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Abstract

Breast cancer is often associated with deregulated activity of two receptor tyrosine kinases (RTKs) from the ERBB family: EGFR and ERBB2 (HER2/Neu). Whereas the EGF receptor is often mutated in breast cancer, ERBB2 is up-regulated in up to 20% of invasive breast tumors and plays a triggering role in cell proliferation, invasion and metastasis. Patients overexpressing ERBB2 are treated with targeted therapies and in particular with the monoclonal antibody trastuzumab, which binds an extracellular domain of the receptor and abrogates receptor-kinase activation. However, ERBB2-positive breast cancer patients overexpress ERBB2 to different extent. The expression range is very wide, raising the question whether the tumors expressing moderate, high and very high ERBB2 levels have different outcome and patients carrying them can be treated as one entity or not. To better understand the impact of different ERBB2 levels I developed stable cell line pools overexpressing ERBB2 at different levels. I observed that in 3D culture as well as in matrigel-based invasion assays, cells expressing very high ERBB2 levels showed more invasive properties than those expressing moderate ERBB2 levels, even in the absence of EGF. This was accompanied by disruption of cell polarity in 3D-grown spheroids and anchorage-independent growth which occured only when ERBB2 expression was very high. These phenotypes could be at least partially explained by ERBB2-dose dependent epithelial-mesenchymal transition (EMT), an increase in HB-EGF transcription and constitutive ERK and AKT signaling pathway activation at the very high ERBB2 levels. As recently several miRNAs have been reported to regulate EMT-related processes and because their expression is frequently deregulated in cancer, I also hypothesized that their expression would change with the ERBB2 expression level in 3D culture. To verify my hypothesis, I performed small RNA sequencing from stable cell line pools grown in matrigel. This identified several miRNAs which are ERBB2-level dependent. I focused then on the functional characterization of miR-301b and miR-130b whose expressions were ERBB2-dependent in 3D, but not in 2D culture. Overexpression of these miRNAs phenocopied ERBB2 effects on cell invasion and proliferation as well as induction of EMT, as did overexpression of the related miR-301a.