LeftyA decreases Actin Polymerization and Stiffness in Human Endometrial Cancer Cells

www.nature.com/scientificreports OPEN received: 04 November 2015 accepted: 16 June 2016 Published: 11 July 2016 LeftyA decreases Actin Polymerization and Stiffness in Human Endometrial Cancer Cells Madhuri S. Salker1,*, Nicolas Schierbaum2,*, Nour Alowayed1,*, Yogesh Singh1, Andreas F. Mack3, Christos Stournaras4,#, Tilman E. Schäffer2,# & Florian Lang1,# LeftyA, a cytokine regulating stemness and embryonic differentiation, down-regulates cell proliferation and migration. Cell proliferation and motility require actin reorganization, which is under control of rasrelated C3 botulinum toxin substrate 1 (Rac1) and p21 protein-activated kinase 1 (PAK1). The present study explored whether LeftyA modifies actin cytoskeleton, shape and stiffness of Ishikawa cells, a well differentiated endometrial carcinoma cell line. The effect of LeftyA on globular over filamentous actin ratio was determined utilizing Western blotting and flow cytometry. Rac1 and PAK1 transcript levels were measured by qRT-PCR as well as active Rac1 and PAK1 by immunoblotting. Cell stiffness (quantified by the elastic modulus), cell surface area and cell volume were studied by atomic force microscopy (AFM). As a result, 2 hours treatment with LeftyA (25 ng/ml) significantly decreased Rac1 and PAK1 transcript levels and activity, depolymerized actin, and decreased cell stiffness, surface area and volume. The effect of LeftyA on actin polymerization was mimicked by pharmacological inhibition of Rac1 and PAK1. In the presence of the Rac1 or PAK1 inhibitor LeftyA did not lead to significant further actin depolymerization. In conclusion, LeftyA leads to disruption of Rac1 and Pak1 activity with subsequent actin depolymerization, cell softening and cell shrinkage. LeftyA, also known as endometrial bleeding-associated factor (EBAF), is a known regulator of stemness and embryonic differentiation1. It has previously been shown that LeftyA can reprogram cancer cells2 leading to inhibition of cell proliferation, stimulation of apoptosis and thereby, suppression of tumor growth2,3. Together, these lines of data indicate LeftyA is a strong suppressor of tumor cell activity4–6. LeftyA has a powerful negative effect on Na+/H+ exchanger 1(NHE1) activity7, which is expected to compromise survival of tumor cells8–10. Regulators of NHE1 activity include the small G protein ras-related C3 botulinum toxin substrate 1 (GTPase Rac1)11 which is a member of the Rho GTPases family. Rac1 is a key regulator of the actin cell cytoskeleton12 and promotes the formation of lamellipodia13, which is essential for cell motility14. The regulatory proteins of the actin cytoskeleton play a pivotal role for the motility of cancer cells and contribute to most steps during cancer progression15,16. The ability of cancer cells to invade the surrounding tissue, crossing the endothelial barrier to metastasize at a secondary site requires a highly dynamic reorganization of the actin cytoskeleton17. Rac1 and other Rho GTPases were found to be overexpressed in many types of cancer18–20. Down-regulation of Rac1 activity suppresses tumor growth and Rac1 was therefore identified as a potential therapeutic target for cancer cell treatment21,22. As the actin cytoskeleton provides the structural scaffold of a cell and mainly determines its mechanical properties23,24 alteration of actin polymerization is in turn anticipated to modify cell stiffness25,26. Here we report that treatment of human endometrial carcinoma cells with LeftyA leads to dynamic change in mechanical cellular properties in tumor cells. We further provide evidence that in Ishikawa cells LeftyA decreases Rac1 activity, p21 protein-activated kinase 1 (PAK1) phosphorylation, actin polymerization, cell stiffness, area and volume. 1 Department of Cardiology, Vascular Medicine and Physiology, Eberhard Karls University of Tübingen, Germany. Institute of Applied Physics, Eberhard Karls University of Tübingen, Germany. 3Institute of Anatomy Eberhard Karls University of Tübingen, Germany. 4Department of Biochemistry, University of Crete Medical School, Heraklion, Greece. *These authors contributed equally to this work. #These authors jointly supervised this work. Correspondence and requests for materials should be addressed to F.L. (email: ) 2 Scientific Reports | 6:29370 | DOI: 10.1038/srep29370 1 www.nature.com/scientificreports/ Figure 1. AFM analysis of stiffness and shape of Ishikawa cells with and without LeftyA treatment. Representative optical phase contrast image (a), AFM contact height image (b) and AFM stiffness image (c) of an untreated human endometrial cancer Ishikawa cell. Representative optical phase contrast image (d), AFM contact height image (e) and AFM stiffness image (f) of an Ishikawa cell treated for 2 hours with 25 ng/ml LeftyA. Representative optical phase contrast image (g), AFM contact height image (h) and AFM stiffness image (i) of an Ishikawa cell 15 min after addition of 10 μM cytochalasin D. Mean cell stiffness 〈E〉 (j), mean cell area 〈A〉 (k) and mean cell volume 〈V 〉 (l) of untreated, LeftyA and cytochalasin D treated Ishikawa cells (〈E〉Contr = 0.73 kPa, 〈E〉LeftyA = 0.40 kPa, 〈E〉CytoD = 0.21 kPa, 〈A〉Contr = 2084 μm2, 〈A〉LeftyA = 1579 μm2, 〈A〉CytoD = 1580 μm2, 〈V〉Contr = 2934 μm3, 〈V〉LeftyA = 2403 μm3, 〈V〉CytoD = 1974 μm3). Error bars represent SEM of geometric mean (j) and SEM of arithmetic mean (k,l). *P < 0.05; **P < 0.01; ***P < 0.001 using one-way ANOVA followed by two-tailed Tukey’s test. Results Impact of LeftyA on the stiffness and the shape of Ishikawa cells. We recently have shown that LeftyA can decrease expression and activity of the NHE17. NHE1 in turn contributes to the stabilization and localization of actin. We hypothesized that NHE1 inhibition could alter the cytoskeleton necessary for maintaining cell structure. Filamentous actin (F-actin), a cytoskeleton protein known to have an important role in maintaining cellular and tissue structure26, is affected by changes in cytosolic pH (pHi)27. To determine whether LeftyA impacts on cell shape and mechanical stiffness of human endometrial cancer Ishikawa cells, atomic force microscopy (AFM) was performed on live Ishikawa cells after a 2 hours treatment with LeftyA (25 ng/ml). The effect of LeftyA was compared to that of the cytoskeletal drug cytochalasin D, which induces rapid actin depolymerization. The cells exhibited large spatial variations of the local stiffness (Fig. 1c,f,i). The calculated single cell stiffness was averaged for a large number of cells to obtain a representative mean stiffness 〈E〉. LeftyA and cytochalasin D treated Ishikawa cells were significantly softer than the control cells (Fig. 1j; ***P = 1 × 10−10, ***P = 4 × 10−9). Histograms of the single cells stiffness values showed an approximately log-normal distribution, which was shifted to lower stiffness values for LeftyA and cytochalasin D treated Ishikawa cells, as compared to untreated Ishikawa cells (Supplementary Fig. S1 (...truncated)