Amplification and overexpression of aurora kinase A (AURKA) in immortalized human ovarian epithelial (HOSE) cells

Immortalization is an early and essential step of human carcinogenesis. Amplification of chromosome 20q has been shown to be a common event in immortalized cells and cancers. We have previously reported that gain and amplification of chromosome 20q is a non-random and common event in immortalized human ovarian surface epithelial (HOSE) cells. The chromosome 20q harbors genes including TGIF2 (20q11.2-q12), AIB1 (20q12), PTPN1 (20q13.1), ZNF217 (20q13.2), and AURKA (20q13.2-q13.3), which were previously reported to be amplified and overexpressed in ovarian cancers. Some of these genes may be involved in immortalization of HOSE cells and represent crucial premalignant changes in ovarian surface epithelium. Investigation of the involvement of these genes was examined in four pairs of pre-crisis (preimmortalized) and post-crisis (immortalized) HOSE cells. Overexpression of AURKA (Aurora kinase A), also known as BTAK and STK15, by both real time-quantitative polymerase chain reaction (RT-QPCR) and Western blotting was detected in all the four immortalized HOSE cells examined while overexpression of AIB1 and ZNF217 was observed in two of four immortalized HOSE cells examined. Overexpression of TGIF2 and PTPN1 was not significant in our immortalized HOSE cell systems. The degree of overexpression of AURKA was shown to be closely associated with the amplification of chromosome 20q in immortalized HOSE cells. Fluorescence in situ hybridization (FISH) with labeled P1 artificial clone (PAC) confirmed the amplification of the chromosomal region (20q13.2-13.3) where AURKA resides. DNA amplification of AURKA was also confirmed using semi-quantitative PCR. Our study showed that amplification and overexpression of AURKA is a common and significant event during immortalization of HOSE cells and may represent an important premalignant change in ovarian carcinogenesis.     

Medulloblastomas overexpress the p53-inactivating oncogene <Emphasis Type="Italic">WIP1/PPM1D</Emphasis>

Medulloblastoma is the most common malignant brain tumor of childhood. Despite numerous advances, clinical challenges range from recurrent and progressive disease to long-term toxicities in survivors. The lack of more effective, less toxic therapies results from our limited understanding of medulloblastoma growth. Although TP53 is the most commonly altered gene in cancers, it is rarely mutated in medulloblastoma. Accumulating evidence, however, indicates that TP53 pathways are disrupted in medulloblastoma. Wild-type p53-induced phosphatase 1 (WIP1 or PPM1D) encodes a negative regulator of p53. WIP1 amplification (17q22-q23) and its overexpression have been reported in diverse cancer types. We examined primary medulloblastoma specimens and cell lines, and detected WIP1 copy gain and amplification prevalent among but not exclusively in the tumors with 17q gain and isochromosome 17q (i17q), which are among the most common cytogenetic lesions in medulloblastoma. WIP1 RNA levels were significantly higher in the tumors with 17q gain or i17q. Immunoblots confirmed significant WIP1 protein in primary tumors, generally higher in those with 17q gain or i17q. Under basal growth conditions and in response to the chemotherapeutic agent, etoposide, WIP1 antagonized p53-mediated apoptosis in medulloblastoma cell lines. These results indicate that medulloblastoma express significant levels of WIP1 that modulate genotoxic responsiveness by negatively regulating p53.     

Genome-wide linkage scan for prostate cancer susceptibility in Finland: evidence for a novel locus on 2q37.3 and confirmation of signal on 17q21-q22

Genome-wide linkage studies have been used to localize rare and highly penetrant prostate cancer (PRCA) susceptibility genes. Linkage studies performed in different ethnic backgrounds and populations have been somewhat disparate, resulting in multiple, often irreproducible signals because of genetic heterogeneity and high sporadic background of the disease. Our first genome-wide linkage study and subsequent fine-mapping study of Finnish hereditary prostate cancer (HPC) families gave evidence of linkage to one region. Here, we conducted subsequent scans with microsatellites and SNPs in a total of 69 Finnish HPC families. GENEHUNTER-PLUS was used for parametric and nonparametric analyses. Our microsatellite genome-wide linkage study provided evidence of linkage to 17q12-q23, with a heterogeneity LOD (HLOD) score of 3.14 in a total of 54 of the 69 families. Genome-wide SNP analysis of 59 of the 69 families gave a highest HLOD score of 3.40 at 2q37.3 under a dominant high penetrance model. Analyzing all 69 families by combining microsatellite and SNP maps also yielded HLOD scores of > 3.3 in two regions (2q37.3 and 17q12-q21.3). These significant linkage peaks on chromosome 2 and 17 confirm previous linkage evidence of a locus on 17q from other populations and provide a basis for continued research into genetic factors involved in PRCA. Fine-mapping analysis of these regions is ongoing and candidate genes at linked loci are currently under analysis.     

Gain of chromosome arm 17q is associated with unfavourable prognosis in neuroblastoma, but does not involve mutations in the somatostatin receptor 2(SSTR2) gene at 17q24

Deletion of chromosome arm 1p and amplification of the MYCN oncogene are well-recognized genetic alterations in neuroblastoma cells. Recently, another alteration has been reported; gain of the distal part of chromosome arm 17q. In this study 48 neuroblastoma tumours were successfully analysed for 17q status in relation to known genetic alterations. Chromosome 17 status was detected by fluorescence in situ hybridization (FISH). Thirty-one of the 48 neuroblastomas (65%) showed 17q gain, and this was significantly associated with poor prognosis. As previously reported, 17q gain was significantly associated with metastatic stage 4 neuroblastoma and more frequently detected than both deletion of chromosome arm 1p and MYCN amplification in tumours of all stages. 17q gain also showed a strong correlation to survival probability (P = 0.0009). However, the most significant correlation between 17q gain and survival probability was observed in children with low-stage tumours (stage 1, 2, 3 and 4S), with a survival probability of 100% at 5 years from diagnosis for children with tumours showing no 17q gain compared to 52.5% for those showing 17q gain (P = 0.0021). This suggests that 17q gain as a prognostic factor plays a more crucial role in low-stage tumours. Expression of the somatostatin receptor 2 (SSTR2), localized in chromosome region 17q24, has in previous studies been shown to be positively related to survival in neuroblastoma. A point mutation in the SSTR2 gene has earlier been reported in a human small-cell lung cancer. In this study, mutation screening of the SSTR2 gene in 43 neuroblastoma tumours was carried out with polymerase chain reaction-based single-stranded conformation polymorphism/heteroduplex (SSCP/HD) and DNA sequencing, and none of the tumours showed any aberrations in the SSTR2 gene. These data suggest that mutations in the SSTR2 gene are uncommon in neuroblastoma tumours and do not correlate with either the 17q gain often seen or the reason some tumours do not express SSTR2 receptors. Overall, this study indicates that gain of chromosome arm 17q is the most frequently occurring genetic alteration, and that it is associated with established prognostic factors.     

Induction of centrosome amplification and chromosome instability in human bladder cancer cells by p53 mutation and cyclin E overexpression

Centrosome amplification frequently occurs in human cancers and is a major cause of chromosome instability (CIN). In mouse cells, centrosome amplification can be readily induced by loss or mutational inactivation of p53. In human cells, however, silencing of endogenous p53 alone does not induce centrosome amplification or CIN, although high degrees of correlation between p53 mutation and CIN/centrosome amplification in human cancer can be detected, suggesting the presence of additional regulatory mechanism(s) in human cells that ensures the numeral integrity of centrosomes and genomic integrity. Cyclin E, a regulatory subunit for CDK2 that plays a key role in centrosome duplication, frequently is overexpressed in human cancers. We found that cyclin E overexpression, together with loss of p53, efficiently induces centrosome amplification and CIN in human bladder cancer cells but not by either cyclin E overexpression or loss of p53 alone. We extended these findings to bladder cancer specimens and found that centrosome amplification is strongly correlated with concomitant occurrence of cyclin E overexpression and p53 inactivation but not with either cyclin E overexpression or p53 inactivation alone. Because cyclin E expression is strictly controlled in human cells compared with mouse cells, our findings suggest that this stringent regulation of cyclin E expression plays an additional role underlying numeral homeostasis of centrosomes in human cells and that deregulation of cyclin E expression, together with inactivation of p53, results in centrosome amplification.     

Status of c-erbB-2 in gastric adenocarcinoma: a comparative study of immunohistochemistry, fluorescence in situ hybridization and enzyme-linked immuno-sorbent assay

c-erb-2 amplification and overexpression are currently attracting a great deal of attention because a new adjuvant therapy using an antibody against the c-erbB-2 gene product, trastuzumab (Herceptin; Genentech, Inc., South San Francisco, CA), has proved effective in treating breast cancer with amplification and/or overexpression of c-erbB-2. Aberrations of c-erbB-2 have also been detected in ovarian, endometrial and gastric carcinomas at varied frequencies. Amplification of the c-erbB-2 locus (17q12-q21.32), overexpression of c-erbB-2 protein (p185) and serum levels of soluble c-erbB-2 protein fragments (p105) were examined in gastric cancer patients using fluorescence in situ hybridization (FISH), immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), respectively. Overexpression of c-erbB-2 protein was found in 29 (8.2%) of the 352 gastric carcinomas analyzed. In FISH analysis, all tumors with 3+ immunostaining and 1 of 5 tumors with 2+ staining showed high-level amplification of c-erbB-2. Pre-operative serum p105 was quantified in serum specimens from 129 patients with gastric cancer and 28 patients with benign diseases. There were no significant differences in the serum p105 levels among 11 patients with c-erbB-2-overexpressing carcinomas, 118 patients with c-erbB-2 non-overexpressing carcinomas and 28 controls, although a single case of gastric carcinoma overexpressing c-erbB-2 with extensive liver metastasis had a higher level than the cut-off value. The mechanisms of overexpression of p185 and high-level amplification of c-erbB-2 in gastric adenocarcinomas seem similar to those well-established in breast cancers. Patients having gastric adenocarcinoma with c-erbB-2 amplification are potential candidates for a new adjuvant therapy using humanized monoclonal antibody.     

Multiple sites of highly amplified DNA sequences detected by molecular cytogenetic analysis in HS-RMS-2, a new pleomorphic rhabdomyosarcoma cell line

A molecular cytogenetic analysis was performed on HS-RMS-2, a cell line established in this laboratory from a rare pleomorphic type of rhabdomyosarcoma. G-banding and multicolor-FISH analyses revealed that the cells have a complex chromosomal composition. Comparative genomic in situ hybridization (CGH) detected eight highly amplified regions at 1p36.1-p36.2, 1p31-p32, 1q21-q31, 8q12-q21, 8q24-qter, 11q12-q13, 12q13-q14 and 18q12-q22, suggesting the co-existence of multiple amplified oncogenes in these tumor cells. Reverse chromosome painting, using a probe regenerated by microdissection of a long marker chromosome, revealed the native location of three of eight possible genes to be on chromosomes 1p31-32, 12q14 and 18q21. FISH using BAC and cosmid probes revealed amplification of JUN (1p31), MYC (8q24), CCND1 (11q13), INT2 (11q13.3), MDM2 (12q14.3-q15) and MALT (18q21). These findings indicate that at least eight amplified oncogenes may contribute to the pathogenesis of a rare pleomorphic type of rhabdomyosarcoma. This new cell line should prove useful for in vitro preclinical studies of molecularly targeted therapies.     

Frequent overexpression of the genes FXR1, CLAPM1 and EIF4G located on amplicon 3q26-27 in squamous cell carcinoma of the lung

Previously, we reported gene amplification at chromosome 3q26-27 in more than one third of squamous cell carcinomas of the lung. Frequent amplification of eukaryotic translation initiation factor 4G on 3q27.1 indicated a possible role of this amplification in translation initiation. The analysis of 61 squamous cell lung carcinomas shows that the percentage of carcinomas with a 3q27.1 amplification increases in higher malignant tumors. Non-invasive (T1) and minimal-invasive (T2) tumor stages showed similar percentages of amplified and non-amplified tumors, whereas locally-invasive (T3) tumors revealed a statistically significant (p < 0.05) increased percentage of amplified tumors. Microarrays were used to analyze the expression pattern of genes mapping in the amplified domain and its flanking regions (3q25-28) as well as the expression of genes directly or indirectly associated with translation initiation in squamous cell carcinoma, large cell carcinoma, adenocarcinoma and small cell carcinoma. Three genes, namely FXR1, CLAPM1 and EIF4G, are most frequently overexpressed in the center of the amplified domain in squamous cell carcinomas. The eukaryotic translation initiation factors 4A1, 2B and 4B as well as the poly(A)-binding protein PABPC1 where found to be overexpressed in all lung cancer entities. We found, however, no overexpression of eIF4E. Our results contribute to the understanding of the frequent amplification processes in squamous cell carcinomas of the lung and to the understanding of the translation initiation that appears not to require eIF4E in lung carcinogenesis.     

Suppression of hydroxyurea-induced centrosome amplification by NORE1A and down-regulation of NORE1A mRNA expression in non-small cell lung carcinoma

The candidate tumor suppressor NORE1A is a nucleocytoplasmic shuttling protein, and although a fraction of the NORE1A in cells is localized to their centrosomes, the role of centrosomal NORE1A has not been elucidated. In this study we investigated the role of NORE1A in the numerical integrity of centrosomes and chromosome stability in lung cancer cells. Exposure of p53-deficient H1299 lung cancer cell line to hydroxyurea (HU) resulted in abnormal centrosome amplification (to 3 or more centrosomes per cell) as determined by immunofluorescence analysis with anti-γ-tubulin antibody, and forced expression of wild-type NORE1A partially suppressed the centrosome amplification. The nuclear export signal (NES) mutant (L377A/L384A) of NORE1A did not localize to centrosomes and did not suppress the centrosome amplification induced by HU. Fluorescence in situ hybridization analyses with probes specific for chromosomes 2 and 16 showed that wild-type NORE1A, but not NES-mutant NORE1A, suppressed chromosome instability in HU-exposed H1299 cells that was likely to have resulted from centrosome amplification. We next examined the status of NORE1A mRNA expression in non-small cell lung carcinoma (NSCLC) and detected down-regulation of NORE1A mRNA expression in 25 (49%) of 51 primary NSCLCs by quantitative real-time-polymerase chain reaction analysis. These results suggest that NORE1A has activity that suppresses the centrosome amplification induced by HU and that NORE1A mRNA down-regulation is one of the common gene abnormalities in NSCLCs, both of which imply a key preventive role of NORE1A against the carcinogenesis of NSCLC.     

Isolation of a novel candidate oncogene within a frequently amplified region at 3q26 in ovarian cancer

Amplification of 3q25-q26 was one of the most frequent chromosomal alterations in human ovarian carcinoma. A chromosome microdissection-hybrid selection method was applied to isolate transcribed sequences from a primary ovarian cancer containing high-copy-number amplification of 3q26 using 3q26 band-specific DNAs generated by chromosome microdissection. Using this method, we have isolated a novel candidate oncogene eIF-5A2 (eukaryotic initiation factor 5A2). eIF-5A2 shares 82% identity of amino acid sequence with eIF-5A including the minimum domain needed for eIF-5A maturation by hypusine modification at lysine-50 residue. Amplification and overexpression of eIF-5A2 was frequently detected in primary ovarian cancers and ovarian cancer cell lines. The proliferation-related function of eIF-5A supports that eIF-5A2 is a candidate oncogene related to the development of ovarian cancer.     

HER-2 and TOP2A coamplification in urinary bladder cancer

HER-2/NEU is one of the most frequently amplified oncogenes and a potential therapeutic target in bladder cancer. In breast cancer, the adjacent TOP2A gene, the molecular target for several anticancer drugs, is frequently coamplified together with HER-2. To study the amplification and expression of TOP2A and HER-2 and associations with tumor phenotype and clinical outcome in bladder cancer, a tissue microarray containing 2,317 bladder tumor samples was analyzed by FISH and immunohistochemistry. Overall amplification frequencies were 6.3% for HER-2 and 1.5% for TOP2A. Amplifications were most frequently seen in advanced-stage (pT2-4) tumors (HER-2 13.8%, TOP2A 3.4%). Of HER-2-amplified tumors, 56% also had alterations of TOP2A, including 14.7% coamplifications, 33.3% gains and 8% deletions. Only 17.6% of TOP2A amplifications occurred independently of HER-2 alterations. Both HER-2 and TOP2A amplifications were significantly associated with advanced tumor stage (HER-2 p < 0.0001, TOP2A p = 0.0218), high grade (p < 0.0001 for both) and protein overexpression (p < 0.0001 for both). TOP2A amplification and overexpression were linked to shortened survival in muscle-invasive tumors (p = 0.0042 and 0.0077, respectively). In summary, our data suggest that HER-2 amplifications are frequently linked to alterations of the TOP2A gene in bladder cancer. The anatomy of the 17q12-q21 amplicon may be important for response to therapies targeting HER-2 or TOP2A.     

Evidence for various 20q status using allelotyping,CGH arrays,and quantitative PCR in distal CIN colon cancers

The genomic aberration profile of chromosome 20q in distal CIN colon carcinomas was analysed using allelotyping and CGH arrays. Allelotyping revealed carcinomas with allelic imbalance along the full long arm, and carcinomas with fully non-aberrant 20q. Oligonucleotide-based CGH showed that among the carcinomas without allelic imbalance, 47% had in fact a gain. In this subgroup, quantitative PCR for the TOPI gene (20q12) confirmed this gain, and fluorescence in situ hybridization showed that the chromosome 20q gain resulted from tetra/polysomy instead of aneusomy. The 20q gain correlated with a high frequency of aberrations, with allelic imbalance at TP53 locus but not at APC locus, and carcinomas with a disomic 20q showed low frequency of genomic aberrations and were significantly associated to mucinous phenotype. The prognostic value of 20q amplification was not demonstrated in this study. These results indicate that on the basis of aberration frequency, chromosome 20q and TP53/APC locus status, distal CIN carcinomas harbor a high degree of genetic heterogeneity suggesting several pathways for carcinogenesis. This study also indicates that allelotyping needs to be carried out with a complementary technique, such as quantitative PCR.     

Centrosome amplification, chromosome instability and cancer development

During mitosis, two centrosomes form spindle poles and direct the formation of bipolar mitotic spindles, which is an essential event for accurate chromosome segregation into daughter cells. The presence of more than two centrosomes (centrosome amplification), severely disturbs mitotic process and cytokinesis via formation of more than two spindle poles, resulting in an increased frequency of chromosome segregation errors (chromosome instability). Destabilization of chromosomes by centrosome amplification aids acquisition of further malignant phenotypes, hence promoting tumor progression. Centrosome amplification occurs frequently in almost all types of cancer, and is considered as the major contributing factor for chromosome instability in cancer cells. Upon cytokinesis, each daughter cell receives one centrosome, and thus centrosome must duplicate once, and only once, before the next mitosis. If centrosomes duplicate more than once within a single cell cycle, centrosome amplification occurs, which is frequently seen in cells harboring mutations in some tumor suppressor proteins such as p53 and BRCA1. The recent studies have provided critical information for understanding how loss of these proteins allows multiple rounds of centrosome duplication. In this review, how centrosome amplification destabilizes chromosomes, how loss of certain tumor suppressor proteins leads to centrosome amplification, and the role of centrosome amplification in cancer development will be discussed.     

Overexpression of Aurora-A potentiates HRAS-mediated oncogenic transformation and is implicated in oral carcinogenesis

Aurora kinases are known to play a key role in maintaining mitotic fidelity, and overexpression of aurora kinases has been noted in various tumors. Overexpression of aurora kinase activity is thought to promote cancer development through a loss of centrosome or chromosome number integrity. Here we observed augmentation of G12V-mutated HRAS-induced neoplastic transformation in BALB/c 3T3 A31-1-1 cells transfected with Aurora-A. Aurora-A-short hairpin RNA (shRNA) experiments showed that the expression level of Aurora-A determines susceptibility to transformation. Aurora-A gene amplification was noted in human patients with tongue or gingival squamous carcinoma (4/11). Amplification was observed even in pathologically normal epithelial tissue taken at sites distant from the tumors in two patients with tongue cancer. However, overexpression of Aurora-A mRNA was observed only within the tumors of all patients examined (11/11). Our data indicate that Aurora-A gene amplification and overexpression play a role in human carcinogenesis, largely due to the effect of Aurora-A on oncogenic cell growth, rather than a loss of maintenance of centrosomal or chromosomal integrity.     

Molecular mechanisms of pancreatic carcinogenesis

Pancreatic ductal adenocarcinoma is one of the most fatal malignancies. Intensive investigation of molecular pathogenesis might lead to identifying useful molecules for diagnosis and treatment of the disease. Pancreatic ductal adenocarcinoma harbors complicated aberrations of alleles including losses of 1p, 6q, 9p, 12q, 17p, 18q, and 21q, and gains of 8q and 20q. Pancreatic cancer is usually initiated by mutation of KRAS and aberrant expression of SHH. Overexpression of AURKA mapping on 20q13.2 may significantly enhance overt tumorigenesity. Aberrations of tumor suppressor genes synergistically accelerate progression of the carcinogenic pathway through pancreatic intraepithelial neoplasia (PanIN) to invasive ductal adenocarcinoma. Abrogation of CDKN2A occurs in low-grade/early PanIN, whereas aberrations of TP53 and SMAD4 occur in high-grade/late PanIN. SMAD4 may play suppressive roles in tumorigenesis by inhibition of angiogenesis. Loss of 18q precedes SMAD4 inactivation, and restoration of chromosome 18 in pancreatic cancer cells results in tumor suppressive phenotypes regardless of SMAD4 status, indicating the possible existence of a tumor suppressor gene(s) other than SMAD4 on 18q. DUSP6 at 12q21-q22 is frequently abrogated by loss of expression in invasive ductal adenocarcinomas despite fairly preserved expression in PanIN, which suggests that DUSP6 works as a tumor suppressor in pancreatic carcinogenesis. Restoration of chromosome 12 also suppresses growths of pancreatic cancer cells despite the recovery of expression of DUSP6; the existence of yet another tumor suppressor gene on 12q is strongly suggested. Understanding the molecular mechanisms of pancreatic carcinogenesis will likely provide novel clues for preventing, detecting, and ultimately curing this life-threatening disease.     

Induction of centrosome amplification during arsenite-induced mitotic arrest in CGL-2 cells

Arsenite-induced mitotic abnormalities result in mitotic death in several cancer cell lines. However, how arsenite induces these effects is not known. We have previously shown that arsenite induces mitotic arrest, mitotic abnormalities, and mitotic death in CGL-2 cells. To further delineate the mechanism of action of arsenite, we examined its effect on centrosome duplication and the possible link between centrosome dysregulation and arsenite-induced mitotic death. Immunofluorescence staining of gamma-tubulin revealed that centrosome amplification was induced in arsenite-arrested mitotic cells but not in nocodazole-arrested cells. When S phase-enriched cells were treated with arsenite, they progressed into and arrested at mitosis and then formed supernumerary centrosomes. A further increase in arsenite-induced centrosome amplification was seen during the prolonged mitotic arrest. The arsenite-induced supernumerary centrosomes might result from uneven fragmentation of centrosome, overexpression of pericentriolar materials, and inhibition of centrosomal coalescence during mitosis. Furthermore, termination of mitotic arrest by treatment of arsenite-arrested mitotic cells with cyclin-dependent kinase 1 inhibitors or by suppression of spindle checkpoint function by small interfering RNA-mediated silencing of BubR1 or Mad2 markedly reduced the induction of centrosome amplification and mitotic death in arsenite-treated cells. These results indicate that centrosome amplification is induced in arsenite-arrested mitotic CGL-2 cells in a spindle checkpoint-dependent manner and is involved in the induction of arsenite-induced mitotic death.     

Polysomy of chromosome 17 in breast cancer tumors showing an overexpression of ERBB2: a study of 175 cases using fluorescence <Emphasis Type="Italic">in situ</Emphasis>hybridization and immunohistochemistry

Introduction  

One of the most common genetic aberrations associated with breast cancer is the amplification and overexpression of the ERBB2 proto-oncogene located at chromosome 17, bands q12-21. The amplification/overexpression occurs in 25 to 30% of all breast cancers. In breast cancer, aneusomy of chromosome 17, either monosomy or polysomy, is frequently observed by conventional cytogenetics and fluorescence in situ hybridization (FISH). The aim of this study was to discover whether or not numerical aberrations on chromosome 17 have a correlation to the amplification or overexpression of the ERBB2 gene and to analyze their clinical implications in subgroups showing 2+ or 3+ positive scores by immunohistochemistry (IHC).