Ovo-like transcriptional repressor 1 (OVOL1) is a key mediator of epithelial lineage determination and mesenchymal-epithelial transition (MET). The cytokines transforming growth factor-beta (TGF... Show moreOvo-like transcriptional repressor 1 (OVOL1) is a key mediator of epithelial lineage determination and mesenchymal-epithelial transition (MET). The cytokines transforming growth factor-beta (TGF-beta) and bone morphogenetic proteins (BMP) control the epithelial-mesenchymal plasticity (EMP) of cancer cells, but whether this occurs through interplay with OVOL1 is not known. Here, we show that OVOL1 is inversely correlated with the epithelial-mesenchymal transition (EMT) signature, and is an indicator of a favorable prognosis for breast cancer patients. OVOL1 suppresses EMT, migration, extravasation, and early metastatic events of breast cancer cells. Importantly, BMP strongly promotes the expression of OVOL1, which enhances BMP signaling in turn. This positive feedback loop is established through the inhibition of TGF-beta receptor signaling by OVOL1. Mechanistically, OVOL1 interacts with and prevents the ubiquitination and degradation of SMAD family member 7 (SMAD7), which is a negative regulator of TGF-beta type I receptor stability. Moreover, a small-molecule compound 6-formylindolo(3,2-b)carbazole (FICZ) was identified to activate OVOL1 expression and thereby antagonizing (at least in part) TGF-beta-mediated EMT and migration in breast cancer cells. Our results uncover a novel mechanism by which OVOL1 attenuates TGF-beta/SMAD signaling and maintains the epithelial identity of breast cancer cells. Show less
Transforming growth factor beta (TGF(beta) induces epithelial-mesenchymal transition (EMT), which correlates with sternness and invasiveness. Mesenchymal-epithelial transition (MET) is induced by... Show moreTransforming growth factor beta (TGF(beta) induces epithelial-mesenchymal transition (EMT), which correlates with sternness and invasiveness. Mesenchymal-epithelial transition (MET) is induced by TGF beta withdrawal and correlates with metastatic colonization. Whether TGF beta promotes sternness and invasiveness simultaneously via EMT remains unclear. We established a breast cancer cell model expressing red fluorescent protein (RFP) under the E-cadherin promoter. In 2D cultures, TGF beta induced EMT, generating RFPlow cells with a mesenchymal transcriptome, and regained RFP, with an epithelial transcriptome, after MET induced by TGF beta withdrawal. RFPlow cells generated robust mammospheres, with epithelio-mesenchymal cell surface features. Mammospheres that were forced to adhere generated migratory cells, devoid of RFP, a phenotype which was inhibited by a TGF beta receptor kinase inhibitor. Further stimulation of RFPlow mammospheres with TGF beta suppressed the generation of motile cells, but enhanced mammosphere growth. Accordingly, mammary fat-pad-transplanted mammospheres, in the absence of exogenous TGF beta treatment, established lung metastases with evident MET (RFPhigh cells). In contrast, TGF beta-treated mammospheres revealed high tumour-initiating capacity, but limited metastatic potential. Thus, the biological context of partial EMT and MET allows TGF beta to differentiate between pro-sternness and pro-invasive phenotypes. Show less
Li, C.; Ma, J.; Groenewoud, A.; Ren, J.; Liu, S.; Snaar-Jagalska, B.E.; Dijke, P. ten 2022
Simple Summary Anti-angiogenesis agents have shown anti-cancer activity by preventing blood vessel ingrowth, thereby limiting tumour growth and metastasis. Although these molecules lead to... Show moreSimple Summary Anti-angiogenesis agents have shown anti-cancer activity by preventing blood vessel ingrowth, thereby limiting tumour growth and metastasis. Although these molecules lead to prolonged overall survival of cancer patients, therapy resistance is easily acquired. Therefore, novel inhibitors against other signaling pathways mediating angiogenesis are needed to achieve more efficient and sustainable targeting of the angiogenesis process. Here, we synthesized and identified two compounds belonging to a new class of small molecules termed macrocyclics that selectively inhibit bone morphogenetic protein receptor kinase activity. One compound also inhibits vascular endothelial growth factor-induced signalling. Treatment studies using in vitro cultured cells and zebrafish embryos revealed that both compounds impaired endothelial cell function and decreased normal and tumour-induced angiogenesis. Both compounds might provide a steppingstone for the development of novel-angiogenesis therapeutic agents. Angiogenesis, i.e., the formation of new blood vessels from pre-existing endothelial cell (EC)-lined vessels, is critical for tissue development and also contributes to neovascularization-related diseases, such as cancer. Vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) are among many secreted cytokines that regulate EC function. While several pharmacological anti-angiogenic agents have reached the clinic, further improvement is needed to increase clinical efficacy and to overcome acquired therapy resistance. More insights into the functional consequences of targeting specific pathways that modulate blood vessel formation may lead to new therapeutic approaches. Here, we synthesized and identified two macrocyclic small molecular compounds termed OD16 and OD29 that inhibit BMP type I receptor (BMPRI)-induced SMAD1/5 phosphorylation and downstream gene expression in ECs. Of note, OD16 and OD29 demonstrated higher specificity against BMPRI activin receptor-like kinase 1/2 (ALK1/2) than the commonly used small molecule BMPRI kinase inhibitor LDN-193189. OD29, but not OD16, also potently inhibited VEGF-induced extracellular regulated kinase MAP kinase phosphorylation in ECs. In vitro, OD16 and OD29 exerted strong inhibition of BMP9 and VEGF-induced ECs migration, invasion and cord formation. Using Tg (fli:EGFP) zebrafish embryos, we found that OD16 and OD29 potently antagonized dorsal longitudinal anastomotic vessel (DLAV), intra segmental vessel (ISV), and subintestinal vessel (SIV) formation during embryonic development. Moreover, the MDA-MB-231 breast cancer cell-induced tumor angiogenesis in zebrafish embryos was significantly decreased by OD16 and OD29. Both macrocyclic compounds might provide a steppingstone for the development of novel anti-angiogenesis therapeutic agents. Show less
Transforming growth factor-beta (TGF beta) family members are structurally and functionally related cytokines that have diverse effects on the regulation of cell fate during embryonic development... Show moreTransforming growth factor-beta (TGF beta) family members are structurally and functionally related cytokines that have diverse effects on the regulation of cell fate during embryonic development and in the maintenance of adult tissue homeostasis. Dysregulation of TGF beta family signaling can lead to a plethora of developmental disorders and diseases, including cancer, immune dysfunction, and fibrosis. In this review, we focus on TGF beta, a well-characterized family member that has a dichotomous role in cancer progression, acting in early stages as a tumor suppressor and in late stages as a tumor promoter. The functions of TGF beta are not limited to the regulation of proliferation, differentiation, apoptosis, epithelial-mesenchymal transition, and metastasis of cancer cells. Recent reports have related TGF beta to effects on cells that are present in the tumor microenvironment through the stimulation of extracellular matrix deposition, promotion of angiogenesis, and suppression of the anti-tumor immune reaction. The pro-oncogenic roles of TGF beta have attracted considerable attention because their intervention provides a therapeutic approach for cancer patients. However, the critical function of TGF beta in maintaining tissue homeostasis makes targeting TGF beta a challenge. Here, we review the pleiotropic functions of TGF beta in cancer initiation and progression, summarize the recent clinical advancements regarding TGF beta signaling interventions for cancer treatment, and discuss the remaining challenges and opportunities related to targeting this pathway. We provide a perspective on synergistic therapies that combine anti-TGF beta therapy with cytotoxic chemotherapy, targeted therapy, radiotherapy, or immunotherapy. Show less
TGF beta-SMAD3 signaling is a major driving force for cancer metastasis, while BMP-SMAD1/5 signaling can counteract this response. Analysis of gene expression profiles revealed that an increased... Show moreTGF beta-SMAD3 signaling is a major driving force for cancer metastasis, while BMP-SMAD1/5 signaling can counteract this response. Analysis of gene expression profiles revealed that an increased TGF beta-SMAD3 and a reduced BMP-SMAD1/5 targeted gene expression signature correlated with shortened distant metastasis free survival and overall survival of patients. At molecular levels, we discovered that TGF beta abolished BMP-induced SMAD1/5 activation in the highly-invasive breast cancer MDA-MB-231 cells, but to a less extent in the non-invasive cancer and normal breast cells. This suggests an inverse correlation between BMP signaling and invasiveness of tumor cells and TGF beta signaling acts in a double whammy fashion in driving cancer invasion and metastasis. Sustained ERK activation by TGF beta was specifically observed in MDA-MB-231 cells, and MEK inhibitor (MEKi) treatment restored BMP-SMAD1/5 signaling while not affecting SMAD2/3 activation. FK506 potently activated BMP, but not TGF beta signaling in breast cancer cells. MEKi or FK506 alone inhibited MDA-MB-231 extravasation in a zebrafish xenograft cancer model. Importantly, when administrated at suboptimal concentrations MEKi and FK506 strongly synergized in promoting BMP-SMAD1/5 signaling and inhibiting cancer cell extravasation. Furthermore, this combination of suboptimal concentrations treatment in a mouse tumor model resulted in real-time reduction of BMP-SMAD1/5 signaling in live tumors, and consequently potently inhibited tumor self-seeding, liver and bone metastasis, but not lung and brain metastasis. Mechanistically, it is the first time to identify BMP-SMAD1/5 signaling as an underlying molecular driver for organ-specific metastasis. Combining of MEKi and FK506, or their analogues, may be explored for clinical development of breast cancer. Show less
Barone, M.; Muller, M.; Chiha, S.; Ren, J.; Albat, D.; Soicke, A.; ... ; Kuhne, R. 2020
Battling metastasis through inhibition of cell motility is considered a promising approach to support cancer therapies. In this context, Ena/VASP-depending signaling pathways, in particular... Show moreBattling metastasis through inhibition of cell motility is considered a promising approach to support cancer therapies. In this context, Ena/VASP-depending signaling pathways, in particular interactions with their EVH1 domains, are promising targets for pharmaceutical intervention. However, protein-protein interactions involving proline-rich segments are notoriously difficult to address by small molecules. Hence, structure-based design efforts in combination with the chemical synthesis of additional molecular entities are required. Building on a previously developed nonpeptidic micromolar inhibitor, we determined 22 crystal structures of ENAH EVH1 in complex with inhibitors and rationally extended our library of conformationally defined prolinederived modules (ProMs) to succeed in developing a nanomolar inhibitor (K-d = 120 nM, MW = 734 Da). In contrast to the previous inhibitor, the optimized compounds reduced extravasation of invasive breast cancer cells in a zebrafish model. This study represents an example of successful, structure-guided development of low molecular weight inhibitors specifically and selectively addressing a proline-rich sequence-recognizing domain that is characterized by a shallow epitope lacking defined binding pockets. The evolved high-affinity inhibitor may now serve as a tool in validating the basic therapeutic concept, i.e., the sup pression of cancer metastasis by inhibiting a crucial protein- protein interaction involved in actin filament processing and cell migration. Show less
This thesis elucidated the possibility of manipulating BMP/TGFβ signaling to achieve inhibition of breast cancer metastasis, including boosting BMP signaling via blockade of the BMP antagonist... Show moreThis thesis elucidated the possibility of manipulating BMP/TGFβ signaling to achieve inhibition of breast cancer metastasis, including boosting BMP signaling via blockade of the BMP antagonist Grem1 extracellularly or via stimulation of small-molecule compounds intracellularly, preventing TGFβ signaling to allow accumulation of pro-oncogenic stimuli. We also highlight the importance of selecting appropriate cancer types when adopting dual inhibition of PD-L1 and TGFβ signaling. I hope my research will aid in more efficient clinical cancer therapies. Show less
Neckmann, U.; Wolowczyk, C.; Hall, M.; Almaas, E.; Ren, J.; Zhao, S.; ... ; Holien, T. 2019
