TRANSFORMING GROWTH FACTOR ß1: IMPLICATIONS IN ADRENOCORTICAL TUMORIGENESIS
G. Arnaldi, S. Freddi, T. Mancini, B. Kola, F. Mantero. Division of Endocrinology, Dept. Internal Medicine - University of Ancona. Ancona. ITALY
ABSTRACT
TGFß1, a multifunctional growth modulator, inhibits the proliferation of epithelial cells. TGFß1 signaling is dependent on the heterodimerization of the TGFß1 receptor II (TGFß1RII) with the TGFß1 receptor I (TGFBIRI). The cytoplasmic proteins Smads are the mediators of the TGFß1 signal. TGFß1 regulates adult and fetal adrenal growth and function. Previously we have shown by Northern analysis that TGFß1mRNA was well expressed in normal adrenal and in adrenocortical adenomas but reduced in carcinomas. To investigate whether TGFß1 receptors may act as tumor suppressors of adrenal tumorigenesis, 16 adenomas and 12 carcinomas were studied. We have used SSCP analysis to scan for inactivating mutations in carcinomas. All tumor samples were negative for somatic alterations of both genes. A competitive RT-PCR system was developed to compare the levels of expression of TGFß1, TGFBIR-I and TGFBIR-II, Smad-2 and Smad-4 genes in all tumors. In our study, we confirmed the presence of reduced levels of TGFß1 in carcinomas. On the contrary, Smad-4 gene levels were elevated in carcinomas when compared to that of adenomas. No significant differences were observed in gene expression of TGFBIRI and Smad-2. Our results suggest that mutations of TGFß1 receptors appear not to be involved in adrenal tumorigenesis. Adrenal carcinomas showed a significant reduction of the TGFß1 mRNA levels but on the contrary Smad 4 mRNA levels were significantly increased.
INTRODUCTION
The TGFß1 belongs to a family of multifunctional cytokines and acts predominantly to inhibit cell growth. Loss of cellular sensitivity to TGFß1- mediated growth inhibition may contribute directly to carcinogenesis (1) Although four receptors have been cloned, only two of them, type 1 (TGFBIRI) and type 2 (TGFBIRII), have been conclusively proven to mediate TGFß1 signaling. These receptors belong to a large family of serine/threonine kinases. Upon TGFß1 binding to the TGFß1RII, the TGFß1RI is recruited into a complex containing both receptors and ligand. Activated TGFBRI initiate intracellular signaling through activation of specific Smad proteins (2, 3, 4). Phosphorilated Smad-2 or Smad-3 binds to Smad-4, the common Smad; the complex moves into the nucleus where it regulates gene transcription.
Loss of TGFB1 responsiveness could depend from the deregulation of TGFß1 signaling: many of the events downstream of TGFß1 signaling have been shown to be mutated in cancers. Mutations or loss of expression of the TGFß signaling pathway are found in a number of human cancers (1, 2).
The role of TGFß1 in adrenal function and growth is of great interest (5, 6). TGFß1, normally synthesized by fetal and adult human adrenal, is a potent inhibitor of steroid production in culture cells (5, 7). TGFB1 inhibits proliferation and steroid production by fetal and definitive zone cell. Furthermore, it play a regulatory role in human fetal adrenal remodeling by inducing apoptosis of fetal zone (8, 9,). The effects of TGFß1 in the adult adrenal growth are, at present, poorly understood. Previously we have shown by Northern analysis that TGFß1mRNA was well expressed in normal adrenal and in adrenocortical adenomas but reduced in carcinomas (10)
The aim of this study was to explore if quantitative and/or qualitative molecular abnormalities of the TGFß1 signaling components may contribute to the multifactorial mechanisms involved in adrenal tumorigenesis
MATERIALS AND METHODS
We have studied 28 human adrenocortical tumors including 16 adenomas (4 nonfunctioning) and 12 carcinomas (6 nonfunctioning). RNA and DNA extraction was performed according standard methods.
In all 12 carcinomas, the intracellular kinase domain region (GS domain) of the human TGFBIRI and the microsatellite instability of PolyA of the human TGFBIRII were investigated for inactivating mutations by Single Strand Conformational Polymorphism (SSCP) analysis.
We have also developed a quantitative/competitive RT-PCR system to compare the expression of TGFB1, TGFBIRI, TGFB1RII, Smad-2 and Smad-4 genes. We designed a competitor (termed multi-mutant) that can be used with various primers to quantify the expression of several genes from a single cDNA preparation. This competitor was synthesized by successive PCR reactions introducing the various primers at the extremity of a core sequence taken from pBluescript SK. The final PCR product was subcloned in pT7 blue and nucleotide sequence determined. The recombinant plasmid was trascribed in vitro with T7 RNA polymerase. The lenght of the competitor differs 80-110 bp from the length of the different known genes of interest. After validation of the multi-mutant system, increasing amounts of competitor were mixed with fixed amounts of tumoral RNA. The quantity of genes was established when the ratio of synthetic/tumoral gene band intensities was equal to 1.
RESULTS AND DISCUSSION
All 12 carcinomas were negative for somatic mutations in the kinase domain regions of TGFBRI and TGFØRII. Therefore, mutations of TGFB receptors appear not to be involved in the pathogenesis of adrenal carcinomas.
Copies / µg Total RNA x 10 3
70
p= 0.002 ** *
1
60
Adenomas
50
40
30
p= 0.0011
20
10
0
TGFB1
TGFBRI
TGFBRII
Smad 2
Smad 4
However, other mutations interfering with TGFß signaling pathway have been described in human neoplasia (i.e. Smad2 e Smad4). Further studies are warranted to evaluated their role in adrenal tumorigenesis.
The expression of TGFß1 and TGFB1 signaling components were measured by a strict quantitative RT-PCR analysis in a large series of human adrenocortical tumors. The mRNA of all genes were readly detectable in all tissues investigated with an individual considerable variability. Absolute mRNA values appeared the highest for Smad4 and the lowest for TGFBIRI.
We confirmed the reduced (2-3 fold) levels of TGFß1 mRNA in carcinomas compared to adenomas (p<0.001). On the contrary, the mean levels of Smad-4 mRNA were increased (2 fold) in all carcinomas (p<0.001).
No significative differences were observed in gene expression of TGFØRI, TGFØRII and Smad-2 in different tumors. The gene expression was also similar in functioning and nonfunctioning tumors.
The biological role of these quantitative abnormalities remain unclear. Considering the role of Smad 4 that traslocates into the nucleus where it controls the transcription of target genes, its overexpression in adrenal carcinomas seems to be interesting. Moreover, Smads appears to function not only as nuclear effector of TGFß family members but as signal integrators within an extensive intracellular network (3).
Our preliminary results suggest that quantitative abnormalities of TGFß1 signaling pathway (decreases of TGFß1 and increases of Smad4) may play an autocrine/paracrine role in adrenal tumorigenesis and contribute to malignant progression.
ACKNOWLEDGEMENTS
This work was supported in part by grant AIRC, MURST 9706151106 and MURST 9806261488
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