NCAPG promotes the oncogenesis and progression of non-small cell lung cancer cells through upregulating LGALS1 expression

Abstract

Background: Numerous common oncogenic driver events have been confirmed in non-small cell lung cancer (NSCLC). Although targeted therapy has revolutionized NSCLC treatment, some patients still do not respond. NCAPG, also known as non-SMC condensin I complex subunit G, was positively associated with proliferation and migration in several tumor types.

Methods: We used transcriptional sequencing and TCGA database analysis to identify NCAPG as a new therapeutic target for NSCLC. The oncogenic roles of NCAPG in NSCLC tumor growth and metastasis were detected in vitro and in vivo. Ncapg^+/+ or Ncapg^+/- mice with urethane treatment were analyzed for oncogenesis of NSCLC.

Results: We investigated NCAPG as a new oncogenic driver which promoted NSCLC tumorigenesis and progression. We used transcriptome sequencing and the Cancer Genome Atlas (TCGA) database analysis to screen and found that NCAPG was negatively correlated with NSCLC survival. Using immunohistochemistry, we demonstrated that NCAPG overexpression was an independent risk factor for NSCLC survival. Functionally, NCAPG knockdown inhibited proliferation, migration, and invasion of NSCLC cells in vitro and in vivo. We exposed wildtype or Ncapg^+/- mice to urethane and discovered that urethane-induced lung tumors were reduced in Ncapg^+/- mice. Mechanistically, the function of NCAPG in promoting initiation and progression of NSCLC was closely related to LGALS1, which was also upregulated in NSCLC and might interact directly with NCAPG.

Conclusions: This study indicates that NCAPG is one of the essential factors for NSCLC oncogenesis and progression, providing a new target for prognosis prediction and treatment of NSCLC.

Keywords: LGALS1; NCAPG; NSCLC; Oncogenesis; Progression.

Fig. 1

Identification of NCAPG expression in NSCLC. A Gene sequencing volcano plots of NSCLC tumor tissues and adjacent normal tissues (blue indicates downregulated genes; red indicates upregulated genes). B GO-BP analysis of upregulated genes in tumor tissues. C Heatmaps of all 39 upregulated genes involved in cell cycle, mitotic division, cell division, and mitotic cell cycle. D The 13 upregulated genes highly expressed in NSCLC tumor tissues from the TCGA database. E The relationship between NCAPG expression and survival in NSCLC from the TCGA database. F, G NCAPG expression level in the TCGA and GEO databases. (*** p < 0.001)

Fig. 2

Overexpression of NCAPG in NSCLC tissues and cell lines. A NCAPG expression in NSCLC tumor tissues by IHC. Left: negative; middle & right: positive; top: adenocarcinoma; bottom: squamous cell carcinoma. B The relationship between NCAPG expression and survival in NSCLC patients. C-E This negative correlation between NCAPG expression and survival was identified in elderly patients (≥ 60 years old) (n = 84, p = 0.0022), adenocarcinoma (n = 92, p = 0.0239), and squamous cell carcinoma (n = 43, p = 0.0296). F, G NCAPG mRNA (F) and protein expression (G) in NSCLC tumor tissues (n =21) (T: tumor tissues, N: matched adjacent normal tissues). H, I NCAPG mRNA (N: Average NCAPG mRNA expression in adjacent normal tissues (n = 21)) and protein (N: Representative protein level of NCAPG in adjacent normal tissues) expression in NSCLC cell lines

Fig. 3

The effects of NCAPG knockdown in vitro and in vivo. A, B The decreased mRNA and protein expression of NCAPG in A549 cells following NCAPG knockdown were analyzed using RT-qPCR and Western blot. C-E The inhibited abilities of proliferation (C), migration (D), and invasion (E) of A549 cells after NCAPG knockdown were shown. (*** p < 0.001). F Tumors dissected from subcutaneous axilla of mice injected with LV-shNCAPG H1299 cells and LV-shCtrl H1299 cells, and the expression of NCAPG, SPARC, and Ki-67 of tumors was tested using immunohistochemistry. G, H Tumor volumes (G) and tumor weights (H) of xenografts in LV-shNCAPG and LV-shCtrl group. I The photograph of pulmonary tumors formed by injecting LV-shNCAPG or LV-shCtrl H1299 cells into caudal vein. J The number of metastatic lung tumors in LV-shNCAPG and LV-shCtrl group

Fig. 4

Urethane-induced lung tumor in Ncapg^+/+ and Ncapg^+/− mice. A The strategy of spontaneous lung tumor induced by urethane. B NCAPG mRNA expression analysis of the main organs in Ncapg^+/+ and Ncapg^+/− mice. C The photograph of urethane-induced lung tumor and representative images of hematoxylin-eosin staining in Ncapg^+/+ and Ncapg^+/− mice. D, E Number (D) and Volume (E) of lung tumors induced by urethane in Ncapg^+/+ and Ncapg^+/−mice. (* p < 0.05)

Fig. 5

LGALS1 was identified as an important NCAPG-interacting protein. A The SDS-PAGE image with Coomassie Blue Staining before mass spectrometry identified NCAPG-interacting proteins in A549 cells. B The highest score of NCAPG-interacting protein network was measured with biological function enrichment analysis. C LGALS1 was co-precipitated by NCAPG in A549 cells. D For each panel, the images from left to right showed PLA signal in A549 cells (red), cell nuclei stained by DAPI (blue), and overlays of the two images. Scale bar 20 μm. E The histogram displays the relative fluorescent value of PLA punctuates and nuclei. F PLA signal of A549 cells for different conditions by flow cytometer. G Western blot analysis for LGALS1 protein after LGALS1 knockdown in A549 cells. H-M The abilities of proliferation, migration, and invasion were decreased after LGALS1-knockdown in H1299 and A549 cells. Scale bar 50 μm. (** p < 0.01, *** p < 0.001)

Fig. 6

The proliferation and metastasis of NSCLC cells depended on NCAPG/LGALS1/SPARC axis. A Western blot was performed in A549 and H1299 cells with NCAPG knockdown and/or LGALS1 overexpression. B, C CCK8 analysis was used to detect the proliferation of A549 and H1299 cells. D-F The proliferation of A549 and H1299 cells was tested by flow cytometer. G-K The abilities of migration and invasion were measured through transwell analysis with NCAPG knockdown and/or LGALS1 overexpression. Scale bar 50 μm. (** p < 0.01, *** p < 0.001)