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Ezrin Expression Is an Independent Prognostic Factor in Gastro-intestinal Cancers

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Journal of Gastrointestinal Surgery Aims and scope

Abstract

Background

Ezrin, a member of the ezrin–radixin–moesin (ERM) family of plasma membrane–cytoskeleton linker proteins, has been associated with metastatic behavior.

Methodology

Microarrayed pathological tissues of surgically resected colorectal cancer liver metastasis (CRLM) and whole tissue sections of cancer of the ampulla of Vater (CAV) were analyzed to determine ezrin expression levels and correlation with survival. The requirement of ezrin in invasive capability was assessed using in vitro assays.

Results

Surgically resected CAV showing a low ezrin score have a better 5-year disease-specific survival than those showing a high ezrin score (P < 0.0001). Similarly, high ezrin expression at the invasive front of CRLM resulted in poor disease-free survival (P = 0.05). Multivariate analysis demonstrated high ezrin expression to be an independent adverse prognostic factor for CAV (hazard ratio (HR) 15.22 (95 % confidence interval (CI) 1.98–117.03), P < 0.01) and CRLM (HR 6.42 (95 % CI 1.01–52.43), P = 0.05), among other clinically relevant variables such as lymph node metastasis (for CAV) and the presence of extrahepatic disease, large hepatic metastases (>5 cm), and close surgical resection margins (<5 mm) (all for CRLM). In vitro experiments indicated that ezrin expression was vital for cellular processes such as adhesive and invasive activity.

Significance

High ezrin expression indicates an adverse prognosis in primary CAV and CRLM.

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Acknowledgments

This work was funded by Cancer Research UK (PA), European Society of Surgical Oncology International Fellowship (SP), and NIHR Clinician Scientist Fellowship (HMK) as well as AIRC Regionale Veneto, Associazione Italiana Ricerca sul Cancro, Italy; the European Community Grant FP7 “EPC-TM-NET”; and Ministero della Salute, Rome, Italy.

Conflict of interest

There are no potential conflicting interests and no editorial assistance was obtained.

Author information

Authors and Affiliations

  1. Centre for Tumour Biology, John Vane Science Centre, Barts Cancer Institute—a CR-UK Centre of Excellence, Queen Mary University of London, Charterhouse Square, London, EC1M 6BQ, UK

    Prabhu Arumugam, Stefano Partelli, Stacey J. Coleman, Fieke E. M. Froeling & Hemant M. Kocher

  2. Barts and the London HPB Centre, The Royal London Hospital, London, E1 1BB, UK

    Prabhu Arumugam, Stefano Partelli, Nilukushi Wijesuriya, Jo-Anne Chin Aleong & Hemant M. Kocher

  3. Surgical Department, Pancreatic Unit, University of Ancona, Verona, Italy

    Stefano Partelli

  4. Department of Pathology, University of Verona, Verona, Italy

    Ivana Cataldo & Aldo Scarpa

  5. ARC-NET Research Center for Applied Research on Cancer, University of Verona, Verona, Italy

    Stefania Beghelli & Aldo Scarpa

  6. Department of Surgery, Pancreas Institute, University of Verona, Verona, Italy

    Claudio Bassi

Authors
  1. Prabhu Arumugam
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  2. Stefano Partelli
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  3. Stacey J. Coleman
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  5. Stefania Beghelli
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  7. Nilukushi Wijesuriya
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  9. Fieke E. M. Froeling
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  11. Hemant M. Kocher
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Corresponding author

Correspondence to Hemant M. Kocher.

Additional information

Prabhu Arumugam, Stefano Partelli, and Stacey J Coleman contributed equally to this work.

Electronic supplementary material

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Supplementary Table 1

Summary of recent studies demonstrating a role for Ezrin in ability to independently prognosticate survival in various GI and non-GI cancers as indicated by clinico-pathological and cell biological evidence. (DOC 258 kb)

Supplementary Figure 1

Overview images of slides (A-C) with liver metastasis and surrounding liver stained for Ezrin expression demonstrate absence of Ezrin stain in normal liver as well as the center of the metastatic deposit (B,C) but pronounced Ezrin stain in the periphery of the metastatic deposit (A,C). Furthermore representative TMA core images demonstrate that whilst Ezrin expression is minimal in the core from the center of the metastatic deposit (D-F), it is abundant in the periphery (G-I), where the surrounding normal liver is uniformly negative for Ezrin expression. Scale bar: 200 μm (JPEG 248 kb)

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Supplementary Figure 2

Ezrin down-regulation in cancer cells. Two separate siRNA sequences could reproducibly knock-down Ezrin expression in SW707 and HCT116 cancer cell lines (highest expression of Ezrin). Western blots of Ezrin expression along with HSC70 (loading control) and densitometric quantitative analysis of three separate experiments for both cell lines are shown after transfection with either Scrambled (Scr, grey) or Ezrin RNAi (1st or 2nd sequence, white). *, P < 0.05, Student t-test. (JPEG 54 kb)

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Supplementary Figure 3

Ezrin down-regulation affects cellular processes in cancer cells. Confocal z-stacks of SW707 cell line after transfection with either Scr or Ezrin RNAi and staining with Ezrin (green) and actin (red) demonstrated that, when cells were plated on fibronectin, there are Ezrin-containing lamellipodia (arrowhead) which are absent in Ezrin RNAi treated cells. Representative images from bottom of the cells shows abundance of Ezrin, where cells are in contact with substrate (fibronectin, ventral aspect), whilst in the Ezrin knockdown cells most of the residual Ezrin expression is on the opposite (dorsal) aspect of the cell. X, Y and Z axes are marked with respective Z-cross-sections (on top (green line) and side (red line) panels) demonstrated to show the cells’ bottom (right panel), middle (middle panel) and top (left panel) aspects. Scale bar: 5 μm. (JPEG 101 kb)

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Supplementary Figure 4

Ezrin down-regulation affects sheet migration of cancer cells. Reduction in the ability to close the wound within 48 hours of a scratch assay of transfected HCT116 cells with either Scrambled (Scr, grey) or Ezrin RNAi (1st or 2nd sequence, white) Representative images are shown at the start and end-point of the assay along with quantification of the % of wound closure. *, P < 0.05, Student t-test. (JPEG 182 kb)

High resolution image

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Supplementary Figure 5

Ezrin over-expression increases migration of cancer cells. SW122 cells transfected with GFP-tagged Ezrin migrated in higher numbers compared to untransfected cells. Western blotting confirms GFP expression (A), whilst quantification of blots confirms higher Ezrin intensity in cells transfected with GFP-Ezrin (B). Transwell migration analysis confirms that SW122 cells migrated in higher numbers over 24 hours when over expressing Ezrin. ***, P < 0.001; **, P < 0.01; Student t-test (JPEG 39 kb)

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(TIFF 447 kb)

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Arumugam, P., Partelli, S., Coleman, S.J. et al. Ezrin Expression Is an Independent Prognostic Factor in Gastro-intestinal Cancers. J Gastrointest Surg 17, 2082–2091 (2013). https://doi.org/10.1007/s11605-013-2384-1

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