Cyclin D1 and estrogen receptor messenger RNA levels are positively correlated in primary breast cancer

The CCND1 gene, encoding the cell cycle regulatory protein cyclin D1, maps to chromosome 11q13, a locus that is amplified in about 13% of breast cancers. Because several studies have indicated a relationship between 11q13 amplification and markers of phenotype including estrogen receptor (ER) status, we tested the relationship between CCND1 and ER gene expression in 364 primary breast cancers using Northern blot analysis. Seventy-three % of samples were positive for ER mRNA, and cyclin D1 mRNA levels in the ER-positive group were significantly higher than those in the ER-negative group (P = 0.0001). When the samples were divided into quartiles of cyclin D1 expression, 58% of samples were ER positive in the lowest quartile and 87% in the highest quartile. The tumors expressing the highest levels of cyclin D1 (7%) were all ER positive. Furthermore, ER mRNA levels in the half with lower cyclin D1 mRNA were significantly less than in the half with higher cyclin D1 levels (P = 0.0001). Using simple regression analysis, there was a significant positive correlation between cyclin D1 and ER mRNA levels in the total population (P = 0.0001). This study demonstrates that cyclin D1 mRNA and ER mRNA are positively correlated in primary breast cancer, but the functional relationship between these genes remains to be elucidated.     

Cyclin DI amplification is not associated with reduced overall survival in primary breast cancer but may predict early relapse in patients with features of good prognosis

Amplification of chromosome 11q13 is frequently observed in human malignancies, including breast cancers. A candidate oncogene at this locus is the CCND1 gene, which encodes the cell cycle regulatory protein cyclin D1. Because published data on the relationship between 11q13 amplification and prognosis in breast cancer have been controversial, we investigated the clinical significance of CCND1 amplification and its association with established clinicopathological features of prognosis in 1014 primary breast cancer patients. Amplification of the CCND1 gene and the INT-2/FGF-3 gene, which also maps to 11q13, was 10% and 17%, respectively. There were no associations between CCND1 or INT-2 amplification and patient age, tumor size, tumor grade, axillary lymph node status, HER/neu amplification, MIB-1 monoclonal antibody to Ki67 antigen count, or p53 expression. CCND1 amplification was predominantly observed in hormone receptor-positive tumors; at a copy number >/=3, CCND1 amplification was significantly correlated with both estrogen receptor (ER; P = 0.036) and progesterone receptor (P = 0.012) positivity. After a median follow-up period of 66 months, CCND1 or INT-2 amplification was not associated with significant increases in relapse or death from breast cancer. However, in the node-negative and ER-positive subgroups, there was a trend for an increased relapse rate in patients with INT-2 or CCND1 amplification. Thus, in this study, assessment of CCND1 or INT-2 amplification at 11q13 by slot-blot hybridization was of little use in determining phenotype or disease outcome in the whole group of patients but had a potential role in identifying a subset of poor-prognosis patients within the node-negative or ER-positive, good-prognosis groups. Because the prevalence of CCND1 amplification is much lower than the reported prevalence of cyclin D1 overexpression, additional studies are required to determine the true prognostic significance of altered cyclin D1 expression in breast cancer.     

DNA amplifications at 20q13 and MDM2 define distinct subsets of evolved breast and ovarian tumours

DNA amplification seems to be particularly frequent in human breast tumours and has been associated with cancer evolution and aggressiveness. Recent data indicate that new events should be added to the list, such as the amplifications at chromosome 20q13 or the MDM2 gene. The present work aimed at determining the incidence and clinicopathological signification of these amplifications in a large series of breast and ovarian tumours. We tested 1371 breast and 179 ovarian tumours by Southern blotting and observed amplification of 20q13 in 5.4% breast and 2.8% ovarian carcinomas, whereas MDM2 was found amplified in 5.3% and 3.8% of breast and ovarian tumours respectively. MDM2 RNA expression levels were analysed in a subset of 57 breast tumours and overexpression was observed in 4/57 (7%) of the tumours. Elevated expression levels coincided with amplification of the gene. In breast cancer, 20q13 and MDM2 amplifications seem to define subsets of aggressive tumours. Indeed, 20q13 was correlated to axillary nodal involvement and occurred preferentially in younger patients (< 50 years). Furthermore, 20q13 correlated, as did MDM2 amplification, to aneuploidy. In parallel, we had also tested our tumour DNAs for amplification of CCND1, ERBB-2 and MYC, which made it possible to test for correlations with 20q13 or MDM2 amplifications. Whereas 20q13 showed a very strong correlation to CCND1 amplification, that of MDM2 was prevalent in MYC-amplified tumours. Interestingly, 20q13 and MDM2 amplifications showed some degree of correlation to each other, which may possibly be owing to the fact that both events occurred preferentially in aneuploid tumours. In ovarian cancer, no statistically significant correlation was observed. However, 20q13 amplification occurred preferentially in stage 3 tumours and MDM2 was correlated to ERBB-2 amplification. This may suggest that in ovarian tumours also, 20q13 and MDM2 amplifications occur in late or aggressive cancers.     

EMS1 gene expression in primary breast cancer: relationship to cyclin D1 and oestrogen receptor expression and patient survival

The EMS1 and CCND1 genes at chromosome 11q13 are amplified in about 15% of primary breast cancers but appear to confer different phenotypes in ER positive and ER negative tumours. Since there are no published data on EMS1 expression in large series of breast cancers we examined the relationship of EMS1 expression with EMS1 gene copy number and expression of mRNAs for cyclin D1 and ER. In a subset of 129 patients, where matched tumour RNA and DNA was available, EMS1 mRNA overexpression was associated predominantly with gene amplification (P = 0.0061), whereas cyclin D1 mRNA overexpression was not (P = 0.3142). In a more extensive series of 351 breast cancers, there was no correlation between cyclin D1 and EMS1 expression in the EMS1 and cyclin D1 overexpressors (P = 0.3503). Although an association between EMS1 mRNA expression and ER positivity was evident (P = 0.0232), when the samples were divided into quartiles of EMS1 or cyclin D1 mRNA expression, the increase in the proportion of ER positive tumours in the ascending EMS1 mRNA quartiles was not statistically significant (P = 0.0951). In marked contrast there was a significant stepwise increase in ER positivity in ascending quartiles of cyclin D1 mRNA (P = 0.030). A potential explanation for this difference was provided by the observation that in ER positive breast cancer cells oestradiol treatment resulted in increased cyclin D1 gene expression but was without effect on EMS1. The relationship between EMS1 expression and clinical outcome was examined in a subset of 234 patients with median follow-up of 74 months. High EMS1 expression was associated with age > 50 years (P = 0.0001), postmenopausal status (P = 0.0008), lymph node negativity (P = 0.019) and an apparent trend for worse prognosis in the ER negative subgroup. These data demonstrate that overexpression of EMS1 mRNA is largely due to EMS1 gene amplification, is independent of cyclin D1 and ER expression and, in contrast to cyclin D1, is not regulated by oestrogen. Independent overexpression of these genes may confer different phenotypes and disease outcomes in breast cancer as has been inferred from recent studies of EMS1 and CCND1 gene amplification.     

Independent amplification and frequent co-amplification of three nonsyntenic regions on the long arm of chromosome 20 in human breast cancer

DNA amplification at 20q13.2 is common in breast cancer, correlates with poor prognosis, and may reflect location of an important oncogene. Recently, other regions along 20q were also found to undergo amplification. Here, amplification levels and patterns of co-amplification were analyzed by interphase fluorescence in situ hybridization at 14 loci along 20q in 146 uncultured breast carcinomas and 14 cell lines. Three regions were independently amplified in uncultured tumors: RMC20C001 region at 20q13.2 (highly amplified in 9.6% of the cases), PTPN1 region 3 Mb proximal (6.2%), and AIB3 region at 20q11 (6.2%). Co-amplifications involving two or three of these regions were seen in 11 of the 19 highly amplified tumors. The results suggest that three distinct nonsyntenic regions along 20q may be important and that complex chromosomal rearrangements underlie their frequent co-amplification in breast cancer.     

Methylation of estrogen and progesterone receptor gene 5' CpG islands correlates with lack of estrogen and progesterone receptor gene expression in breast tumors

Hormonal factors have a profound influence on the development, treatment, and outcome of breast cancer. The absence of steroid hormone receptors is highly correlated with resistance to antihormonal treatments. Work in cultured human breast cancer cell lines has shown that the absence of estrogen receptor (ER) gene expression in ER- cells is associated with extensive methylation of the ER gene 5' CpG island, and treatment with agents that demethylate the ER gene CpG island results in the production of functional ER protein. The current study shows that CpG islands in the 5' region of the ER and progesterone receptor (PR) genes are methylated in a significant fraction of primary human breast cancer tissues. The ER CpG island is methylated at the methylation-sensitive NotI restriction site in 9 of 39 (25%) of primary ER- breast cancers but remains unmethylated in 53 ER+ breast cancers and 9 normal breast specimens. Three methylation-sensitive restriction sites in the PR gene CpG island are not methylated in normal breast specimens and PR+ human breast cancers but are hypermethylated in 40% of PR- human breast tumors. These data demonstrate that methylation of the ER and PR gene CpG islands is associated with the lack of ER and PR gene expression in a significant fraction of human breast cancers.     

Estrogen receptor mediated inhibition of cancer cell invasion and motility: an overview

In this overview of results from our laboratory, we address the question of the role of estrogens during early steps of metastasis, involving cell invasion through the basement membrane and cell motility. The motility of several estrogen receptor (ER) positive breast (MCF7, T47D) and ovarian (BG-1, SKOV3, PEO4) cancer cell lines was studied using a modified Boyden chamber assay. We observed, in all cases, estradiol induced inhibition of cancer cell invasion and motility. A similar inhibitory effect of estradiol was found when the wild-type ER alpha was stably transfected in the ER-negative MDA-MB231 cells and 3Y1-Ad12 cancer cells. The mechanism of this inhibitory effect is unknown. In ovarian cancer, however, it may involve intermediary proteins such as fibulin-1, an extracellular matrix protein that strongly interacts with fibronectin and which is induced by estrogen and secreted by ovarian cancer cells. We conclude that estrogens in ER-positive breast and ovarian cancers have a dual effect, since they stimulate tumor growth but inhibit invasion and motility. This may be consistent with the good initial prognostic value of ER-positive breast cancers compared to ER negative breast cancers noted in several clinical studies.     

Mapping of DNA amplifications at 15 chromosomal localizations in 1875 breast tumors: definition of phenotypic groups

DNA amplification is frequent in breast cancer and has been associated with specific clinicopathological parameters and/or worsened course of the disease. In the present work, we were interested in further defining the association linking the occurrence of DNA amplification to the emergence of specific breast tumor phenotype. To this aim, we studied by Southern blotting a total of 1875 breast tumor DNAs with 26 probes mapping at 15 distinct chromosomal localizations. Of the 26 loci tested, 11 loci showed elevated levels of amplification, 9 loci showed occasional and/or low level of DNA copy number increase, and 6 loci showed very rare or no variation. This allowed us to define six amplified domains mapping at 8p12, 8q24, 11q13, 12q13, 17q12, and 20q13.2, respectively. Over 60% of the tumors analyzed presented at least one amplification at one of these localizations. Amplifications often covered large regions of DNA and bore complex patterns involving coamplification of several colocalized markers. Statistical analysis revealed correlations associating DNA amplification with breast tumor phenotype, as well as sets of preferential coamplifications. Based on these correlations, we defined three subsets of breast cancer according to their patterns of DNA amplification. The first subset (group A) was organized around the amplifications at 11q13 and/or 8p12 and was predominantly composed of estrogen receptor-positive tumors and presented a large proportion of lobular cancers. The second subset (group B) was organized around the amplifications of ERBB2 and/or MYC. These tumors were mostly estrogen receptor-negative and of the ductal invasive type. The third subset (group C) corresponded to tumors in which no amplification was detected in the present screen. Tumors in this group were largely diploid and of low histopathological grading.     

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The gene for Cyclin D1 (CCND1) lies within chromosomal region 11q13 and codes for a cell cycle regulator essential for G1 phase progression. This G1-cyclin is a putative protooncogene whose clonal rearrangement and/or amplification and mRNA overexpression occurs in several types of human neoplasias, including head and neck squamous cell carcinomas. Data from the literature suggest that amplification and overexpression of the CCND1 gene could lead to destabilisation of cell cycle control mechanisms and uncontrolled cell proliferation. We developed a differential PCR system for the determination of CCND1 gene amplification in head and neck squamous cell carcinomas. A 115 bp CCND1 fragment and a 150 bp gamma-interferon fragment are amplified simultaneously in the same reaction tube under optimized conditions. Statistical analysis of amplification data obtained by differential PCR revealed excellent correlation with amplification data obtained by conventional Southern hybridization.     

Glucocorticoid and estrogen receptors have elevated activity in human endometrial and ovarian tumors as compared to the adjacent normal tissues and recognize sequence elements of the H-ras proto-oncogene

We examined the level of receptor binding in H-ras elements, using nuclear extracts derived from human endometrial and ovarian lesions and from adjacent normal tissue in gel retardation assays. We found increased binding of the glucocorticoid receptor (GR) to the H-ras GR element in more than 90% of endometrial tumors and in all ovarian tumors tested, as compared to the corresponding adjacent normal tissue. Additionally, we found elevated binding of the estrogen receptor (ER) in H-ras ER element in all pairs of ovarian tumor/normal tissue tested, whereas in ER-negative control breast tumor/normal tissue pairs, no differences in ER DNA-binding levels were observed. These results suggest that steroid hormone receptor binding could directly activate the H-ras oncogenic potency in human endometrial and ovarian lesions, providing additional evidence for the role of H-ras expression in hormonally responsive human cancers.     

Amplification of the genes BCHE and SLC2A2 in 40% of squamous cell carcinoma of the lung

Gene amplification is a common genetic change in human cancer cells. Previously, we provided the first evidence for gene amplification at chromosome band 3q26 in squamous cell lung carcinoma. In this study, the following analyses were performed: (a) we evaluated biopsies and paraffin-embedded tissues of 16 additional squamous cell lung carcinomas for gene amplification using reverse chromosome painting. Of the 16 tumors, 3 tumors showed an amplification of the entire long arm of chromosome 3, and 3 tumors showed various amplifications on 3q, all of which involved chromosome band 3q26; (b) we tested eight genes encompassing region 3q25-qter in two different tumors to identify amplified genes on chromosome 3q. The genes SI, BCHE, and SLC2A2 were amplified in both tumors; and (c) we analyzed 15 additional paraffin-embedded tissues to determine the amplification frequency of these genes. Of the 15 squamous cell lung carcinomas, 6 showed amplification for at least 1 of the genes, with BCHE and SLC2A2 as the genes most frequently amplified. Together, our reverse chromosome painting data and our PCR analysis indicate gene amplification at 3q26 in 40% of all squamous cell lung carcinomas with BCHE and SLC2A2 as possible target genes of the amplification unit in squamous cell lung carcinoma.     

Classification of breast cancer cells on the basis of a functional assay for estrogen receptor

BACKGROUND: The receptor (ER) for estrogen (E2) is routinely assayed as a marker to determine the feasibility of anti-hormone therapy against breast cancer because ER-positive (ER+) tumors are much more likely to respond to anti-hormone therapy than are ER-negative (ER-). However 40% of ER+ breast cancer patients do not respond to anti-hormone therapy. We suggest that this unpredictability of therapeutic responses lies in the current ER assays, which measure only an initial component of the E2-responsive pathway, and that the difference depends upon altered downstream processes. We propose a functional criterion that subclassifies breast cancers on the basis of specific binding of ER to its cognate DNA sequence, the estrogen response element (ERE). MATERIALS AND METHODS: ER was identified in breast cancer cell lines by immunofluorescence assay, Western blot analysis, identification of ER-specific mRNA, and by interaction of the ER-ERE complex with three different ER-specific antibodies. ER-ERE complex formation was measured by electrophoretic mobility shift assay (EMSA). Transactivation of the E2-responsive gene was studied by transfection of cells with fusion gene construct with the promoter-containing ERE sequence and assay of reporter gene activity in the cell extracts. RESULTS: The growth of ER+ T47D cells was sensitive to tamoxifen, ICI-182,780, and ethynyl estradiol (EE2), whereas another ER+ breast cancer cell line, 21 PT, was resistant to these compounds. The estrogen receptor (ER) in the nuclear extracts of MCF-7 and T47D demonstrated hormone-dependent interaction with the response element (ERE) and also downstream transactivation of the E2-responsive PS2 promoter. But in the 21 PT cell line that was designated as ER- on the basis of ligand-binding assay and was found to be ER+ by all the other ER assays, ER-ERE interaction and PS2 promoter transactivation were independent of hormone. CONCLUSIONS: On the basis of the downstream functional assay of ER interaction with ERE, ER+ breast tumor cells can be subclassified into two categories. The first is E2-dependent (ERd+) and these cells should respond to anti-hormone therapy. The second type of ER interacts with ERE independent of E2 (ERi+) and constitutively transactivates responsive genes. It is predicted that the latter type of breast cancers will not respond to antihormone therapy.     

Antisense estrogen receptor RNA expression increases epidermal growth factor receptor gene expression in breast cancer cells

In human breast cancer, progression to a more malignant phenotype is often accompanied by decreased expression of estrogen receptor (ER) and increased expression of epidermal growth factor receptor (EGFR). Higher levels of this receptor tyrosine kinase are found in tumors lacking ER, and a quantitative, inverse relationship exists between the level of ER and EGFR mRNA in human breast cell lines. Antisense ER (ASER) RNA was used to evaluate the consequence of decreased ER expression in breast cancer cells, specifically to determine whether ER is involved in the regulation of EGFR gene expression. ER-positive MCF-7 human breast cancer cells were transfected with ASER, and clones constitutively expressing ASER RNA had decreased ER and up to a 3-fold increase in the expression of EGFR mRNA. To confirm that this observation was a direct consequence of ASER expression, a metal-inducible ASER expression construct was transfected into MCF-7 cells, and transfected clones were isolated and characterized. Northern analysis revealed an induction of ASER RNA within 1 h of the addition of zinc, which was followed by a 4-fold increase in EGFR mRNA levels, maximal at 6-12 h. The basal level of expression of the glucocorticoid receptor is also inversely related to that of ER among breast cancer cell lines, but neither constitutive nor inducible expression of ASER affected the expression of glucocorticoid receptor. These data support the hypothesis that the level of expression of ER specifically influences the expression of EGFR in human breast cancer cells and provides a potential link between loss of steroid sensitivity and the acquisition of autonomous growth.     

Dysregulated cyclin D1 expression early in head and neck tumorigenesis: in vivo evidence for an association with subsequent gene amplification

Cyclin D1 proto-oncogene is a key regulator of the mammalian cell-cycle acting at the restriction point in late G1. Amplification of the cyclin D1 locus, located on chromosome 11q13, as well as cyclin D1 protein overexpression have been reported in several human malignancies. The purpose of this study was to evaluate cyclin D1 gene copy status and protein expression during the multistep process of head and neck tumorigenesis, using a combination of fluorescence in situ hybridization and immunohistochemistry techniques. From 29 selected patients presenting with head and neck squamous carcinoma and whose tumor cytospins had been previously screened for presence (16 cases) or absence (13 cases) of amplification at the 11q13 band, we analysed 46 paraffin-embedded tissue specimens that demonstrated, besides the primary tumor, the presence of contiguous adjacent normal tissue and/or premalignant lesions. Of the 16 amplified cases, nine demonstrated a continuous progression from premalignant to invasive carcinoma and seven (77.7%) of these cases showed cyclin D1 gene amplification in premalignant lesions prior to development of invasive carcinoma. Increased cyclin D1 protein expression was observed in all 16 amplified tumors and five of the 13 (38.4%) non-amplified tumors. Interestingly, dysregulated cyclin D1 expression was also found in the premalignant lesions adjacent to all 16 amplified tumors, and it appeared to precede cyclin D1 gene amplification. In contrast no dysregulated expression was detected in the premalignant lesions of the non-amplified tumors. In conclusion, these findings provide strong evidence for early dysregulation of cyclin D1 expression during the tumorigenesis process and suggest that dysregulated increased expression precedes and possibly enables gene amplification.     

FISH characterization of head and neck carcinomas reveals that amplification of band 11q13 is associated with deletion of distal 11q

In order to characterize homogeneously staining regions (HSR) and other 11q13 rearrangements identified cytogenetically, we performed fluorescence in situ hybridization (FISH) using a CCND1 cosmid and five YAC clones spanning chromosomal bands 11q13-14 on metaphase cells from 14 primary and one metastatic head and neck carcinomas. At the cytogenetic level, a total of 17 HSR were detected in ten cases: five were in derivative chromosomes 11 in band 11q13, and 12 were located in other derivative chromosomes. Other forms of 11q13 rearrangements were observed in five cases, whereas two cases had normal chromosomes 11. FISH analysis demonstrated that all HSR but two were derived from the 11q13 band. The size of the amplicon varied from case to case, but the amplification always included the region covered by YAC 55G7, which contains the CCND1 locus. The amplification of CCND1 was confirmed by use of a CCND1 cosmid. We also showed that most of the cases (9 of 11) with 11q13 amplification had lost material from distal 11q. The breakpoints were mapped by FISH and were shown to cluster to the region between YACs 55G7 and 749G2. We conclude that loss of gene(s) in distal 11q may be as important as amplification of genes in 11q13 for the biological aggressiveness of head and neck carcinomas.