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. The most recent efforts have examined the relationship of TGF-beta 1 and cell cycle genes in lung carcinogenesis. Because of the limitations imposed by the advanced stages of human lung biopsy specimens and cell culture systems, we have complemented and expanded our studies with whole animal model systems. We developed the AJBL6 TGF-beta 1 heterozygous mouse by mating A/J mice with C57BL/6 TGF-beta 1 heterozygous mice that shows increased carcinogen-induced lung lesions with decreased latency to examine progressive events in lung tumorigenesis. Mouse cDNA macroarrays were used to identify cell cycle genes that are differentially regulated in ethyl carbamate-induced lung adenocarcinomas compared to normal lung tissue in AJBL6 TGF-beta 1 heterozygous mice using probes that were generated from tissues isolated using laser capture microdissection. While expression of the genes for cyclin D1, CDK4, and E2F1 increased in lung adenocarcinomas relative to normal lung, expression of p15, p16, p21, p27, p57, and pRb genes decreased in comparison. Competitive RT-PCR showed that the levels of cyclin D1 and CDK4 mRNAs were 2- and 3-fold higher, respectively, in lung adenocarcinomas than in normal lung, while the mRNAs for p15, p16, p21, p27, and pRb were 3- to 4-fold lower in adenocarcinomas than in normal lung, thus validating the macroarray findings. Competitive RT-PCR of microdissected lesions also showed that the levels of cyclin D1 and CDK4 mRNAs increased significantly, while the mRNAs for p15 and p27 decreased significantly as lung tumorigenesis progressed. Immunohistochemical staining for cyclin D1 and CDK4 showed staining in more than 80% of nuclei in adenocarcinomas compared to fewer than 20% of nuclei staining positively in normal lung. In contrast, while more than 60% of normal lung cells showed immunostaining for p15, p16, p21, p27, and pRb, staining for these proteins decreased in hyperplasias, adenomas, and adenocarcinomas. Our findings show that multiple components of the cyclin D1/CDK4/p16/pRb signaling pathway are frequently altered early in lung lesions of AJBL6 TGF-beta 1 heterozygous mice that are induced by ethyl carbamate as a function of progressive lung carcinogenesis, suggesting that components of this pathway may be potential targets for gene therapy. To complement our animal model systems, the TGF-beta 1 sensitive epithelial lung cancer cell line NCI-H727, whose growth can be inhibited by TGF-beta 1, was used as a model system to identify potential genes involved in TGF-beta 1 growth inhibition. Comparative cDNA expression patterns between NCI-H727 cells treated with TGF-beta 1 or vehicle alone were generated by differential mRNA display analysis. Among the several cDNA fragments that represent genes that are potentially differentially regulated by TGF-beta 1, we identified a novel gene that we named Differentially Expressed Nucleolar TGF-?1 Target (DENTT) that is a novel member of the TSPY/TSPY-L/SET/NAP-1 (TTSN) superfamily. We have investigated the expression of DENTT protein in the adult mouse. By western analysis, DENTT is highly expressed in the pituitary gland, and moderately in the adrenals, brain, testis, and ovary. Immunohistochemical staining analysis for DENTT showed differential cytoplasmic and nuclear staining patterns in several cell types. The pituitary gland showed the highest level of immunostaining for DENTT, with strong cytoplasmic immunoreactivity in the anterior lobe, moderate levels in the posterior lobe, and a few cells showing nuclear staining in the intermediate lobe. In contrast, the intermediate lobe of the pituitary showed intense cytoplasmic staining for TGF-beta 1. Nuclear and cytoplasmic staining for DENTT was present in the islets of Langerhans in the pancreas. Cytoplasmic staining for DENTT was particularly intense in the cortex of the adrenal gland, while the medulla showed weak nuclear staining. In the nervous system, the choroid plexus showed the highest immunoreactivity, with cortical motoneurons and Purkinje cells having relatively high levels of staining for DENTT as well. DENTT immunoreactivity was found in Leydig interstitial cells, Sertoli cells, and primary spermatocytes in the testis. In the female reproductive system, DENTT immunoreactivity was present in oocytes, thecal cells, and corpora lutea. The bronchial epithelium of the lung showed moderate levels of staining for DENTT localized to the cell nucleus. Additionally, three rodent pituitary cell lines (AtT20, GH3, and aT3-1, representing corticotropes, lactotropes, and gonadotropes, respectively) showed expression of DENTT. Addition of TGF-?1 or serum to AtT20 cells increased DENTT protein production by 4 h, and after reaching maximal levels at 2.4-fold above basal level by 8 h, decreased, while no more than a 1.5-fold increase in DENTT protein occurred in GH3 or aT3-1 cells. Transient transfection studies showed that ectopic DENTT expression significantly increased the level of p3TP-Lux reporter transcription in AtT20 cells, but not in GH3 or aT3-1 cells. Interestingly, addition of TGF-?1 had no significant effect on the ability of DENTT expression to influence p3TP-Lux reporter transcription in AtT20 cells. This is the first detailed immunohistochemical examination of a member of the TTSN superfamily in the adult mouse. Expression of DENTT in endocrine tissues, nervous system, lung, oocytes, and thecal cells, in addition to the testis, suggests new roles for the TTSN superfamily. The differential patterns of expression of DENTT and TGF-beta 1 in some tissues, including the pituitary, suggest that other factors are likely to be regulators of DENTT besides TGF-beta 1. The significance of this project is to determine the role of TGF-beta in cancer and the manner in which the signaling pathway of TGF-beta and its target genes is altered from the normal condition. The identification of other important proteins with which TGF-beta 1 and its target genes interact in normal cells will be important in defining and determining how this interaction may be altered in cancer and carcinogenesis.
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