The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC

The survival of osteosarcoma patients is connected to metastasis. The ezrin expression is associated with the development of metastasis and poor outcome in osteosarcoma. Ezrin is present in the cytoplasm and after phosphorylation assumes an active form and links F-actin to the cell membrane. This study evaluated ezrin and phosphorylated ezrin at site Tyr354 and Thr567 expression and its subcellular localization in osteosarcoma. We studied 50 osteosarcoma patients (mean follow-up 9.8 years). Ezrin expression was assessed using immunohistochemical and immunofluorescence analysis on tissue microarray and cultured cells of human osteosarcoma 143B. The western blot analysis was carried out on cultured cells. The majority of osteosarcomas, showing cytoplasmic positivity for ezrin, phosphorylated and unphosphorylated, were associated with membranous and nuclear positivity for phosphorylated ezrin Thr567 and phosphorylated ezrin Tyr354, respectively. Ezrin expression was associated with high-grade osteosarcoma (P ¼ 0.04), with metastasis (P ¼ 0.04) and with tumors that developed metastasis (P ¼ 0.04); phosphorylated ezrin Thr567 expression was present mostly in tumors with metastasis (P ¼ 0.01) and in osteosarcomas that did not develop metastasis (P ¼ 0.002). The osteosarcoma patients with ezrin expression have a short survival. The cytoplasmic ezrin expression in osteosarcoma matches its role of membrane-cytoskeleton linker protein. The subcellular trafficking of ezrin is not blocked and it is linked to ezrin phosphorylation, also in cancer. The phosphorylated ezrin Tyr354 nuclear localization suggests its possible role as a nuclear factor in osteosarcoma. The phosphorylated ezrin Thr567 phosphorylation may not be necessary in osteosarcoma metastatic progression but it was modulated. The ezrin expression is associated with more aggressive osteosarcomas and with metastasis.

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“…Ren et al 29 has recently shown that the Thr 567-phosphorylated ezrin expression decreased in osteosarcoma metastatic lesions.…”

Section: Discussionmentioning

confidence: 99%

Phosphorylated ezrin is located in the nucleus of the osteosarcoma cell

et al. 2010

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“…K7M2 is a metastatic osteosarcoma cell line. These cells express high levels of the cytoskeletal linker protein ezrin, which accounts for the invasive properties of this cell line 13,14 . When HUVEC cells are challenged with K7M2 cells, there is a drop in electrical resistance within 6 hours.…”

Section: Representative Resultsmentioning

confidence: 99%

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Rahim

Üren

2011

JoVE

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Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host [1][2][3] . Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors 4,5 . The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phasecontrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process 6 . Here, we describe an electricalimpedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells. Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes 7,8 . The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance [9][10][11][12] . The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods. Video LinkThe video component of this article can be found at https://www.jove.com/video/2792/ Protocol 1. Preparation 1. All steps should be performed under sterile conditions in a tissue culture hood. 2. The xCELLigence station is placed in a 37°C incubator in the presence of 5% CO 2 . 3. Use low passage HUVEC cells, preferably no more than passage 6. Also, make sure tha...

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“…Recent studies suggest that ezrin was dynamically phosphorylated during metastatic progression in osteosarcoma [22]. However, it is currently unclear whether ezrin phosphorylation contributes to the malignancy phenotype of a tumor by promoting tumor cells to internalize NK cells.…”

Section: Discussionmentioning

confidence: 99%