Whitney R. Grither

Mechanical signals regulate and activate SNAIL1 protein to control the fibrogenic response of cancer-associated fibroblasts.

ABSTRACT Increased deposition of collagen in extracellular matrix (ECM) leads to increased tissue stiffness and occurs in breast tumors. When present, this increases tumor invasion and metastasis. Precisely how this deposition is regulated and maintained in tumors is unclear. Much has been learnt about mechanical signal transduction in cells, but transcriptional responses and the pathophysiological consequences are just becoming appreciated. Here, we show that the SNAIL1 (also known as SNAI1) protein level increases and accumulates in nuclei of breast tumor cells and cancer-associated fibroblasts (CAFs) following exposure to stiff ECM in culture and in vivo. SNAIL1 is required for the fibrogenic response of CAFs when exposed to a stiff matrix. ECM stiffness induces ROCK activity, which stabilizes SNAIL1 protein indirectly by increasing intracellular tension, integrin clustering and integrin signaling to ERK2 (also known as MAPK1). Increased ERK2 activity leads to nuclear accumulation of SNAIL1, and, thus, avoidance of cytosolic proteasome degradation. SNAIL1 also influences the level and activity of YAP1 in CAFs exposed to a stiff matrix. This work describes a mechanism whereby increased tumor fibrosis can perpetuate activation of CAFs to sustain tumor fibrosis and promote tumor metastasis through regulation of SNAIL1 protein level and activity. Highlighted article: SNAIL1 is a mechanoresponsive regulator of the fibrogenic response of tumors. The work provides insight into the transcriptional response of tumors to their microenvironment and CAF activation to sustain tumor fibrogenesis.

The Action of Discoidin Domain Receptor 2 in Basal Tumor Cells and Stromal Cancer-Associated Fibroblasts Is Critical for Breast Cancer Metastasis

High levels of collagen deposition in human and mouse breast tumors are associated with poor outcome due to increased local invasion and distant metastases. Using a genetic approach, we show that, in mice, the action of the fibrillar collagen receptor discoidin domain receptor 2 (DDR2) in both tumor and tumor-stromal cells is critical for breast cancer metastasis yet does not affect primary tumor growth. In tumor cells, DDR2 in basal epithelial cells regulates the collective invasion of tumor organoids. In stromal cancer-associated fibroblasts (CAFs), DDR2 is critical for extracellular matrix production and the organization of collagen fibers. The action of DDR2 in CAFs also enhances tumor cell collective invasion through a pathway distinct from the tumor-cell-intrinsic function of DDR2. This work identifies DDR2 as a potential therapeutic target that controls breast cancer metastases through its action in both tumor cells and tumor-stromal cells at the primary tumor site.

Inhibition of tumor–microenvironment interaction and tumor invasion by small-molecule allosteric inhibitor of DDR2 extracellular domain

Significance To effectively prevent cancer spread from primary tumor sites, new treatments need to target tumor cells, the cells and extracellular matrix within the tumor environment, and communicating pathways between these sites simultaneously. The collagen receptor discoidin domain receptor 2 (DDR2) has been implicated as such a target. Here, we describe the identification and characterization of a small molecule inhibitor of DDR2 that uniquely acts in an allosteric manner via the extracellular domain to selectively inhibit the action of DDR2 in tumor cells and tumor stromal cancer-associated fibroblasts. In experimental mouse models of breast cancer, WRG-28 inhibits DDR2 signaling and tumor cell invasion. The action of the collagen binding receptor tyrosine kinase (RTK) discoidin domain receptor 2 (DDR2) in both tumor and tumor stromal cells has been established as critical for breast cancer metastasis. Small molecule inhibitors that target the extracellular domain of RTKs are rare, as they have classically been regarded as too small to block binding with large polypeptide ligands. Here, we report the identification and characterization of a selective, extracellularly acting small molecule inhibitor (WRG-28) of DDR2 that uniquely inhibits receptor–ligand interactions via allosteric modulation of the receptor. By targeting DDR2, WRG-28 inhibits tumor invasion and migration, as well as tumor-supporting roles of the stroma, and inhibits metastatic breast tumor cell colonization in the lungs. These findings represent an approach to inhibiting tumor–stromal interactions and support the development of allosteric inhibitors of DDR2, such as WRG-28, as a promising approach to antimetastasis treatment.

TWIST1 induces expression of discoidin domain receptor 2 to promote ovarian cancer metastasis

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. Using patient derived tumor cells and established human ovarian tumor cell lines, we find that the Epithelial-to-Mesenchymal Transition inducing factor TWIST1 drives expression of discoidin domain receptor 2 (DDR2), a receptor tyrosine kinase (RTK) that recognizes fibrillar collagen as ligand. 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. Additionally, DDR2 stabilizes SNAIL1, allowing for sustained mesenchymal phenotype. In patient derived ovarian cancer specimens, DDR2 expression correlated with enhanced invasiveness. DDR2 expression was associated with advanced stage ovarian tumors and metastases. In vivo studies demonstrated that the presence of DDR2 is critical for ovarian cancer metastasis. These findings indicate that the collagen receptor DDR2 is critical 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.

Discoidin Domain Receptors and Disease

The discoidin domain receptors have been implicated as contributing to normal developmental and pathologic conditions in humans. Mutations in the DDR genes have been described in a number of human diseases. Some of these are now being modeled in mice, which have afforded a better understanding for the cellular basis for the action of DDRs in various pathologic states. Herein, we first discuss the role(s) that DDR1 and DDR2 play in mouse development. Then we discuss pathologic conditions in humans where the action of DDRs has been associated with or correlated with pathologic and genomic analyses. Accumulated data indicate that the action of DDRs in various cell types have both cell-intrinsic roles as well as affecting the nature of the extracellular matrix produced or present in pathologic conditions.

Abstract PR01: Disruption of DDR2-collagen interactions in tumor and stroma with a novel small molecule inhibitor blunts cancer metastasis

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. In tumor cells DDR2 expression, which is absent in normal breast epithelium, is induced during Epithelial Mesenchymal transition (EMT), and serves to stabilize protein levels of the EMT inducing transcription factor, Snail1. DDR2 expression is present in a majority of human invasive ductal breast carcinomas, and expression is predominately localized to the tumor-stroma boundary. In these tumors DDR2 is acting in a positive feedback manner to maintain Snail1 levels and activity at the leading, invasive edge of the tumors, where cells have undergone EMT and come into contact with extracellular matrix (ECM) collagen I. This allows continued invasion through the ECM and contributes to breast cancer metastasis. Interestingly, in genetic models of breast cancer in mice, selectively eliminating DDR2 in either the tumor (MMTV-PyMT; DDR2 fl/fl; K14-Cre) or the fibroblasts within the stroma (MMTV-PyMT; DDR2 fl/fl; FSP-Cre) leads to a dramatic inhibition of tumor metastasis. This indicates that in addition to maintaining EMT in the tumor cells, DDR2 functions within the stromal compartment to facilitate metastasis as well. In culture, the matrix synthesized and remodeled by cancer associated fibroblasts (CAFs) is organized very differently than that of normal fibroblasts. When DDR2 is depleted from CAFS, the matrix more closely resembles that produced by normal fibroblasts, with less organization of matrix proteins. Therefore, it appears that DDR2 engagement by collagen provides a means for both tumor and stromal cells to enhance metastatic potential. As such, DDR2 is a novel RTK target for the treatment of breast cancer metastasis, and we have developed potent and selective small molecule inhibitors of DDR2. These compounds provide a means of blunting breast cancer metastasis by targeting the tumor and stromal cells, thereby disrupting DDR2 signaling in both compartments. The developed inhibitors have nanomolar cellular potency at blocking DDR2 activation. Unlike traditional tyrosine kinase inhibitors (TKIs) that target the intracellular kinase domain, the inhibitors that we have developed act on the extracellular domain of DDR2 and disrupt DDR2 signaling by accelerating the disassembly of the DDR2-collagen complex. Therefore these inhibitors have a unique mode of action that is distinct from the canonical classes of TKIs. These compounds have the ability 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 syngeneic transplant model. Treating CAFs with the developed inhibitors in culture reverts the organizational phenotype of the resulting matrix to that resembling the matrix produced by normal fibroblasts. 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 to 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 nonsmall cell lung carcinoma. This abstract is also presented as Poster B13. Citation Format: Whitney R. Grither, Gregory D. Longmore. Disruption of DDR2-collagen interactions in tumor and stroma with a novel small molecule inhibitor blunts cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR01.