Scrib, a tumor suppressor gene involved in cell polarity

Polarity is a fundamental feature of all organisms both during development and in the adult. This reflects the key role of cell polarity during basic fundamental processes such as cell division, cell differentiation and cell migration. The control of cell polarity relies on functionally conserved proteins. Among these, Scribble, initially identified as a tumor suppressor gene in Drosophila, has been first involved in epithelial polarity. More recently Scribble function has been implicated in neuronal polarity and polarized cell migration. Scribble joins the growing family of tumor suppressors that play a key and conserved function in cell polarity. Scribble illustrates the more and more obvious link between regulation of cell polarity, cell transformation and tumor formation.     

Persistent expression of the tumor suppressor gene DCC is essential for neuronal differentiation.

Cell differentiation is associated either with a complete loss of proliferative potential or with a change in growth requirements. Neoplastic transformation may result from the activation of oncogenes that support growth or from inactivation or loss of tumor suppressor genes, which are thought to regulate differentiation. To examine the relationship between tumor suppressor genes and cell differentiation, we chose the gene "deleted in colorectal cancer" (DCC) and studied its role in a pheochromocytoma cell line, PC-12, using antisense RNA as well as antisense oligonucleotides to DCC. When exposed to nerve growth factor for several days, PC-12 cells develop long dendrites. This morphological change follows the transient expression of immediate early genes and is associated with an up-regulation of DCC. Interestingly, if the up-regulation of DCC was counteracted using an antisense RNA technique, the morphological changes were prevented, but the other parameters of the nerve growth factor response were unaffected. Moreover, when DCC expression was inhibited by antisense oligonucleotides to DCC in nerve growth factor-differentiated cells, the neuron-like phenotype was reversed. Our results demonstrate that the gene DCC is involved in a distal segment of neural differentiation and provide the first direct evidence that a tumor suppressor gene plays a role in cell differentiation.     

The human homolog of yeast SEP1 is a novel candidate tumor suppressor gene in osteogenic sarcoma

The hSEP1 gene is the human homolog of yeast SEP1. Yeast SEP1 is a multifunctional gene that regulates a variety of nuclear and cytoplasmic functions including homologous recombination, meiosis, telomere maintenance, RNA metabolism and microtubule assembly. The function of hSEP1 is not known. We show loss or reduced expression of hSEP1 messenger RNA (mRNA) in three of four primary osteogenic sarcoma (OGS)-derived cell lines and in eight of nine OGS biopsy specimen. In addition, we find a heterozygous missense mutation (Valine(1484)>Alanine) at a conserved amino acid in the primary OGS-derived cell line U2OS. Importantly, we identified a homozygous missense mutation involving a CG-dinucleotide leading to a change in a conserved amino acid, aspartic acid(1137) >asparagine, in the primary OGS-derived cell line, TE85. hSEP1 mRNA expression was nearly undetectable in TE85 and low in U2OS cell lines. None of these mutations were identified in 20 normal samples consisting of bone, cartilage and fibroblast. The hSEP1 gene is located in chromosome 3 at 3q25-26.1 between markers D3S1309 and D3S1569. An adjacent locus defined by the polymorphic markers D3S1212 and D3S1245 has previously been reported to undergo loss of heterozygosity (LOH) at a >70% frequency in OGS and claimed to harbor an important tumor suppressor gene in osteosarcoma. The homozygous mutation in the hSEP1 mRNA in TE85 cell line suggest that this gene itself is subject to LOH. Taken together, these results suggest that hSEP1 acts as a tumor suppressor gene in OGS.     

Analysis of the candidate tumor suppressor Ris-1 in primary human breast carcinomas

Frequent chromosome 3 losses have been described in several tumors types, which strongly suggest the presence of one or several tumor suppressor genes. Recently, a novel candidate tumor suppressor gene termed Ris-1 (for Ras-induced senescence 1) has been identified at chromosomal position 3p21.3. Ris-1 has been proposed to participate in anti-tumor responses that resemble cellular senescence and that are elicited by oncogenes such as Ras. To analyze the role of Ris-1 as a putative tumor suppressor gene in human breast cancer, we have performed a real-time quantitative analysis of its mRNA expression in 60 patients. Moreover, we carried out a first approach to evaluate the most common inactivation mechanism that can affect expression levels of tumor suppressor genes (mutation, promoter hypermethylation and allelic losses). Furthermore, a correlation study between expression as well as inactivating mechanisms of Ris-1 and several clinico-pathological parameters of the tumors was designed, with the objective of appraising the prognostic value of Ris-1 status. Decreased expression of Ris-1 was observed in 23% of the cases and overexpressed Ris-1 was detected in 15% of the primary breast tumors. Our data showed high frequency of LOH (30%) at one of the markers used. Nevertheless, a polymorphism related with the expression levels was described. Statistically significant correlations were found between decreased Ris-1 expression and negative progesterone receptors, as well as between overexpressing Ris-1 tumors and high histological grade. Despite all these data, we conclude that the suggested role of Ris-1 as tumor suppressor gene is not evident, at least in breast cancer. Future and larger series studies in different tumor types are necessary to clarify Ris-1 function in human cancer.     

The tumor suppressor protein TSLC1 is involved in cell-cell adhesion

TSLC1 is a tumor suppressor gene encoding a member of the immunoglobulin (Ig) superfamily. The significant homology of its extracellular domain with those of other Ig superfamily cell adhesion molecules (IgCAMs) has raised the possibility that TSLC1 participates in cell-cell interactions. In this study, the physiological properties of TSLC1 were investigated in Madin-Darby canine kidney (MDCK) cells expressing TSLC1 tagged with green fluorescent protein (GFP) as well as in the cells that express endogenous TSLC1. Biochemical analysis has revealed that TSLC1 is an N-linked glycoprotein with a molecular mass of 75 kDa and that it forms homodimers through cis interaction within the plane of the cell membranes. Confocal laser scanning microcopy of the cells expressing TSLC1 showed the localization patterns characteristic to adhesion molecules. At the beginning of cell attachment, TSLC1 accumulated in interdigitated structures at cell-cell boundaries, but, when cells reached a confluence, TSLC1 was distributed all along the cell membranes. In polarized cells, TSLC1 was recruited to the lateral membrane, implying trans interaction of TSLC1 between neighboring cells. In support of this notion, MDCK cells expressing TSLC1-GFP showed a significant level of cell aggregation in the absence or presence of Ca(2+) and Mg(2+). Taken together, these results indicate that TSLC1 mediates intracellular adhesion through homophilic interactions in a Ca(2+)/Mg(2+)-independent manner.     

ATM gene expression is associated with differentiation and angiogenesis in infiltrating breast carcinomas

The product of the ATM gene, mutated in the human genetic disorder ataxia-telangiectasia (A-T) plays a key role in the detection and repair of DNA double-strand breaks. A-T is defined by progressive cerebellar ataxia, telangiectasia, sensitivity to ionising radiation and genomic instability with cancer predisposition. On the other hand, increased angiogenesis is essential for tumor growth and metastasis. The aim of this study was to investigate ATM expression in breast carcinomas and its relationship to neoangiogenesis. METHODS AND RESULTS: Fifty-two breast tumors from 51 patients, 38 of them with concomitant in situ component (CIS), were analyzed by immunohistochemistry for the expression of ATM. CD34 expression was used for the morphometric evaluation of vasculature. ATM was positive in 1 to 10% of normal epithelial cells. ATM expression was reduced in 55.8% of infiltrating carcinomas, non-reduced in 34.6%, and increased in 9.6%. Expression of ATM in CIS was similar to the infiltrating component in 71% of cases and reduced in 23.7% of them. High-grade ductal infiltrating carcinomas showed lower ATM expression than low-grade ones. Reduced ATM expression also correlated with increased microvascular area. CONCLUSIONS: Reduced ATM expression in breast carcinomas correlated with tumor differentiation and increased microvascular parameters, supporting its role in neoangiogenesis and tumor progression in breast carcinogenesis.     

NIRF constitutes a nodal point in the cell cycle network and is a candidate tumor suppressor

In biological networks, a small number of “hub” proteins play critical roles in the network integrity and functions. The cell cycle network orchestrates versatile cellular functions through interactions between many signaling modules, whose defects impair diverse cellular processes, often leading to cancer. However, the network architecture and molecular basis that ensure proper coordination between distinct modules are unclear. Here, we show that the ubiquitin ligase NIRF (also known as UHRF2), which induces G₁ arrest, interacts with multiple cell cycle proteins including cyclins (A2, B1, D1 and E1), p53 and pRB, and ubiquitinates cyclins D1 and E1. Consistent with its versatility, a bioinformatic network analysis demonstrated that NIRF is an intermodular hub protein that is responsible for the coordination of multiple network modules. Notably, intermodular hubs are frequently associated with oncogenesis. Indeed, we detected loss of heterozygosity of the NIRF gene in several kinds of tumors. When a cancer outlier profile analysis was applied to the Oncomine database, loss of the NIRF gene was found at statistically significant levels in diverse tumors. Importantly, a recurrent microdeletion targeting NIRF was observed in non-small cell lung carcinoma. Furthermore, NIRF is immediately adjacent to the single nucleotide polymorphism rs719725, which is reportedly associated with the risk of colorectal cancer. These observations suggest that NIRF occupies a prominent position within the cell cycle network, and is a strong candidate for a tumor suppressor whose aberration contributes to the pathogenesis of diverse malignancies.Key words: NIRF, UHRF2, cell cycle network, systems biology, ubiquitin ligase, COPA, tumor suppressor, glioblastoma, non-small cell lung carcinoma, rs719725     

NIRF constitutes a nodal point in the cell cycle network and is a candidate tumor suppressor

In biological networks, a small number of "hub" proteins play critical roles in the network integrity and functions. The cell cycle network orchestrates versatile cellular functions through interactions between many signaling modules, whose defects impair diverse cellular processes, often leading to cancer. However, the network architecture and molecular basis that ensure proper coordination between distinct modules are unclear. Here, we show that the ubiquitin ligase NIRF (also known as UHRF2), which induces G1 arrest, interacts with multiple cell cycle proteins including cyclins (A2, B1, D1 and E1), p53 and pRB, and ubiquitinates cyclins D1 and E1. Consistent with its versatility, a bioinformatic network analysis demonstrated that NIRF is an intermodular hub protein that is responsible for the coordination of multiple network modules. Notably, intermodular hubs are frequently associated with oncogenesis. Indeed, we detected loss of heterozygosity of the NIRF gene in several kinds of tumors. When a cancer outlier profile analysis was applied to the Oncomine database, loss of the NIRF gene was found at statistically significant levels in diverse tumors. Importantly, a recurrent microdeletion targeting NIRF was observed in non-small cell lung carcinoma. Furthermore, NIRF is immediately adjacent to the single nucleotide polymorphism rs719725, which is reportedly associated with the risk of colorectal cancer. These observations suggest that NIRF occupies a prominent position within the cell cycle network, and is a strong candidate for a tumor suppressor whose aberration contributes to the pathogenesis of diverse malignancies.     

The expression of tumor suppressor gene Cyld is upregulated by histone deacetylace inhibitors in human hepatocellular carcinoma cell lines

CYLD is a deubiquitinating enzyme that exerts a tumor suppressive function. Its downregulation or inactivation has been associated with the development of several types of malignancies including hepatocellular carcinoma (HCC). HCC cells display significantly lower Cyld expression compared to primary human hepatocytes, and Cyld downregulation can contribute to apoptotic resistance of HCC cells. Little is known about the mechanism of Cyld downregulation in human HCC cells. In the present study we explored the possible regulation of Cyld expression by histone deacetylases (HDACs) in human HCC cell lines. We demonstrated that the HDAC inhibitors suberoylanilide hydroxamic acid, sodium butyrate, and trichostatin A induced the upregulation of both mRNA and protein levels of CYLD in two different HCC cell lines, HepG2 and Huh7. Our results demonstrate the involvement of HDACs in the downregulation of Cyld expression in HCC cells and support and may improve the use of HDAC inhibitors for the treatment for HCC.     

The Wilms' tumor suppressor gene (wt1) product regulates Dax-1 gene expression during gonadal differentiation

Gonadal differentiation is dependent upon a molecular cascade responsible for ovarian or testicular development from the bipotential gonadal ridge. Genetic analysis has implicated a number of gene products essential for this process, which include Sry, WT1, SF-1, and DAX-1. We have sought to better define the role of WT1 in this process by identifying downstream targets of WT1 during normal gonadal development. We have noticed that in the developing murine gonadal ridge, wt1 expression precedes expression of Dax-1, a nuclear receptor gene. We document here that the spatial distribution profiles of both proteins in the developing gonad overlap. We also demonstrate that WT1 can activate the Dax-1 promoter. Footprinting analysis, transient transfections, promoter mutagenesis, and mobility shift assays suggest that WT1 regulates Dax-1 via GC-rich binding sites found upstream of the Dax-1 TATA box. We show that two WT1-interacting proteins, the product of a Denys-Drash syndrome allele of wt1 and prostate apoptosis response-4 protein, inhibit WT1-mediated transactivation of Dax-1. In addition, we demonstrate that WT1 can activate the endogenous Dax-1 promoter. Our results indicate that the WT1-DAX-1 pathway is an early event in the process of mammalian sex determination.     

Hedgehog is involved in prostate basal cell hyperplasia formation and its progressing towards tumorigenesis

The role of Hedgehog signaling in human basal cell hyperplasia formation and its progressing towards tumorigenesis was investigated. Hedgehog signaling members including PTCH1, GLI1, GLI2, and GLI3 were found co-localized with p63 expression in most hyperplastic basal cells, but rarely in normal basal cells, suggesting Hedgehog involvement in basal cell hyperplasia formation. Both CK-14 and CK-8 markers were found co-localized in the majority of hyperplastic basal cells, but relatively few in the normal basal cells, indicating a Hedgehog-promoted transitory differentiation. Furthermore, CK-14 and PTCH1 were found co-localized with CD44 in the hyerplastic basal cells, in a way similar to the CD44 co-localization with PTCH1 and GLI1 in the cancer cells. Together, the present study indicated Hedgehog involvement in forming basal cell hyperplasia and its progressing towards cancer, presumably by transforming the normal basal stem cells into the cancer stem cells where persistent Hedgehog activation might be mandatory for tumorigenesis.     

Hoxc8 downregulates Mgl1 tumor suppressor gene expression and reduces its concomitant function on cell adhesion

Hoxc8 is a homeobox gene family member, which is essential for growth and differentiation. Mgl1, a mouse homologue of the Drosophila tumor suppressor gene lgl, was previously identified as a possible target of Hoxc8. However, the biological effects and underlying molecular mechanism of Hoxc8 regulation on Mgl1 has not been fully established. The endogenous expression patterns of Hoxc8 were inversely correlated with those of Mgl1 in different types of cells and tissues. Here we showed that Hoxc8 overexpression downregulated the Mgl1 mRNA expression. Characterization of the ∼2 kb Mgl1 promoter region revealed that the upstream sequence contains several putative Hox core binding sites and chromatin immunoprecipitation assay confirmed that Hoxc8 directly binds to the 5′ upstream region of Mgl1. The promoter activity of this region was diminished by Hoxc8 expression but resumed by knockdown of Hoxc8 using siRNA against Hoxc8. Functional study of Mgl1 in C3H10T1/2 cells revealed a significant reduction in cell adhesion upon expression of Hoxc8. Taken together, our data suggest that Hoxc8 downregulates Mgl1 expression via direct binding to the promoter region, which in turn reduces cell adhesion and concomitant cell migration.     

The anti-proliferative gene TIS21 is involved in osteoclast differentiation

The remodeling process of bone is accompanied by complex changes in the expression levels of various genes. Several approaches have been employed to detect differentially-expressed genes in regard to osteoclast differentiation. In order to identify the genes that are involved in osteoclast differentiation, we used a cDNAarray-nylon membrane. Among 1,200 genes that showed a measurable signal, 19 genes were chosen for further study. Eleven genes were up-regulated; eight genes were downregulated. TIS21 was one of the up-regulated genes which were highly expressed in mature osteoclasts. To verify the cDNA microarray results, we carried out RT-PCR and real-time RT-PCR for the TIS21 gene. The TIS21 mRNA level was higher in differentiated-osteoclasts when compared to undifferentiated bone-marrow macrophages. Furthermore, the treatment with 1 mM of a TIS21 antisense oligonucleotide reduced the formation of osteoclasts from the bone-marrow-precursor cells by approximately 30%. These results provide evidence for the potential role of TIS21 in the differentiation of osteoclasts     

Dolichos biflorus agglutinin-binding site expression in basal keratinocytes is associated with cell differentiation

A basal layer of squamous epithelia such as epidermis contains stem cells, transit amplifying cells as well as postmitotic differentiating cells. A detailed knowledge of the transition among these cell types in the course of epidermal renewal is important. It would help in better understanding of many pathological processes, including cancer, and in employment of epidermal cells for therapeutic purposes. In this study we analyzed the possible role of Dolichos biflorus agglutinin (DBA)-reactive alpha-N-acetylgalactosamine glycosylation in behavior of the human epidermal basal cells under in vivo and in vitro conditions. The data received from porcine epidermis were also included. Part of basal cells was positive for DBA-binding sites and these cells exhibited a lower presence of beta1 integrin in their basal surface connected to the basement membrane. The perinuclear Golgi-like accumulation of beta1 integrin was observed in some cultured keratinocytes. The co-localization of integrin with DBA-binding sites and 58 kDa protein suggests that alpha-N-acetylgalactosamine glycosylation could be related to beta1 integrin retention in the endoplasmatic reticulum Golgi intermediate compartment (ERGIC) at the beginning of the secretory pathway. The lack of anchorage in culture elevated the number of DBA-binding site positive cells without significant influence on cell growth when cells isolated directly from epidermis were employed in study. Some role of DBA-reactive glycoligand expressions in a suprabasal movement of differentiated basal cells can be hypothesized.     

Pretreatment dietary intake is associated with tumor suppressor DNA methylation in head and neck squamous cell carcinomas

Diet is associated with cancer prognosis, including head and neck cancer (HNC), and has been hypothesized to influence epigenetic state by determining the availability of functional groups involved in the modification of DNA and histone proteins. The goal of this study was to describe the association between pretreatment diet and HNC tumor DNA methylation. Information on usual pretreatment food and nutrient intake was estimated via food frequency questionnaire (FFQ) on 49 HNC cases. Tumor DNA methylation patterns were assessed using the Illumina Goldengate Methylation Cancer Panel. First, a methylation score, the sum of individual hypermethylated tumor suppressor associated CpG sites, was calculated and associated with dietary intake of micronutrients involved in one-carbon metabolism and antioxidant activity, and food groups abundant in these nutrients. Second, gene specific analyses using linear modeling with empirical Bayesian variance estimation were conducted to identify if methylation at individual CpG sites was associated with diet. All models were controlled for age, sex, smoking, alcohol and HPV status. Individuals reporting in the highest quartile of folate, vitamin B12 and vitamin A intake, compared with those in the lowest quartile, showed significantly less tumor suppressor gene methylation, as did patients reporting the highest cruciferous vegetable intake. Gene specific analyses identified differential associations between DNA methylation and vitamin B12 and vitamin A intake when stratifying by HPV status. These preliminary results suggest that intake of folate, vitamin A and vitamin B12 may be associated with the tumor DNA methylation profile in HNC and enhance tumor suppression.     

Pretreatment dietary intake is associated with tumor suppressor DNA methylation in head and neck squamous cell carcinomas

Diet is associated with cancer prognosis, including head and neck cancer (HNC), and has been hypothesized to influence epigenetic state by determining the availability of functional groups involved in the modification of DNA and histone proteins. The goal of this study was to describe the association between pretreatment diet and HNC tumor DNA methylation. Information on usual pretreatment food and nutrient intake was estimated via food frequency questionnaire (FFQ) on 49 HNC cases. Tumor DNA methylation patterns were assessed using the Illumina Goldengate Methylation Cancer Panel. First, a methylation score, the sum of individual hypermethylated tumor suppressor associated CpG sites, was calculated and associated with dietary intake of micronutrients involved in one-carbon metabolism and antioxidant activity, and food groups abundant in these nutrients. Second, gene specific analyses using linear modeling with empirical Bayesian variance estimation were conducted to identify if methylation at individual CpG sites was associated with diet. All models were controlled for age, sex, smoking, alcohol and HPV status. Individuals reporting in the highest quartile of folate, vitamin B12 and vitamin A intake, compared with those in the lowest quartile, showed significantly less tumor suppressor gene methylation, as did patients reporting the highest cruciferous vegetable intake. Gene specific analyses identified differential associations between DNA methylation and vitamin B12 and vitamin A intake when stratifying by HPV status. These preliminary results suggest that intake of folate, vitamin A and vitamin B12 may be associated with the tumor DNA methylation profile in HNC and enhance tumor suppression.     

Nitric oxide regulates synthesis of gene products involved in keratinocyte differentiation and ceramide metabolism

During terminal differentiation of keratinocytes the expression of various proteins, which are required for the formation of the epidermal water barrier in the skin of land dwelling animals, is upregulated. Using a cell culture model for the differentiation of human keratinocytes and real-time PCR, we quantified the mRNA levels of several proteins involved in differentiation and ceramide metabolism. A calcium shift (1.1 mM CaCl2, 10 microM linoleic acid) for 8 days triggered an increase in mRNA levels of keratin 10 (75-fold), profilaggrin (55-fold), glucosylceramide synthase (40-fold), beta-glucocerebrosidase (30-fold), prosaposin (15-fold), acid sphingomyelinase (5-fold), and serine palmitoyltransferase (SPTLC2, 4-fold). However, mRNA levels of keratin 14 and acid ceramidase did not change significantly. On the other hand nitric oxide added at concentrations lower than 0.25mM stimulates proliferation of keratinocytes (Krischel et al., J. Invest. Dermatol. 111, 286-291, 1998). Accordingly, the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 0.2 mM) had no effect on the morphology of cultured keratinocytes, whereas in cultured human fibroblasts apoptosis was induced. The expression patterns obtained suggest that keratinocytes remain in a basal proliferative state, with a 3-fold increase in keratin 14 expression, a marked decrease in mRNA levels of differentiation markers and of most ceramide-metabolizing enzymes to negligible levels. The inhibitor of the NO synthase, N(G)-nitro-L-arginine-methyl ester (L-NAME, 10 mM), induced a transient increase in ceramide formation, followed by apoptosis in keratinocytes but not in fibroblasts. Both, SNAP and L-NAME, decreased the mRNA levels of all proteins involved in ceramide metabolism.