TGF-β Antagonists Research Articles

Bone Sialoprotein Mediates the Tumor Cell–Targeted Prometastatic Activity of Transforming Growth Factor β in a Mouse Model of Breast Cancer

Transforming growth factor betas (TGF-beta) play a dual role in carcinogenesis, functioning as tumor suppressors early in the process, and then switching to act as prometastatic factors in late-stage disease. We have previously shown that high molecular weight TGF-beta antagonists can suppress metastasis without the predicted toxicities. To address the underlying mechanisms, we have used the 4T1 syngeneic mouse model of metastatic breast cancer. Treatment of mice with a monoclonal anti-TGF-beta antibody (1D11) significantly suppressed metastasis of 4T1 cells to the lungs. When metastatic 4T1 cells were recovered from lungs of 1D11-treated and control mice, the most differentially expressed gene was found to be bone sialoprotein (Bsp). Immunostaining confirmed the loss of Bsp protein in 1D11-treated lung metastases, and TGF-beta was shown to regulate and correlate with Bsp expression in vitro. Functionally, knockdown of Bsp in 4T1 cells reduced the ability of TGF-beta to induce local collagen degradation and invasion in vitro, and treatment with recombinant Bsp protected 4T1 cells from complement-mediated lysis. Finally, suppression of Bsp in 4T1 cells reduced metastasis in vivo. We conclude that Bsp is a plausible mediator of at least some of the tumor cell-targeted prometastatic activity of TGF-beta in this model and that Bsp expression in metastases can be successfully suppressed by systemic treatment with anti-TGF-beta antibodies.

Abrogation of Transforming Growth Factor-β Signaling by SMAD7 Inhibits Collagen Gel Contraction of Human Dermal Fibroblasts

Human fibroproliferative disorders like hypertrophic scarring of the skin are characterized by increased contractility and excess extracellular matrix synthesis. A beneficial role of transforming growth factor (TGF)-beta in wound healing was proposed; however, chronic stimulation by this cytokine leads to fibrosis. In the present report, the intracellular TGF-beta signaling in fibroblasts derived from hypertrophic scars and normal skin was examined. In an attempt to intervene in profibrogenic TGF-beta functions, ectopic expression of Smad7 or dominant negative Smads3/4 completely inhibited contractility of scar-derived and normal fibroblasts after suspension in collagen gels. Both cell types displayed constitutive Smad2/3 phosphorylation and (CAGA)9-MLP-Luc activity with expression and phosphorylation of Smad3 being predominant in hypertrophic scar-derived fibroblasts. Down-regulation of intrinsic signaling with various TGF-beta antagonists, e.g. soluble TGF-beta receptor, latency-associated peptide, and anti-TGF-beta1 antibodies, confirms autocrine TGF-beta stimulation of both cell populations. Further, Smad7 expression inhibited alpha1 (I) collagen and alpha-smooth muscle actin expression. In summary, our data indicate that autocrine TGF-beta/Smad signaling is involved in contractility and matrix gene expression of fibroblasts from normal and hypertrophic scars. Smad7 inhibits these processes and may exert beneficial effects on excessive scar formation.

A microscale assay for the identification of TGF-β antagonists based on functional coupling of the heterodimeric TGF-β receptor to STAT6-driven promoter activation

Inadequate function of Transforming Growth Factor- (TGF-) β is involved in numerous disease states including immune disorders and cancer, rendering this polypeptide and its receptor an attractive target for pharmaceutical interference. We have developed a novel microscale functional test system suited for the investigation of ligand-induced receptor activation and the automatable evaluation of potential agonists and antagonists. Hybrid receptors were constructed from the cytoplasmic domain of the human interleukin-4 (IL-4) receptor α-chain and extracellular domains of a TGF-β type I and type II receptor, respectively. These chimeras were stably introduced into the factor-dependent murine cell line Ba/F3 along with an IL-4-inducible luciferase reporter gene construct, yielding a reporter cell line which responds to productive ligand?receptor interactions by specific luciferase activity in a dose-dependent fashion. A model experiment employing inhibitory peptides demonstrates that the devised reporter cell provides a rational readout for TGF-β activity on target cells and its impairment by specific antagonists.

Development and possible clinical use of antagonists for PDGF and TGF-β

Platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) are examples of signaling molecules which control the growth, survival motility and differentiation of cells. PDGF stimulates the growth mainly of connective tissue cells, whereas TGF-β inhibits the growth of most cell types. PDGF and TGF-β exert their cellular effects by vinding to receptors equipped with tyrosine and serine/threonine kinase activities, respectively. Both factors have important roles e.g. during the embryonal development and in wound healing.Overactivity of PDGF or PDGF receptors contributes to the development of certain diseases characterized by excessive cell growth including fibrotic disorders, atherosclerosis and malignancies. Overactivity of TGF-β also contributes to fibrotic conditions, since TGF-β promotes accumulation of extracellular matrix molecules. In cancer, TGF-β is initially a tumor suppressor due to its ability to inhibit cell growth, however, at later stages of tumor progression TGF-β has tumor promoting activity by enhancing the invasive properties of tumor cells and by suppressing the immune system and promoting angiogenesis.The involvement of PDGF in TGF-β in serious diseases makes clinically useful antagonists highly desirable. A low molecular weight receptor kinase inhibitor of the PDGF receptor kinase is now tested clinically, and TGF-β antagonists are under development. The present review discusses the development and possible clinical use of antagonsts for PDGF and TGF-β.

Use of mouse models to validate and therapeutically target transforming growth factor beta as an important player in breast cancer progression

Transforming growth factor betas (TGF-βs) play key roles in embryogenesis, maintenance of adult homeostasis and response to injury. In epithelial carcinogenesis the TGF-β play complex roles, functioning as tumor suppressors early in the process, but as pro-oncogenic factors in late-stage metastatic disease, when TGF-β ligands are frequently overexpressed. To probe this complexity in vivo, and determine whether TGF-β might be a viable therapeutic target, we developed a transgenic mouse overexpressing a soluble TGF-β antagonist. This antagonist ('SR2F') consisted of the extracellular domain of the type II TGF-β receptor fused to the Fc domain of human IgG1. The SR2F was secreted into the circulation and distributed to all organs except the brain. To determine the effect of this TGF-β antagonist on breast carcinogenesis, the mouse mammary tumor virus (MMTV)-SR2F and wild-type control mice were crossed with the MMTV-neu transgenic mouse model of metastatic breast cancer. The neu/SR2F bigenic mice showed a significant threefold decrease in the incidence of lung metastases compared with mice expressing neu alone. A similar suppression of metastasis was seen in using a tail vein injection model of metastatic melanoma. Importantly, the SR2F did not accelerate primary tumorigenesis, despite the fact that TGF-β has been shown to function as a tumor suppressor in the MMTV-neu model [1]. Furthermore, none of the pathology that is usually associated with TGF-β loss, such as autoimmune disease and increased spontaneous tumorigenesis, was observed on prolonged exposure to SR2F. The mechanistic basis for the unexpected selectivity of the SR2F in antagonizing the pro-metastatic effects of TGF-β while sparing effects on tumor suppression and normal homeostasis is currently not clear, but it does not seem to be a dosage effect. Overall, our data suggest that high molecular weight TGF-β antagonists might have promise in the clinic for prevention of metastasis. This study demonstrates the utility of a transgenic approach for testing expensive protein-based therapeutics in long-term realistic models of cancer progression.

Role of CD80, CD86, and CTLA4 on mouse CD4+ T lymphocytes in enhancing cell-cycle progression and survival after activation with PMA and ionomycin

Cell surface interactions between the T cell costimulatory receptors, CD28 and cytotoxic T-lymphocyte antigen-4 (CTLA4), with their cognate ligands, CD80 and CD86, on antigen-presenting cells play an important role in T cell activation. Although CD80 and CD86 are induced on T cells after activation, not much is known about their role in modulating T cell function. We show that CD80, CD86, and CTLA4 are induced on purified CD4(+) T cells after in vitro activation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, and they play an essential role for proliferation and survival. Blockade of CTLA4-CD80/CD86 interactions greatly reduces PMA and ionomycin-mediated mouse CD4(+) T cell activation. The three key features of this inhibition of activation are: First, late events in T cell activation (after 18 h) are affected; second, these cells do not undergo anergy; and third, CD4(+)CD25(+) regulatory T cells are not responsible. Activation of T cells with PMA and ionomycin together with CTLA4-CD80/CD86 blockade results in decreased induction of CD25 and Bcl-X(L), reduced interleukin (IL)-2, and enhanced transforming growth factor-beta (TGF-beta) production. Furthermore, extended CTLA4-CD80/CD86 blockade results in decreased cell-cycle progression and enhanced apoptosis in a large proportion of cells. This inhibition of T cell proliferation can be rescued completely with anti-CD28 or IL-2 and partially with TGF-beta antagonists. This study reveals a functional role for CD80, CD86, and CTLA4 on CD4(+) T lymphocytes and sheds light on the mechanisms by which these molecules enhance activation and survival with PMA and ionomycin.

Expression of TGFβ2 but Not TGFβ1 Correlates with the Deposition of Scar Tissue in the Lesioned Spinal Cord

Transforming growth factor-βs (TGFβs) are implicated in fibrotic pathologies. TGFβ1 and -β2 expression is increased around the glial/fibrotic scar in the injured brain. Moreover, local injection of TGFβ antagonists into cerebral wounds reduces glial scarring. Here, we monitored expression of TGFβ1 and -β2 mRNA and protein in the spinal cord after transection of the dorsal funiculi. Levels of TGFβ1 mRNA were most elevated over the acute inflammatory phase, while TGFβ2 mRNA levels were raised locally about the wound, particularly in astrocytes and neovascular endothelial cells, over the subacute period of scarring. TGFβ protein production also increased after injury. Both TGFβ1 and TGFβ2 were found in hematogenous inflammatory cells, while TGFβ1 was also neuron-associated, and high levels of TGFβ2 were localized to multiple cell types in the wound, including reactive astrocytes, during the period of glial/collagen scar formation. The cellular localization and temporal pattern of expression of TGFβ after spinal cord injury suggest that TGFβ1 modulates the inflammatory and neuronal responses, while TGFβ2 regulates glial/collagen scarring.

Expression of Recombinant Extracellular Domain of the Type II Transforming Growth Factor-β Receptor: Utilization in a Modified Enzyme-Linked Immunoabsorbent Assay to Screen TGF-β Agonists and Antagonists

TGF-β is a ubiquitous protein that exhibits a broad spectrum of biological activity. The prokaryotic expression and purification of the extracellular domain of the type II TGF-β receptor (TβR-II-ED), without the need for fusion protein cleavage and refolding, is described. The recombinant TβR-II-ED fusion protein bound commercially available TGF-β1 and displayed an affinity of 11.1 nM. In a modified ELISA, receptor binding to TGF-β1 was inhibited by TGF-β3. The technique lends itself to high-throughput screening of combinatorial libraries for the identification of TGF-β agonists and antagonists and this, in turn, may have important therapeutic implications.

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The results of our study suggest that inactivation of the TGF-β signaling pathway is a common and important event in colon cancer pathogenesis and that mutation of TGF-βRII is the responsible mechanism in one third of all colon cancer cases. The mechanism causing TGF-β resistance in the remaining cases is still unknown and is an important area for active investigation. Mutations in the Smad family members, hSmad2, 3, and 4, may explain a portion of the additional TGF-β–resistant tumors; however, the low incidence thus far detected of hSmad 2, 3, and 4 mutations in human colon cancer implies hSmad mutations alone will not account for all the remaining cases. The results of our study raise the question of what pathophysiological advantage accrues to tumors from inactivating the TGF-β signaling pathway. Work from our group has shown RII mutations develop late in the adenoma to carcinoma sequence concurrent with the acquisition of malignant behavior of the tumor (Cancer Res 1998;58:3101-3104). This observation suggests TGF-βRII mutations promote cancer formation through mechanisms beyond release from TGF-β–induced growth inhibition, perhaps by promoting neoangiogenesis and/or tumor invasiveness. Strikingly, colon cancers often produce significant amounts of TGF-β. In an informative breast cancer model, Yin et al. recently reported that the cancer, although resistant to TGF-β growth inhibition, preserved autocrine TGF-β–mediated induction of PTHrp expression that favored the formation of bony metastatic disease (J Clin Invest 1999;103:197-205). In contrast, we find that colon cancers that are resistant to TGF-β–mediated growth inhibition have also lost TGF-β–induced transcriptional responses. Nonetheless, secreted TGF-β could contribute to the pathogenesis of TGF-β–resistant colon cancer via paracrine effects on neighboring stromal cells that could alter extracellular matrix, induce the growth of new blood vessels, and suppress the local activity of immune cells in the tumor environment. If such a model ultimately is verified, it would paradoxically imply a potential role for TGF-β antagonists as therapeutic agents in the treatment of TGF-β–resistant colon cancer. More directly, the discovery of RII mutations in a substantial portion of MSS as well as MSI colon cancers suggests the opportunity for novel therapeutic interventions such as gene reconstitution with TGF-βRII or the development of pharmaceutical agents that could reactivate postreceptor TGF-β signaling elements in these cancers. Unpublished observations in our laboratory suggest that gene reconstitution strategies may prove most successful in MSS colon cancer, because the MSI tumors rapidly mutate the reconstituted wild-type RII tumor-suppressor gene. Currently, we have developed a mutation-resistant version of wild-type RII and are evaluating its effectiveness as a tumor-suppressor of MSI colon cancer. The design of pharmaceutical agents that could reactivate postreceptor TGF-β signaling will become a potential reality as progress continues to be made in elucidating the gene targets that mediate TGF-β–induced growth inhibition and the mechanisms causing TGF-β resistance in colon cancers in which receptors are still wild-type. In addition to potential new treatment approaches, diagnostic assays that exploit the presence of mutations on RII could be developed. The mutant RII found in MSI colon cancer results from a frameshift mutation in the RII gene that prematurely truncates the protein, resulting in loss of the intracytoplasmic and transmembrane component of the receptor. Consequently, the mutant protein is predicted to be secreted by the tumor cells and should be detectable in the blood. Thus, the identification of RII mutations in colon cancer has not only provided insight into the mechanisms that contribute to the pathogenesis of colon cancer but also offers the promise of new therapeutic and diagnostic strategies to help treat this common malignancy.