Date of Degree
PhD (Doctor of Philosophy)
Molecular Physiology and Biophysics
Michael D. Henry
The interplay between cancer cells and the extracellular matrix (ECM) remains a critical regulator of both normal tissue organization and cancer cell invasion. Proteins that function as ECM receptors function to link the cell with the ECM. Abberations in either the structure of the ECM or the expression of ECM receptors leads to disrupted interaction and downstream signaling effects. Dystroglyan (DG) is an ECM receptor that is expressed in a variety of tissue types and functions to mediate sarcolemma stability, epithelial polarity, and is critical in the early formation of basement membranes. However, DG has primarily been studied in muscle where loss of its function is linked to a host of muscular dystrophies. In the epithelium, the role of DG remains enigmatic. While DG has repeatedly been shown to lose function during cancer development and progression, the mechanism and functional consequence of its loss are currently unknown.
In order to increase our understanding of DG in cancer development, we analyzed its expression and glycosylation, a functional requirement for DG, in a range of prostate cancer cell lines. Previous work has shown DG to be downregulated in prostate cancer, but the mechanism by which this occurs has remained largely unclear. We found that DG expression is maintained while its glycosylation was heterogeneous in the cell lines. Further investigation revealed that lines with hypoglycosylated DG strongly associated with the loss of expression of the glycosyltransferase LARGE2. Further this enzyme is frequently downregulated in human cancers and appears to serve as a required enzyme in DG glycosylation within prostate epithelium. This is the first work to demonstrate the functional requirement of LARGE2 for DG, and the only work to implicate loss-of-function of LARGE2 in cancer progression.
To determine whether loss of LARGE2 is found in other tumor types, we analyzed human clear cell renal cell carcinoma (ccRCC) samples by immunohistochemistry and via in silico analysis with the Cancer Genome Atlas (TCGA). Our work demonstrated a frequent and significant downregulation of LARGE2 expression and its association with DG hypoglycosylation. Additionally, we found the loss of LARGE2 strongly associated with increased mortality. Thus, we again demonstrated a functional requirement of LARGE2 but also found a clinical correlate with increased mortality.
Finally, we examined the functional outcome of DG hypoglycosylation or loss of expression in both a mouse model of prostate cancer and a variety of cell lines models. We found that while loss of DG expression does not increase prostate cancer growth or metastasis in one model of cancer, loss of its glycosylation does seem to mediate downstream metabolic changes within cells. The mechanism for this change remains unclear.
In summary, these studies have contributed to our understanding of DG glycosylation and function in both prostate and renal carcinoma. Additionally, we have shown a novel mechanism by which DG glycosylation is lost with downregulation of LARGE2 expression. Finally, while we were unable to demonstrate a clear mechanism by which signaling changes arose, we were able to demonstrate a strong correlation between DG hypoglycosylation and increased mortality in ccRCC. These insights could be used to improve treatment of multiple cancer types as our understanding of DG function continues to improve.
Cancer remains a major cause of mortality even while healthcare quality continues to improve. While there are many reasons that tumors result in mortality, one of the most common is metastatic spread of the tumor and disruption of normal bodily processes. There are a number of ways by which tumors metastasize, but one of the earliest steps is interruption of the normal connections that exist between a cell and the surrounding acellular material, the extracellular matrix (ECM). One of the proteins that mediates this connection is dystroglycan (DG). DG sits on the cell surface and interacts with the ECM to keep the cell attached and in its designated space. When cancer causes cells to grow abnormally, the production and processing of DG is decreased such that it no longer functions to bind the cells to the ECM. Our work has focused on understanding the changes that lead to decreased DG function and the effects that has on tumor progression and patient prognosis. We found that one of thirteen proteins known to modify DG is frequently downregulated in advanced disease, and the loss of this protein, LARGE2, leads to a loss of DG function in prostate and renal cell carcinomas. Additionally, loss of LARGE2 leads to a significant reduction in overall survival of patients with renal cell carcinoma. While we were unable to identify the mechanism by which DG disruption leads to decreased survival, our work lays the groundwork for future experiments and possible therapeutics in cancer patients.
publicabstract, Dystroglycan, Glycosylation, LARGE2, Prostate Adenocarcinoma, Renal Cell Carcinoma
xii, 152 pages
Includes bibliographical references (pages 131-152).
Copyright 2015 Michael Miller