*2.6. KLF5 Is Crucial for Androgen*/*AR to Promote Cell Proliferation and Tumor Growth in PCa Cells*

Based on the necessity of KLF5 for AR to regulate genes including *MYC* and *CCND1*, we tested whether KLF5 is indeed involved in the pro-proliferative function of AR in PCa cells. In LNCaP or C4-2B cells, colony and sphere formation assays demonstrated that silencing KLF5 by RNAi in normal medium significantly reduced colony-forming efficiency in 2-D culture (Figure 6a,b) and sphere formation in Matrigel (Figure 6c,d). Inhibition of AR signaling by enzalutamide treatment strongly suppressed both colony and sphere formation (Figure 6a–d). Further, under the condition of AR inhibition, KLF5 silencing had a weaker yet detectable effect (Figure 6a–d).

We also tested whether KLF5 is necessary for AR to promote xenograft tumorigenesis. C4-2B cells with stable knockdown of KLF5 were inoculated into immunosuppressed female BABL/c nude mice, with or without enzalutamide treatment (10 mg/kg, administered via oral gavage once a day for up to 21 days, for the tumorigenesis assays). Consistent with colony and sphere formation results, KLF5 silencing reduced tumor growth, as indicated by tumor images and tumor weights at excision (Figure 6e,f). Enzalutamide treatment also reduced tumor growth, and KLF5 silencing and enzalutamide had an additive effect on tumor growth (Figure 6e,f).

In the tumor xenografts, IHC staining demonstrated enzalutamide treatment reduced the expression of both KLF5 and AR, which is consistent with in vitro findings (Figure 1g–l), and KLF5 silencing also downregulated AR expression (Figure 6g,h). Similarly, IHC staining demonstrated that both KLF5 silencing and enzalutamide treatment reduced the expression of Ki67, a cell proliferation marker; the number of Ki67-positive cells; and the expression of cyclin D1 and MYC (Figure 6i,j). These results indicate that KLF5 is crucial for AR to function in PCa cells.

**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.
