Targeting the transforming growth factor-β signalling pathway in metastatic cancer

Transforming growth factor (TGF)-β signalling plays a dichotomous role in tumour progression, acting as a tumour suppressor early and as a pro-metastatic pathway in late-stages. There is accumulating evidence that advanced-stage tumours produce excessive levels of TGF-β, which acts to promote tumour growth, invasion and colonisation of secondary organs. In light of the pro-metastasis function, many strategies are currently being explored to antagonise the TGF-β pathway as a treatment for metastatic cancers. Strategies such as using large molecule ligand traps, reducing the translational efficiency of TGF-β ligands using antisense technology, and antagonising TGF-β receptor I/II kinase function using small molecule inhibitors are the most prominent methods being explored today. Administration of anti-TGF-β therapies alone, or in combination with immunosuppressive or cytotoxic therapies, has yielded promising results in the preclinical and clinical settings. Despite these successes, the temporal- and context-dependent roles of TGF-β signalling in cancer has made it challenging to define patient subgroups that are most likely to respond, and the therapeutic regimens that will be most effective in the clinic. Novel mouse models and diagnostic tools are being developed today to circumvent these issues, which may potentially expedite anti-TGF-β drug development and clinical application.     

Hypoxia-inducible factor-1α and vascular endothelial growth factor expression in circulating tumor cells of breast cancer patients

Introduction  

The detection of peripheral blood circulating tumor cells (CTCs) and bone marrow disseminated tumor cells (DTCs) in breast cancer patients is associated with a high incidence of disease relapse and disease-related death. Since hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) play an important role in angiogenesis and tumor progression, the purpose of the current study was to investigate their expression in CTCs.     

Targeting transforming growth factor-β signaling in liver metastasis of colon cancer

Despite a primary tumor suppressor role, there is compelling evidence suggesting that TGF-β can promote tumor growth, invasion and metastasis in advanced stages of colorectal cancer. Blocking these tumor-promoting effects of TGF-β provides a potentially important therapeutic strategy for the treatment of colorectal cancer. However, little is known about how the inhibitors of TGF-β receptor kinases affect colorectal carcinogenesis in vivo. Here, we have observed that a novel dual kinase inhibitor of TGF-β type I and type II receptors, LY2109761, inhibits TGF-β-mediated activation of Smad and non-Smad pathways in CT26 colon adenocarcinoma cells having K-Ras mutation. The inhibitor attenuates the oncogenic effects of TGF-β on cell migration, invasion and tumorigenicity of CT26 cells. Furthermore, LY2109761 decreases liver metastases and prolongs survival in an experimental metastasis model. These findings suggest that the dual kinase inhibitor LY2109761 has potential therapeutic value for metastatic colorectal cancer.     

Interaction of transforming growth factor-β (TGF-β) and epidermal growth factor (EGF) in human glioma cells

Gliomas are characterized by a deregulation of growth factor production and growth factor receptors expression, e.g. overproduction of the cytokine transforming growth factor- (TGF-) and overexpression/constitutive activation of receptors for the epidermal growth factor (EGF). Potential interactions of such growth factors and their signaling cascades could enhance the malignancy of these tumors. Therefore, we investigated the effects of TGF- and EGF alone and in combination on the proliferation of glioma cells cultivated from eight solid human WHO grade IV gliomas and one glioma cell line, analyzed the expression and intactness of the TGF--signaling molecules Samd-4 and -2, and the phosphorylation of the EGF-signaling kinases ERK 1/2. The effects were divergent and complex: Whereas EGF mostly stimulated glioma cell proliferation, TGF- either enhanced, inhibited or had no significant effect on proliferation. In combination, co-stimulation and inhibition of the EGF-induced mitogenic activity could be observed. Smad-4/-2 were expressed in all glioma cells, one point mutation at base 1595 in Smad-4 did not affect its protein sequence. In part of the glioma cells, reduced phosphorylation of ERK 1/2 and expression of cyclin-dependent kinase inhibitor 1 or p21 was observed in co-stimulation experiments. These experiments show that TGF- can inhibit EGF-mediated effects only in some gliomas, whereas it enhances it in others. The interaction of both factors is very complex and varies between different gliomas.     

Effect of irradiation on transforming growth factor-β secretion by malignant glioma cells

Glioblastoma cells secrete transforming growth factor- (TGF-), whichhas a variety of immunosuppressive properties. We investigatedthe effect of irradiation TGF- secretion by malignantglioma cells. Three malignant glioma cell lines (T98G,A172, KG-1-C) were cultured and irradiated using 10and 50 Gy Linac radiation. After further culturefor 36 hours in serum-free culture medium, thesupernatants were collected. The TGF- activity in theculture supernatants was determined using a specific bioassay.The levels of the active form and totalTGF- in the supernatants from irradiated malignant gliomacells decreased compared to those from un-irradiated cells.However, since irradiation inhibited the growth of tumorcells, the amount of TGF- secretion per cellin irradiated cells tended to increase after irradiation.These results suggest that malignant glioma cells canstill secrete TGF- and activate latent TGF- evenafter large dose irradiation, despite the inhibition oftumor growth.     

Are basic fibroblast growth factor and vascular endothelial growth factor prognostic indicators in pediatric patients with malignant solid tumors?

Angiogenesis plays an important role in the growth, progression, and metastasis of solid tumors. Among angiogenic factors, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) appear to be useful markers in adults with cancer. The aim of this pilot study was to determine the levels of VEGF in serum and bFGF in serum and urine of children with solid tumor at diagnosis (as measured by ELISA), and to investigate whether these parameters provide prognostic information. Forty consecutive patients with different types of cancer were prospectively included in this study. Median values of all studied angiogenic factors were higher in patients than in controls (n = 40), and the differences were statistically significant for bFGF in serum and urine: 10 versus 3 pg/ml (P = 0.0004) and 6406 versus 0 pg/g of creatinine (P < 0.0001), respectively. Among patients, median serum values of bFGF and VEGF were higher in children with metastatic disease (n = 14) than in those with localized disease (n = 26). The difference was statistically significant for serum bFGF: 17.5 versus 6 pg/ml (P = 0.02). Serum angiogenic factor levels correlated with outcome. The estimated event-free survival at 3 years was 79% for patients with normal bFGF values (n = 13) versus 42% (n = 26; P = 0.02) for those with high levels, and 71% in case of normal VEGF values (n = 20) versus 38% (n = 19; P = 0.04) for those with high levels. No benefit of normal urinary bFGF values was observed. Our results provide a rationale for exploring the clinical interest of bFGF and VEGF measurements in body fluids of a larger group of children with cancer.     

Transforming growth factor-β signalling controls human breast cancer metastasis in a zebrafish xenograft model

Introduction

The transforming growth factor beta (TGF-β) signalling pathway is known to control human breast cancer invasion and metastasis. We demonstrate that the zebrafish xenograft assay is a robust and dependable animal model for examining the role of pharmacological modulators and genetic perturbation of TGF-β signalling in human breast tumour cells.

Methods

We injected cancer cells into the embryonic circulation (duct of cuvier) and examined their invasion and metastasis into the avascular collagenous tail. Various aspects of the TGF-β signalling pathway were blocked by chemical inhibition, small interfering RNA (siRNA), or small hairpin RNA (shRNA). Analysis was conducted using fluorescent microscopy.

Results

Breast cancer cells with different levels of malignancy, according to in vitro and in vivo mouse studies, demonstrated invasive and metastatic properties within the embryonic zebrafish model that nicely correlated with their differential tumourigenicity in mouse models. Interestingly, MCF10A M2 and M4 cells invaded into the caudal hematopoietic tissue and were visible as a cluster of cells, whereas MDA MB 231 cells invaded into the tail fin and were visible as individual cells. Pharmacological inhibition with TGF-β receptor kinase inhibitors or tumour specific Smad4 knockdown disturbed invasion and metastasis in the zebrafish xenograft model and closely mimicked the results we obtained with these cells in a mouse metastasis model. Inhibition of matrix metallo proteinases, which are induced by TGF-β in breast cancer cells, blocked invasion and metastasis of breast cancer cells.

Conclusions

The zebrafish-embryonic breast cancer xenograft model is applicable for the mechanistic understanding, screening and development of anti-TGF-β drugs for the treatment of metastatic breast cancer in a timely and cost-effective manner.     

Bistable switch in migration stimulating factor expression: regulation by the concerted signalling of transforming growth factor-β1 and the extracellular matrix

Migration stimulating factor (MSF) is an oncofetal motogenic/angiogenic cytokine constitutively expressed by epithelial and stromal cells in fetal and neoplastic tissues. Fibroblasts derived from healthy adult skin do not express MSF but can be induced to do so by treatment with transforming growth factor-β1 (TGF-β1). As the bioactivities of both MSF and TGF-β1 are modulated by the extracellular matrix, we investigated whether the induction of MSF expression by TGF-β1 is also matrix dependent. We now report that adult fibroblasts are induced to express MSF by a transient treatment with TGF-β1 (as short as 2 hr) but only when the cells are adherent to a "wound" matrix, such as denatured type I collagen, fibrin or plastic tissue culture dishes. Unexpectedly, this induction of MSF expression persists unabated for the entire subsequent lifespan of the treated cells in the absence of further TGF-β1 and irrespective of the substratum. Such "activated" MSF expression may, however, be persistently switched off again by a second transient exposure to TGF-β1 but this time only when the cells are adherent to a "healthy" matrix of native type I collagen. Significantly, the constitutive expression of MSF by fetal and cancer patient fibroblasts could also be persistently switched off by this means. We conclude that TGF-β1 may both switch on and switch off MSF expression in a manner critically determined by the nature of the matrix substratum and suggest that this may be a possible mechanism underlying the observed dual functionality of TGF-β1 as both a tumour suppressor and tumour promoter.     

Expression of adhesion molecules and transforming growth factor-β in pleomorphic carcinomas of the lung

Pleomorphic carcinoma (PC) of the lung consists of an epithelial component showing the histology of poorly differentiated non-small cell carcinoma of the lung and a sarcomatous component, that is more aggressive compared to non-small cell carcinoma of the lung. To determine the differences between an epithelial component of PC and poorly differentiated non-small cell carcinoma, the expression of adhesion molecules (E-cadherin, β-catenin and N-cadherin) and transforming growth factor-β (TGF-β) was compared immunohistochemically among 14 poorly differentiated adenocarcinomas of the lung (PDAs) and 14 PCs of the lung, with an epithelial component, showing the histology of PDA. Expression levels of E-cadherin and β-catenin were significantly lower in epithelial or sarcomatous components of PCs than in PDAs while that of TGF-β was significantly higher in epithelial components of PCs than in PDAs. No significant difference was found in incidences of the expression of these molecules between epithelial and sarcomatous components of PCs. No significant difference was noted in the expression level of N-cadherin among PDAs and epithelial and sarcomatous components of PCs. The present results showed that E-cadherin and β-catenin expression is reduced and TGF-β expression is increased in epithelial components of PCs with the same histology as PDA when compared to PDAs, suggesting that an epithelial component of PC is distinct from non-small cell carcinoma with the same histology.     

Mechanistic basis and clinical relevance of the role of transforming growth factor-β in cancer

Transforming growth factor-β (TGF-β) is a key factor in cancer development and progression. TGF-β can suppress tumorigenesis by inhibiting cell cycle progression and stimulating apoptosis in early stages of cancer progression. However, TGF-β can modulate cancer-related processes, such as cell invasion, distant metastasis, and microenvironment modification that may be used by cancer cells to their advantage in late stages. Corresponding mechanisms include angiogenesis promotion, anti-tumor immunity suppression, and epithelial-to-mesenchymal transition (EMT) induction. The correlation between TGF-β expression and cancer prognosis has also been extensively investigated. Results suggest that TGF-β pathway can be targeted to treat cancer; as such, the feasibility of this treatment is investigated in clinical trials.KEYWORDS : Transforming growth factor-β (TGF-β), neoplasms, prognosis, therapeutics     

Lack of transforming growth factor-β signaling promotes collective cancer cell invasion through tumor-stromal crosstalk

ABSTRACT: INTRODUCTION: Transforming growth factor beta (TGF-β) has a dual role during tumor progression, initially as a suppressor and then as a promoter. Epithelial TGF-β signaling regulates fibroblast recruitment and activation. Concurrently, TGF-β signaling in stromal fibroblasts suppresses tumorigenesis in adjacent epithelia, while its ablation potentiates tumor formation. Much is known about the contribution of TGF-β signaling to tumorigenesis, yet the role of TGF-β in epithelial-stromal migration during tumor progression is poorly understood. We hypothesize that TGF-β is a critical regulator of tumor-stromal interactions that promote mammary tumor cell migration and invasion. METHODS: Fluorescently labeled murine mammary carcinoma cells, isolated from either MMTV-PyVmT transforming growth factor-beta receptor II knockout (TβRII KO) or TβRIIfl/fl control mice, were combined with mammary fibroblasts and xenografted onto the chicken embryo chorioallantoic membrane. These combinatorial xenografts were used as a model to study epithelial-stromal crosstalk. Intravital imaging of migration was monitored ex ovo, and metastasis was investigated in ovo. Epithelial RNA from in ovo tumors was isolated by laser capture microdissection and analyzed to identify gene expression changes in response to TGF-β signaling loss. RESULTS: Intravital microscopy of xenografts revealed that mammary fibroblasts promoted two migratory phenotypes dependent on epithelial TGF-β signaling: single cell/strand migration or collective migration. At epithelial-stromal boundaries, single cell/strand migration of TβRIIfl/fl carcinoma cells was characterized by expression of α-smooth muscle actin and vimentin, while collective migration of TβRII KO carcinoma cells was identified by E-cadherin+/p120+/β-catenin+ clusters. TβRII KO tumors also exhibited a twofold greater metastasis than TβRIIfl/fl tumors, attributed to enhanced extravasation ability. In TβRII KO tumor epithelium compared with TβRIIfl/fl epithelium, Igfbp4 and Tspan13 expression was upregulated while Col1α2, Bmp7, Gng11, Vcan, Tmeff1, and Dsc2 expression was downregulated. Immunoblotting and quantitative PCR analyses on cultured cells validated these targets and correlated Tmeff1 expression with disease progression of TGF-β-insensitive mammary cancer. CONCLUSION: Fibroblast-stimulated carcinoma cells utilize TGF-β signaling to drive single cell/strand migration but migrate collectively in the absence of TGF-β signaling. These migration patterns involve the signaling regulation of several epithelial-to-mesenchymal transition pathways. Our findings concerning TGF-β signaling in epithelial-stromal interactions are important in identifying migratory mechanisms that can be targeted as recourse for breast cancer treatment.     

Interleukin 8 and vascular endothelial growth factor -- prognostic factors in human gastric carcinomas?

Gastric carcinoma cells express potent angiogenic factors including vascular endothelial growth factor (VEGF). We previously reported that interleukin-8 (IL-8) acts as an angiogenic factor for human gastric carcinomas. More recently, we found that IL-8 upregulates matrix metalloproteinase-9 (MMP-9) expression and increases invasive activity of gastric carcinoma cells. The purpose of this study was to determine whether the expression of IL-8 and VEGF correlates with clinicopathological parameters in human gastric carcinomas. IL-8 and VEGF expression levels were measured by an enzyme-linked immunosorbent assay (ELISA) in 56 gastric carcinomas and the surrounding normal mucosa. Macroscopic and histopathological tumour findings, presence of metastasis and prognosis were obtained from the patient records and endoscopic, surgical and pathological reports. IL-8 protein levels were higher in most neoplasms than in the corresponding normal mucosal tissue. In contrast, VEGF expression in the tumours was similar to that in normal mucosa. The IL-8 level in the neoplasms correlated significantly with the depth of invasion, venous invasion and lymphatic invasion. VEGF expression in the tumours correlated well with the depth of invasion and lymph node metastasis. No correlation between IL-8 and VEGF expression in the tumours was observed. The survival rates of patients with tumours displaying high IL-8 and VEGF expression levels were significantly lower (P<0.05) than those of patients with tumours displaying low IL-8 and VEGF expression. The results suggest that IL-8 and VEGF may be independent and important prognostic factors in human gastric carcinomas.     

SMAD3 is essential for transforming growth factor-β1-induced urokinase type plasminogen activator expression and migration in transformed keratinocytes

Transforming growth factor-β1 (TGF-β1) stimulates the extracellular matrix degrading proteases expression and cell migration in order to enhance cancer cells malignancy. In the present study, we analysed the role of TGF-β1-induced Smad3 activation in the urokinase type plasminogen activator (uPA) production, as well as in cell migration and E-cadherin downregulation in transformed PDV keratinocyte cell line. TGF-β1 signalling was interfered by the chemical inhibitor of the TGF-β1-receptor 1 (ALK5), SB505124, and the specific Smad3 inhibitor, SiS3. Our results showed that TGF-β1 stimulates uPA expression directly through ALK5 activation. The inhibition of Smad3 strongly reduced the capacity of TGF-β1 to stimulate uPA expression, in parallel decreasing the uPA inhibitor plasminogen activator inhibitor type 1 (PAI-1) expression. In addition, the transient expression of dominant negative Smad3 mutant inhibited the TGF-β1-induced uPA promoter transactivation. Moreover, Smad3-/- mouse embryonic fibroblasts were refractory to the induction of uPA by TGF-β1. The inhibition of both ALK5 and Smad3 dramatically blocked the TGF-β1-stimulated E-cadherin downregulation, F-actin reorganisation and migration of PDV cells. Taken together, our results suggest that the TGF-β1-induced activation of Smad3 is the critical step for the uPA upregulation and E-cadherin downregulation, which are the key events preceding the induction of cell migration by TGF-β1 in transformed cells.     

Wnt signalling via the epidermal growth factor receptor: a role in breast cancer?

Recent data have suggested the epidermal-growth-factor receptor (EGFR) as a point of convergence for several different classes of receptor. Civenni and colleagues have now demonstrated crosstalk between Wnt signalling and the EGFR, showing that in breast epithelial cells Wnts activate downstream targets of the EGFR, including cyclin D1. Given the role of members of these pathways in the aetiology of breast cancer and as markers of outcome and potential therapeutic targets in breast cancer, this observation has a number of potential implications important for both the basic biology of breast cancer and the clinical management of the disease.     

Wnt signalling via the epidermal growth factor receptor: a role in breast cancer?

Recent data have suggested the epidermal-growth-factor receptor (EGFR) as a point of convergence for several different classes of receptor. Civenni and colleagues have now demonstrated crosstalk between Wnt signalling and the EGFR, showing that in breast epithelial cells Wnts activate downstream targets of the EGFR, including cyclin D1. Given the role of members of these pathways in the aetiology of breast cancer and as markers of outcome and potential therapeutic targets in breast cancer, this observation has a number of potential implications important for both the basic biology of breast cancer and the clinical management of the disease.