KLF5 Is Crucial for Androgen-AR Signaling to Transactivate Genes and Promote Cell Proliferation in Prostate Cancer Cells

Abstract

Androgen/androgen receptor (AR) signaling drives both the normal prostate development and prostatic carcinogenesis, and patients with advanced prostate cancer often develop resistance to androgen deprivation therapy. The transcription factor Krüppel-like factor 5 (KLF5) also regulates both normal and cancerous development of the prostate. In this study, we tested whether and how KLF5 plays a role in the function of AR signaling in prostate cancer cells. We found that KLF5 is upregulated by androgen depending on AR in LNCaP and C4-2B cells. Silencing KLF5, in turn, reduced AR transcriptional activity and inhibited androgen-induced cell proliferation and tumor growth in vitro and in vivo. Mechanistically, KLF5 occupied the promoter of AR, and silencing KLF5 repressed AR transcription. In addition, KLF5 and AR physically interacted with each other to regulate the expression of multiple genes (e.g., MYC, CCND1 and PSA) to promote cell proliferation. These findings indicate that, while transcriptionally upregulated by AR signaling, KLF5 also regulates the expression and transcriptional activity of AR in androgen-sensitive prostate cancer cells. The KLF5-AR interaction could provide a therapeutic opportunity for the treatment of prostate cancer.

Keywords: KLF5; androgen receptor; cell proliferation; prostate cancer; tumorigenesis.

Figure 1

Androgen-androgen receptor (AR) signaling upregulates the transcription of KLF5 in PCa cells. (a–d) R1881 induced the expression of KLF5 at both protein (a, c) and RNA (b, d) levels in LNCaP (a, b) and C4-2B (c, d) cells. After 24-hour culture in phenol red–free RPMI-1640 medium containing 10% charcoal-stripped (CS) FBS, cells were treated with R1881 for 24 h at the indicated concentrations. Western blotting and real-time qPCR were performed to detect protein and mRNA respectively. (e,f) R1881 induced the expression of KLF5 at both protein (e) and RNA (f) levels at the indicated times in C4-2B cells. Cell culture conditions and the detection of KLF5 protein and mRNA were the same as in panels a-d. After 24-hour culture, cells were treated with R1881 (10 nM) for the indicated times. (g–j) Enzalutamide inhibited the expression of KLF5 at both protein (g, i) and RNA (h, j) levels in LNCaP (g, h) and C4-2B (i, j) cells. Cells were cultured in complete media for 24 h and treated with enzalutamide at the indicated concentrations for 24 h. (k,l) Enzalutamide inhibited the expression of KLF5 at both protein (k) and RNA (l) levels at the indicated times in C4-2B cells. Cell culture conditions and the detection of KLF5 protein and mRNA were the same as in panels g-j. After 24-hour culture, cells were treated with enzalutamide (Enz, 10 µM) for the indicated times. (m,n) RNAi-mediated silencing of AR prevented R1881 from upregulating KLF5 expression at both protein (m) and mRNA (n) levels in C4-2B cells. Cell culture conditions and the detection of KLF5 protein and mRNA were the same as in panels a-d. Transfection of siRNAs was for 6 h before R1881 treatment (10 nM). Enzalutamide (Enz, 10 µM) was used as a control. siCtrl, control siRNA; siAR, AR siRNA. (o) Knockdown of AR also prevented R1881 from inducing transcriptional activity of the KLF5 promoter in C4-2B cells, as detected by the promoter luciferase reporter activity assay. Experimental conditions were the same as in panels i and j except that the reporter plasmid was co-transfected with siRNAs. ns, not significant; *, p < 0.05; **, p < 0.01; ***p < 0.001.

Figure 2

KLF5 is crucial for the transcriptional activity of AR in PCa cells. (a–d) Knockdown of KLF5 reduced the expression of AR transcriptional target genes PSA, TMPRSS2, and FKBP5. Gene expression was detected for protein by western blotting (a, b) and real-time qPCR for mRNA (c, d). LNCaP (a, c) and C4-2B (b, d) cells in full medium were transfected with siRNAs (a, c) or infected with shRNA lentiviruses (b, d) to silence KLF5. One group of cells were treated with enzalutamide (10 µM, 24 h) to inhibit AR function, which served as a control. siCtrl and shCtrl are control siRNA and shRNA respectively. (e,f) Knockdown of KLF5 reduced the activities of two androgen-responsive promoters, PSA and MMTV, in the same cells with the same treatments as in panels a-d, except that the PSA– or MMTV–luciferase reporter plasmid and Renilla-luciferase reporter plasmid were transfected for 24 h before enzalutamide treatment. (g) Binding of AR to the promoters of PSA and FKBP5 and the enhancer of TMPRSS2 was detected after the knockdown of KLF5 in C4-2B cells, as detected by ChIP and regular PCR (left) or real-time qPCR (right). Cells were infected with lentiviruses expressing shRNAs against KLF5 (shKLF5) or control (shCtrl) to knock down KLF5. (h) KLF5 binds to the promoter of PSA but not the promoter of FKBP5 or the enhancer of TMPRSS2 in C4-2B cells in full medium, as detected by ChIP and regular PCR (left) or real-time qPCR (right). Cells were treated with enzalutamide (10 µM, 24 h), with DMSO as a control. ns, not significant; *, p < 0.05; **, p < 0.01; ***p < 0.001.

Figure 3

KLF5 is required for the transcription of AR in PCa cells. (a–d) Knockdown of KLF5 decreased AR expression at both protein (a, c) and RNA (b, d) levels in LNCaP (a-b) and C4-2B cells (c–d). Cells were cultured in complete media for 24 h and transfected with siRNAs for 48 h in LNCaP cells or infected with lentiviruses expressing shRNA targeting KLF5 (shKLF5) in C4-2B cells. Western blotting and real-time qPCR were performed to detect protein and mRNA respectively. (e) The AR promoter contains multiple potential KLF5 binding sites, as predicted by aligning the 2-Kb immediate promoter sequence of AR to the consensus KLF5 binding sequence (top) defined in the JASPAR database. Location of these sequences relative to the transcription initiation site (+1) is shown at left. (f) Schematic of the AR promoter region (−2000 to +200) with the locations of predicted KLF5 binding sites (empty oval) and primers used for PCR amplification of 5 regions (A, B, C, B1, B2) of the AR promoter spanning the potential binding sites. (g) Knockdown of KLF5 decreased AR promoter activity in C4-2B cells, as detected by the luciferase activity assay. The pGL3 vector was used to express full-length AR promoter (g, pGL3-AR, from −2000 to +200) and two shorter AR promoter fragments (pGL3-AR-1, −2000 to −500; pGL3-AR-2, −500 to +200), with their luciferase readings normalized by that of the pGL3-Basic vector control. (h) Detection of KLF5-bound AR promoter DNA in C4-2B cells using ChIP and PCR. ns, not significant; *, p < 0.05; **, p < 0.01; ***p < 0.001.

Figure 4

KLF5 physically associates with AR in epithelial cells. (a,b) HEK293T cells were transiently transfected with expression plasmids of vector control, Flag-tagged (a) or HA-tagged KLF5 (b), and pSG5-AR (a) or FLAG-tagged AR (b), and then subjected to co-IP with FLAG antibody and western blotting with indicated antibodies. (c) HEK293T cells transfected with KLF5 and/or AR as in panel a for 24 h were treated with 10 µM enzalutamide for 24 h, and then subjected to co-IP and western blotting with indicated antibodies. (d) Mapping of interacting KLF5 regions that interact with AR by co-IP with Flag antibody and western blotting with indicated antibodies in HEK293T cells expressing AR and different fragments of KLF5. (e,f) Detection of endogenous KLF5-AR association in C4-2B cells by co-IP with KLF5 (e) or AR antibody (f) and western blotting with indicated antibodies. WCL is whole cell lysates before IP. IgG was used as a negative control for co-IP. (g,h) Detection of the Klf5-Ar association in mouse prostates by co-IP with KLF5 (g) or AR antibody (h) and western blotting with indicated antibodies.

Figure 5

Knockdown of KLF5 attenuates AR-mediated expression of MYC and cyclin D1 in androgen-responsive PCa cells. (a–d) RNAi-mediated KLF5 silencing attenuated R1881-promoted expression of cyclin D1 and MYC in LNCaP (a) and C4-2B (b) cells, as detected by western blotting for protein (a, b) and by real-time qPCR for mRNA (c, d). One group of cells were treated with 10 μM enzalutamide for 72 h. (e) Binding of AR to the promoters of MYC and CCND1 was detected by ChIP and PCR (top) or real-time qPCR (bottom) in C4-2B cells expressing shRNAs against KLF5 (shKLF5) and control (shCtrl). (f) Binding of KLF5 to the promoters of MYC and CCND1 was detected by ChIP and PCR (top) or real-time qPCR (bottom) in C4-2B cells treated with or without enzalutamide (10 µM, 24 h). ns, not significant; *, p < 0.05; **, p < 0.01; ***p < 0.001.

Figure 6

KLF5 is also crucial for androgen/AR signaling to promote cell proliferation and tumor growth in PCa cells. (a–d) Knockdown of KLF5 by siRNA in LNCaP cells or by shRNA in C4-2B cells reduced colony forming efficiency in 2-D culture (a, b) and sphere formation in Matrigel (c, d). Cells with KLF5 knockdown were seeded onto 6-well plates at 2000 cells/well for LNCaP and 1000 cells/well for C4-2B in regular medium for colony formation assay, and at 4000 cells/well for LNCaP and 2000 cells/well for C4-2B cells for sphere formation assay. Regular media were used, and enzalutamide (10 µM) treatment was applied. The culture time was 2 weeks for C4-2B and 3 weeks for LNCaP in both assays. Images of colonies or spheres were taken (left), and their numbers were counted (right). Only spheres with a diameter greater than 80 µm were counted. Scale bars, 200 μm. (e,f) Knockdown of KLF5 attenuated tumor growth of C4-2B cells in nude mice, as indicated by tumor images (e) and tumor weights at excision (f). (g,h) Knockdown of KLF5 reduced AR expression in xenograft tumors of C4-2B cells, as detected by immunohistochemical (IHC) staining with anti-KLF5 (g) and anti-AR (h) antibodies. (i–j) Knockdown of KLF5 reduced cell proliferation, as indicated by the Ki67 index, and the expression of cyclin D1 and MYC in tumor xenografts of C4-2B cells, as detected by IHC staining (i) and quantitation of positive cells (j). Scale bars, 100 μm. ns, not significant; *, p < 0.05; **, p < 0.01; ***p < 0.001.