Splice-Variant Knock-Out of TGFβ Receptors Perturbates the Proteome of Ovarian Carcinoma Cells

The aim of this study was to analyze the biological role of different transforming growth factor-β (TGFβ) receptor splice variants in ovarian carcinoma (OC). Specific receptor variant knockouts (KO) were prepared using the CRISPR/Cas9 genome editing system in two OC cell lines, TβRI variant 1 (TβRIv1) KO in ES-2 cells and TβRII variant 1 (TβRIIv1) KO in OVCAR-8 cells. Control and KO cells were compared by proteomic analysis, functional tests, analysis of epithelial–mesenchymal transition (EMT) drivers, and Western blot of signaling proteins. Proteomic analysis revealed significant changes in protein pathways in the KO cells. TβRIv1 KO resulted in a significant reduction in both cellular motility and invasion, while TβRIIv1 KO significantly reduced cellular motility and increased Reactive Oxygen Species (ROS) production. Both receptor variant KOs reduced MET protein levels. Of the EMT drivers, a significant decrease in TWIST protein expression, and increase in SNAIL protein and MALAT1 mRNA levels were observed in the TβRIIv1 KO compared to control. A significant decrease in JNK1 and JNK2 activation was found in the TβRIv1 KO compared to control cells. These findings provide new insight regarding the biological role of the TGFβ receptor variants in the biology and potentially the progression of OC.

Huntingtin CAG expansion impairs germ layer patterning in synthetic human 2D gastruloids through polarity defects

ABSTRACT Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expansion of the CAG repeats in the huntingtin gene (HTT). Although HD has been shown to have a developmental component, how early during human embryogenesis the HTT-CAG expansion can cause embryonic defects remains unknown. Here, we demonstrate a specific and highly reproducible CAG length-dependent phenotypic signature in a synthetic model for human gastrulation derived from human embryonic stem cells (hESCs). Specifically, we observed a reduction in the extension of the ectodermal compartment that is associated with enhanced activin signaling. Surprisingly, rather than a cell-autonomous effect, tracking the dynamics of TGFβ signaling demonstrated that HTT-CAG expansion perturbs the spatial restriction of activin response. This is due to defects in the apicobasal polarization in the context of the polarized epithelium of the 2D gastruloid, leading to ectopic subcellular localization of TGFβ receptors. This work refines the earliest developmental window for the prodromal phase of HD to the first 2 weeks of human development, as modeled by our 2D gastruloids.

Divergence in chondrogenic potential between in vitro and in vivo of adipose- and synovial-stem cells from mouse and human

Background Somatic stem cell transplantation has been performed for cartilage injury, but the reparative mechanisms are still conflicting. The chondrogenic potential of stem cells are thought as promising features for cartilage therapy; however, the correlation between their potential for chondrogenesis in vitro and in vivo remains undefined. The purpose of this study was to investigate the intrinsic chondrogenic condition depends on cell types and explore an indicator to select useful stem cells for cartilage regeneration. Methods The chondrogenic potential of two different stem cell types derived from adipose tissue (ASCs) and synovium (SSCs) of mice and humans was assessed using bone morphogenic protein-2 (BMP2) and transforming growth factor-β1 (TGFβ1). Their in vivo chondrogenic potential was validated through transplantation into a mouse osteochondral defect model. Results All cell types showed apparent chondrogenesis under the combination of BMP2 and TGFβ1 in vitro, as assessed by the formation of proteoglycan- and type 2 collagen (COL2)-rich tissues. However, our results vastly differed with those observed following single stimulation among species and cell types; apparent chondrogenesis of mouse SSCs was observed with supplementation of BMP2 or TGFβ1, whereas chondrogenesis of mouse ASCs and human SSCs was observed with supplementation of BMP2 not TGFβ1. Human ASCs showed no obvious chondrogenesis following single stimulation. Mouse SSCs showed the formation of hyaline-like cartilage which had less fibrous components (COL1/3) with supplementation of TGFβ1. However, human cells developed COL1/3+ tissues with all treatments. Transcriptomic analysis for TGFβ receptors and ligands of cells prior to chondrogenic induction did not indicate their distinct reactivity to the TGFβ1 or BMP2. In the transplanted site in vivo, mouse SSCs formed hyaline-like cartilage (proteoglycan+/COL2+/COL1−/COL3−) but other cell types mainly formed COL1/3-positive fibrous tissues in line with in vitro reactivity to TGFβ1. Conclusion Optimal chondrogenic factors driving chondrogenesis from somatic stem cells are intrinsically distinct among cell types and species. Among them, the response to TGFβ1 may possibly represent the fate of stem cells when locally transplanted into cartilage defects.

A Role for TGFβ Signaling in Preclinical Osteolytic Estrogen Receptor-Positive Breast Cancer Bone Metastases Progression

While tumoral Smad-mediated transforming growth factor β (TGFβ) signaling drives osteolytic estrogen receptor α-negative (ER-) breast cancer bone metastases (BMETs) in preclinical models, its role in ER+ BMETs, representing the majority of clinical BMETs, has not been documented. Experiments were undertaken to examine Smad-mediated TGFβ signaling in human ER+ cells and bone-tropic behavior following intracardiac inoculation of estrogen (E2)-supplemented female nude mice. While all ER+ tumor cells tested (ZR-75-1, T47D, and MCF-7-derived) expressed TGFβ receptors II and I, only cells with TGFβ-inducible Smad signaling (MCF-7) formed osteolytic BMETs in vivo. Regulated secretion of PTHrP, an osteolytic factor expressed in >90% of clinical BMETs, also tracked with osteolytic potential; TGFβ and E2 each induced PTHrP in bone-tropic or BMET-derived MCF-7 cells, with the combination yielding additive effects, while in cells not forming BMETs, PTHrP was not induced. In vivo treatment with 1D11, a pan-TGFβ neutralizing antibody, significantly decreased osteolytic ER+ BMETs in association with a decrease in bone-resorbing osteoclasts at the tumor-bone interface. Thus, TGFβ may also be a driver of ER+ BMET osteolysis. Moreover, additive pro-osteolytic effects of tumoral E2 and TGFβ signaling could at least partially explain the greater propensity for ER+ tumors to form BMETs, which are primarily osteolytic.

Dual inhibition of TGFβ and AXL as a novel therapy for human colorectal adenocarcinoma with mesenchymal phenotype

A subset of colorectal cancer (CRC) with a mesenchymal phenotype (CMS4) displays an aggressive disease, with an increased risk of recurrence after surgery, reduced survival, and resistance to standard treatments. It has been shown that the AXL and TGFβ signaling pathways are involved in epithelial-to-mesenchymal transition, migration, metastatic spread, and unresponsiveness to targeted therapies. However, the prognostic role of the combination of these biomarkers and the anti-tumor effect of AXL and TGFβ inhibition in CRC still has to be assessed. To evaluate the role of AXL and TGFβ as negative biomarker in CRC, we conducted an in-depth in silico analysis of CRC samples derived from the Gene Expression Omnibus. We found that AXL and TGFβ receptors are upregulated in CMS4 tumors and are correlated with an increased risk of recurrence after surgery in stage II/III CRC and a reduced overall survival. Moreover, we showed that AXL receptor is differently expressed in human CRC cell lines. Dual treatment with the TGFβ galunisertib and the AXL inhibitor, bemcentinib, significantly reduced colony formation and migration capabilities of tumor cells and displayed a strong anti-tumor activity in 3D spheroid cultures derived from patients with advanced CRC. Our work shows that AXL and TGFβ receptors identify a subgroup of CRC with a mesenchymal phenotype and correlate with poor prognosis. Dual inhibition of AXL and TGFβ could represent a novel therapeutic strategy for patients with this aggressive disease.

Huntingtin CAG expansion impairs germ layer patterning in synthetic human gastruloids through polarity defects

Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by an expansion of the CAG repeats in the Huntingtin gene (HTT). While HD has been shown to have a developmental component, how early during human embryogenesis the HTT-CAG expansion can cause embryonic defects remains unknown. Here, we demonstrate a specific and highly reproducible CAG length-dependent phenotypic signature in a synthetic model for human gastrulation derived from human embryonic stem cells (hESCs). Specifically, we observed a reduction in the extension of the ectodermal compartment that is associated with enhanced ACTIVIN signaling. Surprisingly, rather than a cell-autonomous effect, tracking the dynamics of TGFβ signaling demonstrated that HTT-CAG expansion perturbs the spatial restriction of ACTIVIN response. This is due to defects in the apicobasal polarization in the context of the polarized epithelium of the gastruloid, leading to ectopic subcellular localization of TGFβ receptors. This work refines the earliest developmental window for the prodromal phase of HD to the first two weeks of human development as modeled by our gastruloids.

Abstract 16737: Inhibition of Tgf-β1 Signaling Protect Against Cell-Cell Decoupling and Asynchronous Automaticity of Tbx18-ipms

Background: We have previously demonstrated that TBX18 suffices to reprogram postnatal ventricular cardiomyocytes to induced pacemaker cells (TBX18-iPMs). However, the nascent automaticity appeared to wane over time, characterized by loss of syncytial pacing but preservation of single cell automaticity. Hypothesis: Based on increase in Tgfβ ligands in TBX18-iPMs and the known role of Tgfβ signaling in electrical remodeling, we hypothesized that loss of syncytial automaticity is due to electrical decoupling of the iPMs mediated by Tgfβ signaling. Methods: Adenoviral vectors expressing either human TBX18 or GFP were used for gene transfer into neonatal rat ventricular myocytes (NRVMs) or adult rat ventricular myocardium in vivo. Results: NRVM monolayers transduced with GFP were mostly quiescent, interspersed by paroxysmal contractions. In contrast, TBX18 transduced NRVM monolayers showed spontaneous and rhythmic contractions, but the syncytial pacing wavered over the next 5-7 days although numerous iPMs continued to beat asynchronously. Treatment of TBX18-iPMs with an inhibitor of Tgfβ receptors, A83-01, preserved syncytial pacing, suggesting that electrical remodeling cues from Tgfβ signaling led to loss of iPM-iPM electrical coupling. We examined the molecules that are integral to the function of gap junction in the myocardium, namely Cx43, N-cadherin and β-catenin. Cx43 and N-cadherin were downregulated in TBX18-iPMs compared to control by 77±16% and 43±11% (p<0.01, n=6), respectively. Total β-catenin protein level was unaltered, but its distribution decreased at the sarcolemmal and increased in the nuclei of TBX18-iPMs compared to control. Inhibition of Tgfβ with A83-01 restored Cx43 protein level in TBX18-iPMs (2.2-folds higher) compared to untreated TBX18-iPMs. Similarly, adult rats that received intramyocardial injection of TBX18 showed increased Cx43 expression at the boundary between the iPMs and the host myocardium when the animals were treated with systemic delivery of A83-01 for one week compared to TBX18 animals treated with DMSO. Conclusions: TBX18 activates Tgfβ signaling which suppresses cell-cell electrical coupling. Inhibition of Tgfβ signaling in TBX18-iPMs preserves gap junction components and syncytial pacing.

Abstract 16644: Durable Septal Pacing Induced by Non-viral Tbx18 Gene Transfer and Tgfβ Inhibition in a Porcine Model of Complete Atrioventricular Block

Introduction: Cardiac pacing can be created by myocardial delivery of TBX18. However, evidence for overt fibrosis at the injection site and the use of viral vectors impede clinical translation of this concept. We hypothesized that non-viral gene transfer of TBX18 combined with prevention of fibrosis with a Tgfβ inhibitor enables durable ventricular pacing in the porcine model of CAVB. Methods: All pigs underwent transvenous RF ablation of the AV node to achieve complete heart block on day-1. The pigs were implanted with a backup pacemaker (VVI=50 bpm) and with osmotic pumps designed to release A83-01, an inhibitor of Tgfβ receptors. Synthetic mRNA encoding either TBX18 (n=7) or GFP (n=2) in saline was injected in the high interventricular septum with a NOGA-MYOSTAR™ catheter. Continuous and ambulatory ECG, blood pressure (BP) and activity were recorded for the 4-week study period with telemetry. Results: The control pigs were dependent on backup pacing throughout 4 weeks with retrograde conduction from the RV apex. In contrast, TBX18 injected pigs showed higher heart rate (HR) compared to control (4-week mean HR of 58±7 vs 52±7, p <0.05), exhibiting antegrade conduction from the high septum with narrow QRS complexes. Max. HR was higher in TBX18 pigs compared to control with a 4-week average of 70±9 vs 55±11 bpm ( p <0.05). TBX18 pigs exhibited diurnal HR oscillations that directly correlated with their physical activity, indicating chronotropic competence. Likewise, mean BP correlated tightly with mean HR in TBX18 injected pigs ( p <0.01) but not in control ( p =0.164), indicating superior hemodynamic measures. Radiopaque agent-enabled, real-time fluoroscopic visualization revealed that the injected biologic spread beyond the intended delivery site in 2 of 7 TBX18 pigs. The mean and max. HR of the 2 pigs were slower than the other five TBX18 pigs, suggesting that conventional imaging can be leveraged to identify redosing criteria, and minimize non-responders. No increase in spontaneous or induced arrhythmias was observed in TBX18 pigs compared to control. Conclusions: mRNA delivery of TBX18 combined with Tgfβ inhibition achieved durable septal pacing for 4 weeks in a porcine model chronic heart block, demonstrating therapeutic efficacy with greater safety profiles.

Abstract 16793: Smad7 Induction in Activated Infarct Myofibroblasts Protects From Adverse Remodeling by Restraining TGF-beta-Mediated and Receptor Tyrosine Kinase Signaling

Cardiac repair is dependent on myofibroblast TGF-b/Smad3 signaling and subsequent formation of an organized scar. However, to prevent prolonged activation and fibrosis, TGFβ effects are tightly regulated through induction of suppressive signals, such as the inhibitory Smads, that restrain TGFβ cascades. We hypothesized that the inhibitory Smad7 may be induced in infarct myofibroblasts, protecting from adverse remodeling and fibrosis. Moreover, we dissected the molecular signals modulated by Smad7. Smad7 is markedly induced in infarct myofibroblasts through a TGFβ/Smad3 pathway. To investigate the role of endogenous Smad7 in post-infarction remodeling, we generated mice with myofibroblast-specific Smad7 loss (MFS7KO). Following non-reperfused infarction, MFS7KO mice had increased late mortality that was not due to rupture, but was associated with worse heart failure. Surviving MFS7KO mice had accentuated adverse remodeling, worse systolic/diastolic dysfunction and increased collagen deposition in the infarct border zone, in comparison to Smad7 fl/fl animals. In isolated cardiac fibroblasts, Smad7 overexpression attenuated myofibroblast conversion and profibrotic gene expression, whereas Smad7 knockdown promoted matrix synthesis. In Smad7 KO fibroblasts, overactive fibrogenic activity was associated with enhanced Smad2/3, Erk and Akt signaling, but comparable TβRI/TβRII phosphorylation, suggesting that Smad7 acts downstream of the TGFβ receptors. To dissect the mechanisms for Smad7 actions, we compared the transcriptome of Smad7 KO and WT fibroblasts, in presence/absence of TGFβ. Surprisingly, Smad7 loss had more prominent effects on receptor tyrosine kinase (RTK) cascades than on TGFβ-inducible genes. An RTK protein array identified Erbbs as targets of Smad7 in fibroblasts. Western blot showed that Smad7 restrains Erbb1 and Erbb2 signaling through effects independent of TGFβ. In conclusion, Smad7 induction in infarct myofibroblasts restrains fibrosis, by inhibiting Smad2/3, Erk and Akt signaling via actions downstream of TβRs, and through TGFβ-independent interactions with Erbb1/2. Protective effects of Smad7 in cardiac remodeling may have important therapeutic implications for heart failure patients.