CDCA2 promotes lung adenocarcinoma cell proliferation and predicts poor survival in lung adenocarcinoma patients

Abstract

Cell division cycle associated 2(CDCA2) is overexpressed in neuroblastoma and oral squamous cell carcinoma, and its overexpression positively correlates to tumor progression. However, the biological and clinical significance of CDCA2 in lung adenocarcinoma(LAC) has never been investigated. We determined the expression profile and clinical significance of CDCA2 using The Cancer Genome Atlas(TCGA) and tissue microarray(TMA). Furthermore, we explored the biological function of CDCA2 both in vitro and in vivo. A great upregulation of CDCA2 was observed in LAC tissues compared with adjacent normal tissues. Importantly, Cox regression analysis indicated that high level of CDCA2 was an independent risk factor for overall survival(OS) in LAC patients (TCGA: HR = 1.720, p = 0.004; TMA: HR = 1.971, p = 0.023). Inhibition of CDCA2 suppressed the proliferation of LAC cells via G1 phase arrest by downregulating cyclin E1(CCNE1), while overexpression of CDCA2 promoted LAC cells proliferation by upregulating CCNE1. Moreover, the oncogenic activity of CDCA2 was also confirmed in vivo. In conclusion, CDCA2 promotes proliferation of LAC cells and predicts poor prognosis in LAC patients. CDCA2 might play a significant role in LAC progression.

Keywords: CDCA2; TCGA; lung adenocarcinoma; prognosis; proliferation.

Figure 1. CDCA2 is upregulated in LAC tissues and correlates with more aggressive clinical characteristics in TCGA dataset

(A) CDCA2 is over-expressed in 94.7% (54 of 57) of LAC tissues compared with para-tumor normal tissues. (B and C) CDCA2 was found to be positively correlated with Ki-67 (r = 0.8189, p < 0.0001) and DNA ploidy (p < 0.0001) in LAC tissues. (D) Kaplan-Meier survival analysis indicated that higher CDCA2 expression is associated with worse overall survival in LAC patients (HR = 1.919, p = 0.0002).

Figure 2. Tissue microarray analysis

(A) Representative images of CDCA2 staining in different histological differentiation levels (from well to poor). (B) Representative images of CDCA2 staining in different tumor sizes (from T1 to T4). (C) CDCA2 staining score was significantly increased in LAC tissues compared to adjacent normal tissues. (D) CDCA2 staining score was significantly increased along with worse differentiation. (E) CDCA2 staining score was significantly increased along with advanced T stage. (F) Kaplan-Meier survival analysis and log-rank test indicated that high level of CDCA2 expression was associated with poor overall survival (HR = 2.073, p = 0.005). *p < 0.05, **p < 0.01, ***p < 0.001, ^#No significance.

Figure 3. Knockdown of CDCA2 inhibited LAC cell lines proliferation and induced G1 phase arrest in vitro

(A and B) A549 and H1299 cell lines were chosen as appropriate cellular models to knockdown CDCA2 for further investigation. (C and D) CCK-8 assays showed knockdown of CDCA2 inhibited both A549 and H1299 cells proliferation. (E and F) Colony numbers of A549 and H1299 cells transfected with siRNA-CDCA2 were less than those transfected with siRNA-NC. (G and H) H1299 and A549 cells transfected with siRNA-CDCA2 exhibited more arrest at G1 phase than those transfected with siRNA-NC. (I and J) No difference of apoptosis was observed between siRNA-CDCA2 group and siRNA-NC group in H1299 and A549 cells. *p < 0.05, **p < 0.01, ***p < 0.001, ^#No significance.

Figure 4. Knockdown of CDCA2 influences CCNE1 expression

(A) Genes co-expressed with CDCA2 are enriched in the “cell cycle” pathway using GO enrichment analysis. (B and C) qRT-PCR showed that CCNE1 mRNA levels decreased after siRNA-CDCA2 transfection in both H1299 and A549 cells, while CCND1, p21 or p27 was not significantly altered. (D) Western bolt also showed that CCNE1 protein levels decreased after siRNA-CDCA2 transfection in both H1299 and A549 cells, while CCND1, p21 or p27 was not significantly altered. (E) Pearson test showed that CDCA2 positively correlated with CCNE1 (r = 0.6491, p < 0.0001, n = 511) in LAC tissues in TCGA dataset. ***p < 0.001, ^#No significance.

Figure 5. Overexpression of CDCA2 enhanced LAC cells proliferative ability in vitro

(A) CCK-8 assays showed that overexpression of CDCA2 promoted H1975 and SPCA-1 cells proliferation compared to empty vector (EV) control. (B) Transfection efficiency of CDCA2 overexpression was measured using western blot. CCNE1 was increased after CDCA2 expression was upregulated. (C and D) Colony numbers of H1975 and SPCA-1 cells transfected with CDCA2 plasmid were significantly more than those in EV groups. (E and F) H1975 and SPCA-1 cells transfected with CDCA2 plasmid exhibited less percentage at G1 phase than those in EV groups. (G and H) No difference of apoptosis was observed between CDCA2-overexpression group and EV group in H1975 and SPCA-1 cells. **p < 0.01, ***p < 0.001, ^#No significance.

Figure 6. Enforced overexpression of CCNE1 partly rescues the malignant phenotypes in CDCA2-knockdown cells

(A) Enforced overexpression of CCNE1 (oe-CCNE1) could partially reverse the shRNA-CDCA2-mediated proliferation inhibition of A549 cells. (B) Transfection efficiency of CCNE1 was determined by western blot. (C and D) The colony formation ability in shRNA-CDCA2-mediated A549 cells was partially recovered after enforced oe-CCNE1 treatment (E and F) Flow-cytometry analysis showed that enforced oe-CCNE1 significantly alleviated shRNA-CDCA2-mediated G1-phase arrest in A549 cells. **p < 0.01, ***p < 0.001.

Figure 7. Knockdown of CDCA2 inhibits tumor growth in vivo

(A) Xenograft model in nude mices. (B) Nodules harvested from sh-NC group and sh-CDCA2 group. (C and D) Tumor nodules derived from sh-CDCA2-transfected A549 cells are significantly smaller than those in sh-NC group. (E and F) Immunohistochemistry showed that CCNE1 and Ki-67 staining was weaker in sh-CDCA2 group, as well as CDCA2.