EXPRESSION OF EZRIN mRNA AND PROTEIN IN OSTEOSARCOMA:

RELATIONSHIP TO PATIENT CLINICAL

CHARACTERISTICS

Taiqiang Yan, Rita Kandel, Nalan Gokgoz, Taiqiang Yan, Rita Kandel, Nalan Gokgoz, Robert Bell, Irene Andrulis & Jay WunderRobert Bell, Irene Andrulis & Jay Wunder

University Musculoskeletal Oncology Unit, Samuel Lunenfeld Research Institute,

Mount Sinai Hospital, Toronto, Canada

Khanna C et al. Cancer Research 2001, 61: 3750-3759

Khanna C et al. Nature Medicine 2004, 10: 182-186

• Ezrin was overexpressed in a highly metastatic murine Osteosarcoma cell line

• Ezrin overexpression was associated with enhanced motility, adherence

• In an orthotopic murine tumor model, metastases were reduced by Ezrin suppression and increased by transfection of full-length Ezrin constructs

• Ezrin overexpression was associated with poor outcome in canine and pediatric Osteosarcoma

Ezrin

Background

• Ezrin is the best characterized ERM (ezrin-radixin-moesin) protein family member

• ERM proteins act as linkers between the cell membrane and the actin cytoskeleton

• ERM proteins are involved in signal transduction through growth factor receptors and adhesion molecules

Study Goals

•Examine level of Ezrin mRNA and protein in biopsy specimens from patients with newly diagnosed Osteosarcoma

•Compare Ezrin mRNA and protein levels

•Correlate Ezrin expression with patient clinicopathologic data and outcome

Patients and Tumors

• 39 patients with high grade intramedullary Osteosarcoma

• 14 patients with mets at diagnosis 25 patients with localized tumors

• operative biopsy before chemotherapy• fresh frozen tumor from biopsy

paraffin embedded tissue from biopsy• patient follow-up

Quantitative Real-Time RT-PCR

• ABI prism 7900HT system

• TaqMan probe

• House keeping gene asparagine synthetase as internal control

• mRNA levels normalized to a control cell line

Immunohistochemistry

• Paraffin-embedded tissue blocks from Osteosarcoma biopsy specimens prior to chemotherapy

• Avidin-biotinylated peroxidase complex method

• Sigma 3C12 monoclonal antibody• Assessed % positive staining cells• Assessed localization- membrane

- cytoplasmic

Patient Characteristics

Characteristics Metastasis at diagnosis

No metastasis at diagnosis

P value

Gender Male: 8Female: 6

Male: 16Female: 9

0.31

Mean Age (Years) 24.2 27.5 0.51

Tumor Location Proximal: 11Distal: 3

Proximal: 12Distal: 13

0.09

Mean Tumor Size (cm)

14.2 9.1 0.0001

Relative value (ezrin/AS) Number of tumors

0.3-0.99 12

1.0-1.99 10

2.0-2.99 6

3.0-8.8 11

1. Ezrin mRNA expression in Osteosarcoma by Real-Time RT-PCR

Ezrin mRNA expression by Real-Time RT-PCR and patient characteristics at

diagnosis

•No correlation between Ezrin mRNA expression and patient clinical characteristics

•Level of Ezrin mRNA was significantly lower in tumors from patients with metastases at diagnosis compared to patients who presented with localized disease (p=0.039)

Ezrin mRNA expression and clinical outcome for “curable” patients

•For 25 patients with localized Osteosarcoma at diagnosis, there was no difference in the level of Ezrin mRNA expression between 9 patients who developed metastases during follow-up and 16 patients who remain free of disease (p=0.28)

Ezrin: Cellular Localization

• Ezrin protein exists in the cytoplasm in an Inactive form

• Phosphorylation leads to Ezrin Activation and membrane translocation

•Diffuse Ezrin IHC staining Cytoplasm (arrow) and Membrane staining (Low power)

•Membrane Ezrin IHC staining (High power)

2. Ezrin Immunohistochemisty

Ezrin Immunohistochemisty

Ezrin staining (> 10% cells)

Metastasis at Diagnosis

No Metastasis at Diagnosis

Membrane only

4/14 4/25

Membrane + Cytoplasm

2/14 1/25

Cytoplasm only

1/14 8/25

• 23/39 (59%) tumors had positive Ezrin IHC staining

Ezrin protein expression by IHC and clinical outcome in “curable” patients

•Patients with localized tumors at diagnosis

Ezrin Membrane IHC was positive (>10% cells) in 2/9 (22%) tumors which later metastasized vs 3/16 (19%) tumors without systemic spread

•We did not identify any correlation between Ezrin IHC and patient characteristics at diagnosis or outcome - 10% IHC staining

- any staining - membrane, cytoplasm

or both

3. Ezrin mRNA vs protein expression

•the level of Ezrin mRNA expression did not correlate with protein by IHC

•6 of 12 tumors with Ezrin Cytoplasm staining also had mRNA overexpression

•None of 8 tumors with Ezrin Membrane staining had mRNA overexpression

•p=0.042

Discussion

• Ezrin Membrane and Cytoplasm staining by IHC in Osteosarcoma

• A distinctive Membranous Ezrin IHC pattern was seen more commonly in tumors from patients with metastases at diagnosis

• Ezrin activation and binding to the cell membrane may facilitate early invasion and metastasis in Osteosarcoma

• Cytoplasmic Ezrin staining was more common in tumors localized at diagnosis

• Ezrin mRNA expression only correlated with cytoplasmic staining

Discussion

Lack of Ezrin mRNA – Ezrin protein correlation:

Different mechanisms of transcription/translation• Overactivation of post-translational modification

in tumors with metastases at diagnosis• Different Ezrin isoforms due to alt. splice variants

- Original cDNA cloning paper (Gould et al, EMBO 1989)

- Genomic structure

Acknowledgements

• Ontario Cancer Research Network (OCRN)• National Cancer Institute of Canada

(NCIC)• Canadian Institutes of Health Research (CIHR)

Interdisciplinary Health Research Team (IHRT) in Musculoskeletal Neoplasia

• Rubinoff-Gross Chair in Orthopaedic Oncology at Mount Sinai Hospital, University of Toronto

Hunter K. Ezrin, a key component in tumor metastasis. Trends in Molecular Medicine, 2004, 10: 201-204

• Regulate cellular activities, such as survival, adhesion, migration and invasion

• Cell-cell interaction

• Cell-matrix interaction

• MAPK signal transduction

• Rho GTPase signal transduction

• Akt mediated cellular apoptosis

• There is a significant correlation between increased ezrin immunoreactivity and higher histological grade in astrocytoma.

Geiger KD, etc. Ezrin immunoreactivity is associated with increasing malignancy of

astrocytic tumors but is absent in oligodendrogliomas. Am J Pathol 2000, 157:1785-93.

• Ezrin expression correlates with tumor thickness and level of invasion in primary cutaneous melanoma.

Ilmonen S, etc. Ezrin in primary cutaneous melanoma. Mod Pathol 2005, 18:503-10.

• Ezrin has been reported to be involved in dissemination of soft tissue sarcoma, and could be valuable as an additional prognostic marker.

Weng WH, etc. Prognostic impact of immunohistochemical expression of ezrin in highly malignant soft tissue sarcomas. Clin Cancer Res, 2005, 11: 6198-6204.

• Ezrin was also indicated as a key metastatic regulator in rhabodomysarcoma and breast cancer.

Yu Y, etc. Expression profiling identifies the cytoskeletal organizer ezrin and the developmental homeoprotein Six-1 as key metastatic regulators. Nat Med 2004, 10:175-81.

Elliott BE, etc. The membrane cytoskeletal crosslinker ezrin is required for metastasis of breast carcinoma cells. Breast Cancer Res 2005, 7:R365-73.

Future DirectionsFuture Directions

• Ezrin isoform characterization

• Growth inhibition by siRNA to confirm whether Ezrin is involved in cell proliferation

• Antibody to phosphorylated Ezrin to determine functional status of protein subcellular distribution

Khanna C, et al. The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis, Nature Medicine, 2004, 10: 182-186

•Canine OSA (n=83) •Pediatric OSA (n=19)

•No Mets at Diagnosis •Mets at Diagnosis

Microarray Analysis of Osteosarcoma

Ezrin•Yan et al, CTOS 2004•Khanna et al, Cancer Res 2001•Leonard et al, Br J Cancer 2003