LRIG1 expression and colorectal cancer prognosis

Background To make the right treatment decisions about colorectal cancer (CRC) patients reliable predictive and prognostic data are needed. However, in many cases this data is not enough. Some studies suggest that LRIG1 gene (leucine-rich repeats and immunoglobulin-like domains1) has prognostic implications in different kinds of cancers. Methods One hundred and two patients with colorectal cancer were retrospectively analyzed for LRIG1 expression at both mRNA and protein levels. SYBR Green Real-Time RT-PCR technique was used for mRNA expression analyses and Glyceraldehyde-3-Phosphate Dehydrogenase gene (GAPDH) was considered as a reference gene for data normalization. LRIG1 protein expression was analyzed using Immunohistochemistry. Additionally, appropriate statistic analyses were used to assess the expression of LRIG1 in test and control groups. The prognostic significance of LRIG1 expression was analyzed using the univariate and multivariate analyses. Results The data revealed that the expression of LRIG1 in both mRNA and protein levels was down regulated in colorectal tumor tissues (P < 0.01) but is not clinically relevant prognostic indicator in CRC. Conclusions Therefore, it is suggested that LRIG1 expression analyses may not be considered as an important issue when making informed and individualized clinical decisions regarding the management of colorectal cancer patients.


Background
Colorectal Cancer (CRC) is considered as one of the most significant cancers worldwide, with about 1 million new cases and more than 550,000 deaths per year [1]. Despite the significant advances in the diagnosis of CRC, the survival rate decreases for patients diagnosed with metastatic and regional disease [2]. The reported overall median survival time of CRC is only 1.1 years [3]; therefore, understanding about biological factors with impact on CRC is very important.
The lack of predictive and prognostic biomarkers with the ability to predict therapy response and recurrence of the disease is an important issue that needs to be addressed. The LRIG1 (the leucine-rich repeats and immunoglobulin-like domains 1) as an emerging tumor suppressor and its paralogs LRIG2 (the leucine-rich repeats and immunoglobulin-like domains 2) and LRIG3 (the leucine-rich repeats and immunoglobulin-like domains 3), are considered to have prognostic significance in various kinds of cancers, such as head-and-neck [4,5], prostate [6], breast [7,8], uterine cervical cancer [9][10][11], and cutaneous squamous cell carcinoma [12], and glioma [13,14].
The locus of LRIG1 is located on chromosome 3p14.3. The encoding protein is a transmembrane protein consisting of an extracellular region including three immunoglobulin (Ig)-like domains and fifteen leucinerich repeats. The leucine-rich repeats and immunoglobulin-like domains have interactions with all four extracellular region binding protein B receptor family members leading to the regulation of receptor levels by subsequent lysosomal degradation and increasing ubiquitination, independent of ligands [15][16][17]. In addition the LRIG1 is considered as a marker of human epithelial stem cells in a quiescent non-proliferative state [18]. The enhanced proliferation associated with epithelial hyper-proliferation in vivo and stem cell expansion in vitro is the result of the genetic erosion of the leucine-rich repeats and immunoglobulin-like domains [18,19]. It is recommended by lineage tracing that the leucine-rich repeats and immunoglobulin-like domains mark non-cycling, long-lived stem cells of the ‫‬ 4 quiescent intestinal stem cell niche in the crypt [20]; and also progenitor cells in the stomach that are involved in restoring gastric cell mucosa after DMP-777 induced acute damage [21].
Although the LRIG1 plays vital role in cancers, little is known about its association with clinico-patho-physiology characteristics of CRC patients. Here the LRIG1 expression in the lesions of CRC patients was studied in order to evaluate its relationship with the major clinicohistological predictive factors and its respective impacts on patient prognosis hoping to improve the approaches for colorectal cancer management. Therefore, the main goal of the present study was to compare and analyze the expression levels of the LRIG1 gene in samples of tumor and normal colorectal tissues of CRC patients by quantitative real-time RT-PCR and immunohistochemical (IHC) techniques. Moreover, to estimate the prognostic indicator of the mentioned gene expression levels, we surveyed their correlations with clinicopathological parameters, as well as the overall survival (OS) of patients with CRC.

Patient information
A total of 102 cases of colorectal cancer from Imam Khomeini Hospital, Tehran, Iran were selected. The average age was 55.0 ± 10.0 years. The inclusion criteria were post-operative diagnosis of primary CRC based on histopathology. The study was approved by the Ethics Committee of the NIGEB based on the Helsinki declaration. All the patients signed informed consent. The ethics code number is IR.NIGEB.1395.11.10.E. The patients' characteristics are presented in Table 1. Overall survival (OS) was defined as the time from the date of primary treatment to the date of death from any cause or until the date of the last follow-up. Disease-free survival (DFS) for patients with CRC was defined as the time from the date of primary treatment to the date of diagnosis for recurrence or disease or to the date of the last follow-up.

Immunohistochemical analysis
Surgical specimens were formaldehyde fixed paraffin embedded and sectioned at a thickness of 4 μm followed by xylene dewaxing, ethanol gradient rehydration and harnessed to high pressure and temperature for antigen retrieval. The slices were incubated in H 2 O 2 harnessed to the primary antibody, rinsed with phosphate buffered saline (PBS), then harnessed to secondary and mouse anti-human LRIG1 monoclonal antibody, respectively. The slices incubated with PBS instead of the primary antibody were used as the negative control. The sections were assessed using an Olympus BX41 light microscope (Olympus, Tokyo, Japan) by a pathologist. The scale based on the reaction intensity were used to assess immunoreactivity in enterocytes or cancer cells of the studied sections (0, no reaction; 10, up to 10%; 30, 11-30%; 60, 31-60%; 80, 61-80%; and 100, > 80%) (Fig. 1).

RNA extraction, cDNA synthesis and LRIG1 mRNA expression analysis
Total RNA was isolated from the colorectal tissue using YTzol kit (Yekta Tajhiz Azma Co, Tehran, Iran) according to the manufacturer's protocol. cDNA was synthesized following the manufacturer's instructions (Cinaclon Co, Tehran, Iran) and stored at − 20 °C until analyzed. The primer sequences for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and LRIG1 genes Other metastasis 22 (22) were designed using primer 3 software (https ://prime r3.ut.ee/) and then blasted using https ://www.ncbi. nlm.nih.gov/tools /prime r-blast /. The designed primer sequences are shown in Table 2. The Real-time RT-PCR amplifications were conducted in a final volume of 15 μl reaction mixture containing 1 μl of cDNA, 7.5 μl RealQ plus 2 × master mix green (Ampliqon, Denmark), 0.6 μl (10 μmol/l) of each primer and 5.3 μl sterilized water, using the Rotor-Gene Q System (QIAGEN Hilden, Germany). The cycling conditions were as follows: 15 min at 95 °C followed by 40 cycles of denaturation at 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s for the LRIG1 and also the GAPDH, which was used as a normalizer. Experiments were performed in triplicates for each data point. The linear standard curve (from 0.1 to 1000 ng) assessed by ultraviolet spectrophotometer was used for amplification efficiency determination of each primer pair. The standard curves showed good linearity and amplification (100%). The data was presented as the fold change in gene expression normalized to an endogenous reference gene relative to the controls using 2 −ΔΔCT method.

Statistical analysis
Graphpad Prism 8.0.2 (California Corporation, USA) and SAS computer software version 9.1 (SAS Institute Inc., Cary, NC, USA) were used to analyze the data. The Mann-Whitney U test and Kruskal-Wallis test were performed for numerical data and the Chi-square test was used to analyze the relationship between parameter data. Numerical data are presented as the mean ± standard deviation (SD). Differences were considered as statistically significant if P < 0.05. The Cox proportional-hazards model was used for univariate and multivariate analyses to identify the independent prognostic factors for OS, DFS.

LRIG1 expression and clinicopathological features LRIG1 mRNA expression
The LRIG1 mRNA expression was significantly downregulated in Colorectal cancerous tissues compared with normal control (P < 0.01). The mean of LRIG1 relative expression in cancerous tissues compared with normal control was 0.57 ± 0.24 with a range of 0.23 to 1.2. About 40% of cancerous samples showed the relative expression < 0.5 that was considered as downregulation. As shown in Fig. 2, there were no significant differences between different demographical and clinicopatological characteristics of CRC patients and LRIG1 expression (P > 0.05).

LRIG1 protein expression
The LRIG1 immunoreactivity was found in the cytoplasm of enterocytes as well as cancer cells of the analyzed tissues (Fig. 1). The tumor LRIG1-positive rate in colorectal cancer tumors was 37.4%, which was significantly lower than that in control tissues (57.7%, P < 0.05). There was no significant correlation in the expression of the LRIG1 with tumor size, tumor diameter, tumor differentiation, age, and the number of positive mesenteric lymph nodes or vascular cancer embolus (P > 0.05) ( Table 3).

Univariate and multivariable analyses of survival impact of LRIG1 expression in patients with CRC
The univariate and multivariate analyses were performed to investigate independent prognostic factors for Overall survival (OS) and Disease-free survival (DFS) in patients with colorectal cancer using the Cox proportional-hazards model ( Table 4). The analysis results demonstrated no significant difference for parameters in terms of OS (P > 0.05) and DFS (P > 0.05).

Discussion
Cancer is one of the most important and prevalent diseases with poor prognosis and there is no effective method to treat and predict the procedure of tumorigenesis. Finding the appropriate biomarkers of cancer prediction or prognosis will have huge importance in cancer management. Nowadays the new strategies searching for informative biomarkers in cancer management have attracted good attention in the world. The increasing evidences have demonstrated the leucine-rich repeats and immunoglobulin-like domain as an independent prognosis factor and predictive biomarker of clinicopathology in variety of tumors. Due to inconsistency on the effect of the LRIG1 in different types of tumors, the present study was carried out to investigate the prognostic importance of the LRIG1 expression and its relationship with clinicopathological significance in CRC. In the present study, the expression of LRIG1 at both mRNA and  protein levels was significantly decreased in CRC tumors compared with normal control but, the high levels of leucine-rich repeats and immunoglobulin-like domains expression were not significantly associated with longer overall survival, which was consistent with the conclusion of subgroup analysis. These results suggested that the LRIG1 was not a prognostic marker in CRC tumors. Meanwhile, the LRIG1 expression was significantly lower in cancer tissues than normal ones and the same result was detected with no heterogeneity in subgroup analysis based on the type of tumor. The higher levels of LRIG1 expression was not also related to positive HPV status and tumor progression assessed by its association with degree of differentiation. Also, there was no association between the LRIG1 expression and lymphatic metastasis. Some genes, such as K-ras and epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) are reported to be involved in the progress and development of colorectal cancer [22][23][24][25], but the LRIG1 roles in colorectal cancer have not been well studied and remained contradictory. Some studies showed that distal and proximal colon cancers differ in terms of molecular, pathological, and clinical features [26,27]. The present data revealed that there was not any correlation between LRIG1 expression and bilateral and peritoneal CRC metastasis (P > 0.05) and also with age, synchronous or metachronous CRC or primary tumor location (P > 0.05). Although, earlier studies proposed that LRIG1 expression was associated with a good prognosis in terms of overall survival (OS) and might act as a predictive factor for characteristics of cancer patients [28], whether the LRIG1 expression could predict a lower risk of CRC remains doubtful.

Conclusions
In conclusion, our studies revealed that although LRIG1 was down regulated in CRC and primary tumors of CRC patients but, its expression in both mRNA and protein levels, was not clinically relevant prognostic indicator in CRC. Therefore, it is suggested that LRIG1 expression analyses may not be important when making informed and individualized clinical decisions regarding the management of colorectal cancer patients.