The effect of epigenetic silencing and TP53 mutation on the expression of DLL4 in human cancer stem disorder

Abstract

The Li-Fraumeni Syndrome (LFS), a genetically rare heterogeneous cancer syndrome, is characterized primarily by a germline p53 (TP53) gene mutation. We recently discovered a balanced reciprocal chromosomal translocation t(11;15)(q23;q15) in the non-cancerous skin fibroblasts of a bilateral breast cancer patient in LFS family. This prompted us to investigate the breakpoint region of the translocation, which uncovered a gene that encodes a Notch ligand, DLL4, (locus at 15q15.1), a key target in tumor vasculature. We analyzed DLL4 gene expression and protein level in LFS non-cancerous skin fibroblast cell lines and non-LFS cancer cell lines. DLL4 is abrogated in all the LFS cells and drastically down-regulated in breast (MCF7) and brain (IMR32) cancer cells and tumor tissue samples. However, DNA methylation studies revealed that DLL4 promoter is silenced only in MCF7 but not in LFS cells. We further investigated the regulation of DLL4 gene expression by ChIP assays, which demonstrated that p53 binds to DLL4 promoter through its association with CTCF, a chromosomal networking protein CCCTC binding factor. This implies a possible karyotype-phenotype correlation with respect to DLL4 in LFS and breast cancer initiation and progression. The drastic reduction or absence in the expression of DLL4 in LFS as well as breast and brain cancer cells is significant and supports the concept that this ligand may also play a role in cancer immune-surveillance; and its resuscitation in the tumor microenvironment may stimulate T-cell immunity and suppress tumor growth. Therefore, DLL4 may provide a strong platform as an immuno-therapeutic target in LFS and cancer patients.

Keywords: DLL4; Notch; angiogenesis; epigenetics; p53.

Figure 1. Analysis of gene expression in the breakpoint region of chromosome 15q15 and determination of the corresponding protein levels in the normal skin fibroblasts of Li-Fraumeni Syndrome (LFS) patients and unrelated cancer cell lines

A. A schematic representation of several genes located in chromosome 15q15. B. mRNA levels of the genes located in chromosome 15q15 in LFS cell lines and breast cancer cell line, MCF7 as well as neuroblastoma cell line IMR32, compared with normal human foreskin fibroblast cell line, HS27. C. DLL4 mRNA and corresponding protein levels in LFS and other cancer cell lines displayed in B. RT-PCR (upper panel) and immunoblot (lower panel) showing decreased expression of DLL4 in LFS cell lines, MCF7 as well as IMR32, in contrast to normal human foreskin fibroblast cell line, HS27. Densitometric analyses and the results shown in panel C reflect a mean ±S.E.M. from three independent experiments, performed in triplicates. Significance: ***P < 0.001 compared with control values, determined by t-test.

Figure 2. Immunohistochemistry analyses of DLL4 expression in normal, benign hyperplasia and tumor tissues

A. Breast, B. Kidney, C. Prostate, D. Lung.

Figure 3. Immunohistochemistry and densitometric analyses of DLL4 expression in normal, cirrhotic and tumor tissues

A. Immunohistochemistry analyses of DLL4 expression in normal, cirrhotic and tumor liver tissues. B. Immunoblot analyses of human normal tissue and tumor tissue samples including colon, stomach and lung. Densitometric analyses and the results shown in lower panel reflect a mean ±S.E.M. from three independent experiments, performed in triplicates. Significance: ***P < 0.001 compared with control values, determined by t-test.

Figure 4. DNA methylation pattern of DLL4 gene promoter in LFS, breast and brain cancer cell lines

A. Schematic representation of DLL4 promoter and CpG islands; B. Methylation status of the DLL4 promoter in LFS cell lines and cancer cell lines as detected by MS-PCR; C. Methylation status of the DLL4 promoter in human normal tissue and tumor tissue samples including colon, stomach and lung. D. Marked Reactivation of DLL4 gene expression in MCF7 but neither in the two DLL4-negative LFS cell lines nor in the normal cell line, HS27 by the DNA methylation inhibitor, 5-aza-dC.

Figure 5. Role of TP53 and CTCF in regulation of DLL4 gene expression

A. Ideogram representing primers used in this ChIP assay for DLL4 proximal promoter region (upper panel) and regulation of DLL4 gene expression in LFS and MCF7 cells by ChIP assay. HS27 cells were used as control (lower panel). B. Ideogram representing primers used in this ChIP assay for DLL4 distal promoter region (upper panel) and regulation of DLL4 gene expression in LFS and MCF7 cells by ChIP assay in DLL4 proximal promoter region. HS27 cells were used as control (lower panel). C. TP53 and CTCF protein levels in LFS, MCF7 and IMR32 cancer cell lines. D. Determining the interaction between TP53 and CTCF by co-immunoprecipitation.

Figure 6. Role of DNA methylation, TP53 and CTCF in regulation of DLL4 gene expression

A. silenced DLL4 by DNA methylation in MDA231 cell line is reactivated by inhibitor of DNA methylation 5′-aza-dC. I) DLL 4 expression level in MDA231 cell line is compared with HS27 and MCF7 by RT-PCR assay. II) Reactivation of silenced DLL4 gene in MDA231 by the DNA methylation inhibitor, 5-aza-dC. III) Methylation status of the DLL4 promoter in MDA231 cell lines treated with 5-aza-dC and detected by MS-PCR. IV) Phase-contrast photomicrographs showing morphologic changes in MDA231 cells treated with 2, 5μM, 10μM and 25μM of 5-aza-dC for 3 days, compared with untreated control cells maintained for 3 days. B. The effect of DNA methylation on interaction between CTCF and DLL4 promoter by ChIP assay C. Reactivation of DLL4 gene expression in LFS cell line, 3335, by CTCF and TP53 siRNA treatment. D. A schematic representation of presumable mechanism of regulation of DLL4 gene expression.