Early diagnostic value of survivin and its alternative splice variants in breast cancers.

Abstract

35Background: The inhibitor of apoptosis (IAP) protein Survivin and its splice variants are differentially expressed in breast cancer tissues and have recently been shown to be released from tumor cells via small membrane-bound vesicles called exosomes. Tumor-derived exosomes play multiple roles in tumor growth and metastasis and may produce these functions by impacting immune escape, tumor invasion and angiogenesis. We, therefore, hypothesize that analysis of exosomal Survivin and its splice variants may provide a novel biomarker for early diagnosis of breast cancer in addition to current recommended methods. Methods: Twenty paired breast cancer patient’s sera and tumor tissue, and ten normal control sera were used for analysis. ELISA was performed to quantitate serum levels of Survivin. Exosomes were isolated from the sera using Exoquick. RT-PCR, western blots with densitometry, and immunohistochemistry followed by confocal microscopy were then performed. Results: For each breast cancer patient serum, Survivin levels were significantly higher compared to control (p<0.05). While Survivin and the DEx3 splice variant expression and localization were similar, differential expression of Survivin and the 2B splice variant protein and mRNA existed in the exosomes and tissue samples. Survivin and -DEx3 proteins were the predominant forms detected in 100% (20/20) of the breast cancer tissues evaluated, whereas a more variable expression of Survivin-2B, with enhanced levels found in areas of necrosis. We also for the first time here show the exosomal localization of Survivin and its splice variants DEx3 and 2B in sera from breast cancer patients. Conclusions: The result of the proposed project supports our hypothesis that differential expression of exosomal-Survivin and its alternative splice variants may serve as a diagnostic marker in breast cancer patients. For future direction, we plan to study the prognostic value of exosomal-Survivin and its splice variants on a large panel of primary breast cancers within a setting of well-followed clinical outcomes.