*2.2. KLF5 is Crucial for Maintaining the Transcriptional Activity of AR in PCa Cells*

To determine whether androgen-upregulated KLF5 has a functional role in androgen/AR signaling, we evaluated whether knockdown of KLF5 affects AR's transcriptional activity in LNCaP cells with KLF5 silencing by siRNA and C4-2B cells with KLF5 silencing by shRNA. Cells were cultured in regular media, which contains hormones as regular FBS was used. Enzalutamide treatment was applied to inhibit AR signaling activity. KLF5 silencing clearly decreased the expression of PSA, a classic transcriptional target of AR [40], at both protein and mRNA levels in both LNCaP and C4-2B cell lines (Figure 2a,b). With enzalutamide treatment, expression of both PSA and AR was dramatically reduced, and the effect of KLF5 knockdown on PSA expression was weakened at the protein and mRNA level (Figure 2a–d). The mRNA expression of two additional AR target genes, *TMPRSS2* and *FKBP5* [41,42], was also decreased by KLF5 silencing, as detected by real-time qPCR (Figure 2c,d). We noticed that KLF5 silencing also decreased AR expression in both cell lines (Figure 2a,b), which is further addressed in Figure 3.

**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.

We also analyzed the activities of two androgen-responsive promoters, *PSA* and *MMTV* [43], in the same cells with the same treatments. The activities of both *PSA* and *MMTV* promoters were significantly decreased by KLF5 knockdown, and enzalutamide treatment eliminated both the promoter activities and the effect of KLF5 silencing on promoter activities (Figure 2e,f).

To test whether KLF5 directly binds to the promoters and enhancer of AR target genes, we performed ChIP assay using both AR and KLF5 antibodies. In AR-precipitated DNA, the *PSA* and *FKBP5* promoters and the *TMPRSS2* enhancer were detected by PCR in C4-2B cells, as expected, and KLF5 silencing reduced the promoter and enhancer DNA (Figure 2g). In KLF5-precipitated DNA, while the promoter DNA of *PSA* was detected, neither the *FKBP5* promoter nor the *TMPRSS2* enhancer was detected (Figure 2g), and the *PSA* promoter was eliminated by the inhibition of AR signaling by enzalutamide (Figure 2h). These results suggest that KLF5 is crucial for the transcriptional activity of AR.

**Figure 3.** *Cont.*

**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.

#### *2.3. KLF5 also Promotes Transcription of the AR Gene in PCa Cells*

Analyzing the effect of KLF5 on AR's gene transactivating function, we noticed that knockdown of KLF5 reduced AR expression in both LNCaP and C4-2B cells (Figure 2a,b). We thus tested whether KLF5 modulates the expression of AR. Knockdown of KLF5 by siRNA in LNCaP cells or by shRNA in C4-2B cells clearly reduced AR expression at both protein and mRNA levels (Figure 3a–d).

To further test whether KLF5 directly promotes AR transcription, we analyzed the 2-Kb immediate AR promoter sequence for potential KLF5 binding sites using the JASPAR database, in which the consensus KLF5 binding sequences were defined by ChIP-Seq study [40]. Multiple such sites were predicted, and the 5 with the highest binding scores were all located within the immediate 350-bp AR promoter (Figure 3e).

We then constructed a promoter-luciferase reporter plasmid with the entire 2-Kb immediate AR promoter sequence in the pGL3 vector (pGL3-AR, Figure 3f). Two additional AR promoter luciferase reporter plasmids were also constructed, one with the upper 1.5-Kb and the other with the lower 0.5-Kb of the full 2-kb promoter sequence (pGL3-AR1 and pGL3-AR2, Figure 3f). Both pGL3-AR and pGL3-AR2 also contained 0.2-Kb sequence of exon 1 (Figure 3f). Interestingly, knockdown of KLF5 significantly decreased the activities of pGL3-AR and pGL3-AR2, both of which contained potential KLF5 binding sites, but not that of pGL3-AR1, which did not contain a KLF5 binding site (Figure 3g). Therefore, it is likely that KLF5 directly promotes AR transcription via promoter binding.

To test whether KLF5 directly binds to AR promoter, ChIP was performed with KLF5 antibody in C4-2B cells and PCR performed with primers to amplify the AR promoter in pGL3-AR2 in three fragments, A, B, and C (Figure 3f). Fragment B, which contained three potential KLF5 binding sites (Figure 3f), was detected in the DNA pulled down by KLF5 antibody, but fragments A and C were not (Figure 3h). Further analysis showed that fragment B2, containing the sites from −107 to −93, was detected in the DNA pulled down by KLF5 antibody, but fragment B1 was not. Therefore, KLF5 can directly bind the AR promoter via the sites from −107 to −93.
