Mutant p53 disrupts mammary tissue architecture via the mevalonate pathway.

p53 is a frequent target for mutation in human tumors, and mutant p53 proteins can actively contribute to tumorigenesis. We employed a three-dimensional culture model in which nonmalignant breast epithelial cells form spheroids reminiscent of acinar structures found in vivo, whereas breast cancer cells display highly disorganized morphology. We found that mutant p53 depletion is sufficient to phenotypically revert breast cancer cells to a more acinar-like morphology. Genome-wide expression analysis identified the mevalonate pathway as significantly upregulated by mutant p53. Statins and sterol biosynthesis intermediates reveal that this pathway is both necessary and sufficient for the phenotypic effects of mutant p53 on breast tissue architecture. Mutant p53 associates with sterol gene promoters at least partly via SREBP transcription factors. Finally, p53 mutation correlates with highly expressed sterol biosynthesis genes in human breast tumors. These findings implicate the mevalonate pathway as a therapeutic target for tumors bearing mutations in p53.

The life history of 21 breast cancers.

Cancer evolves dynamically as clonal expansions supersede one another driven by shifting selective pressures, mutational processes, and disrupted cancer genes. These processes mark the genome, such that a cancer's life history is encrypted in the somatic mutations present. We developed algorithms to decipher this narrative and applied them to 21 breast cancers. Mutational processes evolve across a cancer's lifespan, with many emerging late but contributing extensive genetic variation. Subclonal diversification is prominent, and most mutations are found in just a fraction of tumor cells. Every tumor has a dominant subclonal lineage, representing more than 50% of tumor cells. Minimal expansion of these subclones occurs until many hundreds to thousands of mutations have accumulated, implying the existence of long-lived, quiescent cell lineages capable of substantial proliferation upon acquisition of enabling genomic changes. Expansion of the dominant subclone to an appreciable mass may therefore represent the final rate-limiting step in a breast cancer's development, triggering diagnosis.

Mutational processes molding the genomes of 21 breast cancers.

All cancers carry somatic mutations. The patterns of mutation in cancer genomes reflect the DNA damage and repair processes to which cancer cells and their precursors have been exposed. To explore these mechanisms further, we generated catalogs of somatic mutation from 21 breast cancers and applied mathematical methods to extract mutational signatures of the underlying processes. Multiple distinct single- and double-nucleotide substitution signatures were discernible. Cancers with BRCA1 or BRCA2 mutations exhibited a characteristic combination of substitution mutation signatures and a distinctive profile of deletions. Complex relationships between somatic mutation prevalence and transcription were detected. A remarkable phenomenon of localized hypermutation, termed "kataegis," was observed. Regions of kataegis differed between cancers but usually colocalized with somatic rearrangements. Base substitutions in these regions were almost exclusively of cytosine at TpC dinucleotides. The mechanisms underlying most of these mutational signatures are unknown. However, a role for the APOBEC family of cytidine deaminases is proposed.

Author Correction: Landscape of somatic mutations in 560 breast cancer whole-genome sequences.

In the Methods section of this Article, 'greater than' should have been 'less than' in the sentence 'Putative regions of clustered rearrangements were identified as having an average inter-rearrangement distance that was at least 10 times greater than the whole-genome average for the individual sample. '. The Article has not been corrected.

Cis-regulatory mutations associate with transcriptional and post-transcriptional deregulation of gene regulatory programs in cancers.

Most cancer alterations occur in the noncoding portion of the human genome, where regulatory regions control gene expression. The discovery of noncoding mutations altering the cells' regulatory programs has been limited to few examples with high recurrence or high functional impact. Here, we show that transcription factor binding sites (TFBSs) have similar mutation loads to those in protein-coding exons. By combining cancer somatic mutations in TFBSs and expression data for protein-coding and miRNA genes, we evaluate the combined effects of transcriptional and post-transcriptional alterations on the regulatory programs in cancers. The analysis of seven TCGA cohorts culminates with the identification of protein-coding and miRNA genes linked to mutations at TFBSs that are associated with a cascading trans-effect deregulation on the cells' regulatory programs. Our analyses of cis-regulatory mutations associated with miRNAs recurrently predict 12 mature miRNAs (derived from 7 precursors) associated with the deregulation of their target gene networks. The predictions are enriched for cancer-associated protein-coding and miRNA genes and highlight cis-regulatory mutations associated with the dysregulation of key pathways associated with carcinogenesis. By combining transcriptional and post-transcriptional regulation of gene expression, our method predicts cis-regulatory mutations related to the dysregulation of key gene regulatory networks in cancer patients.

Landscape of somatic mutations in 560 breast cancer whole-genome sequences.

We analysed whole-genome sequences of 560 breast cancers to advance understanding of the driver mutations conferring clonal advantage and the mutational processes generating somatic mutations. We found that 93 protein-coding cancer genes carried probable driver mutations. Some non-coding regions exhibited high mutation frequencies, but most have distinctive structural features probably causing elevated mutation rates and do not contain driver mutations. Mutational signature analysis was extended to genome rearrangements and revealed twelve base substitution and six rearrangement signatures. Three rearrangement signatures, characterized by tandem duplications or deletions, appear associated with defective homologous-recombination-based DNA repair: one with deficient BRCA1 function, another with deficient BRCA1 or BRCA2 function, the cause of the third is unknown. This analysis of all classes of somatic mutation across exons, introns and intergenic regions highlights the repertoire of cancer genes and mutational processes operating, and progresses towards a comprehensive account of the somatic genetic basis of breast cancer.

The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups.

The elucidation of breast cancer subgroups and their molecular drivers requires integrated views of the genome and transcriptome from representative numbers of patients. We present an integrated analysis of copy number and gene expression in a discovery and validation set of 997 and 995 primary breast tumours, respectively, with long-term clinical follow-up. Inherited variants (copy number variants and single nucleotide polymorphisms) and acquired somatic copy number aberrations (CNAs) were associated with expression in ~40% of genes, with the landscape dominated by cis- and trans-acting CNAs. By delineating expression outlier genes driven in cis by CNAs, we identified putative cancer genes, including deletions in PPP2R2A, MTAP and MAP2K4. Unsupervised analysis of paired DNA­RNA profiles revealed novel subgroups with distinct clinical outcomes, which reproduced in the validation cohort. These include a high-risk, oestrogen-receptor-positive 11q13/14 cis-acting subgroup and a favourable prognosis subgroup devoid of CNAs. Trans-acting aberration hotspots were found to modulate subgroup-specific gene networks, including a TCR deletion-mediated adaptive immune response in the 'CNA-devoid' subgroup and a basal-specific chromosome 5 deletion-associated mitotic network. Our results provide a novel molecular stratification of the breast cancer population, derived from the impact of somatic CNAs on the transcriptome.

The landscape of cancer genes and mutational processes in breast cancer.

All cancers carry somatic mutations in their genomes. A subset, known as driver mutations, confer clonal selective advantage on cancer cells and are causally implicated in oncogenesis, and the remainder are passenger mutations. The driver mutations and mutational processes operative in breast cancer have not yet been comprehensively explored. Here we examine the genomes of 100 tumours for somatic copy number changes and mutations in the coding exons of protein-coding genes. The number of somatic mutations varied markedly between individual tumours. We found strong correlations between mutation number, age at which cancer was diagnosed and cancer histological grade, and observed multiple mutational signatures, including one present in about ten per cent of tumours characterized by numerous mutations of cytosine at TpC dinucleotides. Driver mutations were identified in several new cancer genes including AKT2, ARID1B, CASP8, CDKN1B, MAP3K1, MAP3K13, NCOR1, SMARCD1 and TBX3. Among the 100 tumours, we found driver mutations in at least 40 cancer genes and 73 different combinations of mutated cancer genes. The results highlight the substantial genetic diversity underlying this common disease.

Assessment of TP53 Polymorphisms and MDM2 SNP309 in Premenopausal Breast Cancer Risk.

Germline polymorphic variants in cancer predisposition genes such as TP53 have been shown to impact the risk of premenopausal cancer. Accordingly, the aim of this study was to assess the spectrum of polymorphisms in TP53 and its negative regulatory gene, MDM2 (SNP309:T>G) in patients with premenopausal breast cancer. Our findings in a cohort of 40 female patients demonstrate no significant correlation between the studied polymorphisms and risk of premenopausal breast cancer. Although one polymorphism is found in high frequency in this cohort (rs1800372:A>G, 9.0%), it was not associated with the risk of developing cancer before the age of 35 years in an extended cohort of 1,420 breast cancer cases. Functional studies of the rs1800372:A>G polymorphic allele reveal that it does not affect p53 transactivation function. Further study of variants or mutations in other cancer susceptibility genes is warranted to refine our understanding of the germline contribution to premenopausal breast cancer susceptibility.

Complex landscapes of somatic rearrangement in human breast cancer genomes.

Multiple somatic rearrangements are often found in cancer genomes; however, the underlying processes of rearrangement and their contribution to cancer development are poorly characterized. Here we use a paired-end sequencing strategy to identify somatic rearrangements in breast cancer genomes. There are more rearrangements in some breast cancers than previously appreciated. Rearrangements are more frequent over gene footprints and most are intrachromosomal. Multiple rearrangement architectures are present, but tandem duplications are particularly common in some cancers, perhaps reflecting a specific defect in DNA maintenance. Short overlapping sequences at most rearrangement junctions indicate that these have been mediated by non-homologous end-joining DNA repair, although varying sequence patterns indicate that multiple processes of this type are operative. Several expressed in-frame fusion genes were identified but none was recurrent. The study provides a new perspective on cancer genomes, highlighting the diversity of somatic rearrangements and their potential contribution to cancer development.

AMG 900, pan-Aurora kinase inhibitor, preferentially inhibits the proliferation of breast cancer cell lines with dysfunctional p53.

Aurora kinases play important roles in cell division and are frequently overexpressed in human cancer. AMG 900 is a novel pan-Aurora kinase inhibitor currently being tested in Phase I clinical trials. We aimed to evaluate the in vitro activity of AMG 900 in a panel of 44 human breast cancer and immortalized cell lines and identify predictors of response. AMG 900 inhibited proliferation at low nanomolar concentrations in all cell lines tested. Response was further classified based on the induction of lethality. 25 cell lines were classified as highly sensitive (lethality at 10 nM of AMG 900 >10 %), 19 cell lines as less sensitive to AMG 900 (lethality at 10 nM of AMG 900 <10 %). Traditional molecular subtypes of breast cancer did not predict for this differential response. There was a weak association between AURKA amplification and response to AMG 900 (response ratio = 2.53, p = 0.09). mRNA expression levels of AURKA, AURKB, and AURKC and baseline protein levels of Aurora kinases A and B did not significantly associate with response. Cell lines with TP53 loss of function mutations (RR = 1.86, p = 0.004) and low baseline p21 protein levels (RR = 2.28, p = 0.0004) were far more likely to be classified as highly sensitive to AMG 900. AMG 900 induced p53 and p21 protein expression in cell lines with wt TP53. AMG 900 caused the accumulation of cells with >4 N DNA content in a majority of cell lines independently of sensitivity and p53 status. AMG 900 induced more pronounced apoptosis in highly sensitive p53-dysfunctional cell lines. We have found that AMG 900 is highly active in breast cancer cell lines and that TP53 loss of function mutations as well as low baseline expression of p21 protein predict strongly for increased sensitivity to this compound in vitro.

Obesity-associated changes in molecular biology of primary breast cancer.

Obesity is associated with an increased risk of developing breast cancer (BC) and worse prognosis in BC patients, yet its impact on BC biology remains understudied in humans. This study investigates how the biology of untreated primary BC differs according to patients' body mass index (BMI) using data from >2,000 patients. We identify several genomic alterations that are differentially prevalent in overweight or obese patients compared to lean patients. We report evidence supporting an ageing accelerating effect of obesity at the genetic level. We show that BMI-associated differences in bulk transcriptomic profile are subtle, while single cell profiling allows detection of more pronounced changes in different cell compartments. These analyses further reveal an elevated and unresolved inflammation of the BC tumor microenvironment associated with obesity, with distinct characteristics contingent on the estrogen receptor status. Collectively, our analyses imply that obesity is associated with an inflammaging-like phenotype. We conclude that patient adiposity may play a significant role in the heterogeneity of BC and should be considered for BC treatment tailoring.

Functional characterization of the 19q12 amplicon in grade III breast cancers.

INTRODUCTION: The 19q12 locus is amplified in a subgroup of oestrogen receptor (ER)-negative grade III breast cancers. This amplicon comprises nine genes, including cyclin E1 (CCNE1), which has been proposed as its 'driver'. The aim of this study was to identify the genes within the 19q12 amplicon whose expression is required for the survival of cancer cells harbouring their amplification. METHODS: We investigated the presence of 19q12 amplification in a series of 313 frozen primary breast cancers and 56 breast cancer cell lines using microarray comparative genomic hybridisation (aCGH). The nine genes mapping to the smallest region of amplification on 19q12 were silenced using RNA interference in phenotypically matched breast cancer cell lines with (MDA-MB-157 and HCC1569) and without (Hs578T, MCF7, MDA-MB-231, ZR75.1, JIMT1 and BT474) amplification of this locus. Genes whose silencing was selectively lethal in amplified cells were taken forward for further validation. The effects of cyclin-dependent kinase 2 (CDK2) silencing and chemical inhibition were tested in cancer cells with and without CCNE1 amplification. RESULTS: 19q12 amplification was identified in 7.8% of ER-negative grade III breast cancer. Of the nine genes mapping to this amplicon, UQCRFS1, POP4, PLEKHF1, C19ORF12, CCNE1 and C19ORF2 were significantly over-expressed when amplified in primary breast cancers and/or breast cancer cell lines. Silencing of POP4, PLEKHF1, CCNE1 and TSZH3 selectively reduced cell viability in cancer cells harbouring their amplification. Cancer cells with CCNE1 amplification were shown to be dependent on CDK2 expression and kinase activity for their survival. CONCLUSIONS: The 19q12 amplicon may harbour more than a single 'driver', given that expression of POP4, PLEKHF1, CCNE1 and TSZH3 is required for the survival of cancer cells displaying their amplification. The observation that cancer cells harbouring CCNE1 gene amplification are sensitive to CDK2 inhibitors provides a rationale for the testing of these chemical inhibitors in a subgroup of patients with ER-negative grade III breast cancers.

Association of the germline TP53 R72P and MDM2 SNP309 variants with breast cancer survival in specific breast tumor subgroups.

The tumor suppressor gene TP53 and its regulator MDM2 are both important players in the DNA-damage repair "TP53 response pathway". Common germline polymorphisms in these genes may affect outcome in patients with tumors characterized by additional somatic changes in the same or a related pathway. To evaluate this hypothesis, we determined the effect of the common germline TP53 R72P and MDM2 SNP309 polymorphisms on breast cancer survival in a consecutive cohort of breast cancer patients (age at diagnosis <53 years, n = 295) with gene expression data available. Patients were classified in subgroups according to their tumor TP53 mutation status and three gene expression profiles; a TP53 mutation status expression signature, a PTEN/PI3K pathway signature and the 70-gene prognosis profile. Survival analyses were performed using Cox regression models adjusting for clinico-pathological characteristics and treatment. An increase in breast cancer-specific mortality was observed for carriers of the germline MDM2 SNP309 rare GG-genotype (range hazard ratios: 2-3) or TP53 R72P heterozygous GC-genotype (range hazard ratios: 1-2) compared to those having the common genotypes within subgroups of tumors displaying a "more aggressive phenotype" gene expression profile. There was no evidence of such an effect on survival within the TP53-mutated tumor group for TP53 R72P carriers but a suggestion of an effect for MDM2 SNP309 carriers (GG vs. TT-genotype HR 2.99, P = 0.06). These results indicate that common polymorphisms in specific pathways may add to the worse prognosis of patients with tumors in which these pathways are affected by somatic alterations.

Epithelial-stromal interaction 1 (EPSTI1) substitutes for peritumoral fibroblasts in the tumor microenvironment.

Tumor cells can activate stroma, yet the implication of this activation in terms of reciprocal induction of gene expression in tumor cells is poorly understood. Epithelial Stromal Interaction 1 (EPSTI1) is an interferon response gene originally isolated from heterotypic recombinant cultures of human breast cancer cells and activated breast myofibroblasts. Here we describe the first immunolocalization of EPSTI1 in normal and cancerous breast tissue, and we provide evidence for a role of this molecule in the regulation of tumor cell properties and epithelial-mesenchymal transition. In general, no EPSTI1 staining was observed in normal breast epithelial cells from reduction mammoplasties (n=25). However, in carcinomas, staining was positive in 22 of 40 biopsies and inversely correlated with the level of differentiation. To address the function of EPSTI1, we expressed EPSTI1 ectopically in one cell line and silenced endogenous EPSTI1 by RNA interference in another. Irrespective of the experimental approach, EPSTI1 expression led to an increase in tumorsphere formation-a property associated with breast stem/progenitor cells. Most remarkably, we show that EPSTI1, by conveying spread of tumor cells, can replace peritumoral activated fibroblasts in a tumor environment assay. These observations implicate EPSTI1 as a hitherto unappreciated regulator of tumor cell properties.

BRCA1-mutated and basal-like breast cancers have similar aCGH profiles and a high incidence of protein truncating TP53 mutations.

BACKGROUND: Basal-like breast cancers (BLBC) are aggressive breast cancers for which, so far, no targeted therapy is available because they typically lack expression of hormone receptors and HER2. Phenotypic features of BLBCs, such as clinical presentation and early age of onset, resemble those of breast tumors from BRCA1-mutation carriers. The genomic instability of BRCA1-mutated tumors can be effectively targeted with DNA-damaging agents and poly-(ADP-ribose) polymerase 1 (PARP1) inhibitors. Molecular similarities between BLBCs and BRCA1-mutated tumors may therefore provide predictive markers for therapeutic response of BLBCs. METHODS: There are several known molecular features characteristic for BRCA1-mutated breast tumors: 1) increased numbers of genomic aberrations, 2) a distinct pattern of genomic aberrations, 3) a high frequency of TP53 mutations and 4) a high incidence of complex, protein-truncating TP53 mutations. We compared the frequency of TP53 mutations and the pattern and amount of genomic aberrations between BRCA1-mutated breast tumors, BLBCs and luminal breast tumors by TP53 gene sequencing and array-based comparative genomics hybridization (aCGH) analysis. RESULTS: We found that the high incidence of protein truncating TP53 mutations and the pattern and amount of genomic aberrations specific for BRCA1-mutated breast tumors are also characteristic for BLBCs and different from luminal breast tumors. CONCLUSIONS: Complex, protein truncating TP53 mutations in BRCA1-mutated tumors may be a direct consequence of genomic instability caused by BRCA1 loss, therefore, the presence of these types of TP53 mutations in sporadic BLBCs might be a hallmark of BRCAness and a potential biomarker for sensitivity to PARP inhibition. Also, our data suggest that a small subset of genomic regions may be used to identify BRCA1-like BLBCs. BLBCs share molecular features that were previously found to be specific for BRCA1-mutated breast tumors. These features might be useful for the identification of tumors with increased sensitivity to (high-dose or dose-dense) alkylating agents and PARP inhibitors.

DNA copy number motifs are strong and independent predictors of survival in breast cancer.

Somatic copy number alterations are a frequent sign of genome instability in cancer. A precise characterization of the genome architecture would reveal underlying instability mechanisms and provide an instrument for outcome prediction and treatment guidance. Here we show that the local spatial behavior of copy number profiles conveys important information about this architecture. Six filters were defined to characterize regional traits in copy number profiles, and the resulting Copy Aberration Regional Mapping Analysis (CARMA) algorithm was applied to tumors in four breast cancer cohorts (n = 2919). The derived motifs represent a layer of information that complements established molecular classifications of breast cancer. A score reflecting presence or absence of motifs provided a highly significant independent prognostic predictor. Results were consistent between cohorts. The nonsite-specific occurrence of the detected patterns suggests that CARMA captures underlying replication and repair defects and could have a future potential in treatment stratification.

EZH2 and BMI1 inversely correlate with prognosis and TP53 mutation in breast cancer.

INTRODUCTION: PolycombGroup (PcG) proteins maintain gene repression through histone modifications and have been implicated in stem cell regulation and cancer. EZH2 is part of Polycomb Repressive Complex 2 (PRC2) and trimethylates H3K27. This histone mark recruits the BMI1-containing PRC1 that silences the genes marked by PRC2. Based on their role in stem cells, EZH2 and BMI1 have been predicted to contribute to a poor outcome for cancer patients. METHODS: We have analysed the expression of EZH2 and BMI1 in a well-characterised dataset of 295 human breast cancer samples. RESULTS: Interestingly, although EZH2 overexpression correlates with a poor prognosis in breast cancer, BMI1 overexpression correlates with a good outcome. Although this may reflect transformation of different cell types, we also observed a functional difference. The PcG-target genes INK4A and ARF are not expressed in tumours with high BMI1, but they are expressed in tumours with EZH2 overexpression. ARF expression results in tumour protein P53 (TP53) activation, and we found a significantly higher proportion of TP53 mutations in tumours with high EZH2. This may explain why tumours with high EZH2 respond poorly to therapy, in contrast to tumours with high BMI1. CONCLUSIONS: Overall, our data highlight that whereas EZH2 and BMI1 may function in a 'linear' pathway in normal development, their overexpression has different functional consequences for breast tumourigenesis.

TP53 mutation status and gene expression profiles are powerful prognostic markers of breast cancer.

INTRODUCTION: Gene expression profiling of breast carcinomas has increased our understanding of the heterogeneous biology of this disease and promises to impact clinical care. The aim of this study was to evaluate the prognostic value of gene expression-based classification along with established prognostic markers and mutation status of the TP53 gene (tumour protein p53) in a group of breast cancer patients with long-term (12 to 16 years) follow-up. METHODS: The clinical and histopathological parameters of 200 breast cancer patients were studied for their effects on clinical outcome using univariate/multivariate Cox regression. The prognostic impact of mutations in the TP53 gene, identified using temporal temperature gradient gel electrophoresis and sequencing, was also evaluated. Eighty of the samples were analyzed for gene expression using 42 K cDNA microarrays and the patients were assigned to five previously defined molecular expression groups. The strength of the gene expression based classification versus standard markers was evaluated by adding this variable to the Cox regression model used to analyze all samples. RESULTS: Both univariate and multivariate analysis showed that TP53 mutation status, tumor size and lymph node status were the strongest predictors of breast cancer survival for the whole group of patients. Analyses of the patients with gene expression data showed that TP53 mutation status, gene expression based classification, tumor size and lymph node status were significant predictors of survival. Breast cancer cases in the 'basal-like' and 'ERBB2+' gene expression subgroups had a very high mortality the first two years, while the 'highly proliferating luminal' cases developed the disease more slowly, showing highest mortality after 5 to 8 years. The TP53 mutation status showed strong association with the 'basal-like' and 'ERBB2+' subgroups, and tumors with mutation had a characteristic gene expression pattern. CONCLUSION: TP53 mutation status and gene-expression based groups are important survival markers of breast cancer, and these molecular markers may provide prognostic information that complements clinical variables. The study adds experience and knowledge to an ongoing characterization and classification of the disease.

Somatic sequence alterations in twenty-one genes selected by expression profile analysis of breast carcinomas.

INTRODUCTION: Genomic alterations have been observed in breast carcinomas that affect the capacity of cells to regulate proliferation, signaling, and metastasis. Re-sequence studies have investigated candidate genes based on prior genetic observations (changes in copy number or regions of genetic instability) or other laboratory observations and have defined critical somatic mutations in genes such as TP53 and PIK3CA. METHODS: We have extended the paradigm and analyzed 21 genes primarily identified by expression profiling studies, which are useful for breast cancer subtyping and prognosis. This study conducted a bidirectional re-sequence analysis of all exons and 5', 3', and evolutionarily conserved regions (spanning more than 16 megabases) in 91 breast tumor samples. RESULTS: Eighty-seven unique somatic alterations were identified in 16 genes. Seventy-eight were single base pair alterations, of which 23 were missense mutations; 55 were distributed across conserved intronic regions or the 5' and 3' regions. There were nine insertion/deletions. Because there is no a priori way to predict whether any one of the identified synonymous and noncoding somatic alterations disrupt function, analysis unique to each gene will be required to establish whether it is a tumor suppressor gene or whether there is no effect. In five genes, no somatic alterations were observed. CONCLUSION: The study confirms the value of re-sequence analysis in cancer gene discovery and underscores the importance of characterizing somatic alterations across genes that are related not only by function, or functional pathways, but also based upon expression patterns.