Independence of HIF1a and androgen signaling pathways in prostate cancer

Abstract

Background: Therapeutic targeting of the androgen signaling pathway is a mainstay treatment for prostate cancer. Although initially effective, resistance to androgen targeted therapies develops followed by disease progression to castrate-resistant prostate cancer (CRPC). Hypoxia and HIF1a have been implicated in the development of resistance to androgen targeted therapies and progression to CRCP. The interplay between the androgen and hypoxia/HIF1a signaling axes was investigated.

Methods: In vitro stable expression of HIF1a was established in the LNCaP cell line by physiological induction or retroviral transduction. Tumor xenografts with stable expression of HIF1a were established in castrated and non-castrated mouse models. Gene expression analysis identified transcriptional changes in response to androgen treatment, hypoxia and HIF1a. The binding sites of the AR and HIF transcription factors were identified using ChIP-seq.

Results: Androgen and HIF1a signaling promoted proliferation in vitro and enhanced tumor growth in vivo. The stable expression of HIF1a in vivo restored tumor growth in the absence of endogenous androgens. Hypoxia reduced AR binding sites whereas HIF binding sites were increased with androgen treatment under hypoxia. Gene expression analysis identified seven genes that were upregulated both by AR and HIF1a, of which six were prognostic.

Conclusions: The oncogenic AR, hypoxia and HIF1a pathways support prostate cancer development through independent signaling pathways and transcriptomic profiles. AR and hypoxia/HIF1a signaling pathways independently promote prostate cancer progression and therapeutic targeting of both pathways simultaneously is warranted.

Keywords: Androgen signaling; HIF1a signaling; Hypoxia; Prostate cancer.

Fig. 1

HIF1a overexpression in the androgen dependent LNCaP cell line increased proliferation and resistance to androgen deprivation therapy. a, stable HIF1a expression increased cell proliferation compared to the LNCaP/Empty control cells when cells were treated with the ethanol vehicle control or synthetic androgen R1881 (two way multiple comparison ANOVA; *p < 0.05, **p < 0.01). b, stable HIF1a expression led to resistance to bicalutamide treatment (two-tail t-test, * p = 0.058). Data points represent the mean of three intra-assay and two biological repeats ± SEM. Differences in growth rates are due to cells grown in media containing charcoal stripped serum (androgen depleted media) for 96 h prior to treatment (a) or in standard RPMI median with FBS that contains androgen and growth stimulants (b)

Fig. 2

HIF1a accelerated tumor growth in non-castrated (full) and castrated mice. A, tumor xenografts derived from the LNCaP/HIF1a clone 1 cell line showed accelerated growth in full and castrated mice however LNCaP/HIF1a tumors grew significantly slower in the castrated mouse compared to the full mouse model. In the castrated mice LNCaP/HIF1a tumors continued to grow whilst the empty control clone regressed (data points represent the mean ± SEM, two-way ANOVA; **p < 0.01)

Fig. 3

Genes upregulated by androgen (R1881), hypoxia and HIF1a in LNCaP cells. a, 47 genes upregulated by androgen (LNCaP vehicle control vs. LNCaP R1881, right circle) were independently upregulated by hypoxia (LNCaP normoxia vs. LNCaP 1% hypoxia, left circle). b, 7 genes upregulated by HIF1a overexpression (LNCaP Empty vs. LNCaP HIF1a, left circle) were also independently upregulated by androgen (LNCaP Empty vehicle control vs. LNCaP Empty R1881, right circle). Three genes were independently upregulated by and androgen, hypoxia and HIF1a (SPRED1, NDRG1 and IGFBP)

Fig. 4

Conservation of AR and HIF binding sites. a, The majority of AR binding sites were conserved following androgen treatment. Of the 35,320 AR called peaks in the normoxic ethanol vehicle control 86% were conserved in the normoxic R1881 treated cells (left). Of the 18,404 AR called peaks in the hypoxic ethanol vehicle control 79% were conserved in the hypoxic androgen treated cells under (right). b, HIF binding sites were not conserved upon hypoxic exposure. Of the 523 HIF called peaks in the normoxic androgen cells 3% were conserved in the hypoxic androgen treated cells under (left). Of the 1181 HIF called peaks in the normoxic ethanol vehicle control 6% were conserved in the hypoxic ethanol vehicle control cells (right)

Fig. 5

The global genomic distribution of the histone markers, H3K4me1 and H3K4me3, in the LNCaP ChIP-seq analysis. The distribution of the H3K4me1 marker did not change with androgen treatment or hypoxia (a-d). Hypoxia decreased H3K4me3 markers within promoter regions (e vs g). Synthetic androgen R1881 increased the location of H3K4me3 markers within promoter regions under normoxia (e vs f) and hypoxia (g vs h)