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. 2020 Feb 26;12(3):539.
doi: 10.3390/cancers12030539.

Enhanced CXCR4 Expression Associates with Increased Gene Body 5-Hydroxymethylcytosine Modification but not Decreased Promoter Methylation in Colorectal Cancer

Alexei J Stuckel  1 , Wei Zhang  2 , Xu Zhang  3 , Shuai Zeng  4   5 , Urszula Dougherty  6 , Reba Mustafi  6 , Qiong Zhang  1 , Elsa Perreand  1 , Tripti Khare  1 , Trupti Joshi  4   7   8 , Diana C West-Szymanski  6 , Marc Bissonnette  6 , Sharad Khare  1   9
Affiliations

Enhanced CXCR4 Expression Associates with Increased Gene Body 5-Hydroxymethylcytosine Modification but not Decreased Promoter Methylation in Colorectal Cancer

Alexei J Stuckel et al. Cancers (Basel). .

Erratum in

Abstract

In colorectal cancer (CRC), upregulation of the C-X-C motif chemokine receptor 4 (CXCR4) is correlated with metastasis and poor prognosis, highlighting the need to further elucidate CXCR4's regulation in CRC. For the first time, DNA methylation and 5-hydroxymethylcytosine aberrations were investigated to better understand the epigenetic regulation of CXCR4 in CRC. CXCR4 expression levels were measured using qPCR and immunoblotting in normal colon tissues, primary colon cancer tissues and CRC cell lines. Publicly available RNA-seq and methylation data from The Cancer Genome Atlas (TCGA) were extracted from tumors from CRC patients. The DNA methylation status spanning CXCR4 gene was evaluated using combined bisulfite restriction analysis (COBRA). The methylation status in the CXCR4 gene body was analyzed using previously performed nano-hmC-seal data from colon cancers and adjacent normal colonic mucosa. CXCR4 expression levels were significantly increased in tumor stromal cells and in tumor colonocytes, compared to matched cell types from adjacent normal-appearing mucosa. CXCR4 promoter methylation was detected in a minority of colorectal tumors in the TCGA. The CpG island of the CXCR4 promoter showed increased methylation in three of four CRC cell lines. CXCR4 protein expression differences were also notable between microsatellite stable (MSS) and microsatellite instable (MSI) tumor cell lines. While differential methylation was not detected in CXCR4, enrichment of 5-hydroxymethylcytosine (5hmC) in CXCR4 gene bodies in CRC was observed compared to adjacent mucosa.

Keywords: 5-hydroxymethylcytosine; CXCR4 gene expression; DNA methylation; colorectal cancer; epigenetic regulation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
C-X-C motif chemokine receptor 4 (CXCR4) transcript and protein expression in tumor and adjacent normal tissue. RT-PCR analysis of CXCR4 transcript abundance in normal and tumor tissue: (a) stromal cells and (b) colonocytes after multiple testing correction (Tukey post hoc test, Supplementary Table S1). Note the increase of CXCR4 transcripts in both tumor stromal cells and colonocytes compared to adjacent matched tissue controls (q-value < 0.05). (c) CXCR4 protein expression in tumor colonocytes (T) and normal colonocytes adjacent to tumor (adj). Note the increased CXCR4 protein expression in tumor colonocytes compared to adjacent colonocytes from normal appearing mucosa (n = 5; p-value < 0.05, paired Student’s t-test, Supplementary Table S2).
Figure 2
Figure 2
Representational combined bisulfite restriction analysis (COBRA) results of 3 regions of interest located in CXCR4 from one colorectal cancer (CRC) patient: (a) (#492) and one healthy individual (Supplementary Figure S3) (b) (#578) (Supplementary Figure S4). Each sample was run on two lanes: lane 1) bisulfite-treated PCR amplified DNA without restriction enzyme digestion, which served as a reference control for unmethylated CpG and lane 2) bisulfite-treated PCR amplified DNA digested with a restriction enzyme recognizing amplicons containing a 5’mCpG sequence. Region #3 shows complete PCR product digestion, indicating that region #3 possesses cytosine methylation (5mC) CXCR4 in tumor colonocytes (T# patient ID number), adjacent normal colonocytes (C# patient ID number), and proximal/distal colon of healthy individuals (H# patient ID number P/D proximal/distal colon). Restriction enzyme digestion was not evident in regions #1 or #2 in any tumor or normal colonocytes from any CRC patient or controls and therefore unmethylated in both regions. PCR product treated with restriction enzyme lane = +; PCR product from untreated sample lane = −; Restriction enzyme sensitive digestion was not detected for the amplicons of the intragenic CpG island region #2 or promoter region #1 in either tumor or control samples, indicating the absence of 5mC in those regions (Figure 2a). In addition to CRC samples, regions #1–#3 were assessed in colonocytes from proximal and distal colon in five healthy control individuals. The 5mC results of CXCR4 for colonocytes from healthy control individuals were identical to those of malignant colonocytes from CRC patients, with 5mC present in region #3, but absent in regions #1 or #2 (Figure 2b). Whole COBRA gel images of region #1–#3 for the other CRC and normal control colons tested for CXCR4 5mC are shown in Supplementary Figures S3 and S4, respectively.
Figure 3
Figure 3
5 Boxplots displaying methylation beta-values (Y-axis) of 5 probes within promoter CpG island region #1, extracted from CRC patients classified in the Tumor, Node, Metastasis (TNM) staging system as M1 = evidence of metastasis and M0 = no evidence of metastasis from the TCGA registry. The [CG########] ID on the X-axis of each boxplot represents the unique ID of each probe within the 450K-array data set. A student t-test was conducted in finding mean beta-value differences for two CG probes between M0 and M1 cohorts (p-value < 0.05).
Figure 4
Figure 4
Combined bisulfite restriction analysis (COBRA) results of 3 regions of interest located in CXCR4 from: (a) 4 CRC cell lines: HT29, SW480, HCT116, and RKO cells and (b) 4 untransformed colon cell lines: CCD-841, CCD-18Co, FHC, and FHs 74 cells. Region #3 shows complete PCR product digestion in CRC cell lines: HT29, SW480, and HCT116 cells and untransformed cell lines: FHC and FHs 74 cells, indicating that region #3 is fully methylated. Incomplete PCR product digestion in RKO cells and untransformed colon cell lines: CCD-841 and CCD-18Co cells indicate partial methylation in region #1. Incomplete PCR digestion was only evident in RKO cells in region #2 indicating partial methylation. 3 of 4 CRC cell lines: HT29, HCT116, and RKO cells displayed incomplete PCR digestion in region #1, whereas no PCR digestion was evident in any untransformed colon cell line cells. (c) RT-PCR analysis of CXCR4 transcript abundance in 4 CRC cell lines: HCT116, SW480, Caco2, and DLD1 cells (q-value < 0.05) after multiple testing correction (Tukey post hoc test, Supplementary Table S1). Note the increase of CXCR4 transcripts in SW480 cells compared to HCT116, Caco2, and DLD1 cells. (d) Western blot analysis of CXCR4 in 9 CRC cell lines: Caco2, HCA-7, HCT116, HT29, DLD1, Lovo, LS174T, RKO, and SW480 cells (Supplementary Table S2). PCR product treated with restriction enzyme lane = +; PCR product untreated lane = −.
Figure 5
Figure 5
Demethylating agent 5-aza-2’deoxycytidine increases CXCR4 transcript abundance. RT-PCR results display a decreased normalized Ct value (average) and therefore reflects the increased CXCR4 mRNA expression in colorectal cancer cell lines (a) HCT116 and (b) HT29 after treatment with 5 µM concentration of demethylating agent 5-aza-2’-deoxycytidine.
Figure 6
Figure 6
5-hydroxymethylcytosine (5hmC) abundance in CXCR4 gene bodies in colon cancers and matched adjacent mucosa (n = 42 samples, p < 0.01, paired Student’s t-test): (a) Counts per million reads at CXCR4 gene, (b) plus promoter (upstream 3kb region) in matched tumors and adjacent healthy tissue in 30 colorectal cancer patients. The moving averages at 0.01 smoother span are shown. black bars mark exons.
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