Abstract
Extracellular matrix (ECM)-adhesion proteins and actin cytoskeleton are pivotal in cancer cell invasion. Ras Suppressor-1 (RSU-1), a cell-ECM adhesion protein that interacts with PINCH-1, thus being connected to Integrin Linked Kinase (ILK), alpha-parvin (PARVA), and actin cytoskeleton, is up-regulated in metastatic breast cancer (BC) samples. Apart from the originally-identified gene (RSU-1L), an alternatively-spliced isoform (RSU-1-X1) has been reported. We used non-invasive MCF-7 cells, expressing only RSU-1L, and highly invasive MDA-MB-231-LM2 expressing both isoforms and generated stable shRNA-transduced cells lacking RSU-1L, while the truncated RSU-1-X1 isoform was depleted by siRNA-mediated silencing. RSU-1L depletion in MCF-7 cells resulted in complete abrogation of tumor spheroid invasion in three-dimensional collagen gels, whereas it promoted MDA-MB-231-LM2 invasion, through a compensatory upregulation of RSU-1-X1. When RSU-1-X1 was also eliminated, RSU-1L-depletion-induced migration and invasion were drastically reduced being accompanied by reduced urokinase plasminogen activator expression. Protein expression analysis in 23 human BC samples corroborated our findings showing RSU-1L to be upregulated and RSU-1-X1 downregulated in metastatic samples. We demonstrate for the first time, that both RSU-1 isoforms promote invasion in vitro while RSU-1L elimination induces RSU-1-X1 upregulation to compensate for the loss. Hence, we propose that both isoforms should be blocked to effectively eliminate metastasis.
Highlights
Breast cancer (BC) is the most frequent type of cancer in women
Intrigued by our previous work showing that siRNA-mediated silencing of Ras Suppressor-1 (RSU-1), using a commercially available pool of siRNAs that targets both isoforms, leads to reduced invasive capacity of breast cancer (BC) cells accompanied by inhibition of urokinase Plasminogen Activator, and metalloproteinase-13 (MMP-13)[22], we set out to further investigate the role of RSU-1 isoforms in BC cell metastasis
RSU-1 was connected to Ras-mediated oncogenic transformation[9,14], its expression in cancer tissues, and its association to metastasis are still vague
Summary
Breast cancer (BC) is the most frequent type of cancer in women. When BC is characterized as non-invasive or in situ, the prognosis is far better than when it is characterized as metastatic or invasive. Integrins and extracellular matrix (ECM)-related adhesion proteins play an important role in all stages of the metastatic process facilitating the communication between cells and the ECM4. They act as adaptor proteins being involved in multiple protein-protein interactions[5], while being connected directly or indirectly to the actin cytoskeleton. Meta-analysis of Affymetrix microarray gene expression data from 5143 BC patients showed that elevated RSU-1 mRNA expression was not correlated with overall survival, it was correlated with poor prognosis both in terms of distant metastasis-free survival and remission-free survival[22] These data indicate that RSU-1 may be involved in BC metastasis, the underlying mechanism is still vague. We validated our findings using human BC samples that expressed differential levels of the two RSU-1 isoforms
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