ROR2/Wnt5a Signaling Regulates Directional Cell Migration and Early Tumor Cell Invasion in Ovarian Cancer.

Adhesion to and clearance of the mesothelial monolayer are key early events in metastatic seeding of ovarian cancer. ROR2 is a receptor tyrosine kinase that interacts with Wnt5a ligand to activate noncanonical Wnt signaling and has been previously shown to be upregulated in ovarian cancer tissue. However, no prior study has evaluated the mechanistic role of ROR2 in ovarian cancer. Through a cellular high-throughput genetic screen, we independently identified ROR2 as a driver of ovarian tumor cell adhesion and invasion. ROR2 expression in ovarian tumor cells serves to drive directed cell migration preferentially toward areas of high Wnt5a ligand, such as the mesothelial lined omentum. In addition, ROR2 promotes ovarian tumor cell adhesion and clearance of a mesothelial monolayer. Depletion of ROR2, in tumor cells, reduces metastatic tumor burden in a syngeneic model of ovarian cancer. These findings support the role of ROR2 in ovarian tumor cells as a critical factor contributing to the early steps of metastasis. Therapeutic targeting of the ROR2/Wnt5a signaling axis could provide a means of improving treatment for patients with advanced ovarian cancer. IMPLICATIONS: This study demonstrates that ROR2 in ovarian cancer cells is important for directed migration to the metastatic niche and provides a potential signaling axis of interest for therapeutic targeting in ovarian cancer.

Stromal DDR2 Promotes Ovarian Cancer Metastasis through Regulation of Metabolism and Secretion of Extracellular Matrix Proteins.

Ovarian cancer is the leading cause of gynecologic cancer-related deaths. The propensity for metastasis within the peritoneal cavity is a driving factor for the poor outcomes associated with this disease, but there is currently no effective therapy targeting metastasis. In this study, we investigate the contribution of stromal cells to ovarian cancer metastasis and identify normal stromal cell expression of the collagen receptor, discoidin domain receptor 2 (DDR2), that acts to facilitate ovarian cancer metastasis. In vivo, global genetic inactivation of Ddr2 impairs the ability of Ddr2-expressing syngeneic ovarian cancer cells to spread throughout the peritoneal cavity. Specifically, DDR2 expression in mesothelial cells lining the peritoneal cavity facilitates tumor cell attachment and clearance. Subsequently, omentum fibroblast expression of DDR2 promotes tumor cell invasion. Mechanistically, we find DDR2-expressing fibroblasts are more energetically active, such that DDR2 regulates glycolysis through AKT/SNAI1 leading to suppressed fructose-1,6-bisphosphatase and increased hexokinase activity, a key glycolytic enzyme. Upon inhibition of DDR2, we find decreased protein synthesis and secretion. Consequently, when DDR2 is inhibited, there is reduction in secreted extracellular matrix proteins important for metastasis. Specifically, we find that fibroblast DDR2 inhibition leads to decreased secretion of the collagen crosslinker, LOXL2. Adding back LOXL2 to DDR2 deficient fibroblasts rescues the ability of tumor cells to invade. Overall, our results suggest that stromal cell expression of DDR2 is an important mediator of ovarian cancer metastasis. IMPLICATIONS: DDR2 is highly expressed by stromal cells in ovarian cancer that can mediate metastasis and is a potential therapeutic target in ovarian cancer.

Detection of a somatic GREB1-NCOA1 gene fusion in a uterine tumor resembling ovarian sex cord tumor (UTROSCT).

Uterine tumor resembling ovarian sex cord tumor (UTROSCT) is a rare uterine neoplasm of uncertain malignant potential. We present the case of a 69-year-old woman who underwent hysterectomy for postmenopausal bleeding and was found to have a myometrial UTROSCT. RNA-sequencing identified a somatic GREB1-NCOA1 fusion, supporting the diagnosis. Next-generation sequencing is increasingly being adopted in diagnostic pathology laboratories. This report highlights the value of RNA-sequencing in identifying rare fusion events to support pathologic diagnoses.

DDR2 controls breast tumor stiffness and metastasis by regulating integrin mediated mechanotransduction in CAFs.

Biomechanical changes in the tumor microenvironment influence tumor progression and metastases. Collagen content and fiber organization within the tumor stroma are major contributors to biomechanical changes (e., tumor stiffness) and correlated with tumor aggressiveness and outcome. What signals and in what cells control collagen organization within the tumors, and how, is not fully understood. We show in mouse breast tumors that the action of the collagen receptor DDR2 in CAFs controls tumor stiffness by reorganizing collagen fibers specifically at the tumor-stromal boundary. These changes were associated with lung metastases. The action of DDR2 in mouse and human CAFs, and tumors in vivo, was found to influence mechanotransduction by controlling full collagen-binding integrin activation via Rap1-mediated Talin1 and Kindlin2 recruitment. The action of DDR2 in tumor CAFs is thus critical for remodeling collagen fibers at the tumor-stromal boundary to generate a physically permissive tumor microenvironment for tumor cell invasion and metastases.

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

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.

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.

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

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.

Comparison of experimental and computationally predicted sulfoxide bond dissociation enthalpies.

The accurate estimation of S-O bond dissociation enthalpies (BDE) of sulfoxides by computational chemistry methods has been a significant challenge. One of the primary causes for this challenge is the well-established requirement of including high-exponent d functions in the sulfur basis set for accurate energies. Unfortunately, even when high-exponent d functions were included in Pople-style basis sets, the relative strength of experimentally determined S-O BDE was incorrectly predicted. The aug-cc-pV(n+d)Z basis sets developed by Dunning include an additional high-exponent d function on sulfur. Thus, it was expected that the aug-cc-pV(n+d)Z basis sets would improve the prediction of sulfoxide S-O BDE. This study presents the S-O BDE predicted by B3LYP, CCSD, CCSD(T), M05-2X, M06-2X, and MP2 combined with aug-cc-pV(n+d)Z, aug-cc-pVnZ, and Pople-style basis sets. The accuracy of these predictions was determined by comparing the computationally predicted values to the experimentally determined S-O BDE. Values within experimental error were obtained for dialkyl sulfoxides when the S-O BDEs were estimated using an isodesmic oxygen transfer reaction at the M06-2X/aug-cc-pV(T+d)Z level of theory. However, the S-O BDE of divinyl sulfoxide was overestimated by this method.

Computational investigation of the reaction mechanisms of nitroxyl and thiols.

Nitroxyl, or nitrosyl hydride, (HNO) is a pharmacologically relevant molecule whose physiological responses have been thought to result from modification of intracellular thiols. The reaction of HNO with thiols has been shown to lead to disulfides and sulfinamides. The free energies of reaction (DeltaG) and activation (DeltaG(++)) were determined for the reaction pathways of HNO and five different thiols using computational methods. The methods employed included B3LYP, MP2, and CBS-QB3, as well as IEF-PCM to approximate implicit water solvation. The five examined thiols were hydrogen sulfide, methanethiol, trifluoromethanethiol, thiophenol, and cysteine. A putative N-hydroxysulfenamide intermediate was the initial product for the reaction of HNO with a thiol. Analysis of the Wiberg bond indices indicated that the formation of the S-N bond was concerted with the proton transfers that led to the intermediate. The calculated pK(a) of protonated N-hydroxysulfenamide was approximately 13, and from the protonated N-hydroxysulfenamide intermediate, two irreversible reactions that lead to either the disulfide or sulfinamide were found. The calculated values of DeltaG(++) indicated the preferred reaction pathway would be dependent upon the hydrophobicity of the environment, the availability of a local base, and the identity of the thiol substituent. In a hydrophobic environment, the formation of the disulfide was kinetically favored. Formation of the sulfinamide product was expected to occur upon the protonation of the hydroxy group of the N-hydroxysulfenamide intermediate.

Photodeoxygenation of dibenzothiophene S-oxide derivatives in aqueous media.

The use of atomic oxygen (O((3)P)) as potent oxidant in water has suffered from the lack of a facile, efficient source. The photodeoxygenation of aromatic sulfoxides to the corresponding sulfides in organic solvents has been suggested to produce O((3)P) in low quantum yields. The photolysis of 4,6-dihydroxymethyldibenzothiophene S-oxide and 2,8-dihydroxymethyldibenzothiophene S-oxide in water results in deoxygenation at significantly higher quantum yields than in organic solvents. Depending upon conditions, a variable amount of oxidation of the hydroxymethyl substituent into an aldehyde was observed to accompany deoxygenation. Analysis of the photoproducts indicated the deoxygenation occurred by at least two different pH-sensitive mechanisms. Under basic conditions, photoinduced electron transfer yielding a hydroxysulfuranyl radical that decomposed by heterolytic S-O cleavage was thermodynamically feasible. The thermodynamics of photoinduced electron transfer were expected to become increasingly unfavorable as the pH of the solution decreased. Thus, at neutral and acidic pH, an S-O bond scission mechanism was suspected. The observed increase in the photodeoxygenation quantum yields was consistent with charge separation accompanying S-O bond scission. Oxidative cleavage of alkenes in aerobic conditions suggested O((3)P) was produced during photolysis in these conditions; however, the formation of discrete O(*-)/HO(*) may occur, particularly at low pH.