Introduction: Colorectal Cancer(CRC) metastasis to distant organ sites is a major cause of CRC-related death. To date, there are no effective anti-metastatic therapies available. The purpose of this study was to use a proteomics-based approach to identify and characterize molecular targets associated with CRC metastasis.

Methods: GFP-labeled GEO human CRC cells were used to generate subcutaneous xenografts in BALB/c nude mice, which in turn were orthotopically implanted. This model allows for reproducible quantitative analysis of metastasis to liver and lungs. Primary and liver metastases were collected and a 2D-DIGE proteomics approach was used to characterize the global protein expression in the primary tumors and the corresponding liver metastases. Tissues were labeled with CyDye DIGE fluors and analyzed on a single multiplexed gel. DeCyder “in-gel” analysis software was used to compare all protein spots and protein expression ratios between the primary tumor and liver metastasis were generated. Differential spots were selected, in-gel digested and peptide mass fingerprints were obtained. Gene microarray analysis using the Illumina platform was also performed on primary and metastatic tumors. Westerns were performed to confirm protein expression. Survival signaling in GEO cells is dependent on the IGF1R pathway and thus, an IGF1R monoclonal antibody, MK-0646 was used to assess downstream effects.

Results: Differential expression of Ezrin was identified, as it was increased in liver metastases compared to primary tumors. This was validated by microarrays and westerns. Ezrin, a protein belonging to the Ezrin-Radixin-Moesin (ERM) family plays important roles in cell motility, invasion, tumor progression and metastasis. We also observed that T567 hyperphosphorylation of Ezrin was correlated to CRC metastasis by western blot analysis of primary tumor with liver metastases. This was further confirmed by IHC on human CRC patient specimens. Moreover, inhibition of the IGF-1R signaling pathway by mAb MK-0646 in mouse GEO CRC subcutaneous xenografts resulted in inhibition of Ezrin phosphorylation at T567, indicating that Ezrin signaling is downstream of the IGF-1R signaling pathway. SiRNA knockdown of Ezrin significantly downregulated XIAP, which is a metastasis gene in this model. Therefore, XIAP is downstream of IGF-1R-Ezrin signaling.

Conclusions: Using a proteomics approach, we identified ezrin as a potential biomarker for metastasis in CRC. We show for the first time that Ezrin activation at T567 is downstream of the IGF-1R signaling pathway and contributes to cell survival in CRC metastasis. Thus, ezrin might be a novel target that could be utilized for the development of effective anti-metastatic therapies against CRC.

Citation Format: Premila Devi Leiphrakpam, Ashwani Rajput, Michael G. Brattain, Sanjib Chowdhury. The expression and clinical significance of Ezrin in colorectal cancer metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3887. doi:10.1158/1538-7445.AM2013-3887