Three-dimensional modelling identifies novel genetic dependencies associated with breast cancer progression in the isogenic MCF10 model
The initiation and progression of breast cancer from the transformation of the normal epithelium to ductal carcinoma in situ (DCIS) and invasive disease is a complex process involving the acquisition of genetic alterations and changes in gene expression, alongside microenvironmental and recognized h...
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| Vydané v: | The Journal of pathology Ročník 240; číslo 3; s. 315 - 328 |
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| Hlavní autori: | , , , , , , , , , , , , |
| Médium: | Journal Article |
| Jazyk: | English |
| Vydavateľské údaje: |
Chichester, UK
John Wiley & Sons, Ltd
01.11.2016
Wiley Subscription Services, Inc |
| Predmet: | |
| ISSN: | 0022-3417, 1096-9896, 1096-9896 |
| On-line prístup: | Získať plný text |
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| Shrnutí: | The initiation and progression of breast cancer from the transformation of the normal epithelium to ductal carcinoma in situ (DCIS) and invasive disease is a complex process involving the acquisition of genetic alterations and changes in gene expression, alongside microenvironmental and recognized histological alterations. Here, we sought to comprehensively characterise the genomic and transcriptomic features of the MCF10 isogenic model of breast cancer progression, and to functionally validate potential driver alterations in three‐dimensional (3D) spheroids that may provide insights into breast cancer progression, and identify targetable alterations in conditions more similar to those encountered in vivo. We performed whole genome, exome and RNA sequencing of the MCF10 progression series to catalogue the copy number and mutational and transcriptomic landscapes associated with progression. We identified a number of predicted driver mutations (including PIK3CA and TP53) that were acquired during transformation of non‐malignant MCF10A cells to their malignant counterparts that are also present in analysed primary breast cancers from The Cancer Genome Atlas (TCGA). Acquisition of genomic alterations identified MYC amplification and previously undescribed RAB3GAP1–HRAS and UBA2–PDCD2L expressed in‐frame fusion genes in malignant cells. Comparison of pathway aberrations associated with progression showed that, when cells are grown as 3D spheroids, they show perturbations of cancer‐relevant pathways. Functional interrogation of the dependency on predicted driver events identified alterations in HRAS, PIK3CA and TP53 that selectively decreased cell growth and were associated with progression from preinvasive to invasive disease only when cells were grown as spheroids. Our results have identified changes in the genomic repertoire in cell lines representative of the stages of breast cancer progression, and demonstrate that genetic dependencies can be uncovered when cells are grown in conditions more like those in vivo. The MCF10 progression series therefore represents a good model with which to dissect potential biomarkers and to evaluate therapeutic targets involved in the progression of breast cancer. © 2016 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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| Bibliografia: | istex:C95E00281AEDA81005F3641F347E84D6D72D1AC0 ArticleID:PATH4778 Supplementary materials and methodsSupplementary figure legendsFigure S1: Variant allele frequency plots of acquired mutations Variant allele frequency (VAF) plots of predicted driver mutations acquired in malignant cells, depicting the cell line (x-axis) and VAF (y-axis). The size of the dots corresponds to the depth of coverage.Figure S2: IGV screenshots of acquired mutations in the MCF10 progression series Screenshots from Integrative Genomics Viewer (IGV) of driver genes acquired from in malignant cells, showing the presence or absence of mutant reads in the cell lines. Reads are sorted according to mutant reads.Figure S3: Copy number alterations in the MCF10 progression series Genome plots showing normalised coverage of reads (y-axis) from whole genome sequencing are plotted according to the genomic position in the genome (x-axis).Figure S4: Focal amplifications in the MCF10 progression series (A) Chromosome 10 and (B) Chromosome 17 plots showing focal amplifications acquired during progression. Normalised coverage (y-axis) is plotted according to genomic position (x-axis)Figure S5: RNAi modulation of the isogenic MCF10 model in cancer cell line spheroids (A) Micrograph images of MCF10DCIS.com cells subsequent to transfection of siRNA. Images were taken at day 7. (B) Heatmap of spheroid growth after siRNA mediated silencing and gene expression on plastic (2D). Relative spheroid growth is measured by the survival fraction of treated cells relative to siControl. (C) Micrograph images of spheroids at day 1, 4 and 7 of the MCF10 progression series, demonstrating good growth kinetics. (D) Images of spheroids stained with H&E and antibodies against Ki67, phosphor-AKT, and TP53.Figure S6: Oligo deconvolution in MCF10Ca1a cells in spheroids and on plastic. (A) Barplots showing relative mRNA levels after gene silencing. Statistically significant alterations in mRNA expression were calculated using Student's t test (*p ≤ 0.05) (B) Barplots of relative cell growth in 2D and as 3D spheroids with individual oligos. Relative spheroid growth is measured by the survival fraction of treated cells relative to siControl. Statistical comparisons were performed using Student's t-test (*p ≤ 0.05)Table S1 Details of primer sequences, siRNA sequences and RT-qPCR assays.Table S2 Mutations identified in the MCF10 progression series.Table S3 Gains and losses and focal amplifications and deletions from GISTIC.Table S4 Expressed fusion genes identified from RNA-sequencing of cell lines grown as spheroids identified by both DeFuse and Chimerascan algorithms.Table S5 Differentially expressed genes between pre-invasive and invasive cells grown on plastic and as spheroids.Table S6 Pathway analysis of differentially expressed genes between pre-invasive and invasive cell lines grown on plastic and as spheroids.Table S7 Summary of siRNA screen of cells grown as spheroids and on plastic. Breast Cancer Now ark:/67375/WNG-QNHCR42H-J ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 Equal contributions. No conflicts of interest were declared. |
| ISSN: | 0022-3417 1096-9896 1096-9896 |
| DOI: | 10.1002/path.4778 |