Date of Award

5-1-2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Field of Study

Biomedical Sciences

Department

Graduate School of Biomedical Sciences

First Advisor

Jamboor K. Vishwanatha

Second Advisor

Abbot F. Clark

Third Advisor

Ignacy Gryczynski

Abstract

Early detection of cancer using circulating biomarkers is a realistic possibility with the discovery of exosomes. Cells under both physiological and pathological conditions secrete a wide array of membranous vesicles containing specific protein and RNA signatures. Exosomes, which are 40-100 nm in size, comprise a major portion of these vesicles. Exosomes play important roles in promoting tumor progression and metastasis, but the mechanisms by which they act are not yet understood. Annexin A2 (AnxA2) is a 36 kDa calcium dependent phospholipid-binding protein up-regulated in many cancer types that promotes tumorigenesis and angiogenesis. Although AnxA2 is highly expressed in exosomes, its function has never been characterized. In this study first we characterized exosomal AnxA2 (exo-AnxA2) expression in a breast cancer progression model. We found that exo-AnxA2 expression is significantly higher in malignant cells than normal and premetastatic cells. Next we explored the correlation and functionality of exo-AnxA2 in angiogenesis and breast cancer metastasis. In vitro and in vivo angiogenesis studies showed that exo-AnxA2 is an important mediator of angiogenesis and targeting the N-terminus of AnxA2 with a competitive hexapeptide greatly reduce the angiogenic effects induced by exo-AnxA2. To study the role of exo-AnxA2 in breast cancer metastasis we used MDA-MB-231 breast cancer cell line and its organ specific metastatic variants MDA-MB-831 (brain metastatic) and MDA-MB-4175 (lung metastatic) breast cancer cells. By using exosome priming in a mouse model, we show that priming with exosomes from metastatic breast cancer cells creates a favorable microenvironment for metastasis, and priming with exo-AnxA2 depleted exosomes leads to reduction of brain as well as lung metastasis. Detailed analysis of the signaling pathways revealed that p38MAPK/NF-κB and STAT3 pathways were up-regulated in the cancer exosomes primed animals than AnxA2 depleted exosome primed animals. These data demonstrate an important role for exo-AnxA2 in breast cancer pathogenesis. Finally, to validate whether exo-AnxA2 can be developed as s potential biomarker, we screened 50 breast cancer serum samples and 50 age matched control samples. Clinical analysis of the serum samples revealed that exo-AnxA2 is over expressed in breast cancer serum samples compared to normal samples. Detailed analysis of the exo-AnxA2 levels among breast cancer subtypes showed that more aggressive breast cancer subtype TNBC has higher exo-AnxA2 levels than HER2+ samples. In summary, our data suggest that exo-AnxA2 plays a major role in promoting angiogenesis and breast cancer metastasis. Further, breast cancer serum samples have higher exo-AnxA2 expression than control samples. Thus, exo-AnxA2 can be a potential diagnostic and prognostic marker in breast cancer patients to detect and monitor metastasis.

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