Introduction

Primary liver cancer is the fourth leading cause of cancer-related death worldwide, with estimated 906,000 new cases and 830,000 deaths in 2020 [1]. Hepatocellular carcinoma (HCC) accounts for the majority of primary liver cancer, integrated studies have demonstrated that HCC is highly heterogeneous, with alternations in genetics, molecules, and cell signaling pathways among patients and even among different compartments within one tumor [2, 3]. Although considerable progress has been achieved in previous studies [48]. mTOR inhibitors have been proved to decrease HCC recurrence rate after liver transplantation [49, 50]. Our research suggests that the combination of mTOR inhibitors and KIF18B inhibitors may synergistically enhance their preventing recurrence and anti-tumor effect.

In summary, the results of this study demonstrate that KIF18B is a direct target of FOXM1 and promotes HCC recurrence through activating mTORC1 signaling. Mechanistically, KIF18B interacts with γ-Actin. They synergistically modulate lysosome positioning, subsequently promote mTORC1 translocation to lysosome membrane, and prevent p70 S6K from entering lysosomes for degradation (Fig. 6F). Our study highlights the potential of KIF18B as a therapeutic target for the treatment of HCC.

Materials and methods

Cell lines and clinical samples

Hep3B, Huh1, Huh7, LO2 and HEK293T were obtained from American Type Culture Collection. HCCLM3, BEL-7402 and SMMC-7721 were purchased from Cell Bank of Chinese Academy of Sciences. The cell lines HCCLM3, BEL-7402, Hep3B, Huh1, Huh7 and 293 T were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Biological Industries, 04-001-1), 100 U/ml penicillin and 0.1 mg/ml streptomycin. LO2 and SMMC-7721 cell lines were maintained in Roswell Park Memorial Institute-1640 (RPMI-1640) medium supplemented with 10% FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin. All of these cells were incubated at 37 °C in an atmosphere of 5% CO2. A total of 60 HCC tissues were obtained from the affiliated hospital of Hangzhou Normal University. Written informed consent was obtained from each patient, and the study was approved by the Biomedical Ethics Committee of the affiliated hospital of Hangzhou Normal University. This study was performed in accordance with the Declaration of Helsinki.

Lentivirus infection and in vivo models

HCCLM3 or BEL-7402 cells were infected with lentiviruses in presence of polybrene and selected with 2 μg/mL and 1 μg/mL puromycin, respectively, for 7 days. For the subcutaneous xenograft model, five-week-old male BALB/c nude mice were used. All animal care and handling procedures were performed in accordance with the National Institutes of Health guide for the care and use of Laboratory animals and approved by the Animal Care and Use Committee of Hangzhou Normal University. The control and experimental HCCLM3 cells (5 × 106) were suspended in 100 μL PBS and then injected subcutaneously into the flanks of the nude mice. Tumor growth was examined over the course of 4 weeks and tumor volume was measured with calipers and calculated using the formula: Volume (mm3) = length × width2/2 as previously reported [51]. After 4 weeks, the mice were sacrificed and the tumors were weighed, fixed, and paraffin-embedded for further analysis.

Statistical analysis

Spearman test was used to analyze the correlation between KIF18B and p-mTOR expression in HCC tissues. The Kaplan-Meier method was used to analyze patient survival. The Student, t test was used to for the comparisons between two groups. Statistical analysis and graphs were generated using the SPSS 20.0 software and GraphPad Prism 7 software. P < 0.05 was considered as significant.