Ezrin expression and cell survival regulation in colorectal cancer

Abstract

Colorectal cancer (CRC) is the second largest cause of cancer deaths in the United States. A key barrier that prevents better outcomes for this type of cancer as well as other solid tumors is the lack of effective therapies against the metastatic disease. Thus there is an urgent need to fill this gap in cancer therapy. We utilized a 2D-DIGE proteomics approach to identify and characterize proteins that are differentially regulated between primary colon tumor and liver metastatic deposits of the IGF1R-dependent GEO human CRC xenograft, orthotopically implanted in athymic nude mice that may serve as potential therapeutic targets against CRC metastasis. We observed increased expression of ezrin in liver metastasis in comparison to the primary colonic tumor. Increased ezrin expression was further confirmed by western blot and microarray analyses. Ezrin, a cytoskeletal protein belonging to Ezrin-Radixin-Moesin (ERM) family plays important roles in cell motility, invasion and metastasis. However, its exact function in colorectal cancer is not well characterized. Establishment of advanced GEO cell lines with enhanced liver-metastasizing ability showed a significant increase in ezrin expression in liver metastasis. Increased phosphorylation of ezrin at the T567 site (termed here as p-ezrin T567) was observed in liver metastasis. IHC studies of human CRC patient specimens showed an increased expression of p-ezrin T567 in liver metastasis compared to the primary tumors of the same patient. Ezrin modulation by siRNA, inhibitors and T567A/D point mutations significantly downregulated inhibitors of apoptosis (IAP) proteins XIAP and survivin that have been linked to increased aberrant cell survival and metastasis and increased cell death. Inhibition of the IGF1R signaling pathway by humanized recombinant IGF1R monoclonal antibody MK-0646 in athymic mouse subcutaneous xenografts resulted in inhibition of p-ezrin T567 indicating ezrin signaling is downstream of the IGF1R signaling pathway. We identified increased expression of p-ezrin T567 in CRC liver metastasis in both orthotopically implanted GEO tumors as well as human patient specimens. We report for the first time that p-ezrin T567 is downstream of the IGF1R signaling and demonstrate that ezrin regulates cell survival through survivin/XIAP modulation.

Ezrin Inhibition Up-regulates Stress Response Gene Expression

“…Ezrin has been implicated in the regulation of many essential cellular functions including cell adhesion, motility, regulation of ion channels, maintenance and determination of cell shape, morphogenesis, cell proliferation, and apoptosis (2). A growing body of evidence suggests that high ezrin expression correlates with metastatic behavior and poor clinical outcome in a variety of tumors including melanoma, colorectal cancer, endometrioid carcinoma, astrocytic tumors, soft tissue sarcomas, and adenocarcinomas of lung and breast (3)(4)(5)(6)(7)(8)(9)(10)(11). Ezrin has also been directly implicated in tumor progression and metastatic process in pediatric solid tumors of mesenchymal origin including osteosarcoma and rhabdomyosarcoma (12)(13)(14).…”

TGFβ and IGF1R signaling activates protein kinase A through differential regulation of ezrin phosphorylation in colon cancer cells

“…In addition, ezrin plays a key role in cell survival of colon cancer cells and regulates expression of Inhibitor of Apoptosis Proteins (IAP), XIAP and survivin, which have been shown to be involved in cell survival and metastasis (5,22). We have previously shown that IGF1R signaling regulates ezrin phosphorylation at T567 (5). Other studies have shown that ezrin is a downstream effector of the PI3K/AKT pathway (23,24).…”

“…Recently, we demonstrated that transient knockdown of ezrin using siRNA leads to downregulation of XIAP and survivin expression (5). To understand the underlying molecular mechanism, stable knockdown (KD) of ezrin expression was performed using GIFZ lentiviral shRNA#1 and #3 in GEO and FET colon cancer cells.…”

“…Ezrin plays an important role in cell motility and invasion and has been implicated in metastasis of several types of cancer including CRC (16)(17)(18)(19)(20)(21). In addition, ezrin plays a key role in cell survival of colon cancer cells and regulates expression of Inhibitor of Apoptosis Proteins (IAP), XIAP and survivin, which have been shown to be involved in cell survival and metastasis (5,22). We have previously shown that IGF1R signaling regulates ezrin phosphorylation at T567 (5).…”

“…Our recent studies have demonstrated that expression of ezrin is upregulated in liver metastases when compared to primary tumors in an orthotopic model of colon cancer; and that ezrin expression is increased in primary tumors from colon cancer patients (5). Ezrin, a member of the ezrin radixin moesin (ERM) family (6)(7)(8)(9), exists in two conformations, an active open form mainly localized at the plasma membrane and a dormant closed form largely resides in the cytoplasm.…”