As a leading cause of cancer-related deaths in Americans, cancer of colon and rectum (CRC) remains in third position for new estimated cases and mortality according to American Cancer Society in 2020 [1]. Current diagnosis of CRC is based on tumor-node-metastasis (TNM) stage, which lacks the interpretation of epigenetic background and genetic variants. Therefore, identification of new biologic markers is a promising approach to improve detection of aggressive phenotypes and provide better guidelines for clinicians towards CRC treatment.

MHC class I polypeptide-related sequence A (MICA) is a cell surface protein overexpressed under stress conditions [2]. Upon interaction with natural killer group 2D (NKG2D) receptors, MICA promotes activation of natural killer (NK) cells, IFN-gamma secretion, and degranulation of perforin and granzymes, leading to apoptosis of cells expressing high levels of MICA [3, 4]. MICA functions as a component of the immunosurveillance system to combat infections by foreign pathogens and conditions that lead to cellular stress, including cancer [5]. MICA has a soluble form (sMICA), which is highly expressed in aggressive forms of cancer and reduces the cytotoxic activity of NK cells [6]. Therefore, MICA has been proposed as a relevant player of the tumor microenvironment (TME) [7], worth to be explored as a factor of tumorigenesis.

Aberrant expression of MICA has been described in different types of cancers, including those of the prostate, lung, stomach, and cholangiocarcinoma [8]. Despite the level of information, the role of MICA expression in tumorigenesis is not clear. In carcinoma of the prostate [9], gastric cancer [10], and non-small cell lung cancer [11], higher expression of MICA relates to a better prognosis. On the other hand, higher expression in patients with pancreatic cancer [12], breast cancer [13], hepatocellular carcinoma [14], and non-small cell lung cancer [15] predicts for poor outcomes. In relation to CRC, elevated expression of MICA has been found in tumor tissue as compared to normal specimens [16]. However, improved disease-specific survival is reported for patients with high expression of MICA [17, 18].

Since data suggest that MICA is a molecule of the TME with an emergent role as a marker of aggressive disease, further investigations are needed to establish its prognostic value in CRC. Herein, we determined the levels of MICA (RNA and protein) in CRCs. Additionally, we established correlations between MICA expression and clinical characteristics. Publicly available gene expression data and bioinformatics tools were used for validation purposes. Our findings agree with published literature for higher expression of MICA in CRCs, however, contrary to prior reports for CRC [17, 18], they point to a poor prognosis for patients whose CRCs exhibit high MICA expression. In sum, our findings suggest that additional work is needed to establish the role of MICA expression as a discriminator of aggressive CRC.

Bioinformatics analyses of RNA expression of MICA in CRC tissues: Inspection of uninvolved tissues (N = 41) vs. primary CRC (N = 286) (Fig. 1A) in the TCGA database, available through UALCAN, revealed a 30% increase of expression of MICA mRNA in CRCs relative to normal epithelia (p = 1.794E-07). Furthermore, we found elevated expression of MICA in CRCs of patients for both sexes (males N = 156 and females N = 127, p 0.0001 for each comparison) (Fig. 1B). Further analysis of MICA transcripts based on individual cancer stages; stage 1 (N = 45), stage 2 (N = 110), stage 3 (N = 80), and stage 4 (N = 39), revealed high expression for all cancer stages. relative to normal tissue (p 0.0001) for each comparison (Fig. 1C); however no differences were observed between individual stages. Next, we determined the association of MICA mRNA with patient’s race. MICA transcripts were elevated regardless of race in CRC, when tumors of Caucasian (N = 193), p 0.0001; African-American (N = 55), p = 0.002; and Asian (N = 11), p = 0.022 CRC patients were compared to uninvolved tissue (Fig. 1D). When analyzed by age (Fig. 1E), levels of MICA mRNA were not different between normal tissues and tumors obtained from 12 individuals aged 21–40 years old (p = 0.06). MICA transcripts, however, were significantly elevated for all older groups [41–60 years (N = 90), p 0.0001; 61–80 years (N = 149), p 0.0001; and 81–100 years (N = 32), p = 0.002], relative to normal epithelium. Further analysis showed that MICA RNA expression based on histological subtypes was higher in CRCs than in uninvolved tissues (Fig. 1F). Expression was high, for adenocarcinomas (N = 243), p 0.0001 and for mucinous adenocarcinomas (N = 37), p 0.0001 relative to normal tissue. However, no differences in transcript levels were noted between adenocarcinomas and mucinous tumors. In addition, MICA expressions in three distinct nodal metastasis status [N0 (N = 166), N1 (N = 70), and N2 (N = 47); p 0.0001 for each comparison] were all upregulated, but comparable, as compared to non-tumorous tissue (Fig. 1G). Likewise, MICA expression based on p53-status was elevated in CRCs. It was found that 160 CRC patients with p53-wild type and 122 patients with p53-mutated status exhibited higher MICA expression (Fig. 1H), p 0.0001 for each case. Transcripts of MICA, however were not different between tumors from patients with p53-wild type or p53-mutated status.

Fig. 1.

MICA RNA expression in CRC tissues. (A) Box plots showing relative expression of MICA mRNA in uninvolved tissues and CRC (Student’s t-test). The mRNA expression levels were normalized as transcripts per million reads. (B) MICA expression in CRCs on the basis of patient sex, (C) various stages of CRC, (D) race, (E) age, (F) tumor histologic types, (G) nodal metastatic status, and (H) p53 mutation status (B–H, one-way ANOVA with Dunnett’s multiple comparisons test). Pairwise comparisons relative to normal tissue. CRC, colorectal cancer; MICA, MHC class I polypeptide-related sequence A; TCGA, The Cancer Gene Atlas.

4.1 Association between expression of MICA transcripts and survival of CRC patients

Using the prognostic database PROGgeneV2, we retrieved data and performed survival analyses on the datasets GSE41258 and GSE29621, using the median value as threshold. The dataset GSE41258 consisted of 390 expression arrays, including primary colon adenocarcinomas, adenomas, metastasis, and corresponding normal mucosae, from patients who presented at Memorial Sloan-Kettering Cancer Center from 1992 and 2004. Only 182 expression arrays, from primary adenocarcinomas, were used for survival analysis. The dataset GSE29621 consisted of 65 mRNA samples extracted from primary tumors of colon cancer patients for microarray analysis.

In both datasets, there was a significantly poorer prognosis for patients with high MICA mRNA levels relative to those with low MICA mRNA (log rank, p = 0.014, HR: 2.15, 95% CI: 1.17–3.94 for GSE41258 and log rank, p = 0.003, HR: 9.87, 95% CI: 2.18–44.69 for GS29621) (Fig. 2A,B).

Fig. 2.

Survival analysis for patients with CRC according to the expression of MICA mRNA. Plots generated using the prognostic database PROGgeneV2 to analyze the datasets GSE41258 (A) and GSE29621 (B), using the mean value as threshold. In both datasets, a poor prognosis was evident for patients with higher expression of MICA mRNA (log rank, p = 0.014, HR: 2.15, 95% CI: 1.17–3.94 for GSE41258 and log rank, p = 0.003, HR: 9.87, 95% CI: 2.18–44.69 for GSE29621).

4.2 MICA protein expression by immunohistochemical (IHC) profiling of normal colonic and tumor tissues

Of 384 cores, 74 were unsuitable and excluded from analysis due to loss of tissue or lack of viable cells within the core. Higher MICA expression was observed as globular/nuclear or cytoplasmic in cells from normal tissues (Fig. 3A). Nuclear staining was observed in 11.6% (8 of 69) positively stained uninvolved cores. Cytoplasmic immunostaining was observed in 40.6% (28 of 69) normal cores. In both the basal and luminal portions of colonic crypts, staining was evident mainly in the cytoplasm of epithelial cells and the peripheral cytoplasm of Goblet cells, with negative reactivity for mucous glands. In CRCs, MICA staining was predominantly cytoplasmic, as noted in 84.9% (73 of 86) of the positively stained cores (Fig. 3A). Globular staining was present in 32.6% (28 of 86) of positively stained specimens (Table 2). High expression was also observed in mucinous tumors (Fig. 3B) as well as in moderately (Fig. 4A) and poorly differentiated adenocarcinomas (Fig. 4B). Analysis of nuclear immunostaining revealed a 3.5-fold higher combined intensity score for CRCs (1.4 $\pm$ 2.5) relative to normal glandular samples (0.4 $\pm$ 1.4), p = 0.002. Likewise, cytoplasmic immunostaining was 2.4-fold higher when the combined intensity score in CRCs (3.4 $\pm$ 2.8) was compared to normal glandular samples (1.4 $\pm$ 2.1), p 0.0001 (Fig. 5 and Table 2). Due to the higher prevalence of cytoplasmic immunostaining in CRCs, we used this value to evaluate data for further analyses.

Fig. 3.

IHC staining of MICA in contiguous normal-tumor tissue and mucinous adenocarcinomas. (A) Representative microphotographs from three different adenocarcinoma cases to show MICA staining in TMA tissues using IHC analysis. The 4X images (left panel) have contiguous normal (denoted by N) and tumor components (denoted by T) from adjacent sections of same patient while 10X images are of normal (middle panel) and tumor (right panel) sections obtained from the TMAs. MICA glandular expression was nuclear and cytoplasmic in both normal and tumor tissues. Scale bar; 4X—100 $\mu$m, 10X—200 $\mu$m. (B) 4X image shows MICA staining in mucinous adenocarcinoma tissues with contiguous normal (denoted by N) section as well as tumor (denoted by T) components.

Table 2.Frequency of MICA expression in colonic tissue according to tissue type and localization.

Tissue/Localization	Frequency	MICA average combining stage score
Normal/Cytoplasmic	28/69	1.4 $\pm$ 2.1*${}^{\mathrm{\#}}$
	(40.6%)
Tumor/Cytoplasmic	73/86	3.4 $\pm$ 2.8*${}^{\mathrm{\#}}$
	(84.9%)
Normal/Nuclear	8/69	0.4 $\pm$ 1.4*${}^{\mathrm{\#}}$
	(11.6%)
Tumor/Nuclear	27/86	1.4 $\pm$ 2.5*${}^{\mathrm{\#}}$
	(31.4%)
*p 0.05 when comparing between different tissue types in the same cellular sub-localization. ${}^{\mathrm{\#}}$p 0.05 when comparing between different cellular sub-localization in the same tissue type.

Fig. 4.

IHC staining of MICA in moderately and poorly differentiated CRC tissues. Representative microphotographs from different adenocarcinoma cases to show MICA staining in CRC tissues based on (A) moderate and (B) poor differentiation.

Fig. 5.

Comparison of MICA nuclear and cytoplasmic staining in CRC tissues. Stronger MICA immunoreaction was observed in tumor glandular cells relative to normal tissue, represented in the scatter plot. Mann-Whitney U test for non-matched data. *p 0.05 when comparing between different tissue types in the same cellular sub-localization. ${}^{\mathrm{\#}}$p 0.05 when comparing between different cellular sub-localization in the same tissue type.

CRC mortality rates are elevated worldwide. Even though the five-year survival of CRC patients has improved due to early detection, close to 25% of patients still are diagnosed with stage 4 disease. As the relative 5-year survival rate of patients with metastatic CRC (mCRC) remains poor [22], there is an urgent, unmet need to develop more effective treatments for patients suffering from this disease. PD1 inhibitors have been a successful immunotherapy approach for a specific subgroup of mCRC, those that are mismatch-repair-deficient and microsatellite instability-high [23]. Ongoing research is focused on looking for treatments for other subgroups of mCRC. Emerging approaches include targeting of the TME, which might complement immune checkpoint inhibition. To this end, in the current study, we evaluated MICA as a potential TME marker for aggressive disease. Analysis of CRC in the UALCAN database suggested that MICA expression was closely associated with individual cancer stages. In addition, MICA expression in the UMMC CRC cohort, assessed by IHC, was increased in CRCs and was associated to features of aggressive disease.

Expressed in various malignancies, MICA is considered a component of tumor immunosurveillance by interacting with the receptor NKG2D, activating NK cells, and co-stimulating subtypes of T-cells [24, 25, 26]. Our results showed increased expression of MICA in CRC compared to uninvolved tissue. Higher MICA expression was significantly associated to increased tumor stage (T3 and T4), suggesting the potential of MICA as a marker for aggressive CRC.

A mechanistic rationale for the high levels of MICA in cancers may relate to its role in cell homeostasis [27]. High rates of cell proliferation, as observed in tumors during inflammatory processes, lead to MICA overexpression [3, 6]. This upregulation can be interpreted as a means to restore cellular homeostasis. As tumors secrete sMICA, binding of sMICA to the NKG2D receptor triggers downregulation of NKG2D, inactivation of NK/T cells [28, 29], and tumor immunoescape. Additional considerations to the tumor immune evasion mechanism mediated by MICA include those inherent to its highly polymorphic nature as a contributor of differential susceptibility for proteolytic shedding by metalloproteases in the TME [30]. These may be reasons for overexpression of MICA in CRCs as observed in the current study. On the contrary, results [9, 10] demonstrating poor outcomes for patients with low expression of MICA may be due to the presence of MICA alleles that have a tendency to shed or release as extracellular vesicles.

In the literature, there is disagreement about the association between MICA expression and the prognosis for cancer patients. High tumor levels of MICA were previously associated with a good prognosis for prostate cancer and cervical cancer [31]. However, elevated MICA was reported as an indicator of poor prognosis for pancreatic cancer [12] and breast cancer [13]. Survival analysis performed for our cohort suggests, in CRCs, a possible association of poor prognosis to higher expression of MICA, supported by the PROGgeneV2 survival analysis in two distinct cohorts. In disagreement with our findings, two independent studies indicated better prognosis for patients with expression of MICA in CRC [17, 18]. Because this controversy has been also found for other tumors such as non-small cell lung cancer [15, 32], and gastric cancer [10, 33], the matter of expression of MICA and its association with outcome remains an issue of active debate.

In various tumors, MICA sheds from the cell surface into the circulation as sMICA. Binding of sMICA to the NKG2D receptor, without activation or co-stimulation of the effector cells, promotes tumor escape. Unfortunately, we did not have access to plasma to assess circulating levels of sMICA or NKG2D levels in NK cells. Access to these data would help us interpret our results on scope of the described tumor immunoevasion strategy mediated by MICA. In aggressive pancreatic carcinoma, there is an inverse correlation between expression levels of sMICA and NKG2D [12]. Moreover, findings of a recent study concluded that high levels of MICA in serum are associated with a poor prognosis for hepatocellular carcinoma (HCC) [34]. Findings from this study also suggested that MICA blocks the NKG2D signaling pathway by mediating tumor immune escape in HCC [34]. In CRC, this pathway would protect tumor cells from NK cell-mediated cytotoxicity. Further studies are needed to evaluate expression of NKG2D in CRC, its functional association with MICA, and the mechanistic basis of the interaction between these two molecules. Benefits include development of innovative TME-based immunotherapy strategies.

MICA expression has been previously reported in various cellular localizations, such as cell membrane, intracellular space, exosome surface, and soluble form in plasma or supernatant [35]. As discussed before, cell-surface MICA is important for NKG2D interaction and NK cell activation, and sMICA has been reported to impair this mechanism. In a similar manner, exosomes with surface MICA decrease NK cell cytotoxic activity in prostate cancer cell lines [35]. Intracellular localization of MICA has been previously reported, but its biological function has not been fully elucidated. For example, intracellular retention and low surface expression of MICA, presented by melanoma cell lines, contributed to protect tumor cells from cytotoxicity mediated by NK cells [36]. Perinuclear expression of MICA has been reported in mucosal samples of coeliac disease patients; however, nuclear expression has not been addressed [37]. Our study is the first to report nuclear expression of MICA in CRCs. Further investigation is necessary to identify possible DNA binding sites and to address other biological functions of nuclear MICA. Another limitation of the present study is not analyzing the sMICA. Tumor-associated MICA is not the only protein form involved in CRC. Since defined cellular functions are reported for sMICA, future analyses of sMICA will allow elucidation of its role in growth and progression of CRC.

For CRC patients, immune checkpoint blockade therapy has achieved limited success. To improve the outcome for patients with aggressive forms of the disease, current research is focusing on combined treatment with immunotherapy, including chemoimmunotherapy, immunotherapy with radiation therapy, and other strategies. A potential option is stimulation of NK cells and cytotoxic T cells through lowering of MICA expression and neutralization of sMICA. Additional research is needed to clarify the divergent information related to the expression of MICA in CRC as well as its prognostic value and mechanistic involvement in disease aggressiveness.

Our study provides evidence for up-regulation of MICA in CRC and suggests a poor prognosis for CRC patients exhibiting high MICA expression. We believe that the relevance of our findings is high due to similar patterns of high MICA expression identified in large, publicly available omics databases, and the potential of MICA as an actionable molecule of the TME.

IE, SA, MS, VS, WSO, and SAD contributed to the conceptualization of the research concept, performing the experiments, formal analyses, and writing the original draft. AP, SV, UM, and CRG contributed to the conceptualization of the research, study design, supervision, reviewing and editing the original draft, and funding acquisition.

The study was approved by the UMMC Institutional Review Board. This study was a retrospective protocol that was exempt from participant consent. The study was approved by the University of Mississippi Medical Center (UMMC) Institutional Review Board under protocol # 2012-0205.

We thank Elizabeth Tarsi, Tara Craft, Eldrin Bhanat, Jaswinder Kaur, and Joy King for establishing and maintaining the databases. We thank Amit Reddy at the University of Mississippi Medical Center for technical assistance and Donald Hill at the University of Alabama at Birmingham for editing the manuscript.

This study was supported by the Office of Research and Sponsored Programs, University of Mississippi Medical Center (IE and CRG); Coordination for the Improvement of the Higher Education Personnel (CAPES) Foundation, Scholarship #13603-13-2 (MJS), and the Impact Funds from the School of Medicine and the Department of Pathology, University of Alabama at Birmingham (UM).

The authors declare no conflict of interest.

Not applicable.

Datasets utilized for bioinformatics analysis are publicly available (GSE41258: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE41258; GSE29621: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE29621). Due to regulatory aspects related to protection of privacy of human subjects, we are unable to access the clinical raw data of the individual patients from these publicly available data sets. Histopathology data or our cohort are available from the corresponding author on reasonable request.

Dr. Gomez contributed to this article as an employee of the University of Mississippi Medical Center. The views expressed are his own and do not necessarily represent the views of the National Institutes of Health or the US Government.