Molecular characterization of transforming growth factor-beta3

Normal tissue homeostasis is controlled by a critical balance of positive and negative modulators. Chapter 2 gives an overview of the molecular aspects of growth control, in particular the role of growth factors and oncogene and anti-oncogene products. Uncontrolled growth of cancer cells may result from either an abrogation of growth stimulatory or a deficiency of growth inhibitory pathways. Mediators of growth inhibition include secretory polypeptide growth inhibitors, like transforming growth factor β(TGF-β) and nuclear proteins, like the retinoblastoma gene product. Early studies on organogenesis suggested the presence of growth inhibitors (challones) to regulate the growth of organs. Postulating that growth inhibitory proteins might have potential in cancer therapy, we began to analyze human tissues for the presence of novel tumor inhibitory factors. Purification of these activities and physico-chemical characterization suggested a relatedness to TGF-β. The biochemistry and cell biology of (TGF-β) will be reviewed in Chapter 3.At the start of the investigation, only one (TGF-β) had been identified. Our subsequent results indicated that a family of TGF-βproteins exists. Conventional purification of these TGF-βlike activities provided only limited quantities of material for analysis. We therefore adopted an alternative strategy which included the isolation of the cDNAs for TGF-β-like factors using TGF-β1 as a probe, assuming that related molecules might possess sufficient sequence similarity to cross-hybridize to a TGF-β1 probe. Differential hybridization of a Southern blot with human genomic DNA probed with TGF-β1 cDNA suggested the presence of a related gene, which we termed TGF-β3. The research described in this thesis includes the molecular cloning and expression of TGF-β3. Furthermore, experiments were carried out to gain insight into the effects of TGF-β3 on cell growth and differentiation and its mechanism of action, including initial studies to gauge the potential therapeutic uses of this factor.In Chapter 4, we report the cloning of the human TGF-β3 cDNA and the encoded TGF-β3 protein is compared with other members of the TGF-βfamily. In Chapter 5 the interspecies conservation of TGF-B3 is examined and the chromosomal location of the human TGF-β3 gone is determined. In Chapter 6, the recombinant expression and purification of TGF-β3 is described. The purified TGF-β3 protein has potent growth modulating effects on a number of normal as well as tumor cells. The studies in Chapter 7 were performed to assess the effect of TGF-β3 on osteoblasts and to characterize the specific binding of TGF-β3 to bone cells. TGF-β3 appears to be a potent regulator of functions associated with bone formation. Crosslinking studies showed that TGF-β3 and TGF-β1 associate in a similar fashion with three cell surface binding proteins, which have been characterized as putative receptor types I and II and a membrane-bound proteoglycan, termed betaglycan. The different (TGF-β) isoforms appear to have different potencies on Mv1Lu mink lung epithelia] and fetal bovine heart endothelial cells. In Chapter 8, we investigate the role of TGF-β. receptors and serum factors as determinants of the cell-specific responsiveness to the three homodimeric isoforms. The induction of mesoderm in Xenopus laevis animal cap explants by TGF-β3 is discussed in Chapter 9. Finally, in Chapter 10 we review the therapeutic applications of growth factors for wound healing

Targeting TGF beta signal transduction for cancer therapy

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 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.Cancer Signaling networks and Molecular Therapeutic

E3 ubiquitin ligases: key regulators of TGF beta signaling in cancer progression

Transforming growth factor beta (TGF beta) is a secreted growth and differentiation factor that influences vital cellular processes like proliferation, adhesion, motility, and apoptosis. Regulation of the TGF beta signaling pathway is of key importance to maintain tissue homeostasis. Perturbation of this signaling pathway has been implicated in a plethora of diseases, including cancer. The effect of TGF beta is dependent on cellular context, and TGF beta can perform both anti- and pro-oncogenic roles. TGF beta acts by binding to specific cell surface TGF beta type I and type II transmembrane receptors that are endowed with serine/threonine kinase activity. Upon ligand-induced receptor phosphorylation, SMAD proteins and other intracellular effectors become activated and mediate biological responses. The levels, localization, and function of TGF beta signaling mediators, regulators, and effectors are highly dynamic and regulated by a myriad of post-translational modifications. One such crucial modification is ubiquitination. The ubiquitin modification is also a mechanism by which crosstalk with other signaling pathways is achieved. Crucial effector components of the ubiquitination cascade include the very diverse family of E3 ubiquitin ligases. This review summarizes the diverse roles of E3 ligases that act on TGF beta receptor and intracellular signaling components. E3 ligases regulate TGF beta signaling both positively and negatively by regulating degradation of receptors and various signaling intermediates. We also highlight the function of E3 ligases in connection with TGF beta's dual role during tumorigenesis. We conclude with a perspective on the emerging possibility of defining E3 ligases as drug targets and how they may be used to selectively target TGF beta-induced pro-oncogenic responses.Cancer Signaling networks and Molecular Therapeutic

BMP type II receptor as a therapeutic target in pulmonary arterial hypertension

Cancer Signaling networks and Molecular Therapeutic

Cancer associated-fibroblast-derived exosomes in cancer progression

To identify novel cancer therapies, the tumor microenvironment (TME) has received a lot of attention in recent years in particular with the advent of clinical successes achieved by targeting immune checkpoint inhibitors (ICIs). The TME consists of multiple cell types that are embedded in the extracellular matrix (ECM), including immune cells, endothelial cells and cancer associated fibroblasts (CAFs), which communicate with cancer cells and each other during tumor progression. CAFs are a dominant and heterogeneous cell type within the TME with a pivotal role in controlling cancer cell invasion and metastasis, immune evasion, angiogenesis and chemotherapy resistance. CAFs mediate their effects in part by remodeling the ECM and by secreting soluble factors and extracellular vesicles. Exosomes are a subtype of extracellular vesicles (EVs), which contain various biomolecules such as nucleic acids, lipids, and proteins. The biomolecules in exosomes can be transmitted from one to another cell, and thereby affect the behavior of the receiving cell. As exosomes are also present in circulation, their contents can also be explored as biomarkers for the diagnosis and prognosis of cancer patients. In this review, we concentrate on the role of CAFs-derived exosomes in the communication between CAFs and cancer cells and other cells of the TME. First, we introduce the multiple roles of CAFs in tumorigenesis. Thereafter, we discuss the ways CAFs communicate with cancer cells and interplay with other cells of the TME, and focus in particular on the role of exosomes. Then, we elaborate on the mechanisms by which CAFs-derived exosomes contribute to cancer progression, as well as and the clinical impact of exosomes. We conclude by discussing aspects of exosomes that deserve further investigation, including emerging insights into making treatment with immune checkpoint inhibitor blockade more efficient.Cancer Signaling networks and Molecular Therapeutic

Therapeutic targeting of TGF-β in cancer: hacking a master switch of immune suppression

Cancers may escape elimination by the host immune system by rewiring the tumour microenvironment towards an immune suppressive state. Transforming growth factor-β (TGF-β) is a secreted multifunctional cytokine that strongly regulates the activity of immune cells while, in parallel, can promote malignant features such as cancer cell invasion and migration, angiogenesis, and the emergence of cancer-associated fibroblasts. TGF-β is abundantly expressed in cancers and, most often, its abundance associated with poor clinical outcomes. Immunotherapeutic strategies, particularly T cell checkpoint blockade therapies, so far, only produce clinical benefit in a minority of cancer patients. The inhibition of TGF-β activity is a promising approach to increase the efficacy of T cell checkpoint blockade therapies. In this review, we briefly outline the immunoregulatory functions of TGF-β in physiological and malignant contexts. We then deliberate on how the therapeutic targeting of TGF-β may lead to a broadened applicability and success of state-of-the-art immunotherapies.Cancer Signaling networks and Molecular Therapeutic

SUMO-triggered ubiquitination of NR4A1 controls macrophage cell death

Cancer Signaling networks and Molecular Therapeutic

TGF-beta-mediated endothelial to mesenchymal transition (EndMT) and the functional assessment of EndMT effectors using CRISPR/Cas9 gene editing

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-beta), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-beta-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-beta-induced EndMT. Using these techniques, we provide evidence that TGF-beta 2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.Cancer Signaling networks and Molecular Therapeutic

Metabolic reprogramming of mammary epithelial cells during TGF-beta-induced epithelial-to-mesenchymal transition

The cytokine transforming growth factor-beta (TGF-beta) can induce normal breast epithelial cells to take on a mesenchymal phenotype, termed epithelial-to-mesenchymal transition (EMT). While the transcriptional and proteomic changes during TGF-beta-induced EMT have been described, the metabolic rewiring that occurs in epithelial cells undergoing EMT is not well understood. Here, we quantitively analyzed the TGF-beta-induced metabolic reprogramming during EMT of non-transformed NMuMG mouse mammary gland epithelial cells using nuclear magnetic resonance (NMR) spectroscopy. We found that TGF-beta elevates glycolytic and tricarboxylic acid (TCA)-cycle activity and increases glutaminolysis. Additionally, TGF-beta affects the hexosamine pathway, arginine-proline metabolism, the cellular redox state, and strongly affects choline metabolism during EMT. TGF-beta was found to induce phosphocholine production. A kinase inhibitor RSM-93A that inhibits choline kinase alpha (CHK alpha) mitigated TGF-beta-induced changes associated with EMT, i.e., increased filamentous (F)-actin stress fiber formation and N-Cadherin mesenchymal marker expression.Cancer Signaling networks and Molecular Therapeutic