Identification of cellular and genetic drivers of breast cancer heterogeneity in genetically engineered mouse tumour models
The heterogeneous nature of mammary tumours may arise from different initiating genetic lesions occurring in distinct cells of origin. Here, we generated mice in which Brca2, Pten and p53 were depleted in either basal mammary epithelial cells or luminal oestrogen receptor (ER)‐negative cells. Basal...
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| Published in | The Journal of pathology Vol. 233; no. 2; pp. 124 - 137 |
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| Main Authors | , , , , , , , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
Chichester, UK
John Wiley & Sons, Ltd
01.06.2014
Wiley Subscription Services, Inc |
| Subjects | |
| Online Access | Get full text |
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| Summary: | The heterogeneous nature of mammary tumours may arise from different initiating genetic lesions occurring in distinct cells of origin. Here, we generated mice in which Brca2, Pten and p53 were depleted in either basal mammary epithelial cells or luminal oestrogen receptor (ER)‐negative cells. Basal cell‐origin tumours displayed similar histological phenotypes, regardless of the depleted gene. In contrast, luminal ER‐negative cells gave rise to diverse phenotypes, depending on the initiating lesions, including both ER‐negative and, strikingly, ER‐positive invasive ductal carcinomas. Molecular profiling demonstrated that luminal ER‐negative cell‐origin tumours resembled a range of the molecular subtypes of human breast cancer, including basal‐like, luminal B and ‘normal‐like’. Furthermore, a subset of these tumours resembled the ‘claudin‐low’ tumour subtype. These findings demonstrate that not only do mammary tumour phenotypes depend on the interactions between cell of origin and driver genetic aberrations, but also multiple mammary tumour subtypes, including both ER‐positive and ‐negative disease, can originate from a single epithelial cell type. This is a fundamental advance in our understanding of tumour aetiology. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. |
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| Bibliography: | ArticleID:PATH4345 istex:2F8302F61E9437561F659892FBE7ABDD15D50189 ark:/67375/WNG-G90SCWGD-3 Appendix S1. Supplementary materialExpression of Trp53 and Brca2 in Brca2f/f:p53f/f mouse tumours. (A, B) Trp53 expression in tumour versus matched (A) or average (B) spleens, determined with a probe against (floxed) exon 4. (C) Brca2 expression levels in tumour versus matched spleens determined with probes against exon 6 and (floxed) exon 11. Gene expression levels were determined by qPCR on triplicate samples from each tumour and are shown as mean gene expression levels ± 95% confidence limits, relative to the comparator sample. *No detectable expressionCopy number analysis for floxed versus unfloxed alleles, determined by droplet digital PCR results. (A) Detection of Brca2 alleles in Brca2 p53 tumours. (B) Detection of Trp53 alleles in Brca2:p53 tumours. (C) Detection of the Trp53 alleles in Pten:p53 tumours. Reference copy number value is two copies, as predicted by gene reference (Tfrc) and further confirmed by normal mammary gland (MG) cells. Error bars indicate the Poisson 95% confidence intervals for each copy number determination; x axis crosses y at reference copy number value 2. Note that all tumours consistently show a lower number of copies of the floxed alleles (*) as compared with the unfloxed alleles. Pten analysis could not be performed, due to technical limitations to assess copy number data in the floxed allele (see Supplementary experimental procedures)Expression of Pten and Trp53 in Ptenf/f and Ptenf/f:p53f/f mouse tumours. (A, B) Pten expression levels in tumour versus matched spleens, determined with probes against (floxed) exon 4 and exon 11 in Ptenf/f (A) and Ptenf/f:p53f/f (B) tumours. (C) Trp53 expression in tumour versus matched spleens, determined with a probe against (floxed) exon 4. Gene expression levels were determined by qPCR on triplicate samples from each tumour and are shown as mean gene expression levels ± 95% confidence limits relative to the comparator samplePapillary features in benign and malignant AMEs of Pten tumours. (A-F) Histological features of a benign BlgCre:Ptenf/f papillary adenomyoepithelioma: (A) low-power H&E of benign tumour; (B) high-power H&E showing tumour cells with palisade-like nuclei tumour cells, typical of papillary tumours; (C) K14 staining in basal cells; (D) K18 staining in luminal cells; (E) p63 expression in basal cells; (F) ER expression in neoplastic cells located in the luminal cells of gland-like structures. (G-L) Histological features of a malignant BlgCre:Ptenf/f papillary adenomyoepithelioma: (G) low-power H&E of tumour multifocal necrosis and mixed borders; (H) high-power H&E showing tumour cells with pleomorphic palisade-like nuclei tumour cells; (I) K14 staining in basal cells; (J) K18 staining in luminal cells; (K) p63 expression in basal cells; (L) neoplastic cells in both luminal and basal regions express ER. Bars = (A, B) 1.5 mm; (B-F and H-L) 100 µm. Inset boxes are magnified ×3Features of benign Pten and Pten:p53 tumours and comparison of ER staining in malignant Pten and Pten:p53 tumours. (A-E) Percentage of tumour cells positive for K14 (A), K18 (B), p63 (C) and ER (D) in benign Pten and Pten:p53 tumours; (E) percentage of tumour cells positive for ER in malignant Pten and Pten p53 tumours. (F) Percentage of tumour cells positive for PRA in malignant Pten and Pten:p53 tumours. (G-I) Staining of a BlgCre:Ptenf/f:p53f/f tumour for ER (G), PRA (H) and PRB (I). Bars (G-I) = 50 µm. Insets are magnified ×2.5Co-localization of p63 and ER in malignant but not benign adenomyoepitheliomas. (A) Dual immunofluorescence staining of benign (top, A) and malignant (bottom, B) AME for p63 (green) and ER (red). Sections are counterstained with DAPI. Note co-localization of p63 and ER in the malignant tumour (eg white arrows), while occasional p63+ ER− nuclei (green arrows) and p63−ER+ nuclei (red arrows) demonstrate that the double staining was not due to cross-reactivity of antibodies. Bar = 70 µm. Inset panels magnified ×3Affymetric gene expression analysis is not confounded by batch variation. Unsupervised hierarchical clustering of gene expression data from qPCR analysis of 22 lineage-associated genes (A) and from Affymetrix gene expression data (see Figure 5) of the same 22 genesMouse mammary tumour molecular signatures are differentially correlated with human breast cancer molecular subtypes. (A) Bar plots of the mean expression values for both up- (top) and down-regulated (bottom) mouse gene lists across the human subtypes in the TCGA dataset . Error bars indicate the Poisson 95% confidence intervals (standard t-test in pairwise comparisons). (B) Spearman rank correlation values for each signature plotted against dataset molecular phenotypes from additional human breast cancer datasets . (C) As in (A), but tested in a dataset in which claudin-low tumours have been annotated . (D) As in (B), but again for additional human breast cancer datasets in which claudin-low tumours have been annotated . (E) As in (B), but considering human familial breast cancers datasets . Human samples were BRCA1-mutated, BRCA2-mutated, non-BRCA1/2 mutated and sporadic breast cancer casesMouse mammary metaplastic spindle cell tumours have molecular signatures similar to the human claudin-low breast cancer subtype. From low (left) to high (right) expression, bar plots indicate the average expression of genes in the down-regulated gene set of the human 'claudin-low' breast cancer subtype . Bars are colour-coded by genotype and histological phenotypes are indicated below the x axis. Note that all metaplastic spindle cell tumours in this series (blue boxes) have low expression for genes characteristically down-regulated in human claudin-low tumoursImmunostaining of human tumours. (A-C) CLDN4 staining of: (A) a human triple-negative IDC-NST showing strong staining; (B) a human triple-negative IDC-NST showing reduced staining; and (C) a human metaplastic spindle cell tumour showing absence of CLDN4 staining in spindle cells. (D-L) Analysis of PTEN expression in human histological subtypes: (D) PTEN staining of normal human breast; (E) H&E of benign AME; and (F) PTEN staining of benign AME; (G) H&E of malignant AME; and (H) PTEN staining of malignant AME; (I) triple-negative IDC-NST showing PTEN staining; (J) triple-negative IDC-NST showing absence of PTEN staining in tumour cells; (K) PTEN-positive metaplastic spindle cell tumour; (L) PTEN-negative metaplastic spindle cell tumour. Bars = (A-C) 50 µm; (D-L) 25 µm(A) Primers used for genotyping (B) TaqMan gene expression assaysFull malignant tumour histological features and immunohistochemical findingsFull benign tumour histological features and immunohistochemical findingsGenes up-regulated in tumour molecular clusters determined by SAM pair-wise comparisons and analysed by Gene Ontology (GO) and KEGG Pathway AnalysisDetailed PAM analysis results; correlations for each tumour, comparing tumour molecular profiles human molecular subtypes .Staining of human tumour samples for PTEN, CLDN3, CLDN4 and CDH1 These authors contributed equally to this study. No conflicts of interest were declared. ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 |
| ISSN: | 0022-3417 1096-9896 |
| DOI: | 10.1002/path.4345 |