Epithelial-mesenchymal plasticity in breast cancer model systems: mplications for metastatic progression of clinical breast cancer

Epithelial-mesenchymal plasticity (EMP) can provide carcinoma cells with the invasive ability to leave the primary tumour, enter into the circulation as circulating tumour cells (CTCs), arrive at a distant organ and ultimately form a metastatic deposit. Basal A, triple-negative, MDA-MB-468 human breast cancer cells exhibit mesenchymal change in response to EGF or hypoxia in vitro. In vivo, they exhibit spontaneous epithelial mesenchymal transition (EMT) at the stromal periphery and in the hypoxic region at the necrotic interface. Vascular inclusions at the primary site or in lymph node and lung metastases were predominantly epithelial, however CTCs exhibited a mixed phenotype with both epithelial and mesenchymal gene expression, a finding confirmed in the ED-03 BC PDX system. Mesenchymal enforcement through shRNA suppression of E-cadherin or miR-200c knockdown dramatically reduced growth rate in vitro and in vivo, promoted constitutive mesenchymal change, and stimulated lymph node colonisation.

We have also bioinformatically selected an EMP-sensitive gene set predicted to have low/no expression in blood cells and reasonably high levels of expression in breast cancer (BC) cells, for tandem-nested RT-qPCR assay of clinical samples. EMP sensitivity of this gene set was validated in public BC cell line data. We have assessed publically-available BC and BC-CTC gene expression data, as well as cohorts of early stage and metastatic breast cancers in which CTCs have been enriched using a combination EpCAM/EGF receptor-directed immunobead approach or microfluidics using the Clearbridge Fx spiral system. Strongly positive cases are rare, but exhibit a predominantly epithelial signature.

Molecular characterisation of BC CTCs may provide avenues for novel therapeutic approaches that can be developed and tested in cell line and PDX xenograft models. This work was supported by the NHMRC #1027527 and the National Breast Cancer Foundation (NBCF#CG-10-04 EMPathy Breast Cancer Network; NBCF#PF-15-053; NBCF/Cancer Australia #628720).