Lysine-Specific Demethylase 1 (LSD1/KDM1A) Contributes to Colorectal Tumorigenesis via Activation of the Wnt/Β-Catenin Pathway by Down-Regulating Dickkopf-1 (DKK1)

<div><p>We collected paired samples of tumor and adjacent normal colorectal tissues from 22 patients with colorectal carcinoma to compare the differences in the expression of lysine specific demethylase 1 (LSD1) in these two tissues. The results showed that in 19 paired samples (86.4%), LSD1 is more highly expressed in tumor tissue than in normal tissue. To explore the role of LSD1 in colorectal tumorigenesis, we used somatic cell gene targeting to generate an LSD1 knockout (KO) HCT 116 human colorectal cancer cell line as a research model. The analysis of phenotypic changes showed that LSD1 KO colorectal cancer cells are less tumorigenic, both in vivo and in vitro. The differential expression analysis of the cells by mRNA sequencing (RNA-Seq) yielded 2,663 differentially expressed genes, and 28 of these genes had highly significant differences (Q <0.01). We then selected the 4 colorectal cancer-related genes ADM, DKK1, HAS3 and SMURF2 for quantitative real-time PCR verification. The results showed that the differences in the expression of ADM, DKK1 and HAS3 were consistent with those measured using the RNA-Seq data. As DKK1 was the gene with the most significant differential expression, we analyzed the key proteins of the DKK1-related Wnt/β-catenin signaling pathway and found that, after knocking out LSD1, the amount of free β-catenin translocated to the nucleus was significantly reduced and that the transcription of the signaling pathway target gene c-Myc was down-regulated. Our studies show that LSD1 activates the Wnt/β-catenin signaling pathway by down-regulating the pathway antagonist DKK1, which may be one of the mechanisms leading to colorectal tumorigenesis.</p></div

Mechanisms of LSD1-induced colorectal tumorigenesis mediated by down-regulating DKK1.

<p>(A) Western blot analysis of key proteins of the Wnt/β-catenin pathway in WT and KO cell lines. GAPDH was used as an internal control. (B) ELISA analysis of DKK1 in culture media of WT and KO cell lines. (C) Western blot analysis of β-catenin in the cytoplasm and the nucleus of WT and KO cell lines. GAPDH was used as an internal control for the cytosolic proteins, and CREB was used as an internal control for the nuclear proteins. The intensity of each band was quantified using AlphaEase®FC software after densitometric scanning of the films.</p

LSD1 KO colorectal cancer cells are less tumorigenic both in vivo and in vitro.

<p>(A) Proliferation assays. Approximately 1,000 cells per well of the indicated cell lines were seeded in 96-well plates. The proliferation of each clone was measured daily for 6 consecutive days by the CCK-8 method (measuring the absorbance at 450 nm) and plotted. (B) Plate colony formation. Approximately 200 cells per well of the indicated cell lines were seeded in 6-well plates. After 10 days in culture, the cells were stained with crystal violet, the colonies were counted and the number of colonies was plotted for each of the clones. The experiments were performed in triplicate; representative wells are shown. (C) Soft agar colony formation. Approximately 1.0×10⁴ cells per well of the indicated cell lines were suspended in soft agar medium in 6-well plates. After 10 days in culture, the colonies were then counted, and the number of colonies was plotted for each of the clones. The experiments were performed in triplicate; representative wells are shown. (D) Xenograft experiments. LSD1 KO colorectal cancer cells are less tumorigenic in vivo. Athymic nude mice were injected subcutaneously with cells from the indicated clones. Tumor sizes were measured every 3 days for approximately 3 weeks and plotted. The mice were then sacrificed, and the tumors were harvested and weighed. Each mouse grew two tumors. #1∼#6, mouse 1∼6 in each group.</p

Generation of the LSD1 KO colorectal cancer cell line.

<p>(A) Selection of colorectal cancer cell lines. (B) Diagram of the targeting strategy. The endogenous LSD1 locus is shown, along with the AAV targeting vector and the targeted allele before and after Cre-mediated recombination. PCR primers P1∼P10 were described in Materials and Methods. Three STOP codons TGA were added at the end of the left HA to ensure premature termination of the transcript. L-ITR and R-ITR, left and right inverted terminal repeats, respectively; HA, homology arm; TK, TK promoter; Neo, neomycin resistance gene; polyA, polyadenylation signal. (C) Identification of LSD1 null cell lines by genomic PCR. PCR detected the WT and targeted alleles from the indicated cells using genomic DNA as template. Two independent clones for the KO are shown. The lower row with the band designated “WT” indicates that the lower band was amplified from wild-type allele. The upper row with the band designated “KO” shows that the upper band was amplified from the allele with part of exon 7 of the LSD1 knocked out and LoxP inserted. (D) Confirmation of LSD1 null cell lines by Western blot analysis. β-actin was used as an internal control.</p

Expression differences of LSD1 between tumor and adjacent normal colorectal tissues.

<p>GAPDH was used as an internal control. #1∼#22, paired tissue samples 1∼22.</p

A diagram of the Wnt/β-catenin signaling pathway.

<p>We propose that LSD1 down-regulates the Wnt/β-catenin signaling pathway antagonist, DKK1, and that this down-regulation causes free β-catenin to avoid degradation and accumulate in the cytoplasm. The free β-catenin then translocated to the nucleus, activates the transcription of the signaling pathway target gene c-Myc, and finally leads to the aberrant proliferation of cells and tumorigenesis.</p

Re-expression of LSD1 in the KO cell restored the phenotype.

<p>(A) Proliferation assays. (B) Plate colony formation. (C) Soft agar colony formation.</p

RNA-Seq differential gene expression analysis of the cells before and after knocking out LSD1. Q values <0.01.

<p>RNA-Seq differential gene expression analysis of the cells before and after knocking out LSD1. Q values <0.01.</p