Prosaposin activates the androgen receptor and potentiates resistance to endocrine treatment in breast cancer

Abstract

Introduction: HOX genes play vital roles in growth and development, however, atypical redeployment of these genes is often associated with steroidal adaptability in endocrine cancers. We previously identified HOXC11 to be an indicator of poor response to hormonal therapy in breast cancer. In this study we aimed to elucidate genes regulated by HOXC11 in the endocrine resistant setting.

Methods: RNA-sequencing paired with transcription factor motif-mapping was utilised to identify putative HOXC11 target genes in endocrine resistant breast cancer. Validation and functional evaluation of the target gene, prosaposin (PSAP), was performed in a panel of endocrine sensitive and resistant breast cancer cell lines. The clinical significance of this finding was explored in clinical cohorts at both mRNA and protein level.

Results: PSAP was shown to be regulated by HOXC11 in both tamoxifen and aromatase inhibitor (AI) resistant cell lines. Transcript levels of HOXC11 and PSAP correlated strongly in samples of primary breast tumours (r = 0.7692, n = 51). PSAP has previously been reported to activate androgen receptor (AR) in prostate cancer cells. In a panel of breast cancer cell lines it was shown that endocrine resistant cells exhibit innately elevated levels of AR compared to their endocrine sensitive counterparts. Here, we demonstrate that stimulation with PSAP can drive AR recruitment to a hormone response element (HRE) in AI resistant breast cancer cells. Functionally, PSAP promotes cell migration and invasion only in AI resistant cells and not in their endocrine sensitive counterparts. In a cohort of breast cancer patients (n = 34), elevated serum levels of PSAP were found to associate significantly with poor response to endocrine treatment (p = 0.04). Meta-analysis of combined PSAP and AR mRNA are indicative of poor disease-free survival in endocrine treated breast cancer patients (hazard ratio (HR): 2.2, P = 0.0003, n = 661).

Conclusion: The HOXC11 target gene, PSAP, is an AR activator which facilitates adaptation to a more invasive phenotype in vitro. These findings have particular relevance to the development of resistance to AI therapy which is an emerging clinical issue. PSAP is a secreted biomarker which has potential in identifying patients failing to exhibit sustained response to hormonal treatment.

Fig. 1

Prosaposin (PSAP) was identified as a putative HOXC11 target gene from RNA-sequencing (RNA-seq) of endocrine-resistant breast cancer cells in which HOXC11 was knocked down. a (i) RNA-seq was performed on mRNA from endocrine-resistant breast cancer cells (LY2) in which HOXC11 had been silenced (n = 2): 1,919 hits (cutoff >= 0.95), corresponding to 1,243 Entrez gene IDs were identified. TFFind, a motif mapping programme, was used to search for the HOXC11 motif (consensus sequence: GTCGTAAA) in all annotated human genes from UCSC (26,648 sequences). The analysis of these data resulted in the identification of 711 genes putatively regulated by HOXC11. (ii) Breakdown of RNA subtype percentages identified in RNA-seq screening. b De novo motif identification was performed on sequences of HOXC11 targets that harbour hormone response elements using the MEME program resulting in the identification of a novel motif (e-value: 1.3e-1337). The novel motif has significant similarity to androgen receptor (AR) (p value: 2.26e-6) and NR3C1 (glucocorticoid receptor (GR)) (p value: 2.81e-4). c AR motif analysis of total HOXC11 target genes shows that approximately 60 % contain an androgen response element (ARE) in the proximal promoter region. d Merging RNA-sequencing and HOXC11 transcription factor motif-mapping datasets yielded a total of 29 genes, which were then ranked by magnitude of fragments per Kilobase of transcript per million. e Validation of the putative HOXC11 target gene, PSAP, was confirmed by performing chromatin immunoprecipitation to determine recruitment of HOXC11 to the PSAP promoter in LY2 (tamoxifen resistant) cells cultured in the presence of tamoxifen versus vehicle. Results are representative of three separate experiments

Fig. 2

HOXC11 is recruited to the prosaposin (PSAP) promoter when estrogen signalling is disrupted. a HOXC11 is present on the PSAP promoter in letrozole-resistant (LetR) cells cultured in androstenedione but not estrogen. HOXC11 is not present on the PSAP promoter in LY2 (tamoxifen (Tam) resistant) cell cultures in estrogen. Cycle threshold (ct) values were not detected for distal-promoter control reactions for any sample. Error bars are representative of mean ± standard error of the mean from three separate experiments (LetR) and two separate experiments (LY2). b In summary, HOXC11 is recruited to the PSAP promoter in endocrine-resistant breast cancer cells in which estrogen signalling has been disrupted. c (i) Expression of PSAP mRNA is significantly reduced in LY2 cells following silencing of HOXC11 by siRNA. (ii) Expression of PSAP mRNA is significantly reduced in LetR cells following silencing of HOXC11 by siRNA. Results are representative of three separate experimental replicates. d HOXC11 and PSAP mRNA levels were evaluated by qRT-PCR from RNA extracted from primary breast cancer specimens (n = 51). Spearman’s rank test was used to evaluate correlation between PSAP and HOXC11 transcripts (r = 0.7745: p <0.0001). *p <0.05, **p <0.001, ***p <0.0001). Andro androstenedione, HRE hormone response element

Fig. 3

Prosaposin (PSAP), a known androgen receptor (AR) activator, is readily detectable in breast cancer cells that have high endogenous levels of HOXC11 and AR protein. a Secreted PSAP protein levels were evaluated in conditioned media harvested from a range of breast cancer cell lines that were designated endocrine-sensitive (MCF7, Aro) or endocrine-resistant (LY2, letrozole-resistant (LetR) and anastrozole-resistant (AnaR)). PSAP ELISA detected significant levels of protein in the endocrine-resistant cells. b (i) Protein levels of AR were determined by western blot analysis of nuclear protein cell lysate extracted from breast cancer cells (MCF7, LetR, MDA-MB-453, LY2 and SKBR3). (ii) Endocrine-resistant cells LY2 and LetR exhibited >2 fold increase in AR nuclear protein expression compared to their endocrine-sensitive MCF7 counterparts. Image representative of three experimental replicates. Error bars are representative of mean ± standard error of the mean (SEM) of three separate experiments. c (i) Transient overexpression of HOXC11 in MCF7 cells resulted in a significant increase in the level of nuclear AR. Image representative of three experimental replicates. Error bars are representative of mean ± SEM of three separate experiments. c (ii) Representative images of AR in MCF7 cells with transient overexpression of pSPORT empty vector versus pSPORT HOXC11. d Elevated levels of PSAP, HOXC11 and AR in endocrine-resistant cells. ^§McIlroy et al., 2010. *p <0.05 **p <0.001. Tam tamoxifen, AI aromatase inhibitor

Fig. 4

Androgen receptor (AR) protein is upregulated and transcriptionally activated in letrozole-resistant (LetR) cells treated with recombinant prosaposin (PSAP); the effect of PSAP can be diminished by co-treatment with the anti-AR drug enzalutamide (Enza). a (i) Western blot analysis was used to quantify AR protein levels in LetR cells treated with recombinant human PSAP (rhPSAP) (10 ng/ml) versus control (Tris–HCl). (ii) Densitometry values from separate experiments. Error bars are representative of mean ± standard error of the mean (SEM) of three separate experiments. b (i) Modified TransAM assay was used to evaluate AR recruitment to a direct AR binding sequence 5′ – TGTTCT – 3′ when LetR cells are cultured in the presence of rhPSAP. Results are representative of two separate experiments (ii) In LetR cells the AR binds the direct AR consensus sequence in the presence of either R1881 or rhPSAP c. Nuclear translocation assays were used to observe the trafficking of AR in aromatase-inhibitor (AI)-resistant LetR cells, as visualised by the detection of immunofluorescently labelled AR within the nucleus. c (i) rhPSAP treatment significantly increased AR nuclear translocation in LetR cells (p <0.0001). rhPSAP and Enza combination treatment significantly decreased AR nuclear translocation in LetR cells (p <0.05). Error bars are representative of mean ± SEM of three separate experiments. Anti-AR drug treatment (Enza) significantly decreased AR nuclear translocation in LetR cells (p <0.05). (ii) There was no change in AR nuclear translocation in MCF7 cells following treatment with either rhPSAP or Enza individually or with a combination of both rhPSAP and Enza. Error bars are representative of mean ± SEM of three separate experiments. (iii) Representative images of AR nuclear translocation in LetR cells following individual treatments with rhPSAP and Enza and combination treatments: *p <0.05, **p <0.001, ***p <0.0001. HRE hormone response element, Veh vehicle, DAPI 4′, 6-diamidino-2-phenylindole

Fig. 5

Prosaposin (PSAP) increases cell motility and invasiveness of letrozole-resistant (LetR) breast cancer cells with negligible impact on the function of endocrine-sensitive cells. a (i) In vitro scratch assays were used to evaluate the impact of recombinant human PSAP (rhPSAP) on cell migration. Cell migration was significantly enhanced in LetR cells exposed to increasing doses of rhPSAP with maximal impact observed with 10 ng/ml PSAP treatment (p <0.0001) (ii), conversely, there were no changes in cell migration observed in MCF7 cells (not significant (n.s.). Error bars are representative of mean ± standard error of the mean (SEM) of three separate experiments. b (i-ii) Using matrigel invasion chambers it was determined that the invasive potential of LetR cells was greatly amplified when cells were cultured in the presence of rhPSAP (10 ng/ml) (40 hours) compared to their endocrine-sensitive counterparts, MCF7 which were unresponsive to treatment (iii-iv). b (v) Bar-chart representing data from rhPSAP invasion assay in LetR and MCF7. Error bars are representative of mean ± SEM of three separate experiments. c Transfection of LetR cells with siRNA-HOXC11 significantly inhibits cell migration (i-ii), furthermore, treatment of cells with rhPSAP (10 ng/ml) (60 hours) does not stimulate cell invasion when HOXC11 is knocked down (iii-iv). c (v) Data from rhPSAP invasion assay in LetR +/− rhPSAP. Error bars are representative of mean ± SEM from four separate experiments: *p <0.05, **p <0.001, ***p <0.0001. Scram veh scrambled vehicle

Fig. 6

Inhibition of androgen receptor (AR) can diminish letrozole-resistant (LetR) cell proliferation and the pro-migratory impact of recombinant human prosaposin (rhPSAP). a Scratch assays were utilised to investigate the impact of rhPSAP on the migratory capacity of aromatase inhibitor (AI)-resistant LetR cells. Anti-AR, bicalutamide (Bica), treatment did not significantly impact on the migratory capacity of AI-resistant LetR cells over 48 hours (i); rhPSAP treatment (10 ng/ml) significantly increased LetR cell migration over 48 hours (ii). Combination treatment with rhPSAP and bicalutamide significantly inhibited rhPSAP-mediated cell migration in AI-resistant LetR cells over 48 hours (iii). b (i) MTS and (ii) colony forming assays were performed to assess the impact of bicalutamide on AI cell proliferation. Treatment of AI-resistant cells with the anti-AR drug, bicalutamide, reduces cell proliferation in LetR. Error bars are representative of mean ± standard error of the mean from three separate experiments. *p <0.05, **p <0.001. VEH vehicle, COMBO combination

Fig. 7

Pre-operative serum levels of prosaposin (PSAP) are significantly higher in breast cancer patients whose disease subsequently recurred. High levels of both PSAP and androgen receptor (AR) mRNA are very strongly associated with poor disease-free survival (DFS) in endocrine-treated breast cancer. a A commercially available ELISA kit for human PSAP was used to quantify serum levels of the secreted protein in sera from breast cancer patients. PSAP levels were evaluated in a subset of endocrine-resistant patients versus age-matched controls. (i) The non-recurrent control group was used to generate a training set to establish a cutoff value of 0.84 ng/μl (median + 2 SD) to distinguish between patients sensitive and resistant to endocrine therapy (dashed horizontal line). The median follow up of the cohort was 35 months. Preliminary findings indicate approximately 35 % of postmenopausal breast cancer patients who had recurrent disease whilst on endocrine therapy expressed PSAP serum levels elevated above the cutoff (p = 0.04). (ii) Levels of PSAP detected in sera from patients with recurrent breast cancer was significantly higher than those of endocrine-responsive patients (p = 0.03). b (i) Breast cancer patients (The Cancer Genome Atlas dataset (TGCA) with high androgen receptor (AR) expression (upper quartile) were selected and Spearman’s correlation between HOXC11 and PSAP was calculated for each subtype: gray luminal A, blue luminal B, red basal, green HER2 (n = 199). (ii) Tabulated results show Spearman’s correlation between HOXC11 and PSAP mRNA in AR-high breast cancers per subtype. c (i) Meta-analysis of transcript levels in breast cancer patients (n = 661) indicates that PSAP mRNA is a significant predictor of poor response to endocrine treatment in endocrine-treated patients (p = 0.03; hazard ratio (HR) 1.4). (ii) Patients expressing high mRNA levels of both PSAP and AR experience much shorter periods of DFS reflected in an HR of 2.2 (p = 0.0003). BrCa breast cancer, HER2 human epidermal growth factor 2