Breast cancer, cancer cell, cancer hormone therapy, cancer incidence, cancer patient, cell invasion, cell migration, cohort analysis, disease free survival, estrogen responsive element, gene, gene targeting, hormone responsive element, HOXC11 gene, human, in vitro study, phenotype, protein motif, RNA sequence, treatment response
Breast Cancer Campaign (UK) Health Research Board (Ire) Breast Cancer Ireland Scholarship.
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.
Medicine and Health Sciences | Surgery
Ali A, Creevey L, Hao Y, McCartan D, O'Gaora P, Hill A, Young L, McIlroy M. Prosaposin activates the androgen receptor and potentiates resistance to endocrine treatment in breast cancer. Breast Cancer Research. 2015;17(1):123.
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License.
a Modified TransAM assay was used to evaluate androgen receptor (AR) recruitment to a direct AR binding sequence 5′ – TGTTCT – 3′. LNCaP steroid-dependent prostate cancer cells were evaluated as a positive control. Both LNCaP and letrozole-resistant (LetR) cells were cultured in the presence of R1881 (10 nmol/L), recombinant human prosaposin (rhPSAP) (10 ng/ml) and androstenedione (100 nmol/L). b Optimal AR antibody concentration was evaluated using R1881 (10 nmol/L) LNCaP nuclear lysate. An antibody dilution of 1:250 provided optimal signal-noise ratio. HRE human response element.
Additional file 2.pdf (1500 kB)
Table: HOXC11 KD downregulated genes. HOXC11 differentially expressed genes (DEGs) downregulated after HOXC11 knockdown, ranked by p value.
Additional file 3.pdf (259 kB)
Table: HOXC11 KD upregulated genes. HOXC11 differentially expressed genes (DEGs) upregulated after HOXC11 knockdown, ranked by p value.
Additional file 4. Figure S2.pdf (209 kB)
a(i) Validation of HOXC11 target gene, GREB1, in LY2 cells in which HOXC11 was knocked down by siRNA. (ii) Levels of concomitant GREB1 decrease when HOXC11 is silenced is comparable to alterations observed in HOXC11 RNA-seq data. b The top novel motif returned from MEME was then compared to all known motifs annotated in the JARSPAR database (JARSPAR CORE 2014) using the TOMTOM program. The top matched motif is shown to be androgen receptor (AR) (p value: 2.26e-6) and the second most similar motif is NR3C1 glucocorticoid receptor (GR) (p value: 2.81e-4).
Additional file 5.pdf (804 kB)
Table: Androgen receptor (AR) motif results p <0.001. A 400-bp-sized window surrounding starting sites of HOXC11 target genes was selected for AR motif searching. The searching process was performed using the FIMO program available in MEME-suite with the p value significant cutoff set at 0.001.
Additional file 6.pdf (198 kB)
Table: Estrogen receptor (ER) motif results ( p <0.001). A 400-bp-sized window surrounding starting sites of HOXC11 target genes was selected for ER motif searching. The searching process was performed using the FIMO program available in MEME-suite with the p value significant cutoff set at 0.001.
Additional file 7. Figure S3.pdf (114 kB)
Representative images of androgen receptor (AR) nuclear translocation in MCF7 cells following individual treatments with recombinant human prosaposin (rhPSAP) and enzalutamide (Enza) and combination treatments
Additional file 8. Figure S4.pdf (204 kB)
a Hazard ratio (HR) curve was generated for prosaposin (PSAP) using the TCGA dataset. PSAP expressions between 25 and 75 % quantiles have a HR consistently >1. b Androgen receptor (AR) mRNA does not associate with poor disease-free survival (DFS) in endocrine-treated breast cancer. Kaplan-Meier survival curves were generated to assess the impact of high androgen receptor transcript levels on survival of endocrine-treated patients with breast cancer (n = 661).
Additional file 9. Figure S5.pdf (86 kB)
An MTS assay was utilised to assay the impact of increasing doses of recombinant human prosaposin (rhPSAP) on letrozole-resistant (LetR) cell proliferation after 24 hours treatment. No change in LetR cell proliferation was detected following increased doses in rhPSAP (n = 3).
Additional file 10. Figure S6.pdf (138 kB)
a Letrozole-resistant (LetR) cells were treated with increasing doses of recombinant human prosaposin (rhPSAP). p-AKT expression in these cells showed a dose-dependent increase with rhPSAP treatment (n = 3). b In LetR cells, there is increased protein expression of p-AKT when treated with rhPSAP. However, when treated with BEZ235 (PI3K inhibitor), there is marked reduction in p-AKT expression. This reduction is also similarly seen in the combination group of rhPSAP and BEZ235 treatments (n = 3).