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. 2021 Jul 28:11:656852.
doi: 10.3389/fonc.2021.656852. eCollection 2021.

Gankyrin as Potential Biomarker for Colorectal Cancer With Occult Liver Metastases

Chengxing Wang  1 , Xiaoping Li  1 , Liangliang Ren  2 , Changyi Ma  3 , Meimei Wu  2 , Weijun Liang  1 , Jinglin Zhao  1 , Shangren Li  1 , Qunying Tan  1 , Yuehua Liao  4 , Lixia Sun  4 , Xin Zhang  2 , Yaoming He  1
Affiliations

Gankyrin as Potential Biomarker for Colorectal Cancer With Occult Liver Metastases

Chengxing Wang et al. Front Oncol. .

Abstract

The majority of occult liver metastases cannot be detected by computed tomography (CT), magnetic resonance imaging (MRI) or other traditionally morphological imaging approaches since the lesions are too small or they have not yet formed cancer nodules. Gankyrin is a small molecular protein composed of seven ankyrin domains. In this study, the expression of Gankyrin in colorectal cancer (CRC) patients with liver metastases was investigated to determine its prognosis value. Gankyrin expression in CRC patients was initially analyzed using data from The Cancer Genome Atlas (TCGA) database and bioinformatics tools. RT-qPCR, western blotting, immunohistochemistry (IHC) and transwell migration and invasion assays were then performed to verify the expression and function of Gankyrin in CRC cell line, CRC tissues and matched non-tumor tissues of clinical patients. General clinicopathological information including TNM stage as well as preoperative and postoperative imaging results were collected. The main outcome indicator was overall survival (OS), referring to the length of time from surgery to either death or the last visit. Statistical analyses included chi-squared tests, Cox analyses, progression free survival (PFS) rates and OS rates. Elevated Gankyrin expression was confirmed in CRC patients. The upregulated Gankyrin expression was positively correlated with the progression of disease and liver metastasis in CRC patients. OS analysis revealed that prognosis was worse in CRC patients with high Gankyrin expression compared to those with low expression. CRC patients with higher Gankyrin expression also had a higher risk of occult liver metastases and a lower PFS rate. Therefore, Gankyrin can be used as a potential biomarker for early diagnosis of CRC with occult liver metastasis.

Keywords: Gankyrin; biomarker; colorectal cancer; liver metastasis; occult.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Bioinformatic analyses suggested that Gankyrin was upregulated in colorectal cancer (CRC). (A) The expression of Gankyrin (PSMD10) in human CRC tissues and matched non-tumor colorectal tissues was analyzed using The Cancer Genome Atlas (TCGA) database (COAD [colorectal adenocarcinoma] and READ [rectum adenocarcinoma])-based Gene Expression Profiling Interactive Analysis (GEPIA) platform. Box plot graph presented statistical results. (B) Using six independent studies from ONCOMINE database, comparison of the relative mRNA expression of Gankyrin in CRC with that in non-tumor colorectal tissue. (C) A meta-analysis of Gankyrin using five data sets is represented by forest plot.
Figure 2
Figure 2
Gankyrin was overexpressed in colorectal cancer (CRC) tissues. (A) Gankyrin expression in CRC and matched non-tumor colorectal tissues were measured by RT-qPCR (n = 40), and values were presented as mean ± standard deviation (SD). (B) The expression of Gankyrin in CRC and matched non-tumor colorectal tissues was detected by western blot (n = 10), and numerical values were expressed as mean ± SD. Scatter plot displayed the difference of Gankyrin expression level. (C) Immunohistochemistry (IHC) staining of Gankyrin in CRC and matched non-tumor colorectal tissues (n = 150; scale bars, 50 μm and 200 μm), numerical values were expressed as mean ± SD. Scatter plot indicated the H-score of Gankyrin staining intensity. **p < 0.01.
Figure 3
Figure 3
High Gankyrin expression was associated with liver metastasis of CRC. (A) Detection of Gankyrin expression in non-tumor colorectal tissues and matched CRC with or without liver metastasis tissues by RT-qPCR (n = 20). (B) The expression of Gankyrin in non-tumor colorectal tissues and matched CRC with or without liver metastasis tissues were detected by western blot (n = 9). Scatter plot showed the difference of Gankyrin expression level. (C) Immunohistochemistry (IHC) staining of Gankyrin in non-tumor colorectal tissues and matched CRC with or without liver metastasis tissues sections (n = 60; scale bars, 50 μm and 200 μm). Scatter plot indicated the H-score of Gankyrin IHC staining intensity. (D) SW480 cells were transfected with lenti-virus overexpressing Gankyrin (SW480-Gankyrin) or empty lentiviral vector (SW480-vector). 48 hours after transfection, total protein extracts were analyzed on Western blot for E-cadherin, N-cadherin and Gankyrin. (E) Matrigel invasion assay was used to measure CRC invasive ability, after 48 hours incubation, invaded cells were stained by hematoxylin and counted under a light microscope. The data represent the means of three independent experiments with SEM bars. In this series of statistical analysis, numerical value was expressed as the mean ± standard deviation (SD). *p < 0.05, **p < 0.01.
Figure 4
Figure 4
X-tile analysis of survival data. (A) The optimum cut-off points for Gankyrin was 226 (H-score) according to the X-tile program. (B) Kaplan-Meier overall survival (OS) analysis of colorectal cancer (CRC) patients with high and low expression levels of Gankyrin. (C) OS analysis of CRC patients with localized disease (stages I + II). (D) OS analysis of CRC patients with advanced disease (stages III + IV).
Figure 5
Figure 5
The expression of Gankyrin association with occult liver metastasis. Through the comparison of gankyin in non-tumor tissues, paracancerous tissues and cancerous tissues of colorectal cancer (CRC) patients, it is revealed that partial patients with high expression of Gankyrin are more likely to be in the state of occult liver metastasis, and then develop into CRC liver metastasis.
Figure 6
Figure 6
The progression free survival (PFS) of colorectal cancer (CRC) patients. The PFS was compared according to the expression of Gankyrin and the presence of liver metastasis, which characterized three groups: low Gankyrin expression without liver metastasis; high Gankyrin expression without liver metastasis; and high Gankyrin expression with liver metastasis.
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