Reduction of Raf-1 Kinase Inhibitor Protein (rkip) Expression in Breast Cancer Metastases

INTRODUCTION Raf-1 Kinase Inhibitor Protein (RKIP) is physiological endogenous inhibitor of the Raf-MEK-ERK pathway (1). Alterations in this pathway are associated with tumor development and progression. Recent work has shown that RKIP is diminished in some metastatic cancers. We studied lymph node negative cancers and node-positive patients, with paired lymph node metastases and show highly significant downregulation of RKIP in lymph node metastases (p<0.001), compared to matched primary tumors. No significant correlations were found between RKIP expression and established clinico-pathological prognostic indicators, suggesting that RKIP expression is an independent marker of breast cancer lymph node metastases. METHOD Paraffin-embedded tumors from 103 patients (52 node-negative and 51 node-positive patients, with 51 paired lymph node metastases) underwent immunohistochemical staining for RKIP using the streptavidin–biotin method and the chromogen 3, 3diaminobenzidine. Paraffin-embedded sections of prostate cancer served as positive RKIP controls and omission of primary antibody served as negative controls. RKIP expression levels were scored using the visual grading system. RESULTS All of the primary breast tumors analyzed in this study demonstrated RKIP immunoreactivity (except for one node-negative tumor), and the distribution of RKIP was found to be mainly cytoplasmic. RKIP expression was predominantly moderate in node-positive tumors (31/51 cases, 60.8%) and was weakly positive in 20/51 (39.2%) cases. By contrast, in the matched lymph node metastases RKIP expression was considerably diminished and, in some cases, was entirely absent (9/51, 17.7%), and this decrease was found to be highly significant (p=0.000003; Fig 1). In total, 30/51 (58.8%) of lymph node cases were weakly positive and 12/51 cases (23.5%) were moderately positive for RKIP expression. Of the node-negative tumors, RKIP expression was moderate in 37/52 cases (71.2%), weakly positive in 14/52 cases (26.9%) and negative in 1/52 cases (1.9%). A trend was observed for more intense RKIP staining of node-negative breast tumors than for node-positive tumors, but this was not found to be statistically significant.