TUBB4A interacts with MYH9 to protect the nucleus during cell migration and promotes prostate cancer via GSK3β/β-catenin signalling

Abstract

Human tubulin beta class IVa (TUBB4A) is a member of the β-tubulin family. In most normal tissues, expression of TUBB4A is little to none, but it is highly expressed in human prostate cancer. Here we show that high expression levels of TUBB4A are associated with aggressive prostate cancers and poor patient survival, especially for African-American men. Additionally, in prostate cancer cells, TUBB4A knockout (KO) reduces cell growth and migration but induces DNA damage through increased γH2AX and 53BP1. Furthermore, during constricted cell migration, TUBB4A interacts with MYH9 to protect the nucleus, but either TUBB4A KO or MYH9 knockdown leads to severe DNA damage and reduces the NF-κB signaling response. Also, TUBB4A KO retards tumor growth and metastasis. Functional analysis reveals that TUBB4A/GSK3β binds to the N-terminal of MYH9, and that TUBB4A KO reduces MYH9-mediated GSK3β ubiquitination and degradation, leading to decreased activation of β-catenin signaling and its relevant epithelial-mesenchymal transition. Likewise, prostate-specific deletion of Tubb4a reduces spontaneous tumor growth and metastasis via inhibition of NF-κB, cyclin D1, and c-MYC signaling activation. Our results suggest an oncogenic role of TUBB4A and provide a potentially actionable therapeutic target for prostate cancers with TUBB4A overexpression.

Fig. 1. TUBB4A expression in normal prostate and prostate cancer tissues.

Differential expression of TUBB4A A between normal prostate and prostate cancer tissues, B between tumor tissues with different Gleason scores, and C between AA and EA tumor tissues from TCGA dataset. Numbers in parentheses indicate the sample size. D DNA methylation of TUBB4A promoter (TCGA dataset). E Correlation of mRNA expression levels with DNA methylation levels in the CpG island of TUBB4A in prostate cancer samples (TCGA dataset). F Kaplan–Meier survival curves for TUBB4A expression with low and high levels in TCGA prostate cancer patients. G Representative IHC staining in normal prostate and prostate cancer tissues with anti-human TUBB4A mAb (Abcam, ab11315) from UAB prostate cancer specimens. Scale bars, 100 μm or 200 μm. This experiment was repeated two times. H–J Quantitative H-scores for prostate cancer samples from European and African American ancestry and tumor stages and Gleason scores. Data are presented as the medians and interquartile ranges as determined with a Kruskal–Wallis (B–D) or Mann–Whitney U (A–D) test. Data are presented as the means and standard deviation (SD) as determined with an ANOVA followed by Tukey’s post hoc t test (H–J). Source data are provided as a Source data file. AA: African-American; EA: European-American; NAT: Normal prostate tissue adjacent to the tumor.

Fig. 2. TUBB4A KO reduces proliferation and migration of prostate cancer cells.

A–D Confirmation of TUBB4A KO, cell growth, clone formation, and statistical analysis of PC3 cells. Cell growth was measured by live cell kinetic imaging with walk-away automation using a Lionheart™ FX cell imager. A colony was considered to be 50 cells or more as determined microscopically. E confirmation of TUBB4A KO and rescue of DU145 cells. F–H Cell growth, clone formation, and statistical analysis of DU145 cells. I, J Confirmation of TUBB4A knockdown (KD) and cell growth of LNCaP cells. K Cell growth assay of LNCaP cells transfected with TUBB4A siRNAs in androgen-depleted culture medium. L, M 3D soft-agar colony formation and statistical analysis of PC3 cells. A 3D colony was counted based on the capacity of single cells to grow to colonies consisting of at least 50 cells. Scale bar, 500 μm. N, O Transwell assay and statistical analysis of PC3 cells. Scale bar, 100 μm. P, Q 3D soft-agar colony formation and statistical analysis of DU145 cells. Scale bar, 500 μm. R, S Transwell assay and statistical analysis of DU145 cells. Scale bar, 100 μm. Data are replicated 3 (J, K, M, Q), 4 (B, F), 6 (D, H), and 10 (O, S) times. Data are presented as the means and SD with a repeated measures ANOVA (B, F, J, K) or an ANOVA followed by Tukey’s post hoc t test (D, H, M, O, Q, S). Source data are provided as a Source data file. Scr, scramble; KO, knockout; NC, negative control siRNA; siRNA, small interfering RNA; NS, no significant difference.

Fig. 3. TUBB4A KO and MYH9 KD increase DNA damage and cell death during migration of prostate cancer cells.

A Immunofluorescence (IF) staining of TUBB4A and F-actin in DU145 and PC3 cells with or without TUBB4A. Scale bar, 50 μm. B, C IF staining of γH2AX and 53BP1 in DU145 cells with or without TUBB4A during migration in 3D extracellular matrix. Yellow arrows indicate cells with DNA damage. Scale bar, 50 μm. D, E Death rate of DU145 cells with or without TUBB4A after migration in 3D collagen gels. Scale bar, 500 μm. F, G IF staining of γH2AX and 53BP1 in DU145 cells with or without TUBB4A after migration in 3D collagen gels. Scale bar, 10 μm. H–K IF staining of γH2AX and 53BP1 in scrambled, TUBB4A KO, and rescued DU145 and PC3 cells during migration in a 3D extracellular matrix. Scale bar, 25 μm. L–O IF staining of γH2AX and 53BP1 in scramble control and MYH9 KD DU145 and PC3 cells during migration in a 3D extracellular matrix. Scale bar, 25 μm. Data are replicated 3 (I, K, M, O) and 10 (C, G) times. Data are presented as the means and SD with a two-tailed t test (C, G, M, O) or an ANOVA followed by Tukey’s post hoc t test (I–K). Source data are provided as a Source data file. Scr, scramble; KO, knockout; NC, negative control siRNA; siRNA, small interfering RNA; KD, knockdown; NS, no significant difference.

Fig. 4. TUBB4A interacts with MYH9 and their relevant DNA damage response and signaling pathways in prostate cancer cells.

A Bands on an SDS-PAGE gel after TUBB4A protein pull-down in DU145 cells. Red and blue arrows indicate the bands presumed to be MYH9 and TUBB4A, respectively, in the pulldown. B TUBB4A-interacting proteins as determined by mass spectrometry analysis. Red arrow indicates MYH9 and its potential site on SDS-PAGE gel. C, D Co-immunoprecipitation (IP) assay of TUBB4A and MYH9. The input was diluted as 1/10. E Co-localization of TUBB4A and MYH9 by IF staining. Scale bar, 100 μm. F Co-localization of TUBB4A and MYH9 at the forefront of the nucleus during migration in 3D collagen gels determined by IF staining. Scale bar, 10 μm. G, H Western blotting showing changes in proteins of DNA damage response and proteins of the NF-κB p65 and IKK complex in DU145 and PC3 cells with or without TUBB4A and MYH9. I Western blotting showing changes in proteins of DNA damage response and proteins of the intrinsic apoptosis pathway in DU145 cells with or without TUBB4A and MYH9. J Western blotting showing changes in proteins of NF-kB-relevant signaling pathways in DU145 cells with or without TUBB4A and MYH9. Source data are provided as a Source data file. Scr, scramble; KO, knockout; NC, negative control siRNA; siRNA, small interfering RNA; NS, no significant difference.

Fig. 5. TUBB4A/MYH9-regulated GSK3β/β-catenin signaling pathway in prostate cancer cells.

A, B Co-IP assay of TUBB4A and MYH9 in DU145 cells. The input was diluted at 1/10. C, D Co-IP assay of TUBB4A and N-terminal (N-MYH9) or C-terminal (C-MYH9) of MYH9-Flag in DU145 cells. E Western blotting for changes in proteins of the GSK3β/β-catenin signaling pathway and EMT markers in DU145 cells with or without TUBB4A and MYH9. F Western blotting showing the effect of TUBB4A KO on MYH9 and GSK3β stability in DU145 cells incubated with cycloheximide (CHX) at the indicated time points. G Ubiquitination of GSK3β in DU145 cells with or without TUBB4A and MYH9. Cells were either untreated or treated with proteasome inhibitor MG132 (10 μM) for 12 h. Equal aliquots of the cellular lysates were immunoprecipitated with anti-GSK3β antibodies. Source data are provided as a Source data file. Scr, scramble; KO, knockout; NC, negative control siRNA; siRNA, small interfering RNA.

Fig. 6. TUBB4A KO reduces xenograft tumor growth in NSG mice.

A Schematic diagram of S.C. xenograft tumors in NSG mice followed up to 30 days. B Luciferase imaging of xenografts after implantation. C Tumor volumes after injection (n = 6/group). D, E Representative tumor masses and mean tumor weights on day 30. F H/E, IHC, and IF staining of xenograft tumors by specific antibodies to TUBB4A, Ki67, cyclin D1, c-MYC, p-IKKα/β, and p-p65. Scale bar, 50 μm or 100 μm. G Statistical analysis of Ki67+ cells for scrambled and KO groups. H, I IF staining and statistical analysis of xenograft tumors by the DNA damage response marker γH2AX. Scale bar, 50 μm. J, K IF staining and statistical analysis of xenograft tumors by the apoptotic marker, TUNEL. Scale bar, 50 μm. Data are replicated 6 times (C, E, G, I, K). Data are presented as the means and SD with a repeated measures ANOVA (C) or an ANOVA followed by Tukey’s post hoc t test (E, I, K) or a two-tailed t test (G). Source data are provided as a Source data file. Scr, scramble; KO, knockout; s.c., subcutaneous injection; luc, luciferase.

Fig. 7. TUBB4A KO decreases tumor metastasis in the lungs of NSG mice.

A Schematic diagram of S.C. xenograft tumors followed for up to 50 days in NSG mice. B Luciferase imaging of xenograft tumors and metastases after implantation. C, D Statistical analysis of tumor nodules and burdens of lung metastasis at day 50. E H/E and IF staining of metastatic tumors. Scale bar, 100 μm. F IHC staining of c-MYC, p-IKKα/β, and NF-κB (p-p65) of lung metastatic tumors in DU145 xenografts. Scale bar, 100 μm. G Schematic diagram of S.C. xenograft tumors followed for up to 90 days in NSG mice. H Representative tumor metastases in the lung at day 50. Scale bar, 500 μm. I, J Statistical analysis of tumor nodules and burdens of lung metastasis at day 50. K IHC staining of TUBB4A and E-cadherin in scrambled/KO mixed S.C. xenografts. Scale bar, 50 μm. L IHC staining of TUBB4A in scrambled/KO mixed lung metastatic tumors at day 90. Scale bar, 100 μm or 500 μm. M Statistical analysis of tumor nodules of lung metastasis at day 90. Data are replicated 10 times (C, D, I, J, M). Data are presented as the medians and interquartile ranges with a Mann–Whitney U (C, D) or Kruskal–Wallis followed by a Benjamini–Hochberg false discovery rate test for multiple comparisons (I, J). Data are presented as the means and SD with a two-tailed t test (M). Source data are provided as a Source data file. Scr, scramble; KO, knockout; s.c., subcutaneous injection; luc, luciferase.

Fig. 8. Prostate-specific deletions of Tubb4a and Pten and tumor progression in mouse prostate.

A Schematic diagram of spontaneous developed prostate tumors followed for up to 50 weeks of age in genetically engineered mouse models. B Representative mouse prostates at 25 and 35 weeks of age. C Weights of prostates in the mice at 35 weeks of age. D Representative H/E staining of mouse prostates at 35 weeks of age. Scale bar, 200 µm. E Kaplan–Meier curves of mPIN incidences for up to 50 weeks of age. At 20, 25, 30, 35, 40, 45, and 50 weeks of age, 5 mice/per time point were sacrificed for pathologic analysis. F Representative immunostaining for TUBB4A, AR, Ki67, c-MYC, p-IKK, and p-p65 in the prostates of mice at 35 weeks of age. Scale bar, 100 µm. G The percentage of Ki67 cells as an indicator of proliferating cells among the mouse prostate or tumor tissues. H Relative mRNA levels of c-Myc, Ccnd1, and Vim genes as a percentage of Hprt expression in microdissected prostate epithelial cells as determined by qPCR at 35 weeks of age. Data are replicated 5 (H), 10 (G), 23–55 (C), and 40 (E) times. Data are presented as the means and SD with a two-tailed t test (G, H) or an ANOVA followed by Tukey’s post hoc t test (C). Source data are provided as a Source data file. AP, anterior prostate; DP, dorsal prostate; LP, lateral prostate; VP, ventral prostate; cKO, prostate conditional knockout; mPIN, mouse prostatic intraepithelial neoplasia; AR, androgen receptor; i.p., intraperitoneal injection.

Fig. 9. Prostate-specific deletions of Tubb4a and prostate tumor progression and metastasis in TRAMP models.

A Schematic diagram of spontaneously developed prostate tumors followed for up to 50 weeks of age in genetically engineered mouse models. B, C Kaplan–Meier curves of palpable prostate tumors and survival for up to 40 and 50 weeks of age, respectively. D, E Representative prostate tumor growth and metastasis and H/E staining at 50 weeks of age. Scale bar, 100 μm, 200 μm, or 500 μm. F Weights of prostates at 30 weeks of age. G Heatmap of prostate tumor progression and metastasis at 30 weeks of age. H Representative immunostaining for TUBB4A, AR, c-MYC, p-IKK, and p-p65 in the prostates of mice at 30 weeks of age. Scale bar, 100 µm. I Statistical analysis of Ki67+ cells for TRAMP and Tubb4a-cKO TRAMP groups. Data are replicated 10 (I), 20–32 (B, C), and 12–28 (F) times. Data are presented as the medians and interquartile ranges with a Kruskal–Wallis followed by a Dunn’s post hoc test (F). Data are presented as the means and SD with a two-tailed t test (I). Source data are provided as a Source data file. AP, anterior prostate; VP, ventral prostate; cKO, prostate conditional knockout; mPIN, mouse prostatic intraepithelial neoplasia; Mod/well, moderately/well-differentiation grades; LN, lymph node; AR, androgen receptor; i.p., intraperitoneal injection; LN, lymph nodes.