Tumor Suppressor Fat Research Articles

DNA methylation biomarkers for lung cancer

Changes in DNA methylation patterns are an important characteristic of human cancer including lung cancer. In particular, hypermethylation of CpG islands is a signature of malignant progression. Methylated CpG islands are promising diagnostic markers for the early detection of cancer. However, the full extent and sequence context of DNA hypermethylation in lung cancer has remained unknown. We have used the methylated CpG island recovery assay and high-resolution microarray analysis to find hypermethylated CpG islands in squamous cell carcinomas (SCC) and adenocarcinomas of the lung. Each tumor contained several hundred hypermethylated CpG islands. In an initial microarray screen, 36 CpG islands were methylated in five of five (=100%) of the SCC tumors tested and 52 CpG islands were methylated in at least 75% of the adenocarcinomas tested (n=8). Using sodium-bisulfite-based approaches, 12 CpG islands (associated with the BARHL2, EVX2, IRX2, MEIS1, MSX1, NR2E1, OC2, OSR1, OTX1, PAX6, TFAP2A, and ZNF577 genes) were confirmed to be methylated in 85% to 100% of the squamous cell carcinomas and 11 CpG islands (associated with the CHAD, DLX4, GRIK2, KCNG3, NR2E1, OSR1, OTX1, OTX2, PROX1, RUNX1, and VAX1 genes) were methylated in >80% of the adenocarcinomas. From the list of genes that were methylated in lung adenocarcinomas, we identified the gene FAT4 and found that this gene was methylated in 39% of the tumors. FAT4 is the closest mammalian homologue of the Drosophila tumor suppressor Fat which is an important component of the Hippo growth control pathway. Many of these newly discovered methylated CpG islands hold promise for becoming biomarkers for the early detection of lung cancer.

Dual Processing of FAT1 Cadherin Protein by Human Melanoma Cells Generates Distinct Protein Products

The giant cadherin FAT1 is one of four vertebrate orthologues of the Drosophila tumor suppressor fat. It engages in several functions, including cell polarity and migration, and in Hippo signaling during development. Homozygous deletions in oral cancer suggest that FAT1 may play a tumor suppressor role, although overexpression of FAT1 has been reported in some other cancers. Here we show using Northern blotting that human melanoma cell lines variably but universally express FAT1 and less commonly FAT2, FAT3, and FAT4. Both normal melanocytes and keratinocytes also express comparable FAT1 mRNA relative to melanoma cells. Analysis of the protein processing of FAT1 in keratinocytes revealed that, like Drosophila FAT, human FAT1 is cleaved into a non-covalent heterodimer before achieving cell surface expression. The use of inhibitors also established that such cleavage requires the proprotein convertase furin. However, in melanoma cells, the non-cleaved proform of FAT1 is also expressed at the cell surface together with the furin-cleaved heterodimer. Moreover, furin-independent processing generates a potentially functional proteolytic product in melanoma cells, a persistent 65-kDa membrane-bound cytoplasmic fragment no longer in association with the extracellular fragment. In vitro localization studies of FAT1 showed that melanoma cells display high levels of cytosolic FAT1 protein, whereas keratinocytes, despite comparable FAT1 expression levels, exhibited mainly cell-cell junctional staining. Such differences in protein distribution appear to reconcile with the different protein products generated by dual FAT1 processing. We suggest that the uncleaved FAT1 could promote altered signaling, and the novel products of alternate processing provide a dominant negative function in melanoma.

Phosphorylation of the Tumor Suppressor Fat Is Regulated by Its Ligand Dachsous and the Kinase Discs Overgrown

The Drosophila tumor suppressor gene fat encodes a large cadherin that regulates growth and a form of tissue organization known as planar cell polarity (PCP). Fat regulates growth via the Hippo kinase pathway, which controls expression of genes promoting cell proliferation and inhibiting apoptosis (reviewed in). The Hippo pathway is highly conserved and is implicated in the regulation of mammalian growth and cancer development. Genetic studies suggest that Fat activity is regulated by binding to another large cadherin, Dachsous (Ds). The tumor suppressor discs overgrown (dco)/Casein Kinase I delta/epsilon also regulates Hippo activity and PCP. The biochemical nature of how Fat, Ds, and Dco interact to regulate these pathways is poorly understood. Here we demonstrate that Fat is cleaved to generate 450 kDa and 110 kDa fragments (Fat(450) and Fat(110)). Fat(110) contains the cytoplasmic and transmembrane domain. The cytoplasmic domain of Fat binds Dco and is phosphorylated by Dco at multiple sites. Importantly, we show Fat forms cis-dimers and that Fat phosphorylation is regulated by Dachsous and Dco in vivo. We propose that Ds regulates Dco-dependent phosphorylation of Fat and Fat-associated proteins to control Fat signaling in growth and PCP.

The Fat and Warts signaling pathways: new insights into their regulation,mechanism and conservation

A cassette of cytoplasmic Drosophila tumor suppressors, including the kinases Hippo and Warts, has recently been linked to the transmembrane tumor suppressor Fat. These proteins act within interconnected signaling pathways, the principal functions of which are to control the growth and polarity of developing tissues. Recent studies have enhanced our understanding of the basis for signal transduction by Fat and Warts pathways, including the identification of a DNA-binding protein at the end of the pathway, have established the conservation of Fat and Warts signaling from flies to mammals, and have given us new insights into their regulation and biological functions.

Genes Affecting Cell Competition in Drosophila

Cell competition is a homeostatic mechanism that regulates the size attained by growing tissues. We performed an unbiased genetic screen for mutations that permit the survival of cells being competed due to haplo-insufficiency for RpL36. Mutations that protect RpL36 heterozygous clones include the tumor suppressors expanded, hippo, salvador, mats, and warts, which are members of the Warts pathway, the tumor suppressor fat, and a novel tumor-suppressor mutation. Other hyperplastic or neoplastic mutations did not rescue RpL36 heterozygous clones. Most mutations that rescue cell competition elevated Dpp-signaling activity, and the Dsmurf mutation that elevates Dpp signaling was also hyperplastic and rescued. Two nonlethal, nonhyperplastic mutations prevent the apoptosis of Minute heterozygous cells and suggest an apoptosis pathway for cell competition . In addition to rescuing RpL36 heterozygous cells, mutations in Warts pathway genes were supercompetitors that could eliminate wild-type cells nearby. The findings show that differences in Warts pathway activity can lead to competition and implicate the Warts pathway, certain other tumor suppressors, and novel cell death components in cell competition, in addition to the Dpp pathway implicated by previous studies. We suggest that cell competition might occur during tumor development in mammals.

FAT is a component of glomerular slit diaphragms

Slit diaphragms are intercellular junctions of podocytes of the renal glomerulus. The molecular composition of slit diaphragms is still elusive. Slit diaphragms are characterized by the presence of a wide intercellular space. The morphological feature is shared by desmosomes and adherens junctions, which contain members of the cadherin superfamily. Thus, we have hypothesized that some components of slit diaphragms belong to the cadherin superfamily. Consequently, we have isolated cDNA encoding FAT from reverse-transcribed (RT) glomerular cDNA by homology polymerase chain reaction (PCR) using primers based on conserved sequences in cadherin molecules. FAT is a novel member of the cadherin superfamily with 34 tandem cadherin-like extracellular repeats, and it closely resembles the Drosophila tumor suppressor fat. Expression of FAT was examined in glomeruli of the adult rat kidney by the ribonuclease protection assay and in situ hybridization. To localize the FAT protein in podocytes minutely, we prepared affinity-purified antibody against FAT by immunizing rabbits against an oligopeptide corresponding to the C-terminal 20 amino acids. Expression of FAT mRNA was detected in total RNA from glomeruli. In situ hybridization revealed significant signals in podocytes. Western blot analysis using solubilized glomeruli showed a single band, in which the molecular weight was more than 500 kD. Immunostaining of cultured epithelial cells from rat kidney (NRK52E) revealed FAT accumulation in cell-cell contact sites. In the glomerulus, FAT staining was observed distinctly along glomerular capillary walls. Double-label immunostaining using monoclonal antibody against slit diaphragms (mAb 5-1-6) showed identical localization of anti-FAT antibody and mAb 5-1-6. Furthermore, the double-label immunogold technique with ultrathin cryosections demonstrated that gold particles for FAT cytoplasmic domain were located at the base of slit diaphragms labeled by mAb 5-1-6 and that the cytoplasmic domain of FAT colocalized with ZO-1, a cytoplasmic component associated with slit diaphragms. The molecular structure of FAT and its colocalization with 5-1-6 antigen and ZO-1 indicate that FAT is a component of slit diaphragms.

Multiple Cadherins Are Expressed in Human Fibroblasts

Although the cell-cell adhesiveness of fibroblasts is thought to be related to wound healing, the molecular basis of this adhesiveness is still unknown. We isolated five kinds of cadherin fragments from the cDNA of human fibroblasts by polymerase chain reaction (PCR). Two of the five were known cadherins: PC43, a protocadherin containing six cadherin repeats in the extracelluar domain, and human Fat, which is the human homologue of the Drosophila tumor suppressor Fat. The other three were novel cadherin fragments, and we named them cadherins FIB1, FIB2, and FIB3. The expressions of cadherins including E-, P-, and N-cadherin, PC43, human Fat, and cadherins FIB1, FIB2, and FIB3 were compared in human fibroblasts, human melanocytes, and human epidermal keratinocytes. The latter six cadherins were expressed in human fibroblasts, and cadherins FIB1 and FIB2 were fibroblast-specific. These results suggest that diverse cadherin molecules may contribute to the cell-cell adhesion in human fibroblasts.

A family of laminin-related proteins controlling ectodermal differentiation in Drosophila

It is shown that the proteins encoded by the tumor suppressor fat gene, the neurogenic slit gene and crumbs gene of Drosophila contain domains homologous with modules identified previously in laminin A. These proteins of Drosophila have a number of features in common; they have large extracellular regions containing laminin A modules linked to epidermal growth factor-like domains, and they are all involved in cell—cell interactions that are crucial for correct morphogenesis or ectodermal tissues (development of midline neuroepithelia, organization of epihelial tissues etc.) It is suggested that the laminin A-type modules of these proteins play important roles in the interactions that control ectodermal differentiation.