LMTK3 promotes tumorigenesis in bladder cancer via the ERK/MAPK pathway

Abstract

Lemur tyrosine kinase 3 (LMTK3) is a key member of the serine-threonine tyrosine kinase family. It plays an important role in breast cancer tumorigenesis and progression. However, its biological role in bladder cancer remains elusive. In this study, we demonstrated that LMTK3 was overexpressed in bladder cancer and was positively correlated with bladder cancer malignancy. High LMTK3 expression predicted poor overall survival. Knockdown of LMTK3 in bladder cancer cells triggered cell-cycle arrest at G2/M phase, suppressed cell growth, and induced cell apoptosis in bladder cancer cells. Furthermore, Transwell assays revealed that reduction of LMTK3 decreased cell migration by regulating the epithelial-to-mesenchymal transition pathway. Conversely, LKTM3 overexpression was shown to promote proliferation and migration of bladder cancer cells. We assessed phosphorylation of MEK and ERK1/2 in bladder cancer cells depleted of LMTK3 and demonstrated a reduced phosphorylation status compared with the control group. Using an MAPK signaling-specific inhibitor, U0126, we could rescue the promotion of proliferation and viability in LMTK3-overexpressing cells. In conclusion, we extend the status of LMTK3 as an oncogene in bladder cancer and provide evidence for its function via the activation of the ERK/MAPK pathway. Thus, targeting LMTK3 may hold potential as a diagnostic and prognostic biomarker and as a possible future treatment for bladder cancer.

Keywords: biomarker; bladder cancer; lemur tyrosine kinase-3; weighted gene coexpression network analysis.

Fig. 1

Bioinformatics analysis of LMTK3 in bladder cancer. (A) Dendrogram of all differentially expressed genes clustered based on a dissimilarity measure (1‐Topological Overlap Measure). (B) Heatmap of the correlation between MEs and different clinical information of bladder cancer (invasiveness, stage, grade and age). (C) Distribution of the average gene significance and errors in the modules associated with the stage of bladder cancer. (D) The comparison of LMTK3 expression in 188 bladder cancer tumor tissues and 68 paracarcinomatous tissues. Comparison was performed with Student’s t‐test. Data are presented as the mean ± SD. (E) Diagnostic value analysis of LMTK3 in bladder cancer with excellent specificity and sensitivity.

Fig. 2

The altered types and the regulatory network of LMTK3. (A) The main altered types of LMTK3 were amplification and mRNA up‐regulation in patients with bladder cancer. (B) The generated network by GeneMANIA showed that there were numerous interactions and coexpression between LMTK3 and other cell‐cycle highly relevant genes.

Fig. 3

LMTK3 is a diagnostic and prognostic factor. LMTK3 expression and prognostic value were analyzed based on 68 bladder cancer tissues and matched paracancerous tissues from the Department of Urology in our hospital. (A) LMTK3 mRNA expression is up‐regulated in 68 bladder cancer tissues compared with matched paracancerous tissues. (B) Western blot results show that LMTK3 protein expression is up‐regulated in three bladder cancer tissues and paired paracancerous tissues. (C, D) Immunohistochemical staining results show the protein expression of LMTK3 in 68 bladder cancer tissues and paired paracancerous tissues. (E) Kaplan–Meier survival analysis reveals that LMTK3 expression predicts a poor prognosis in 68 patients with bladder cancer. (F) ROC curves analysis of LMTK3. Data are presented as the mean ± SD; statistical analysis was performed, and comparison was performed with Student’s t‐test. Scale bar: 400 μm.

Fig. 4

Knockdown LMTK3 gene with siRNA. (A, B) Real‐time quantitative PCR analysis of the knockdown efficiency of LMTK3 siRNA in EJ and UMUC3 cells. (C) Western blot assay revealed a significantly decreased protein abundance of LMTK3 by the si‐LMTK3 knockdown as compared with si‐control treatment. (D) Representative immunofluorescence staining of LMTK3 (red) in the si‐LMTK3 and si‐control groups. Data represent the mean ± SD of three separate experiments. Differences between two groups were compared using Student’s t‐test. *P < 0.01. Scale bar: 200 μm.

Fig. 5

Down‐regulation of LMTK3 represses bladder cancer cell proliferation and migration. (A, B) MTT assay was used to detect the viability of bladder cancer cells in the si‐LMTK3 and si‐control groups. (C) Cell colony formation assay detects alteration of cell survival for EJ and UMUC3 in the si‐LMTK3 and si‐control groups. Clone number in each well was counted and statistically analyzed in the cell colony formation assay. (D) Cell migration analysis of the si‐LMTK3 and si‐control groups in bladder cancer cells. (E) Statistical analysis of cell migration. (F) Western blot analysis of proteins involved in the EMT pathway. Data represent the mean ± SD of three separate experiments. Differences between two groups were compared using Student’s t‐test. *P < 0.05, **P < 0.001. Scale bar: 200 μm.

Fig. 6

Knockdown of LMTK3 induces cell apoptosis and cell‐cycle arrest at G2/M phase. (A, B) Cell‐cycle analysis of si‐LMTK3 and si‐control groups in bladder cancer cells. (C) Western blot analysis showed the variety of proteins involved in the G2/M cell cycle. (D, E) Flow cytometry analysis of cell apoptosis of si‐LMTK3 and si‐control groups in bladder cancer cells. (F) Western blot analysis showed the variety of proteins involved in the cell apoptosis. All values are presented as the mean ± SD from three independent research results; comparison was performed with Student’s t‐test.

Fig. 7

Overexpression of LMTK3 promotes proliferation and migration of bladder cancer cells. (A, B) Real‐time quantitative PCR and western blot analyses verified the overexpressed efficiency of LMTK3. (C) Cell colony formation of NC and LMTK3‐OE groups in EJ cells. (D) MTT assay of NC and LMTK3‐OE groups in EJ cells. (E) Transwell migration assay of NC and LMTK3‐OE groups in EJ cells. Statistical analysis was performed. All of the values shown are mean ± SD of triplicate measurements; comparison was performed with Student’s t‐test. Scale bars: 200 μm. GAPDH, glyceraldehyde‐3 phosphate dehydrogenase; OE, overexpression; ** <0.01.

Fig. 8

LMTK3 promotes bladder cancer cells proliferation and migration through the ERK/MAPK signaling pathway. (A) The protein levels of LMTK3 and ERK/MAPK markers in si‐LMTK3 and si‐control bladder cancer cells were estimated by western blot. (B) The protein levels of LMTK3 and ERK/MAPK signaling pathway‐related markers in LMTK3‐overexpression EJ cells [treated with MAPK inhibitor U0126 (10 μm)] were assessed by western blot. (C–E) Rescue experiments of LMTK3 overexpression by using MAPK inhibitor U0126 (10 μm): cell colony formation, MTT and Transwell migration assay. Data represent the mean ± SD of three separate experiments; comparison was performed with one‐way ANOVA followed by Tukey’s post hoc test. Scale bar: 200 μm.