CIP4 Expression is Associated With The Prognosis in Colorectal Cancer

Background: This study was conducted to detect the expression of Cdc42 interacting protein 4 (CIP4) in patients with colorectal cancer (CRC), and explore the role of CIP4 in prognosis of CRC patients. Methods: The expression of CIP4 mRNA was determined by quantitative real-time PCR (qRT-CPR) and compared by student’s t-test between groups. Relationships of clinical characteristics and CIP4 expression were analyzed by Chi-square test. Kaplan-Meier curves were used to estimate the overall survival of CRC patients. And Cox regression analysis was conducted to identify the prognostic biomarkers for CRC patients. Results: The qRT-PCR results showed that CRC tissues were detected with signicantly high CIP4 mRNA expression compared with adjacent normal controls (P<0.0001). The overexpression of CIP4 in CRC tissues was inuenced by distant metastasis (P=0.021), lymphatic invasion (P=0.012) and TNM stage (P=0.006). But, other clinical factors including age, gender, differentiation and tumor site were proved to have no obvious effects on CIP4 expression (all, P>0.05). The survival curves showed that patients with high CIP4 expression generally lived shorter than those with low CIP4 expression (P<0.001). In addition, the multivariate analysis revealed that differentiation (P=0.044, HR=1.631, 95%CI=1.013-2.626) and CIP4 expression (P=0.000, HR=5.283, 95%CI=3.138-8.893) were of great prognostic signicance for CRC patients. Conclusion: Taken together, up-regulation of CIP4 in CRC tissues represented poor prognosis for patients.


Background
Colorectal cancer (CRC) is one of the most common malignancies worldwide with increasing incidence rate, especially in developed counties [1,2]. According to the global cancer statistics, CRC is proved to be the third common type of cancer and the fourth leading cause of cancer-related death in the world [3,4]. Moreover, it has been reported that there are more than 1 million newly diagnosed CRC cases annually and CRC is responsible for more than 600,000 death cases each year in the word [5,6]. At present, the treatment strategies for CRC patients are mainly surgical resection, which is the optimal method [7,8].
However, the prognosis of CRC patients is still signi cantly poor because of the recurrence, metastasis and advanced stages [9,10]. The 5-year overall survival rate of CRC patients has been claimed to be less than 60%, and even 10-15% in certain metastasis cases [11,12]. Although some prognostic factors have been used for the prognosis of CRC, the survival time varies widely in patients with different TNM stage and grade. As a result, it is essential to nd e cient biomarker to predict and treat CRC.
Cdc42-interacting protein 4 (CIP4), also known as TRIP10, is a member of the F-BAR (Fes-CIP4 homology-Bin/Amphyphysin/Rvs) protein family, which consists of a N-terminal F-BAR domain, a HR1 (PKN homology region-1) domain and a C-terminal SH3 (SRC homology 3) domain [13,14]. It has been demonstrated that CIP4 protein is composed of 545 amino acids, interacts with Cdc42 protein as a downstream effector of Cdc42 [15]. What's more, numerous studies have suggested that CIP4 is implicated in a variety of biological regulation progresses, such as allergic response, glucose metabolism, membrane deformation and tubulation, endocytosis, vesicle scission and remodeling of actin cytoskeleton [16][17][18]. The aberrant expression of CIP4 has been investigated in various cancer, including breast cancer and CRC [16,19]. However, the clinical role of CIP4 in CRC prognosis was still unclear.
In the present study, we were engaged in determining the expression of CIP4 in CRC and estimating its prognosis value in patients with this disease.

Methods
Patients and specimens A total of 117 CRC patients who were subjected to surgical resection in the Southwest Hospital, Army Medical University were enrolled in our study. Patients with preoperative chemotherapy or radiotherapy were excluded from our investigation. Clinical information of the patients was recorded, including age, gender, differentiation, tumor size, distant metastasis, TNM stage and lymphatic invasion. The CRC tissue samples and the adjacent non-carcinoma tissues were obtained by surgery and immediately put into liquid nitrogen then stored at -80℃ for use. All patients were followed up through telephone calls in a 5year duration. Moreover, the study was supported by the Ethics Committee of Southwest Hospital, Army Medical University. And each patient had provided the written the informed consents in advance.

Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from CRC tissues and adjacent normal controls with Trizol reagent (Invitrogen) following the manufacture's instructions. Then the rst strand of cDNA was synthesized by iScript cDNA Synthesis Kit (BioRad). Finally quantitative real-time PCR was conducted using iQ SYBR Green reagent (BioRad) with a MyiQ Color Real-Time PCR Detection System (BioRad). Expression of CIP4 mRNA was normalized to GAPDH. Each sample was treated in triplicate.

Statistical analysis
All analyses were carried out with SPSS 18.0 (SPSS, Inc., Chicago, IL, USA) and Sigmaplot 12.5 (Systat Software Inc.) softwares. The difference of CIP4 expression in CRC tissues and controls was compared using the student's t-test. Relationships of clinical factors with CIP4 expression were analyzed by Chisquare test. The overall survival rate of patients was analyzed by Kaplan-Meier survival curves. And the Cox regression analysis was used to evaluate the prognostic signi cance of clinical factors in CRC. The results were considered to be statistically signi cant when P was less than 0.05.

Increased expression of CIP4 mRNA in CRC tissues
The expression of CIP4 mRNA was determined in 117 pairs of CRC tissue samples and controls using qRT-PCR. As shown in Fig. 1, the expression level of CIP4 mRNA was signi cantly higher in CRC tissues than in the controls (3.42 ± 0.80 vs 1.91 ± 0.51). Signi cant difference was found between the two groups (P < 0.0001).

Association between CIP4 expression and clinical characteristics of CRC patients
According to the median expression level of CIP4 (3.45), all patients were divided into two groups manually: the high expression group (n = 59) and the low expression group (n = 58). As shown in Table 1, overexpression of CIP4 was signi cantly related with distant metastasis (P = 0.021), lymphatic invasion (P = 0.012) and TNM stage (P = 0.006). However, no signi cant relationship was observed between CIP4 expression and other clinical parameters, including age (P = 0.074), gender (P = 0.079), differentiation (P = 0.079) and tumor size (P = 0.116).  Fig. 2, patients with high expression of CIP4 had lower survival time than those with low CIP4 expression (P < 0.001). In addition, Cox univariate analysis suggested that differentiation, distant metastasis, lymphatic invasion, TNM stage and CIP4 expression were related with prognosis of CRC patients (Table 2). Furthermore, the multivariate analysis further revealed that differentiation (P = 0.044, HR = 1.631, 95%CI = 1.013-2.626) and CIP4 expression (P = 0.000, HR = 5.283, 95%CI = 3.138-8.893) were two independent biomarkers for CRC patients prognosis.

Discussion
CRC is one of the most common malignant tumors in the world with increasing incidence rate year by year [20,21]. In recent years, with the progress of surgical treatment and the advent of new chemotherapy drugs as well as the popularity of endoscopic diagnosis technology, the curative effects of patients are signi cantly improved to a certain extent. However the 5-year survival of patients is still unsatis ed, especially for those with advanced stage. This is mainly caused by lack of knowledge about survivalrelated factors and lack reasonable prognostic evaluation system. It is apparent that the understanding on tumors has been deepened with the development of molecular biology. Therefore, the molecular biology will be bene t for early diagnosis and prevention of CRC to explore the prognostic biomarkers and treat these markers with positive intervention.
CIP4 is a skeleton protein of CDC42, which is widely present in human organs, such as brain, trachea, liver, kidney, colon, heart, lung and prostate. It has been revealed that CIP4 is involved in the process of epithelial-mesenchymal transition (EMT), which is important for the embryonic development, chronic in ammation and cancer metastasis, through regulating the endocytosis of E-cadherin via different signaling pathways [22,23]. Besides, CIP4 is reported to mainly regulate the polymerization of actin and dynamics of cell membrane and to stabilize the tono laments to recombine ctyoskeleton. Moreover, CIP4 also plays an important role in cell morphology, cell polarity, cell adhesion, intracellular transport, and signal transduction. So far, up-regulation of CIP4 has been investigated in various diseases, indicating CIP4 might be related with disease progression. For example, Malet-Engra et al. showed that the expression of CIP4 was at high level in chronic lymphocytic leukemia [24]. In the study of Otto et al., they found that in human invasive breast cancer, high CIP4 level was signi cantly associated with tumor progression and promoting disease metastasis [25].
In the present study, we determined the expression of CIP4 in CRC tissues and then investigated its role in prognosis of CRC patients. The expression of CIP4 in CRC samples was signi cantly higher than that in paired normal controls. Besides, the Chi-square test demonstrated that CIP4 overexpression was closely related with distant metastasis, lymphatic invasion and advanced TNM stage. The above results con rmed the previous assumption, indicating CIP4 might be involved in the development and progression of CRC. Based on the above results and hypothesis, we further explored the prognostic signi cance of CIP4 for CRC patients. The survival curves showed that patients with high CIP4 level were more easily to die than those with low CIP4 expression, concluding that CIP4 up-regulation represented unfavorable prognoses in CRC. Meanwhile, the Cox regression analysis suggested CIP4 expression was a prognostic marker for CRC patients.
It is well known that invasion and metastasis are two important features of malignant tumors, and also main causes for cancer-related deaths. There are evidences proving that CIP4 expression is signi cantly related with cell metastasis and invasion. Truesdull et al. demonstrated that CIP4 expression was greatly elevated and could promote tumor metastasis in lung adenocarcinoma [26]. Besides previous study also proved that CIP4 expression was signi cantly elevated and could promote cell metastasis in triplenegative breast cancer [25]. These might provide theoretical foundations for us to further investigate the mechanisms of CIP4 on CRC development and progression.

Conclusions
In conclusion, CIP4 was highly expressed in CRC tissues compared with paired normal controls. Upregulation of CIP4 was signi cantly related to distant metastasis, lymphatic invasion and TNM stage. From the survival curve, patients with low CIP4 expression had more favorable survival than those with high CIP4 expression. Cox regression analysis revealed CIP4 expression was a promising candidate marker for CRC prognosis.

Declarations
Ethics approval and consent to participate This study was supported by the Ethics Committee of Southwest Hospital, Army Medical University and also has been carried out in accordance with the World Medical Association Declaration of Helsinki.
The subjects had been informed the objective. Certainly, written consents were signed by every subject in this study.

Consent for publication
We obtaining permission from participants to publish their data.
Availability of data and materialsAll data generated or analysed during this study are included in this published article.Competing interestsThe authors declare that they have no competing interests.Authors' contributions K.Z. and Z.W. design of the work; T.M. and Z.H. the acquisition, analysis, X.W. and Y.P. interpretation of data; Y.C. and Y.D. the creation of new software used in the work; Z.R. have drafted the work or substantively revised it. All authors read and approved the nal manuscript.

Figure 1
Expression of CIP4 mRNA was detected by qRT-PCR in CRC tissues and paired normal controls. The result claimed a higher level of CIP4 mRNA in CRC tissues than normal controls (P<0.0001).