cyclin-d1 and Tuberous-Sclerosis

During the past two years, new molecular targets have been discovered which link cell cycle, cell proliferation and cellular growth. It has become more and more evident that whereas gain-of-function mutations in specific genes can lead to cancer, genomic instability plays also an important role in tumour progression. With examples taken from the recent literature, we describe in this short review crucial findings on the molecular mechanisms controlling cell cycle and proliferation. We illustrate how specific combinations of proto-oncogenes alterations can result in tissue-specific tumours. Finally, impairment of the interactions of a cancer cell with its surrounding neighbours is also shown to participate in the progression toward aggressive phenotypes.

Topics: Animals; Antineoplastic Agents, Phytogenic; CDC2-CDC28 Kinases; Cell Cycle; Cell Division; Cell Transformation, Neoplastic; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Disease Progression; Drug Resistance; Fusion Proteins, bcr-abl; Humans; Mice; Mutation; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms; Neovascularization, Pathologic; Neural Cell Adhesion Molecules; Paclitaxel; Protein Serine-Threonine Kinases; Radiation Tolerance; Rats; Thrombospondins; Transforming Growth Factor beta; Tuberous Sclerosis

The loss of tuberin, the tuberous sclerosis-2 (Tsc-2) gene product, is associated with cytoplasmic mislocalization of p27 in uterine leiomyomas derived from Eker rats (Tsc-2(EK/+)) and in human metastatic renal cell carcinoma tissue. Signaling associated with cytoplasmic mislocalization of p27 in renal cancer is relatively unknown. Renal tumors derived from 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ)-treated Tsc-2(EK/+) rats, and null for tuberin, display elevated nuclear and cytosolic p27, with parallel increases in cytosolic cyclin D1 levels. Similar changes are observed in TGHQ-transformed renal epithelial cells derived from Tsc-2(EK/+) rats (QTRRE cells), which, in addition to the cytoplasmic mislocalization of p27 and cyclin D1, exhibit high ERK, B-Raf, and Raf-1 kinase activity. Renal tumor xenografts, derived from subcutaneous injection of QTRRE cells into nude mice, also display increases in cytosolic mislocalization of p27 and cyclin D1. Dibutyryl cAMP and/or phosphodiesterase inhibitors (PIs; pentoxifylline or theophylline) increase Rap1B activation, B-Raf kinase activity, and cytosolic p27/cyclin D1 protein levels in QTRRE cells. Inhibition of Raf kinases with either sorafenib or B-Raf small interfering RNA (siRNA) caused a mitogen-activated protein kinase-mediated downregulation of p27. Moreover, decreases in cyclin D1 were also associated with p27 siRNA knockdown in QTRRE cells. Finally, theophylline-mediated increases in p27 and cyclin D1 were attenuated by sorafenib, which modulated Raf/MEK/ERK signaling. Collectively, these data suggest that the cAMP/Rap1B/B-Raf pathway modulates the expression of p27 and the cytoplasmic mislocalization of p27-cyclin D1 in tuberous sclerosis gene-regulated-renal cancer. Therefore, the loss of tuberin and engagement of the cAMP pathway may independently direct p27-cyclin D1 cytosolic stabilization during renal tumor formation.

Topics: Animals; Benzenesulfonates; Bucladesine; Carcinoma, Renal Cell; Cell Line; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Cytosol; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Glutathione; Humans; Hydroquinones; Kidney Neoplasms; Male; Mice; Mice, Nude; Mitogen-Activated Protein Kinases; Niacinamide; Pentoxifylline; Phenylurea Compounds; Phosphodiesterase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-raf; Pyridines; Rats; RNA, Small Interfering; Signal Transduction; Sorafenib; Theophylline; Tuberous Sclerosis; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

The tuberous sclerosis complex (TSC) is caused by defects in one of two tumor suppressor genes, TSC-1 or TSC-2. TSC-2 gene encodes tuberin, a protein involved in the pathogenesis of kidney tumors, both angiomyolipomas and renal cell carcinomas. Loss of heterozygosity at the 8-oxoG-DNA glycosylase (OGG1) allele is found in human kidney clear cell carcinoma identifying loss of OGG1 function as a possible contributor to tumorigenesis in the kidney. Tuberin regulates OGG1 through the transcription factor NF-YA in cultured cells. The purpose of this study is to determine the effect of tuberin-deficiency on OGG1 protein and mRNA levels as well as on 8-oxodG levels in kidney tumors from patients with TSC. In addition we evaluated the phophorylation level of downstream targets of mTOR, phospho-S70K, in kidney tumor tissue from TSC patients.. Kidney angiomyolipoma tissue from TSC patients expresses significant levels of phopho-tuberin and low levels of tuberin compared to control kidney tissue. The increase in tuberin phosphorylation and the decrease tuberin expression are associated with decrease in OGG1 protein and mRNA levels in tumor samples compared to normal kidney samples. The decrease OGG1 expression is also associated with significant decrease in the transcription factor, NF-YA, expression in tumor samples compared to normal tissues. In addition, the levels of 8-oxodG are 4-fold higher in tumors compared to control samples. The significant increase of phospho-tuberin expression is associated with increase phosphorylation of S6K in tumor samples compared to controls. Cyclin D1 expression is also 3-fold higher in increase in the tumor tissues compared to normal kidney tissues.. These data indicate that tuberin deficiency in angiomyolipoma enhances mTOR activation by phosphorylation of S6K and downregulation of protein and mRNA expression of OGG1 resulted in accumulation of oxidized DNA in patients with TSC. These data suggest that tuberin and OGG1 are important proteins in the pathogenesis of angiomyolipoma in TSC patients.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Angiomyolipoma; Animals; CCAAT-Binding Factor; Cell Line; Cyclin D1; Deoxyguanosine; DNA Damage; DNA Glycosylases; Humans; Immunoblotting; In Vitro Techniques; Kidney; Mice; Oxidative Stress; Phosphorylation; Protein Kinases; Rats; Reverse Transcriptase Polymerase Chain Reaction; TOR Serine-Threonine Kinases; Tuberous Sclerosis; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

Tuberous sclerosis (TS), autosomal dominant disorder manifested by the formation of usually benign tumors in the brain, heart, kidneys and skin, results from an inactivating mutation in one of two tumor suppressor genes TSC1 or TSC2. Protein products of these genes, hamartin and tuberin, respectively, have been shown to participate in the mTOR pathway controlling translation of approx. 10-15% of all proteins. In the current paper, we aimed at verifying whether hamartin and tuberin may also be implicated in the control of gene transcription. Very recently it has been hypothesized that the pathway triggered by WNT, one of embryonic growth factors involved in cell differentiation and migration, could be disturbed in TS. In order to test this hypothesis we evaluated samples of four subependymal giant cell astrocytomas (SEGAs), brain tumors developing in the progress of TS. We found that beta-catenin, transcription factor and mediator of WNT pathway activity is indeed present and active in SEGAs. mRNA transcripts for c-Myc and N-Myc, proteins whose transcription is regulated by beta-catenin, were upregulated in two of four SEGAs, while cyclin D1 mRNA was significantly higher in three SEGAs. At the same time, c-Myc and N-Myc proteins were detected in the same two samples. Thus, we show for the first time that aberrant WNT signaling may contribute to the pathogenesis of TS-associated SEGAs.

Topics: Astrocytoma; beta Catenin; Blotting, Western; Brain Neoplasms; Cell Nucleus; Cyclin D1; Genes, myc; Humans; Immunohistochemistry; Nerve Tissue Proteins; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transcription, Genetic; Translocation, Genetic; Tuberous Sclerosis; Up-Regulation; Wnt Proteins

The pathology associated with tuberous sclerosis complex (TSC) shows diverse phenotypes that suggest abnormal signaling of multiple pathways. Besides the negative regulatory role of the TSC1/TSC2 proteins on mTOR, we have reported an effect on beta-catenin signaling at the level of the degradation complex in vitro. The TSC1/TSC2 complex associates with GSK3 and Axin and promotes beta-catenin degradation to inhibit Wnt-stimulated TCF/LEF-dependent transcription. Here, we show that beta-catenin and its effectors, cyclin D1 and connexin 43, were up-regulated in TSC-related angiomyolipomas and lymphangioleiomyomatosis. This was supported by the failure of three disease-causing TSC2 missense mutants to inhibit Wnt signaling. Further, the interaction between TSC1/TSC2 and components of the beta-catenin degradation complex was dependent on Wnt stimulation such that binding of tuberin to GSK3 and Axin was reduced in the presence of Wnt whereas the tuberin-Dishevelled interaction was increased. GSK3 activity played a role in regulating the assembly/stability of the degradation complex. Inhibition of GSK3 by lithium chloride reduced its association with TSC1 whereas disruption of GSK3-phosphorylation sites in TSC1 reduced interaction between TSC2 and TSC1. Collectively, our data provide further evidence that beta-catenin signaling plays a role in TSC pathogenesis in vivo and suggest a novel role of GSK3 in modulating the TSC1/TSC2 complex through TSC1 phosphorylation.

Topics: Adaptor Proteins, Signal Transducing; Angiomyolipoma; Animals; Axin Protein; beta Catenin; Connexin 43; Cyclin D1; Cytoskeletal Proteins; Dishevelled Proteins; Glycogen Synthase Kinase 3; Humans; Immunohistochemistry; Immunoprecipitation; Intercellular Signaling Peptides and Proteins; Lymphangioleiomyomatosis; Mice; Mutation, Missense; Phosphoproteins; Phosphorylation; Rats; Repressor Proteins; Signal Transduction; Trans-Activators; Tuberous Sclerosis; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins; Wnt Proteins