ONCOLOGY REPORTS 13: 37-44, 2005 Carcinoma cell-specific Mig-7: A new potential marker for circulating and migrating cancer cells TROY M. PHILLIPS and J. SUZANNE LINDSEY Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA Received April 27,2004; Accepted June 17,2004 Abstract. Identification of genes that are expressed in a cancer cell-specific manner can provide markers for detection, diagnosis, and disease progression. We have previously reported that receptor tyrosine kinase ligands in concert with ligation of avl35 integrin induce expression of Mig-7 restricted to carcinoma cells. Because of this highly specific expression, we hypothesized that Mig-7 could be used as a marker of occult tumor cells. The objective of this study was to begin to test this hypothesis by generating Mig-7 specific antisera and RT-PCR methods for detection of Mig-7 expression in tissues and blood from cancer patients as compared to those from normal subjects. By immunohistochemistry and by RT- PCR, we detected Mig-7 mRNA in lymph nodes from 7 out of 9 (77.8%) endometrial carcinoma xenograft mice but not from any of the 5 negative control animals. Mig-7 expression was more specific than Met expression, the RTK that binds Scatter factor and is used as a marker of poor progression, in endometrial carcinoma as compared to normal endometrial tissue samples. In 87.3% of tumors from various tissues including breast, lung, colon and ovary, we detected Mig-7 expression. Blood samples from untreated metastatic cancer patients also displayed Mig-7 mRNA in contrast to a lack of expression in chemotherapy treated or normal individuals. In conclusion, we report the first immunohistochemical and RT- PCR assays for Mig-7 and discuss its highly specific localization to cancer cells in contrast to an absence in normal cells. Our preliminary data indicate that Mig-7 may be a potential early marker of migrating and circulating carcinoma cells. Introduction Existing markers, such as c-Met, cytokeratins 18, 19, 20, carcinoembryonic antigen and Mucin-1 that detect circulating Correspondence to: Dr J. Suzanne Lindsey, Present address: Dept. of Pharmaceutical Sciences, Wegner Hall, Rm 305A, Washinton State University, College of Pharmacy, Pullman, WA 99164-6534, USA E-mail: lindseys@wsu.edu Key words: carcinoma specific, occult tumor cells, biomarker, receptor tyrosine kinase, integrin carcinoma cells, also detect normal cells resulting in false- positive assays (1-3). In addition, tissue specific markers such as PSA, HE4, CA125 and tyrosinase, are limited to specific carcinomas and detect normal cells because these markers are not carcinoma-specific (1,4). Also, tissue specific markers and epithelial markers may be down-regulated in undifferentiated circulating and migrating cancer cells resulting in false negative assays (1). For example, cytokeratin has been shown to be replaced by vimentin in cells undergoing epithelial to mesenchyme transition, a result of receptor tyrosine kinase (RTK) activation (5). Thus, using cytokeratin as a marker could decrease or prevent detection of migrating and circulating carcinoma cells depending on their level of differentiation. Using a 'finger- print' of several genes expressed in metastatic cells is difficult to evaluate due to false-positives, false-negatives, complexity of the assay and resulting analyses. As an example, using a typical microarray that represents at least 10,000 genes, the expected number of false positive expressed genes with a 5% significance level is 500 (6). Development of molecular markers that are highly specific for invasive carcinoma cells in addition to a sensitive assay to detect occult tumor cells are, required for management of this disease. Recently, we reported a novel, carcinoma cell-specific cDNA called Mig-7, which is induced by an RTK ligand, hepatocyte growth factor also known as scatter factor (SF) in concert with avo5 integrin (avl35) signal transduction, before and during carcinoma cell migration (7). Tyrosine kinase receptor-induced cancer cell migration and dissemination requires avB5 signaling in vivo (8) and in vitro (9,lO). In addition, when Mig-7 expression is inhibited by Mig-7 specific antisense treatment in vitro, endometrial carcinoma cell migration is decreased by 82.21%+_3.18 (p<0.05) (7). The cysteine-rich Mig-7 protein is 22 kDa without the FLAG tag and is found predominantly in the membrane fraction (7). Expression of Mig-7 before and during migration (7) implies that it may be an early marker of carcinoma cells with invasive potential. In this study, we used a cancer profiling array, normal and cancer endometrial tissue samples, blood samples from cancer and normal individuals, and a xenograft nude mouse model of metastasis, to initiate the testing of the hypothesis that Mig-7 expression can be used as a biomarker for occult carcinoma cells.