P4-01-06: Global Characterization of the SRC-1 Transcriptome Identifies Disintegrin C as an ER-Independent Mediator of Endocrine Resistant Breast Cancer.
Authors: Bolger JC , McCartan DP , McIlroy M , Byrne C , Fagan A , Xu J , O'Gaora P , Hill ADK , Young LS 
Published: June 2012

Background: The development of breast cancer resistance to endocrine therapy results from an increase in cellular plasticity leading to the development of a steroid independent tumour. The p160 steroid coactivataor protein SRC-1 drives this tumour adaptability. SRC-1 is central to the development of the endocrine resistant phenotype and is an independent predictor of disease free survival in tamoxifen-treated patients. Here, using discovery studies we identify Disintegrin C (DC), a multidomain transmembrane glycoprotein as a direct, ER-independent target of SRC-1.

Methods: SRC-1-ChIP sequencing was performed in endocrine resistant (LY2) cells. Microarray experiments were performed on LY2 cells transfected with scrambled siRNA or SRC-1 siRNA, +/−4-OHT to assess SRC-1-dependent gene expression. Combining the ChIP-seq and expression array datasets, a total of 2,065 genes were significantly downregulated (p&lt;0.05) following SRC-1 knockdown. DC was selected for validation as a potential novel metastatic oncogene. DC transcipt levels were measured following knockdown and over-expression of SRC-1 and ERα. Mouse xenograft and SRC-1 knockout models were used to examine DC expression in vivo. A TMA comprising 560 patients was stained for DC. Functional validation studies were performed using 3D culture and migration assays, with transient knockdown of DC and treatment with JC1, an inhibitory peptide against DC.

Results: Treatment with 4-OHT significantly increased the number of ChIP-enriched intervals identified. The majority of peaks in the 4-OHT treated sample were close to the transcriptional start site, in the promoter, first exon or upstream of the promoter. As a defined ER coactivator, an overlap between ER binding sites and those identified for SRC-1 is expected. 43% of high confidence SRC-1 peaks in 4-OHT-treated LY2 cells contained an ERE binding motif. This raises the possibility that, in endocrine resistance, SRC-1 can drive transcription independent of ER. Treatment with 4-OHT resulted in substantial recruitment of SRC-1 to the DC promoter. In primary tumours there was a significant association between transcript levels of SRC-1 and DC. Cells derived from mammary tumours of the SRC-1-/−/PyMT mouse lacked DC, but there was high expression in the wild type PyMT mouse. Knockdown of ERα in LY2 cells did not alter DC expression. Knockdown of DC reduced cell migration and restored cell differentiation in resistant cells. In xenograft studies treatment with 4-OHT increased tumour volume in the resistant tumours and induced DC expression. Treatment with recombinant JC1 reduced cellular migration in endocrine resistant cells. Kaplan Meier estimates of disease free survival show that DC is a strong predictor of disease free survival in breast cancer (p&lt;0.0001). SRC-1 and DC are significant independent predictors of disease recurrence (odds ratios 2.18 and 2.4).

Conclusion: Our discovery studies have uncovered a steroid independent SRC-1 mediated network in endocrine resistant breast cancer. We identified a new SRC-1 target, DC. DC represents a rational new therapeutic target with a robust companion biomarker for treatment of endocrine resistant tumours.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-06.