Capicua homology protein inhibits the progression of gastric cancer through the PI3K/AKT signaling pathway

Purpose Capicua homolog protein (CIC) played a broad role in the development of cancer in humans, however, its role in the progression of gastric cancer (GC) specically has been unclear. This study aimed to explore the expression of CIC and its potential clinical value in patients with GC. Methods The CIC levels in GC tissues and cell lines were examined by quantitative real-time polymerase chain reaction (qRT-PCR). And the in-vitro effects of CIC expression in MGC-803 cells on their proliferation, invasion, and the progression of epithelial-mesenchymal transition were assessed by CCK-8 assays, Matrigel-invasion analysis, qRT-PCR and Western blot assays, separately. In addition, the effects of downregulation of CIC on the activation of PI3K/AKT signaling pathway were measured using Western-blot analysis. Results The results showed CIC levels were lower in GC tissues and GC cell lines, and these lower CIC levels were correlated with tumor differentiation, Helicobacter pylori infection, TNM stage, and patient survival. In addition, CIC overexpression could promote cell proliferation, invasion, and progression of epithelial-mesenchymal transition in MGC-803 cells. Notably, exotic expression of CIC inactivated the phosphoinositide 3-kinase/protein kinase B signaling pathway. Conclusions In conclusion, our nding suggested CIC could serve as a potential diagnostic and prognostic biomarker and a probable therapy target for GC. MAPK, mitogen-activated protein qRT-PCR, mesenchymal phosphoinositide 3-kinase/protein kinase


Background
Although the morbidity and mortality of gastric cancer (GC) has declined, this disease remains the third most lethal cancer worldwide (1). Because the symptoms of early GC are not readily apparent or easily observed, most patients are already in the advanced stages when diagnosed; therefore, it is critical to investigate a molecular marker for an early diagnosis and treatment of this disease.
Capicua homology protein (CIC) is a member of the SOX-related high-mobility group (HMG) subfamily of proteins that is highly conserved among different species (2). As a transcriptional repressor, CIC contains two highly conserved domains-the HMG-box which mediates DNA binding and nuclear location, and a Cterminal motif C1, which cooperates with the HMG-box for DNA binding (2)(3)(4)(5). Recent studies have reported that CIC functions as a transcriptional regulator and is linked to several cancer and non-cancer diseases, including neurodegenerative disease, T cell lymphoblastic leukemia, and hepatocellular carcinoma (6)(7)(8)(9). In addition, evidence has implicated that CIC functions as a determinant of the sensitivity to inhibitors of the epidermal growth factor receptor and mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase pathway (10), which suggests that CIC plays a broad role in human cancer; however, the function and mechanism of CIC in GC have remained unclear. The epithelialmesenchymal transition (EMT), a complex cellular process, has been found to promote the migration and invasion of various cancer cells (11)(12)(13), including GC (14,15). Previous studies have proved a variety of molecules, including microRNA (16), long non-coding (Lnc)RNA (17,18), and messenger (m)RNA (19,20), contribute to multiple biochemical changes in epithelial cells (21); therefore, it is important to explore the molecules that drive the EMT process to be able to identify a speci c drug that could target the metastasis potential of GC. The aim of the present study was to investigate the function of CIC in metastatic GC and its underlying molecular mechanisms of action in hopes of shedding new light on target therapy for patients with this disease.

Patients and tissue samples
This study was approved and supervised by the Research Ethics Committee of the A liated Hospital of Nantong University (Nantong, China). All patients provided their signed written informed consent. Paired GC and adjacent normal gastric tissues were obtained in 2017 and 2018 from 20 patients who underwent primary surgical GC resection at the hospital. All tissue samples were immediately frozen in liquid nitrogen until use.
The GC specimens were obtained from 183 GC patients at the A liated Hospital of Nantong University (Nantong, China) from 2012 to 2017. All patients had received their rst diagnosis of GC but had not received any other treatment, including chemotherapy, before surgery. The clinicopathological data on the 183 GC patients comprised age, sex, degree of differentiation, histological type, lymph node metastasis, distant metastasis, and TNM stage (Table 1). reaction (qRT-PCR) analysis for the indicated molecules was conducted using SYBR Green Supermix (Thermo Fisher Scienti c). The primer sequences are shown in Table 2; β-actin was used as the internal control. The relative expression of each gene was quanti ed using the 2 −ΔΔCt method as reported (22).

Statistical analyses
Statistical analyses were conducted using SPSS 19.0 (SPS Inc., Chicago, IL, USA). Patient survival rates were calculated using the Kaplan-Meier survival analysis method. Statistically signi cant differences were found using one-way analysis of variance. The results are expressed as the mean ± SD. P < 0.05 was considered statistically signi cant.

CIC is downregulated in human GC tissues and GC cell lines
To reveal the function of CIC in GC tissues and GC cell lines, CIC expression was detected using qRT-PCR and Western blotting. The results indicated that CIC expression in GC tissues was lower than that in adjacent normal tissues in both mRNA and proteins (Fig. 1). In addition, the MKN-45 and MGC-803GC cell lines showed decreased levels of CIC compared with that in GES-1 cells (Fig. 1). Furthermore, low expression of CIC was associated with poor differentiation, lymph node metastasis, and an advanced clinical stage of GC, but not with age, sex, tumor diameter, or tumor location (Tables 3, 4). In addition, the survival time of patients with low CIC expression was shorter than that in those with high CIC expression (Fig. 2).  To investigate the function of CIC on GC progression, the CCK-8 assay revealed that overexpressed CIC signi cantly inhibited cell growth (Figs. 4A, B) after 48 h, and that ectopic expression of CIC in MKN-45 cells inhibited the number of invasive cells compared with that in the control cells (Fig. 4C). These data indicated that CIC was also involved in the invasion of GC expect for proliferation.

CIC inhibited the progression of EMT and activation of the phosphoinositide 3-kinase/protein kinase B signaling pathway
Because EMT is associated with GC metastasis (19,24), the function of CIC was observed on the progression of EMT in MKN-45 cells. As shown in Fig. 3, the mRNA and protein levels of N-cadherin decreased; whereas, E-cadherin increased in the pcDNA-CIC group compared with those in the controls.
When CIC was downregulated, the mRNA and protein levels of N-cadherin increased and that of Ecadherin decreased compared with those in the controls. These results indicated that CIC suppressed EMT progression in the GC cell lines; however, the mechanism remained unclear.
It has been well documented that the phosphoinositide 3-kinase/protein kinase B signaling pathway (PI3K/AKT) signaling pathway plays an important role in the proliferation, invasion, and EMT metastasis of GC (25,26); therefore, we hypothesized whether CIC affects PI3K/AKT signaling. As shown in Fig. 5, CIC signi cantly decreased phosphorylation levels of p-PI3K and p-AKT (T308) but not PI3K and AKT in MKN-45 cells, which suggested that CIC might also play a role in the inactivation of the PI3K/AKT pathway in GC cells.

Discussion
CIC has been proved to be a tumor suppressor and participate in the progression of many cancers, including leukemia(6) and hepatocellular carcinoma (7). Previous studies have revealed that low expression of CIC is associated with tumor differentiation and lymph node metastasis, and to our knowledge, CIC is reported to be involved in several important processes, including organ growth (27), stem-cell proliferation (28), and cell proliferation (29); however, CIC's actual biological functions and its underlying mechanisms have not yet been well elucidated. We rst addressed this issue using MGC-803 and MKN-45 cells, both of which contained lower levels of CIC compared with that in GES-1 cells.
Our results indicated that cells transfected with CIC overexpressed plasmids led to the e cient increased levels of CIC in both mRNA and protein in vitro. In addition, low levels of CIC were associated with poor differentiation, lymph node metastasis, and advanced clinical stages of GC, but not with age, sex, tumor diameter, or tumor location, which suggested that CIC may be a dependent probable prognostic marker in the disease. Moreover, CIC upregulation signi cantly reduced the invasive and proliferative capacity of MKN-45 cells, which suggested that CIC may be associated with GC cell metastasis. Because EMT is a critical process during cancer progression, many molecules have been found to be involved in EMT regulation in cancers. In this study, ectopic CIC resulted in elevated levels of E-cadherin and vimentin and reduced levels of N-cadherin in GC cells, which was the rst time that CIC was demonstrated to act as an inhibitor of EMT modulation and participate in GC progression.
Previous data have shown that CIC is involved in the activation of several signaling pathways, including MAPK (30), Toll/IL-1 (31), and epidermal growth factor(32); however, its involvement in the activation of the PI3K/AKT signaling pathway in GC has not been reported. In the current study, we observed that CIC overexpression triggered inactivation of AKT and PI3K in GC cells, which suggested that activation of PI3K/AKT is regulated by CIC in these cells. It is well known that the activated PI3K/AKT signaling pathway directly modulates the growth, migration, and invasion of several types of cancer cells, including GC cells (33)(34)(35); therefore, it is reasonable to hypothesize that decreased cell proliferation and invasion rates observed in CIC-overexpressed plasmid-transfected GC cells were partly the result of decreased PI3K/AKT activity. While there were some limitations to our study. First, the role of CIC in normal gastric tissues must be further investigated, which would help to better illustrate the expression of CIC in GC.
Second, although our ndings suggested that CIC inhibits the proliferation and invasion of GC cells in vitro, there is no evidence on the roles of CIC in vivo; therefore, we would conduct in vivo experiments to further explore the functions of CIC in GC cells in future.

Conclusions
In conclusion, our current study implicated that CIC, as a new prospect as a diagnostic marker, remarkably inhibits cell proliferation, invasion, migration, and EMT in vitro. In addition, CIC overexpression suppressed the activation of the PI3K/AKT signaling pathway, which might provide valuable information for developing targeted therapeutic mechanisms against GC. Availability of data and materials The datasets used and/ or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.