Fig. 6.

p-MYH9 (Ser1943) is a therapeutic target for stemness and LR, and predicts poor prognosis and LR in patients with HCC. a HuH-7-LR and SNU-387-LR cells were exposed to the indicated concentrations of CX-4945. Western blot was used to assess expression of p-MYH9 (Ser1943), MYH9 and HIF-1α (b) The effect of CX-4945 treatment on the relative luciferase activity of HRE in HCC LR cells was also investigated. The influence of CX-4945 on HCC LR cell stemness was used to assess in vitro self-renewal (c), migration (d), the mRNA expression of stemness markers (e) and colony formation (f). g HuH-7-LR and SNU-387-LR cells were treated with DMSO or 2 μM CX-4945 combined with the indicated concentration of lenvatinib. Differences in sensitivity to lenvatinib and CX-4945 were observed through cell count using trypan blue staining. h HuH-7-LR cell xenografted nude mice were administrated with vehicle, Len (lenvatinib, 30 mg/kg), CX-4945 (20 mg/kg) and their combination daily for 3 weeks after the tumor volume reached an average size of 50–100 mm³ (n = 5 per group). Then, the mice were sacrificed and the isolated tumors were photographed. i Tumor volume of each group was measured twice a week. j IHC was used to detect the expression of HIF-1α and p-MYH9 (Ser1943) in the xenografts following treatment. k HuH-7-LR cell was injected into the sub-capsular space of right liver lobe nude mice to establish the orthotopic tumor model. After 1 week, mice were treated with vehicle, Len (lenvatinib, 30 mg/kg), CX-4945 (20 mg/kg) and their combination daily for 3 weeks (n = 5 per group). Then, the mice were sacrificed and the isolated livers were photographed. l Tumor volume was calculated. m p-MYH9 (Ser1943) expression was detected by IHC in normal adjacent to tumor tissue (NAT) and HCC samples from 171 patients. Based on IHC staining of p-MYH9 (Ser1943), patients were categorized into two groups, and subsequent overall survival curves (n) were plotted. o Patients with HCC treated with lenvatinib were separated into two groups depending on their p-MYH9 (Ser1943) protein expression, and subsequent progression-free survival curves were plotted. p Positive correlation between p-MYH9 (Ser1943) and HIF-1α IHC results in 215 HCC samples. The data of cell functional assays, Luciferase reporter assay and RT-qPCR analysis were presented as mean ± SD of three individual experiments, and the data of animal experiments were presented as mean ± SEM. The Student’s t-test was used for comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 μm. MYH9 non-muscle myosin heavy chain 9, LR lenvatinib resistance, HIF-1α hypoxia-inducible factor-1α, HRE hypoxia-responsive element, HCC hepatocellular carcinoma, IHC immunohistochemistry, DMSO dimethyl sulfoxide, ns nonsignificant