The transforming growth factor-betas (TGF-betas) are multifunctional regulators of cellular growth and function and potent inhibitors of epithelial cell proliferation. The widespread expression of TGF-beta indicates a pivotal role in epithelial homeostasis. These features make the TGF-betas attractive candidates for new therapeutic intervention approaches to the prevention and treatment of cancer. TGF-beta plays a major role in adult physiology, as well as in the control of differentiation and morphogenesis in embryonic development. The tissue distribution pattern of the TGF-betas, which include TGF-betas 1, 2, and 3 in mammals, has possible significance for signaling roles in epithelial-mesenchymal interactions during embryogenesis, as well as in cancer and carcinogenesis. TGF-beta is secreted by a variety of normal and malignant cells. The TGF-betas function through a set of cell surface protein receptors that includes TGF?beta type I (RI) and type II (RII). TGF-beta RII can bind TGF-beta directly to form a complex, which then is able to bind TGF-beta RI, and TGF-beta RII is then able to phosphorylate TGF-beta RI, which is necessary for signal transduction. The TGF-beta signaling system has been implicated as a tumor suppressor pathway in several organ systems. Loss of functional TGF-beta RI or RII contributes to loss of TGF-beta responsiveness, resulting in tumor progression. Defects in responsiveness to TGF-beta have been implicated in the pathogenesis of several human epithelial cancers, suggesting that TGF-beta has tumor suppressor properties. But, many advanced tumors show increased expression of TGF-beta, and parallel poor prognosis, suggesting that TGF-beta also has properties of a tumor suppressor. The complex mechanisms of action of TGF-beta in its capacity as a tumor suppressor and a tumor promoter and the target genes that are regulated by TGF-beta in these different capacities must be clearly defined to be able to exploit this gene for clinical therapeutic intervention purposes. Our broad goal is to determine how TGF-beta signaling regulates the development and malignant transformation of epithelial cells, now with a primary emphasis on lung epithelial cells. Our approach is based on the hypotheses that (1) Signaling pathways for TGF-beta occur that are separate from the growth inhibitory pathway and these pathways may be operational in lung cancer cells that are resistant to growth inhibition by TGF-beta; (2) The integrity of the TGF-beta signaling pathways is important for the normal regulation of downstream target molecules of TGF-beta and dysregulation of the activity of these signaling pathways during progressive stages of lung tumorigenesis impacts on the regulation of these target genes; (3) The tumor suppressor and tumor promoter activities of TGF-beta differentially regulate target genes that contribute to these activities. Our research efforts are focused around the proposal that there is a delicate balance between the tumor suppressor and tumor promoter roles of TGF-beta in epithelial tissues. Our premise is that the ability of TGF-beta to act as a tumor suppressor is primary to that of a tumor promoter in normal epithelial cells, and that the ability of TGF-beta to act as a tumor promoter takes on added significance as cells that are sensitive to TGF-beta become transformed and eventually resistant to TGF-beta, expand clonally, and ultimately progress to malignancy. Awareness of the timing of the phenotypic switch from TGF-beta sensitivity to TGF-beta resistance that occurs during tumorigenesis is likely to be important in designing and applying strategies for tumor prevention and treatment. Our most recent efforts have examined the Nkx2.1 homeobox gene lung tumorigenesis in TGF-beta 1 heterozygous (HT) mice. In addition to altered levels of genes that regulate the cell cycle in the lungs of HT mice, our use of suppression subtraction hybridization also showed decreased expression of the Nkx2.1 homeobox gene, a gene that can be detected throughout mouse lung development in the respiratory epithelial cells and that is essential for organogenesis and differentiation of the lung (37), in the lungs of AJBL6 HT mice compared to wildtype (WT) mice. Although NKX2.1 has been used a marker of advanced human lung carcinomas, immunostaining for NKX2.1 has been reported to vary widely among patients with lung tumors, from 27-76%. Furthermore, it is not known whether NKX2.1 participates in early pre-malignant stages of lung tumorigenesis. We used the AJBL6 HT mouse model of lung carcinogenesis to examine Nkx2.1 in progressive lung tumorigenesis. Northern and western analyses showed significantly lower levels of Nkx2.1 mRNA and protein, respectively, in lungs of normal HT mice compared to WT mice. Competitive RT-PCR amplification of RNAs from microdissected normal lung and EC-induced lung lesions showed that Nkx2.1 mRNA decreased significantly in adenomas and adenocarcinomas compared to normal lung in HT mice. Furthermore, Nkx2.1 showed weaker immunostaining in adenomas and adenocarcinomas compared to normal lung bronchiolar epithelium. The reduction of Nkx2.1 in progressive lung tumorigenesis is similar to reported decreased NKX3.1 in progressive human prostate cancer. In vitro studies showed that addition of TGF-beta 1 to TGF-beta 1-responsive normal immortalized mouse lung epithelial E10 cells co-transfected with Sp1 plasmid and NKX2.1Luc Luciferase reporter resulted in a significant increase in NKX2.1Luc transcription similar to human lung cells. Co-transfection of Sp3 and dominant negative TGF- RII plasmids negated the effect of Sp1. Co-transfected Sp1 plasmid with either dominant negative Smad2 or Smad3 or Smad4 plasmids significantly decreased NKX2.1Luc transcription. Electrophoretic mobility shift assays revealed binding of Sp1 and Smad4 to the NKX2.1 promoter. Our findings show reduced Nkx2.1 in lungs of HT mice compared to WT mice, that is further reduced in carcinogenesis, and that correlates with reduction of Sp1, Sp3, Smad2, Smad3, and Smad4 in lung adenocarcinomas. Our in vitro data suggest that regulation of Nkx2.1 by TGF-beta 1 occurs through TGF-beta/Smad signaling and Sp1 and Sp3 in mouse lung cells. During our study, it was reported that TGF-beta 1 inhibits Surfactant Protein-B (SP-B) gene transcription through Smad3 interactions with NKX2.1 in human lung cancer cells. This substantiates our findings and shows that Nkx2.1 interacts with TGF-1 signaling components in human and mouse lung cancer. Significance: Although our studies do not allow us to distinguish whether reduced Nkx2.1 is a cause or a consequence of lung tumorigenesis, our findings do suggest that reduced Nkx2.1 and TGF-beta 1 signaling components may contribute to tumorigenesis in the lungs of TGF-beta 1 HT mice. To learn more about the biology of adrenomedullin (AM) in the absence of the effects of TGF-beta 1 in vivo, we examined AM in TGF-1 knockout mice and compared this with TGF-beta 1 HT and WT mice. In an earlier report, we showed that the expression of TGF-beta 1 and AM, a hypotensive polypeptide that has been shown to stimulate cyclic AMP and intracellular free Ca2+, is regulated spatially and temporally such that overlapping patterns of expression of TGF-beta 1 and AM occur in several tissues at the same stage of development and in the same cellular location in rodent embryogenesis. Because of their co-localization, we hypothesized that TGF-beta 1 and AM may be able to coordinately act to influence development, differentiation, inflammation, and cancer. Significance: Our findings show that expression of AM is reduced in tissues of the developing embryonic TGF-beta 1 knockout mouse compared to HT and WT mice, but increases in postnatal development in TGF-beta 1 knockout mice.

Agency
National Institute of Health (NIH)
Type
Intramural Research (Z01)
Project #
1Z01SC000166-13
Application #
7066747
Study Section
(CCBB)
Project Start
Project End
Budget Start
Budget End
Support Year
13
Fiscal Year
2004
Total Cost
Indirect Cost
Name
Clinical Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Ozbun, Laurent L; Martinez, Alfredo; Jakowlew, Sonia B (2005) Differentially expressed nucleolar TGF-beta1 target (DENTT) shows tissue-specific nuclear and cytoplasmic localization and increases TGF-beta1-responsive transcription in primates. Biochim Biophys Acta 1728:163-80
Andreola, Fausto; Calvisi, Diego F; Elizondo, Guillermo et al. (2004) Reversal of liver fibrosis in aryl hydrocarbon receptor null mice by dietary vitamin A depletion. Hepatology 39:157-66
Bodegas, Elena; Martinez, Alfredo; Ozbun, Laurent L et al. (2004) Depressed adrenomedullin in the embryonic transforming growth factor-beta1 null mouse becomes elevated postnatally. Int J Dev Biol 48:67-70
Kang, Yang; Hebron, Haroun; Ozbun, Laurent et al. (2004) Nkx2.1 transcription factor in lung cells and a transforming growth factor-beta1 heterozygous mouse model of lung carcinogenesis. Mol Carcinog 40:212-31
Zhang, F; Pao, W; Umphress, S et al. (2004) Serum levels of surfactant protein D are increased in mice with lung tumors. Chest 125:109S
Zhang, Feijie; Pao, William; Umphress, Sarah M et al. (2003) Serum levels of surfactant protein D are increased in mice with lung tumors. Cancer Res 63:5889-94
Jakowlew, Sonia B; Mariano, Jennifer (2003) Detection of the transcripts and proteins for the transforming growth factor-beta isoforms and receptors in mouse lung tumorigenesis using in situ hybridization and immunohistochemistry in paraffin-embedded tissue sections. Methods Mol Med 74:145-66
Ozbun, Laurent L; Martinez, Alfredo; Angdisen, Jerry et al. (2003) Differentially expressed nucleolar TGF-beta1 target (DENTT) in mouse development. Dev Dyn 226:491-511
Kang, Y; Prentice, M A; Mariano, J M et al. (2000) Transforming growth factor-beta 1 and its receptors in human lung cancer and mouse lung carcinogenesis. Exp Lung Res 26:685-707
Farley, J; Gray, K; Nycum, L et al. (2000) Endocervical cancer is associated with an increase in the ligands and receptors for transforming growth factor-beta and a contrasting decrease in p27(Kip1). Gynecol Oncol 78:113-22

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