosis and quadrant Q4 An+/PI--early apoptosis) increased signi cantly in a concentration-dependent manner in cell line after the exposure to two drugs. Our results indicate the pro-apoptotic activity of DHA and Nocodazole against A375 cells.

TUBB4A inhibitors modulate melanoma cell cycle progression
We also examined the effect of two TUBB4A inhibitors on cell cycle progression in A375 cells, using propidium iodide staining. The A375 cells were treated by DHA for 48h and Nocodazole for 24h, respectively. With the increase of DHA concentration, the cell cycle of A375 cells (Fig. 8a, b) was signi cantly blocked in G2/M phase. As shown in Fig. 8c, d, Nocodazole exerted signi cant tumor cell cycle arrest, and the dosages effect of 0.075μM and 0.15μM differ slightly. The results revealed that DHA and Nocodazole inhibited the proliferation of A375 cells by inducing cell cycle arrest.

Discussion
Despite the remarkable advancement made in melanoma therapeutic strategies, the long-term prognosis of melanoma patients remains poor due to limited understanding of the underlying mechanisms of tumor initiation and development. This study aimed to explore the function and signi cance of TUBB4A and its inhibition agents in cutaneous melanoma. We demonstrated that TUBB4A was highly expressed in SKCM, and the results of survival analysis showed that the expression level of TUBB4A have signi cant effects on the viability of melanoma cells. Next, we validated and con rmed the functions of TUBB4A in tumorigenesis and development in melanoma. TUBB4A knockdown by siRNA in the A375 and B16-F10 melanoma cells signi cantly inhibited the proliferation and metastasis of cancer cell. Finally, we found that two TUBB4A inhibitors, Dihydroartemisinin and Nocodazole, promoted the cell apoptosis and induced cell cycle arrest in G2/M phase in vitro.
Class III beta-tubulin (β3-tubulin) has been reported that it correlates with enhanced neoplastic cell survival, metastasis and resistance to chemotherapy [34]. But there are few studies focusing on the role of Class IV beta-tubulin (β4-tubulin) in tumor progression. Previous studies have provided the rst evidence that the upregulation of TUBB4 and TUBB3 is coupled with increased cell migration in endothelial-mesenchymal transition-induced human microvascular endothelial cells (HMEC-1) [35]. Also, TUBB4 are necessary for the transport and proper localization of N-cadherin within the plasma membrane [35]. Moreover, non-adherent culture induces paclitaxel resistance in H460 lung cancer cells via ERK-mediated up-regulation of bIVa-tubulin [17]. Above studies have shown that TUBB4A plays crucial role in tumorigenesis. Thus, it is of signi cance to explore the role of TUBB4A in cancer metastasis, as well as its potential functions in the acquirement of drug resistance.
Furthermore, we explored the the transcription factors (TFs) of TUBB4A, and retrieved four TFs from KnockTF database [36], including HOXA1, IKZF2, TFAP2C and TP53. Many studies have provided evidence that these TFs are colsely coupled with TUBB4A in several carcinoma. For example, HOXA1 drives melanoma tumor growth and metastasis involving in diverse cytokine pathways that include the TGFβ signaling axis. It also down-regulates the expression of microphthalmia-associated transcription factor and other genes required for melanocyte differentiation [37]. Maeda et al. also reported that the expression level of HOXA1 in melanoma with distant metastasis was higher than those in melanoma without it [38]. It is reported that microRNA-214 contributes to melanoma tumor progression through suppression of TFAP2C [39]. Differences in methylation between the primary melanoma lesions and multiple metastases were evident for TFAP2C gene [40]. Tubulin acetylation favors the molecular chaperone Hsp90 recruitment to microtubules, and stimulates the binding and signaling function of the kinase Akt/PKB and transcription factor P53 [41]. Furthermore, Arai et al. reported that the tubulin inhibitor vinca alkaloid enhanced expression of class II beta-tubulin isotype (mTUBB2) in mouse B16F10 melanoma cells via alteration of the tumor suppressor P53 protein [42]. Other studies found that P53 transcriptionally downregulated microtubule-associated protein 4 [43], and that human breast carcinoma cells with a mutated P53 gene displayed an increased level of class IV β-tubulin [44,45].
We also investigated the downstream factors of TUBB4. Proximity ligation assay (PLA) and immunoprecipitation studies performed on human glioblastoma patient specimen con rm GLUT1 interaction with TUBB4. Treatment of glioblastoma cells with TUBB4 inhibitor, CR-42-24, reduces the expression of GLUT1 however, TUBB4 expression is unaltered upon GLUT1 inhibitor fasentin treatment [14]. Here indicated that GLUT1 may be a downstream target gene of TUBB4A. Also, one of the isoform of Intersectin1 ITSN1-L was able to strengthen cell-cell adhesion by upregulating N-cadherin expression and its re-localization to membrane by ANXA2 and TUBB3/TUBB4 [46].
A limitation of our study mainly lies in that, although enrichment analysis has suggested several related pathways, and extensive literature was conducted to illustrate the mechanism of TUBB4A, there is still a lack of wet-lab experiments to reveal the related celluar mechanism. Despite this limitation, our ndings demonstrated the biological function of TUBB4A in SKCM for the rst time, suggesting it as a promising prognostic marker and therapeutic target.

Conclusion
Overall, the ndings indicate that TUBB4A functions as a driver factor in increasing melanoma cell proliferation and motility, and the targeted drugs of TUBB4A signi cantly induced melanoma cell apoptosis and G2/M cell cycle arrest. We would draw the conclusion that TUBB4A may be a potential prognostic marker and therapeutic target of melanoma.    TUBB4A knockdown suppressed proliferation of SKCM cells. a, b Knockdown of TUBB4A was con rmed by qRT-PCR analysis in A375 and B16-F10 cells. c, d CCK8 cell proliferation assays in A375 and B16-F10 cells with or without TUBB4A knockdown. *P < 0.05, **P < 0.01, ***P < 0.001. TUBB4A knockdown suppressed migration of SKCM cells by transwell assay. Transwell assay was applied in A375 (a, b) and B16-F10 (c, d) cells with or without TUBB4A knockdown. *P < 0.05, **P < 0.01, ***P < 0.001.