Abstract P5-01-25: Potential prognostic value of exosomal Beta Protein 1 in breast cancer
Authors: Lou Yaoxian , Goldsmith Paul , Rheey Jinguen , Berg Patricia E. 
Published: March 2020

Abstract
Background: Beta protein 1 (BP1), discovered in our lab, is an isoform protein of the DLX4 gene that is a member of the homeobox gene family. Previous work showed BP1 mRNA was activated in 80% of invasive ductal breast (IDC) tumors, where 100% of estrogen receptor (ER) negative tumor tissues and 73% of ER positive tumor tissues were BP1 positive. Moreover, high BP1 levels were associated with tumor cell survival, breast cancer aggressiveness and metastasis. BP1 overexpression was found to stimulate known oncogenes including BCL2 and c-Myc. Exosomes are nano-sized membrane-bound vesicles released by various cells and play important roles in intercellular communication. In addition, researchers found that exosomal proteins have unique characteristics compared to traditional biomarkers for carcinoma diagnosis and prognosis. Therefore, we hypothesized that BP1 protein may be packed in exosomes, and analysis of exosomal BP1 protein could provide a novel biomarker for diagnosis or prognosis of breast cancer.
Materials and Methods: Exosomes were isolated using the commercially available Exosome Precipitation Solution. In cell line experiments, fluorescent immunohistochemistry was used to detect the location of BP1 protein. Levels of BP1 protein were determined in cell extracts (CE) and conditioned media (CM) using Western Blot analysis. The hypothesis that exosomal BP1 protein may be related to the development of breast cancer was investigated with clinical serum samples purchased from Capital Bioscience. Total exosomal protein levels in the serum were analyzed using BCA assay, and exosomal BP1 protein levels were determined using Western blot. In order to develop a method that could be easily used in the clinic, an ELISA assay was also designed to assess exosomal BP1 protein concentrations in the serum from metastatic breast cancer patients and normal controls.
Results: It was observed that BP1 protein is localized to the nucleus, the cytoplasm and the conditioned media of MCF-7 cells. In addition, experiments with cell lines showed that the secreted BP1 protein could be internalized by cells and exhibited mitogenic activity, which is related to cancer metastasis. Moreover, it was observed that exosomal BP1 is included in secreted BP1 protein in the conditioned media. Experiments with serum samples demonstrated that the total exosomal protein levels have no significant differences between that in breast cancer patients and in normal controls. However, the results of Western blot and ELISA both showed exosomal BP1 protein levels were significantly higher in breast cancer samples compared to normal controls (P &lt; 0.05). Moreover, Western blot results suggested that ER positive patients have much more (P &lt; 0.05) exosomal BP1 protein in the serum compared to ER negative patients.
Conclusion: It was shown for the first time that BP1 protein is exosomally packaged in breast cancer patients’ serum. The significant difference in the exosomal BP1 protein levels between the serum from women with metastatic breast cancer and that from normal controls indicated that exosomal BP1 protein in the serum could be a potential prognostic biomarker for breast cancer. Furthermore, the ELISA assay, designed to be easily used for exosomal BP1 protein quantification, may have many uses in the clinic through aiding in prognosis, monitoring therapy and early detection.
Citation Format: Yaoxian Lou, Paul Goldsmith, Jinguen Rheey, Patricia E. Berg. Potential prognostic value of exosomal Beta Protein 1 in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-25.