Nils-Erik Heldin

Identification of Smad7, a TGFβ-inducible antagonist of TGF-β signalling

TGF-β signals from the membrane to the nucleus through serine/threonine kinase receptors and their downstream effectors, termed SMAD proteins. The activated TGF-β receptor induces phosphorylation of two such proteins, Smad2 and Smad3 (refs 2, 3, , 5, 6), which form hetero-oligomeric complex(es) with Smad4/DPC4 (refs 5, 6, 7, 8, 9, 10) that translocate to the nucleus,,,, where they then regulate transcriptional responses,. However, the mechanisms by which the intracellular signals of TGF-β are switched off are unclear. Here we report the identification of Smad7, which is related to Smad6 (ref. 13). Transfection of Smad7 blocks responses mediated by TGF-β in mammalian cells, and injection of Smad7 RNA into Xenopus embryos blocks activin/TGF-β signalling. Smad7 associates stably with the TGF-β receptor complex, but is not phosphorylated upon TGF-β stimulation. TGFβ-mediated phosphorylation of Smad2 and Smad3 is inhibited by Smad7, indicating that the antagonistic effect of Smad7 is exerted at this important regulatory step. TGF-β rapidly induces expression of Smad7 mRNA, suggesting that Smad7 may participate in a negative feedback loop to control TGF-β responses.

Transforming growth factor-beta and epidermal growth factor synergistically stimulate epithelial to mesenchymal transition (EMT) through a MEK-dependent mechanism in primary cultured pig thyrocytes

Enhancement of tumor cell growth and invasiveness by transforming growth factor-β (TGF-β) requires constitutive activation of the ras/MAPK pathway. Here we have investigated how MEK activation by epidermal growth factor (EGF) influences the response of fully differentiated and growth-arrested pig thyroid epithelial cells in primary culture to TGF-β1. The epithelial tightness was maintained after single stimulation with EGF or TGF-β1 (both 10 ng/ml) for 48 hours. In contrast, co-stimulation abolished the transepithelial resistance and increased the paracellular flux of [3H]inulin within 24 hours. Reduced levels of the tight junction proteins claudin-1 and occludin accompanied the loss of barrier function. N-cadherin, expressed only in few cells of untreated or single-stimulated cultures, was at the same time increased 30-fold and co-localised with E-cadherin at adherens junctions in all cells. After 48 hours of co-stimulation, both E- and N-cadherin were downregulated and the cells attained a fibroblast-like morphology and formed multilayers. TGF-β1 only partially inhibited EGF-induced Erk phosphorylation. The MEK inhibitor U0126 prevented residual Erk phosphorylation and abrogated the synergistic responses to TGF-β1 and EGF. The observations indicate that concomitant growth factor-induced MEK activation is necessary for TGF-β1 to convert normal thyroid epithelial cells to a mesenchymal phenotype.

Smad7 mediates apoptosis induced by transforming growth factor β in prostatic carcinoma cells

Transforming growth factor beta (TGF-beta) is an important regulator of apoptosis in some cell types, but the underlying molecular mechanisms are largely unknown. TGF-beta signals through type I and type II receptors and downstream effector proteins, termed Smads. TGF-beta induces the phosphorylation of Smad2 and Smad3 (receptor-activated Smads) which associate with Smad4 and translocate to the nucleus, where they regulate gene transcription [1]. Smad7 protein is induced by TGF-beta1 and has been classified as an inhibitory Smad. Smad7 prevents phosphorylation of receptor-activated Smads, thereby inhibiting TGF-beta-induced signaling responses [1]. Smad7 expression is increased in rat prostatic epithelial cells undergoing apoptosis as a result of castration [2]. Here we have shown that TGF-beta1 treatment or ectopic expression of Smad7 in human prostatic carcinoma cells (PC-3U) induces apoptosis. Furthermore, TGF-beta1-induced apoptosis was prevented by inhibition of Smad7 expression, by antisense mRNA in stably transfected cell lines or upon transient transfection with antisense oligonucleotides in several investigated cell lines. These findings provide evidence for a new effector function for Smad7 in TGF-beta1 signaling.

Mechanisms for 2-methoxyestradiol-induced apoptosis of prostate cancer cells

Prostate and breast carcinomas are sex hormone‐related carcinomas, which are known to be associated with an over‐expression of the proto‐oncogene Bcl‐2. Here, we report that 2‐methoxyestradiol (2‐ME), an endogenous metabolite of estrogen that does not bind to nuclear estrogen receptors, effectively induces apoptosis in Bcl‐2‐expressing human prostate and breast carcinoma cells in vitro and in a rat prostate tumor model in vivo. In several cell lines derived from prostate, breast, liver and colorectal carcinomas, 2‐ME treatment led to an activation of c‐Jun N‐terminal kinase (JNK) and phosphorylation of Bcl‐2, which preceded the induction of apoptosis. In summary, our data suggest that 2‐ME induces apoptosis in epithelial carcinomas by causing phosphorylation of JNK, which appears to be correlated with phosphorylation of Bcl‐2.

Collagen-binding proteoglycan fibromodulin can determine stroma matrix structure and fluid balance in experimental carcinoma

Research on the biology of the tumor stroma has the potential to lead to development of more effective treatment regimes enhancing the efficacy of drug-based treatment of solid malignancies. Tumor stroma is characterized by distorted blood vessels and activated connective tissue cells producing a collagen-rich matrix, which is accompanied by elevated interstitial fluid pressure (IFP), indicating a transport barrier between tumor tissue and blood. Here, we show that the collagen-binding proteoglycan fibromodulin controls stroma structure and fluid balance in experimental carcinoma. Gene ablation or inhibition of expression by anti-inflammatory agents showed that fibromodulin promoted the formation of a dense stroma and an elevated IFP. Fibromodulin-deficiency did not affect vasculature but increased the extracellular fluid volume and lowered IFP. Our data suggest that fibromodulin controls stroma matrix structure that in turn modulates fluid convection inside and out of the stroma. This finding is particularly important in relation to the demonstration that targeted modulations of the fluid balance in carcinoma can increase the response to cancer therapeutic agents.

N‐cadherin‐mediated adhesion and aberrant catenin expression in anaplastic thyroid‐carcinoma cell lines

The role of cadherins and catenins in the progression of thyroid carcinoma is unclear. We have investigated α‐, β‐ and γ‐catenins and p120ctn in relation to the expression of cadherins in human anaplastic thyroid‐carcinoma cell lines (HTh7, HTh74, C643 and SW1736) with Western blotting and immunofluorescence. E‐cadherin was lacking except in SW1736, which consisted of E‐cadherin‐positive (approx. 5%) and ‐negative cells. The α‐ and β‐catenin levels were similar to those of primary cultured non‐neoplastic (E‐cadherin‐positive) human thyrocytes. In contrast, the expression of γ‐catenin was low and variable, correlating with the different levels of cytokeratin in the same cells (HTh74 > SW1736 > C643 > HTh7). p120ctn resolved as a doublet in Western blots; the approximately 100‐kDa band also found in non‐neoplastic epithelial cells was reduced whereas the approximately 115‐kDa band, corresponding to the fibroblast‐type isoform of p120ctn, was neo‐expressed. A DNA‐demethylating agent, 5‐aza‐2′‐deoxycytidine, up‐regulated E‐cadherin in SW1736 and γ‐catenin in SW1736 and C643, whereas the other cell lines were unresponsive; other catenins were not affected. The catenins were generally distributed along the cell borders. Immunostaining, cell‐surface biotinylation and co‐immunoprecipitation revealed that all cell lines expressed N‐cadherin in connection with β‐catenin at the plasma membrane. Incubation with an N‐cadherin antibody disrupted cell‐cell adhesion. We conclude that E‐cadherin‐negative anaplastic thyroid‐carcinoma cell lines display functional N‐cadherin/β‐catenin complexes, partial or complete loss of γ‐catenin, and isoform shift of p120ctn. The unequal expression of E‐cadherin and γ‐catenin and the variable response to DNA de‐methylation suggest that anaplastic thyroid carcinoma is not a uniform entity. Int. J. Cancer 83:692–699, 1999.

Inhibition of carcinoma cell-derived VEGF reduces inflammatory characteristics in xenograft carcinoma

The stroma of carcinomas shares several characteristics with inflamed tissues including a distorted vasculature, active angiogenesis and macrophage infiltration. In addition, the tumor interstitial fluid pressure (PIF) of the stroma is pathologically elevated. We show here that bevacizumab [rhuMab vascular endothelial growth factor (VEGF), Avastin], a monoclonal antibody to VEGF, at a dose of 5 mg/kg modulated inflammation in KAT‐4 xenograft human anaplastic thyroid carcinoma tissue. At this dose, bevacizumab reduced the density of macrophages, MHC class II antigen expression by macrophages and IL‐1β mRNA expression. Furthermore, bevacizumab lowered tumor extracellular fluid volume, plasma protein leakage from tumor vessels, the number of CD31‐positive structures and tumor PIF. The tumor plasma volume and the number of α‐smooth muscle actin‐positive vessels, however, remained unchanged. Our data suggest that carcinoma cell‐derived VEGF either directly or indirectly participates in maintaining an inflammatory microenvironment in experimental KAT‐4 carcinoma. Furthermore, our data indicate that the reduction of inflammation resulting in reduced vascular permeability and decrease in the tumor extracellular fluid volume by bevacizumab contributes to reduced tumor PIF.

Interference with TGF‐β1 and ‐β3 in tumor stroma lowers tumor interstitial fluid pressure independently of growth in experimental carcinoma

A high tumor interstitial fluid pressure (TIFP) is a pathologic characteristic distinguishing the stroma of carcinomas from normal interstitial loose connective tissues. The role of TGF‐β1 and ‐β3 in generating a high TIFP was investigated in xenografted experimental anaplastic thyroid carcinoma (ATC) derived from the human ATC cell line KAT‐4. A single intravenous injection of a soluble recombinant TGF‐β receptor type II‐murine Fc:IgG2A chimeric protein that specifically inhibits TGF‐β1 and ‐β3, significantly lowered TIFP in a time and concentration dependent manner but did not change total tissue water content in the tumors. Tumor growth rate was higher in tumors treated with the TGF‐β1 and ‐β3 inhibitor compared to control tumors during the first 10 days after administration of the inhibitor. The apoptotic index of carcinoma cells, and expression of the cell cycle inhibitor p27Kip1, were, however, increased in TGF‐β1 and ‐β3 inhibitor‐treated tumors. Prolonged treatment periods and administration of a second dose of the inhibitor decreased tumor growth rate. The TGF‐β1 and ‐β3 inhibitor did not affect proliferation or expression of phosphorylated Smad2 protein in KAT‐4 cells cultured in vitro. Our results indicate that members of the TGF‐β family are potential targets for novel anti‐cancer treatment directed to the stroma. First by controlling TIFP and by that potentially the uptake of anticancer drugs into tumors and second by their suggested role in maintaining a supportive tumor stroma.

Expression of transforming growth factor-beta1, activin A, and their receptors in thyroid follicle cells: negative regulation of thyrocyte growth and function.

Expression of transforming growth factor-beta1, activin A, and their receptors in thyroid follicle cells: negative regulation of thyrocyte growth and function.

Inhibition of TGF-beta modulates macrophages and vessel maturation in parallel to a lowering of interstitial fluid pressure in experimental carcinoma.

A pathologically elevated interstitial fluid pressure (IFP) is a characteristic of both clinical and experimental carcinoma. The soluble TGF-β receptor type II-murine Fc:IgG2A chimeric protein (Fc:TβRII) lowers IFP in the KAT-4 experimental model for anaplastic thyroid carcinoma. Analyses of messenger RNA (mRNA) expressions by Affymetrix microarrays and RNase protection assays, as well as of protein expressions identified tumor macrophages as targets for Fc:TβRII. Treatment with Fc:TβRII reduced albumin extravasation, increased coverage of α-smooth muscle actin-positive cells and reduced expression of NG2, a marker of activated pericytes, in KAT-4 carcinoma blood vessels. Specific inhibition of interleukin-1 (IL-1), a major cytokine produced by activated macrophages, lowered carcinoma IFP to a similar degree as Fc:TβRII but had no significant effect on the parameters of blood vessel maturation. Neither Fc:TβRII nor inhibition of IL-1 changed blood vessel density. Finally, pretreatment of KAT-4 carcinomas with Fc:TβRII increased the antitumor efficacy of doxorubicin. Our data emphasize a potential role of tumor macrophages in carcinoma physiology and identify these cells as potential stromal targets for treatment aimed to improve efficacy of chemotherapy.