Prognostic and therapeutic potential of Adenylate kinase 2 in lung adenocarcinoma

Abstract

Adenylate kinase 2 (AK2), an isoenzyme of the AK family, may have momentous extra-mitochondrial functions, especially in tumourigenesis in addition to the well-known control of energy metabolism. In this study, we provided the first evidence that AK2 is overexpressed in lung adenocarcinoma. The positive expression of AK2 is associated with tumor progression, and poor survival in patients with pulmonary adenocarcinoma. Knockdown of AK2 could suppress proliferation, migration, and invasion as well as induce apoptosis and autophagy in human lung adenocarcinoma cells. Remarkably, silencing AK2 exerted the greater tumor suppression roles when combined with hydroxychloroquine, an effective autophagy inhibitor, in vitro and in xenografts mouse models. Our data have probably provided preclinical proof that systematic inhibition of AK2 and autophagy could be therapeutically effective on lung cancer.

Figure 1

The relative expression of AK2 and prognostic significance in lung adenocarcinoma (LAD). (A) Expressionof AK2 in human LAD of different subtypes analyzed with immunohistochemistry (IHC) staining. Representative immunostaining micrographs of AK2 in LAD at IIASLC/ATS/ERS classification were shown. (magnification, ×200). (B) Semi-quantitative analysis of the stained sections was performed using light microscopy to calculate the immunoreactive score (see methods). (C) Respective percentage of high and low expression of AK2 in five LAD subtypes. (D) Relative AK2 expression was analysed by bioinformatics methods using 548 cases of lung cancer (Ca) and paracancerous tissues (Pa) from TCGA database. (E) Comparison of PFS between the low expression and high expression groups. (F) Comparison of OS between the low expression and high expression groups. Error bars represent the mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01 vs. control group.

Figure 2

Effects of silencing AK2 on cell proliferation, cycle arrest and apoptosis. (A) ΔCt valueof AK2 was examined by qRT-PCR in four LAD cell lines and human normal bronchial epithelial cell line 16HBE. (B) Expression of AK2was detected by qRT-PCR in AK2-shRNA (sh-AK2) or scrambled shRNA(sh-NC) transfectants. (C,D) Impaired cell proliferation in sh-AK2- or sh-NC-transfectedLAD cells was detected by CellomicsArrayScan assay. Growth of A549 and H1299 cells infected by sh-NC (top) or sh-AK2 (bottom) at different time points were shown. (E,F) Cell cycle analysis was applied to examine the effect of AK2 interference on LAD cellscycle. (G,H) Flow cytometric analysis was empolyed to detect the effect of AK2 knockdown on LAD cellapoptosis. Error bars represent the mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01 vs. control group.

Figure 3

Knockdown of AK2 was associated with cell motility and could reverse EMT to MET of LAD cells. (A–D) Transwell assays were utilized to exam the function of AK2 on metastasis and invasion ability of LAD cells. Western blotting assays were performed to detect the change of EMT markers in sh-AK2-transfected H1299 and A549 cells. Error bars represent the mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01 vs. control group. (E) Expression of EMT marker was analyzed by western blot in sh-NC- or sh-AK2-transfected A549 and H1299 cells.After 48 h of the transfection, total proteins were isolated, and EMT marker protein was detected by specific antibody. GAPDH was used as an internal control. The grouping of blots were cropped from different gels. (F,G) Quantification anslyses of EMT marker protein in sh-NC or sh-AK2-transfected A549 and H1299 cells. Error bars represent the mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01 vs. control group.

Figure 4

The effects of AK2 knockdown in combination with HCQ on human A549 cells proliferation in vitro and in nude mice. (A) AK2Knockdown increased punctate LC3 expression in LAD cells. A549 and H1299 cells were respectively co-transfected with an LC3-expressing vector (RFP-GFP-tagged LC3) and sh-NC or sh-AK2. Then cells were fixed and increased green, red, and yellow fluorescent puncta were observed by fluorescent microscopy. (B) Changes of autophagosome-associated proteins in LAD cells infected with lentivirus expressing sh-NC or sh-AK2. A549 and H1299 cells were treated for 72 h with sh-NC or sh-AK2 and cell extracts were analyzed by immunoblots with antibodies against LC3 and p62. Blots were also probed for GAPDH.The grouping of blots were cropped from different gels. (C) After infected with lentivirus expressing sh-NC or sh-AK2 for 72 h, A549 and H1299 cells were treated with or without 10 µM hydroxychloroquine (HCQ) for 24 h. Colonies were stained with 0.1% crystal violet after an additional 10 days’ culture. (D) Quantitative analysis of the number of clones ofsh-NC- or sh-AK2-transfected A549 and H1299 cells combined with or without HCQ. (E) Tumors from nude mice in each treatment group. (F) Tumor volume in each group. Data are expressed as the mean ± SEM, and were analyzed by 2-way ANOVA. (G) Tumor weight in each group. Data were expressed as the mean ± SD, and were analyzed by 2-way ANOVA. Data in B and D are expressed as the mean ± SD, *p < 0.05, **p < 0.01 vs. control group.