Survey Part- A ] Serine/Threonine and Tyrosine Protein Kinases in "Fibroblast" of Stroma (cancer) Microenvironment

b) TGF-Beta: Serine/threonine protein kinases: TKL group(TGFBR2 family - Transforming growth factor, beta receptor II) and associated proteins:


Transforming growth factor-beta (TGF-beta) plays complex dual roles as an inhibitor and promoter of tumor progression. Although the influence of the stromal microenvironment on tumor progression is well recognized, little is known about the functions of TGF-beta signaling in the stroma during tumor progression. In a co-xenograft model, the authors have demonstrated that TGF-bR2(FspKO) fibroblasts enhance mammary carcinoma growth and metastasis in mice while increasing hepatocyte growth factor (HGF) expression and c-Met signaling downstream pathways including signal transducers and activators of transcription 3 (Stat3) and p42/44 mitogen-activated protein kinase (MAPK). The results show that TGF-beta signaling in fibroblasts suppresses tumor metastasis by antagonizing HGF/c-Met signaling within tumor epithelial cells. Furthermore, this co-xenograft model represents a unique context to study stromal TGF-beta and HGF signaling in mammary tumorigenesis(1).

Independent studies:
In Crouzon's syndrome: demonstrated about the in vitro differences between normal and Crouzon fibroblasts may be due to an imbalance in TGF beta and bFGF levels which alters the microenvironment where morphogenesis takes place. Further studies also proved that a TM domain (transmembrane domains of receptor tyrosine kinases (RTKs)) pathogenic mutation is the Ala391-->Glu mutation in fibroblast growth factor receptor 3 (FGFR3), linked to Crouzon syndrome with acanthosis nigricans, as well as to bladder cancer(2, 3)

In idiopathic pulmonary fibrosis (IPF): is characterized by fibroblast expansion and extracellular matrix accumulation. Some secreted matrix metalloproteinases (MMPs) as MMP2 are highly upregulated in IPF lungs(4), also a most common lung disease predisposing lung cancer (5) where also showed the cross-talk of epithelial abnormalities and the involvement of up-regulated p63-jag1 pathway (5).

In lung fibroblasts, TGF-beta1 induced a strong upregulation of MT3-MMP, both at the gene and protein level. This effect was blocked by genistein, a protein tyrosin kinase inhibitor and partially repressed by SB203580 a p38 MAP kinase inhibitor. Interestingly MT3-MMP (Type 3 transmembrane) that was found in fibroblastic foci was upregulated in vitro by TGF-beta1 a potent profibrotic mediator(4)

In skin tumor: Keloids are abnormal fibrous growths of the dermis that develop only in response to wounding and represent a form of benign skin tumor. Previous studies have shown increased protein levels of TGF-beta in keloid tissue, suggesting a strong association with keloid formation. Further immunoblotting analysis demonstrated that p38 MAPK was phosphorylated within 15 min and was maintained at a high level in keloid human fibroblasts (KFs) but not in normal human fibroblasts (NFs). The transcription factors activating transcription factor-2 and Elk-1 are activated by p38 MAPK, and also showed rapid and prolonged phosphorylation kinetics in KFs but not in NFs. In conclusion, increased TGF-beta2 transcription in response to serum stimulation in KFs appears to be mediated by the p38 MAPK pathway. This suggests the mechanism of keloid pathogenesis may be due in part to an inherent difference in how the fibroblasts respond to wounding(6)

Epidermal growth factor (EGF) Vs Gangliosides:
Gangliosides are shed by tumor cells and can bind to normal cells in the tumor microenvironment and affect their function. Exposure of fibroblasts to exogenous gangliosides increases epidermal growth factor (EGF)-induced fibroblast proliferation and enhances EGF receptor (EGFR)-mediated activation of the mitogen-activated protein kinase signaling pathway (Li, R., Liu, Y., and Ladisch, S. (2001) J. Biol. Chem. 276, 42782-42792).

The authors concluded that membrane ganglioside enrichment of normal fibroblasts (such as by tumor cell ganglioside shedding) facilitates receptor-receptor interactions (possibly by altering membrane topology), causing ligand-independent EGFR dimerization and, in turn, enhanced EGF signaling(7).

References:

1Cheng N etal Enhanced hepatocyte growth factor signaling by type II transforming growth factor-beta receptor knockout fibroblasts promotes mammary tumorigenesis Cancer Res. 2007 May 15;67(10):4869-77

2Baroni T etal Crouzon's syndrome: differential in vitro secretion of bFGF, TGFbeta I isoforms and extracellular matrix macromolecules in patients with FGFR2 gene mutation Cytokine. 2002 Jul 21;19(2):94-101.

3 Li E etal FGFR3 dimer stabilization due to a single amino acid pathogenic mutation J Mol Biol. 2006 Feb 24;356(3):600-12

4García-Alvarez J etal Membrane type-matrix metalloproteinases in idiopathic pulmonary fibrosis Sarcoidosis Vasc Diffuse Lung Dis. 2006 Mar;23(1):13-21

5 Murata K etal p63 - Key molecule in the early phase of epithelial abnormality in idiopathic pulmonary fibrosis Exp Mol Pathol. 2007 Apr 10;

6 Xia W etal P38 MAP kinase mediates transforming growth factor-beta2 transcription in human keloid fibroblasts Am J Physiol Regul Integr Comp Physiol. 2006 Mar;290(3):R501-8.

7 Liu Y etal Exogenous ganglioside GD1a enhances epidermal growth factor receptor binding and dimerization J Biol Chem. 2004 Aug 27;279(35):36481-9.

Abstracts for basic references/facts:

i) Alvarez RJ etal Biosynthetic and proliferative characteristics of tubulointerstitial fibroblasts probed with paracrine cytokines Kidney Int. 1992 Jan;41(1):14-23

Fibroblasts in parenchymal organs potentially contribute extracellular matrix to local fibrogenic processes. This contribution, in some circumstances, may be initiated by cytokines disseminated from inflammatory lesions. Different populations of fibroblasts, however, might respond distinctively to this cytokine bath depending on the microenvironment in which they reside. We have begun to explore this issue using syngeneic, low-passage fibroblasts cultured in serum-free media that were derived originally from the dermis (DFBs) and from tubulointerstitium (TFBs) of the kidney. Our findings indicate that, while fibroblasts from each compartment appear similar at the ultrastructural level, there are a variety of functional differences which distinguish their proliferative response, and their collagen secretory response (types I, III, IV, and V) following challenge with various doses of immune-relevant cytokines (TGF beta, EGF, IL-1, IL-2 and gamma IFN) in culture. DFBs, for example, express more surface EGF receptors than do TFBs, and, as a consequence, exhibit a more robust proliferative response to EGF in serum-free media. Unstimulated DFBs also secrete more collagen types I and III than TFBs, while unstimulated TFBs secrete more types IV and V. The expression of these collagens in TFBs was confirmed by Northern blot hybridization. When these sets of fibroblasts were further stimulated by cytokines, some of the cytokines not only differentially effect the secretion of various species of collagens within the same group of cells, but also between cells from populations which are anatomically distinct. DFBs, furthermore, at mid-level doses of cytokine, demonstrated a general trend towards less secretion of all types of collagen (particularly for TGF beta, EGF, and IL-2), while TFBs seemed less repressive. In TFBs the cytokine-induced responses for collagen types I and III tended to be discordant, and for types I and IV EGF inhibited, while TGF beta stimulated the secretory process. These findings speak collectively for the presence of a functional heterogeneity among organ-based populations of syngeneic fibroblasts in normal tissues.

ii) Chesi M etal Activated fibroblast growth factor receptor 3 is an oncogene that contributes to tumor progression in multiple myeloma Blood. 2001 Feb 1;97(3):729-36.


The t(4;14) translocation occurs frequently in multiple myeloma (MM) and results in the simultaneous dysregulated expression of 2 potential oncogenes, FGFR3 (fibroblast growth factor receptor 3) from der(14) and multiple myeloma SET domain protein/Wolf-Hirschhorn syndrome candidate gene 1 from der(4). It is now shown that myeloma cells carrying a t(4;14) translocation express a functional FGFR3 that in some cases is constitutively activated by the same mutations that cause thanatophoric dysplasia. As with activating mutations of K-ras and N-ras, which are reported in approximately 40% of patients with MM, activating mutations of FGFR3 occur during tumor progression. However, the constitutive activation of ras and FGFR3 does not occur in the same myeloma cells. Thus the activated forms of these proteins appear to share an overlapping role in tumor progression, suggesting that they also share the signaling cascade. Consistent with this prediction, it is shown that activated FGFR3-when expressed at levels similar to those seen in t(4;14) myeloma-is an oncogene that acts through the MAP kinase pathway to transform NIH 3T3 cells, which can then generate tumors in nude mice. Thus, FGFR3, when overexpressed in MM, may be not only oncogenic when stimulated by FGF ligands in the bone marrow microenvironment, but is also a target for activating mutations that enable FGFR3 to play a ras-like role in tumor progression.

iii) Kerry A Brenner Regulation of fibronectin matrix assembly by activated Ras in transformed cells Oncogene (2000) 19, 3156-3163

Fibronectin extracellular matrix plays a critical role in the microenvironment of cells. Loss of this matrix frequently accompanies oncogenic transformation, allowing changes in cell growth, morphology, and tissue organization. The HT1080 human fibrosarcoma cell line is deficient in formation of fibronectin matrix fibrils but assembly can be induced by the glucocorticoid dexamethasone. Here we show that fibronectin assembly can also be restored by stimulation of alpha5beta1 integrin with activating antibody or with Mn2+ suggesting that integrin activity is reduced in these cells. While dexamethasone promoted actin stress fiber formation, actin filaments remained cortical following Mn2+ treatment showing that the dexamethasone effect is not due solely to cytoskeletal changes. HT1080 cells have one activated allele of N-ras and PD98059 inhibition of signaling from Ras through ERK increased fibronectin matrix accumulation. Conversely, the p38 MAP kinase inhibitor SB203580 blocked induction of matrix and increased ERK phosphorylation. Thus, two MAP kinase pathways contribute to the control of integrin-mediated fibronectin assembly. ERK activity and fibronectin assembly were linked in three different ras-transformed cell lines but not in SV40- or RSV-transformed cells indicating that oncogenic Ras uses a distinct mechanism to down-regulate cell-fibronectin interactions.