Abstract
Colorectal cancer (CRC) is the third most common cancer and one of the main causes of death around the world. Multiple lines of evidence have suggested the role of the corticotropin-releasing hormone (CRH) family in CRC induction, including the low expression of corticotropin-releasing hormone receptor 2 (CRHR2), which is an angiogenesis inhibitor and inflammatory modulator. Previous research suggests that CRHR2 expression in colonic intestinal cells can regulate migration, proliferation and apoptosis through the modulation of several pathways. The aim of this study was to analyze the association of the rs10250835, rs2267716 and rs2267717 variants of CRHR2 gene with CRC in the Mexican population in order to consider its predictive value in CRC. This cross-sectional study included a group of 187 unrelated patients with sporadic CRC and a control group of 191 healthy blood donors. DNA extraction from peripheral blood was carried out using the Miller method. Identification of the rs10250835 variant was performed using PCR-restriction fragment length polymorphism (RFLP) and the rs2267716 and rs2267717 variants using TaqMan allelic discrimination assay. The minor allele homozygous CC of the rs2267716 variant of CRHR2 showed significant difference between CRC and control group (p=0.025), as well as the GCA haplotype (p=0.007), corresponding to the rs10250835, rs2267716 and rs2267717 variants, respectively. Our results suggest that the rs2267716 variant and GCA haplotype of CRHR2 represent a risk factor for CRC development in Mexican patients.
Significance of this study
What is already known about this subject?
Corticotropin-releasing hormone receptor 2 (CRHR2) regulates migration, proliferation and apoptosis pathways.
CRHR2 had been previously associated with prostate, pancreatic ductal, lung, kidney, hepatocellular cancer and colorectal cancer (CRC).
Downregulation of CRHR2 is associated with hepatocellular cancer and CRC, favoring the progression and poor prognosis of the neoplasia.
Different variants of CRHR2 have been associated mainly with mental disorders.
What are the new findings?
It is the first study where the rs2267716 variant and the GCA haplotype, corresponding to the rs10250835, rs2267716 and rs2267717 variants of CRHR2, are associated with CRC in Mexican population.
How might these results change the focus of research or clinical practice?
The analysis of the CRHR2 gene should be an excellent biomarker to prognosis and progression in CRC.
The results found suggest that the rs2267716 variant could be considered as a prognosis biomarker in CRC.
Introduction
Colorectal cancer (CRC) is the third most common cancer worldwide and the third leading cause of cancer death after breast, prostate and lung cancer.1 CRC is a complex multifactorial disease and has been classified into three types: sporadic, which accounts for 70% of cases, familial with 25% and hereditary with 5% of cases. According to Vogelstein, CRC is the result of multiple steps, initiated by a driver mutation, which generates chromosomal instability and microsatellite instability. Another mechanism described in colorectal carcinogenesis is the CpG island methylator phenotype, involved in the inactivation of tumor suppressor genes by methylation of promoter regions, which is responsible for about 35% of both sporadic and hereditary CRC.2–5 Recently, a strong relationship between the brain and gut axis was reported, which is maintained by the hypothalamic-pituitary-adrenal axis and its main mediator the corticotropin-releasing hormone (CRH). CRH in conjunction with urocortins (UCN1, UCN2 and UCN3) and the corticotropin-releasing hormone receptors 1/2 (CRHR1/CRHR2) are modulators of stress-related behavior as well as being responsible for anatomic and visceral changes under stress.6 7 CRHR2 is located in chromosome 7p14.3 and has 16 exons. According to its alternative splicing in exon 1, CRHR2 gives rise to three isoforms: CRHR2α (411 amino acids), CRHR2β (438 amino acids) and CRHR2γ (397 amino acids).8 9 CRHR2 is widely expressed in the central nervous system principally, and in several peripheral tissues, such as the endometrium, ovary, testis, gut, lung, liver, among others.6 8 CRHR2 gene expression variations have been associated with major depression, post-traumatic stress disorder, suicide, drug susceptibility and with CRC associated with ulcerative colitis. Additionally, previous studies have demonstrated the important role of CRHR2 in the progression, metastasis and survival of several types of cancer, mostly prostate, pancreatic ductal, lung, kidney, hepatocellular cancer and CRC.7 10–18 It has been demonstrated that one of the main processes involved in CRC invasion and metastasis is an abnormal angiogenesis, which is promoted by the release of various proangiogenic factors that activate different signaling pathways. In this regard, CRHR2 is a potent angiogenic suppressor mediated through phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), phospholipase C gamma (PLCγ) and vascular endothelial growth factor A (VEGF-A) pathways.19–22 The anti-inflammatory response of CRHR2 has been associated with the modulation of toll-like receptor 4 expression as part of innate immunity.23 In addition, CRHR2 expression can regulate the migration, proliferation and apoptosis of intestinal cells in the colon through downregulation of interleukin (IL)-6 and its receptor IL-6R. This results in the inhibition of STAT3 in CRC cells, thereby blocking the expression of all STAT3-regulated genes, which are associated with the inhibition of cell cycle and epithelial-mesenchymal transition.7 12 24 It is suggested that CRHR2 expression is of great clinical significance in the development of CRC.6 7 12 23 24 In a recent study, Panagopoulou et al observed that CRHR2 hypermethylation patterns may be associated with decreased expression of the receptor, which in turn may contribute to CRC progression and metastasis. Additionally, it had been suggested that CRHR2 variants may affect splicing levels, resulting in protein expression modifications that favoring the CRC develop.25
Studies on CRHR2 variants have been mainly associated with mental disorders, such as the rs3779250 variant associated with depression, negative emotions and irritable bowel syndrome,9 26 and the rs2190242, rs2284217 and rs2014663 variants associated with suicide.14 The rs2267717 variant located in the second intron of the CRHR2 gene at chromosomal position 30,677,427 was previously associated with openness personality, however, it was not associated with preterm birth.27 28 Finally, the rs2267716 variant, which is also located in the second intron of the CRHR2 gene in chromosomal position 30,677,027, was associated with food addiction and as a contributing factor to variability in acute bronchodilator response in Caucasian asthmatic individuals.29 30 However, there is little information about CRHR2 variants and cancer. To our knowledge, there are two reports in which the rs2267716 variant is associated with susceptibility to hepatocellular carcinoma (HCC) in hepatitis B virus-positive patients in Chinese population.8 31 Gu et al suggest that the presence of the rs2267716 variant might cause an alternative splicing regulating the gene expression, or change the conformational gene structure, modifying its affinity to bind to transcription factors.8
Due to limited information regarding cancer and CRHR2 variants, in the present study, we aimed to analyze the association of the three intronic variants rs10250835, rs2267716 and rs2267717 of the CRHR2 gene in peripheral blood of patients with CRC.
Materials and methods
Patients and samples
This retrospective cross-sectional study included 187 DNA samples from unrelated patients with CRC without a family history of cancer who had not previously received chemotherapy; 71 (38%) were females and 116 (62%) were males with a mean age of 58 years. The control group included 191 DNA samples from randomly selected blood donors, of whom 81 (43%) were females and 109 (57%) were males. The samples were collected at the Juan I. Menchaca and Fray Antonio Alcalde Hospitals in Guadalajara, Jalisco, Mexico and the Alfredo Pumarejo Hospital in Matamoros, Tamaulipas, Mexico during 2017–2019.
Blood samples were collected in EDTA tubes and the DNA extraction was performed by the Miller method.32 DNA concentration and purity were determined with the NanoDrop One/One C Microvolume UV-Vis spectrophotometer. DNA aliquots were stored at −80°C for subsequent genotyping. The CRHR2 gene variants selection was performed considering a minor allele frequency >0.10 in Mexican population from Los Angeles, California according to the Haplotype Map Project (http://hapmap.ncbi.nlm.nih.gov/index.html.en). The variants rs10250835, rs2267716 and rs2267717 were selected.
Genotyping
Genotyping of the rs10250835G>A variant was performed by PCR-RFLP method, using the specific primers 5’-GCA GCC TCT ACA TCC ACC AT 3’ (forward) and 5’-TAG TTT CGT CGC CTG CTT TT 3’ (reverse). Reaction conditions were performed in a volume of 25 µL, containing 17.85 µL of sterile distilled water, 0.75 µL of MgCl2 (1.5 mM), 0.5 µL of deoxyribose nucleotide triphosphate (dNTPs) (100 µM), 1 µL of each primer (100 µM), 2.5 µL of buffer 10X (1X), 0.4 µL of Taq DNA polymerase (200 U/µL) and 1 µL of genomic DNA (50 ng). The parameters used were as follows: 94°C×2 min; followed by 30 cycles of 94°C×30 s, 64°C×30 s, 72 °C×30 s and a final extension of 72°C×5 min. After DNA amplification, the 160 bp PCR product was digested with the restriction endonuclease BsrDI (New England BioLabs, Beverly, Massachusetts, USA), which identifies the ancestral allele G, generating fragments that were separated by electrophoresis and visualized with AgNO3. The three different genotypes were identified in accordance with the following fragments: G/G, 160 bp fragment; G/A, 160, 85 and 75 bp fragments and A/A, 85 and 75 bp fragment (figure 1). For the rs2267716T>C and rs2267717G>A variants, genotypes were identified by TaqMan allelic discrimination assay following the manufacturing conditions. We used the TaqMan Universal PCR Master Mix, TaqMan SNP Genotyping Assays (ID C_2570970_1_ Applied Biosystems to rs2267716T>C variant and ID C_15872907_10 to rs227717G>A variant) and 50 ng of genomic DNA. Cycling parameters were as follows: 95°C for 10 min, 35 cycles of 92°C for 15 s, 35 cycles of 60°C for 1.5 min and a final extension of 60°C for 2 min. All results were automatically called by the thermocycler RealPlex, Eppendorf Real-Time PCR System software.
Silver-stained polyacrylamide gel of the rs10250835 polymorphism. Lane 1: 25 bp molecular weight marker; lane 2: blank; lanes 3, 4, 5, 6, 7, 8, 11, 14, 15 and 16: G/G genotype; lanes 9 and 13: G/A genotype; lanes 10 and 12: no result.
Statistical analysis
Statistical analysis was performed with the SPSS V.23.0 (IBM, Chicago, Illinois, USA). Qualitative variables are presented by frequency and percentage, and quantitative variables as mean±SD. Allelic and genotypic frequencies in both groups were compared with χ2 test, while the association analysis was carried out by OR and 95% CI values. Hardy-Weinberg equilibrium (HWE) was tested using the χ2 test. Linkage disequilibrium (LD) and haplotypes analyses were carried out using SHEsis online software (http://analysis.bio-x.cn/myAnalysis.php).33 The criterion for statistical significance was p<0.05. For each variant, in silico predictive analysis was done with the Human Splicing Finder software.34
Results
It was possible to obtain information regarding tumor site in only 160 cases of the 187 samples of CRC: 96 (60%) tumors were located in the descending colon and 64 (40%) in the rectum. Allelic and genotypic frequencies of CRHR2 variants are shown in table 1. Allele frequencies in both groups were consistent with HWE. When genotypic frequencies were compared between the control group and CRC groups, a significant difference was found for the rs2267716 variant (OR=2.410 (95% CI=1.115 to 5.072); p=0.025).
Allelic and genotype frequencies of rs10250835, rs2267716 and rs2267717 variants of CRHR2 in patients with CRC and control group
LD and haplotype analyses of all variants among CRC and the control group showed that the rs10250835 and rs2267716 variants as well as the rs2267716 and rs2267717 variants were medium LD with D’ values of 0.572 and 0.453, respectively. However, both LD had extremely low r2 values (figure 2). We have identified seven haplotypes, where the GCA haplotype (rs10250835/rs2267716/rs2267717) emerged as a risk factor (OR=5.613 (95% CI=1.602 to 19.667); p=0.007) for CRC in this population (table 2). In silico analysis only revealed that the rs2267716 variant generated a donor site, creating a new (weak to medium) splicing site (70.59>80.16).
Haplotype analysis of rs10250835, rs2267716 and rs2267717 variants of CRHR2 in patients with CRC and control group
Linkage disequilibrium (LD) plots illustrating the degree of linkage disequilibrium of CRHR2 variants in this study. (A) Values in the LD blocks represented the D’ in percentage. (B) Values in the LD blocks represented the r2 in percentage.
Discussion
There is evidence for the role of the neurotransmitters and neuropeptides in the pathogenesis of inflammation associated with CRC development, and one of the most important mechanisms is the role of chronic stress and regulation of inflammation, which have the potential for carcinogenesis. The CRH family of neuropeptides, which include UCN1, UCN2, UCN3, CRH-binding protein and their receptors CRHR1 and CRHR2, have recently been associated with CRC.6 7 18 23 35
Most analyses of CRHR2 variants have focused on human behavioral diseases due to the abundance of expression in the brain, mainly of the subcortical structures. However, CRHR2 has also been observed to be expressed in several peripheral tissues in humans, including fat tissue, the heart, skeletal muscle, myometrium, adrenal gland, small intestine and colon.23 34 In the current study, we identified that the CC genotype of the rs2267716 variant of CRHR2 as well as the GCA haplotype are associated with the development of CRC in the Mexican population. According to in silico analysis, the CC genotype of rs2267716 variant creates a donor site, which likely affects protein expression.34 The minor allele C of rs2267716 has previously been associated with two chronic diseases, asthma and hepatocellular cancer. The asthma study was carried out to identify the effect of the rs2267716 variant of CRHR2 on bronchodilator response in 152 subjects who were enrolled in the Clinical trial of Low-Dose Theophylline and Montelukast. In this study, the author suggested three possible hypotheses, with the first referring to the CRHR2 pathway as able to desensitize adrenoceptor beta 2 (β2-AR) function. The second proposed mechanism was that a reduction in CRHR2 expression results in increased production of anti-inflammatory cytokines, which is sufficient to downregulate the pathway through β2-AR. The last hypothesis was that β2-AR and CRHR2 act as a synergic team, relaxing bronchial smooth muscle and stimulating 3',5'-cyclic adenosine monophosphate (cAMP) production to generate a protection mechanism in patients with asthma. Thus, patients carrying the rs2267716 variant generate a decrease in biological activity, resulting in a weak bronchial response.30 The second chronic disease that CRHR2 variants have been associated with is hepatocellular cancer, a concept first studied by Kato et al, who evaluated 188 Japanese patients with chronic hepatitis C virus, including patients with HCC. In this research, they observed that a specific haplotype of the SCYB14 and GFRA1 genes, and the presence of rs2267716 variant of CRHR2 are associated with HCC in patients with chronic hepatitis C virus infection.31 A second study investigated the association between rs2267716 variant in CRHR2 and its susceptibility to HCC related to hepatitis B virus in a Chinese population, suggesting that patients carrying the variant influenced the inflammatory response to hepatitis B virus infection and, therefore, modified the risk for HCC development, all due to the reduced inhibitory capacity of CRHR2 for angiogenesis.8 CRHR2 is a very intriguing protein regarding its expression and functions, probably because it has 5’ promoter elements bound by several transcription factors, such as EGR1 and RXRA, which can activate different signaling pathways.36
Most studies of CRHR2, however, have focused on expression profiling, demonstrating that low CRHR2 protein expression is associated with CRC development. Changes in the expression of CRHR2 and its ligands have been shown to be associated with progression and poor prognosis in several types of cancer.37 38 In this regard, Ducarouge et al demonstrated that CRHR2α and UCN3 expression are in concordance with tumor grade, showing that CRHR2 expression is increased by 33% in low-grade CRC samples and up to 72% in high-grade CRC tissues.39 At the same time, this study demonstrated that CRHR2 induced disruption of adherent cell junctions due to E-cadherin endocytosis, exhibiting an important role of CRHR2 in cell migration and differentiation. UCN3 was also demonstrated to regulate transcription of the matrix metalloproteases (MMP)3 and MMP7, which are responsible for cell migration and invasion into different tissues by degrading extracellular matrix elements.39
Some reports describe the roles of UCN1 and CRHR2 in enteric enterochromaffin and neuronal cells to mediate inhibitory and stimulatory gastric motility through an autocrine or paracrine mechanism and have suggested their involvement in regulating cellular processes such as apoptosis, angiogenesis and inflammation.36 40 However, little is known regarding the impact of CRHR2 expression on cancer development and prognosis in humans, suggesting that CRHR2 may play an important role in the regulation of different pathways associated with the modulation of the local immunological profile, which is one of the main mechanisms associated with carcinogenesis.23 Another study performed in mice with CRC tumors detected that UCN2 can regulate CRHR2 mRNA levels, which are substantially lower than those observed in normal tissue, suggesting its association with poor prognosis, which is valued as a dramatical reduction in survival and metastasis in human CRC. One of the functions of CRHR2 is to inhibit cell division through the downregulation of IL-6 and its receptor IL-6R in addition to a decrease of STAT3 phosphorylation.7 Recently, it has been shown that CRC development associated with ulcerative colitis may be related to CRHR2 downregulation by hypermethylation.12
In this sense, we consider that this study was limited by the relatively small sample size and the absence of tumorous tissue to be able to evaluate the expression of CRHR2, as well as some of the genes involved in angiogenesis and immunological pathways, which are targets of CRHR2 in order to assess the real contribution on CRC development.
In conclusion, this is the first report where association of the CC genotype of the rs2267716 variant and the GCA haplotype (rs10250835/rs2267716/rs2267717) of CRHR2 increased the risk of CRC in a Mexican population. However, it is important to consider that the development of CRC is a complex disease that involves the mutation of several genes and environmental factors such as lifestyle and the ethnicity of the participant.
Data availability statement
All data relevant to the study are included in the article or uploaded as supplementary information. All relevant data can be requested to the corresponding author to the mail nellymacias_2000@yahoo.com.mx.
Ethics statements
Patient consent for publication
Ethics approval
This study involves human participants and was approved by Ethic committee of Centro Universitario del Sur, Universidad de Guadalajara (ID number CB-018/2017). Participants gave informed consent to participate in the study before taking part.
Acknowledgments
The authors would like to thank Consejo Nacional de Ciencia y Tecnología (CONACYT) for the scholarship granted to the first author for doctoral studies. The authors would also like to thank Dr María de la Luz Ayala-Madrigal, Dr Jorge Peregrina-Sandoval and Ruth Ramírez-Ramírez for their support with sample recollection.
Footnotes
Contributors AAR-G, COG-V, EL-U, MG-A, MR-F, ID-E and NMM-G have contributed to design of the work and interpretation of data. Moreover, they have approved the final version. AAR-G, COG-V and NMM-G have worked with acquisition, analysis of molecular data and guarantor.
Funding The project was supported by Universidad de Guadalajara, México.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
References
