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

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.


HIGHLIGHTS INTRODUCTION
Colorectal cancer is the third most common cancer diagnosed and the second leading cause of cancer deaths in the United States (Siegel et al., 2019) and China . Since many factors contribute to CRC tumorigenesis, including genetic factors, epigenetic factors, race, gender, drug, type II diabetes, inflammatory bowel disease, and others (Dekker et al., 2019), CRC therapy has been a challenge for clinicians and researchers (Ferrari de Andrade et al., 2018;Wang et al., 2018;Courau et al., 2019). Although the pathogenesis of CRC is still unclear, it is believed that multiple genetic factors contribute to CRC susceptibility (Zarour et al., 2017;Dekker et al., 2019).
The human major histocompatibility complex (HLA) class I chain-related protein A (MICA) controls the immune process by interacting with its receptor, NKG2D (Zingoni et al., 2018) to regulate the activities of natural killer cells, γδ T-cells, αβ CD8 T-cells, and immunosuppressive CD4 T-cells (Choy and Phipps, 2010;Risti and Bicalho, 2017). The gene consists of 6 exons, in which exon 1 encodes the leader peptides, exons 2-4 encode three extracellular globular domains, exon 5 encodes the transmembrane (TM) domain, and exon 6 encodes the cytoplasmatic tail (Risti and Bicalho, 2017). MICA is one of the necessary negative regulators in cancer immunology (Glas et al., 2001), and both MICA and its corresponding receptor are highly expressed in carcinomas and inflammatory lesions (Zhou et al., 2014). High expression of MICA and NKG2D is strongly linked to tumor immunosurveillance (Groh et al., 2002;Choy and Phipps, 2010) while low levels of MICA are associated with a poor prognosis in patients receiving aggressive chemotherapy for CRC (Chen and Gyllensten, 2014).
MICA is a highly polymorphic gene (Glas et al., 2001) with about a total of 109 named alleles according to IPD-IMGT/HLA database 1 . In addition to these variants, MICA also contains tri-nucleotide microsatellite polymorphisms (GCT)n, which are designated as An, that start at codon 295 in the TM domain; a five GCT repetition can also coexist with a guanosine insertion that is designated as A5.1 (Zhou et al., 2014).
Although the associations of MICA polymorphisms with genetic predisposition to different cancer types have been 1 http://www.ebi.ac.uk/imgt/hla/align.html investigated in candidate gene-based studies (Chen and Gyllensten, 2014;Ghadially et al., 2017), these previous studies predominantly focused on polymorphisms within the TM.
Therefore, the associations of MICA polymorphisms in exons 2-5 with CRC have not been reported. In this study, we investigated the genetic associations between molecular and clinical phenotypes of CRC and MICA alleles in a cohort of patients with CRC and healthy controls from Southern China and validated our results via functional analysis in CRC cell lines carrying KRAS driver gene mutation transfected with MICA * 012:01 and * 008. Preliminary analysis of disease-free survival time in patients with and without MICA * 012:01 may support the clinical relevance of this allele for CRC prognosis. Figure 1A is a flowchart of study procedures for MICA allele determination, association studies, and functional analysis in the context of CRC cell lines and patient samples. PCR sequencing analysis and comparison of base variations between 87 CRC tissue samples and corresponding paracancerous normal tissues did not detect any somatic mutations in MICA ( Figure 1B). However, allele detection of the sampled population identified twelve MICA alleles in patients with CRC and fourteen alleles in the control group. Among these, MICA * 007:02 was only allele to occur in CRC samples while MICA * 009:02, * 018:01, and * 033 only occurred in controls. Frequency analysis revealed that MICA * 009:01 or * 049 were significantly decreased in CRC compared to control samples [p = 0.0041 (odds ratio) OR = 0.35, Table 1].

Association of MICA Alleles With Driver Gene Mutations of CRC
Some driver genes' mutations such as KRAS, NRAS, and BRAF can lead to CRC. Therefore, preliminary association analysis of MICA codon 295 polymorphisms and alleles to driver gene mutations, specifically KRAS codon 12 mutation, in CRC samples indicated that MICA * 012:01 was significantly associated with an increased OR for KRAS codon 12 mutation (p = 0.027, OR = 3.33, Table 2). MICA * A4 had a marginal but not significant association with the mutation of KRAS codon 12 (p = 0.105, OR = 2.25; Supplementary Table S1).  (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.

Association of MICA Alleles With Molecular Typing of CRC
when accounting for age and gender variations in the sampled population (Supplementary Table S3).

MICA Alleles Were Associated With Clinical Phenotypes of CRC
We conducted further association studies of MICA codon 295 polymorphisms and MICA alleles with tumor characteristics such as differential status, UICC stage, tumor size, invasion depth, lymph nodes metastasis, and gross classification, and discovered that A5.1 was significantly increased in highly differentiated CRC compared to that in medium state (p = 0.019; Supplementary

Association of MICA Alleles With Protein
Expression of Immune Checkpoint PD-L1 and Tumor Biomarkers CEA, CA19-9, and CYFRA21-1 Programmed Death-Ligand 1 expression is a key factor in cancer immune therapy response. CEA, CA19-9, and CYFRA21-1 are widely used biomarkers in clinical practice for CRC diagnosis and progression. Though the expression of PD-L1 was not   Through a CCK-8-based proliferation assay, we found that the MICA * 012:01 overexpressed group had a higher proliferation rate than MICA * 008 overexpressed group, untransfected control, and empty plasmid groups in SW480, HCT116, and DLD1 cells ( were markedly more invasive than those overexpressing MICA * 008, untransfected control, and empty plasmid groups in SW480, DLD1, and HCT116 cells (p = 0.0188, 0.0076, and 0.0050, respectively). Western blot analysis of extracellular matrix degrading enzyme matrix metalloproteinase-9 (MMP-9) and cell-cell adhesion protein E-cadherin revealed notably increased MMP-9 expression in the MICA * 012:01 overexpressed group compared to MICA * 008, and transfection control groups in SW480, DLD1, and HCT116 cells (Figures 3E,G,I,K). In contrast, the expression level of E-cadherin was significantly decreased in the MICA * 012:01 overexpressed group than the remaining groups in all cell lines tested (Figures 3E,F,H,J).
Taken together, our data demonstrate that overexpression of MICA * 012:01 allele significantly enhanced the invasiveness and metastatic ability of CRC cell lines carrying KRAS mutations.
To validate the functional role of MICA * 012:01 in CRC malignancy, we analyzed the disease-free survival time (DFS) of patients with CRC that carry the KRAS codon 12 mutation and contain MICA * 012:01 to those that do not have MICA * 012:01. Kaplan Meyer survival curve analysis did not reveal a statistically significant difference in DFS by the presence of MICA * 012:01 (p = 0.103) though we note that our sample size is underpowered for this analysis. However, CRC patients with MICA * 012:01 had a tendency to experience relapse or metastasis in the first 20 months after the surgery than those with no MICA * 012:01. Therefore, MICA * 012:01 allele expression may be a predictive marker for poor prognosis in patients with KRAS codon 12 mutated CRC.

DISCUSSION
MICA is commonly expressed in tumor cells as a tumorassociated antigen that works in tandem with NKG2D receptor to regulate immunosurveillance (Groh et al., 2002;Choy and Phipps, 2010). Analysis of MICA polymorphisms indicates that MICA is strongly associated with immunologic escape of tumors by MICA shedding or other mechanisms (Groh et al., 2002;Chen and Gyllensten, 2014;Ji et al., 2015). Our genetic association studies herein indicate that MICA * 009:01 or * 049 were associated with decreased frequency of certain CRC subtypes, and MICA * 012:01 was increased in patient samples carrying KRAS codon 12 mutation. Functional studies of SW480, DLD1, and HCT116 cell lines transfected with MICA alleles indicated that MICA * 012:01 allele enhanced the malignant phenotype of CRC in cell lines that express KRAS mutation. Examination of tumor tissues did not show any somatic mutations in exons 2-5 of the MICA gene which may indicate that certain MICA alleles are associated with increased susceptibility to CRC progression.
In addition to specific MICA alleles, polymorphisms in the TM region of the gene, especially in * An, also showed specific associations in Chinese patients with CRC. The frequency of MICA * A5.1 was decreased in medium or low differention state in patient samples compared to those with highly differented CRC (Supplementary Tables S4, S5). On the other hand, the frequencies of MICA * A6 and * A9 that carry relatively longer repetitions of GCT are significantly less frequent in Chinese patients with CRC, and MICA * A4, which carries the shortest repetition of GCT, was increased in Chinese patients with CRC carrying a KRAS mutation at codon 12 (Supplementary Table S1). These results are in contrast to Kopp et al. (2009), who previously reported that MICA TM polymorphisms had no difference in the normal colonic mucosa and colorectal tumor tissue with MSI and KRAS codon 12 mutation subtypes in a European population (Kopp et al., 2009). This inconsistency may be due to variation in genotype distribution of MICA among different races. For instance, MICA * 008:01, * 004, and * 009:02 are the most common alleles in a French population (Carapito et al., 2017), yet according to Zarour et al. (2017) MICA * 002:01, * 008:01 or 04, and * 010:01 are the most prevalent alleles in the Chinese population. However, it is worth noting that MICA codon 295 polymorphism * A5.1 results in a premature stop codon that leads to a break in TM region of MICA to produce a soluble MICA (sMICA) (Zhou et al., 2014) that interacts with NKG2D, resulting in NKG2D internalization and subsequent suppression or impairment of NKG2D-mediated immune response (Groh et al., 2002) that may alter susceptibility to CRC development. Whether the GCT repetition length can affect stability and/or functional changes of MICA is still unknown.
MMP-9 is a matrix remodeling enzyme expressed in alveolar macrophages, granulocytes and mast cells that plays essential roles in leukocyte migration and tumor cell metastasis among others (Tsai et al., 2014;Huttlin et al., 2017;Yang et al., 2017). In contrast, E-cadherin, which belongs to the Ca 2+ dependent cell adhesion molecule family (Guilford et al., 1998), regulates cell-cell adhesions, mobility, and proliferation of epithelial cells and is a potent invasive suppressor for tumor cells (Ma et al., 2010;Satow et al., 2014;Indra et al., 2018). Our cell line based functional studies indicate that overexpression of MICA * 012:01 correlated with increased expression of MMP-9 and corresponding downregulation of E-cadherin ( Figure 3E). Moreover, in sharp contrast to the common allele MICA * 008, overexpression of MICA * 012:01 allele significantly exacerbated the malignant phenotype of KRAS mutated CRC similar to results from other groups (Simanshu et al., 2017;Janes et al., 2018;Li et al., 2018;Brandt et al., 2019;Serebriiskii et al., 2019 ; Figures 2, 3).
Analysis of DFS in clinical patients with and without MICA * 012:01 did not reveal a statistically significant difference in DFS though it should be noted that the sample size was underpowered to detect a significant difference for a low effect allele. Future studies will incorporate additional data from more patient samples.
Taken together, we provide evidence that MICA * 012:01, * 009:01 or * 049, and MICA * A4 are important genetic factors associated with CRC, CRC with KRAS mutation, and MSI mutation subtypes of CRC in a Chinese population. Overexpression of MICA * 012:01 allele enhanced the malignant phenotype of CRC with KRAS mutation specifically proliferation, invasion, and metastasis in cultured CRC cell line which may suggest that MICA * 012:01 enhances the metastatic potential of CRC by interacting with the NKG2D to evade immune surveillance.

CRC and Control Samples
A total of 104 patients with CRC were enrolled from the Department of General Surgery at the Affiliated Hospital of Nantong University, Jiangsu Province, China. Tumors were classified according to TNM/UICC criteria (Provenzale et al., 2015) following histopathological examination. All patients were enrolled under the screening and diagnosis criteria of CRC (Provenzale et al., 2015). A total of 536 control samples (whole blood) were obtained from a study project of Chinese population genetics in Fudan University, Shanghai, China (Zhou et al., 2014), and these patients were enrolled from clinics and hospitals in southern cities of China. Samples were collected from individuals of Southern Han Chinese ancestry. The average ages of CRC patients and controls were 66 and 46, respectively and gender ratios varied between groups from 69% men and 31% women with CRC, and 51% men versus 49% women for control samples. Postoperative follow-up was performed according to recommendations from the Chinese Society of Clinical Oncology (CSCO).

MICA Sequencing and Genotyping
Genomic DNA was extracted from peripheral blood cells from control samples and from intestinal epithelial cells from colorectal tumors and paracancerous normal intestine tissue that is > 10 sm distant from the edge of cancer, respectively. MICA was genotyped by PCR sequencing of exons 2, 3, 4, and 5 using bidirectional Sanger sequencing methods (Zhou et al., 2014). Sequencing data were analyzed by Chromas 2.4.1 software (technelysium, Au). Allelic genotypes of MICA in each sample were obtained according to reference sequence of specific MICA alleles 2 . For quality control in DNA typing, all sequencing processes were performed using robotic automation system to minimize sample mislabeling and misplacing. We also had blind duplicates in each sample plate.

Screening for Tumor Microsatellite Instability and KRAS, NRAS, and BRAF Oncogene Mutations
Analysis of tumor MSI was performed using fluorescence in situ hybridization (FISH) to detect MLH1, MSH2, MSH6, and PMS2 (Sepulveda et al., 2017). The positive expression of all four genes was judged as MSS or CIN while three or less gene positive expression was considered as MSI.
Driver mutations in codons 12, 13, 59, 61, 117, and 146 of KRAS and NRAS genes, and the mutation in codon 600 of BRAF gene (Sepulveda et al., 2017) in patient samples were analyzed by FastTarget next-generation sequencing .

Plasmid Vectors and Transfection
pEGFP-C3 plasmid vector was developed and maintained by our laboratory. The full-length cDNA of MICA * 012:01 and * 008 were chemically synthesized by RuiMian Bio (Shanghai, China) and inserted into thepEGFP-C3 plasmid after digestion with restriction enzymes KpnI and EcoRI (Fermantas lnc., Burlington, ON, Canada). The plasmid was then ligated to form pEGFP-MICA * 012 and pEGFP-MICA * 008 and transfected into CRC cells by DNA transfection agent Entranster TM H4000 (Engreen, Beijing, China).

Cell Proliferation Assay With CCK-8 Detection
Cell proliferation assays were performed using Cell Counting Kit-8 (CCK-8; Keygen, China) according to the manufacturer's instructions. Briefly, SW480, DLD1, and HCT116 cells were seeded in 96-well plates in DMEM or McCoy's 5A with 0.5% FBS at a density of 2 × 10 3 cells/well and transfected with pEGFP-MICA * 012 and control. At 24, 48, 72, and 96 h, the optical density at 450 nm wavelength, which correlates to the number of viable cells, was measured and the results expressed as mean of OD450 ± SEM.

Detection of Cell Colony Formation Ability
Cells were seeded in a 24-well ultra-low attachment round bottom plate (Excell, United States) at a density of 300 cells per well in 10% FBS DMEM/McCoy's 5A medium supplemented with pEGFP-C3 plasmid, pEGFP-MICA * 008, or pEGFP-MICA * 012 transfection, respectively, and cultured for 8 days. Cultured cells were washed with phosphate buffered saline (PBS), fixed with 200 µL absolute methanol for 10 min, and then stained with Giemsa dye. The number of violet cell spheres was counted manually. Cell colony-formatting efficiency was calculated as: PE (Planting efficiency)% = Cell clone/Inoculation cell number × 100%.

Cell Invasion Assay With Transwell Experiment
The cell invasion assay was performed using invasion chambers consisting of a 12-well tissue culture plate with 24-well sized inserts (Corning, NY, United States). Cell suspensions (0.5 × 10 6 cells/mL) were transfected with pEGFP-MICA * 012:01, pEGFP-MICA * 008, or medium alone were added to the interior of the inserts with 300 µL serum-free media and 500 µL of media containing 10% FBS as a chemoattractant to the lower chamber and incubated for 72 h. Invasive cells on the lower surface of the membrane were stained by crystal violet for 10 min and imaged under the high magnification field (HMF, 400 fold) of the microscope.

Statistical Analysis
Correlations of MICA alleles with histopathological, genetic, and other molecular parameters were evaluated. Parameters include tumor invasion, lymph node involvement, histological type, presence of distant metastasis, UICC stages, MSI, driver gene mutation, immune checkpoint PD-L1, and diagnostic biomarkers. Exact p-values were obtained via Fisher's test or Mantel-Haenszel stratification test from 2 × 2 tables of allele counts. 3-year disease-free survival times were calculated by Kaplan-Meier curves and compared by the log rank test. Two-tailed t tests and ANOVA were used to the functional experiments analysis. A p-value of less than 0.05 was used to indicate statistical significance. Statistical analysis was performed using SPSS statistical software (SPSS version 20.0).

DATA AVAILABILITY STATEMENT
The datasets generated during the current study available from the corresponding authors on reasonable request.

ETHICS STATEMENT
The study was approved by the Ethics Committee of Affiliated Hospital of Nantong University, and patients' informed consent was obtained.