Abstract
BackgroundOsteosarcoma (OS) is the most common primary bone cancer in adolescents and lung metastasis is the leading cause of death in patients with OS. However, the molecular mechanisms that promote OS growth and metastasis remain unknown.MethodsWe investigated the expression of myosin light chain kinase family members between metastasis and non-metastasis patients in the TARGET database and ensured that only myosin light chain kinase family member 4 (MYLK4) had higher expression in metastatic osteosarcoma patients. Then we confirmed the results by immunohistochemistry (IHC) and Western blotting (WB) of OS tissues. The effect of MYLK4 on the metastasis and proliferation of OS cells was investigated by wound healing, Transwell and colony-formation assays. Mass spectrum analysis was used to ensure the new binding protein of MYLK4. Tissue microarrays analysis was used to show the correlation between MYLK4 and pEGFR (Y1068). A series of in vivo experiments were conducted to reveal the mechanisms by which MYLK4 modulated the metastasis and proliferation of OS.ResultsMyosin Light Chain Kinase Family Member 4 (MYLK4) was significantly upregulated in metastatic human OS tissues. Growth and metastasis of OS could be accelerated by MYLK4 overexpression, whereas silencing MYLK4 expression resulted in decreased cell growth and metastasis. Mechanistically, mass spectrum analysis showed that MYLK4 interacted with the epidermal growth factor receptor (EGFR) in osteosarcoma cells and promoted growth and metastasis via the EGFR signaling pathway. Tissue microarrays analysis also showed that MYLK4 expression had a positive correlation with the expression of pEGFR (Y1068). Moreover, the EGFR inhibitor gefitinib could partially reverse the effect of cell proliferation and metastasis caused by MYLK4 overexpression. Importantly, the combination of MYLK4 and EGFR inhibitors had synergistic effects on growth and metastasis of OS in vitro and in vivo.ConclusionOur results indicate that MYLK4 promotes OS growth and metastasis by activating the EGFR signaling pathway and can be a novel therapeutic target for the treatment of OS patients.
Highlights
Osteosarcoma (OS) is the most common primary bone cancer in adolescents and lung metastasis is the leading cause of death in patients with OS
Myosin Light Chain Kinase Family Member 4 (MYLK4) is upregulated in metastatic OS tissues In order to illuminate the function and clinical importance of myosin light chain kinase family members (MYLK, MYLK2, MYLK3 and MYLK4) in OS, we first analyzed Myosin light chain kinase (MYLK), MYLK2, MYLK3, and MYLK4 expression using the Therapeutically Applicable Research to Generate Effective Treatments database to understand the function and clinical importance of the MYLK family
We found that only MYLK4 had a higher expression in patients with OS during metastasis (P = 0.0415) (Fig. 1a; Supplementary Fig. 1A-C)
Summary
Osteosarcoma (OS) is the most common primary bone cancer in adolescents and lung metastasis is the leading cause of death in patients with OS. Cytoskeletal myosin II phosphorylated by MYLK increases the metastatic potential of tumor cells, and MYLK-dependent cytoskeleton rearrangement adjusts the angiogenesis of the vascular endothelial barrier, which is an inevitable step in cancer metastasis [7]. Bao et al found through bioinformatics analysis that MYLK2 was associated with poor prognosis in triple-negative breast cancer [8]. For those with hepatocellular carcinoma, Wang et al indicated that MYLK2 was upregulated in tumor tissues and was linked with a lower survival rate [9]. MYLK family members were found to be associated with the development of tumors in previous studies, how they are involved in the growth and metastasis of OS remains unclear
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