Ezrin is key regulator of Src-induced malignant phenotype in three-dimensional environment

Abstract

The oncogenic tyrosine kinase Src has a role in cancer development, especially by promoting invasive and metastatic behavior. It is, however, unclear which of the Srcregulated signaling cascades induce malignant phenotype in three-dimensional environment. One of Src substrates is ezrin, a cytoskeletal organiser and regulator of signal transduction. Ezrin expression correlates with poor outcome of diverse cancers and is essential in experimental metastatic osteosarcoma. We reconstituted genetically ezrin-deficient cells with wild-type (WT) or phosphorylation-deficient Y477F ezrin together with constitutively active Src. In two-dimensional cultures, Src induced malignant features regardless of the presence or absence of ezrin. In contrast, only WT ezrin-expressing cells grew efficiently in soft agar or in suspension. In Matrigel, only WT ezrin significantly promoted growth and invasion, and was targeted to specific regions on the plasma membrane. WT and Y477F ezrin-expressing cells showed marked differences only when growing or scattering in threedimensional matrix. Additional experiments showed that Y477-phosphorylated ezrin is also needed for the growth of Src-transformed epithelial cells in three-dimensional matrix. Cells lacking functional ezrin had reduced cyclin D levels and fewer cells in G2 þ S phase, possibly as a consequence of abnormal mTOR signaling, as ezrin Y477F cells showed lower expression of phosphorylated intermediates downstream of mTOR than WT cells. We conclude that the pathways activated by Src depend on the type of environment and that ezrin is a crucial element of Src-induced malignant features in cells growing inside three-dimensional environment.

The metastatic cancer cell cortex: An adaptation to enhance robust cell division in novel environments?

“…In doing so, rounding makes mitosis robust to the environment. Moreover, this may explain the identification of key regulators of mitotic rounding, particularly Ect2 and the ERM family proteins, as oncogenes associated with metastasis and capable of transforming cells to grow in soft agar [28,30]. If these ideas are borne out in experiments, it will be important to reassess the roles of the many other actin cytoskeletal regulators that have been implicated in cancer progression.…”

“…Ect2 is up-regulated in a number of cancers including lung, brain, ovarian and bladder [27], and when silenced by RNAi, leads to reduced invasion, tumorigenicity and growth in soft agar in lung cancer cells [28]. Similarly, Ezrin is over-expressed in a huge range of cancer types [29] and has been shown to be essential for metastasis and anchorage independent growth in soft agar [30]. Yet despite well-established links with tumorigenesis, the molecular function of Ect2 or Ezrin in the context of cancer has not been addressed in great detail.…”

Cytoplasmic Ezrin and Moesin Correlate with Poor Survival in Head and Neck Squamous Cell Carcinoma

“…Altered expression of a subset of 4.1 family proteins is believed to contribute to carcinogenesis and metastasis, as exemplified by the following: merlin has been shown to function as a tumor suppressor [9]; ezrin is believed to play a role in the development of metastasis [10,11]; moesin has been implicated in oral squamous cell carcinomas [12][13][14]; and willin has been shown to antagonize some of the functions of the YAP oncogene [6]. We have shown previously that a high level of cytoplasmic ezrin correlates or that high levels of cytoplasmic ezrin correlate with poor survival in head and neck squamous cell carcinoma [15], and recent studies show that increased expression of ezrin is also associated with poor clinical outcome in a variety of human cancers [16][17][18][19][20][21].…”

FLT3-driven redox-modulation of Ezrin regulates leukaemic cell migration

“…Further evidence links phosphorylation on Ezrin Y146 to Src-dependent adhesion signalling promoting cell proliferation (Srivastava et al, 2005) and nominates another Src-phosphorylated residue Y477 as the effector of a malignant phenotype in Src-transformed cells (Heiska and Carpen, 2005;Heiska et al, 2011), supported again by a recent report from Mak et al (2012) proposing a role for phosphorylation of Y477 in invasion and migration.…”

“…Ezrin is also of interest from a cancer perspective, with an established role in metastasis (Brambilla and Fais, 2009;Fais, 2004;Federici et al, 2009;Zhou et al, 2010a) and an emerging role in proliferative signalling (Gautreau et al, 1999;Heiska et al, 2011;Kishore et al, 2005;Srivastava et al, 2005). Ezrin has been previously shown to be increased in leukaemic cells (Monni et al, 2008) and has been identified as a target of oncogenic signalling from extracellular signalling mediators PDGF, EGF and HGF as well as intracellular oncogenic kinases Src, Lck and Rho (Crepaldi et al, 1997;Fazioli et al, 1993;Monni et al, 2008).…”