Abstract
Triple-negative breast cancer (TNBC) accounts for 10-20% of primary invasive breast cancer diagnoses, globally. With a higher risk of developing visceral and brain metastasis than other breast cancer subtypes, and limited targeted therapy options, TNBC patients have an overall poorer prognosis than other breast cancer patients. The reasons for organ selectivity and rapid metastatic progression are unclear, so in this study, we sought to elucidate molecular mechanisms for preferential organ metastasis in TNBC. As a model for organ selectivity, we leveraged a lung metastatic in vitro TNBC cell line, previously derived by clonal expansion from the heterogenous parental MDA-MB-231 cell line.
We first aimed to compare the in vitro cell phenotypes and biologic behaviour of the lung metastatic MDA-MB-231-LM2 (LM2) cell line and the parental MDA-MB-231 cell line. When cultured in 3D artificial extracellular matrix, we found the LM2 line to form loosely connected invasive cell clusters, while the parental line formed well-differentiated and demarcated spheroids. There was no significant difference in cell motility, measured through a scratch wound assay, however, cell proliferation and viability assays both indicated a significantly greater proliferation rate in the LM2 cell line. Our second aim was to identify differentially expressed genes in the lung metastatic LM2 cell line through RNA-seq analysis. Differential gene expression analysis of ultra-deep RNA-seq datasets using DESeq2 revealed 7,331 differentially expressed (p≤0.05) genes between the LM2 and parental cell line, with 3,470 genes being upregulated in the LM2 cell line – of which, 472 were non-coding; and 3,861 down-regulated genes, with 298 of those being non-coding genes. Of interest, we have identified one highly overexpressed novel long non-coding RNA transcript in the LM2 cell line that warrants future investigation. Subsequent GAGE analysis found the cytokine receptor interaction, neuroactive ligand-receptor interaction, and IL-17 signalling KEGG pathways to have both up- and down-ward perturbations in gene expression.
The differences in 3D spheroid morphology, proliferation rate, and gene expression profile between the LM2 and MB231 cell line collectively suggests a more malignant phenotype in the LM2 cell line. Findings from our interrogation of the transcriptomes of the cell lines corroborate the observed differences in morphological and cell biologic characteristics. We have found unique molecular features to underpin organ selective metastasis in TNBC. These findings support further investigation into identifying and characterising uniquely or differentially expressed genes in the organ selective TNBC cell lines, to find candidate biomarkers and therapeutic targets for improved clinical management of TNBC.