Proteomic and Transcriptomic Profiling Identifies Mediators of Anchorage-Independent Growth and Roles of Inhibitor of Differentiation Proteins in Invasive Lobular Breast Cancer

Abstract

BACKGROUNDInvasive lobular carcinoma (ILC) is a histological subtype of breast cancer with distinct molecular and clinical features from the more common subtype invasive lobular carcinoma (IDC). We have previously shown that human ILC cells lines have a remarkably unique ability to grow in ultra-low attachment (ULA) suspension cultures as compared to IDC cells, the mediators of which remain unknown. METHODSUsing flow cytometry and immunoblotting in human ILC and IDC cell lines, we measured levels of apoptosis and cell proliferation in attached (2D) and suspension (ULA) cultures. siRNA-mediated knockdown and pharmacological inhibitors were utilized to assess the effects of known regulators of anchorage-independence. Reverse Phase Protein Arrays and RNA-Sequencing were performed to identify novel proteomic and transcriptomic mediators of ULA growth in ILC cells. RESULTSWe show that human ILC cell lines exhibit enhanced anoikis resistance and cell proliferation in ULA cultures as compared to IDC cells. Transient restoration of E-cadherin did not impact the 2D or ULA growth of human ILC cell lines, while transient E-cadherin knockdown in IDC cells partially rescued their growth defect in ULA culture. Inhibition of the Rho/ROCK, p120-catenin or YAP/Hippo pathways previously implicated in anoikis resistance did not have a major effect on the ULA growth of ILC cells. Proteomic comparison of ILC and IDC cell lines identified unique induction of PI3K/Akt and p90-RSK pathways in ULA culture in ILC cells. Transcriptional profiling uncovered unique upregulation of the Inhibitors of Differentiation family transcription factors ID1 and ID3 in ILC ULA culture, the knockdown of which diminished anchorage-independent growth. We find that ID1 and ID3 expression is higher in human ILC tumors as compared to IDC and correlated with a worse disease-specific survival uniquely in the ILC cohort. CONCLUSIONOur comprehensive study of 2D and ULA growth in human ILC cell lines revealed anoikis resistance, cell proliferation and novel mediators of anchorage-independence and provides possible mechanistic insights and clinical implications for metastatic dissemination of ILC. High expression in human ILC tumors and association with clinical outcome implicate ID1 and ID3 as novel drivers and therapeutic targets for lobular breast cancer.