Objective To investigate the role of kinesin family member 18A (KIF18A) in breast cancer and its impact on cellular biological behaviors, and explore its potential as a novel molecular target for triple negative breast cancer (TNBC).
Methods A total of 138 TNBC surgical specimens were collected from the Foshan First People's Hospital between August 2012 and December 2016. Tissue microarray was used to detect KIF18A expression, and its impact on patients' overall survival was analyzed. KIF18A-overexpressing and KIF18A-knockdown TNBC cell lines were constructed [overexpression: H23570 group (experimental group) and overexpression NC group (control group); knockdown: Y20559 group (experimental group) and knockdown NC group (control group)]. Cell proliferation, apoptosis, invasion and migration abilities were assessed using CCK-8 assay, flow cytometry, Transwell assay and wound healing assay, respectively. Western blot analysis was performed to detect the expression of related signaling pathway proteins and explore the potential mechanism of KIF18A. The chi-square test was used to analyze the correlation between KIF18A expression and clinical indicators. Survival analysis was performed using the Kaplan-Meier method and log-rank test. Univariate and multivariate Cox proportional hazards regression models were used to evaluate independent prognostic factors. Measurement data conforming to a normal distribution were presented as
±
s, and comparisons between two groups were made using
t test. One-way analysis of variance (ANOVA) was used to compare migration width, and factorial design ANOVA was used to analyze the time-group interaction effect on scratch wound area.
Results The expression of KIF18A differed significantly between patients of different ages (≤48 years vs >48 years, 71.4% vs 49.1%, χ2=4.478, P=0.034) and tumor sizes (≤3 cm vs >3 cm, 67.9% vs 43.0%, χ2=8.111, P=0.004). The OS of KIF18A low expression group (n=64) was significantly higher than that of KIF18A high expression group (n=74) at all time points, with distinct separation of survival curves between two groups (HR=4.330, 95%CI: 2.277-6.252, P<0.001). High KIF18A expression (HR=3.080, 95%CI: 1.374-6.906, P=0.006) and advanced TNM stage (HR=2.551, 95%CI: 1.204-5.402, P=0.014) were independent risk factors for OS in TNBC patients. The mRNA expression levels of KIF18A in MDA-MB-231, MDA-MB-468, MDA-MB-453 and BT474 cell lines were 9.042±0.074, 5.340±0.108, 6.040±0.171 and 7.068±0.259, respectively, with significant differences among groups (F=274.67, P<0.0001). Compared with the corresponding control group, KIF18A overexpression promoted cell proliferation (t=5.450, P=0.031), whereas KIF18A knockdown had no significant effect on cell proliferation (t=1.708, P=0.163). Cell proliferation was significantly inhibited by the KIF18A inhibitor Sovilesib at a concentration of 18.77 μmol/L. KIF18A overexpression mainly suppressed early apoptosis (P<0.05), while KIF18A knockdown increased the overall apoptotic level (P<0.001). In the overexpression group, the migration rate of H23570 cells was significantly higher than that of the overexpression NC group [(80.18±2.96) % vs (38.18±4.32) %, t=13.901, P<0.001]. KIF18A knockdown did not significantly affect cell migration ability initially, but after 72 hours, the migration ability of TNBC cells with KIF18A knockdown was significantly lower than that of knockdown NC group (P<0.001). KIF18A overexpression enhanced cell invasion ability (t=29.502, P<0.001), while KIF18A knockdown exerted an inhibitory effect on cell invasion (t=20.210, P<0.001). Western blot results demonstrated that compared with the control group, KIF18A overexpression upregulated the expression levels of mTOR, PD-L1, and CDK4 (t=7.471, 9.729, 4.064, all P<0.05) and downregulated PARP1 expression (t=12.310, P<0.05) in the H23570 group. Conversely, KIF18A knockdown reduced the expression levels of mTOR, PI3K, Akt, PD-L1, Cyclin D1, and CDK4 (t=2.792, 6.035, 4.091, 15.750, 12.940, 3.979, all P<0.05) in the Y20559 group.
Conclusion High expression of KIF18A is correlated with poor prognosis and malignant biological behaviors of TNBC, and KIF18A may play its role by regulating the expression of proteins such as CDK4/cyclin D1.