Abstract
Using our data set (GSE50760) previously established by RNA sequencing, the present study aimed to identify upregulated genes associated with colorectal cancer (CRC) liver metastasis (CLM) and verify their biological behavior. The potential roles of candidate genes in tumors were assessed using cell proliferation and invasion assays. Tissue samples were collected from 18 CRC patients with synchronous CLM and two CRC cell lines (SW480 and SW620) were used for transfection and cloning. The roles of the genes identified in CLM were verified using immunohistochemistry in 48 nude mice after intrasplenic transplantation of CRC cells. mRNA and protein expression was determined by quantitative real-time reverse transcription polymerase chain reaction and western blot, respectively. Nine genes were initially selected according to the relevance of their molecular function and biological process and, finally, ALDH1A1 and IGFBP1 were chosen based on differential mRNA expression and a positive correlation with protein expression. The overexpression of ALDH1A1 and IGFBP1 significantly and time-dependently decreased cell proliferation (p ≤ 0.001–0.003) and suppressed invasiveness by ≥3-fold over control cells (p < 0.001) in the SW480 cell line, whereas they had a slight effect on reducing SW620 cell proliferation. The protein expression levels of E-cadherin, N-cadherin, claudin-1, and vimentin were significantly higher in CLM than in primary tumor tissues (p < 0.05). However, the cadherin switch, namely, N-cadherin overexpression with reduced E-cadherin expression, was not observed in CLM tissues and transfected CRC cells. Irrespective of reduced proliferation and invasion found on in vitro cell assays, persistent overexpression of β-catenin, vimentin, and ZO-1 in IGFBP1-overexpressing SW480 cells possibly contributed to CLM development in mice implanted with IGFBP1-overexpressing SW480 cells (CLM occurrences: SW480/IGFBP1-transfected mice vs. SW480/vector- and SW480/ALDH1A1-transfected mice, 4/8 vs. 0/10, p = 0.023). In conclusion, ALDH1A1 and IGFBP1 are differentially overexpressed in CLM and may play a dual role, functioning as both tumor suppressors and metastasis promoters in CRC.
Highlights
Liver metastasis frequently occurs in colorectal cancer (CRC), resulting in the survival of disseminated tumor cells in the liver
These genes were narrowed down to nine genes according to the relevance of their molecular function and biological process [15], namely, cell growth and proliferation, extracellular matrix, epithelial-mesenchymal transition (EMT), and/or cancer stem cell, angiogenesis, chemotaxis, and apoptosis, as determined by the Gene Ontology Consortium: IGFBP1, hepatocyte growth factor activator (HGFAC), chemokine ligand 16 (CCL16), inhibin beta E (INHBE), activating transcription factor 5 (ATF5), proteoglycan 4 (PRG4), cadherin 2 type 1 (CDH2), ALDH1A1, and ERBB receptor feedback inhibitor 1 (ERRFI1) (Fig 1)
The relative mRNA expression was significantly higher in CLM (2ΔCtCLM-ΔCtNCE) than in primary CRC (PCC) (2ΔCtPCC-ΔCtNCE) samples, and the expression of six genes differed by >1-fold (p 0.001–0.003)
Summary
Liver metastasis frequently occurs in colorectal cancer (CRC), resulting in the survival of disseminated tumor cells in the liver. In liver metastasis of CRC (CLM), the fate of tumor cells is primarily determined by their interactions with hepatic sinusoidal/extrasinusoidal cells [2]. Hepatic stellate cells play a major role in CLM by releasing various factors that promote CLM, including growth factors [transforming growth factor-β (TGF-β), epidermal growth factor, vascular endothelial growth factor, and insulin-like growth factor (IGF)-I] and metalloproteinases [3]. The six members of the IGF-binding protein (IGFBP) family were initially characterized as passive reservoirs of circulating IGFs but were later shown to play diverse roles in intracellular and pericellular compartments in the regulation of cell growth and survival [4]. Previous studies that investigated the relationships between altered serum IGFBP levels and the presence or risk of various cancers had inconclusive and contradictory results [4,5]
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