KDM5B Is Essential for the Hyperactivation of PI3K/AKT Signaling in Prostate Tumorigenesis

Abstract

KDM5B (lysine[K]-specific demethylase 5B) is frequently upregulated in various human cancers including prostate cancer. KDM5B controls H3K4me3/2 levels and regulates gene transcription and cell differentiation, yet the contributions of KDM5B to prostate cancer tumorigenesis remain unknown. In this study, we investigated the functional role of KDM5B in epigenetic dysregulation and prostate cancer progression in cultured cells and in mouse models of prostate epithelium-specific mutant Pten/Kdm5b. Kdm5b deficiency resulted in a significant delay in the onset of prostate cancer in Pten-null mice, whereas Kdm5b loss alone caused no morphologic abnormalities in mouse prostates. At 6 months of age, the prostate weight of Pten/Kdm5b mice was reduced by up to 70% compared with that of Pten mice. Pathologic analysis revealed Pten/Kdm5b mice displayed mild morphologic changes with hyperplasia in prostates, whereas age-matched Pten littermates developed high-grade prostatic intraepithelial neoplasia and prostate cancer. Mechanistically, KDM5B governed PI3K/AKT signaling in prostate cancer in vitro and in vivo. KDM5B directly bound the PIK3CA promoter, and KDM5B knockout resulted in a significant reduction of P110α and PIP3 levels and subsequent decrease in proliferation of human prostate cancer cells. Conversely, KDM5B overexpression resulted in increased PI3K/AKT signaling. Loss of Kdm5b abrogated the hyperactivation of AKT signaling by decreasing P110α/P85 levels in Pten/Kdm5b mice. Taken together, our findings reveal that KDM5B acts as a key regulator of PI3K/AKT signaling; they also support the concept that targeting KDM5B is a novel and effective therapeutic strategy against prostate cancer. SIGNIFICANCE: This study demonstrates that levels of histone modification enzyme KDM5B determine hyperactivation of PI3K/AKT signaling in prostate cancer and that targeting KDM5B could be a novel strategy against prostate cancer.

Figure 1.. Kdm5b inactivation suppresses prostate tumorigenesis of Pten-null mice in vivo.

(A)Top panel, a schematic showing the strategy of generating Pten/Kdm5b mutant mice. Bottom panel, the actual sizes of representative biopsies of anterior prostates (AP) from Wt, Pten^pc–/–, Kdm5b^pc–/–, and Pten^pc–/–; Kdm5b^pc–/– mice at 6 months of age. (B) Quantification of AP weights from indicated genotypes of mice at 6 months of age. The averages of AP weight and numbers of mice for each cohort are indicated. (C) H&E staining of AP from indicated genotypes of mice at 6 months of age (Magnification: 4X and 20X). (D) Comparison of the onset of PIN in prostate glands between Pten^pc–/– and Pten^pc–/–; Kdm5b^pc–/– mice. Error bars represent means ± SD (3 mice/group).

Figure 2.. Kdm5b deficiency abrogates AKT signaling through reducing PI3K levels in vivo in mouse prostates.

(A) Immunohistochemical (IHC) staining for pAKT(S473), Ki67 and TUNEL in prostate tissues from indicated genotypes of mice at 6 months of age (Magnification: 20X). (B) Quantification of the prostate cells positive for pAKT(S473), Ki67 and TUNEL in (A). Error bars represent means ± SD from 3 mice for each group. (C) Western blotting analysis of protein levels of Pten, Kdm5b, pAKT, P110α, and P85 in prostate tissues of mice with indicated genotypes. Two mouse prostate samples for each genotype. (D) Quantification of protein levels for pAKT, P110α, and P85 between Pten/Kdm5b double null and Pten null mice. Error bars represent means ± SD of triplicates.

Figure 3.. Kdm5b is essential for the hyperactivation of the PI3K/AKT signaling pathway in MEFs.

(A) Effects of Kdm5b inactivation on the cell proliferation of MEFs. Error bars represent means ± SD of triplicates. (B) Western blotting analysis of protein levels of Pten, Kdm5b, pAKT, P110α, P85, and β-galactosidase in MEFs with indicated genotypes. (C) Quantification of protein levels for pAKT, P110α, P85, and β-galactosidase between Pten/Kdm5b double null and Pten null MEFs. Error bars represent means ±SD of triplicates. (D) Immunofluorescence (IF) images showing the levels and membrane localization of PIP3 in MEFs with indicated genotypes. (E) Quantification of fluorescence intensities for PIP3 levels in MEFs. Error bars represent means ± SD (20 cells/ group). (F) Images showing the β-galactosidase staining in senescent MEFs. (G) Quantification of the MEFs positive for β-galactosidase. Error bars represent means ± SD (30 cells/ group).

Figure 4.. KDM5B knockout abrogates PI3K/AKT signaling in human prostate cancer cells.

(A) Left panel, western blotting analysis showing the protein levels of KDM5B, pAKT, P110α, and P85 in LNCaP KDM5B-KO and the parental control human PCa cells. Right panel, quantification analysis of pAKT, P110α, and P85 in LNCaP KDM5B-KO and the control cells. (B) A comparison of the cell proliferation between LNCaP KDM5B-KO and the control cells. (C) Immunofluorescence (IF) images and analysis of PIP3 in LNCaP KDM5B-KO cells. Left panel, IF images showing the levels and membrane localization of PIP3 in LNCaP KDM5B-KO cells and the control cells. Right panel, quantification of the fluorescence intensities of PIP3 in LNCaP KDM5B-KO and the control cells. Error bars represent means ± SD (20 cells/group). (D) RNA-Seq peaks showing the changes of KDM5B, IRS1, PIK3CA, and PIK3R1 expression between LNCaP KDM5B-KO and the control cells. (E) Quantitative RT-PCR analysis to show the relative mRNA levels of IRS1, PIK3CA, and PIK3R1 in LNCaP KDM5B-KO cells. (F) Top panel, western blotting analysis showing the protein levels of KDM5B, P110α and P85 in LNCaP KDM5B-KO and the control cells at indicated time points in CHX chase experiments. Bottom panel, quantification of protein remaining for P110α and P85 at indicated time points in KDM5B-KO and the control cells.

Figure 5.. KDM5B controls the PIK3CA expression in human prostate cancer cells.

(A) A schematic showing the positions of 5 amplicons relative to the PIK3CA transcription start site (TSS). (B) ChIP analysis of KDM5B levels at the indicated regions near PIK3CA TSS in LNCaP KDM5B-KO and the parental control cells. (C) ChIP analysis of H3K4me3 levels at the indicated regions near PIK3CA TSS in LNCaP KDM5B-KO and the control cells. (D) Top panel, a schematic showing that KDM5B regulates the transcription of PIK3CA through affecting promoter activities. Bottom panel, comparisons of luciferase activities of the PIK3CA promoter between LNCaP KDM5B-KO cells and the parental control cells. (E) Luciferase activities showing the effects of KDM5B restoration on the PIK3CA promoter activities in LNCaP KDM5B-KO cells.

Figure 6.. The PI3K/AKT signaling and IGF responses are regulated by KDM5B in human prostate cancer cells.

(A) Western blotting analysis showing the increased levels of P110α and pAKT (Ser473/Thr308) in BHPrE1 cells upon KDM5B overexpression. (B) Quantification of the levels for P110α and pAKT in BHPrE1 cells from (A). (C) Western blotting analysis showing that the effects of KDM5B restoration on the levels of P110α, P85 and pAKT (Ser473/Thr308) in LNCaP KDM5B-KO cells. (D) Quantification of the protein levels for pAKT, P110α and P85 in LNCaP KDM5B-KO cells from (C). (E) Western blotting analysis showing the differential responses between PC3 KDM5B-KO and the parental control cells to IGF-1 stimulation. (F) Quantification of protein levels for pAKT (Ser473/Thr308), P110α, and P85 in (E). Error bars represent means ±SD of triplicates.