The CDKN2A/CDKN2B/CDK4/CCND1 pathway is pivotal in well-differentiated and dedifferentiated liposarcoma oncogenesis: an analysis of 104 tumors

The MDM2 and CDK4 genes are the main targets of chromosome 12 amplification in well-differentiated and dedifferentiated liposarcomas. Nevertheless, around 10% of these tumors do not amplify CDK4. To find substitutive alterations of CDK4 amplification, we analyzed a large series of liposarcomas by array-CGH, real-time genomic PCR, gene expression array, and real-time RT-PCR. We demonstrate that an alteration in the CDKN2A/CDKN2B/CDK4/CCND1 pathway is present in almost all cases without CDK4 amplification, thereby confirming the pivotal role of this pathway in liposarcoma oncogenesis. Moreover, we show that cell cycle and differentiation are driven by a subtle and complex balance between members of this pathway. Finally, we demonstrate that in tumors without amplification/overexpression of CDK4, the chromosome 1q21-1q23 region is a preferential partner of chromosome 12 amplicon, suggesting that the mechanism of amplification is slightly different in this group of tumors.     

Absence of CCND1 gene amplification in breast tumours of BRCA1 mutation carriers.

BACKGROUND/AIMS: It was recently reported that significantly fewer breast tumours of BRCA1 mutation carriers overexpressed cyclin D1 and HER2 protein than tumours of age matched breast cancer cases unselected for family history. This study aimed to examine the genetic basis of this reduction by determining the frequency of tumours within this cohort showing DNA amplification of these genes. METHODS: Paraffin wax embedded sections of breast tumours from BRCA1 mutation carriers and age, grade, histological type, and tumour size matched non-familial controls that had previously been stained for cyclin D1 and HER2 protein overexpression were analysed for CCND1 and HER2 gene amplification using fluorescence in situ hybridisation. RESULTS: CCND1 amplification was detected in none of the 30 tumours of the BRCA1 mutation carriers and in 19 of 74 tumours of the matched controls. Of those samples previously determined to overexpress the HER2 protein, HER2 amplification was detected in one of three tumours from BRCA1 mutation carriers and in 13 of 17 tumours of the age matched non-familial cases. CONCLUSION: None of the tumours of BRCA1 mutation carriers showed CCND1 amplification and only one tumour showed HER2 amplification. In contrast, a large proportion of cyclin D1 and HER2 overexpression in tumours of non-familial breast cancer cases could be accounted for by amplification of these genes. These data suggest that breast tumorigenesis in BRCA1 mutation carriers occurs by a molecular mechanism distinct from that of age matched non-familial cases.     

Frequency and distribution of trisomy 11 in multiple myeloma patients: relation with overexpression of CCND1 and t(11;14)

Multiple myelomas (MM) recently have been stratified into five groups (TC1-TC5) on the basis of the presence of the recurrent IgH chromosomal translocation and cyclin D expression. Cyclin D1 is detectable in up to one third of MM patients and plays crucial role in the regulation of G1-S transition in cell cycle. To evaluate the mechanisms of cyclin D1 overexpression, fluorescence in situ hybridization analysis with specific probes for the CCND1 gene and t(11;14)(q13;q32) were performed on highly purified plasma cells from bone marrow samples of 30 MM patients at diagnosis. CCND1 gene overexpression was detected in 14/30 cases (46.6%). Patients with evidence of the t(11;14) showed strong nuclear staining for cyclin D1 (TC1 group) and 7/8 demonstrated CCND1 overexpression. The remaining 7/15 cases with increased CCND1 gene copy numbers lacked the t(11;14) and showed low to negative levels of cyclin D1 protein (TC2 group). Trisomy 11 was demonstrated in 2/8 cases carrying the t(11;14) (TC1), 6/7 overexpressing cyclin D1 without the translocation (TC2), and 4/15 negative for both alterations (TC3-TC5). According to our data, trisomy 11 does not appear to directly cause CCND1 gene overexpression because it was present in 4/15 patients without the overexpression of the CCND1 gene and in 2/8 patients carrying the t(11;14). One patient belonging to the TC2 group overexpressed cyclin D1 and lacked both trisomy and translocation, suggesting that cyclin D1 can be dysregulated by additional mechanisms. In the TC2 group, trisomy 11 may probably be considered as a recurrent polisomy of the hyperdiploid status.     

原发性脑瘤的分子遗传学研究进展

［摘要］ 原发性脑瘤是发生于神经系统常见的疾病,高度恶性的胶质瘤占原发性脑瘤的40%,主要分为I型胶质母细胞瘤（继发性胶质母细胞瘤）和II型胶质母细胞瘤（原发性胶质母细胞瘤）,二者的产生有着不同的遗传学改变。I型胶质母细胞瘤的产生途径是从一种恶性程度较低的星形细胞瘤（II级,主要为p53基因突变和血小板来源的生长因子/受体过表达）至恶性的间变型星型细胞瘤（III级,主要为Rb基因突变,CDK4基因扩增,9p、11p、13q和19q的等位基因缺失）,再到胶质母细胞瘤（IV级,最常见的遗传学改变是7号和20号染色体的获得,10号染色体的丢失以及PTEN和LRRC4基因的缺失,PDGFα及其受体的过表达）的渐进的发展过程;而II型胶质母细胞瘤则是一种没有恶性程度渐进过程的原发性胶质母细胞瘤,表皮生长因子受体基因的扩增是最常见的遗传学改变。     

Amplification of CCND1 and expression of its protein product, cyclin D1, in ductal carcinoma in situ of the breast.

Ductal carcinoma in situ (DCIS) of the breast is a heterogeneous disease clinically and biologically. The few available studies of its natural history implicate DCIS as a non-obligate precursor for invasive carcinoma. We have used fluorescence in situ hybridization (FISH) to detect gene amplification of the cell cycle regulator gene CCND1 in 88 examples of formalin-fixed, paraffin-embedded DCIS. Expression of its protein product cyclin D1 was detected by immunohistochemistry. CCND1 was amplified in 18% of DCIS cases. High grade DCIS was more likely to show amplification than low grade DCIS (32% versus 8%; P = 0.08). Gene amplification was associated with cyclin D1 protein expression (P = 0.001), although cyclin D1 was detected in cases that did not demonstrate gene amplification. Overall, cyclin D1 protein was detected in 50% of DCIS cases. Although only 2 of 23 (8%) cases of low grade DCIS had CCND1 amplification, over 50% (13/23) of these cases expressed cyclin D1 protein. Low grade DCIS had a higher mean percentage of nuclei expressing cyclin D1 than did intermediate or high grade DCIS (P = 0.007). Mechanisms other than gene amplification may be responsible for increased cyclin D1 protein in DCIS, especially in low grade DCIS. Identifying mechanisms that control cell cycle progression in DCIS may yield clues to its biological behavior.     

Alterations of the CCND1 and HER-2/neu (ERBB2) proteins in esophageal and gastric cancers

We evaluated the relationship of amplification and polysomy of both the CCND1 and the ERBB2 (alias HER-2/NEU) genes to the overexpression of their proteins in esophageal and gastric cancers and also their association with clinicopathological features. CCND1 gene amplification (45%) was more prevalent than polysomy (25%) in esophageal carcinoma, but the pattern observed was similar in gastric adenocarcinoma (10% amplification, 15% polysomy). For ERBB2, polysomy was a more frequent mechanism than amplification in both esophageal (32.5 vs. 7.5%) and gastric (15 vs. 5%) cancers. Overexpression of cyclin D1 protein was identified in 37.5% of the specimens of esophageal tumors and 35% of gastric tumors, and overexpression of Her-2/neu protein in 12.5 and 7.5%, respectively. The kappa-statistics revealed a fair agreement in both types of tumors only in overexpression and amplification of the CCND1 gene; the ERBB2 gene showed a fair agreement in amplification and polysomy and the level of protein expression in gastric adenocarcinoma. Thus, polysomy 17 could contribute to a high Her-2/neu protein level, at least in gastric cancer. Our data indicated an association with alcohol consumption and the CCND1 gene or protein levels, in both esophageal and gastric cancers.     

肝细胞癌细胞周期蛋白D1基因的扩增和表达

目的　探讨细胞周期蛋白 (cyclin)D1基因 (CCND1)扩增和cyclinD1在肝细胞癌 (HCC)中的表达情况及其与HCC发生发展的关系。方法　分别应用差异聚合酶链反应 (DPCR)、逆转录(RT) PCR和免疫组织化学法检测 2 0例HCC中cyclinD1基因扩增率、mRNA和蛋白表达状况 ,并分析其与HCC组织学分级的关系。结果　cyclinD1基因扩增结果为 6/ 2 0 ,其mRNA和蛋白过表达分别为9/ 2 0和 14 / 2 0。cyclinD1mRNA表达与基因扩增相关 (P <0 .0 5) ,也和蛋白表达水平相关 (P <0 .0 5)。cyclinD1蛋白表达与HCC组织学分级具有相关性 (P <0 .0 5)。结论　cyclinD1基因扩增在HCC中较常见 ,是引起cyclinD1mRNA和蛋白过表达的主要原因之一。cyclinD1蛋白过表达与HCC的组织学分级相关。cyclinD1基因扩增及过表达可能在肝癌的演进和分化中起重要作用     

Rearrangements and increased expression of cyclin D1 (CCND1) in neuroblastoma

Cyclin D1 regulates G1 cell cycle progression by controlling the phosphorylation of the retinoblastoma protein. This pathway is frequently deregulated in many malignancies. In neuroblastoma, however, no consistent G1 cell cycle checkpoint aberrations have been found. We examined the possible deregulation of cyclin D1 (CCND1) in this tumor. mRNA expression profiles of neuroblastoma generated by SAGE (Serial Analysis of Gene Expression) revealed a high expression of CCND1 in a subset of neuroblastoma cell lines and tumors. The CCND1 expression level can be 0.3% of the total cellular mRNA. Northern blot analysis of CCND1 expression showed a relative overexpression in 16 of 23 neuroblastoma cell lines and 10 of 15 tumor samples. In the majority of cases, the high CCND1 mRNA levels also led to high CCND1 protein levels. In the search for mechanisms causing this relative overexpression, we screened for amplifications and rearrangements of CCND1. Five amplifications were found in 202 neuroblastoma tumors and cell lines. Analysis of the 3'-UTR of CCND1 showed a rearrangement in 1 of 96 tumors. These clonal aberrations of CCND1 together with the high expression suggest a role for deregulated CCND1 activity in neuroblastoma tumorigenesis.     

A Molecular Genetic Model of Astrocytoma Histopathology

As the molecular events responsible for astrocytoma formation and progression are being clarified, it is becoming possible to correlate these alterations with the specific histopathological and biological features of astrocytoma, anaplastic astrocytoma and glioblastoma multiforme. In WHO grade II astrocytomas, autocrine stimulation by the platelet-derived growth factor system coupled with inactiva-tion of the p53 gene may lead to a growth stimulus in the face of decreased cell death with slow net growth ensuing. Such cells would also have defective responses to DNA damage and impaired DNA repair, setting the stage for future malignant change. Such biological scenarios recapitulate many of the clinicopathological features of WHO grade II astrocytomas. Anaplastic astrocytomas further display release of a critical cell cycle brake that involves the CDKN2/p16, RB and CDK4 genes. This results in mitoses seen histologically; clinically, there is more conspicuous, rapid growth. Finally, glioblastomas may emerge from the microenviron-mental outgrowth of more malignant clones in a complex vicious cycle that involves necrosis, hypoxia, growth factor release, angiogenesis and clonal selection; growth signals mediated by activation of epidermal growth factor receptors may precipitate glioblastomas. It is clear as well that glioblastoma multiforme can arise via a number of independent genetic pathways, although the clinical significance of these distinctions remains unclear.     

High-resolution array comparative genomic hybridization analysis of human bronchial and salivary adenoid cystic carcinoma

Adenoid cystic carcinoma (ACC) is a rare but distinctive tumor. Oligonucleotide array comparative genomic hybridization has been applied for cataloging genomic copy number alterations (CNAs) in 17 frozen salivary or bronchial tumors. Only four whole chromosome CNAs were found, and most cases had 2-4 segmental CNAs. No high level amplification was observed. There were recurrent gains at 7p15.2, 17q21-25, and 22q11-13, and recurrent losses at 1p35, 6q22-25, 8q12-13, 9p21, 12q12-13, and 17p11-13. The minimal region of gain at 7p15.2 contained the HOXA cluster. The minimal common regions of deletions contained the CDKN2A/CDKN2B, TP53, and LIMA1 tumor suppressor genes. The recurrent deletion at 8q12.3-13.1 contained no straightforward tumor suppressor gene, but the MIRN124A2 microRNA gene, whose product regulates MMP2 and CDK6. Among unique CNAs, gains harbored CCND1, KIT/PDGFRA/KDR, MDM2, and JAK2. The CNAs involving CCND1, MDM2, KIT, CDKN2A/2B, and TP53 were validated by FISH and/or multiplex ligation-dependent probe amplification. Although most tumors overexpressed cyclin D1 compared with surrounding glands, the only case to overexpress MDM2 had the corresponding CNA. In conclusion, our report suggests that ACC is characterized by a relatively low level of structural complexity. Array CGH and immunohistochemical data implicate MDM2 as the oncogene targeted at 12q15. The gain at 4q12 warrants further exploration as it contains a cluster of receptor kinase genes (KIT/PDGFRA/KDR), whose products can be responsive to specific therapies.     

Copy number defects of G1-cell cycle genes in neuroblastoma are frequent and correlate with high expression of E2F target genes and a poor prognosis

The tightly controlled network of cell cycle genes consists of a core of cyclin dependent kinases (CDKs) that are activated by periodically expressed cyclins. The activity of the cyclin-CDK complexes is regulated by cyclin dependent kinase inhibitors (CDKIs) and multiple signal transduction routes that converge on the cell cycle. Neuroblastoma are pediatric tumors that belong to the group of small round blue cell tumors, characterized by a fast proliferation. Here, we present high throughput analyses of cell cycle regulating genes in neuroblastoma. We analyzed a series of 82 neuroblastomas by comparative genomic hybridization arrays, single nucleotide polymorphism arrays, and Affymetrix expression arrays and analyzed the datasets in parallel with the R2 bioinformatic tool (http://r2.amc.nl). About 30% of the tumors had genomic amplifications, gains, or losses with shortest regions of overlap that suggested implication of a series of G1 cell cycle regulating genes. CCND1 (cyclin D1) and CDK4 were amplified or gained and the chromosomal regions containing the CDKN2 (INK4) group of CDKIs were frequently deleted. Cluster analysis showed that tumors with genomic aberrations in G1 regulating genes over-expressed E2F target genes, which regulate S and G2/M phase progression. These tumors have a poor prognosis. Our findings suggest that pharmacological inhibition of cell cycle genes might bear therapeutic promises for patients with high risk neuroblastoma.     

Recurrent coamplification of cytoskeleton-associated genes EMS1 and SHANK2 with CCND1 in oral squamous cell carcinoma

Chromosomal band 11q13 is frequently amplified in oral squamous cell carcinoma (OSCC) and assumed to be critically involved in tumor initiation and progression by proto-oncogene activation. Though cyclin D1 (CCND1) is supposed to be the most relevant oncogene, several additional putative candidate genes are inside this chromosomal region, for which their actual role in tumorigenesis still needs to be elucidated. To characterize the 11q13 amplicon in detail, 40 OSCCs were analyzed by comparative genomic hybridization to DNA microarrays (matrix-CGH) containing BAC clones derived from chromosomal band 11q13. This high-resolution approach revealed a consistent amplicon about 1.7 Mb in size including the CCND1 oncogene. Seven BAC clones covering FGF3, EMS1, and SHANK2 were shown to be frequently coamplified inside the CCND1 amplicon. Subsequent analysis of tissue microarrays by FISH revealed amplification frequencies of 36.8% (88/239) for CCND1, 34.3% (60/175) for FGF3, 37.4% (68/182) for EMS1, and 36.3% (61/168) for SHANK2. Finally, quantitative mRNA expression analysis demonstrated consistent overexpression of CCND1 in all tumors and of EMS1 and SHANK2 in a subset of specimens with 11q13 amplification, but no expression of FGF3 in any of the cases. Our study underlines the critical role of CCND1 in OSCC development and additionally points to the functionally related genes EMS1 and SHANK2, both encoding for cytoskeleton-associated proteins, which are frequently coamplified with CCND1 and therefore could cooperatively contribute to OSCC pathogenesis.     

Genetic profile of gliosarcomas

There are distinct genetic pathways leading to the glioblastoma, the most malignant astrocytic brain tumor. Primary (de novo) glioblastomas develop in older patients and are characterized by epidermal growth factor (EGF) receptor amplification/overexpression, p16 deletion, and PTEN mutations, whereas secondary glioblastomas that progressed from low-grade or anaplastic astrocytoma develop in younger patients and frequently contain p53 mutations. In this study, we assessed the genetic profile of gliosarcoma, a rare glioblastoma variant characterized by a biphasic tissue pattern with alternating areas displaying glial and mesenchymal differentiation. Single-strand conformation polymorphism followed by direct DNA sequencing revealed p53 mutations in five of 19 gliosarcomas (26%) and PTEN mutations in seven cases (37%). Homozygous p16 deletion was detected by differential polymerase chain reaction in seven (37%) gliosarcomas. The overall incidence of alterations in the Rb pathway (p16 deletion, CDK4 amplification, or loss of pRb immunoreactivity) was 53%, and these changes were mutually exclusive. Coamplification of CDK4 and MDM2 was detected in one gliosarcoma. None of the gliosarcomas showed amplification or overexpression of the EGF receptor. Thus gliosarcomas exhibit a genetic profile similar to that of primary (de novo) glioblastomas, except for the absence of EGFR amplification/overexpression. Identical PTEN mutations in the gliomatous and sarcomatous tumor components were found in two cases. Other biopsies contained p16 deletions, an identical p53 mutation, or coamplification of MDM2 and CDK4 in both tumor areas. This strongly supports the concept of a monoclonal origin of gliosarcomas and an evolution of the sarcomatous component due to aberrant mesenchymal differentiation in a highly malignant astrocytic neoplasm.     

Structural basis of inhibition of CDK-cyclin complexes by INK4 inhibitors

The cyclin-dependent kinases 4 and 6 (Cdk4/6) that drive progression through the G(1) phase of the cell cycle play a central role in the control of cell proliferation, and CDK deregulation is a frequent event in cancer. Cdk4/6 are regulated by the D-type cyclins, which bind to CDKs and activate the kinase, and by the INK4 family of inhibitors. INK4 proteins can bind both monomeric CDK, preventing its association with a cyclin, and also the CDK-cyclin complex, forming an inactive ternary complex. In vivo, binary INK4-Cdk4/6 complexes are more abundant than ternary INK4-Cdk4/6-cyclinD complexes, and it has been suggested that INK4 binding may lead to the eventual dissociation of the cyclin. Here we present the 2.9-A crystal structure of the inactive ternary complex between Cdk6, the INK4 inhibitor p18(INK4c), and a D-type viral cyclin. The structure reveals that p18(INK4c) inhibits the CDK-cyclin complex by distorting the ATP binding site and misaligning catalytic residues. p18(INK4c) also distorts the cyclin-binding site, with the cyclin remaining bound at an interface that is substantially reduced in size. These observations support the model that INK4 binding weakens the cyclin's affinity for the CDK. This structure also provides insights into the specificity of the D-type cyclins for Cdk4/6.