Expression profiling of migrated and invaded breast cancer cells predicts early metastatic relapse and reveals Krüppel-like factor 9 as a potential suppressor of invasive growth in breast cancer
Ridha Limame1,8, Ken Op de Beeck1,2,8, Steven Van Laere3,4, Lieselot Croes1,2,5, Annemieke De Wilde5, Luc Dirix3, Guy Van Camp2, Marc Peeters1,6, Olivier De Wever7, Filip Lardon1, Patrick Pauwels1,5
1 Center for Oncological Research (CORE), University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium
2 Department of Medical Genetics, University of Antwerp and Antwerp University Hospital, Universiteitsplein 1, B-2610 Antwerp, Belgium
3 Translational Cancer Research Unit (TCRU), GZA Hospitals Sint-Augustinus, Oosterveldlaan 24, B-2610 Wilrijk (Antwerp), Belgium
4 Department of Oncology, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium
5 Laboratory of Pathology, Antwerp University Hospital, Wilrijkstraat 10, B-2650 Edegem (Antwerp), Belgium
6 Department of Oncology, Antwerp University Hospital, Wilrijkstraat 10, B-2650 Edegem (Antwerp), Belgium
7 Laboratory of Experimental Cancer Research, Department of Radiotherapy and Experimental Cancer Research, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium
8 These authors equally contributed to this work.
Ridha Limame, email:
Keywords: breast cancer, invasion, migration, KLF9, gene expression
Received: November 28, 2013 Accepted: January 16, 2014 Published: January 18, 2014
Cell motility and invasion initiate metastasis. However, only a subpopulation of cancer cells within a tumor will ultimately become invasive. Due to this stochastic and transient nature, in an experimental setting, migrating and invading cells need to be isolated from the general population in order to study the gene expression profiles linked to these processes. This report describes microarray analysis on RNA derived from migrated or invaded subpopulations of triple negative breast cancer cells in a Transwell set-up, at two different time points during motility and invasion, pre-determined as “early” and “late” in real-time kinetic assessments. Invasion- and migration-related gene expression signatures were generated through comparison with non-invasive cells, remaining at the upper side of the Transwell membranes. Late-phase signatures of both invasion and migration indicated poor prognosis in a series of breast cancer data sets. Furthermore, evaluation of the genes constituting the prognostic invasion-related gene signature revealed Krüppel-like factor 9 (KLF9) as a putative suppressor of invasive growth in breast cancer. Next to loss in invasive vs non-invasive cell lines, KLF9 also showed significantly lower expression levels in the “early” invasive cell population, in several public expression data sets and in clinical breast cancer samples when compared to normal tissue. Overexpression of EGFP-KLF9 fusion protein significantly altered morphology and blocked invasion and growth of MDA-MB-231 cells in vitro. In addition, KLF9 expression correlated inversely with mitotic activity in clinical samples, indicating anti-proliferative effects.