Zbtb7a suppresses prostate cancer through repression of a Sox9-dependent pathway for cellular senescence bypass and tumor invasion

Abstract

Zbtb7a has previously been described as a powerful proto-oncogene. Here we unexpectedly demonstrate that Zbtb7a has a critical oncosuppressive role in the prostate. Prostate-specific inactivation of Zbtb7a leads to a marked acceleration of Pten loss-driven prostate tumorigenesis through bypass of Pten loss-induced cellular senescence (PICS). We show that ZBTB7A physically interacts with SOX9 and functionally antagonizes its transcriptional activity on key target genes such as MIA, which is involved in tumor cell invasion, and H19, a long noncoding RNA precursor for an RB-targeting microRNA. Inactivation of Zbtb7a in vivo leads to Rb downregulation, PICS bypass and invasive prostate cancer. Notably, we found that ZBTB7A is genetically lost, as well as downregulated at both the mRNA and protein levels, in a subset of human advanced prostate cancers. Thus, we identify ZBTB7A as a context-dependent cancer gene that can act as an oncogene in some contexts but also has oncosuppressive-like activity in PTEN-null tumors.

Figure 1. Conditional deletion of Lrf in mouse prostate dramatically promotes Pten-loss-induced prostate tumorigenesis

(a) H&E, anti-Pan-cytokeratin (Pan-K) and anti-smooth muscle actin (SMA) staining of WT, Lrf^flox/flox;Pb-Cre4, Pten^flox/flox;Pb-Cre4, and Pten^flox/flox;Lrf^flox/flox;Pb-Cre4 prostates. (b) Percentage of invasive prostate carcinoma in Pten^flox/flox;Pb-Cre4, and Pten^flox/flox;Lrf^flox/flox;Pb-Cre4 mice. (c) MRI analysis of Pten^flox/flox;Pb-Cre4 and Pten^flox/flox;Lrf^flox/flox;Pb-Cre4 prostates. (d) Tumor volume quantification. (e) Anterior prostate (AP) tumor weight from 3 month-old Pten^flox/flox;Pb-Cre4, and Pten^flox/flox;Lrf^flox/flox;Pb-Cre4 mice (n=5). (f) Ventral prostate (VP) tumor weight from 6, 7, 9 month-old Pten^flox/flox;Pb-Cre4 (n=5, grey bars), and Pten^flox/flox;Lrf^flox/flox;Pb-Cre4 mice (n=5, black bars). (g) Dorsolateral prostate (DLP) tumor weight from 6, 7, 9 month-old Pten^flox/flox;Pb-Cre4 (n=5, grey bars), and Pten^flox/flox;Lrf^flox/flox;Pb-Cre4 mice (n=5, black bars). Data are presented as mean ± standard deviation. (h) Representative prostates from WT, Lrf^flox/flox;Pb-Cre4, Pten^flox/flox;Pb-Cre4 , and Pten^flox/flox;Lrf^flox/flox;Pb-Cre4 mice 1 year old. (i) Representative H&E staining from Pten^flox/flox;Pb-Cre4 , and Pten^flox/flox;Lrf^flox/flox;Pb-Cre4 mice 1 year old. (j) Cumulative survival of WT (n=20), Lrf^flox/flox;Pb-Cre4 (n=20), Pten^flox/flox;Pb-Cre4 (n=20), and Pten^flox/flox;Lrf^flox/flox;Pb-Cre4 (n=11) mice.

Figure 2. Loss of Lrf leads to senescence bypass and increased proliferation

(a) Senescence-associated β-galactosidase staining (SA-β-gal) of WT, Lrf^flox/flox;Pb-Cre4, Pten^flox/flox;Pb-Cre4 , and Pten^flox/flox;Lrf^flox/flox;Pb-Cre4 prostates of 12 week-old mice show a significant reduction of senescence in Pten^flox/flox;Lrf^flox/flox;Pb-Cre4 prostates as compared to Pten^flox/flox;Pb-Cre4 prostates. (b) Percentage of SA-b-gal positive cells in the prostate of 12 week-old Pten^flox/flox;Pb-Cre4, and Pten^flox/flox;Lrf^flox/flox;Pb-Cre4 mice (number of mice=3/genotype, number of cells=1000/field, number of fields=10/lobe). (c) anti-p53, anti-p27 and p19Arf staining in 12 week-old WT, Lrf^flox/flox;Pb-Cre4, Pten^flox/flox;Pb-Cre4 , and Pten^flox/flox;Lrf^flox/flox;Pb-Cre4 prostates. (d) Western blot analysis for Pten, pAkt (Serine 473), Lrf, Smad4, pSmad2, AR, p53, p21, p27, and p19Arf. (e) Ki-67 staining of WT, Lrf^flox/flox;Pb-Cre4, Pten^flox/flox;Pb-Cre4 , and Pten^flox/flox;Lrf^flox/flox;Pb-Cre4 prostates suggests that loss of Lrf and Pten leads to increased proliferation. (f) Percentage of Ki67 positive cells in the three lobes of WT (light grey bars), Lrf^flox/flox;Pb-Cre4 (grey bars), Pten^flox/flox;Pb-Cre4 (dark grey bars), and Pten^flox/flox;Lrf^flox/flox;Pb-Cre4 (black bars) 12-week-old mice (number of mice=3/genotype, total cells/lobe=5000). (g) Western blot analysis for cleaved caspase 3, total caspase 3, and Gapdh of Pten^flox/flox;Pb-Cre4 , and Pten^flox/flox;Lrf^flox/flox;Pb-Cre4 12-week-old mice prostates.

Figure 3. Hyper-activation of Sox9 in Pten;Lrf double-null prostate tumors down-regulates Rb expression through H19/miR675

(a) qRT-PCR analysis of Mia1, Dmbt1, and Sox9 expression in WT (black bars), Lrf^flox/flox;Pb-Cre4 (orange bars), Pten^flox/flox;Pb-Cre4 (red bars), and Pten^flox/flox;Lrf^flox/flox;Pb-Cre4 (blue bars) n=3/genotype 12 week-old mice prostates. (b) Anti-Sox9 staining of WT, Lrf^flox/flox;Pb-Cre4, Pten^flox/flox;Pb-Cre4 , and Pten^flox/flox;Lrf^flox/flox;Pb-Cre4 prostates. (c) Western blot analysis of prostate cells lines for LRF expression. (d) Dual-luciferase assay with a Sox9-reporter in PC3 cells. (e) Co-immunoprecipitation of SOX9 and LRF from PC3 cells, RWPE-1 cells, and mouse prostate. (f) shRNA targeting LRF (grey bars) in RWPE-1 cells leads to an increase in MIA1 and DMBT1 mRNAs expression. Scramble shRNA was used as control (pink bars). (g) Chromatin immunoprecipitation (ChIP) of MIA1 and DMBT1 promoters using anti-LRF (yellow bar), anti-SOX9 (green bar) or rabbit IgG (white bars) antibodies. (h) H19 and miR675 expression in WT (black bar), Lrf^flox/flox;Pb-Cre4 (orange bar), Pten^flox/flox;Pb-Cre4 (red bars), Pten^flox/flox;Lrf^flox/flox;Pb-Cre4 (blue bars). N=3 mice/genotype were used. (i) shRNA targeting LRF (grey bar) in RWPE-1 cells leads to an increase in H19 mRNA expression. Scramble shRNA was used as control (pink bar). Data are presented as mean of 3 independent experiments ± standard deviation. (j) Both LRF (yellow bar) and SOX9 (green bar) bind to H19 promoter as shown by ChIP analysis in RWPE-1 cells. Rabbit IgG were used as control (white bar). All data are presented as mean of 3 independent experiments ± standard deviation. (k-l) Rb protein expression in Pten^flox/flox;Lrf^flox/flox;Pb-Cre4 and Pten^flox/flox;Pb-Cre4 prostates as shown by Western blot analysis (k) and immunohistochemical analysis (l).

Figure 4. LRF down-regulation in human prostate cancers correlates with tumor progression and metastasis

(a-d) LRF mRNA is significantly down-regulated in a subset of primary prostate cancers and metastasis (Oncomine). (e) Expression analysis of miR20, miR106b, and miR93 in normal prostate epithelium (grey bars) versus prostate cancer tissue (red bars). (f) IHC analysis of PTEN and LRF protein expression in high Gleason human prostate cancers demonstrating that loss of LRF is strongly associated with loss of PTEN. (g) Distribution of Pten+/LRF+, PTEN+/LRF−, PTEN−/LRF+, and PTEN−/LRF− samples in the cohort of patients analyzed. (h) Model for the role of LRF in prostate cancer progression.