Cancer enhances the risk of venous thromboembolism, but a hypercoagulant microenvironment also promotes cancer progression. Although anticoagulants have been suggested as a potential anticancer... Show moreCancer enhances the risk of venous thromboembolism, but a hypercoagulant microenvironment also promotes cancer progression. Although anticoagulants have been suggested as a potential anticancer treatment, clinical studies on the effect of such modalities on cancer progression have not yet been successful for unknown reasons. In normal physiology, complex formation between the subendothelial-expressed tissue factor (TF) and the blood-borne liver-derived factor VII (FVII) results in induction of the extrinsic coagulation cascade and intracellular signaling via protease-activated receptors (PARs). In cancer, TF is overexpressed and linked to poor prognosis. Here, we report that increased levels of FVII are also observed in breast cancer specimens and are associated with tumor progression and metastasis to the liver. In breast cancer cell lines, tumor-expressed FVII drives changes reminiscent of epithelial-to-mesenchymal transition (EMT), tumor cell invasion, and expression of the prometastatic genes, SNAI2 and SOX9. In vivo, tumorexpressed FVII enhanced tumor growth and liver metastasis. Surprisingly, liver-derived FVII appeared to inhibit metastasis. Finally, tumor-expressed FVII-induced prometastatic gene expression independent of TF but required a functional endothelial protein C receptor, whereas recombinant activated FVII acting via the canonical TF:PAR2 pathway inhibited prometastatic gene expression. Here, we propose that tumor-expressed FVII and liverderived FVII have opposing effects on EMT and metastasis. Show less
Liu, Y.M.; Paauwe, M.; Nixon, A.B.; Hawinkels, L.J.A.C. 2021
Approximately 30 years ago, endoglin was identified as a transforming growth factor (TGF)-beta coreceptor with a crucial role in developmental biology and tumor angiogenesis. Its selectively high... Show moreApproximately 30 years ago, endoglin was identified as a transforming growth factor (TGF)-beta coreceptor with a crucial role in developmental biology and tumor angiogenesis. Its selectively high expression on tumor vessels and its correlation with poor survival in cancer patients led to the exploration of endoglin as a therapeutic target for cancer. The endoglin neutralizing antibody TRC105 (Carotuximab(R), Tracon Pharmaceuticals (San Diego, CA, USA) was subsequently tested in a wide variety of preclinical cancer models before being tested in phase I-III clinical studies in cancer patients as both a monotherapy and in combination with other chemotherapeutic and anti-angiogenic therapies. The combined data of these studies have revealed new insights into the role of endoglin in angiogenesis and its expression and functional role on other cells in the tumor microenvironment. In this review, we will summarize the preclinical work, clinical trials and biomarker studies of TRC105 and explore what these studies have enabled us to learn and what questions remain unanswered. Show less
Schoonderwoerd, M.J.A.; Hakuno, S.K.; Sassen, M.; Kuhlennaijer, E.B.; Paauwe, M.; Slingerland, M.; ... ; Hawinkels, L.J.A.C. 2021
Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer and is known to have low immunogenicity and an immunosuppressive microenviron-ment. It is also... Show moreBackground: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer and is known to have low immunogenicity and an immunosuppressive microenviron-ment. It is also characterized by high accumulation of dense stroma, composed of mostly cancer-associated fibroblasts (CAFs). Multiple subsets of CAFs are described, with one of them expressing the transforming growth factor (TGF)-beta co-receptor endoglin. In previous work, we and others have shown that endoglin-expressing CAFs stimulate tumor progression and metastasis. Therefore, in this study, we set out to investigate the role of endoglin-expressing CAFs in pancreatic cancer progression. Methods: First, we investigated the expression of endoglin on CAFs in both human tissues as well as a mouse model for PDAC. Since CAF-specific endoglin expression was high, we targeted endoglin by using the endoglin neutralizing antibody TRC105 in the murine KPC model for PDAC. Results: Although some signs of immune activation were observed, TRC105 did not affect tumor growth. Since 90% of the CD8+ T-cells expressed the immune checkpoint PD-1, we investigated the combination with a PD1 checkpoint inhibitor, which did not enhance therapeutic responses. Finally, genetic deletion of endoglin from collagen 1a1 expressing cells also did not affect the growth of the mouse KPC tumors. Conclusion: Our results show that although endoglin is highly expressed on PDAC-CAFs and signaling is efficiently inhibited by TRC105, this does not result in decreased tumor growth in the KPC model for pancreatic cancer. Show less
Schoonderwoerd, M.J.A.; Koops, M.F.M.; Angela, R.A.; Koolmoes, B.; Toitou, M.; Paauwe, M.; ... ; Hawinkels, L.J.A.C. 2020
Purpose: Endoglin is a coreceptor for TGFb ligands that is highly expressed on proliferating endothelial cells and other cells in the tumor microenvironment. Clinical studies have noted increased... Show morePurpose: Endoglin is a coreceptor for TGFb ligands that is highly expressed on proliferating endothelial cells and other cells in the tumor microenvironment. Clinical studies have noted increased programmed cell death (PD)-1 expression on cytotoxic T cells in the peripheral blood of patients with cancer treated with TRC105, an endoglin-targeting antibody. In this study, we investigated the combination of endoglin antibodies (TRC105 and M1043) with an anti-PD1 antibody.Experimental Design: The combination anti-endoglin/anti-PD1 antibodies was tested in four preclinical mouse models representing different stages of cancer development. To investigate the underlying mechanism, Fc-receptor-knockout mice were used complemented with depletion of multiple immune subsets in mice. Tumor growth and the composition of immune infiltrate were analyzed by flow cytometry. Finally, human colorectal cancer specimens were analyzed for presence of endoglin-expressing regulatory T cells (Treg).Results: In all models, the combination of endoglin antibody and PD1 inhibition produced durable tumor responses, leading to complete regressions in 30% to 40% of the mice. These effects were dependent on the presence of Fcg receptors, indicating the involvement of antibody-dependent cytotoxic responses and the presence of CD8(+) cytotoxic T cells and CD4(+) Th cells. Interestingly, treatment with the endoglin antibody, TRC105, significantly decreased the number of intratumoral Tregs. Endoglin-expressing Tregs were also detected in human colorectal cancer specimens.Conclusions: Taken together, these data provide a rationale for combining TRC105 and anti-PD1 therapy and provide additional evidence of endoglin's immunomodulatory role. Show less
Ouahoud, S.; Voorneveld, P.W.; Burg, L.R.A. van der; Jonge-Muller, E.S.M. de; Schoonderwoerd, M.J.A.; Paauwe, M.; ... ; Hardwick, J.C.H. 2020
Patients with the mesenchymal subtype colorectal cancer (CRC) have a poor prognosis, in particular patients with stroma-rich tumors and aberrant SMAD4 expression. We hypothesized that interactions... Show morePatients with the mesenchymal subtype colorectal cancer (CRC) have a poor prognosis, in particular patients with stroma-rich tumors and aberrant SMAD4 expression. We hypothesized that interactions between SMAD4-deficient CRC cells and cancer-associated fibroblasts provide a biological explanation. In transwell invasion assays, fibroblasts increased the invasive capacity of SMAD4-deficient HT29 CRC cells, but not isogenic SMAD4-proficient HT29 cells. A TGF-beta/BMP-specific array showed BMP2 upregulation by fibroblasts upon stimulation with conditioned medium from SMAD4-deficient CRC cells, while also stimulating their invasion. In a mouse model for experimental liver metastasis, the co-injection of fibroblasts increased metastasis formation of SMAD4-deficient CRC cells (p = 0.02) but not that of SMAD4-proficient CRC cells. Significantly less metastases were seen in mice co-injected with BMP2 knocked-down fibroblasts. Fibroblast BMP2 expression seemed to be regulated by TRAIL, a factor overexpressed in SMAD4-deficient CRC cells. In a cohort of 146 stage III CRC patients, we showed that patients with a combination of high stromal BMP2 expression and the loss of tumor SMAD4 expression had a significantly poorer overall survival (HR 2.88, p = 0.04). Our results suggest the existence of a reciprocal loop in which TRAIL from SMAD4-deficient CRC cells induces BMP2 in fibroblasts, which enhances CRC invasiveness and metastasis. Show less
Purpose: Cancer-associated fibroblasts (CAF) are a major component of the colorectal cancer tumor microenvironment. CAFs play an important role in tumor progression and metastasis, partly through... Show morePurpose: Cancer-associated fibroblasts (CAF) are a major component of the colorectal cancer tumor microenvironment. CAFs play an important role in tumor progression and metastasis, partly through TGF-β signaling pathway. We investigated whether the TGF-β family coreceptor endoglin is involved in CAF-mediated invasion and metastasis.Experimental Design: CAF-specific endoglin expression was studied in colorectal cancer resection specimens using IHC and related to metastases-free survival. Endoglin-mediated invasion was assessed in vitro by transwell invasion, using primary colorectal cancer–derived CAFs. Effects of CAF-specific endoglin expression on tumor cell invasion were investigated in a colorectal cancer zebrafish model, whereas liver metastases were assessed in a mouse model.Results: CAFs specifically at invasive borders of colorectal cancer express endoglin and increased expression intensity correlated with increased disease stage. Endoglin-expressing CAFs were also detected in lymph node and liver metastases, suggesting a role in colorectal cancer metastasis formation. In stage II colorectal cancer, CAF-specific endoglin expression at invasive borders correlated with poor metastasis-free survival. In vitro experiments revealed that endoglin is indispensable for bone morphogenetic protein (BMP)-9–induced signaling and CAF survival. Targeting endoglin using the neutralizing antibody TRC105 inhibited CAF invasion in vitro. In zebrafish, endoglin-expressing fibroblasts enhanced colorectal tumor cell infiltration into the liver and decreased survival. Finally, CAF-specific endoglin targeting with TRC105 decreased metastatic spread of colorectal cancer cells to the mouse liver.Conclusions: Endoglin-expressing CAFs contribute to colorectal cancer progression and metastasis. TRC105 treatment inhibits CAF invasion and tumor metastasis, indicating an additional target beyond the angiogenic endothelium, possibly contributing to beneficial effects reported during clinical evaluations Show less
In dit proefschrift worden studies besproken naar de rol van de TGF-β signaleringsroute in de tumor micro-omgeving in colorectaal kanker. Deze studies hebben zich voornamelijk gefocust op de rol... Show moreIn dit proefschrift worden studies besproken naar de rol van de TGF-β signaleringsroute in de tumor micro-omgeving in colorectaal kanker. Deze studies hebben zich voornamelijk gefocust op de rol van kanker-geassocieerde fibroblasten (CAFs) in kanker. Wij hebben aangetoond dat een co-receptor voor TGF-β, endoglin, een grote rol speelt in het CAF-gemedieerd uitzaaien van darmkanker en dat de expressie van endoglin op CAFs een prognostische marker is voor metastase-vrije overleving in vroeg stadium darmkankerpatienten. Daarnaast werd een nieuwe muizenstam ontwikkeld om specifiek op fibroblasten endoglin uit te schakelen, zodat de rol hiervan in het ontstaan van darmkanker bestudeerd kon worden in een chemisch geinduceerd model voor darmkanker. Samengevat laten de studies in dit proefschrift zien dat endoglin expressie op CAFs een belangrijke rol speelt in het metastaseren van colorectaalkanker en opent het deuren naar therapeutische toepassingen. Show less
Endoglin, a transforming growth factor-beta co-receptor, is highly expressed on angiogenic endothelial cells in solid tumors. Therefore, targeting endoglin is currently being explored in clinical... Show moreEndoglin, a transforming growth factor-beta co-receptor, is highly expressed on angiogenic endothelial cells in solid tumors. Therefore, targeting endoglin is currently being explored in clinical trials for anti-angiogenic therapy. In this project, the redundancy between endoglin and vascular endothelial growth factor (VEGF) signaling in angiogenesis and the effects of targeting both pathways on breast cancer metastasis were explored. In patient samples, increased endoglin signaling after VEGF inhibition was observed. In vitro TRC105, an endoglin-neutralizing antibody, increased VEGF signaling in endothelial cells. Moreover, combined targeting of the endoglin and VEGF pathway, with the VEGF receptor kinase inhibitor SU5416, increased antiangiogenic effects in vitro and in a zebrafish angiogenesis model. Next, in a mouse model for invasive lobular breast cancer, the effects of TRC105 and SU5416 on tumor growth and metastasis were explored. Although TRC105 and SU5416 decreased tumor vascular density, tumor volume was unaffected. Strikingly, in mice treated with TRC105, or TRC105 and SU5416 combined, a strong inhibition in the number of metastases was seen. Moreover, upon resection of the primary tumor, strong inhibition of metastatic spread by TRC105 was observed in an adjuvant setting. To confirm these data, we assessed the effects of endoglin-Fc (an endoglin ligand trap) on metastasis formation. Similar to treatment with TRC105 in the resection model, endoglin-Fc-expressing tumors showed strong inhibition of distant metastases. These results show, for the first time, that targeting endoglin, either with neutralizing antibodies or a ligand trap, strongly inhibits metastatic spread of breast cancer in vivo. Show less
Hawinkels, L.J.A.C.; Vinuesa, A.G. de; Paauwe, M.; Kruithof-de Julio, M.; Wiercinska, E.; Pardali, E.; ... ; Dijke, P. ten 2016
Introduction: Although cancer treatment has evolved substantially in the past decades, cancer-related mortality rates are still increasing. Therapies targeting tumor angiogenesis, crucial for the... Show moreIntroduction: Although cancer treatment has evolved substantially in the past decades, cancer-related mortality rates are still increasing. Therapies targeting tumor angiogenesis, crucial for the growth of solid tumors, mainly target vascular endothelial growth factor (VEGF) and have been clinically applied during the last decade. However, these therapies have not met high expectations, which were based on therapeutic efficacy in animal models. This can partly be explained by the upregulation of alternative angiogenic pathways. Therefore, additional therapies targeting other pro-angiogenic pathways are needed. Areas covered: The transforming growth factor (TGF)-β signaling pathway plays an important role in (tumor) angiogenesis. Therefore, components of this pathway are interesting candidates for anti-angiogenic therapy. Endoglin, a co-receptor for various TGF-β family members, is specifically overexpressed in tumor vessels and endoglin expression is associated with metastasis and patient survival. Therefore, endoglin might be a good candidate for anti-angiogenic therapy. In this review, we discuss the potential of using endoglin to target the tumor vasculature for imaging and therapeutic purposes. Expert opinion: Considering the promising results from various in vitro studies, in vivo animal models and the first clinical trial targeting endoglin, we are convinced that endoglin is a valuable tool for the diagnosis, visualization and ultimately treatment of solid cancers. Show less
