AR Expression in Breast Cancer CTCs Associates with Bone Metastases

Abstract

Molecular drivers underlying bone metastases in human cancer are not well understood, in part due to constraints in bone tissue sampling. Here, RNA sequencing was performed of circulating tumor cells (CTC) isolated from blood samples of women with metastatic estrogen receptor (ER)⁺ breast cancer, comparing cases with progression in bone versus visceral organs. Among the activated cellular pathways in CTCs from bone-predominant breast cancer is androgen receptor (AR) signaling. AR gene expression is evident, as is its constitutively active splice variant AR-v7. AR expression within CTCs is correlated with the duration of treatment with aromatase inhibitors, suggesting that it contributes to acquired resistance to endocrine therapy. In an established breast cancer xenograft model, a bone-tropic derivative displays increased AR expression, whose genetic or pharmacologic suppression reduces metastases to bone but not to lungs. Together, these observations identify AR signaling in CTCs from women with bone-predominant ER⁺ breast cancer, and provide a rationale for testing androgen inhibitors in this subset of patients.Implications: This study highlights a role for the AR in breast cancer bone metastasis, and suggests that therapeutic targeting of the AR may benefit patients with metastatic breast cancer. Mol Cancer Res; 16(4); 720-7. ©2018 AACR.

Fig.1. RNA sequencing of CTCs from breast cancer patients with progressive bone versus visceral metastasis

a ) Schematic representation of sample processing through the CTC-iChip. Metastasis-derived CTCs were identified by staining for EpCAM, HER2 and CDH11 (green), whereas white blood cells were identified by staining for CD45, CD16 and CD14 (red). b) Volcano plot showing the fold change versus false discovery rate (FDR) for each individual gene in CTCs from patients with Bone(+) versus Visceral(+) metastasis. c) Bar graph showing all significantly enriched pathways (FDR<0.25) using the PID tool in CTCs from Bone(+) versus Visceral(+) patients. Pathways are grouped thematically into seven classes.

Fig.2. Androgen Receptor Activation in breast CTCs

a ) Boxplots showing AR transcript expression in CTCs from Bone(+) versus Visceral(+) patients. Reads per million (rpm) are computed per-patient (top). Boxplot shows the levels of the AR signature metagene representative of AR activity (see methods section) in CTCs from patients with “Bone(+)” (red) versus “Visceral(+)” (blue) disease (bottom). b) Heatmap showing the presence or absence of AR and AR-v7 reads, as well as the ratio between AR-v7 and AR reads, for each patient. c) Bar graph shows the mean number of days on aromatase inhibitor treatment for patients with AR negative versus AR positive CTCs, and for patients with AR-v7 negative versus AR-v7 positive CTCs. Error bars represent SEM. *p=0.013.

Fig. 3. Androgen Receptor Expression in metastatic breast cancer

a) Representative images of AR (brown) and hematoxylin (nuclei; blue)-stained human breast cancer bone metastasis (left) and soft-tissue metastasis (right) tissues. The bar graph (bottom) shows the number of AR-positive and AR-negative stains within the bone metastasis versus visceral or soft tissue metastasis. b) Boxplot shows AR expression levels in primary breast tumors (n=192) from patients who later developed metastasis in the bone versus other organs. c) Boxplot shows AR expression levels in metastatic breast cancer biopsies derived from lung, liver or bone (n=29).

Fig. 4. The Androgen Receptor contributes to spontaneous bone metastasis in breast cancer

a) Immunoblot shows AR expression levels in parental, lung variant and bone variant MDA-MB-231 cells. Error bars represent SEM. n=3; *P<0.04. b) Schematic of the experiment to determine the propensity of AR expressing bone variant MDA-MB-231 cells to metastasize to the lung or bone (left). Representative images of GFP-stained primary breast tumor, lung and bone metastases (right) derived from the bone variant MDA-MB-231 cells inoculated into the mouse mammary gland. c) The bar graph shows the number of animals with bone and lung metastases in the groups harboring tumors in which AR expression was suppressed with 2 different shRNAs versus control shRNA. n=8; *P=0.048. d) The bar graph shows the number of animals with bone and lung metastases in the groups treated with vehicle or enzalutamide. n=8; **P<0.009 (bottom).