DISC1 overexpression promotes non-small cell lung cancer cell proliferation

Neuropsychiatric disorder-associated disrupted-in-schizophrenia-1 (DISC1) activates Wnt/β-catenin signaling by inhibiting glycogen synthase kinase 3 beta (GSK3β) phosphorylation, and may promote neural progenitor cell and pancreatic β-cell proliferation. The present study found that DISC1 promotes non-small cell lung cancer (NSCLC) cell growth. Western blotting and immunohistochemistry analyses showed that DISC1 was highly expressed in NSCLC cell lines and patient tissues. DISC1 expression was negatively associated with phosphorylated (p-) GSK3β, but positively correlated with a more invasive tumor phenotype and predicted poor NSCLC patient prognosis. siRNA-mediated DISC1 silencing increased p-GSK3β expression and decreased expression of β-catenin and Cyclin D1, while DISC1 upregulation produced the opposite results. DISC1 knockdown also reduced NSCLC cell proliferation rates in vitro. These results suggest that DISC1 promotes NSCLC growth, likely through GSK3β/β-catenin signaling, and that DISC1 may function as an oncogene and novel anti-NSCLC therapeutic target.


INTRODUCTION
Lung cancer is the most frequently occurring cancer type, and the leading cause of cancer death globally [1,2]. Most lung cancer cases are diagnosed at an advanced stage, and approximately 85% are nonsmall cell lung cancer (NSCLC) [1,3]. NSCLC patient prognosis remains poor; five-year disease-specific mortality rates for stages I-IIIA are 30%, 60%, and 75%, respectively [4]. New diagnostic markers are urgently needed to enhance early lung cancer detection and targeted treatment.
The present study assessed DISC1 expression in human NSCLC cell lines and tumor tissues, and evaluated relationships between DISC1 expression, clinicopathological features, and patient prognosis. We also investigated the role of DISC1 in cancer cell proliferation. We demonstrated that high DISC1 expression promotes NSCLC growth, possibly through GSK3β/β-catenin signaling.

DISC1 expression in human NSCLC cell lines and tissues
Western blotting results showed that DISC1 and PCNA (a cancer cell proliferation marker) levels were higher in NSCLC tissues than in paired adjacent nontumor tissues ( Figure 1A-1C), and exhibited similar expression patterns. DISC1 was also highly expressed in the NSCLC cell lines, A549, H1299, and SPCA-1 ( Figure  1D-1E).

DISC1 expression is correlated with NSCLC patient clinicopathological parameters
DISC1 and the Wnt pathway molecules, p-GSK3β (Phosphorylation at Tyr216), β-catenin, Cyclin D1, and Ki-67, were measured in 140 NSCLC patient tissue samples via immunohistochemical (IHC) analysis. DISC1 and β-catenin were expressed in the cytoplasm and nucleus, p-GSK3β was prominently expressed in cytoplasm, and Ki-67 was expressed in the nucleus. DISC1, β-catenin, Cyclin D1, and Ki-67 levels increased from well-to poorly-differentiated NSCLC tissues (Figure 2), and increased with increasing tumor grade. DISC1 and p-GSK3β levels were negatively correlated, indicating that DISC1 might promote NSCLC development through negative regulation of GSK3β.
Only 36% (26/73) of the 140 NSCLC patients analyzed survived from the high DISC1 expression group, while 66% (44/67) survived from the low expression group (Table 2), overall survival was calculated from the date of surgery to death or the date of last follow-up. DISC1 (P<0.01), tumor size (P=0.040), pathology grade (P<0.01) were correlated with patient survival status ( Table 2). Cox's proportional hazards model revealed that DISC1 (P<0.01) and Ki-67 (P=0.030) levels were independent prognostic factors for patient overall survival (Table 3). Kaplan-Meier analysis associated high DISC1 expression with poor patient survival (P<0.001; Figure 4).

Effects of DISC1 on NSCLC cell cycle progression
DISC1 expression was correlated with that of the cell proliferation markers, PCNA and Ki-67, suggesting that DISC1 might regulate NSCLC cell cycle progression. Flow cytometry results showed that A549 cells, which exhibited the highest DISC1 levels of the three tested NSCLC cell lines, were blocked in G0/G1 phase after serum starvation for 72 h. When serum-free medium was replaced with 10% FBS-supplemented medium, cells were released from G1 phase and entered S phase ( Figure 5A). The number of cells in G1 phase decreased from 91.50% to 52.46%, and those in S phase increased gradually from 5.14% to 29.78%. Western blotting analysis showed that DISC1, Cyclin D1, and PCNA levels increased after serum supplementation ( Figure 5B). www.impactjournals.com/oncotarget  Effects of DISC1 silencing on NSCLC cell proliferation DISC1-targeted siRNAs (DISC1-siRNA1, DISC1-siRNA2 and DISC1-siRNA3) and a negative control siRNA (NC-siRNA) were used to assess the impacts of DISC1 on cell proliferation. DISC1 levels decreased in DISC1-silenced A549 cells, as shown by western blotting (Figure 6A-6B). DISC1-siRNA2, which resulted in the greatest level of silencing, was employed in the following experiments. PCNA and Cyclin D1 levels decreased in cells treated with DISC1-siRNA2 ( Figure 6C). DISC1 knockdown also reduced NSCLC cell growth rates as shown by CCK-8 assay ( Figure 6D) and decreased cell colony formation ( Figure 6E-6F).

DISCUSSION
Uncontrolled cell proliferation is a major factor in lung cancer development and progression [24]. A more complete understanding of proliferation mechanisms is necessary for the development of novel therapeutics that target uncontrolled cell growth. Our results showed that DISC1 overexpression in NSCLC tissues correlated with patient clinicopathologic factors and predicted poor prognosis, and high DISC1 expression promoted NSCLC cell proliferation. These results indicated that DISC1 might be a novel oncogene, and could promote NSCLC progression.   DISC1 was first associated with a variety of brain disorders, including schizophrenia, mood disorders, and autism [10][11][12]. DISC1 is an important neural progenitor cell proliferation regulator that positively modulates canonical Wnt signaling by inhibiting GSK3β catalytic activity, which activates β-catenin [14]. We found that DISC1 was overexpressed in NSCLC patient tissues, and hypothesized that it might promote NSCLC cell proliferation. IHC analyses demonstrated that DISC1 expression in NSCLC patient tissues correlated with     that of p-GSK3β, β-catenin, Cyclin D1, and Ki-67, and was associated with clinicopathologic variables. Cox's proportional hazards model indicated that DISC1 could be an independent prognostic factor for NSCLC patient survival, and Kaplan-Meier analysis showed that DISC1 overexpression predicted poor survival. Finally, a serum starvation and release assay indicated that DISC1 promoted A549 cell proliferation and might play a role in cell cycle progression. Neurological studies reported that DISC1 directly interacts with GSK3β, and suggested that GSK3β promotes β-catenin phosphorylation and ubiquitylation-mediated β-catenin degradation in the proteasome [16][17][18][19]. However, DISC1 inhibits GSK3β activity by reducing β-catenin phosphorylation and stabilizing β-catenin during brain development, which in turn activates transcription of β-catenin target genes, including Cyclin D1 [20]. Whether or not DISC1 activates Wnt/β-catenin signaling through GSK3β inhibition in NSCLC remains unknown. Our results indicated that DISC1 might act as an oncogene in NSCLC development through Wnt/β-catenin signaling, and high DISC1 expression correlated with decreased GSK3β phosphorylation. Additionally, DISC1 knockdown increased p-GSK3β expression and decreased that of β-catenin and Cyclin D1, while DISC1 upregulation produced the opposite results. In summary, we identified a novel role for DISC1 as a potential NSCLC oncogene. Our study suggests that DISC1 might promote NSCLC cell proliferation through GSK3β/β-catenin signaling, and could be a novel anti-NSCLC therapeutical target.

Patient tissue samples
Eight paired NSCLC and adjacent non-tumor tissues were collected immediately after surgical removal and stored at -80°C for western blot analysis. Additionally, 140 NSCLC specimens, along with patient demographic information and clinical data (Table 1), were obtained from the Department of Pathology, Affiliated Hospital of Nantong University between 2008 and 2013 for IHC analysis. Informed consent was obtained from all study participants. The Ethics Committee of the Affiliated Hospital of Nantong University provided permission to use tissue sections for research purposes.

siRNAs, plasmid and transfection
siRNAs targeting DISC1 and negative control siRNA (NC-siRNA) were purchased from GenePharma Co., Ltd (China) ( Table 4), and a DISC1 over-expression plasmid (DISC1-Flag) (GeneCopoeia, China) was also used. siRNAs and plasmid were transfected into NSCLC cell lines using Lipofectamine 2000 (Life Technologies, USA) according to the manufacturer's instructions.

Cell cycle assay
A serum starvation and release process was used for cell cycle synchronization. Briefly, A549 cells were cultured in RPMI 1640 medium without FBS for 72 h, and were then replaced by 10% FBS-supplemented medium. Cells were then fixed in 70% ethanol for 1 h at 4°C and incubated with 1 mg/mL RNaseA for 30 min at 37°C. Cells were stained with propidium iodide (PI, 50 μg/mL) (Becton Dickinson, USA) in PBS and 0.5% Triton X-100, and analyzed using a BD FACScan flow cytometer (Becton Dickinson, San Jose, USA) and the CellQuest acquisition and analysis program.

Cell proliferation assay
Cell counting kit-8 (CCK8) assay was performed to evaluate cell proliferation. A549 cells were seeded into 96well cell culture cluster plates (Corning, USA) at 2×10 4 cells/ well, cultured overnight, and then transfected with DISC1-siRNA2 and negative control siRNA. CCK-8 reagents (Dojindo, Japan) were added to each well at different time points, and wells were incubated for an additional 2 h at 37°C in the dark. Absorbency was measured at 450 nm (reference 650 nm) with a microplate reader (Bio-Rad, USA). Experiments were repeated at least three times.

Colony formation assay
Transfected A549 cells were seeded into 6-well cell culture cluster plates (Corning) at 200 cells/well, and incubated at 37°C with 5% CO 2 for 10 d. Surviving colonies (>50 cells/colony) were stained with 0.5% crystal violet and counted. SPSS 19.0 software was used for statistical analysis. DISC1 expression and clinicopathological features were analyzed using the Chi-square (χ 2 ) test. Kaplan-Meier curves were constructed and Log-rank test was performed to analyze survival data. Multivariate analysis was performed using Cox's proportional hazards model. The risk ratio and its 95% confidence interval were recorded for each marker. P<0.05 was considered statistically significant. All values were expressed as means ± SD. Each experiment was repeated at least three times.