HOXB13 promotes androgen independent growth of LNCaP prostate cancer cells by the activation of E2F signaling

Abstract

Background: Androgen signaling plays a critical role in the development of prostate cancer and its progression. However, androgen-independent prostate cancer cells emerge after hormone ablation therapy, resulting in significant clinical problems. We have previously demonstrated that the HOXB13 homeodomain protein functions as a prostate cancer cell growth suppressor by inhibiting androgen-mediated signals. However, the role of the HOXB13 in androgen-independent growth of prostate cancer cells remains unexplained.

Results: In this report, we first demonstrated that HOXB13 was highly overexpressed in hormone-refractory tumors compared to tumors without prostate-specific antigen after initial treatment. Functionally, in an androgen-free environment minimal induction of HOXB13 in LNCaP prostate cancer cells, to the level of the normal prostate, markedly promoted cell proliferation while suppression inhibited cell proliferation. The HOXB13-mediated cell growth promotion in the absence of androgen, appears to be mainly accomplished through the activation of RB-E2F signaling by inhibiting the expression of the p21waf tumor suppressor. Indeed, forced expression of HOXB13 dramatically decreased expression of p21waf; this inhibition largely affected HOXB13-mediated promotion of E2F signaling.

Conclusions: Taken together, the results of this study demonstrated the presence of a novel pathway that helps understand androgen-independent survival of prostate cancer cells. These findings suggest that upregulation of HOXB13 is associated with an additive growth advantage of prostate cancer cells in the absence of or low androgen concentrations, by the regulation of p21-mediated E2F signaling.

Figure 1

Validation of HOXB13-specific antibodies in prostate cancers: Expression of HOXB13 in various PCa cells by Western blot analysis (A) and immunocytochemistry (B). While LNCaP and MDA PCa 2b cells were HOXB13-retained cells, PC3 and DU145 cells were HOXB13-deficient cells. HOXB13 was immunostained in prostates with normal or tumor tissue with routine hematoxyline-eosin staining. Arrows indicate HOXB13 localized to the nucleus of luminal epithelial cells or cancer cells.

Figure 2

Expression of HOXB13 in hormone-refractory prostate cancers: A, HOXB13 was immunostained in both androgen-dependent (AD) and androgen-independent (AI) tumors. AI tumors were acquired through transurethral resection of the prostate. High-powered pictures are shown in inlets of some selected figures. B, Dot plot demonstration of HOXB13 scores from each of the AD and AI tumors. Expression of HOXB13 in each tumor was scored (0, absent; 1, weak; 2, intermediate; and 3, strong). C, scores of HOXB13 are shown in the bar graph. The p value was determined by Pearson's chi square statistics with a value of 11.2707. *. p = 0.0008.

Figure 3

Construction of HOXB13-manipulated LNCaP prostate cancer cells: A, HOXB13 was induced by doxycycline in S2 and S4 cells compared to no expression in the Tet-on control cells. B, Recombinant HOXB13 in S4 the LNCaP cells was quantitated by densitometry. The endogenous HOXB13 level was arbitrarily set as one fold. C, Western blot analysis showed clones of HOXB13-suppressed LNCaP cells in addition to scrambled DNA-transfected LNCaP. D-E, By the reporter transcription assay, the effect on androgen-stimulated AR activity was tested in the Tet-on and S4 cells and HOXB13-suppressed clones. Cells were transiently transfected with 100 ng of pGL-ARE4-Luc and 2 ng of ranilla with or without 10 nM R1881. When needed, doxycycline was added with R1881 to induce HOXB13. Luciferase assays were performed 48 h post-transfection. Values indicate fold induction (RLU with androgen/RLU without androgen). Each bar represents the mean ± S.D.

Figure 4

Effect of HOXB13 on androgen-independent prostate cancer cell growth: S4 and Tet-on cells were grown under androgen-rich conditions (A, C) or androgen-deprived conditions (B, D) for up to 6 days in 100 nM doxycycline. HOXB13-suppressed cells (1-3 and 4-1) with scrambled control cells were also grown under similar conditions (E, F). The cells were stained with MTT reagent and measured at an optical density at 570 nm. The * in the diagram, determined by a two-tailed Student's t test, corresponds to the difference between the two groups (p < 0.05). In E and F, repeated-measures ANOVA revealed very significant interaction effect between groups and days (p < 0.0001).

Figure 5

Effect of HOXB13 on the p21-RB-E2F signaling pathway: A, S4 cells were grown under hormone-deprived conditions for 3 days and induced for HOXB13 with 100nM doxycycline. Extracted lysates were evaluated for Western blot analysis. LNCaP cells were grown under hormone-deprived condition for 3 days. B, Cells were transfected with p21-luc and pFLAG-HOXB13, pCDNA-HOXA13, or pCMV-HOXD13. C, Cells were transfected with pE2F1-luc and pFLAG-HOXB13. D, Cells were transfected with pE2F1-luc, pFLAG-HOXB13, and pCDNA-E2F1. Corresponding control vectors were used to match each DNA. After 48 hours, the cells were evaluated by luciferase assays. Each bar represents the mean ± S.D.

Figure 6

Effect of HOXB13 on p21-dependent E2F1-mediated transactivation: A, LNCaP cells were grown under hormone-deprived conditions for 3 days and transfected with pE2F1(3X)-luc, pFLAG-HOXB13, and pCDNA-E2F1. B, Both HCT116 (p21+/+) and HCT116 (p21-/-) cells were transfected with pE2F1-luc and an increased amount of pFLAG-HOXB13. C, HCT116 (p21+/+) cells were transfected with pE2F1-luc, pFLAG-HOXB13, and an increased amount of pFLAG-p21. Corresponding control vectors were used to match each DNA. After 48 hours, the cells were evaluated for luciferase assays. Each bar represents the mean ± S.D. D, S4 cells were treated with Dox to induce HOXB13 followed by Western blot and RT-PCR analyses.

Figure 7

Model of HOXB13-mediated regulation of E2F signaling: Overexpression of HOXB13 in androgen-refractory prostate tumors inhibits the expression of the p21 ^waf tumor suppressor and subsequently activates cyclin dependent kinase 2 (CDK2) activities. Hyperphosphorylated RB releases E2F transcription factor, which drives the genes involved in cell proliferation, and results in increased cell survivability in the absence of hormone.