Genetic alterations of CDX1, CYLD and CDKN2B genes in CRC

Seyed Mohammad Taghi Hamidian ¹, Rezvan Azadi ², Pooya Rostami ³, Farnaz Azar Shabe ⁴, Zeynab Khazaee Kohparc ⁴^*

¹Babol University of Medical Sciences, Department of Gastroenterology, Babol, Iran

²Shahid Beheshti University of Medical Sciences, Department of Medicine, Tehran, Iran

³New York University, Londgone Medical Center, Brooklyn, NY, USA

⁴Islamic Azad University of Tonekabon Branch, Department of Biology, Tonekabon, Iran

^*Corresponding Author: Zeynab Khazaee Kohparc

* Email: zeynab_zhazaee_kohparc@yahoo.com

Abstract

Introduction: Colorectal cancer (CRC) is the third most frequent type of cancer in the world. In this explanation, genetic variation is associated in all cancers, particularly CRC, and modifications of numerous genes, such as CDX1, CYLD, and CDKN2B, are linked to tumorigenesis in CRC. As a result, this research was conducted in order to determine changes in the expression of these genes.

Materials and Methods: Specimens of CRC from 72 individuals with confirmation of pathology report,were provided and bought from the Bio banks. Real-time PCR was used to examine the expression of CDX1, CYLD, and CDKN2B genes in tumoral and non-tumoral tissues. These genes' histological associations with grading and staging for upregulation and downregulation were examined.

Result: The expression of CYLD (P = 0.01) and CDKN2B (P = 0.02) were upregulated significantly, but the CDX1 (P = 0.03) gene expression was decreased. Correspondingly, there was no significant association between CDX1 downregulation and CDKN2B upregulation with grade, stage, lymph‐node metastasis (P= 0.02) and distant metastasis. Moreover, the CYLD expression was also significantly associated with high grade (P = 0.03), high stage (P = 0.03), lymph‐node metastasis (P= 0.05) and distant metastasis (P= 0.05).

Conclusion: The upregulation of CYLD and CDKN2B genes and downregulation of CDX1 gene in tumoral tissues were impressive. Conclusively, the alteration of these genes expression can be considered as a colorectal cancer biomarker.

Keywords: Colorectal cancer, CDX1, CYLD, and CDKN2B genes, Alterations

Introduction

Colorectal cancer (CRC) is one of the most important causes of cancer mortality in the world (1). The major factor of CRC is the presence of polyps in the colon and also the changes of adenoma to carcinoma process. CRC is the growth of cancer cells in the colon part caused by uncontrolled growth of cells that can proliferate in other tissues irregularly (2). In this way, the term survival of patients with CRC has not been improved in a therapeutic manner. Strongly, there is a vital and emergency requirement for a better understanding in the molecular pathogenesis of CRC in order to recognize the novel biomarkers for prognosis and diagnosis of CRC (3). Correspondingly, molecular genetic methods especially based on DNA and RNA investigating are really practical and useful in diagnostic medicine (4).

CDX1 (caudal-type homeobox 1) is a transcriptional factor and controls enterocyte differentiation in the colon, where its expression is different from the crypt-base stem cell structure. Remarkably, CDX1 is also a keyword to the capacity of a CRC cell line in differentiation, and it is classified as a negative marker of CRC stem cells. CDX1 is required for the actual development of the homeostasis of the intestinal epithelium and also intestinal tract (5). Interestingly, CDX1 is involved in the modulation of a variety of processes comprising cell adhesion, columnar morphology, proliferation, and apoptosis. CDX1 is a primary controller of enterocyte differentiation and its expression is vital for the transcriptional regulation of a large number of intestine-specific genes essential for the maintenance of the intestinal phenotype, differentiation, and intestine development. Many markers in the differentiation process, containing villin and cytokeratin 20, have been indicated to be directly transcriptionally regulated by this gene. Many evidence indicates the loss or down-regulation of CDX1 expression in colon cancer tumors and cell lines (6, 7).

Another important gene in gastrointestinal cancers particularly CRC, is the cylindromatosis (CYLD) gene, which was initially explored as a tumor suppressor mutated for familial cylindromatosis (8). In addition to skin tumors caused by CYLD loss, decreased CYLD expression has been described in several types of human cancers comprising breast cancer, hepatocellular carcinoma, cervical cancer, renal cell carcinoma, lung cancer, gastric cancer and also colon cancer. Remarkably, the expression profile and clinical significance of CYLD in patients with a series of co- colorectal lesions are so important (9-11).

CYLD was recognized identified as a gene mutated in familial cylindromatosis (FC), a genetic case that predisposes patients for the progression of skin tumors, termed cylindroma. Cylindromas are benign tumors that emerge on the scalp and interestingly is to be derived from hair follicles of stem cells (12). The cylindromatosis patients possess heterozygous germ-line mutations in the CYLD gene, but the wild-type CYLD allele undergoes loss of heterozygosity (LOH) and rarely somatic mutations in different tumors as tumor suppressor gene. The human CYLD gene is situated on chromosome 16q12.1 and encodes a protein of 956 amino acids. The C-terminal region of CYLD includes a catalytic domain with sequence homology to USP family members (9, 13). The second important gene is CDKN2B which is referred to the CDKN2A tumor suppressor gene in a region at 9p21 and this gene is regularly mutated and omitted in many different tumors. Considerably, this gene encodes a cyclin-dependent kinase inhibitor, and it is considered as CDKN2B protein, which is a cell cycle regulator (14). The CDKN2B gene encodes for CDKN2B, which is a member of the INK4 class of cell cycle inhibitors. Noticeably, CDKN2B has ankyrin repeats that permit it to bind and interact of cyclin-dependent kinase (CDK) 4/6 with cyclin D, through inhibiting the function of CDK4/6. Given the critical role of CDK4/6 and cyclin D in improving development through the G1 checkpoint, CDKN2B performs as a significant inhibitor of cell cycle and cell proliferation (15, 16).

Materials and Methods

Samples collection

The research was performed on 72 patients (53 female and 19 male) which was confirmed by the pathology department and also an agreement by patients. The histopathological status of patients is shown in Table 2. 72 tumoral and 72 non-tumoral (margins tissues) were provided and bought from the Bio banks. In this way, DEPC (diethylpyrocarbonate) was employed to clean and treat all sampling instruments during providing the biopsies (tumoral and nontumoral tissues) in order to avoid RNAs enzyme. Correspondingly, after sampling operation, all specimens were transferred to liquid nitrogen for deep freezing. Vitimately, tissue samples were stored at − 80 °C for long preservation and study. RNA isolation from human tumoral and nontumoral tissues was performed using a commercial reagent, Trizol (Invitrogen cat no 15596-025, USA.) Less than 1cm of each tissue was crushed in order to powder them by a mortar and pestle in the presence of liquid nitrogen, and 40– 80 mg of powdered tissue was used for RNA isolation according to the manufacture’s protocol. RNA quantity was measured by A260/A280 ratio using NanoDrop spectrophotometer (TC100, USA) and also controlled by electrophoresis on agarose gel 2% in order to observe all RNA bands (5S, 18S and 28S).

Relatively, cDNA synthesis was done in the presence of 1 pg total RNA, 4 μL 5X reaction buffer, 10 mM each of dNTPs, and 1 μL (200 U/ μL) by QuantiTect Reverse Transcription Kit (cat no 20S313, USA) in a final volume of 20 μL, by 60 min incubation at 44°C. Meanwhile, Real-time PCR was done on Exicycler q6, Bioneer, USA by using a universal reverse primer and Universal Taqman-specific probe and also the expression levels of all these genes were normalized against GAPDH, RNA as control. The 20 μL PCR comprised 1μμL RT yeild, 0.25 mM universal-specific probe, 0.5 mM each forward and reverse primers. The PCR reagents were all from Qiagen HotStarTaq reagent set (Qiagen, cat no 203205). The mixtures were incubated at 96 °C for 5 min, followed by 43 cycles of 90 °C for 45 s, and 63 °C for 1 min. All reactions were done in triplicate. The CTs were described as the fractional cycle number.

The primers were designed by Allel ID version 7 software. The first cDNA strand was synthesized. The sequences of forward and reverse primers used are given in Table 1. The Real-time PCR tests were accomplished in a Step one instrument (Applied Biosystem, USA) using cDNA. An amount of 1 μl cDNA from each sample was determined for amplification. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was employed as a housekeeping gene. Amplification occurred in a 20 μl final volume by initial incubation at 96 °C for 5 min, followed by 43 cycles of 95 °C for 30 s and 60 °C for 1 min. The range of up-regulation or down-regulation in each sample was measured using the 2^{-▲▲ ct} method.

Table 1. Sequences of primers employed for Real-time PCR action.

Primer sequence (5′–3′)
Forward CDX1	5´-AAGCCTCCGRRCCGCGAATCA-3´
Reverse CDX1	5'-GGAAGACTCGTGTATGTATGTGY ATATGTG-3'
Forward CYLD	5'-ATGGATAACCCTATTGGCAACTG-3'
Reverse CYLD	5'-GTATCCAGTGCTGCGACCGT-3'
Forward CDKN2B	5'- TGGCCGGAGGTCATGATG -3'
Reverse CDKN2B	5'- GGGCAGCATCATGCACCG -3'

Statistical Analyses

All the acquired data from Real-time PCR were analyzed by exercycle set. Correspondingly, the significant difference was statistically interpreted by paired Student’s t-test. P < 0.05 was considered statistically significant. Analyses were accomplished using commercially available statistical software (SPSS Statistics software, version 25, Chicago).

Results

Gene expression evaluation in tumoral tissues

The analysis of expression levels of tumoral and corresponding non-tumoral tissues for CDX1, CYLD and CDKN2B genes indicated that the CYLD and CDKN2B were down regulated in tumoral tissues in comparison with their non-tumoral counterparts (P = 0.02). On the contrary, CDX1 expression level had decreased significantly in 70% of samples (Figure 1,2,3).

Clinicopathological analysis

Clinicopathological consequences of CDX1, CYLD and CDKN2B genes expression were evaluated in 72 patients diagnosed with adenocarcinoma of the colorectal. Patients’ clinicopathological characteristics are summarized in Table 2. The analysis of different clinicopathological variables and genes expression correlation is presented in Table (up/down). The mean age of patients was 58.9±12.5 years at the time of diagnosis (female to male ratio, 4:1; age range, 37–88 years). In general, more than half of the patients had advanced stage (Stages III–IV), and high-grade histology. Lymph node metastasis and distant metastasis were observed in more than 60% of the patients.

Table 2. Clinicopathological characteristics of colorectal cancer cases.

Total (N=72)

Patients (%)

Characteristics

53 (73.6)

19 (26.4)

Gender

Female

Male

38 (52.8)

34 (47.2)

Age

< 60 years

≥ 60 years

6 (8.3)

24 (33.3)

38 (52.8)

4 (5.6)

Stage

III

4 (5.6)

26 (36.1)

39 (54.1)

3 (4.2)

Grade

Well differentiated

Moderate differentiate

Poorly differentiate

Undifferentiated

45 (62.5)

27 (37.5)

Yes

44 (61.1)

28 (38.9)

Yes

The number of gene expressions of all samples was compared and investigated with the stage, grade, lymph node metastasis and distance metastasis of all patients. The analysis of different clinicopathological variables and genes expression correlation is presented in Table 3. Statistical analyzes were performed using SPSS 25 and also Chi-Square test and T-test.

The expression of CDX1, CYLD and CDKN2B was matched with different clinicopathological data of the colorectal cancer patients (summarized in Table 2). There was no significant association between CDX1 downregulation and CDKN2B upregulation with the grade, stage, lymph‐node metastasis (P= 0.02) and distant metastasis. Moreover, the CYLD expression was also significantly associated with high grade (P = 0.03), high stage (P = 0.03), lymph‐node metastasis (P= 0.05) and distant metastasis (P= 0.05) (figure 4, 5, 6).

Table 3. The association of genes expression with clinicopathological qualification. LM: Lymph node Metastasis, DM: Distance Metastasis; ↓/−: decrease or no change of expression; ↑: increase of gene expression

	*CDX1*		P value		*CYLD*		P value		*CDKN2B*	P value
Tumor Stage I-II III-IV	↓/− 18 33	↑ 12 9	0.7	↓/− 0 0		↑ 30 42	0.03	↓/− 12 7	↑ 18 35	0.5
Tumor Grade I-II III-IV	19 30	11 10	0.1	0 0		30 42	0.03	13 6	17 36	0.6
LM Yes No	30 21	14 7	0.4	0 0		44 28	0.05	24 11	22 15	0.3
DM Yes No	32 19	12 9	0.5	0 0		44 28	0.05	21 15	23 13	0.2

LM: Lymph node Metastasis, DM: Distance Metastasis

The Association of CDX1, CYLD and CDKN2B expression with clinicopathological qualifications

Discussion

Transgenic expression of CDX1 in mouse gastric epithelium causes intestinal transdifferentiation, which protects this consideration that CDX1 is up-regulated in Barrett’s metaplasia of the esophagus. Considerably, many transcriptional targets and effective activities of CDX1 have been recognized, there remains much to learn about the mechanisms by which it encourages differentiation and, also, those by which it inhibits stemness CDX1 action as transcription factors regulate a wide range of cellular mechanisms (6).

Additionally, CDX1, an intestine-specific transcription factor, is a candidate tumor suppressor gene and it manages the intestine-specific gene transcription and regulates the intestinal epithelial cell phenotype. Past investigation illustrated that the murine CDX1 overexpression in rat normal intestinal epithelial cells regulates proliferation as a conclusion of inducing cell cycle arrest. Meaningly, this antiproliferative role may be mediated through down-regulation of the D-type cyclins (17). The CDX1 gene is expressed in a collaborative model during intestinal progression. CDX1 expression will last in the intestinal epithelium throughout life, notably in the crypt. The same model of CDX1 expression was discovered in the human small intestine. Many searches have described that the CDX1 expression is markedly down-regulated in both adenomas and carcinomas of the colon. Little is known about the molecular mechanisms that regulate the developmental and spatial patterns of the CDX1 expression in normal intestine or what induces the down-regulation in colonic adenomas and cancers (18). Wong et al. have shown that the loss or reduction of CDX1 is often induced by promoter methylation. Together, these observations indicate a potential role of CDX1 loss in tumor development (19).

Recently, the expression monitoring of CYLD in many colorectal-related lesions and the clinical significance of CYLD expression in CRC have remained unclear, although, past investigation indicating that both the transcription function and the protein level of CYLD were downregulated in colon cancer in comparison with normal colon tissues. The difference of CYLD expression in the normal colorectal epithelium, benign adenoma, primary CRC and metastatic lesions was explored (20). Of particular interest, we wondered whether CYLD expression played a part in tumor development, progression, or metastasis and whether reduced CYLD expression was a good or poor prognostic factor for CRC patients. These findings strengthened the fact that CYLD functioned as a tumor-suppressor gene not only in the skin tumor but also in CRC. In addition, reduced CYLD expression was an independent factor for poor prognosis of CRC patients. Based on the evidence above, our results also recommended that the downregulation of CYLD might be involved in a series of important biological properties of colorectal cancer cells, such as carcinogenesis, tumor progression and metastasis (21). These findings also have implications on the tumor suppressor function of CYLD, as colonic inflammation in IBD patients is a risk factor for colorectal cancer. The potential association of CYLD gene suppression with colon cancer is more directly suggested by a study showing reduced expression of CYLD in colon cancer cell lines and tissue samples It is currently unknown how the CYLD gene is suppressed in IBD and colon cancer cells. Nevertheless, the mechanistic insight of CYLD gene repression has been provided by studies using other cancer models (22).

In another study, CYLD expression was analyzed in two of the most common human carcinomas worldwide. Colon carcinoma derives from intestinal epithelial cells and HCC derives from hepatocytes. We found reduced CYLD mRNA expression in all three HCC cell lines and eight colon carcinoma cell lines examined compared with normal primary cells. Additionally, reduction or loss of CYLD expression was found in situ in most hepatocellular and colon carcinoma compared with non-neoplastic tissue samples. Analysis on protein level confirmed these findings. Functional assays with CYLD transfected cell lines revealed that CYLD expression decreased NF-κB activity. Thus, functional relevant loss of CYLD expression may contribute to tumor development and progression, and may provide a new target for therapeutic strategies (11). CDKN2B is a cyclin-dependent kinase inhibitor and functions as a cell growth regulator that controls cell cycle G1 progression. Last investigations have acknowledged CDKN2B as a required tumor suppressor, and deletion of its enhancer element is related to many different malignancies. Silencing of CDKN2B gene expression by epigenetic modification characterize in multiple myelomas gastric adenocarcinoma (23). Reexpression of CDKN2B in tumor-derived cells significantly attenuates the tumorigenic potential of the cells and delays tumor progression (24). Fluctuation of CDKN2B's expression has been announced in association with many malignancies particularly, prostate, colorectal, breast, and liver cancer. Considerably, CDKN2B were ubiquitously expressed in colon cancer at different stages of tumorigenesis (25).

CDKN2B encoded by the INK4b-ARF-INK4a locus. It is an acknowledged tumor suppressor gene that can form a complex with CDK4 or CDK6 and inhibits the activation of the cyclin-dependent kinase and progression of the cell cycle. The INK4b-ARF-INK4a locus is organized by Polycomb repressive complexes. In this way, downregulation of CDKN2B was investigated in cancers (26). The epigenetic investigation of these genes alongside gene expression and also a mutation of other genes which are involved in GI cancers is recommended strongly.

Conclusion

It is concluded that the upregulation of CYLD and CDKN2B genes and downregulation of CDX1 gene in tumoral tissues were impressive. Conspicuously, the modification of these genes expression can be accepted as the main biomarker in colorectal cancer.

Author contributions

RZ, PR, and FAS collected data and accomplished some sections of the study and manuscript, SMTH collected all the biopsies directly in Omid clinic and hospital by himself and also confirmed the clinical qualifications of all the patients as a gastroenterologist. ZKK controlled and confirmed the data quality, evaluated and optimized the informatics database, wrote the paper and edited it, some other essential functions containing study design, controlling the project and protocol development and also data analysis. All authors revised the article carefully, read

and acknowledged the final version of the paper.

Acknowledgments

We thank all people who were involved in this project and contributed us.

Conflict of interests

Authors declare no conflict of interest.

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