Abstract A046: MiR-221 and -222 expression elevates cell invasion and allows distinction between different prognostic groups in advanced breast cancers
Authors: Falkenberg Natalie , Anastasov Natasa , Braselmann Herbert , Auer Gert , Huber Michaela , Schmitt Manfred , Höfler Heinz , Aubele Michaela 
Published: May 2015

Abstract
Introduction: The expression of miR-221 and -222 is frequently altered in invasive breast cancer and in breast cancer cell lines. We aimed to determine the prognostic and/or therapeutic impact of miR-221/222 expression and its association with tumor progression and metastasizing processes.
Experimental procedures: We investigated the expression of miR-221 and -222 in 86 formalin-fixed, paraffin-embedded (FFPE) tissue specimens from invasive ductal breast carcinomas (IDC) using quantitative RT-PCR. The prognostic impact of miRNA expression was analyzed using univariate and multivariable analysis for distant metastasis-free survival of patients. Immunohistochemistry (IHC) of the tissue samples was performed for oncoprotein HER2 and proliferation marker Ki-67, and in addition for p27KIP1, estrogen receptor (ER), and PTEN which were already described as targets of these miRNAs. In vitro studies and in silico target analyses were performed for breast cancer cell lines T47D and SKBR3. Here lentiviral particles were produced for overexpression of miRNAs as described (Anastasov, Rad Oncol 7:206,2012) with packaging plasmids and lentiviral precursor expression vectors for miR-221 and miR-222. After viral infection, the cells were analyzed for several signaling markers by Western blot, for cell proliferation, migration, and invasion compared to the corresponding untreated cell lines.
Summary of results: Our results demonstrate a statistically significant direct association between miR-222 and HER1/HER3 expression (p=0.003), and inverse correlations of miR-221 and HER4 (p=0.006), miR-222 and ER (p=0.016), and miR-222 and p27KIP1 (p=0.048). Nuclear grade of tumors was directly associated with Ki-67 expression (p≥0.001) and inversely with p27KIP1 (p=0.03). Both miRNAs are significantly associated with distant metastasis-free survival of patients; in particular miR-221 expression is a powerful discriminator in defining prognostic subgroups in HER2-positive and lymph node-positive breast cancer patients. To further substantiate these results, miR-221 and miR-222 were overexpressed in cell lines (T47D, SKBR3). Here, we could confirm association of the two miRNAs with p27Kip1, ER, and PTEN expression, which have been described as targets of miR-221 and -222. Upregulation of these miRNAs strongly increased tumor cell proliferation and invasion in vitro. Additional in silico and in vitro analyses revealed that tumor invasion processes are influenced by miR-221 and -222 via targeting the soluble variant of the urokinase-type plasminogen activator receptor (uPAR / CD87).
Statement and conclusions: We could demonstrate that elevated expression of miR-221 and miR-222 in ductal breast cancer allows distinguishing between different prognostic subgroups. Our in vitro results following miR-221/-222 overexpression in breast cancer tumor cell lines demonstrated a strong association between miRNAs and cellular invasion. From our in silico analysis there is also a clear hint that this process is modified by targeting of an uPAR isoform.
Citation Format: Natalie Falkenberg, Jr., Natasa Anastasov, Jr., Herbert Braselmann, Gert Auer, Michaela Huber, Manfred Schmitt, Heinz Höfler, Michaela Aubele. MiR-221 and -222 expression elevates cell invasion and allows distinction between different prognostic groups in advanced breast cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A046.