MICA ∗012:01 Allele Facilitates the Metastasis of KRAS-Mutant Colorectal Cancer

Abstract

Major histocompatibility complex (HLA) class I chain-related protein A (MICA) regulates immune surveillance through activation of NKG2D (natural killer group 2D) receptor. However, the genetic association, potential function, and predictive ability of MICA alleles with colorectal cancer (CRC) prognosis remain undefined. In this study, we characterized MICA alleles in tissue samples from 104 patients with CRC and 536 healthy controls and carried out genetic association studies by molecular and clinical CRC phenotypes. Preliminary sequence analysis revealed that MICA ^∗009:01 or ^∗049 alleles were significantly decreased in patients with CRC (p = 0.0049), and further stratification analysis indicated that MICA ^∗012:01 allele was associated with patients with CRC and carrying KRAS codon 12 mutation (p = 0.027). The functional consequences of MICA alleles were examined via transfected CRC cell lines which showed that overexpression of MICA ^∗012:01 enhanced the proliferation, invasion, and metastatic phenotype of CRC. Preliminary analysis of disease-free survival time in patients with and without MICA ^∗012:01 suggest this allele may be predictive for poor prognosis of patients with KRAS codon 12 mutated CRC, as no somatic mutation of MICA gene was detected in CRC tumors compared to paracancerous tissues. Our study indicates that MICA ^∗012:01 allele is associated with KRAS-mutated CRC, facilitates CRC invasion and metastasis, and possibly reduces the survival of patients with KRAS-mutated CRC.

Keywords: MICA polymorphism; MICA ∗012:01 allele; colorectal cancer; immunosurveillance; tumor immunity.

FIGURE 1

The flowchart of MICA genotyping and analysis of genetic association with CRC, as well as functional experiments. (A) Flowchart of study activities. (B) Exon-intron organization of MICA gene and primers for PCR sequencing. MICA, the human major histocompatibility complex class I chain-related gene A; P, primer; E, exon; CRC, colorectal cancer; MSI, microsatellite instability; KRAS, Kirsten rat sarcoma oncogene homolog; NRAS, neuroblastoma RAS viral oncogene homolog; BRAF, v-raf murine sarcoma oncogene homolog; MLH1, mutL homolog l; MSH2, mutS homolog 2; MSH6, mutS homolog 6; PMS2, PMS1 homolog 2.

FIGURE 2

Cell proliferation and colony forming assays of KRAS mutated CRC cell lines with expression of MICA *012:01 or MICA *008 allele. Growth curves were examined by CCK-8 assay in (A) SW480 cells, (B) DLD cells, and (C) HCT116 cells. (D) The proliferative capability of MICA alleles was determined by cell clone formation experiments with corresponding histograms shown in (E–G). Cell colony-formatting efficiency was calculated as: PE (Planting efficiency)% = Cell clone/Inoculation cell number × 100%. Control, untransfected cells; Empty vector, cells transfected with pEGFP-C3 plasmid vector; MICA *008, cells transfected with pEGFP-MICA *008 recombination vector; MICA *012:01, cells transfected with pEGFP-MICA *012:01 recombination vector. ns, no significance; *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 3

The invasive and metastatic properties of KRAS mutated CRC cell lines transfected with MICA *012:01 or MICA *008 allele. The invasive capability of MICA allele overexpressing CRC cell clines was evaluated by transwell assay in (A) SW480 cells, DLD cells, and HCT116 cells. HMF, high magnification field (400 fold) with corresponding histograms (B–D). ns, no significance; *p < 0.05; **p < 0.01. (E) Relative amounts of total MMP-9 and E-cadherin protein levels were analyzed by Western blotting experiments. Representative blots are shown. (F–K) Corresponding histograms of the results are shown. ns, no significance; *p < 0.05; **p < 0.01. (L) The disease-free survival curve of MICA *012:01 and non-MICA *012:01 alleles in CRC patients carrying KRAS codon 12 mutation. Control, untransfected cells; Empty vector, cells transfected with pEGFP-C3 plasmid vector; MICA *008, cells transfected with pEGFP-MICA *008 recombination vector; MICA *012:01, cells transfected with pEGFP-MICA *012:01 recombination vector.