595

CCND1

BCL1|D11S287E|PRAD1|U21B31;cyclin D1;CCND1;cyclin D1

B-cell CLL/lymphoma 1|B-cell lymphoma 1 protein|BCL-1 oncogene|G1/S-specific cyclin D1|G1/S-specific cyclin-D1|PRAD1 oncogene

11q13

protein-coding

In a retrospective analysis we investigated 59 children with de novo acute lymphoblastic leukemia (ALL) and 28 patients with relapsed ALL for the expression of cyclin D1 using RT-PCR. In addition, the relationships of cyclin D1 to the retinoblastoma tumor suppressor gene and the Ki-67-expression were analyzed. Cyclin D1-mRNA was detectable in 42 out of 58 patients with de novo ALL (72%) and in 26 out of 28 relapsed patients (93%). The blast cells of the relapsed patients contained significantly higher levels of cyclin D1-mRNA compared with the de novo group (p = 0.0007). The cyclin D1 expression was independent of the proliferative activity of the cells and inversely correlated to the expression of the retinoblastoma tumor suppressor gene (r = -0.27, p = 0.03). Prognostic considerations using Kaplan Meier estimates for the relapse-free interval showedthat patients with high cyclin D1- mRNA levels had a poorer prognosis (p = 0.046).

Hepatoblastoma (HBL) is the most common malignant liver tumor in young children.Recent reports have shown that the beta-catenin gene was frequently mutated or deleted in HBLS: To elucidate the role of beta-catenin abnormalities in HBLs, wesearched for mutations of beta-catenin and APC as well as expression of the target genes, cyclin D1, c-myc, and fibronectin, in 68 primary HBLS: The mutation analysis revealed that 44 (65%) tumors carried missense mutations or deletions of beta-catenin, ALL of which were somatic and targeted to the exon 3 encoding the amino acid residues involved in its degradation. However, no loss of function mutation of the APC gene was detected by the yeast functional assay. Of interest, beta-catenin mutation was significantly correlated with overexpression of the target genes, cyclin D1 and fibronectin, but not with that of c-myc in HBLs as measured by quantitative real-time reverse transcription-PCR. The immunohistochemical studies in 15 HBLs demonstrated that the nuclear/cytoplasmicaccumulation of beta-catenin was positive in 13 tumors, 9 of which had the deletion or mutation of the gene. The significant correlation between the beta-catenin gene abnormality and the positive staining of cyclin D1 was also confirmed. Furthermore, the nuclear accumulation of beta-catenin was strongly associated with the poorly differentiated tumor cell components as well as with the positive staining of cyclin D1 within the tumor. Thus, our present results suggested that the gain of function mutation of beta-catenin played a crucial role in the malignant progression of HBL in vivo.

We identified patterns of differentially-expressed genes in cell lines derived from several pediatric solid tumors. Affymetrix Human cancer G110 Arrays, carrying 1,700 cancer-associated genes, were applied to a panel of 11 cell linesoriginating from Ewing tumors (ETs), neuroblastomas, and malignant melanoma of soft parts. Hierarchical clustering clearly differentiated these 3 entities and revealed groups of 75, 102, and 36 gene probe-sets exhibiting tumor-type specific up-regulation in these cell lines, respectively. Whereas ET lines demonstrated increased expression of microtubule-associated protein tau (MAPT), protein phosphatase 1 regulatory subunit 1A (PPP1R1A), NIMA (never in mitosis gene a)-related kinase 2 (NEK2), and cyclin D1 (CCND1), neuroblastoma samples exhibited high expression of wingless-type mouse mammary tumor virus integrationsite family member 11 (WNT11), Drosophila frizzled homolog 2 (FZD2), and adenomatous polyposis coli (APC) which are involved in regulating free beta-catenin levels. These genes likely maintain tumor-specific characteristics and participate in key downstream regulatory mechanisms. We also correlated the expression levels of up-regulated genes in ETs with their chromosomal localization and compared these data to the comparative genomic hybridization profiles of the cell lines. We demonstrate that gains of genetic material contribute essentially to differential gene expression.

Using Affymetrix oligonucleotide microarrays, we analyzed mRNA gene expression patterns of 12 primary pediatric rhabdomyosarcomas (RMS) and 11 Ewing's sarcomas(EWS), which belong to the small round blue cell tumors (SRBCTs). Diagnostic classification of these cancers is frequently complicated by the highly similar appearance in routine histology, and additional molecular markers could significantly improve tumor classification. A combination of three independent statistical approaches (t-test, SAM, k-nearest neighborhood analysis) resulted in 101 highly significant probe sets that clearly discriminate between EWS and RMS.We identified novel marker transcripts that have not been previously associated with either RMS or EWS yet, including CITED2, glypican 3 (GPC3), and cyclin D1 (CCND1). expression levels for selected candidate genes were validated by quantitative real-time reverse-transcription PCR. Furthermore, to identify biologically meaningful trends, functional annotations were assigned to 946 genes differentially expressed between EWS and RMS (t-test). Genes involved in proteinbiosynthesis (n = 28) and complex assembly (n = 9), lipid metabolism (n = 23), energy generation (n = 22), and mRNA processing (n = 11) were expressed significantly higher in EWS. Thus, functional annotation of tumor-specific genesreveals detailed insights into tumor biology and differentiation-specific expression patterns and gives important clues related to the possible cellular origin of these pediatric tumors. Supplementary material for this article is available at the International Journal of cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.#CI- Copyright 2004 Wiley-Liss, Inc.

Ionizing radiation is the most established risk factor for meningioma formation.Our aim was to evaluate the main effect of selected candidate genes on the development of meningioma and their possible interaction with ionizing radiationin the causation of this tumor. The total study population included 440 cases and controls: 150 meningioma patients who were irradiated for tinea capitis in childhood, 129 individuals who were similarly irradiated but did not develop meningioma, 69 meningioma patients with no previous history of irradiation, and 92 asymptomatic population controls. DNA from peripheral blood samples was genotyped for single nucleotide polymorphisms (SNP) in 12 genes: NF2, XRCC1, XRCC3, XRCC5, ERCC2, Ki-ras, p16, cyclin D1, PTEN, E-cadherin, TGFB1, and TGFBR2. SNP analysis was done using the MassArray system (Sequenom, San Diego, CA) and computerized analysis by SpectroTYPER. Logistic regressions were applied to evaluate main effect of each gene on meningioma formation and interaction between gene and radiation. Intragenic SNPs in the Ki-ras and ERCC2 genes were associated with meningioma risk (odds ratio, 1.76; 95% confidence interval, 1.07-2.92 and odds ratio, 1.68; 95% confidence interval, 1.00-2.84, respectively). A significant interaction was found between radiation and cyclin D1 and p16 SNPs (P for interaction = 0.005 and 0.057, respectively). Our findings suggest that Ki-ras and ERCC2 SNPs are possible markers for meningioma formation, whereas cyclin D1 and p16 SNPs may be markers of genes that have an inverse effect on the risk to develop meningioma in irradiated and nonirradiated populations.

OBJECT: Although atypical teratoid/rhabdoid tumor (AT/RT) is known to generate through inactivation of the hSNF5/INI1 gene on chromosome 22q, the downstream molecular mechanism remains unclear. We histologically and molecularly reviewed our pediatric brain tumors for unrecognized AT/RTs and evaluated the role of cyclin D1, a potential molecular target of hSNF5/INI1. METHODS: We analyzed 16 tumors under three years of age: seven medulloblastomas, three anaplastic ependymomas (E IIIs), two each of supratentorial primitive neuroectodermal tumors (sPNETs) and choroid plexus carcinomas (CPCs), and one each of neuroblastoma andpineoblastoma. Immunohistochemistry for glial fibrillary acidic protein, vimentin, epithelial membrane antigen, smooth muscle actin and cyclin D1 was performed. Polymerase chain reaction (PCR)-single-strand conformation polymorphism analysis with direct sequencing, differential PCR and microsatellite analysis were conducted for hSNF5/INI1mutation, homozygous deletion and loss of heterozygosity (LOH) on 22q, respectively. Because of the presence of rhabdoid cells and the polyimmunophenotypic features, the diagnosis was revised to AT/RT in five (31%) tumors, namely, two E IIIs and one each of medulloblastoma, CPC and pineoblastoma. Three of them harbored such hSNF5/INI1 aberrations as germline single base deletion (492/6 delC) and missense mutation (C157T) together with LOH 22q or homozygous deletion. Cyclin D1 was overexpressed in those three tumors but not in the two that lacked hSNF5/INI1 inactivation. CONCLUSION: AT/RT can be misdiagnosed as a variety of tumors, including ependymoma that potentially harbors LOH 22q. Our data indicate that cyclin D1 is a target of hSNF5/INI1in primary tumors.

BACKGROUND: childhood ependymoma remains a major therapeutic challenge despite surgery, chemotherapy, and irradiation. We hypothesized that p53 function might be abrogated in ependymomas and implicated in their resistance to anti-cancer therapy. PROCEDURE: Primary ependymomas at diagnosis or relapse from 24 childrenwere analyzed for p53 pathway, using a functional assay in yeast, RT-PCR, Western blot analysis, and/or immunohistochemistry for TP53 mutation, p14(ARF) deletion and promoter hypermethylation, MDM2 and PAX5 expression, respectively. p53-mediated response to radiation-induced DNA damage was evaluated using Western blot and flow cytometry analysis in two ependymoma xenograft models, IGREP37 andIGREP83, derived from primary anaplastic childhood ependymomas. RESULTS: No TP53, MDM2, p14(ARF), PAX5 gene abnormalities were detected in the primary ependymomastumors and xenografts tested. Interestingly, despite the lack of these abnormalities, p53 induced p21-mediated G(1) growth arrest in response to irradiation was altered in the IGREP37 xenograft tumors. Although irradiation induced necrosis and apoptotic cell death, IGREP37 tumors were moderately sensitive to radiation therapy in vivo. In contrast, irradiation yielded significant tumor growth delays and tumor regressions in the p53 functional IGREP83 xenografts. CONCLUSION: Alterations in p53-mediated growth arrest in ependymomas might be implicated in the radio-resistance of these tumors and demand further evaluation.

Rhabdoid tumors are aggressive pediatric malignancies for which, currently, there are no effective or standard treatment strategies. Rhabdoid tumors arise becauseof the loss of the tumor suppressor gene INI1. We have previously demonstrated that INI1 represses Cyclin D1 transcription in rhabdoid cells by directly recruiting histone deacetylase 1 complex to its promoter, leading to G(0)-G(1) arrest. expression of Cyclin D1 overcomes cell cycle arrest mediated by INI1 andCyclin D1 overexpression in human rhabdoid tumors is a common phenomenon. However, it is not clear whether Cyclin D1 is a critical downstream target of INI1 in vivo and whether the derepression of this gene is essential for rhabdoidtumorigenesis. To determine the requirement of Cyclin D1 for genesis of rhabdoidtumors in vivo, we developed Ini1 heterozygous mice by targeted disruption. We found that the tumors developed in these Ini1+/- mice are rhabdoid, defective for Ini1 protein, and like the human tumors, express Cyclin D1. We crossed Ini1+/- mice to Cyclin D1-/- mice and found that Ini1+/- mice with Cyclin D1 deficiency did not develop any spontaneous tumors, in contrast to the parental Ini1+/- mice. These results strongly support the hypothesis that Cyclin D1 is a key mediator in the genesis of rhabdoid tumors. Our results provide an in vivo proof of concept that drugs that target Cyclin D1 expression or activity could be potentially effective as novel therapeutic agents for rhabdoid tumors.

mutations of beta-catenin have been identified in the majority of pediatric hepatic malignancies, including hepatoblastoma (HB) and hepatocellular carcinoma(HCC), suggesting its important contribution in hepatic tumorigenesis in this age group. However, the role of beta-catenin/canonical Wnt signaling pathway in the neoplastic growth of cancer cells has not been directly studied. To address beta-catenin's capability in maintaining the malignant phenotype in established pediatric HB and HCC cell lines, HuH-6 and HepG2, harboring mutated and overexpressed beta-catenin, we carried out a series of in vitro analyses througha transfection of short interfering RNAs (siRNAs) to generate a loss-of-functionmodel. HuH-7, another HB cell line derived from a pediatric patient without a stabilizing mutation was used for comparison. RNA interference successfully manipulated the degradation of overexpressed beta-catenin. In ALL cell lines, beta-catenin mRNA was suppressed by 80-90% after 48 h of transfection, and a reduction of its protein expression was demonstrated. In HuH-6 and HepG2, the pre-existing beta-catenin nuclear accumulation disappeared and reductions of beta-catenin downstream target genes, c-myc and cyclinD1, were also evidenced after the treatment. The in vitro proliferation of both cell lines was transiently inhibited. In contrast, the suppression of beta-catenin in HuH-7 didnot lead to a significant change in the expression of target genes or cellular proliferation. Our data indicate that beta-catenin can be considered a specific target for gene therapy in pediatric hepatic tumors with mutations and overexpression of this gene.

The vast majority of deaths associated with cancer are a consequence of a complex phenotypic behavior, metastasis, by which tumor cells spread from their primary site of origin to regional and distant sites. This process requires the tumor cell to make numerous adjustments, both subtle and dramatic, to successfully reach, survive, and flourish at favorable secondary sites. It has been suggestedthat molecular mechanisms accounting for metastatic behavior can recapitulate those employed during embryogenesis. We have shown that the homeodomain transcription factor Six1, known to be required for normal development of migratory myogenic progenitor cells, is sufficient to promote metastatic spread in a mouse model of the pediatric skeletal muscle cancer rhabdomyosarcoma. Here,we report that Six1 is able to activate the expression of a set of protumorigenic genes (encoding cyclin D1, c-Myc, and Ezrin) that can control cell proliferation, survival, and motility. Although the role of Ezrin in cytoskeletal organization and adhesion has been well studied, the means by which its expression is regulated are poorly understood. We now show that the gene encoding Ezrin is a direct transcriptional target of Six1. Moreover, Ezrin is indispensable for Six1-induced metastasis and highly expressed in a panel of representative pediatric cancers. Our data indicate that Ezrin represents a promising therapeutic target for patients with advanced-stage rhabdomyosarcoma and perhapsother malignancies.

Although mucoepidermoid carcinoma of the salivary gland is relatively common, mucoepidermoid carcinoma arising from the mucous glands of the bronchus is rare.Bronchial mucoepidermoid carcinoma usually presents as an intraluminal mass producing luminal occlusion. Symptoms are airway obstruction and recurrent pneumonia. Macroscopically, mucoepidermoid carcinoma appears as an exophytic intrabronchial mass with intact or ulcerated bronchial mucosa. Microscopically, the tumors are located in the submucosa of the large bronchi. The tumors are usually well differentiated and contain a combination of mucus-secreting, squamous, and intermediate cells. The increased frequency of this tumor in the pediatric population suggests a genetic abnormality. Recent genetic studies havedemonstrated reciprocal chromosomal translocations including t(1;11)(p22;q13), t(11;19)(q14-21;p12), and t(11; 19)(q21;p13). Chromosome 11 in the first translocation appears to have been altered resulting in up-regulation of the cyclin D1 gene and overexpression of cyclin D1. The t(11;19)(q21;p13) encodes a novel fusion product capable of disrupting the Notch signaling pathway.

Rhabdoid tumors (RTs) are an extremely aggressive pediatric malignancy that results from loss of the INI1/hSNF5 tumor suppressor gene. Loss of INI1 results in aberrant expression of Cyclin D1, which supports rhabdoid tumorigenesis and survival. 4-HPR, a synthetic retinoid that down-modulates Cyclin D1, has shown promise in treating various tumors including RTs. In this study, we have generated a chemical library of peptidomimetic derivatives of 4-HPR in an attempt to create a more biologically active compound for use as a therapeutic agent against RTs and other tumors. We have synthesized novel peptidomimetic compound by substituting alkene backbone with a ring structure that retains the biological activity in cell culture models of rhabdoid tumors. We further identified derivative of peptidomimetic compound (11d, IC(50) approximately 3 microM) with approximately five times higher potency than 4-HPR (1, IC(50) approximately 15 microM) based on a survival assay against rhabdoid tumor cells. These studies indicate that peptidomimetic derivatives that retain the cytotoxic activity are promising novel chemotherapeutic agents against RTs and other tumors.

Renal cell carcinoma (RCC) is a rare tumor in the pediatric population. Recently, a phenotypically and genetically distinct kidney carcinoma, mainly prevalent in children and associated with an Xp11.2 translocation or TFE3 gene fusion, has been described. It has been advanced that in this subtype of RCC, there is an accumulation of cyclin D1, cyclin D3, and p21 ((wafl/cip1)). The aim of the present study was to figure out in two pediatric RCC recently diagnosed in our department (one clear cell-type RCC and one TFE3-positive RCC) whether those features are indeed specific of the latter tumor or occur in pediatric RCC irrespective of the tumor type. The following immunostains were performed in both cases: Ki67, p16(ink4a), p21 ((wafl/cip1)), p27(kip1), p53, p63, mdm2, cyclin D1, cyclin D3, TFE3, CD10, vimentin, E-cadherin, and RCC-antigen. We observed in theTFE3-positive carcinoma an intense immunoreaction for p21 ((wafl/cip1)), cyclin D1, and cyclin D3, without expression for p53, p16, p27(kip1), and mdm2, whereasthe immunoexpression profile observed in the classic RCC was similar to that of clear cell, adult-type RCC. Our study confirms that TFE3-positive RCC exhibits aderegulation of the cell cycle apparently unrelated to the young age of the patients.

PURPOSE: Hepatoblastoma represents the most frequent malignant liver tumor in childhood. The phosphatidylinositol-3'-kinase (PI3K)/AKT pathway is crucial in downstream signaling of multiple receptor tyrosine kinases of pathogenic importance in hepatoblastoma. Increased PI3K/AKT signaling pathway activity and activating mutations of PIK3CA, encoding a PI3K catalytic subunit, have been reported in different childhood tumors. The current study was done to analyze the role of PI3K/AKT signaling in hepatoblastoma. EXPERIMENTAL DESIGN: Immunohistochemical stainings of (Ser473)-phosphorylated (p)-AKT protein, its targets p-(Ser9)-GSK-3beta and p-(Ser2448)-mTOR, as well as the cell cycle regulators Cyclin D1, p27(KIP1), and p21(CIP1) were done and the PIK3CA gene wasscreened for mutations. In vitro, two hepatoblastoma cell lines treated with thePI3K inhibitor LY294002 were analyzed for AKT and GSK-3beta phosphorylation, cell proliferation, and apoptosis. Additionally, simultaneous treatments of hepatoblastoma with LY294002 and cytotoxic drugs were carried out. RESULTS: Mosttumors strongly expressed p-AKT, p-GSK-3beta, and p-mTOR; subgroups showed significant Cyclin D1, p27(KIP1), and p21(CIP1) expression. One hepatoblastoma carried an E545A mutation in the PIK3CA gene. In vitro, PI3K inhibition diminished hepatoblastoma cell growth being accompanied by reduced AKT and GSK-3beta phosphorylation. Flow cytometry and 4', 6-diamidino-2-phenylindole stainings showed that PI3K pathway inhibition leads to a substantial increase inapoptosis and a decrease in cellular proliferation linked to reduced Cyclin D1 and increased p27(KIP1) levels. Simultaneous treatment of hepatoblastoma cell lines with LY294002 and cytotoxic drugs resulted in positive interactions. CONCLUSIONS: Our findings imply that PI3K signaling plays an essential role in growth control of hepatoblastoma and might be successfully targeted in multimodal therapeutic strategies.

Abnormality of the cyclin D1/cdk4/p16INK4a/pRb pathway during tumorigenesis has recently been reported. Hepatoblastoma is a rare malignant liver tumor of childhood, but underlying abnormalities of cell-cycle regulating protein remain to be elucidated. The expression of cyclin D1, cdk4, p16 and retinoblastoma geneproduct (pRb) was studied by immunohistochemistry in 17 paraffin-embedded tissues consisting of both tumor and corresponding non-neoplastic tissues. tumor tissuesshowed overexpression of cyclin D1 (13/17, 76%) and cdk4 (15/17, 88%). Eleven cases showed co-overexpression of both cyclin D1 and cdk4. No abnormal p16 or pRb expression was noted. In the group with a high score (+4) for cyclin D1 expression, a positive correlation with tumor recurrence was noted (P = 0.043). These data suggest that overexpressed cyclin D1 and cdk4 protein might play an important role in the tumorigenesis of hepatoblastoma and that in the group withhigh cyclin D1 expression, tumor recurrence may be more frequent.