Distinct epigenetic changes in the stromal cells of breast cancers

Increasing evidence suggests that changes in the cellular microenvironment contribute to tumorigenesis, but the molecular basis of these alterations is not well understood. Although epigenetic modifications of the neoplastic cells in tumors have been firmly implicated in tumorigenesis, it is not known whether epigenetic modifications occur in the non-neoplastic stromal cells. To address this question in an unbiased and genome-wide manner, we developed a new method, methylation-specific digital karyotyping, and applied it to epithelial and myoepithelial cells, stromal fibroblasts from normal breast tissue, and in situ and invasive breast carcinomas. Our analyses showed that distinct epigenetic alterations occur in all three cell types during breast tumorigenesis in a tumor stage- and cell type-specific manner, suggesting that epigenetic changes have a role in the maintenance of the abnormal cellular microenvironment in breast cancer.     

A Robertsonian translocation suppresses a somatic recombination pathway to loss of heterozygosity

In mammals, loss of APC/Apc gatekeeper function initiates intestinal tumorigenesis. Several different mechanisms have been shown or proposed to mediate functional loss of APC/Apc: mutation in APC/Apc, non-disjunction, homologous somatic recombination and epigenetic silencing. The demonstration that, in the C57BL/6 (B6) Apc(Min/+) mouse model of inherited intestinal cancer, loss of Apc function can occur by loss of heterozygosity (LOH) through somatic recombination between homologs presents an opportunity to search for polymorphisms in the homologous somatic recombination pathway. We report that the Robertsonian translocation Rb(7.18)9Lub (Rb9) suppresses the multiplicity of intestinal adenomas in this mouse model. As the copy number of Rb9 increases, the association with the interphase nucleolus of the rDNA repeats centromeric to the Apc locus on Chromosome 18 is increasingly disrupted. Our analysis shows that homologous somatic recombination is the principal pathway for LOH in adenomas in B6 Apc(Min/+) mice. These studies provide additional evidence that neoplastic growth can initiate in the complete absence of canonical genomic instability.     

Mutational and functional analyses reveal that ST7 is a highly conserved tumor-suppressor gene on human chromosome 7q31

Loss of heterozygosity (LOH) of markers on human chromosome 7q31 is frequently encountered in a variety of human neoplasias, indicating the presence of a tumor-suppressor gene (TSG). By a combination of microcell-fusion and deletion-mapping studies, we previously established that this TSG resides within a critical region flanked by the genetic markers D7S522 and D7S677. Using a positional cloning strategy and aided by the availability of near-complete sequence of this genomic interval, we have identified a TSG within 7q31, named ST7 (for suppression of tumorigenicity 7; this same gene was recently reported in another context and called RAY1). ST7 is ubiquitously expressed in human tissues. Analysis of a series of cell lines derived from breast tumors and primary colon carcinomas revealed the presence of mutations in ST7. Introduction of the ST7 cDNA into the prostate-cancer-derived cell line PC3 had no effect on the in vitro proliferation of the cells, but abrogated their in vivo tumorigenicity. Our data indicate that ST7 is a TSG within chromosome 7q31 and may have an important role in the development of some types of human cancer.     

TSLC1 is a tumor-suppressor gene in human non-small-cell lung cancer

The existence of tumor-suppressor genes was originally demonstrated by functional complementation through whole-cell and microcell fusion. Transfer of chromosome 11 into a human non-small-cell lung cancer (NSCLC) cell line, A549, suppresses tumorigenicity. Loss of heterozygosity (LOH) on the long arm of chromosome 11 has been reported in NSCLC and other cancers. Several independent studies indicate that multiple tumor-suppressor genes are found in this region, including the gene PPP2R1B at 11q23-24 (ref. 7). Linkage studies of NSCLC are precluded because no hereditary forms are known. We previously identified a region of 700 kb on 11q23.2 that completely suppresses tumorigenicity of A549 human NSCLC cells. Most of this tumor-suppressor activity localizes to a 100-kb segment by functional complementation. Here we report that this region contains a single confirmed gene, TSLC1, whose expression is reduced or absent in A549 and several other NSCLC, hepatocellular carcinoma (HCC) and pancreatic cancer (PaC) cell lines. TSLC1 expression or suppression is correlated with promoter methylation state in these cell lines. Restoration of TSLC1 expression to normal or higher levels suppresses tumor formation by A549 cells in nude mice. Only 2 inactivating mutations of TSLC1 were discovered in 161 tumors and tumor cell lines, both among the 20 primary tumors with LOH for 11q23.2. Promoter methylation was observed in 15 of the other 18 primary NSCLC, HCC and PaC tumors with LOH for 11q23.2. Thus, attenuation of TSLC1 expression occurred in 85% of primary tumors with LOH. Hypermethylation of the TSLC1 promoter would seem to represent the 'second hit' in NSCLC with LOH.     

Genome-wide analysis of DNA copy-number changes using cDNA microarrays.

Gene amplifications and deletions frequently contribute to tumorigenesis. Characterization of these DNA copy-number changes is important for both the basic understanding of cancer and its diagnosis. Comparative genomic hybridization (CGH) was developed to survey DNA copy-number variations across a whole genome. With CGH, differentially labelled test and reference genomic DNAs are co-hybridized to normal metaphase chromosomes, and fluorescence ratios along the length of chromosomes provide a cytogenetic representation of DNA copy-number variation. CGH, however, has a limited ( approximately 20 Mb) mapping resolution, and higher-resolution techniques, such as fluorescence in situ hybridization (FISH), are prohibitively labour-intensive on a genomic scale. Array-based CGH, in which fluorescence ratios at arrayed DNA elements provide a locus-by-locus measure of DNA copy-number variation, represents another means of achieving increased mapping resolution. Published array CGH methods have relied on large genomic clone (for example BAC) array targets and have covered only a small fraction of the human genome. cDNAs representing over 30,000 radiation-hybrid (RH)-mapped human genes provide an alternative and readily available genomic resource for mapping DNA copy-number changes. Although cDNA microarrays have been used extensively to characterize variation in human gene expression, human genomic DNA is a far more complex mixture than the mRNA representation of human cells. Therefore, analysis of DNA copy-number variation using cDNA microarrays would require a sensitivity of detection an order of magnitude greater than has been routinely reported. We describe here a cDNA microarray-based CGH method, and its application to DNA copy-number variation analysis in breast cancer cell lines and tumours. Using this assay, we were able to identify gene amplifications and deletions genome-wide and with high resolution, and compare alterations in DNA copy number and gene expression.     

Germline mutations in RAD51D confer susceptibility to ovarian cancer

Recently, RAD51C mutations were identified in families with breast and ovarian cancer. This observation prompted us to investigate the role of RAD51D in cancer susceptibility. We identified eight inactivating RAD51D mutations in unrelated individuals from 911 breast-ovarian cancer families compared with one inactivating mutation identified in 1,060 controls (P = 0.01). The association found here was principally with ovarian cancer, with three mutations identified in the 59 pedigrees with three or more individuals with ovarian cancer (P = 0.0005). The relative risk of ovarian cancer for RAD51D mutation carriers was estimated to be 6.30 (95% CI 2.86-13.85, P = 4.8 × 10(-6)). By contrast, we estimated the relative risk of breast cancer to be 1.32 (95% CI 0.59-2.96, P = 0.50). These data indicate that RAD51D mutation testing may have clinical utility in individuals with ovarian cancer and their families. Moreover, we show that cells deficient in RAD51D are sensitive to treatment with a PARP inhibitor, suggesting a possible therapeutic approach for cancers arising in RAD51D mutation carriers.     

Evolution of neoplastic cell lineages in Barrett oesophagus.

It has been hypothesized that neoplastic progression develops as a consequence of an acquired genetic instability and the subsequent evolution of clonal populations with accumulated genetic errors. Accordingly, human cancers and some premalignant lesions contain multiple genetic abnormalities not present in the normal tissues from which the neoplasms arose. Barrett oesophagus (BE) is a premalignant condition which predisposes to oesophageal adenocarcinoma (EA) that can be biopsied prospectively over time because endoscopic surveillance is recommended for early detection of cancer. In addition, oesophagectomy specimens frequently contain the premalignant epithelium from which the cancer arose. Neoplastic progression in BE is associated with alterations in TP53 (also known as p53) and CDKN2A (also known as p16) and non-random losses of heterozygosity (LOH). Aneuploid or increased 4N populations occur in more than 90-95% of EAs, arise in premalignant epithelium and predict progression. We have previously shown in small numbers of patients that disruption of TP53 and CDKN2A typically occurs before aneuploidy and cancer. Here, we determine the evolutionary relationships of non-random LOH, TP53 and CDKN2A mutations, CDKN2A CpG-island methylation and ploidy during neoplastic progression. Diploid cell progenitors with somatic genetic or epigenetic abnormalities in TP53 and CDKN2A were capable of clonal expansion, spreading to large regions of oesophageal mucosa. The subsequent evolution of neoplastic progeny frequently involved bifurcations and LOH at 5q, 13q and 18q that occurred in no obligate order relative to each other, DNA-content aneuploidy or cancer. Our results indicate that clonal evolution is more complex than predicted by linear models.     

Phosphoglycerate dehydrogenase diverts glycolytic flux and contributes to oncogenesis

Most tumors exhibit increased glucose metabolism to lactate, however, the extent to which glucose-derived metabolic fluxes are used for alternative processes is poorly understood. Using a metabolomics approach with isotope labeling, we found that in some cancer cells a relatively large amount of glycolytic carbon is diverted into serine and glycine metabolism through phosphoglycerate dehydrogenase (PHGDH). An analysis of human cancers showed that PHGDH is recurrently amplified in a genomic region of focal copy number gain most commonly found in melanoma. Decreasing PHGDH expression impaired proliferation in amplified cell lines. Increased expression was also associated with breast cancer subtypes, and ectopic expression of PHGDH in mammary epithelial cells disrupted acinar morphogenesis and induced other phenotypic alterations that may predispose cells to transformation. Our findings show that the diversion of glycolytic flux into a specific alternate pathway can be selected during tumor development and may contribute to the pathogenesis of human cancer.     

Null alleles of ABCG2 encoding the breast cancer resistance protein define the new blood group system Junior

The breast cancer resistance protein, also known as ABCG2, is one of the most highly studied ATP-binding cassette (ABC) transporters because of its ability to confer multidrug resistance. The lack of information on the physiological role of ABCG2 in humans severely limits cancer chemotherapeutic approaches targeting this transporter. We report here that ABCG2 comprises the molecular basis of a new blood group system (Junior, Jr) and that individuals of the Jr(a-) blood type have inherited two null alleles of ABCG2. We identified five frameshift and three nonsense mutations in ABCG2. We also show that the prevalence of the Jr(a-) blood type in the Japanese and European Gypsy populations is related to the p.Gln126* and p.Arg236* protein alterations, respectively. The identification of ABCG2(-/-) (Jr(a-)) individuals who appear phenotypically normal is an essential step toward targeting ABCG2 in cancer and also in understanding the physiological and pharmacological roles of this promiscuous transporter in humans.     

Estrogen receptor alpha (ESR1) gene amplification is frequent in breast cancer

Using an Affymetrix 10K SNP array to screen for gene copy number changes in breast cancer, we detected a single-gene amplification of the ESR1 gene, which encodes estrogen receptor alpha, at 6q25. A subsequent tissue microarray analysis of more than 2,000 clinical breast cancer samples showed ESR1 amplification in 20.6% of breast cancers. Ninety-nine percent of tumors with ESR1 amplification showed estrogen receptor protein overexpression, compared with 66.6% cancers without ESR1 amplification (P < 0.0001). In 175 women who had received adjuvant tamoxifen monotherapy, survival was significantly longer for women with cancer with ESR1 amplification than for women with estrogen receptor-expressing cancers without ESR1 amplification (P = 0.023). Notably, we also found ESR1 amplification in benign and precancerous breast diseases, suggesting that ESR1 amplification may be a common mechanism in proliferative breast disease and a very early genetic alteration in a large subset of breast cancers.     

The mitochondrial bottleneck occurs without reduction of mtDNA content in female mouse germ cells

Observations of rapid shifts in mitochondrial DNA (mtDNA) variants between generations prompted the creation of the bottleneck theory. A prevalent hypothesis is that a massive reduction in mtDNA content during early oogenesis leads to the bottleneck. To test this, we estimated the mtDNA copy number in single germline cells and in single somatic cells of early embryos in mice. Primordial germ cells (PGCs) show consistent, moderate mtDNA copy numbers across developmental stages, whereas primary oocytes demonstrate substantial mtDNA expansion during early oocyte maturation. Some somatic cells possess a very low mtDNA copy number. We also demonstrated that PGCs have more than 100 mitochondria per cell. We conclude that the mitochondrial bottleneck is not due to a drastic decline in mtDNA copy number in early oogenesis but rather to a small effective number of segregation units for mtDNA in mouse germ cells. These results provide new information for mtDNA segregation models and for understanding the recurrence risks for mtDNA diseases.     

Recurrent gross mutations of the PTEN tumor suppressor gene in breast cancers with deficient DSB repair

Basal-like breast cancer (BBC) is a subtype of breast cancer with poor prognosis. Inherited mutations of BRCA1, a cancer susceptibility gene involved in double-strand DNA break (DSB) repair, lead to breast cancers that are nearly always of the BBC subtype; however, the precise molecular lesions and oncogenic consequences of BRCA1 dysfunction are poorly understood. Here we show that heterozygous inactivation of the tumor suppressor gene Pten leads to the formation of basal-like mammary tumors in mice, and that loss of PTEN expression is significantly associated with the BBC subtype in human sporadic and BRCA1-associated hereditary breast cancers. In addition, we identify frequent gross PTEN mutations, involving intragenic chromosome breaks, inversions, deletions and micro copy number aberrations, specifically in BRCA1-deficient tumors. These data provide an example of a specific and recurrent oncogenic consequence of BRCA1-dependent dysfunction in DNA repair and provide insight into the pathogenesis of BBC with therapeutic implications. These findings also argue that obtaining an accurate census of genes mutated in cancer will require a systematic examination for gross gene rearrangements, particularly in tumors with deficient DSB repair.     

Low-penetrance susceptibility to breast cancer due to CHEK2(*)1100delC in noncarriers of BRCA1 or BRCA2 mutations

Mutations in BRCA1 and BRCA2 confer a high risk of breast and ovarian cancer, but account for only a small fraction of breast cancer susceptibility. To find additional genes conferring susceptibility to breast cancer, we analyzed CHEK2 (also known as CHK2), which encodes a cell-cycle checkpoint kinase that is implicated in DNA repair processes involving BRCA1 and p53 (refs 3,4,5). We show that CHEK2(*)1100delC, a truncating variant that abrogates the kinase activity, has a frequency of 1.1% in healthy individuals. However, this variant is present in 5.1% of individuals with breast cancer from 718 families that do not carry mutations in BRCA1 or BRCA2 (P = 0.00000003), including 13.5% of individuals from families with male breast cancer (P = 0.00015). We estimate that the CHEK2(*)1100delC variant results in an approximately twofold increase of breast cancer risk in women and a tenfold increase of risk in men. By contrast, the variant confers no increased cancer risk in carriers of BRCA1 or BRCA2 mutations. This suggests that the biological mechanisms underlying the elevated risk of breast cancer in CHEK2 mutation carriers are already subverted in carriers of BRCA1 or BRCA2 mutations, which is consistent with participation of the encoded proteins in the same pathway.     

Analysis of the coding genome of diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common form of human lymphoma. Although a number of structural alterations have been associated with the pathogenesis of this malignancy, the full spectrum of genetic lesions that are present in the DLBCL genome, and therefore the identity of dysregulated cellular pathways, remains unknown. By combining next-generation sequencing and copy number analysis, we show that the DLBCL coding genome contains, on average, more than 30 clonally represented gene alterations per case. This analysis also revealed mutations in genes not previously implicated in DLBCL pathogenesis, including those regulating chromatin methylation (MLL2; 24% of samples) and immune recognition by T cells. These results provide initial data on the complexity of the DLBCL coding genome and identify novel dysregulated pathways underlying its pathogenesis.     

Integrated detection and population-genetic analysis of SNPs and copy number variation

Dissecting the genetic basis of disease risk requires measuring all forms of genetic variation, including SNPs and copy number variants (CNVs), and is enabled by accurate maps of their locations, frequencies and population-genetic properties. We designed a hybrid genotyping array (Affymetrix SNP 6.0) to simultaneously measure 906,600 SNPs and copy number at 1.8 million genomic locations. By characterizing 270 HapMap samples, we developed a map of human CNV (at 2-kb breakpoint resolution) informed by integer genotypes for 1,320 copy number polymorphisms (CNPs) that segregate at an allele frequency >1%. More than 80% of the sequence in previously reported CNV regions fell outside our estimated CNV boundaries, indicating that large (>100 kb) CNVs affect much less of the genome than initially reported. Approximately 80% of observed copy number differences between pairs of individuals were due to common CNPs with an allele frequency >5%, and more than 99% derived from inheritance rather than new mutation. Most common, diallelic CNPs were in strong linkage disequilibrium with SNPs, and most low-frequency CNVs segregated on specific SNP haplotypes.     

Allele losses in the region 17q12-21 in familial breast and ovarian cancer involve the wild-type chromosome.

A predisposing gene for breast and ovarian cancer has recently been mapped to chromosome 17q12-21. If this gene is a tumour suppressor gene, allele losses would be expected in the tumours of affected family members and the losses should affect the wild-type chromosome, reflecting the need for inactivation of the wild-type allele at the predisposing locus. In four multiple case breast-ovarian cancer families, we have found that in each of nine tumours which showed allele losses, the losses were from the wild-type chromosome. This suggests that the putative 'breast-ovarian' cancer gene is indeed a tumour suppressor gene.     

Quantitative mapping of amplicon structure by array CGH identifies CYP24 as a candidate oncogene

We show here that quantitative measurement of DNA copy number across amplified regions using array comparative genomic hybridization (CGH) may facilitate oncogene identification by providing precise information on the locations of both amplicon boundaries and amplification maxima. Using this analytical capability, we resolved two regions of amplification within an approximately 2-Mb region of recurrent aberration at 20q13.2 in breast cancer. The putative oncogene ZNF217 (ref. 5) mapped to one peak, and CYP24 (encoding vitamin D 24 hydroxylase), whose overexpression is likely to lead to abrogation of growth control mediated by vitamin D, mapped to the other.