TGF-β Function Research Articles

PDCD10 promotes the tumor-supporting functions of TGF-β in pancreatic cancer.

The progression of pancreatic ductal adenocarcinoma (PDAC) is significantly affected by transforming growth factor (TGF)-β but targeting TGF-β can also compromize physiological effects in patients. Our study examined the functions of the ubiquitously expressed protein, PDCD10, as a modulator of TGF-β signaling in PDAC. Using in silico analyses we found that in patient samples, PDCD10 is significantly higher expressed in PDAC tumor tissue compared with normal pancreas and it is highly correlated with reduced survival. We created stable KO's of PDCD10 in two PDAC lines, PaTu 8902 (SMAD4 +/+) and PaTu 8988t (SMAD4 -/-), and found that KO lines are more sensitive to 5-FU and Gemcitabine treatment than their wild-type counterparts. Performing viability and wound closure assays we further found that PDCD10 promotes cell survival and proliferation by enhancing specifically the mitogenic functions of TGF-β. The molecular mechanism underlying this effect was further investigated using Western blots and with primary organoid lines derived from patient PDAC tissue samples. The data imply that PDCD10 mediates an increase in p-ERK through a non-SMAD4 pathway, leading to EMT promotion. Furthermore, PDCD10 facilitates deactivation of RB via a SMAD4-dependent pathway, thereby counter-acting the anti-proliferative actions of TGF-β. By performing proximity ligation assays (PLA) we found that PDCD10 associates with the kinase MST4, translocates it intracellularly and thereby facilitates phosphorylations of RB and ERK1/2. Our study indicates that PDCD10 promotes the proliferative function and EMT induction of TGF-β in pancreatic cancer cells. Therefore, targeting PDCD10 in PDAC patients could represent a promising new strategy to optimize TGF-β targeted therapies.

Abstract 1440: Retinoblastoma binding protein 9 regulates intestinal inflammation and inflammation-associated carcinogenesis

Abstract The intestinal epithelium constitutes the mucosal barrier, and its dysfunction promotes microbial invasion from the gut lumen, which further induces the intestinal inflammation. Chronic intestinal inflammation, as observed in poorly controlled inflammatory bowel diseases (IBD), leads to the development of inflammation-associated cancer. Thus, a deeper understanding of the regulatory system that controls intestinal homeostasis is essential. Retinoblastoma-binding protein 9 (RBBP9) is expressed in a range of human cancer cells. RBBP9 was initially reported to have the ability to regulate the cell cycle pathway. More recent studies have identified RBBP9 as a serine hydrolase (SH) that promotes cancer progression by subverting the anti-proliferative function of TGFβ in a pancreatic cancer model. However, little is known about the roles RBBP9 plays in the development of intestinal cancer. In this study, we investigated the role of RBBP9 in inflammation and inflammation-associated cancer in the intestine. Analyses of surgically resected human samples and publicly available datasets demonstrated that RBBP9 expression was reduced in patient samples of ulcerative colitis (UC) and colitis-associated cancer (CAC). Genetically knockout mice for Rbbp9 (Rbbp9−/−) were susceptible to experimental colitis induced by dextran sulfate sodium (DSS). Furthermore, Rbbp9−/− mice exhibited enhanced azoxymethane/DSS-induced tumorigenesis, a model for human CAC. Mechanistically, RNA sequencing of these tumors showed increased enrichment of inflammation-associated signatures, including interferon signaling pathways. Ex vivo organoid experiments revealed the suppressive function of RBBP9 in interferon signaling-mediated apoptosis of the intestinal epithelium. Simultaneous inhibition of RBBP9 and STAT1, a downstream transcription factor of interferon signaling, reverted the apoptotic phenotype of Rbbp9−/− organoids. Collectively, our data provide insight into the understanding of the crucial role of RBBP9 in protecting against intestinal inflammation and inflammation-related tumorigenesis via regulating the interferon signaling-mediated apoptotic pathway. Citation Format: Yuki Nakanishi, Kensuke Hamada, Mayuki Omatsu, Kosuke Iwane, Hiroki Kitamoto, Maokoto Okabe, Yu Muta, Shuji Yamamoto, Akihisa Fukuda, Hiroaki Kasashima, Hiroshi Seno. Retinoblastoma binding protein 9 regulates intestinal inflammation and inflammation-associated carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1440.

HER2 phosphorylation induced by TGF-β promotes mammary morphogenesis and breast cancer progression.

Transforming growth factor β (TGF-β) and HER2 signaling collaborate to promote breast cancer progression. However, their molecular interplay is largely unclear. TGF-β can activate mitogen-activated protein kinase (MAPK) and AKT, but the underlying mechanism is not fully understood. In this study, we report that TGF-β enhances HER2 activation, leading to the activation of MAPK and AKT. This process depends on the TGF-β type I receptor TβRI kinase activity. TβRI phosphorylates HER2 at Ser779, promoting Y1248 phosphorylation and HER2 activation. Mice with HER2 S779A mutation display impaired mammary morphogenesis, reduced ductal elongation, and branching. Furthermore, wild-type HER2, but not S779A mutant, promotes TGF-β-induced epithelial-mesenchymal transition, cell migration, and lung metastasis of breast cells. Increased HER2 S779 phosphorylation is observed in human breast cancers and positively correlated with the activation of HER2, MAPK, and AKT. Our findings demonstrate the crucial role of TGF-β-induced S779 phosphorylation in HER2 activation, mammary gland development, and the pro-oncogenic function of TGF-β in breast cancer progression.

Synergistic efficacy of simultaneous anti-TGF-β/VEGF bispecific antibody and PD-1 blockade in cancer therapy

BackgroundRecently, therapeutic antibodies against programmed cell death 1 (PD-1) and its ligand (PD-L1) have exerted potent anticancer effect in a variety of tumors. However, blocking the PD-1/PD-L1 axis alone is not sufficient to restore normal immune response. Other negative regulators of antitumor immunity, like TGF-β and VEGFA, are also involved in immune escape of tumor cells and induce immunotherapy resistance.MethodsWe developed a novel anti-TGF-β/VEGF bispecific antibody Y332D based on the Nano-YBODY™ technology platform. The CCK-8, flow cytometry, SBE4 luciferase reporter assay, western blotting and transwell assays were used to measure the biological activities of the anti-TGF-β moiety. The NFAT luciferase reporter assay, luminescent cell viability assay and tube formation assay were used to measure the biological activities of the anti-VEGF moiety. The in vivo anticancer efficacy of Y332D alone or in combination with PD-1 blockade was evaluated in H22, EMT-6, 4T1, and AKT/Ras-driven murine hepatocellular carcinoma tumor models. Immunofluorescent staining, flow cytometry, RNA-seq and quantitative RT-PCR were adopted to analyze the alterations in the tumor microenvironment.ResultsY332D could maintain specific binding affinities for TGF-β and VEGFA. Y332D almost entirely counteracted the in vitro biological functions of TGF-β and VEGFA, including immunosuppression, activated TGF-β signaling, epithelial-mesenchymal transition (EMT), activated VEGF/VEGFR signaling, HUVEC proliferation and tube formation. The in vivo experiment data demonstrated that Y332D was more effective in inhibiting tumor growth and metastasis than anti-TGF-β and anti-VEGF monotherapies. In combination therapies, Y332D plus PD-1 blockade exhibited the most potent and durable anticancer effect. Mechanistically, Y332D plus PD-1 blockade upregulated the density and function of tumor-infiltrating lymphocytes and exerted reinvigorated antitumor immunity.ConclusionY332D could simultaneously block TGF-β and VEGF signalings. In comparison with the monotherapies, Y332D combined with PD-1 blockade exerts superior antitumor effect through improving immune microenvironment.

Abstract P2106: Transforming Growth Factor β-dependent Regulation Of Cardiomyocyte Maturation

Transforming growth factor β (TGFβ) is a secreted growth factor that is sequestered to the ECM as a latent complex and released in response to cardiac stress. Once released, TGFβ is required for conversion of quiescent fibroblasts to myofibroblasts that support cardiac repair and fibrosis through production of ECM components. While these functional features of TGFβ are well known, the fibroblast-independent effector functions of TGFβ in the heart are unknown. We hypothesized that TGFβ is a critical ECM-cell cross talk mediator in the heart whereby cardiomyocyte -generated TGFβ communicates with fibroblasts to program the composition and stability of the ECM in a reinforcing feedback network. We found that deletion of TGFβ ligands ( Tgfb1 , Tgfb2 , and Tgfb3 ) from cardiomyocytes with an α-MHC-Cre transgene ( Tgfb123 fl/fl -αMHC-Cre ), but not a fibroblast-specific targeted allele ( Tgfb123 fl/fl -Tcf21-MCM ), results in cardiac dysfunction. These results suggest that cardiomyocytes (CMs) are the predominant source of TGFβ in the heart at baseline and that perhaps celltypes beyond the fibroblast were also involved. Interestingly, fibroblast numbers and ECM deposition are increased in Tgfb123 fl/fl -αMHC-Cre hearts and with acute injury hearts from these mice produce shorter, disorganized collagen fibers. Gene expression profiling of Tgfb123 fl/fl -αMHC-Cre hearts at 6 weeks of age demonstrates defective cardiomyocyte maturation. Indeed, Tgfb123 fl/fl -αMHC-Cre hearts are dysmorphic at neonatal day 1 and cardiomyocytes from these hearts remain in cell cycle for a longer duration with greater levels of mononucleation. However, deleting TGFβ receptors I/II or Smad2/3 from CMs does not recapitulate this phenotype suggesting that TGFβ is not driving CM maturation in an autocrine manner. Instead, these findings indicate that TGFβ functions as a critical cross-talk mediator to fibroblasts to establish proper ECM organization and content that drives CM maturation.

Role of transforming growth factor-β in peripheral nerve regeneration.

Injuries caused by trauma and neurodegenerative diseases can damage the peripheral nervous system and cause functional deficits. Unlike in the central nervous system, damaged axons in peripheral nerves can be induced to regenerate in response to intrinsic cues after reprogramming or in a growth-promoting microenvironment created by Schwann cells. However, axon regeneration and repair do not automatically result in the restoration of function, which is the ultimate therapeutic goal but also a major clinical challenge. Transforming growth factor (TGF) is a multifunctional cytokine that regulates various biological processes including tissue repair, embryo development, and cell growth and differentiation. There is accumulating evidence that TGF-β family proteins participate in peripheral nerve repair through various factors and signaling pathways by regulating the growth and transformation of Schwann cells; recruiting specific immune cells; controlling the permeability of the blood-nerve barrier, thereby stimulating axon growth; and inhibiting remyelination of regenerated axons. TGF-β has been applied to the treatment of peripheral nerve injury in animal models. In this context, we review the functions of TGF-β in peripheral nerve regeneration and potential clinical applications.

Gambogic acid inhibits tumor metastasis through blocking matrix metalloproteinases mediated TGF-β1 activation

ContextTumor metastasis is the major death factor in cancer patients. Transforming growth factor-β (TGF-β) is an important cytokine promoting tumor metastasis and blocking TGF-β shows crucial antitumor application prospects. In the tumor microenvironment (TME), TGF-β is always highly activated and matrix metalloproteinases (MMPs) are an important TGF-β promoting factor. It gives us a crucial inspiration that TGF-β in TME can be selectively inhibited by specifically correcting TGF-β promoters in TME. Gambogic acid (GA) is an anti-tumor natural product with the activity to inhibit tumor metastasis by preventing the expression of MMP-2, 9. In this study, we explored whether GA could inhibit tumor metastasis by blocking MMPs mediated TGF-β activation. MethodsTwo extremely aggressive tumor cell lines, B16F10 melanoma and 4T1 breast carcinoma were chosen for this study. The in vitro protein expression level and mRNA level of MMPs were determined by ELISA and RT-PCR respectively. The cell mobility, invasion rate, and morphological changes with various treatments were depicted via scratches, transwell assay and immunofluorescence respectively. For further in vivo confirmation, animal models of melanoma, as well as breast cancer, were respectively established using the two cell lines, and metastasis together with related protein expression levels were estimated after treatment with GA. ResultsGA could suppress the expression of MMP-2, 3, 9, which further blocked TGF-β activation. Meanwhile, GM6001 presented a consistent effect. Via inhibiting MMPs-mediated TGF-β upregulation, both GA and GM6001 could suppress the migration and invasion ability of tumor cells in vitro and inhibits tumor metastasis in vivo. Also, GA and GM6001 could downregulate TGF-β downstream signaling pathway. Therefore, the present study provide an important enlightenment for tumor control by specifically blocking TGF-β in TME without affecting normal function of TGF-β. ConclusionsGA can inhibit tumor metastasis by blocking MMPs mediated TGF-β activation. Specifically correcting complicated pro-TGF-β factors in TME may be a more appropriate strategy to suppress metastasis without affecting TGF-β normal function.

Targeting nano-regulator based on metal–organic frameworks for enhanced immunotherapy of bone metastatic prostate cancer

Bone metastasis is the main cause of death in patients with prostate cancer (PCa), but there lacks effective treatment method. Immunotherapy shows new hopes for bone metastatic PCa patients, while the efficacy is still unsatisfactory and limited by the unique immunosuppressive microenvironment in metastatic bone site. Here, we developed a bone-targeted nano-delivery system as a nano-regulator to enhance the immunotherapy of bone metastatic PCa. The nanosystem was assembled via coordination between phytic acid (PA) and Fe3+ to form nano-sized metal–organic framework (MOF), through which mitoxantrone (MTO) was encapsulated. At cellular level, the nanosystem showed selective cytotoxicity towards RM-1 PCa cells over immune cells, and could induce tumor cells immunogenic cell death (ICD) to improve the immunogenicity of the tumor. Moreover, the nanosystem was able to induce ubiquitination of TGFβ receptor (TβR) on immune cells to promote its degradation, thus serving as a nano-regulator to block the functions of TGF-β, an abundant cytokine that has a systematically immunosuppressive effect in the tumor microenvironment. Upon intravenous injection, the nanoparticle showed pro-longed blood circulation and targeting accumulation into bone metastatic site, and imposed robust anti-tumor effect in combination with αCTLA-4. In addition, bone destruction was significantly alleviated after treatment to reduce the skeletal-related events. Overall, this work provides a biocompatible nanomedicine to restore immune sensitivity of bone metastatic tumor for enhanced immunotherapy by blocking TGF-β signaling pathway.

Abstract LB097: Armored bicistronic CAR T cells with dominant-negative TGF-β receptor II to alleviate antigenic heterogeneity and suppressive immune microenvironment in glioblastoma

Abstract Introduction: We have completed two CAR T cell clinical trials for glioblastoma (GBM) and have identified several key challenges to therapeutic efficacy, including the inherently heterogenous genomic landscape and the immunosuppressive tumor microenvironment (TME) found in GBM. Our previous study showed that EGFR variant III (EGFRvIII)-targeting monovalent CAR T cells reduced target-positive tumor cell populations, but tumor recurrence resulted from target-negative tumor cells, highlighting the limitation of single-target approaches in heterogenous tumors. With regards to the highly immunosuppressive TME in GBM, we found that transforming growth factor-β (TGFβ) was present in the GBM TME as a major driver of suppression of the anti-GBM response in clinical samples. TGFb is consistently highly expressed in both GBM tumor cell lines and patient tumor tissues. Methods: We used two parallel scFv constructs, independently targeting both IL13Rα2 and EGFRvIII, in combination with a truncated dominant negative (dn) TGFβ receptor II. This trivalent construct was designed to explore possible additive effects in both in vitro and in vivo GBM model systems to limit tumor escape and overcome the immunosuppressive GBM TME. The CART-EGFR-IL13Rα2-dnTGFb construct broadened the targeted tumor cell repertoire, blocked TGFβ signaling, and served as a sink for free TGFβ in the GBM TME to overcome the suppressive function of TGFβ. Results: The tri-modular CAR T construct had an enhanced proliferative response when compared with the CART-EGFR-IL13Rα2 construct, in vitro. In co-culture assays, this construct led to reduced PD-1 expression and increased central memory phenotype, when compared to the bicistronic CAR T construct, which suggested a lower fraction of exhausted T cells. Tri-modular CAR T cells blocked the suppressive pSmad2/3 signaling pathway, leading to the increased tumor killing activity in co-culture experiments with both adherent and suspension GBM cell lines. In an immunodeficient mouse model, tri-modular CAR T cells eradicated tumor cells efficiently and mice had a longer median survival when compared those treated with the bicistronic CART-EGFR-IL13Rα2 cells, lacking the dnTGFb receptor II. Conclusion: Overcoming the adaptive changes in the local TME and addressing antigen heterogeneity will be required to improve the clinical efficacy of CAR T-directed strategies. Our combination work showed that bicistronic CART constructs cooperate with truncated TGFβ receptor II efficiently. In summary, the dominant-negative TGFβ RII CART-EGFR-IL13Rα2 structure is a promising strategy to address the clinical challenges of antigenic heterogeneity and the immunosuppressive TME in GBM we have observed in our two GBM CART cell trials at UPenn. Citation Format: Nannan Li, Jesse Rodriguez, Zev Binder, Donald O’Rourke. Armored bicistronic CAR T cells with dominant-negative TGF-β receptor II to alleviate antigenic heterogeneity and suppressive immune microenvironment in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB097.

Regulation of T Cell Activation and Metabolism by Transforming Growth Factor-Beta.

Transforming growth factor beta (TGFβ) receptor signalling regulates T cell development, differentiation and effector function. Expression of the immune-associated isoform of this cytokine, TGFβ1, is absolutely required for the maintenance of immunological tolerance in both mice and humans, whilst context-dependent TGFβ1 signalling regulates the differentiation of both anti- and pro-inflammatory T cell effector populations. Thus, distinct TGFβ-dependent T cell responses are implicated in the suppression or initiation of inflammatory and autoimmune diseases. In cancer settings, TGFβ signals contribute to the blockade of anti-tumour immune responses and disease progression. Given the key functions of TGFβ in the regulation of immune responses and the potential for therapeutic targeting of TGFβ-dependent pathways, the mechanisms underpinning these pleiotropic effects have been the subject of much investigation. This review focuses on accumulating evidence suggesting that modulation of T cell metabolism represents a major mechanism by which TGFβ influences T cell immunity.

Microvascular significance of TGF-β axis activation in COVID-19.

As 2023 approaches, the COVID-19 pandemic has killed millions. While vaccines have been a crucial intervention, only a few effective medications exist for prevention and treatment of COVID-19 in breakthrough cases or in unvaccinated or immunocompromised patients. SARS-CoV-2 displays early and unusual features of micro-thrombosis and immune dysregulation that target endothelial beds of the lungs, skin, and other organs. Notably, anticoagulation improves outcomes in some COVID-19 patients. The protein transforming growth factor-beta (TGF-β1) has constitutive roles in maintaining a healthy microvasculature through its roles in regulating inflammation, clotting, and wound healing. However, after infection (including viral infection) TGF-β1 activation may augment coagulation, cause immune dysregulation, and direct a path toward tissue fibrosis. Dysregulation of TGF-β signaling in immune cells and its localization in areas of microvascular injury are now well-described in COVID-19, and such events may contribute to the acute respiratory distress syndrome and skin micro-thrombosis outcomes frequently seen in severe COVID-19. The high concentration of TGF-β in platelets and in other cells within microvascular thrombi, its ability to activate the clotting cascade and dysregulate immune pathways, and its pro-fibrotic properties all contribute to a unique milieu in the COVID-19 microvasculature. This unique environment allows for propagation of microvascular clotting and immune dysregulation. In this review we summarize the physiological functions of TGF-β and detail the evidence for its effects on the microvasculature in COVID-19. In addition, we explore the potential role of existing TGF-β inhibitors for the prevention and treatment of COVID-19 associated microvascular thrombosis and immune dysregulation.

TGF-β as A Master Regulator of Aging-Associated Tissue Fibrosis.

Fibrosis is the abnormal accumulation of extracellular matrix proteins such as collagen and fibronectin. Aging, injury, infections, and inflammation can cause different types of tissue fibrosis. Numerous clinical investigations have shown a correlation between the degree of liver and pulmonary fibrosis in patients and telomere length and mitochondrial DNA content, both of which are signs of aging. Aging involves the gradual loss of tissue function over time, which results in the loss of homeostasis and, ultimately, an organism's fitness. A major feature of aging is the accumulation of senescent cells. Senescent cells abnormally and continuously accumulate in the late stages of life, contributing to age-related fibrosis and tissue deterioration, among other aging characteristics. Furthermore, aging generates chronic inflammation, which results in fibrosis and decreases organ function. This finding suggests that fibrosis and aging are closely related. The transforming growth factor-beta (TGF-β) superfamily plays a crucial role in the physiological and pathological processes of aging, immune regulation, atherosclerosis, and tissue fibrosis. In this review, the functions of TGF-β in normal organs, aging, and fibrotic tissues is discussed: TGF-β signalling is altered with age and is an indicator of pathology associated with tissue fibrosis. In addition, this review discusses the potential targeting of noncoding.

TGFβ control of immune responses in cancer: a holistic immuno-oncology perspective.

The immune system responds to cancer in two main ways. First, there are prewired responses involving myeloid cells, innate lymphocytes and innate-like adaptive lymphocytes that either reside in premalignant tissues or migrate directly to tumours, and second, there are antigen priming-dependent responses, in which adaptive lymphocytes are primed in secondary lymphoid organs before homing to tumours. Transforming growth factor-β (TGFβ) - one of the most potent and pleiotropic regulatory cytokines - controls almost every stage of the tumour-elicited immune response, from leukocyte development in primary lymphoid organs to their priming in secondary lymphoid organs and their effector functions in the tumour itself. The complexity of TGFβ-regulated immune cell circuitries, as well as the contextual roles of TGFβ signalling in cancer cells and tumour stromal cells, necessitates the use of rigorous experimental systems that closely recapitulate human cancer, such as autochthonous tumour models, to uncover the underlying immunobiology. The diverse functions ofTGFβ in healthy tissues further complicate the search for effective and safe cancer therapeutics targeting the TGFβ pathway. Here we discuss the contextual complexity of TGFβ signalling in tumour-elicited immune responses and explain how understanding this may guide the development of mechanism-based cancer immunotherapy.

The transforming growth factor beta ligand TIG-2 modulates the function of neuromuscular junction and muscle energy metabolism in Caenorhabditis elegans.

Deciphering the physiological function of TGF-β (the transforming growth factor beta) family ligands is import for understanding the role of TGF-β in animals' development and aging. Here, we investigate the function of TIG-2, one of the ligands in Caenorhabditis elegans TGF-β family, in animals' behavioral modulation. Our results show that a loss-of-function mutation in tig-2 gene result in slower locomotion speed in the early adulthood and an increased density of cholinergic synapses, but a decreased neurotransmitter release at neuromuscular junctions (NMJs). Further tissue-specific rescue results reveal that neuronal and intestinal TIG-2 are essential for the formation of cholinergic synapses at NMJs. Interestingly, tig-2(ok3416) mutant is characterized with reduced muscle mitochondria content and adenosine triphosphate (ATP) production, although the function of muscle acetylcholine receptors and the morphology muscle fibers in the mutant are comparable to that in wild-type animals. Our result suggests that TIG-2 from different neuron and intestine regulates worm locomotion by modulating synaptogenesis and neurotransmission at NMJs, as well as energy metabolism in postsynaptic muscle cells.

Superwettable and injectable GelMA-MSC microspheres promote cartilage repair in temporomandibular joints.

Temporomandibular disorders (TMD) can be treated by promoting cartilage regeneration with biomaterials. However, there are deficiencies in the infiltration function of bone filler biological materials. In this study, stems cells were loaded onto gelatin methacryloyl (GelMA) hydrogel microspheres endowed with superwettable properties and TGF-β sustained-release function, which can quickly infiltrate the irregular surface of the temporomandibular joint (TMJ) bone defect area and accelerate cartilage healing. First, to improve cell adhesion and spreading function, the BMSCs-coated GelMA microspheres were endowed with superwetting property. At the same time, the swelling adsorption characteristics of gelatin microspheres could be used to load recombinant TGF-β within the microspheres, which could in turn promote the chondrogenic differentiation of multi-potent bone marrow mesenchymal stem cells. The SEM imaging demonstrated that BMSCs-coated GelMA microsphere has superwettable and superhydrophilic property, which enabled rapid adaptation to the bone defect surface morphology, which is conducive to tissue repair. Furthermore, the cartilage defect model showed that rBMSCs-coated GelMA microspheres promote temporomandibular joint arthritis repair. In conclusion, our study established that BMSC-coated GelMA microspheres endowed with superwetting properties, can colonize the bone defect repair site better with sustained release of growth factors, thus providing an innovative strategy for promoting cartilage regeneration.

Lack of extracellular matrix switches TGF-β induced apoptosis of endometrial cells to epithelial to mesenchymal transition

The extracellular matrix and the correct establishment of epithelial cell polarity plays a critical role in epithelial cell homeostasis and cell polarity. In addition, loss of tissue structure is a hallmark of carcinogenesis. In this study, we have addressed the role of extracellular matrix in the cellular responses to TGF-β. It is well known that TGF-β is a double-edged sword: it acts as a tumor suppressor in normal epithelial cells, but conversely has tumor-promoting effects in tumoral cells. However, the factors that determine cellular outcome in response to TGF-β remain controversial. Here, we have demonstrated that the lack of extracellular matrix and consequent loss of cell polarity inhibits TGF-β-induced apoptosis, observed when endometrial epithelial cells are polarized in presence of extracellular matrix. Rather, in absence of extracellular matrix, TGF-β-treated endometrial epithelial cells display features of epithelial-to-mesenchymal transition. We have also investigated the molecular mechanism of such a switch in cellular response. On the one hand, we found that the lack of Matrigel results in increased AKT signaling which is sufficient to inhibit TGF-β-induced apoptosis. On the other hand, we demonstrate that TGF-β-induced epithelial-to-mesenchymal transition requires ERK and SMAD2/3 activation. In summary, we demonstrate that loss of cell polarity changes the pro-apoptotic function of TGF-β to tumor-associated phenotype such as epithelial-to-mesenchymal transition. These results may be important for understanding the dual role of TGF-β in normal versus tumoral cells.

Latent transforming growth factor β binding protein 3 controls adipogenesis

Transforming growth factor-beta (TGFβ) is released from cells as part of a trimeric latent complex consisting of TGFβ, the TGFβ propeptides, and either a latent TGFβ binding protein (LTBP) or glycoprotein-A repetitions predominant (GARP) protein. LTBP1 and 3 modulate latent TGFβ function with respect to secretion, matrix localization, and activation and, therefore, are vital for the proper function of the cytokine in a number of tissues. TGFβ modulates stem cell differentiation into adipocytes (adipogenesis), but the potential role of LTBPs in this process has not been studied. We observed that 72 h post adipogenesis initiation Ltbp1, 2, and 4 expression levels decrease by 74–84%, whereas Ltbp3 expression levels remain constant during adipogenesis. We found that LTBP3 silencing in C3H/10T1/2 cells reduced adipogenesis, as measured by the percentage of cells with lipid vesicles and the expression of the transcription factor peroxisome proliferator-activated receptor gamma (PPARγ). Lentiviral mediated expression of an Ltbp3 mRNA resistant to siRNA targeting rescued the phenotype, validating siRNA specificity. Knockdown (KD) of Ltbp3 expression in 3T3-L1, M2, and primary bone marrow stromal cells (BMSC) indicated a similar requirement for Ltbp3. Epididymal and inguinal white adipose tissue fat pad weights of Ltbp3−/- mice were reduced by 62% and 57%, respectively, compared to wild-type mice. Inhibition of adipogenic differentiation upon LTBP3 loss is mediated by TGFβ, as TGFβ neutralizing antibody and TGFβ receptor I kinase blockade rescue the LTBP3 KD phenotype. These results indicate that LTBP3 has a TGFβ-dependent function in adipogenesis both in vitro and possibly in vivo. SignificanceUnderstanding the control of mesenchymal stem cell fate is crucial for the potential use of these cells for regenerative medicine.

Deregulation of Slug-Snail2 and TGF-β crosstalk in airway epithelial stem-progenitor cells: A key link between COPD and lung cancer?

This commentary focuses on two recent publications showing deregulation of the transcription factor Slug/Snail2 and TGF-β function in primary bronchial basal/progenitor cells of patients with Chronic Obstructive Pulmonary Disease (COPD) and the impact on proliferation and the expression of genes involved in stem cell maintenance.

The Role of Transforming Growth Factor Beta in Joint Homeostasis and Cartilage Regeneration.

Transforming growth factor-beta (TGF-β) is an important regulator of joint homeostasis, of which dysregulation is closely associated with the development of osteoarthritis (OA). In normal conditions, its biological functions in a joint environment are joint protective, but it can be dramatically altered in different contexts, making its therapeutic application a challenge. However, with the deeper insights into the TGF-β functions, it has been proven that TGF-β augments cartilage regeneration by chondrocytes, and differentiates both the precursor cells of chondrocytes and stem cells into cartilage-generating chondrocytes. Following documentation of the therapeutic efficacy of chondrocytes augmented by TGF-β in the last decade, there is an ongoing phase III clinical trial examining the therapeutic efficacy of a mixture of allogeneic chondrocytes and TGF-β-overexpressing cells. To prepare cartilage-restoring chondrocytes from induced pluripotent stem cells (iPSCs), the stem cells are differentiated mainly using TGF-β with some other growth factors. Of note, clinical trials evaluating the therapeutic efficacy of iPSCs for OA are scheduled this year. Mesenchymal stromal stem cells (MSCs) have inherent limitations in that they differentiate into the osteochondral pathway, resulting in the production of poor-quality cartilage. Despite the established essential role of TGF-β in chondrogenic differentiation of MSCs, whether the coordinated use of TGF-β in MSC-based therapy for degenerated cartilage is effective is unknown. We herein reviewed the general characteristics and mechanism of action of TGF-β in a joint environment. Furthermore, we discussed the core interaction of TGF-β with principal cells of OA cell-based therapies, the chondrocytes, MSCs, and iPSCs. Impact Statement Transforming growth factor-beta (TGF-β) has been widely used as a core regulator to improve or formulate therapeutic regenerative cells for degenerative joints. It differentiates stem cells into chondrocytes and improves the chondrogenic potential of differentiated chondrocytes. Herein, we discussed the overall characteristics of TGF-β and reviewed the comprehension and utilization of TGF-β in cell-based therapy for degenerative joint disease.

Abstract 1568: PLK activated TGF-β functions as a prognostic biomarker for patients with pancreatic ductal adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer death with a 5-year survival rate of approximately 7%. Transforming growth factor beta (TGF-β) is highly involved in progression and metastasis in multiple cancers including PDAC. TGF-β is highly involved in tumor fibrosis and immune suppression, and several clinical trials are ongoing to evaluate anti-TGF-β therapies for treating patients diagnosed with cancer and for which biomarkers are key for success. TGF-β is produced in a latent complex with latency-associated protein (LAP), which needs to be cleaved by plasma kallikrein (PLK), releasing TGF-β to enable receptor binding and initiating of signaling. We developed a competitive enzyme-linked immunosorbent assay (ELISA) targeting PLK activated TGF-β and measured it in serum from patients with PDAC. Methods: An ELISA based on monoclonal antibodies raised against the C-terminal side of the PLK cleavage site of TGF-β at R’58↓ was developed and validated for quantification of PLK activated TGF-β (L59TGF-β) in serum. To confirm that the ELISA was specifically measuring PLK cleaved TGF-β, L59TGF-β was measured in a TGF-β dilution after incubation with and without kallikrein. L59TGF-β was measured in pretreatment serum samples from patients with PDAC (n=39) and healthy controls (n=20). The prognostic value of L59TGF-β levels was examined with Kaplan-Meier analysis and evaluated for association with overall survival (OS) by multivariate cox regression analysis adjusted for age and cancer stage. Results: L59TGF-β was only detected after incubation with kallikrein, demonstrating that the ELISA specifically detected kallikrein cleaved TGF-β. L59TGF-β was significantly elevated in patients with PDAC compared to healthy controls (p<0.0001). High levels of L59TGF-β (>median) were significantly associated with poor OS (HR: 2.71, 95%CI: 1.23-5.97, p=0.0135) in patients with PDAC, and median OS was 5.5 months (95%CI: 1.50-20.0) compared to 26.3 months (95%CI: 15.3-28.1) for patients with low levels of L59TGF-β. The association between high levels of L59TGF-β and poor OS remained statistically significant after adjusting for age and stage (HR: 2.33, 95%CI: 1.05-5.18, p=0.0372). Conclusion: Quantification of PLK activated TGF-β (L59TGF-β) in serum predicted poor OS for patients with PDAC. The biomarker value of L59TGF-β should be validated and further investigated in a larger cohort to explore the clinical applicability of the biomarker. Citation Format: Rasmus S. Pedersen, Neel I. Nissen, Christina Jensen, Jeppe Thorlacius-Ussing, Majken L. Olesen, Lasse Langholm, Tina M. Jensen, Hadi M. Diab, Lars N. Jørgensen, Morten Karsdal, Nicholas Willumsen. PLK activated TGF-β functions as a prognostic biomarker for patients with pancreatic ductal adenocarcinoma [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 1568.