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Elucidating Purinergic and Pro-inflammatory Signaling Mechanisms in Breast Cancer
AdvisorBuxton, Iain L.O.
Biochemistry and Molecular Biology
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Integration of cancer cell-intrinsic and extrinsic signaling cues supports the emergence of tissue-specific microenvironments that stimulate neoplastic transformation and malignancy. Tumor-acquired hallmarks that maintain functional plasticity frequently result from intrinsic genomic aberrations leading to transcriptional dysregulation. Concomitantly, tumor-stromal interactions promote the accumulation of stimulatory factors that augment the wound healing response, inflammation, and metastatic dissemination. While the convergence of these signaling compartments is known to preface malignancy, the molecular mechanisms that mediate their functional effects remain enigmatic.Presented herein, Chapter One offers context to subsequent investigations into breast cancer cell-intrinsic regulation of inflammation and tumor-extrinsic communication with distant sites of metastasis. Chapter Two begins with an original characterization of breast cancer-secreted exosomes and employs a proteomic approach to identify unique protein cargo associated with malignancy. Application of an in vivo orthotopic breast tumor model and an alginate-based cell encapsulation method confirms spatiotemporal exosome signaling within the lung microenvironment. Further, mass spectrometry analysis of lungs from exosome-treated mice suggests a role for breast cancer-secreted exosomes in facilitating pre-metastatic niche formation.Chapter Three expands on the previous findings by elucidating a purinergic signaling mechanism propagated by breast cancer-secreted exosomes. Evidence presented here shows that the expression and phosphotransferase activity of a pro-angiogenic nucleoside disphosphate kinase (NDPK) are markedly elevated in exosomes secreted by metastatic breast cancer cells. Moreover, these exosomes elicit pro-angiogenic and vascular remodeling events in vitro and in vivo. Inhibition of NDPK phosphotransferase activity or blockade of the endothelium-specific P2Y1 purinoreceptor attenuates exosome-induced endothelial cell migration, monolayer permeabilization, and experimental metastasis. A shift toward breast cancer-intrinsic regulation of NF-κB-mediated inflammation is presented in Chapter Four. Here, an original investigation into two regulators of the NF-κB signaling pathway elucidates context-specific interaction in non-transformed, hormone-responsive, and hormone-independent breast cancer cells. Epigenetic regulation of NF-κB by the histone methyltransferase EZH2 and reciprocal action by the long noncoding RNA NKILA determine cellular response to therapeutic inhibition of NF-κB and EZH2. Thus, this knowledge critically informs the delivery of NF-κB-modulating adjuvants in breast cancer treatment. Finally, these findings and their associated study limitations are discussed in a broader clinical context in Chapter Five.