Stromal interaction essential for vascular endothelial growth factor A-induced tumour growth via transforming growth factor-β signalling

Background:

High vascular endothelial growth factor (VEGFA) levels at the time of diagnosis confer a worse prognosis to multiple malignancies. Our aim was to investigate the role of VEGFA in promoting tumour growth through interaction with its environment.

Methods:

HL-60 cells were transduced with VEGFA165 or control vector using retroviral constructs. Control cells (n=7) or VEGFA165 cells (n=7) were subcutaneously injected into NOD/SCID mice. Immunohistochemistry of markers for angiogenesis (CD31) and cell proliferation (Ki67) and gene expression profiling of tumours were performed. Paracrine effects were investigated by mouse-specific cytokine arrays.

Results:

In vivo we observed a twofold increase in tumour weight when VEGFA165 was overexpressed (P=0.001), combined with increased angiogenesis (P=0.002) and enhanced tumour cell proliferation (P=0.001). Gene expression profiling revealed human genes involved in TGF-β signalling differentially expressed between both tumour groups, that is, TGFBR2 and SMAD5 were lower expressed whereas the inhibitory SMAD7 was higher expressed with VEGFA165. An increased expression of mouse-derived cytokines IFNG and interleukin 7 was found in VEGFA165 tumours, both described to induce SMAD7 expression.

Conclusion:

These results suggest a role for VEGFA-driven tumour growth by TGF-β signalling inhibition via paracrine mechanisms in vivo, and underscore the importance of stromal interaction in the VEGFA-induced phenotype.     

Insulin-like growth factor-1, insulin-like growth factor-binding protein-3, growth hormone, and mammographic density in the Nurses’ Health Studies

Higher circulating insulin-like growth factor I (IGF-1) levels have been associated with higher mammographic density among women in some, but not all studies. Also, few studies have examined the association between mammographic density and circulating growth hormone (GH) in premenopausal women. We conducted a cross-sectional study among 783 premenopausal women and 436 postmenopausal women who were controls in breast cancer case?Ccontrol studies nested in the Nurses?? Health Study (NHS) and NHSII. Participants provided blood samples in 1989?C1990 (NHS) or in 1996?C1999 (NHSII), and mammograms were obtained near the time of blood draw. Generalized linear models were used to assess the associations of IGF-1, IGF-binding protein-3 (IGFBP-3), IGF-1:IGFBP-3 ratio, and GH with percent mammographic density, total dense area, and total non-dense area. Models were adjusted for potential confounders including age and body mass index (BMI), among others. We also assessed whether the associations varied by age or BMI. In both pre- and postmenopausal women, percent mammographic density was not associated with plasma levels of IGF-1, IGFBP-3, or the IGF-1:IGFBP-3 ratio. In addition, GH was not associated with percent density among premenopausal women in the NHSII. Similarly, total dense area and non-dense area were not significantly associated with any of these analytes. In postmenopausal women, IGF-1 was associated with higher percent mammographic density among women with BMI?<25?kg/m², but not among overweight/obese women. Overall, plasma IGF-1, IGFBP-3, and GH levels were not associated with mammographic density in a sample of premenopausal and postmenopausal women.     

Transforming growth factor-beta and breast cancer: Tumor promoting effects of transforming growth factor-β

This cross-sectional investigation in Hawaii explored the relation between soy foods and mammographic characteristics using two food frequency questionnaires and a computer-assisted density assessment method. Japanese and Chinese women reported significantly greater soy food intake than Caucasian women. Whereas soy intake and the size of the dense areas were not related, soy intake and percent mammographic densities were positively associated. The size of the entire breast and the nondense area (ie the fatty part of the breast) were inversely related to soy intake. These results suggest the hypothesis that soy foods by themselves or as part of an Asian dietary pattern may affect the growth of the female breast before adulthood, but the possible mechanisms of action have to be explored in future studies.     

Estimate of tumor growth time for breast cancer local recurrences: rapid growth after wake-up?

To obtain an estimate of the subclinical tumor growth time (TGT) for breast cancer, a series of 31 patients with local recurrence as first event after mastectomy was evaluated. The recurrence diameter was measured, and the minimum growth rate (mGR) consistent with the sequence no detection at the preceding physical examination recurrence of diameter Dr was obtained. The growth rate for uninterrupted exponential growth from surgery (GRu) was also calculated. mGR was significantly higher than GRu (p < 0.0001), and unrelated to the recurrence-free survival (RFS). Therefore, starting points of local recurrence growth curves had to be set at times variously delayed after mastectomy. The estimate of the delay lower limit (mD), which was obtained from mGR and GRu, showed a strong linear correlation with RFS (R=0.984; p < 0.0001). From the regression equation, TGT for local recurrences could be estimated at 30 ± 8 weeks or less. These findings support the concept that the lag time between surgical treatment of breast cancer and clinical evidence of local recurrence may be explained by a period of tumor dormancy followed by a tumor growing phase. The TGT estimate is remarkably shorter than previously believed and could considerably change the current picture of breast cancer history.     

Mutant epidermal growth factor receptor contributes to head and neck cancer growth and resistance to EGFR targeting

Epidermal growth factor receptor (EGFR) is overexpressed in head and neck squamous-cell carcinoma (HNSCC) and its expression levels correlate with decreased patient survival. Nonetheless, therapies aiming at blocking EGFR has shown limited efficacy in a proportion of patients with HNSCC in clinical trials. Sok et al. in a recent paper (Clin Cancer Res, 2006, 12:5064-5073 ) attempted to ascertain whether it is due to mutation of EGFR. As the most common form of mutation of EGFR seen in several other types of cancer is a truncation mutation, EGFR     

Transforming growth factor-β pathway activity in glioblastoma

Transforming growth factor (TGF)-β is a central molecule maintaining the malignant phenotype of glioblastoma. Anti-TGF-β strategies are currently being explored in early clinical trials. Yet, there is little contemporary data on the differential expression of TGF-β isoforms at the mRNA and protein level or TGF-β/Smad pathway activity in glioblastomas in vivo.Here we studied 64 newly diagnosed and 16 recurrent glioblastomas for the expression of TGF-β_1-3, platelet-derived growth factor (PDGF)-B, and plasminogen activator inhibitor (PAI)-1 mRNA by RT-PCR and for the levels of TGF-β_1-3 protein, phosphorylated Smad2 (pSmad2), pSmad1/5/8 and PAI-1 by immunohistochemistry.Among the TGF-β isoforms, TGF-β₁ mRNA was the most, whereas TGF-β₃ mRNA was the least abundant. TGF-β_1-3 mRNA expression was strongly correlated, as was the expression of TGF-β_1-3 mRNA, and of the TGF-β_1-3 target genes, PDGF-B and PAI-1. TGF-β₂ and TGF-β₃ protein levels correlated well, whereas the comparison of the other TGF-βisoforms did not. Positive correlation was also observed between TGF-β₁ and pSmad1/5/8 and between pSmad2 and pSmad1/5/8. Survival analyses indicated that a group of patients with high expression levels of TGF-β₂ mRNA or pSmad1/5/8 protein have inferior outcome.We thus provide potential biomarkers for patient stratification in clinical trials of anti-TGF-β therapies in glioblastoma.     

Integrin-mediated activation of latent transforming growth factor β

Members of the integrin family recognize a variety of spatially-restricted extracellular ligands. Classically, ligation of integrins activates cytoplasmic signals in the integrin-expressing cell and contributes to cell adhesion, migration, proliferation and survival. At least two members of this family, αvβ6 and αvβ8 perform an additional function, activation of latent complexes of transforming growth factor β. In effect, this process allows integrins on one cell to activate signals on adjacent (in the case of αvβ6) or nearby cells (in the case of αvβ8). Integrin-mediated TGFβ activation has been shown to play important roles in modulating tissue fibrosis, acute lung injury and pulmonary emphysema. Given the important roles that TGFβ plays in modulating epithelial cell growth, epithelial-to-mesenchymal transformation and tumor invasion and metastasis, integrin-mediated TGFβ activation is likely to play important roles in tumor growth ad metastasis.     

Inhibitory effect of soluble platelet-derived growth factor receptor β on intraosseous growth of breast cancer cells in nude mice

Bone metastasis is a frequent complication of advanced breast cancer. On the basis of functional and molecular evidence, signaling mediated by the binding of platelet-derived growth factor (PDGF)-BB and -DD to PDGF receptor β (PDGFRβ) is critical for the survival and growth of metastatic breast cancer cells within the bone microenvironment. In this study, we propose a new approach to blocking PDGFRβ signaling using soluble PDGFRβ (sPDGFRβ) as a decoy receptor for PDGF-BB and -DD secreted from tumor cells and bone marrow stromal cells. A bone-seeking TNBCT/Bo cell line was established by in vivo selection from TNBCT human breast cancer cells, which are negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 protein expression. The TNBCT/Bo cells were transfected with a mammalian expression vector encoding the extracellular domain of PDGFRβ. A stable transfectant (TNBCT/Bo-sPDGFRβ) grew at a similar rate to that of control cells under normal culture conditions, although growth stimulation of human fibroblasts with PDGF-BB was neutralized by the culture medium from TNBCT/Bo-sPDGFRβ cells. Intratibial injection of TNBCT/Bo-sPDGFRβ cells into athymic nude mice resulted in a significant decrease in tumor incidence compared with control mice (P < 0.01). This attenuated growth correlated with decreased cancer cell proliferation, angiogenesis, and recruitment of stromal cells, and with an increase in the number of apoptotic cells. These findings suggest that sPDGFRβ is useful for the treatment of breast cancer bone metastasis.     

Transforming growth factor-β in cancer and metastasis

Transforming growth factor-beta (TGF-β) is a multifunctional regulatory polypeptide that is the prototypical member of a large family of cytokines that controls many aspects of cellular function, including cellular proliferation, differentiation, migration, apoptosis, adhesion, angiogenesis, immune surveillance, and survival. The actions of TGF-β are dependent on several factors including cell type, growth conditions, and the presence of other polypeptide growth factors. One of the biological effects of TGF-β is the inhibition of proliferation of most normal epithelial cells using an autocrine mechanism of action, and this suggests a tumor suppressor role for TGF-β. Loss of autocrine TGF-β activity and/or responsiveness to exogenous TGF-β appears to provide some epithelial cells with a growth advantage leading to malignant progression. This suggests a pro-oncogenic role for TGF-β in addition to its tumor suppressor role. During the early phase of epithelial tumorigenesis, TGF-β inhibits primary tumor development and growth by inducing cell cycle arrest and apoptosis. In late stages of tumor progression when tumor cells become resistant to growth inhibition by TGF-β due to inactivation of the TGF-β signaling pathway or aberrant regulation of the cell cycle, the role of TGF-β becomes one of tumor promotion. Resistance to TGF-β-mediated inhibition of proliferation is frequently observed in multiple human cancers, as are various alterations in the complex TGF-β signaling and cell cycle pathways. TGF-β can exert effects on tumor and stromal cells as well as alter the responsiveness of tumor cells to TGF-β to stimulate invasion, angiogenesis, and metastasis, and to inhibit immune surveillance. Because of the dual role of TGF-β as a tumor suppressor and pro-oncogenic factor, members of the TGF-β signaling pathway are being considered as predictive biomarkers for progressive tumorigenesis, as well as molecular targets for prevention and treatment of cancer and metastasis.     

Altered growth control by natural growth inhibitors in the mutant HD33 substrain of the Ehrlich-Lettré ascites mammary carcinoma

The mutant substrain HD33 of the Ehrlich-Lettré ascites mammary carcinoma (EAC) was found to be altered in its ability to respond to and to produce natural growth inhibitors. Cells of this substrain did not respond to (1) a highly purified growth inhibitor from bovine mammary gland, inhibiting the proliferation of two strains of the original EAC already at concentrations of 0.5–2.0 ng/ml, (2) inhibitory activities found in the ascites fluid of these sensitive strains and partially purified. However, from the ascites fluid of the mutant substrain an inhibitory activity was partially purified, which was inhibitory towards this substrain. It also inhibited the cells from an original strain, although less effectively. This new inhibitory activity is similar in its properties to that of the others investigated in that (1) its mol. wt is between 10,000 and 50,000 D, (2) it is heat labile and (3) its activity can be overcome by insulin, epidermal growth factor and 2′-deoxycytidine, (4) the dose-response curve levels off at 35–45% inhibition. The results demonstrate that the regulation of cell proliferation by endogenous (autocrine) inhibitors can be altered by mutagen treatment.     

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-beta and breast cancer: Cell cycle arrest by transforming growth factor-β and its disruption in cancer

Altered responsiveness to extracellular signals and cell cycle dysregulation are hallmarks of cancer. The cell cycle is governed by cyclin-dependent kinases (cdks) that integrate mitogenic and growth inhibitory signals. Transforming growth factor (TGF)-β mediates G₁ cell cycle arrest by inducing or activating cdk inhibitors, and by inhibiting factors required for cdk activation. Mechanisms that lead to cell cycle arrest by TGF-β are reviewed. Loss of growth inhibition by TGF-β occurs early in breast cell transformation, and may contribute to breast cancer progression. Dysregulation of cell cycle effectors at many different levels may contribute to loss of G₁ arrest by TGF-β. Elucidation of these pathways in breast cancer may ultimately lead to novel and more effective treatments for this disease.     

Glucocorticoids suppress tumor angiogenesis and growth of prostate cancer

Hormone-refractory prostate cancer ( HRPC) sometimes is responsive to treatment with glucocorticoids, such as prednisolone, hydrocortisone and dexamethasone, but the underlying mechanisms are not well established. In a recent paper (Clin Cancer Res, 2006, 12:3003-3009), Yano et al. Hypothesized and confirmed that the therapeutic effect of glucocorticoids on HRPC is attributed to inhibition of angiogenesis. A prostate cancer cell line DU145 that expresses glucocorticoid receptor was used to study the effect of dexamethasone (Dex) on the expres-     

Fibroblast growth factor receptor splice variants are stable markers of oncogenic transforming growth factor β1 signaling in metastatic breast cancers

Introduction

Epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET) facilitate breast cancer (BC) metastasis; however, stable molecular changes that result as a consequence of these processes remain poorly defined. Therefore, with the hope of targeting unique aspects of metastatic tumor outgrowth, we sought to identify molecular markers that could identify tumor cells that had completed the EMT:MET cycle.

Methods

An in vivo reporter system for epithelial cadherin (E-cad) expression was used to quantify its regulation in metastatic BC cells during primary and metastatic tumor growth. Exogenous addition of transforming growth factor β1 (TGF-β1) was used to induce EMT in an in situ model of BC. Microarray analysis was employed to examine gene expression changes in cells chronically treated with and withdrawn from TGF-β1, thus completing one full EMT:MET cycle. Changes in fibroblast growth factor receptor type 1 (FGFR1) isoform expression were validated using PCR analyses of patient-derived tumor tissues versus matched normal tissues. FGFR1 gene expression was manipulated using short hairpin RNA depletion and cDNA rescue. Preclinical pharmacological inhibition of FGFR kinase was employed using the orally available compound BGJ-398.

Results

Metastatic BC cells undergo spontaneous downregulation of E-cad during primary tumor growth, and its expression subsequently returns following initiation of metastatic outgrowth. Exogenous exposure to TGF-β1 was sufficient to drive the metastasis of an otherwise in situ model of BC and was similarly associated with a depletion and return of E-cad expression during metastatic progression. BC cells treated and withdrawn from TGF-β stably upregulate a truncated FGFR1-β splice variant that lacks the outermost extracellular immunoglobulin domain. Identification of this FGFR1 splice variant was verified in metastatic human BC cell lines and patient-derived tumor samples. Expression of FGFR1-β was also dominant in a model of metastatic outgrowth where depletion of FGFR1 and pharmacologic inhibition of FGFR kinase activity both inhibited pulmonary tumor outgrowth. Highlighting the dichotomous nature of FGFR splice variants and recombinant expression of full-length FGFR1-α also blocked pulmonary tumor outgrowth.

Conclusion

The results of our study strongly suggest that FGFR1-β is required for the pulmonary outgrowth of metastatic BC. Moreover, FGFR1 isoform expression can be used as a predictive biomarker for therapeutic application of its kinase inhibitors.     

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.     

On the role of transforming growth factor-β in the growth inhibitory effects of retinoic acid in human pancreatic cancer cells

Background  

Retinoids are potent growth inhibitory and differentiating agents in a variety of cancer cell types. We have shown that retinoids induce growth arrest in all pancreatic cancer cell lines studied, regardless of their p53 and differentiation status. However, the mechanism of growth inhibition is not known. Since TGF-β2 is markedly induced by retinoids in other cancers and mediates MUC4 expression in pancreatic cancer cells, we investigated the role of TGF-β in retinoic acid-mediated growth inhibition in pancreatic cancer cells.