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, 7 (20), 28836-48

Setdb1, a Novel Interactor of ΔNp63, Is Involved in Breast Tumorigenesis

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Setdb1, a Novel Interactor of ΔNp63, Is Involved in Breast Tumorigenesis

Carla Regina et al. Oncotarget.

Abstract

ΔNp63 has been recently involved in self-renewal potential of breast cancer stem cells. Although the p63 transcriptional profile has been extensively characterized, our knowledge of the p63-binding partners potentially involved in the regulation of breast tumour progression is limited. Here, we performed the yeast two hybrid approach to identify p63α interactors involved in breast tumorigenesis and we found that SETDB1, a histone lysine methyl transferases, interacts with ΔNp63α and that this interaction contributes to p63 protein stability. SETDB1 is often amplified in primary breast tumours, and its depletion confers to breast cancer cells growth disadvantage. We identified a list of thirty genes repressed by ΔNp63 in a SETDB1-dependent manner, whose expression is positively correlated to survival of breast cancer patients. These results suggest that p63 and SETDB1 expression, together with the repressed genes, may have diagnostic and prognostic potential.

Keywords: SETDB1; breast cancer; histone methyl transferase; p63; proliferation.

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. ΔNp63α binds SETDB1
(A) Semi-endogenous immunoprecipitation of p63α isoforms and SETDB1. Flag-TAp63α and Flag-ΔNp63α expression vectors were transiently transfected in H1299 cells. Cell extracts were immunoprecipitated with anti-Flag antibody and subjected to western blot analysis (lanes 4 to 6) with anti-SETDB1 antibody (upper panel) and anti-Flag antibody (lower panel). Aliquots of total cell extracts from unprocessed cells were also loaded on the gel (lanes from 1 to 3). EV, empty vector. (B) Immunoprecipitation of endogenous p63 with endogenous SETDB1. MCF7 cells extracts were immunoprecipitated with anti-p63 antibody and subjected to western blot analysis (lane 3) with anti-SETDB1 antibody (lower panel) and anti-p63 antibody (upper panel). The aliquot of total cell extract from unprocessed cells (lane 1) and IgG, used as negative control, (lane 2) were also loaded on the gel. Quantification of SETDB1 IP/SETDB1 IgG = 1,8 fold. (C) It is shown wt ΔNp63α containing all domains of the protein: Transactivation domain of ΔN isoforms (TA1, not shown), DNA-binding domain (DBD), oligomerization domain (OD), transactivation domain 2 (TA2, not shown), sterile alpha motif (SAM) and transactivation inhibitory domain (TID); the first mutant contains only the C-terminus including OD, TA2 (not shown), SAM, TID (CT); the second one contains all domains apart from TID (ΔTID); the third one contains all domains apart from SAM and TID (ΔSAM-TID); the fourth one contains all domains apart from OD, TA2, SAM, TID (NT). (D) Coimmunoprecipitation of p63 deletion mutants and SETDB1. HA-ΔNp63α, HA-CT, HA-ΔTID, HA- ΔSAM-TID, HA-NT expression vectors were transiently transfected in H1299 cells together with full lenght Flag-SETDB1. Cell extracts were immunoprecipitated with anti-SETDB1 antibody and subjected to western blot analysis (lanes 7 to 12) with anti-SETDB1 antibody (upper panel) and anti-HA antibody (lower panel). Aliquots of total cell extracts from unprocessed cells were also loaded on the gel (lanes 1 to 6). EV, empty vector; NT, no transfection. (E) It is shown wt SETDB1 containing all domains of the protein: Tudor domains (TUD), methyl-CpG-binding domain (MBD), PRE-SET domain and the bifurcated SET domain (S-ET). Post-SET domain is not shown; the first mutant (1-256 aa) contains only the N-terminus, lacking all functional domains; the second mutant (1- 615 aa) contains the N-terminus and both Tudor domains; the third mutant contains MBD (methyl-CpG-binding domain), PRE-SET and the bifurcated SET domain. (F) Coimmunoprecipitation of SETDB1 deletion mutants and ΔNp63α. Full lenght Flag-SETDB1, Flag-1-256aa, Flag-1-615aa and Flag-528-1307aa expression vectors were transiently transfected in H1299 cells together with full lenght ΔNp63α. Cell extracts were immunoprecipitated with anti-Flag antibody and subjected to western blot analysis (lanes 6 to 10) with anti-Flag antibody (upper panel) and anti-HA antibody (lower panel). Aliquots of total cell extracts from unprocessed cells were also loaded on the gel (lanes 1 to 5). EV, empty vector. Uncropped images of gels are shown in Supplementary Figure S5.
Figure 2
Figure 2. SETDB1 affects p63 protein stability
(A) Western blot showing p63 and SETDB1 protein expression after transient silencing of p63 or SETDB1 in MCF7 cell line. Cells were collected 48 hours, 72 hours, 96 hours after the silencing. β-Actin is shown as loading control. NT, no transfection. One representative experiment of three is shown. (B) Relative quantification of p63 mRNA after SETDB1 transient silencing (48-72-96 hours). One representative experiments of three is shown. (C) Relative quantification of SETDB1 mRNA after p63 transient silencing (48-72-96 hours). One representative experiments of three is shown. Uncropped images of gels are shown in Supplementary Figure S6. (D) Western blot showing p63 and SETDB1 protein expression after transient silencing of p63 or SETDB1 in MCF7 cell line. Cells were collected 72 hours after the silencing and treatment with MG132. β-Actin is shown as loading control. One representative experiment of three is shown. Uncropped images of gels are shown in Supplementary Figure S7.
Figure 3
Figure 3. Expression analysis of SETDB1 in breast cancer
(A) Semi-quantitative reverse transcriptase analysis of SETDB1 mRNA levels in different breast cancer cell lines (basal type: BT-549, MDM-MB231, MDM-MB468; luminal type: MCF-7, MDM-MB453). Human Mammary Epithelial Cells (HMEC) and MCF-10A have been used as normal primary and immortalized breast epithelial cells). β-Actin is shown as loading control. One representative experiments of three is shown. (B) Western blot analysis of SETDB1 in the breast cell lines stated above. β-Actin is shown as loading control. One representative experiments of three is shown. Uncropped images of gels are shown in Supplementary Figure S8. (C) High level of amplification of the histone methyl transferase, SETDB1, in breast cancer datasets obtained from The Cancer Genome Atlas, via cBioPortal.
Figure 4
Figure 4. SETDB1 growth-promoting effects in breast cancer cell lines
(A) MCF-7 colony formation assay (by counting 100 cells plated) comparing si-SETDB1 cells with si-scramble cells. One representative experiment of three is shown. Graphs represent quantification. (B) HCC-1954 colony formation assay (by counting 400 cells plated) comparing si-SETDB1 cells with si-scramble cells. One representative experiment of three is shown. Graphs represent quantification.
Figure 5
Figure 5. Genes repressed by both p63 and SETDB1 in HCC1954
(A) Venn diagram indicating that of the 90 selected genes repressed by ΔNp63 in skin e lung SCCs, 35 genes are not repressed in HCC1954, 25 are repressed in a SETDB1-independent fashion and 30 are repressed in SETDB1-dependent fashion. (B) List of 30 genes repressed both by p63 and SETDB1. The data are shown are fold over control.
Figure 6
Figure 6. Survival analysis of selected genes repressed by p63 and SETDB1
Effect on survival outcome of selected genes (ANXA2 CRIP2 SCNN1A ADCY9) repressed both by p63 and SETDB1. Clinical follow up data of different breast cancer datasets were censored for survival. Kaplan–Meier analysis showed a significant positive correlation with survival in two datasets. 1, RED: high expression; 2, GREEN, low expression. The following datasets were used: (A) ID:GSE25065 (left), ID:GSE25055 (right); (B) GSE25065 (left), GSE25055 (right); (C) GSE30682 (letf), GSE25055 (right); (D) GSE25065 (left), GSE25055 (right) [21, 22].

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