AMIGO2 as a novel indicator of liver metastasis in patients with colorectal cancer

Abstract

Our previous study showed that adhesion molecule with immunoglobulin like domain 2 (AMIGO2) is a pivotal driver gene of liver metastasis via regulating tumor cell adhesion to liver endothelial cells in mouse models. The aim of the present study was to clarify the role of AMIGO2 in liver metastasis in patients the colorectal cancer (CRC). Two human CRC cell lines, Caco-2 (AMIGO2-low) and HCT116 (AMIGO2-high), were used in this study. AMIGO2-overexpressing Caco-2 and AMIGO2-knockdown HCT116 cells were generated by transfection with an AMIGO2 expression vector or AMIGO2 small interfering RNA, respectively. Cell proliferation, invasion and adhesion to human liver endothelial cells were examined in in vitro studies. Immunohistochemical analysis was also performed to evaluate the association between AMIGO2 expression and liver metastasis in patients with CRC. In vitro studies revealed that cell proliferation, invasion and adhesion to liver endothelial cells were accelerated by upregulation of AMIGO2 expression, but suppressed by downregulation of AMIGO2 expression in human CRC cells. Immunohistochemical analysis using clinical CRC specimens revealed that AMIGO2 expression was associated with the frequency of liver metastasis (P<0.01), but not that of pulmonary metastasis (P=0.611) and peritoneal dissemination (P=0.909). In addition, AMIGO2 expression levels in tumor cells were significantly higher in liver metastatic foci than primary lesions (P=0.012). In conclusion, the present results indicated that AMIGO2 expression may contribute to the formation of liver metastasis in CRC.

Establishment of an Antibody Specific for AMIGO2 Improves Immunohistochemical Evaluation of Liver Metastases and Clinical Outcomes in Patients with Colorectal Cancer

“…Keisuke Goto et al 16 In this study, we produced a rat rTNK mAb specific for human AMIGO2, and confirmed that it detects AMIGO2 but does not cross-react with other AMIGO family molecules; that is, AMIGO1 and AMIGO3. By comparing the results previously reported for commercially available sc mAb with rTNK mAb using the same CRC tissues, the following five new facts and the usefulness of rTNK mAb were clarified: (i) the detection rate of liver metastases in CRC patients with high AMIGO2 expression in primary tumors improved from 47.5% for sc mAb (11) to 65.3% for rTNK mAb (Figure 4A); (ii) the association between AMIGO2 expression and liver metastasis by multivariate analysis resulted in predictability with sc mAb (p = 1.707E-5) (11); but it was significantly predictable with rTNK mAb (p = 7.930E-10, Table 3); (iii) AMIGO2 expression and overall survival were not statistically significant when detected with sc mAb (p = 0.107), but was significantly correlated with worse prognosis when detected with rTNK mAb (p = 0.004, Figure 5A); (iv) in disease-specific survival, high AMIGO2 expression resulted in a poor prognosis, which can also be detected with sc mAb (p = 0.001), whereas it had a much higher correlation with rTNK mAb (p = 0.000044, Figure 5B); and (v) factors affecting liver metastasis in multivariate logistic regression analysis using sc mAb were AMIGO2 expression (p = 1.707E-5), lymph node metastasis, vascular invasion, and sex (11). In contrast, analysis using rTNK mAb eliminated the risk of lymph node metastasis and vascular invasion, leaving only AMIGO2 expression (p = 7.930E-10) and sex (p = 0.049).…”

“…Immunohistochemical analysis was performed using paraffin-embedded colorectal cancer samples from 173 patients who underwent proctocolectomies at Tottori University Hospital between January 2007 and December 2015. For 173 cases, excluding the samples that were no longer available, the same samples reported in the previously were used (11).…”

“…In brief, five fields were chosen at random and examined at ´400 magnification. The intensity of immunoreactivity was bisected according to a previous report (11), and the staining intensity of the primary CRC was defined as low (< 30% staining intensity) and high (≥ 30% staining intensity).…”

“…The expression of AMIGO2 was quantified using a visual grading system based on the extent of staining. The cut-off value for AMIGO2 expression was Keisuke Goto et al 14 determined as the staining intensity of the primary CRC at 30%, as in a previous report (11), and the cases were divided into two groups: high and low expression. High expression was defined as an AMIGO2-positive tumor cell proportion of 30% or higher (≥ 30%, Figure 3B and 3D).…”

“…Multivariate analysis showed that AMIGO2 expression in patients with CRC was an independent predictive factor for liver metastasis (11). Furthermore, transcriptionally high levels of AMIGO2 are associated with shortened survival of patients with CRC (11), breast cancer (12), and gastric cancer (13).…”