Lysine-specific histone demethylase 1B (LSD2/KDM1B) represses p53 expression to promote proliferation and inhibit apoptosis in colorectal cancer through LSD2-mediated H3K4me2 demethylation

LSD2 expression levels in human CRC tissues

We first analyzed LSD2 expression levels in CRC using the Oncomine database. As shown in Figure 1A, compared to that in non-cancerous tissues, LSD2 expression was upregulated in CRC tissues. We further verified LSD2 expression in clinical CRC specimens and analyzed its statistical difference. Consistent with the Oncomine results, LSD2 mRNA was also upregulated compared to that in non-cancerous tissues (Figure 1B). We then used western blotting to measure LSD2 protein expression in CRC. We showed that LSD2 protein was preferentially overexpressed in CRC compared to that in normal tissues (Figure 1C, 1D). Next, immunohistochemistry (IHC) was used to detect the expression of LSD2, which was consistent with previous observations that LSD2 is preferentially overexpressed in CRC tissue (Figure 1E, 1F). We compared LSD2 expression levels between colon cancer cell lines (LoVo, HCT-116, COLO-320, SW1116, and Caco2) and colon cell line NCM460 by western blotting. Results showed that LSD2 expression in CRC cell lines was higher than that in normal colon cell lines (Figure 1G). These results suggest the role of LSD2 in CRC progression.

Figure 1. LSD2 expression levels in human colorectal cancer (CRC) tissue and cells. (A) The ONCOMINE database was used to examine mRNA expression of LSD2 in CRC tissue. (B) Real-time Polymerase chain reaction (PCR) to evaluate mRNA expression of LSD2 in clinical CRC specimens relative to normal tissues (n=36). Data were expressed as means ± SEM. (C, D) Western Blot evaluation of LSD2 protein expression in CRC and normal tissue. (E) LSD2 protein expression in normal and CRC tissue was analysed by IHC. (F) Pearson’s χ2 tests to assess the association between colon tissue type and LSD2 expression by IHC(n=36). (G) Western blotting to assess LSD2 expression in CRC cell lines.

Manipulation of LSD2 expression in CRC cells

We constructed pCDF1-LSD2 and pCDF1-vector plasmids and established stable LSD2 cell lines Caco2 and SW1116, which were confirmed by western blot (Figure 2A). We also performed lentivirus-mediated knockdown of LSD2 in LoVo and HCT-116 cells using two independent shRNAs to silence, which was also confirmed by western blotting (Figure 3A). We used these cell lines to explore the function of LSD2 in CRC.

Figure 2. LSD2 over-expression promotes CRC cell proliferation. (A) Western blot detection of LSD2 and GAPDH (loading control) protein expression in Caco2 and SW1116 cells with control or LSD2. (B, C) BrdU/PI assay to detect and quantify DNA synthesis in Caco2 and SW1116 cells with control or LSD2. **p < 0.01. (D) Cell Counting Kit-8 (CCK8) assay to measure the viability of Caco2 and SW1116 cells with control or LSD2. (E, F) Colony formation assays and quantification in approximately 500 Caco2 and SW1116 cells with control or LSD2. Data were expressed as means ± SEM, **p < 0.01.

LSD2 promotes CRC cell proliferation both in vitro and in vivo

Given the abnormal LSD2 expression in CRC, we further evaluated the role of LSD2 in CRC proliferation using Cell Counting Kit-8 (CCK8) assays. The results showed that ectopically upregulated LSD2 promoted Caco2 and SW1116 cell growth (Figure 2D), whereas LSD2 knockdown decreased proliferation of HCT-116 and LoVo cells compared to that in the respective controls (Figure 3D). Additionally, BrdU/PI assays showed that, compared to vector cells, LSD2 upregulation increased DNA synthesis in Caco2 and SW1116 cells (Figure 2B, 2C), whereas LSD2 depletion reduced the number of HCT-116 and LoVo cells that incorporated BrdU (Figure 3B, 3C). These results indicated that LSD2 increased DNA synthesis in CRC cell lines. Soft agar colony formation assays showed that LSD2 upregulation increased the colony size and number of HCT-116 and LoVo cells (Figure 2E, 2F), while LSD2 silencing markedly decreased both in HCT-116 and LoVo cells (Figure 3E, 3F). These results suggest that LSD2 upregulation promotes CRC cell proliferation.

Figure 3. LSD2 down-regulation inhibits CRC cell proliferation. (A) Western blot showing protein expression of LSD2 and GAPDH (loading control) in HCT-116 and LoVo cells with control or sh-LSD2. (B, C) BrdU/PI assay to detect and quantification of DNA synthesis in HCT-116 and LoVo cells with control or sh-LSD2. **p < 0.01. (D) Cell Counting Kit-8 (CCK8) assay to detect the viability of HCT-116 and LoVo cells with control or sh-LSD2. (E, F) Colony formation assays and quantification in approximately 500 HCT-116 and LoVo cells with control or sh-LSD2. Data were expressed as means ± SEM, **p < 0.01.

To further investigate the tumorigenesis function of LSD2 in vivo, a xenograft model of human CRC cells in nude mice was adopted. Sh-LSD2 and vector control LoVo cells were injected subcutaneously into each flank of nude mice. Tumor formation was observed and tumor weight and volume were measured. The sh-LSD2 group exhibited generally smaller tumors (Figure 4A) and displayed less volume (Figure 4B) and weight (Figure 4C) compared to the vector group. This difference was further confirmed following examination of the xenograft by IHC: the tumors developed from sh-LSD2 cells displayed lower Ki-67 staining than the control group (Figure 4D). Taken together, these results indicate that LSD2 plays a vital role in the tumorigenicity and tumor growth of CRC.

Figure 4. LSD2 promoted CRC cells proliferation in vivo. (A) Effects of LSD2 down-expression on tumor growth in a xenograft mouse model. Empty vector or sh-LSD2 was transfected into LoVo cells, which were injected in the nude mice (n = 6), and the tumors were obtained at day 35 and was determined by measuring their tumor volume (B) and tumor weight (C). Data were expressed as means ± SEM. (D) The tumor sections were under IHC staining using antibodies against LSD2, p53, p21,ki-67, *P < 0.05.

LSD2 reduced apoptosis and induced cell cycle progression to promote CRC cell proliferation

Cell cycle dysregulation is one of the most important reasons for cell proliferation in cancer. Flow cytometry was used to explore whether LSD2 promoted CRC proliferation by regulating cell cycle progression. We observed a dramatic increase in G1/S-phase cell cycle arrest in HCT-116 and LoVo cells with sh-LSD2. We also observed a reduced population of S-phase cells (Figure 5A). Annexin V/PI assays to test apoptosis rates showed higher percentages of early and late apoptotic cells in HCT-116 and LoVo cells with sh-LSD2 compared to those in the control cells (Figure 5B, 5C). Moreover, western blot assays showed decreased levels of the apoptosis inhibitor Bcl-2 and significantly increased levels of CL-caspase 3/CL-caspase 9 and the apoptosis sensor BAX in HCT-116 and LoVo cells with sh-LSD2 (Figure 5D). These results indicate that LSD2 regulates the cell cycle in CRC.

Figure 5. LSD2 promotes proliferation by inducing G1-S arrest and reducing apoptosis in colorectal cancer (CRC) cells. (A) HCT-116 and LoVo cells stably expressing vector or sh-LSD2#1 or sh- LSD2#2. The percentage of cells in G0/G1, S. or G2/M phases was tested using a subG1 assay and flow cytometry. (B) Apoptotic rates of HCT-116 and LoVo cells stably expressing vector or sh-LSD2#1 or sh-LSD2#2 measured by flow cytometry in annexin V/PI assays. LR, early apoptotic cells; UR, terminal apoptotic cells. Values represent the means ± SD. (C) Quantitative analysis of DNA apoptosis. Data were expressed as means ± SEM. **p < 0.01. (D) Bcl-2, BAX, CL-caspase 3, and CL-caspase 9 protein expression measured by Western blot with GAPDH protein as the loading control.

LSD2 epigenetically silenced p53 transcription through LSD2-mediated H3K4me2 demethylation

The p53 pathway is an important cell cycle regulator [18]. To explore the effect of LSD2 on p53 expression in CRC cells, real-time PCR was performed to detect changes in p53 mRNA expression in HCT-116 and LoVo cells with sh-LSD2. The results revealed increased p53 expression in sh-LSD2 HCT-116 and LoVo cells compared to that in the control cells (p < 0.01, Figure 6B). Moreover, ectopic expression of LSD2 downregulated p53 expression (Figure 6A). Western blots revealed increased p53 protein levels in HCT-116 and LoVo cells with sh-LSD2 cells (Figure 6D), while LSD2 overexpression decreased p53 protein levels compared to those in control cells (Figure 6C).

Figure 6. LSD2 inhibits p53 expression via demethylation of H3K4me2 through binding to the p53 promoter. (A, B) Real-time PCR assays to detect p53 mRNA expression for vector, LSD2, ShNC, and Sh-LSD2 in HCT-116 and LoVo cells. (C, D) p53 and H3K4me2 protein expression measured by Western blot for vector, LSD2, and ShNC in HCT-116 and LoVo cells. (E, F) P53 promoter activity in vector, LSD2, ShNC and Sh-LSD2 in HCT-116 and LoVo cells measured by luciferase assay. Data were expressed as means ± SEM. (G) ChIP assay in LoVo cells showing the interaction between LSD2 and H3K4me2 at the p53 promoter. **p < 0.01.

LSD2 affects target genes by regulating H3K4me2 methylation levels [7]. To determine how LSD2 regulates p53 expression, we first measured histone 3 protein methylation levels in HCT-116 and LoVo cells. Compared to the control group, LSD2 upregulation inhibited H3K4me2 methylation, while LSD2 knockdown increased H3K4me2 methylation (Figure 6C, 6D).

This evidence suggested that LSD2 could regulate p53 expression by directly binding to p53. To verify this hypothesis, we performed CHIP assays in LoVo cells, finding that anti-LSD2 antibodies directly immunoprecipitated the p53 promoter (Figure 6G). We performed luciferase assays to determine whether LSD2 could activate the p53 promoter. The results showed that p53 promoter activity did not change significantly regardless of LSD2 overexpression (Figure 6E) or in sh-LSD2 CRC cells (Figure 6F).

In summary, our findings indicate that LSD2 represses p53 expression through demethylation of H3K4me2 at the p53 promoter.

LSD2 downregulated p53 expression driving the cell cycle through the p53-p21-Rb pathway

p53 plays a central role in the induction of G1/S phase arrest by inducing p21, an inhibitor of cyclin-dependent kinases (CDKs), leading to dephosphorylation and activation of cell cycle repressor retinoblastoma (Rb) [19]. To confirm whether LSD2 promoted CRC proliferation through the p53-p21-Rb pathway, we overexpressed p53 in HCT-116 cells stably expressing LSD2 (Figure 7A). Western blotting revealed decreased levels of p53 and p21 and increased levels of RB(pSer807/811) in HCT-116 cells with LSD2. The upregulation of p53 can reverse the decrease in p21 and the increase in RB(pSer807/811). We then knocked down both LSD2 and p53 in HCT-116 cells. The expression of p53 and p21 increased, while the expression of RB decreased, which was caused by the knockdown of LSD2, and the effect could be reversed by downregulation of p53 (Figure 7A). BrdU/PI assays revealed that p53 upregulation counterbalanced the promotion of proliferation mediated by LSD2 (p < 0.01) (Figure 7B, 7C).

Figure 7. LSD2 regulates colorectal cancer (CRC) cell proliferation and apoptosis through p53. ShRNA mediates p53 knockdown in HCT-116 LSD2-downregulated cell lines, while p53 over-expression in HCT-116 LSD2-upregulated cell lines was observed. (A) Protein expression of p53, p21, pRb and LSD2 measured by Western blot among the CRC cell lines mentioned above. (B) BrdU/PI assays were performed to measure DNA synthesis in the indicated CRC cell lines. (C) Quantitative analysis of DNA synthesis. (D) Western blot showing p53 and LSD2 protein expression in the p53 LSD2 double-downregulated HCT-116 and LoVo cell lines. (E) Flow cytometry detection of the apoptotic rates of the indicated CRC cell lines. (F) Quantitative analysis of DNA apoptosis. Data were expressed as means ± SEM. **p < 0.01.

We then knocked down p53 in stable HCT-116 and LoVo cells expressing sh-LSD2 (Figure 7D). Annexin V/PI assays showed that knockdown of both LSD2 and p53 partially decreased CRC apoptosis compared to that of LSD2 downregulation alone (Figure 7E, 7F).

We also detected the expression of p53 and p21 by IHC in xenograft animal studies and found that the expression of p53 and p21 elevated in tumors developed from sh-LSD2 cells (Figure 4D), suggesting that LSD2 affects the cell cycle through the p53-p21-Rb pathway in vivo.

These results indicated that LSD2 regulated the CRC cell cycle partly through the p53-p21-Rb pathway.

Cell culture and reagents

LoVo, HCT-116, COLO-320, SW1116, and Caco2 cells were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) or RPMI-1640 (COLO-320) supplemented with 10% fetal bovine serum at 37°C. Normal colonic epithelial cells (NCM460) were cultured in F12 supplemented with 20% fetal bovine.

Antibodies against LSD2 (ab193080 for CHIP and WB, ab234863 for IHC), p53 (ab32389), H3K4me2 (ab7766), p21 (ab227443) and Ki-67(ab15580) were purchased from Abcam (Cambridge, MA, USA), while CL-caspase 9, CL-caspase 3, BAX, Bcl-2, Phospho-Rb (Ser807/811)(9308) and GAPDH antibodies were purchased from Cell Signaling (Danvers, MA, USA).

Stable cell lines

Stable cell lines overexpressing LSD2 were established using the pCDF1 lentivirus system (System Biosciences, SBI) as previously described [32].

Two different LSD2 shRNAs and controls were obtained from SABiosciences (Germantown, MD, USA) and reverse transfected into HCT-116 and LoVo cells. The cells were then sorted by flow cytometry after selection in 800 μg/mL G418 for 4 weeks and confirmed by western blotting.

Clinical samples and analysis of the cDNA microarray database

The expression of LSD2 mRNA in CRC was first examined using the Oncomine database (https://www.oncomine.org/) [33], which compares the expression of genes in different tissues based on previous research results [34].

Human samples were obtained from colorectal carcinoma patients without chemotherapy or radiotherapy after receiving approval from the Ethical Committee of the Medical Faculty of Fujian Medical College (Fuzhou, PR China) [32].

IHC was performed as previously described [35] using antibodies against LSD2.

Western blot analysis

Proteins were extracted from cells and tissues and measured by western blotting using the antibodies mentioned above, as previously described [36].

Cell proliferation assay

BrdU/PI assays were performed as previously described [36]. We also performed cell counting Kit-8 (CCK8) cell proliferation assays. Briefly, 2,000 cells were seeded in 96-well plates for 1 week before 10 μL CCK8 solution was added to each plate and incubated at 37°C for 2 hours. The absorbance at 450 nm of each plate was measured and compared.

Colony formation assay

A total of 500 cells were seeded into 6-well plates containing 1% noble agar. The medium was replaced every 3 days for 2 weeks before colony formation was observed under a microscope.

Cell cycle analysis and annexin V/propidium iodide detection

PI staining and annexin V/PI staining were performed to assess changes in phase distribution based on DNA content as well as apoptosis rates after manipulating LSD2 expression levels by flow cytometry (FACS Vantage; BD Biosciences), as described previously [36].

Real-time polymerase chain reaction (PCR) assays for LSD2 and p53

Total RNA extraction, complementary DNA (cDNA) synthesis, and real-time PCR assays were performed as described previously [19] using the following primer sequences:LSD2-F (5′-AACCGAACCTAGTCCCAAAG-3′) and LSD2-R (5′-GGCTATCTGTGGAGTAAGCT-3), p53-F (5′-ACCTATGGAAACTACTTCCTGAAA-3′) and p53-R (5′-CTGGCATTCTGGGAGCTTCA-3′), GAPDH-F (5′-GGAGCGAGATCCCTCCAAAAT-3′), and GAPDH-R (5′-GGCTGTTGTCATACTTCTCATGG-3′).

Luciferase assay

The transcriptional activity of the p53 promoter was measured using this assay, as previously described [35]. The p53 promoter sequence was cloned into a pGL3-basic luciferase reporter vector and co-transfected with or without LSD2 (sh-LSD2).

Chromatin immunoprecipitation

An EZChIP™ ChIP Kit (Millipore, Bedford, MA, USA) was used to perform this assay. We first sonicated chromatin DNA into fragments ranging in size from 200 to 500 bp, using RNA-polymerase II (binding to GAPDH) as a positive control and normal mouse immunoglobulin G (IgG) as a negative control. The chromatin was then immunoprecipitated with anti-LSD2 (1:1000). The LSD2 antibodies for the ChIP assays were purchased from Cell Signaling Technology. The GAPDH primers were provided in the kit, and the p53 primers used were p53-CF (5′-ATGTTAGTATCTACGGCACCAG-3′) and p53-CR (5′-CAGCCCGAACGCAAAGTG-3′)./

Animal studies

Female BALB/c NOD mice were bred to 4–5 weeks of age. CRC cells (5×10⁶) in 50 μL DMEM were mixed with 50 μl ice-cold Matrigel and then subcutaneously injected into the nude mice. We measured tumor volumes and dimensions every 7 days until day 35, when the animals were sacrificed. The tumor xenografts were then weighed and bisected, analyzed by IHC for LSD2, p53, p21, and Ki-67. Our study was approved by the Animal Experimentation Committee of the Central Institute for Experimental Animals, Fujian Medical University.

Statistical analysis

Data are expressed as mean ± standard error of the mean (SEM). Analysis of variance (ANOVA) and Student’s t-tests were used for comparisons between groups. SPSS version 13.0 was used for statistical analysis. p < 0.05 was considered statistically significant.