Date of Award

Spring 5-15-2019

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Biochemistry)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Discoidin Domain Receptor 2 (DDR2) is a receptor tyrosine kinase that utilizes the extracellular matrix protein collagen as its ligand. Recently, DDR2 was shown to be critical in facilitating breast cancer metastasis. DDR2 stabilizes protein levels of the epithelial-mesenchymal-transition (EMT) inducing transcription factor, SNAIL1 in basal K14+ epithelial cells that are known to be important for invasive leader cell properties during collective invasion/migration. DDR2 expression is present in a majority of human invasive ductal breast carcinomas, and expression is localized to the tumor-stroma boundary. Additionally, in genetic models of breast cancer, deletion of DDR2 within the stroma (MMTV-PyMT; DDR2 fl/fl; FSP-Cre) leads to a dramatic inhibition of tumor metastasis. This indicates that DDR2 also functions within the stromal compartment to facilitate metastasis, presumably through its function in cancer-associated fibroblasts (CAFs). In CAFs DDR2 is critical for fibrillar collagen and ECM production and the organization and architecture of collagen fibers. Additionally, paracrine factors from DDR2 expressing CAFs promote the invasion of surrounding tumor cells. Therefore, it appears that DDR2 engagement by collagen provides a means for both tumor and stromal cells to communicate with the surrounding environment to enhance metastatic potential.

Additionally, we have established the action of DDR2 as critical for metastasis of ovarian cancer. In both breast and ovarian tumor cells DDR2 expression, which is absent in normal epithelium, is induced during EMT. The mesenchymal gene program has been shown to promote the metastatic progression of ovarian cancer; however, specific proteins induced by this program that lead to these metastatic behaviors have not been identified. We found that the EMT transcription factor TWIST1 drives expression of DDR2 upon EMT induction. In ovarian cancer, the expression and action of DDR2 was critical for mesothelial cell clearance, invasion and migration in ovarian tumor cells. It does so, in part, by upregulating expression and activity of matrix remodeling enzymes that lead to increased cleavage of fibronectin and spreading of tumor cells. In vivo studies demonstrated that the presence of DDR2 promotes ovarian cancer metastasis. These findings indicate the critical importance of DDR2 for multiple steps of ovarian cancer progression to metastasis, and thus, identifies DDR2 as a potential new target for the treatment of metastatic ovarian cancer.

As such, DDR2 is a novel RTK target for the treatment of cancer metastasis, leading us to hypothesize that the development of small molecule inhibitors to target DDR2 function in both the tumor and stromal cells could provide a means of blunting cancer metastasis. Using an in vitro binding assay, followed by medicinal chemistry optimization of lead compounds, we identified potent and selective small molecule inhibitors of DDR2. The developed inhibitors have nanomolar potency at blocking DDR2 activation. Unlike traditional tyrosine kinase inhibitors (TKIs) that target the intracellular kinase domain, these inhibitors act on the extracellular domain (ECD) of DDR2, and disrupt DDR2 signaling by accelerating the disassembly of DDR2-collagen complexes, potentially by disrupting oligomerization of the receptor ECD. Therefore, these inhibitors have a unique mode of action and are able to attenuate DDR2 signaling both in cell culture, as well as in vivo as demonstrated by using a Snail1-clic beetle green reporter in a xenograft transplant model. In co-culture assays, treating CAFs with the developed inhibitors reduces the paracrine effect on invading tumor organoids. Furthermore, using mouse models of late stage breast tumor metastasis, we have shown that these inhibitors reduce metastatic burden in the lungs of mice, to a level comparable of that of genetic knockdown of DDR2. Together these data support further investigation of this novel class of DDR2 inhibitors as anti-metastasis agents, potentially for use in combination with standard of care therapy to halt cancer progression and prevent relapse. Further, it will be important to study the role of these inhibitors in other cancers where DDR2 expression has been shown to promote metastasis, including, but not limited to, ovarian, head and neck, and non-small cell lung carcinoma.

Language

English (en)

Chair and Committee

Gregory D. Longmore

Committee Members

Kendall Blumer, Andreas Herrlich, Robert Mecham, Kevin Moeller,

Comments

Permanent URL: https://doi.org/10.7936/w40v-d295

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