Discoidin Domain Receptor 2 Expression Research Articles

DDR2 signaling and mechanosensing orchestrate neuroblastoma cell fate through different transcriptome mechanisms.

The extracellular matrix (ECM) regulates carcinogenesis by interacting with cancer cells via cell surface receptors. Discoidin Domain Receptor 2 (DDR2) is a collagen-activated receptor implicated in cell survival, growth, and differentiation. Dysregulated DDR2 expression has been identified in various cancer types, making it as a promising therapeutic target. Additionally, cancer cells exhibit mechanosensing abilities, detecting changes in ECM stiffness, which is particularly important for carcinogenesis given the observed ECM stiffening in numerous cancer types. Despite these, whether collagen-activated DDR2 signaling and ECM stiffness-induced mechanosensing exert similar effects on cancer cell behavior and whether they operate through analogous mechanisms remain elusive. To address these questions, we performed bulk RNA sequencing (RNA-seq) on human SH-SY5Y neuroblastoma cells cultured on collagen-coated substrates. Our results show that DDR2 downregulation induces significant changes in the cell transcriptome, with changes in expression of 15% of the genome, specifically affecting the genes associated with cell division and differentiation. We validated the RNA-seq results by showing that DDR2 knockdown redirects the cell fate from proliferation to senescence. Like DDR2 knockdown, increasing substrate stiffness diminishes cell proliferation. Surprisingly, RNA-seq indicates that substrate stiffness has no detectable effect on the transcriptome. Furthermore, DDR2 knockdown influences cellular responses to substrate stiffness changes, highlighting a crosstalk between these two ECM-induced signaling pathways. Based on our results, we propose that the ECM could activate DDR2 signaling and mechanosensing in cancer cells to orchestrate their cell fate through distinct mechanisms, with or without involving gene expression, thus providing novel mechanistic insights into cancer progression.

Abstract A075: Stromal cell DDR2 promotes ovarian cancer metastasis

Abstract Ovarian cancer is the leading cause of gynecological 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. Therefore, we wanted to further investigate how various DDR2-expressing stromal cells may individually be contributing to ovarian cancer metastasis. First, we investigated mesothelial cells, as these are one of the first cell types that ovarian cancer cells interact with during metastasis. We find that DDR2 expression in mesothelial cells lining the peritoneal cavity facilitates tumor cell attachment and clearance. To examine the contribution of DDR2 expression in mesothelial cells in vivo, we have generated and validated a mouse model in which Ddr2 is specifically knocked out in mesothelial cells. We have ongoing experiments to determine whether inhibition of DDR2 in just mesothelial cells decreases ovarian tumor cell attachment and tumor burden in a syngeneic model. Following interaction with mesothelial cells, metastatic tumor cells contact fibroblasts. We find that omentum fibroblast expression of DDR2 promotes tumor cell invasion. Mechanistically, 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 in fibroblasts, 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. Citation Format: Angela Schab, Molly Greenwade, Elizabeth Stock, Elena Lomonosova, Kevin Cho, Whitney Grither, Hollie Noia, Daniel Wilke, Zainab Ibitoye, Mary Mullen, Andrea Hagemann, Ian Hagemann, Premal Thaker, Lindsay Kuroki, Carolyn McCourt, Dineo Khabele, Matthew Powell, David Mutch, Peinan Zhao, Leah Shriver, Gary Patti, Gregory Longmore, Katherine Fuh. Stromal cell DDR2 promotes ovarian cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A075.

Decoding the non‐canonical role of Discoidin Domain Receptor 2 and its plausible interaction with Amyloid beta in Alzheimer’s Disease

AbstractBackgroundReceptor tyrosine kinases (RTK) that perform versatile roles in the disease landscape have been extensively studied in proliferative signaling, but their role in degeneration remains quite unexplored. Interestingly several of these RTKs are perturbed in Alzheimer’s Disease (AD), of which Discoidin Domain Receptor 2 (DDR2) is an interesting candidate. DDR2 belongs to a unique set of RTKs that act as sensors of the Extracellular matrix (ECM) and mediate downstream signaling by binding to its ligand collagen.MethodWe investigated the expression and activity of DDR2 in our AD‐like cell model. We also looked for the expression of collagen levels which are well‐established ligands of DDR2. Docking studies were performed between Amyloid beta and DDR2 ectodomain to predict interactions between the two and some important residues were screened which are further being validated by site‐directed mutagenesis. We also performed Immunocytochemistry studies to investigate the localization of DDR2 under disease conditions.ResultThere is a significant upregulation in the DDR2 expression and activity as observed from western blot analysis and qRT PCR. There were no significant changes observed in the mRNA expression level of collagens in the control versus AD scenario. From the Molecular Docking studies between DDR2 and Aβ, we chose the best‐fitted model and screened a few interacting residues in the protein‐protein interface which were validated by site‐directed mutagenesis. Next, from Immunocytochemistry studies, we observed that DDR2 is naturally localized inside the nucleus of neuroblastoma cells and also present in the nucleus in AD‐like scenarios.ConclusionHeightened expression and activity of DDR2 in the absence of collagen, possibly in the presence of Aβ, results in its dysregulated trans‐compartmental distribution across the nucleocytoplasmic interfaces to commence AD‐like pathology. This study overall depicts a preliminary picture of DDR2 playing an essential role in AD and sheds light on its non‐canonical mode of signaling triggered by Aβ which needs further experimental validation.

Metformin Attenuates Hyperglycaemia-Stimulated Pro-Fibrotic Gene Expression in Adventitial Fibroblasts via Inhibition of Discoidin Domain Receptor 2.

Molecular mechanisms underlying the diverse therapeutic effects of anti-diabetic metformin, beyond its anti-hyperglycaemic effects, remain largely unclear. Metformin is reported to reduce the long-term complications of diabetes, including cardiovascular fibrosis and remodelling. Our recent investigations show that Discoidin Domain Receptor 2 (DDR2), a Collagen receptor tyrosine kinase, has an obligate regulatory role in Collagen type I gene expression in cardiac and vascular adventitial fibroblasts, and that it may be a molecular link between arterial fibrosis and metabolic syndrome in rhesus monkeys. Using gene knockdown and overexpression approaches, the present study examined whether DDR2 is a target of metformin and whether, by targeting DDR2, it inhibits Fibronectin and Collagen type I expression in rat aortic adventitial fibroblasts exposed to hyperglycaemic conditions. Metformin was found to attenuate hyperglycaemia-induced increase in DDR2 mRNA and protein expression by inhibiting TGF-β1/SMAD2/3 signalling that mediates the stimulatory effect of hyperglycaemia on DDR2 expression. Metformin also inhibited DDR2-dependent expression of Fibronectin and Collagen type I, indicating that it regulates these matrix proteins via DDR2 inhibition. The findings identify DDR2, a mediator of cardiovascular remodelling, as a molecular target of metformin, thereby uncovering the molecular basis of its protective role in vascular fibrosis and possibly cardiac fibrosis associated with diabetic cardiomyopathy.

Expression of CILP-2 and DDR2 and ultrastructural changes in the articular cartilage of patients with knee osteoarthritis undergoing total knee arthroplasty: a pilot morphological study.

The aim of the study was to correlate the immunohistochemical expression of cartilage intermediate layer protein 2 (CILP-2) and discoidin domain receptor 2 (DDR2), and the ultrastructural changes in the cartilage with the degree of articular cartilage damage in osteoarthritis (OA) patients. Cartilage samples were obtained from twenty patients aged from 46 to 68years undergoing total knee arthroplasty. In each patient, medial and lateral tibial plateau samples were analysed applying OARSI histopathology grading. Positive correlation was noted between the extent of CILP-2 staining intensity and OARSI grades. Abundant staining for CILP-2 was found in the superficial and middle layers and in the pericellular matrix (PCM) of the deep zone. Transmission electron microscopy studies demonstrated strong damage of chondrocytes, the organelles were often diminished or focally aggregated. As a characteristic finding, PCM was frequently expanded, which may reflect a pathogenic step in OA progression. In conclusion, CILP-2 may potentially be a relevant marker of OA progression as its expression correlated better with cartilage damage than the known marker of articular cartilage damage, DDR2.

Special issue "The advance of solid tumor research in China": Discoidin domain receptor 2 promotes colorectal cancer metastasis by regulating epithelial mesenchymal transition via activating AKT signaling.

Tumor metastasis is one of the main reasons for the high mortality rate associated with colorectal cancer (CRC). However, its underlying mechanisms have not been fully understood. Here, we reported that the expression of discoidin domain receptor 2 (DDR2) was significantly upregulated in CRC tissues compared to that in normal adjacent tissues. The expression level of DDR2 was negatively associated with prognosis of CRC patients. Therefore, DDR2 may play an oncogenic role in CRC development. Furthermore, DDR2 induced epithelial mesenchymal transition in CRC cells and regulated their invasive and metastatic capacity in vitro and in vivo. Mechanistically, increased DDR2 expression level activated the AKT/GSK-3β/Slug signaling pathway. In conclusion, these findings showed that DDR2 promoted CRC metastasis and DDR2 inhibition might represent an effective therapeutic strategy for local advanced and metastatic CRC treatment.

Adipose-derived stem cells-derived exosomes facilitate cutaneous wound healing by delivering XIST and restoring discoidin domain receptor 2

BackgroundAdipose-derived stem cells (ADSCs) and their derived exosomes (ADSC-Exos) have shown potential functions in tissue repair. This study focuses on the effects of ADSCs-Exos on cutaneous wound healing and the potential involvement of the long non-coding RNA (lncRNA) XIST/microRNA-96-5p (miR-96-5p)/discoidin domain receptor 2 (DDR2) axis. MethodsExos were isolated from the ADSCs and identified. A mouse model of full-thickness skin wounds was established. The mice were treated with ADSC-Exos to evaluate the function of ADSC-Exos in wound healing. Mouse dermal fibroblasts (MDFs) were co-cultured with the ADSC-Exos for in vitro experiments. The most differentially expressed lncRNAs in mouse skin tissues after ADSC-Exo treatment were screened by microarray analysis. The downstream molecules were analyzed by bioinformatics tools. Gain- and loss-of-function studies were performed to examine the functions of the XIST/miR-96-5p/DDR2 axis in wound healing. ResultsADSC-Exos facilitated wound healing in mice, reduced inflammatory infiltration, and increased collagen deposition in the wound skin tissues. In vitro, the ADSC-Exos promoted proliferation, migration of the MDFs. XIST was the most upregulated lncRNA in MDFs after ADSC-Exo treatment. Downregulation of XIST suppressed the promoting role of ADSC-Exos in wound healing. XIST bound to miR-96-5p to restore the expression of DDR2 mRNA. Either silencing of miR-96-5p or overexpression of DDR2 restored the promoting functions of ADSC-Exos in proliferation and migration of MDFs. ConclusionThis study demonstrates that ADSC-Exos-carried XIST accelerates cutaneous wound healing through suppressing miR-96-5p and restoring the DDR2 expression.

Pharmacologic inhibition of discoidin domain Receptor 2 (DDR2) sensitizes homologous recombination proficient ovarian cancer models to treatment with olaparib (245)

Pharmacologic inhibition of discoidin domain Receptor 2 (DDR2) sensitizes homologous recombination proficient ovarian cancer models to treatment with olaparib (245)

Abstract 3235: The role of DDR2 in chemoresistant ovarian cancer

Abstract Ovarian cancer is the fifth leading cause of cancer death for women in the United States. As many as 80% of ovarian cancer patients experience recurrent disease and the majority of recurrences become resistant to treatment. Once patients no longer respond to chemotherapy, there are minimal treatment options and they succumb to their disease. Therefore, there is a need to create new targeted therapies to overcome chemoresistance in ovarian cancer and improve patient survival. We have assembled a tumor microarray from 164 patient tumors to characterize the changes in protein expression that lead to resistance in clinical samples. We have found using IHC staining, that patients with high tyrosine kinase receptor discoidin domain receptor 2 (DDR2) protein expression (>70%) have significantly shorter overall survival times regardless of stage at diagnosis (HR= 2.108, p=0.002) as well as reduced platinum free interval (p=0.046). Additionally, platinum refractory tumors have higher DDR2 protein expression than platinum sensitive tumors (p=0.036). To further understand the mechanisms of chemoresistance, we created a novel, chemoresistant, syngeneic, mouse ovarian cancer cell line derived from ID8 trp53-/-BRCA2-/- GFP LUC cells from Walton et. al 2016, and have designated them CHRP5. DDR2 expression assessed via western blot increased significantly in the chemoresistance cells, suggesting that DDR2 is associated with chemoresistance. We knocked down DDR2 using a stable shRNA transfection in CHRP5 and used MTS survival assays to determine if decreased DDR2 expression increases sensitivity to chemotherapy. CHRP5 shDDR2 cells were significantly more sensitive to carboplatin than CHRP5 shControl cells (CHRP5 shControl IC50=213.6uM, CHRP5 shDDR2 IC50=128.5uM, p<0.000001), suggesting that DDR2 is involved in regulating chemoresistance in these cells. Additionally, we have submitted matched chemoresistant and chemosensitive mouse tumor samples for RNA-sequencing. We will use this data to determine which chemoresistant mechanisms are utilized, and if DDR2 is involved in regulating those mechanisms. In the future, we plan to rescue DDR2 in the knockdown cells to confirm that off target effects are not influencing the change in sensitivity. We also have access to a specific, allosteric inhibitor of DDR2 and plan to use this drug to determine if inhibiting DDR2 pharmacologically can overcome chemoresistance and reduce tumor burden in vivo and in vitro. In conclusion, DDR2 is more highly expressed in chemoresistant patients, and higher DDR2 expression correlates with reduced survival and platinum free interval. Additionally, knocking down DDR2 in chemoresistant cells increases sensitivity to chemotherapy. Therefore, DDR2 is a potential new target for ovarian cancer therapy and may be targeted to overcome chemoresistant disease and extend patient survival. Citation Format: Alyssa Oplt, Elizabeth Stock, Hollie Noia, Patrick Cannon, Gregory Longmore, Katherine C. Fuh. The role of DDR2 in chemoresistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3235.

Abstract 3190: Investigating tumor-mesothelial cell interactions during ovarian cancer metastasis

Abstract Introduction: Ovarian cancer is the leading cause of gynecological cancer-related deaths. The propensity for metastasis is a driving factor for the poor outcomes associated with this disease, but there is currently no effective therapy targeting metastasis. Work from our lab has demonstrated that stromal cell expression of the receptor tyrosine kinase Discoidin Domain Receptor 2 (DDR2) promotes ovarian cancer metastasis. One stromal compartment that expresses DDR2 is the mesothelial cell layer that surrounds the organs of the peritoneum - the most common location of ovarian cancer metastasis. Although epithelial in origin, mesothelial cells can adopt mesenchymal properties during metastasis and become active participants in the process. Therefore, we hypothesize that mesothelial cell expression of DDR2 promotes stabilization of a mesenchymal phenotype in mesothelial cells, which facilitates ovarian cancer metastasis. Methods: Human peritoneal mesothelial cells (HPMCs) were isolated from patient omentum samples. HPMCs were immortalized using lentivirus expressing SV40 large T antigen followed by short hairpin control and DDR2 knockdown transfections. qPCR was used to assess transcription and immunoblotting was used to assess protein expression. ELISAs were completed to observe changes in MMP-2 secretion. C57BL6/J mice are used for in vivo studies. Results: In vitro we have found that DDR2-expressing human peritoneal mesothelial cells promote tumor cell clearance through the mesothelial cell layer. Further, mesothelial cell DDR2 increases mesenchymal-associated gene transcription and signaling, including the mesenchymal associated transcription factor SNAI1 and collagen 1. Mesothelial cell DDR2 expression also leads to increased secretion of the protease MMP-2, which is known to promote metastasis. To determine whether mesothelial cell DDR2 expression contributes to metastasis, we are creating a mesothelial cell-specific DDR2 knockout mouse and control mouse. To do so, we are breeding WT1CreERT2/+ mice as a mesothelial cell specific driver of Cre recombination with DDR2 floxed mice to knockout DDR2 as a result of Cre recombination, and Ai14 mice as a Cre reporter as they express tdTomato only in the presence of Cre. After validation of the model, syngeneic ovarian cancer cells will be intraperitoneally injected and tumor burden, ECM structure, and signaling changes will be compared between knockout and control mice. Conclusions: This work will increase our understanding of mesothelial cells’ contribution to metastasis with the possibility of identifying inhibition of DDR2 as a therapeutic target for ovarian cancer patients. Citation Format: Angela Schab, Elena Lomonosova, Isaac Cooper, Stephanie Teng, Zainab Ibitoye, Danny Wilke, Hollie Noia, Maggie Mullen, Gregory Longmore, Katherine Fuh. Investigating tumor-mesothelial cell interactions during ovarian cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3190.

Discoidin Domain Receptor 2 Expression as Worse Prognostic Marker in Invasive Breast Cancer.

Discoidin domain receptor 2 (DDR2) is arising as a promising therapeutic target in breast carcinoma (BC). The ability of DDR2 to bind to collagen promotes protumoral responses in cancer cells that influence the tumor microenvironment (TME). Nonetheless, the interrelation between DDR2 expression and TME modulation during BC progression remains poorly known. For this reason, we aim to evaluate the correlation between intratumoral expression of DDR2 and the infiltration of the main TME cell populations, cancer-associated fibroblasts (CAFs), and tumor-associated macrophages (TAMs). First, collagen and DDR2 expression levels were analyzed in human invasive BC samples. Then, DDR2 status correlation with tumor aggressiveness and patient survival were retrieved from different databases. Subsequently, the main pathways, cell types, and tissues correlated with DDR2 expression in BC were obtained through bioinformatics approach. Finally, we studied the association of DDR2 expression with the recruitment of CAFs and TAMs. Our findings showed that, together with the expected overexpression of TME markers, DDR2 was upregulated in tumor samples. Besides, we uncovered that altered TME markers were linked to DDR2 expression in invasive BC patients. Consequently, DDR2 modulates the stromal reaction through CAFs and TAMs infiltration and could be used as a potential worse prognostic factor in the treatment response of invasive BC.

Tyrosine kinase-independent actions of DDR2 in tumor cells and cancer-associated fibroblasts influence tumor invasion, migration and metastasis.

ABSTRACTBoth tumor cell-intrinsic signals and tumor cell-extrinsic signals from cells within the tumor microenvironment influence tumor cell dissemination and metastasis. The fibrillar collagen receptor tyrosine kinase (RTK) discoidin domain receptor 2 (DDR2) is essential for breast cancer metastasis in mouse models, and high expression of DDR2 in tumor and tumor stromal cells is strongly associated with poorer clinical outcomes. DDR2 tyrosine kinase activity has been hypothesized to be required for the metastatic activity of DDR2; however, inhibition of DDR2 tyrosine kinase activity, along with that of other RTKs, has failed to provide clinically relevant responses in metastatic patients. Here, we show that tyrosine kinase activity-independent action of DDR2 in tumor cells can support Matrigel invasion and in vivo metastasis. Paracrine actions of DDR2 in tumor cells and cancer-associated fibroblasts (CAFs) also support tumor invasion, migration and lung colonization in vivo. These data suggest that tyrosine kinase-independent functions of DDR2 could explain failures of tyrosine kinase inhibitor treatment in metastatic breast cancer patients and highlight the need for alternative therapeutic strategies that inhibit both tyrosine kinase-dependent and -independent actions of RTKs in the treatment of breast cancer.This article has an associated First Person interview with the first author of the paper.

Discoidin Domain Receptor 2 (DDR2) expression in combination with mutation status is predictive of survival in patients with ovarian cancer

<h3>Objectives:</h3> Dicoidin Domain Receptor 2 (DDR2) is a tyrosine kinase receptor which binds the most common extracellular matrix protein, fibrillar collagen type 1. Expression of DDR2 is critical for invasion and migration of ovarian cancer tumor cells. Clinically, high DDR2 expression has been shown to be associated with advanced stage disease and decreased survival. It is not known how <i>BRCA</i> mutation status, a known marker for treatment sensitivity, impacts this relationship between DDR2 expression and survival. This study aimed to determine whether DDR2 expression was correlated with <i>BRCA</i> mutation status and to quantify the effects of this relationship on survival. <h3>Methods:</h3> Immunohistochemical (IHC) analysis of DDR2 expression in two ovarian cancer tissue microarrays (TMA) was performed. DDR2 staining on the membrane of tumor cells was scored microscopically according to the intensity and percentage of cells positive for DDR2 expression compared to controls. High DDR2 staining was defined as >70% and low staining <69%. Clinical information, including germline mutational status and survival data, was abstracted from the electronic medical record. Statistical analysis was performed using descriptive statistics and unpaired t-tests. Survival analysis was performed using the Kaplan-Meier method with statistical significance determined using the log-rank test. <h3>Results:</h3> The cohort included 164 patients diagnosed between 2/1994 and 1/2017. The majority had advanced stage disease (79%) and high-grade serous histology (76%). Germline mutation status was known in 93 patients (57%). <i>BRCA1</i> was the most common mutation in 38 patients (64%) followed by <i>BRCA2</i> in 20 patients (34%). 34 patients had negative germline testing. DDR2 expression was significantly associated with mutational status. Patients without a germline mutation had significantly higher DDR2 expression (64%) compared to <i>BRCA1</i> (37%) (p=0.002) and <i>BRCA2</i> had significantly higher DDR2 expression (55%) compared to <i>BRCA1</i> (37%) (p=0.03). DDR2 expression did not vary by stage, grade or platinum sensitive status. In survival analysis, patients with high DDR2 expression had significantly lower overall survival (33mo vs 83mo, HR 1.94, p=0.002) and progression free survival (17mo vs 34mo, HR 1.668, p=0.04) compared to patients with low DDR2 expression. When survival analysis was stratified by mutation status and DDR2 expression, survival differences were seen. In patients without a germline mutation, high DDR2 expression was associated with worse overall survival (42mo vs 147mo, HR=2.818, p=0.04) compared to low DDR2 expression. <h3>Conclusions:</h3> Tumor DDR2 expression is correlated with mutation status and when combined, provides useful prognostic information. Studies are ongoing to identify additional <i>BRCA</i> deficient tumors for further survival analysis.

Co-expression of PD-L1 and DDR2 in cervical cancer

<h3>Objectives:</h3> Many cancers are dependent on programmed cell death ligand (PD-L1) to evade immune destruction. A clinical benefit of PD-1/PD-L1 inhibitors in cervical cancer has been established based on a combined positive score (CPS) of >1. However, cumulative evidence that the level of PD-L1 expression alone is not predictive of response to anti-PD1 axis therapy in cervical cancer suggests that identification of more complex molecular signatures are needed to select patients appropriate for immunotherapy. There is some indication that kinase pathway activation impacts anti-PD1 response, and recently discoidin domain receptor 2 (DDR2) was identified from an unbiased screen as a leading target for enhancement of response to anti-PD1 axis therapy. We hypothesized that DDR2 and PD-L1 co-expression occurs in some cervix cancers, and sought to determine the co-expression pattern of PD-L1 with DDR2 in patients with cervical cancer. <h3>Methods:</h3> A tissue microarray of pathologic specimens from treatment naïve cervical cancer patients was used for analysis of PD-L1 and DDR2 expression utilizing immunohistochemical (IHC) staining, with interpretable scores for 64 patients. A CPS was used to report PD-L1 expression. Positive PD-L1 staining was defined as CPS score ≥1. DDR2 expression was quantified by identifying percent positive staining pixels within the IHC samples and then categorized as low (0-20% positive), moderate (21-40% positive), or high (41-100% positive) expression. Correlation between PD-L1 expression and survival was calculated by the Kaplan-Meier method and log rank test. <h3>Results:</h3> Of the 64 patients, histology was as follows: 56 squamous, 5 adenocarcinoma, 1 adenoid cystic, 1 adenosquamous, 1 MMMT. Positive PD-L1 staining was observed in 58/64 samples. There was no independent association with PD-L1 positivity and recurrence or survival. Sixty-one of the samples were available for concurrent staining of DDR2. 17/61 samples demonstrated moderate DDR2 expression, and 44/61 samples had high DDR2 expression. Of the PD-L1 positive samples, all expressed at least moderate staining of DDR2, and 70% expressed high levels of DDR2. There was no significant correlation between CPS for PD-L1 and level of DDR2 expression. <h3>Conclusions:</h3> In this cohort of cervical cancer patients, the rate of PD-L1 and DDR2 expression was high. These data suggests that a subset of these patients may benefit from dual inhibition with anti-PD1 therapy and DDR2 inhibition, as this combination treatment has been shown to enhance anti-PD1 response in other tumor types. Further work to test this hypothesis in cervical cancer is needed.

Preventing ovarian cancer cell metastasis through inhibition of discoidin domain receptor 2 (DDR2) in the tumor microenvironment: a metabolomic analysis

Objectives: Discoidin domain receptor 2 (DDR2) is a tyrosine kinase activated by fibrillar collagen type 1. DDR2 expression in both cancer cells and stromal cells is critical for metastases in breast cancer. In ovarian cancer, DDR2 stabilizes Snail, a critical factor in the epithelial to mesenchymal transition, which facilitates cancer cell invasion and metastases. Snail has also been shown to reprogram glucose metabolism in breast cancer cells, thereby allowing cancer cell survival under metabolic stress. This study aimed to investigate how stromal DDR2 expression in normal omental and ovarian cancer associated fibroblasts (NOF and CAF) regulates tumor cell invasion by analyzing changes in stromal cell metabolomics. Methods: Matrigel invasion assays were performed using immortalized normal omental fibroblasts (NOF206) and ES2 ovarian cancer cells. NOF206s were transfected with two different siRNAs to produce transient siDDR2 knockdown. Lysates and conditioned media were collected of siControl (DDR2-expressing), Dharmacon siDDR2 and Sigma siDDR2 (DDR2 non-expressing) cells. Mass spectrometry was performed and metabolites were identified. Quantitative RT-PCR was performed to evaluate changes in the expression of mesenchymal markers based on DDR2 expression. Results: DDR2 expressing tumor cells invaded less when co-cultured with DDR2-non expressing NOFs compared to DDR2 expressing NOFs (51 vs 155 cells per hpf, p Conclusions: Genetic inactivation of DDR2 results in marked changes in the metabolism of normal omental fibroblasts resulting in decreased tumor cell invasion. These findings suggest a novel role of DDR2 in metabolism and stromal cell energy generation. The significance of these findings requires further study on the complex relationship between ovarian cancer and the stromal environment.

Increasing sensitivity to olaparib through inhibition of discoidin domain receptor 2 (DDR2) in homologous-recombination proficient ovarian cancer models

<h3>Objectives:</h3> Discoidin Domain Receptor 2 (DDR2) is a tyrosine kinase receptor which binds the most common extracellular matrix protein, fibrillar collagen type 1. DDR2 expression is critical for invasion and migration of ovarian cancer tumor cells, making DDR2 a potential target for new treatments. This study aimed to determine whether genetic inactivation of DDR2 in ovarian cancer cells would increase sensitivity to treatment with PARP inhibitor. <h3>Methods:</h3> Two ovarian cancer cells lines, ES2 and COV362, were used for <i>in vitro</i> assays. Stable hairpin control and DDR2 knockdowns (shSCRM and shDDR2) were used for ES2 and transient small interfering RNA knockdown of DDR2 (siControl and siDDR2) were used for COV362. Homologous recombination (HR) status was determined by quantification of RAD-51 foci relative to controls using immunofluorescence after irradiation. Cell viability and survival after treatment with olaparib was quantified using MTS assays and clonogenics. The DNA damage response after treatment with olaparib was analyzed using immunofluorescence. <i>In vivo</i> studies to evaluate the sensitivity to olaparib in the presence or absence of DDR2 expression were performed in nude mice after intraperitoneal injection with ES2shSCRM or shDDR2 cells. <h3>Results:</h3> ES2 and COV362 cells were found to have a 2-fold increase in RAD51 foci after irradiation indicating HR proficiency. DDR2 genetically inactivated cells (ES2shDDR2) were found to be more HR deficient than DDR2 expressing cells (ES2shSCRM) which remained HR proficient (0.80 vs 4.78 relative RAD51 foci per cell). We found that genetic inactivation of DDR2 in the ES2s and COV362s led to increased sensitivity to olaparib with an 80% (15uM vs 73uM) and 68% (62uM vs 195uM) reduction in IC50s, respectively, compared to DDR2 expressing cells. Additionally, ES2shDDR2 cells had decreased cell survival by clonogenic assay after treatment with olaparib compared to ES2shSCRM cells as measured by absorbance (0.64 vs 0.80, p=0.005). We found that DNA damage and the repair response was altered in DDR2 genetically inactivated cells treated with olaparib with an increase in gamma-H2AX (8 vs 6 foci per cell, p<0.0001) and 53BP1 foci (3 vs 1 foci per cell, p<0.0001) when compared to DDR2 expressing cells. In an intraperitoneal metastatic mouse model with the HR-proficient cell line ES2, we found that genetic inactivation of DDR2 led to decreased tumor burden when treated with olaparib compared to DDR2-expressing cells treated with olaparib (1499mm³ vs 3236mm³, p=0.04). <h3>Conclusions:</h3> DDR2 knockdown sensitizes HR proficient ovarian cancer cells to treatment with PARP inhibitor in both <i>in vitro</i> and <i>in vivo</i> models through a mechanism of induced HR deficiency and increased DNA damage. Future experiments will explore whether a DDR2 inhibitor can improve sensitivity to PARP inhibitor in HR proficient ovarian cancer models.

Tumor elastography and its association with cell-free tumor DNA in the plasma of breast tumor patients: a pilot study.

Breast tumor stiffness, which can be objectively and noninvasively evaluated by ultrasound elastography (UE), has been useful for the differentiation of benign and malignant breast lesions and the prediction of clinical outcomes. Liquid biopsy analyses, including cell-free tumor DNA (ctDNA), exhibit great potential for personalized treatment. This study aimed to investigate the correlations between the UE and ctDNA for early breast cancer diagnosis. Breast tumor stiffness in 10 patients were assessed by shear wave elastography (SWE), and the ctDNA of eight collected plasma specimens with different tumor stiffness were analyzed by whole-genome sequencing (WGS). Subsequently, the distribution of carcinoma-associated fibroblasts (CAFs) was investigated by detecting the expression levels of alpha-smooth muscle actin (α-SMA) in tissues of breast lesions. We validated the function of discoidin domain receptor 2 (DDR2) in breast tumor CAFs by knockout of fibroblast activation protein (FAP) with different tumor stiffness during cancer progression in vitro and vivo. The UE estimates of tumor stiffness positively correlated with CAF-rich (α-SMA+) tumors (P<0.05). Copy number profiles and percent genome alterations were remarkably different between benign and malignant breast lesions. Somatic genomic alterations or structural variants of DDR2, ANTXRL, TPSG1, and TPSB2 genes were identified in ctDNA of plasma from breast lesions with high SWE values and an increase in the CAF content obtained from clinical samples. Deletion of FAP in breast tumor CAFs by CRISPR/Cas9-mediated gene knockout and decreased tumor stiffness resulted in downregulated expression of DDR2 (P<0.05), which in turn led to decreasing the tumor stiffness and carcinogenesis process in vitro and in vivo. These results have established proof of principle that WGS analysis of ctDNA could complement current UE approaches to assess tumor stiffness changes for the early diagnosis and prognostic assessment of breast cancer.

Discoidin Domain Receptor 2 Mediates Lysophosphatidic Acid-Induced Ovarian Cancer Aggressiveness.

Lysophosphatidic acid (LPA), a bioactive lipid produced extracellularly by autotaxin (ATX), has been known to induce various pathophysiological events, including cancer cell invasion and metastasis. Discoidin domain receptor 2 (DDR2) expression is upregulated in ovarian cancer tissues, and is closely associated with poor clinical outcomes in ovarian cancer patients. In the present study, we determined a critical role and signaling cascade for the expression of DDR2 in LPA-induced ovarian cancer cell invasion. We also found ectopic expression of ATX or stimulation of ovarian cancer cells with LPA-induced DDR2 expression. However, the silencing of DDR2 expression significantly inhibited ATX- and LPA-induced ovarian cancer cell invasion. In addition, treatment of the cells with pharmacological inhibitors of phosphoinositide 3-kinase (PI3K), Akt, and mTOR abrogated LPA-induced DDR2 expression. Moreover, we observed that HIF-1α, located downstream of the mTOR, is implicated in LPA-induced DDR2 expression and ovarian cancer cell invasion. Finally, we provide evidence that LPA-induced HIF-1α expression mediates Twist1 expression to upregulate DDR2 expression. Collectively, the present study demonstrates that ATX, and thereby LPA, induces DDR2 expression through the activation of the PI3K/Akt/mTOR/HIF-1α/Twist1 signaling axes, aggravating ovarian cancer cell invasion.

Amplification of DDR2 mediates sorafenib resistance through NF-κB/c-Rel signaling in hepatocellular carcinoma.

Sorafenib was the first systemic therapy approved by the Food and Drug Administration to treat advanced hepatocellular carcinoma (HCC). However, sorafenib therapy is frequently accompanied by drug resistance. We aimed to explore the mechanisms of sorafenib resistance and provide feasible solutions to increase the response to sorafenib in patients with advanced HCC. The expression profile of discoidin domain receptor 2 (DDR2) in HCC tissues and cells was detected using quantitative real-time PCR (qPCR) and western blotting assays. The effects of DDR2 on sorafenib resistance were examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony formation, TdT-mediated dUTP nick end labeling, and flow cytometry assays. The effect of DDR2 on the nuclear factor kappa B (NF-κB) signaling pathway was evaluated by luciferase reporter, immunofluorescence, qPCR and flow cytometry assays. We demonstrated that DDR2 expression was dramatically upregulated in sorafenib-resistant HCC tissues relative to sensitive tissues. Downregulation of DDR2 sensitized HCC cell lines to sorafenib cytotoxicity. Further analysis showed that DDR2 could increase the nuclear location of REL proto-oncogene, a NF-κB subunit, to mediate NF-κB signaling. Blocking NF-κB signaling using the NF-κB signaling inhibitor, bardoxolone methyl, increased the response of HCC cells to sorafenib. Further analysis showed that DNA amplification of DDR2 is an important mechanism leading to DDR2 overexpression in HCC. Our results demonstrated that DDR2 is a potential therapeutic target in patients with HCC, and targeting DDR2 represents a promising approach to increase sorafenib sensitivity in patients with HCC.

Expression of epithelial-mesenchymal transition biomarkers: Discoidin domain receptor 2, Snail-1, and Ovol-2 as predictors of clinical outcome in patients with epithelial ovarian carcinoma.

Discoidin domain receptor 2 (DDR-2), which belongs to the receptor tyrosine kinase family, Snail-1, which is a member of zinc-finger transcription factor family, and Ovol-2, which is a member of Ovol family, are incriminated in epithelial-mesenchymal transition (EMT) during cancer progression. In the current study, we aim to clarify the extent to which EMT biomarkers, DDR-2, Snail-1, and Ovol-2 expression, are involved in the progression of EOC aiming at identification of novel markers for predicting the prognosis of EOC patients. This was a prospective cohort that was performed in the Faculty of Medicine, Zagazig University. We evaluated DDR-2, Snail-1, and Ovol-2 expression in 60 patients of EOC using immunohistochemistry. We followed our patients for about 36 months and analyzed the relationship between markers expression and the prognosis of patients. SPSS program (Statistical Package for the Social Sciences). High expression of both DDR-2 and Snail-1 was related to higher grade (P = 0.006) and advanced FIGO stage of the tumor (P < 0.001). Ovol-2 high expression was associated with lower grade of the tumor (P = 0.002) and early stage of the tumor (P < 0.001). High Ovol-2 and low DDR2 and Snail-1 expression were strongly correlated with better response to therapy (P = 0.003 and 0.005, respectively) and increased 3-year survival rates (P < 0.001). DDR-2 and Snail-1 are markers of poor prognosis in EOC while Ovol-2 is a marker of good prognosis.