The kinesin light chain-2, a target of mRNA stabilizing protein HuR, inhibits p53 protein phosphorylation to promote radioresistance in NSCLC

Abstract

Background: Radioresistance hinders radiotherapy for the treatment of lung cancer. Kinesin light chain-2 (KLC2) has been found to be upregulated in lung cancer and also to be associated with poor prognosis. This study aimed to investigate the effect of KLC2 on radiosensitivity in lung cancer.

Methods: The radioresistant role of KLC2 was determined by colony formation, neutral comet assay, and γH2AX immunofluorescent staining assay. We further verified the function of KLC2 in a xenograft tumor model. The downstream of KLC2 was identified through gene set enrichment analysis and validated by western blot. Finally, we analyzed clinical data from the TCGA database to reveal the upstream transcription factor of KLC2, which was validated by RNA binding protein immunoprecipitation assay.

Results: Here, we found that downregulation of KLC2 could significantly reduce colony formation, increase γH2AX level, and double-stranded DNA breaks in vitro. Meanwhile, overexpressed KLC2 significantly increased the proportion of the S phase in lung cancer cells. KLC2 knockdown could activate P53 pathway, and ultimately promoting radiosensitivity. The mRNA of KLC2 was observed to bind with Hu-antigen R (HuR). The mRNA and protein expression of KLC2 in lung cancer cells was significantly reduced when combined with siRNA-HuR. Interestingly, KLC2 overexpression significantly increased the expression of HuR in lung cancer cells.

Conclusion: Taken together, these results indicated that HuR-KLC2 forms a positive feedback loop, which decreases the phosphorylation of p53 and thereby weaken the radiosensitivity of lung cancer cells. Our findings highlight the potential prognosis and therapeutic target value of KLC2 in lung cancer patients treated with radiotherapy.

Keywords: HuR; NSCLC; kinesin light chain-2; p53; radioresistance.

FIGURE 1

Identification of the effect of kinesin light chain‐2 (KLC2) on radiosensitivity in lung cancer cells. (a) Detection of radiosensitivity changes in lung cancer cells after overexpression or interference with KLC2 by clone formation assay. (b) Formation of γH2AX foci at 24 h after 5 Gy dose irradiation, analyzed by immunofluorescence stating. (c) Neutral comet assay and (d) cell cycle detection. Data are presented as means ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significance.

FIGURE 2

The effect of kinesin light chain‐2 (KLC2) on radiosensitivity in a xenograft tumor model. (a) 2 × 10⁶ H520 cells after overexpression or interference with KLC2 were implanted into the left flank of wild‐type male BALB/c nude mice. Mice received 2Gy × 5F irradiation (IR) of five mice in each group starting 12 days after tumor implantation. (b) Kaplan–Meier survival curves represent each treatment group (n = 5–8). Median survival: 47 days (Lv‐NC), 28 days (Lv‐KLC2), 49 days (Lv‐sh‐NC), 78 days (Lv‐sh‐KLC2). (c) 2 × 10⁶ H520 cells after overexpression or interference with KLC2 were implanted into the bilateral flank of wild‐type male BALB/c nude mice followed by LRT. (d) Tumor size in mice bearing control A549 xenografts or KLC2 overexpression (n = 5) or interference (n = 4) xenografts. Data points show the tumor volume (calculated with the following formula: V = (L × W ²)/2) of each group, bars, SEM. *p < 0.05. (e) Photographs of tumors developing in normal control (Lv‐NC), KLC2 overexpression (Lv‐KLC2), interference normal control (Lv‐sh‐NC), and interference KLC2 (Lv‐sh‐KLC2) mice are presented. (f) Weight of tumors from the mouse model; tumor weights in the KLC2+IR mice were higher than those from the Vector+IR mice (n = 5). On the contrary, tumor weights in the Lv‐sh‐KLC2+IR mice were lower than those of the Vector+IR mice (n = 4). Data points show the mean tumor weight. bars, SD. *p < 0.05, **p < 0.01.

FIGURE 3

Effect of kinesin light chain‐2 (KLC2) on the p53 signaling pathway. (a) Gene set enrichment analysis (GSEA) analysis showed that the expression level of KLC2 was related to the expression of the p53 signal pathway activated gene set. (b) The KEGG enrichment analysis of the upregulated genes was enriched in the “P53 signaling pathway” of lung adenocarcinoma (LUAD) in the Cancer Genome Atlas (TCGA) datasets. (c) The volcano plot showed the Top10 DEGs distributions in both up‐and downregulated genes between LUAD tumors and normal tissues in the TCGA datasets. (d) The KLC2 expression was associated with poor survival and prognosis in patients with non‐small cell lung cancer (NSCLC) using the TCGA database. (e, f) The expression of total P53 and phosphorylated p53 after overexpression or interference with KLC2 in A549 (e) and H520 (f) cell lines.

FIGURE 4

The effect of Hu‐antigen R (HuR) on kinesin light chain‐2 (KLC2) which mediated radiosensitivity in non‐small cell lung cancer (NSCLC) cells. (a) Cancer Genome Atlas (TCGA) database analysis of KLC2 mRNA expression in lung cancer tissues and normal lung tissues shown by the violin plots. Unpaired t‐test: ***p < 0.001. (b) Binding site prediction for proteins associated with KLC2 mRNA using starBase database. (c) TCGA database analysis of correlation between expression of KLC2 mRNA and HuR mRNA in lung cancer tissues. (d) qRT‐PCR detection of the effect of HuR on KLC2 expression. Data are presented as means ± SD, n = 3, *p < 0.05, **p < 0.01. (e) Western blot detection of KLC2 protein expression in A549 and H520 cell lines after knockdown of HuR. (f) Clonogenic survival assays of A549 and H520 cells treated with knockdown of HuR. (g) Western blot detection of KLC2 protein expression in A549 and H520 cell lines after overexpression of HuR. (h) Clonogenic survival assays of A549 and H520 cells treated with overexpression of HuR. Data are presented as means ± SD, n = 3, *p < 0.05, **p < 0.01. (i)–(k) RNA‐binding protein immunoprecipitation (RIP) assays show that KLC2 bind to HuR. (l) Western blot detection of HuR protein expression in 549 and H520 cell lines after overexpression or interference with KLC2. (m) Summary working pattern diagram of radiosensitivity regulated by KLC2.