Maarten van Dinther

Heligmosomoides polygyrus , a mouse parasite, modulates host immunity by secreting modular transforming growth factor‐β (TGFβ) mimics (TGMs). The agonist TGM1 interacts with TGFBR1, TGFBR2, and the co‐receptor CD44 through domains D1/2, D3, and D4/5, respectively. In contrast, the antagonist TGM6, which lacks D1/2, but retains TGFBR2 binding through D3, targets different cells compared to TGM1. The TGM6 co‐receptor is unknown. Using X‐ray crystallography and binding studies, we show that TGM6 preferentially binds mouse TGFBR2 over human TGFBR2, and that this is essential for its antagonistic function. We identified low‐density lipoprotein receptor‐related protein 1 (LRP1) and betaglycan (TGFBR3) as co‐receptors for TGM6. LRP1 enhances TGM6 efficacy and is required for its antagonistic effect by promoting TGFBR2 lysosomal degradation, whereas betaglycan counteracts TGM6 in a TGFBR2‐dependent manner. The modular organization of TGMs enabled us to design TGM1/6 chimeras or TGM‐D3 fusion with an affibody that recognizes a specific cell‐surface receptor, thereby altering cell‐type specificity and functionality. Furthermore, we developed a TGFBR2 nanobody that, on its own, has no inhibitory effect but, when fused to a receptor antibody, antagonizes TGFβ by blocking TGFβ receptor interaction in a cell‐selective manner. Thus, we designed programmable agents that modulate TGFβ signaling only in co‐receptor‐expressing cells.

The transforming growth factor‐β (TGF‐β) mimics TGM1 and TGM4 are modular proteins expressed by the parasite Heligmosomoides polygyrus . Each comprises 5 complement control protein (CCP) domains binding the TGF‐β receptors TGFBR1 (Domains 1 and 2) and TGFBR2 (Domain 3), whereas Domains 4 and 5 bind cell surface co‐receptors including CD44. Despite sharing 86% amino acid identity, TGM1 and TGM4 differ functionally in key respects of affinity for mammalian TGFBRs and interactions with cell surface co‐receptors. Although both induce T cell expression of the transcription factor Foxp3, TGM1 activates intracellular SMAD signaling in fibroblasts, whereas conversely, TGM4 blocks SMAD responses to TGF‐β or TGM1 in fibroblasts. By recombining and/or deleting domains, we identified that the TGFBR1‐binding Domains 1 and 2 of TGM4 confer antagonist activity, which is dependent on the CD44 co‐receptor interacting domains (Domains 4 and 5); antagonism is enhanced but not fully dependent on binding to TGFBR2. TGMs lack interchain disulfide bonds, in distinction to TGF‐β, which is a covalently linked dimeric cytokine. Dimerization of TGM4 as an Fc fusion protein converted it from an inhibitor to an activator of SMAD signaling in fibroblasts and enhanced Foxp3 induction, indicating that high avidity interactions (either monomer/dimer on T cells or only dimer on fibroblasts) drive TGF‐β signaling. However, when TGM4 is below an avidity threshold, it binds receptors in an antagonistic mode. These results advance our understanding of the mechanism of action of these parasite‐encoded products and uncover new tools modulating TGF‐β functions in health and disease.

Cancer therapy would benefit from suppressing cancer cell motility in the process of metastasis. Such directed cell migration relies on the propulsive force established by the filamentous actin network within lamellipodia. Proteins of the Ena/VASP family and the WAVE regulatory complex orchestrate lamellar protrusions and therefore provide promising targets for pharmacological interventions. Here, we report a cross-talk between Ena/VASP proteins and WAVE2 that is important for cancer cell extravasation. Mutating the EVH1 domain recognition motif in WAVE2 abrogates chemotaxis of triple-negative MDA-MB-231 breast cancer cells and reduces their extravasation in a zebrafish model. In pilot experiments, orthotopic implantation of these cells into mice led to a reduction in macrometastasis, resulting in prolonged survival. Similarly, intervention by an Ena/VASP-EVH1 inhibitor also reduced metastasis in vivo. Our results suggest that pharmacological interference with the Ena/VASP–WAVE2 interaction may thus reduce metastasis.

Long-lived parasites evade host immunity through highly evolved molecular strategies. The murine intestinal helminth, Heligmosomoides polygyrus , down-modulates the host immune system through release of an immunosuppressive TGF-β mimic, TGM1, which is a divergent member of the CCP (Sushi) protein family. TGM1 comprises 5 domains, of which domains 1-3 (D1/2/3) bind mammalian TGF-β receptors, acting on T cells to induce Foxp3 ⁺ regulatory T cells; however, the roles of domains 4 and 5 (D4/5) remain unknown. We noted that truncated TGM1, lacking D4/5, showed reduced potency. Combination of D1/2/3 and D4/5 as separate proteins did not alter potency, suggesting that a physical linkage is required and that these domains do not deliver an independent signal. Coprecipitation from cells treated with biotinylated D4/5, followed by mass spectrometry, identified the cell surface protein CD44 as a coreceptor for TGM1. Both full-length and D4/5 bound strongly to a range of primary cells and cell lines, to a greater degree than D1/2/3 alone, although some cell lines did not respond to TGM1. Ectopic expression of CD44 in nonresponding cells conferred responsiveness, while genetic depletion of CD44 abolished enhancement by D4/5 and ablated the ability of full-length TGM1 to bind to cell surfaces. Moreover, CD44-deficient T cells showed attenuated induction of Foxp3 by full-length TGM1, to levels similar to those induced by D1/2/3. Hence, a parasite protein known to bind two host cytokine receptor subunits has evolved a third receptor specificity, which serves to raise the avidity and cell type–specific potency of TGF-β signaling in mammalian cells.

Patients with bladder cancer often have a poor prognosis due to the highly invasive and metastatic characteristics of bladder cancer cells. Epithelial-to-mesenchymal transition (EMT) has been causally linked to bladder cancer invasion. The E3 ubiquitin ligase, tumor necrosis factor receptor–associated factor 4 (TRAF4) has been implicated as a tumor promoter in a wide range of cancers. In contrast, here we show that low TRAF4 expression is associated with poor overall survival in patients with bladder cancer. We show that the TRAF4 gene is epigenetically silenced and that ERK mediates TRAF4 phosphorylation, resulting in lower TRAF4 protein levels in bladder cancer cells. In addition, we demonstrate that TRAF4 is inversely correlated with an EMT gene signature/protein marker expression. Functionally, by manipulating TRAF4 expression, we show that TRAF4 regulates EMT genes and epithelial and invasive properties in bladder cancer cells. Transcriptomic analysis of dysregulated TRAF4 expression in bladder cancer cell lines revealed that high TRAF4 expression enhances the bone morphogenetic protein (BMP)/SMAD and inhibits the NF-κB signaling pathway. Mechanistically, we show that TRAF4 targets the E3 ubiquitin ligase SMURF1, a negative regulator of BMP/SMAD signaling, for proteasomal degradation in bladder cancer cells. This was corroborated in patient samples where TRAF4 positively correlates with phospho-SMAD1/5, and negatively correlates with phospho-NFκb-p65. Lastly, we show that genetic and pharmacologic inhibition of SMURF1 inhibits the migration of aggressive mesenchymal bladder cancer cells.Implications:Our findings identify E3 ubiquitin ligase TRAF4 as a potential therapeutic target or biomarker for bladder cancer progression.

Patients with bladder cancer often have a poor prognosis due to the highly invasive and metastatic characteristics of bladder cancer cells. Epithelial-to-mesenchymal transition (EMT) has been causally linked to bladder cancer invasion. The E3 ubiquitin ligase, tumor necrosis factor receptor–associated factor 4 (TRAF4) has been implicated as a tumor promoter in a wide range of cancers. In contrast, here we show that low TRAF4 expression is associated with poor overall survival in patients with bladder cancer. We show that the TRAF4 gene is epigenetically silenced and that ERK mediates TRAF4 phosphorylation, resulting in lower TRAF4 protein levels in bladder cancer cells. In addition, we demonstrate that TRAF4 is inversely correlated with an EMT gene signature/protein marker expression. Functionally, by manipulating TRAF4 expression, we show that TRAF4 regulates EMT genes and epithelial and invasive properties in bladder cancer cells. Transcriptomic analysis of dysregulated TRAF4 expression in bladder cancer cell lines revealed that high TRAF4 expression enhances the bone morphogenetic protein (BMP)/SMAD and inhibits the NF-κB signaling pathway. Mechanistically, we show that TRAF4 targets the E3 ubiquitin ligase SMURF1, a negative regulator of BMP/SMAD signaling, for proteasomal degradation in bladder cancer cells. This was corroborated in patient samples where TRAF4 positively correlates with phospho-SMAD1/5, and negatively correlates with phospho-NFκb-p65. Lastly, we show that genetic and pharmacologic inhibition of SMURF1 inhibits the migration of aggressive mesenchymal bladder cancer cells.Implications:Our findings identify E3 ubiquitin ligase TRAF4 as a potential therapeutic target or biomarker for bladder cancer progression.

Supplementary Table from TRAF4 Inhibits Bladder Cancer Progression by Promoting BMP/SMAD Signaling

Supplementary Figure from TRAF4 Inhibits Bladder Cancer Progression by Promoting BMP/SMAD Signaling

Supplementary Table from TRAF4 Inhibits Bladder Cancer Progression by Promoting BMP/SMAD Signaling

Supplementary Figure from TRAF4 Inhibits Bladder Cancer Progression by Promoting BMP/SMAD Signaling