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MMTV-cre;Ccn6 knockout mice develop tumors recapitulating human metaplastic breast carcinomas.

ABSTRACT: Metaplastic breast carcinoma is an aggressive form of invasive breast cancer with histological evidence of epithelial to mesenchymal transition (EMT). However, the defining molecular events are unknown. Here we show that CCN6 (WISP3), a secreted matricellular protein of the CCN (CYR61/CTGF/NOV) family, is significantly downregulated in clinical samples of human spindle cell metaplastic breast carcinoma. We generated a mouse model of mammary epithelial-specific Ccn6 deletion by developing a floxed Ccn6 mouse which was bred with an MMTV-Cre mouse. Ccn6fl/fl;MMTV-Cre mice displayed severe defects in ductal branching and abnormal age-related involution compared to littermate controls. Ccn6fl/fl;MMTV-Cre mice developed invasive high grade mammary carcinomas with bona fide EMT, histologically similar to human metaplastic breast carcinomas. Global gene expression profiling of Ccn6fl/fl mammary carcinomas and comparison of orthologous genes with a human metaplastic carcinoma signature revealed a significant overlap of 87 genes (P=5 × 10-11). Among the shared deregulated genes between mouse and human are important regulators of epithelial morphogenesis including Cdh1, Ck19, Cldn3 and 4, Ddr1, and Wnt10a. These results document a causal role for Ccn6 deletion in the pathogenesis of metaplastic carcinomas with histological and molecular similarities with human disease. We provide a platform to study new targets in the diagnosis and treatment of human metaplastic carcinomas, and a new disease relevant model in which to test new treatment strategies.

SUBMITTER: Martin EE

PROVIDER: S-EPMC5398917 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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MMTV-cre;Ccn6 knockout mice develop tumors recapitulating human metaplastic breast carcinomas.

Martin E E EE Huang W W Anwar T T Arellano-Garcia C C Burman B B Guan J-L JL Gonzalez M E ME Kleer C G CG

Oncogene 20161107 16

Metaplastic breast carcinoma is an aggressive form of invasive breast cancer with histological evidence of epithelial to mesenchymal transition (EMT). However, the defining molecular events are unknown. Here we show that CCN6 (WISP3), a secreted matricellular protein of the CCN (CYR61/CTGF/NOV) family, is significantly downregulated in clinical samples of human spindle cell metaplastic breast carcinoma. We generated a mouse model of mammary epithelial-specific Ccn6 deletion by developing a floxe ...[more]

PMID: 27819674

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Project description:BACKGROUND:PIK3CA mutations are frequent in human breast cancer. Pik3caH1047R mutant expression in mouse mammary gland promotes tumorigenesis. TP53 mutations co-occur with PIK3CA mutations in human breast cancers. We previously generated a conditionally activatable Pik3caH1047R;MMTV-Cre mouse model and found a few malignant sarcomatoid (spindle cell) carcinomas that had acquired spontaneous dominant-negative Trp53 mutations. METHODS:A Pik3caH1047R;Trp53R270H;MMTV-Cre double mutant mouse breast cancer model was generated. Tumors were characterized by histology, marker analysis, transcriptional profiling, single-cell RNA-seq, and bioinformatics. Cell lines were developed from mutant tumors and used to identify and confirm genes involved in metastasis. RESULTS:We found Pik3caH1047R and Trp53R270H cooperate in driving oncogenesis in mammary glands leading to a shorter latency than either alone. Double mutant mice develop multiple histologically distinct mammary tumors, including adenocarcinoma and sarcomatoid (spindle cell) carcinoma. We found some tumors to be invasive and a few metastasized to the lung and/or the lymph node. Single-cell RNA-seq analysis of the tumors identified epithelial, stromal, myeloid, and T cell groups. Expression analysis of the metastatic tumors identified S100a4 as a top candidate gene associated with metastasis. Metastatic tumors contained a much higher percentage of epithelial-mesenchymal transition (EMT)-signature positive and S100a4-expressing cells. CRISPR/CAS9-mediated knockout of S100a4 in a metastatic tumor-derived cell line disrupted its metastatic potential indicating a role for S100a4 in metastasis. CONCLUSIONS:Pik3caH1047R;Trp53R270H;MMTV-Cre mouse provides a preclinical model to mimic a subtype of human breast cancers that carry both PIK3CA and TP53 mutations. It also allows for understanding the cooperation between the two mutant genes in tumorigenesis. Our model also provides a system to study metastasis and develop therapeutic strategies for PIK3CA/TP53 double-positive cancers. S100a4 found involved in metastasis in this model can be a potential diagnostic and therapeutic target.

Project description:Metaplastic breast carcinoma is a rare and aggressive histologic type of breast cancer, preferentially displaying a triple-negative phenotype. We sought to define the transcriptomic heterogeneity of metaplastic breast cancers on the basis of current gene expression microarray-based classifiers, and to determine whether these tumors display gene copy number profiles consistent with those of BRCA1-associated breast cancers. Twenty-eight consecutive triple-negative metaplastic breast carcinomas were reviewed, and the metaplastic component present in each frozen specimen was defined (ie, spindle cell, squamous, chondroid metaplasia). RNA and DNA extracted from frozen sections with tumor cell content >60% were subjected to gene expression (Illumina HumanHT-12 v4) and copy number profiling (Affymetrix SNP 6.0), respectively. Using the best practice PAM50/claudin-low microarray-based classifier, all metaplastic breast carcinomas with spindle cell metaplasia were of claudin-low subtype, whereas those with squamous or chondroid metaplasia were preferentially of basal-like subtype. Triple-negative breast cancer subtyping using a dedicated website (http://cbc.mc.vanderbilt.edu/tnbc/) revealed that all metaplastic breast carcinomas with chondroid metaplasia were of mesenchymal-like subtype, spindle cell carcinomas preferentially of unstable or mesenchymal stem-like subtype, and those with squamous metaplasia were of multiple subtypes. None of the cases was classified as immunomodulatory or luminal androgen receptor subtype. Integrative clustering, combining gene expression and gene copy number data, revealed that metaplastic breast carcinomas with spindle cell and chondroid metaplasia were preferentially classified as of integrative clusters 4 and 9, respectively, whereas those with squamous metaplasia were classified into six different clusters. Eight of the 26 metaplastic breast cancers subjected to SNP6 analysis were classified as BRCA1-like. The diversity of histologic features of metaplastic breast carcinomas is reflected at the transcriptomic level, and an association between molecular subtypes and histology was observed. BRCA1-like genomic profiles were found only in a subset (31%) of metaplastic breast cancers, and were not associated with a specific molecular or histologic subtype.

Project description:Metaplastic breast cancers (MBC) are aggressive, chemoresistant tumors characterized by lineage plasticity. To advance understanding of their pathogenesis and relatedness to other breast cancer subtypes, 28 MBCs were compared with common breast cancers using comparative genomic hybridization, transcriptional profiling, and reverse-phase protein arrays and by sequencing for common breast cancer mutations. MBCs showed unique DNA copy number aberrations compared with common breast cancers. PIK3CA mutations were detected in 9 of 19 MBCs (47.4%) versus 80 of 232 hormone receptor-positive cancers (34.5%; P = 0.32), 17 of 75 HER-2-positive samples (22.7%; P = 0.04), 20 of 240 basal-like cancers (8.3%; P < 0.0001), and 0 of 14 claudin-low tumors (P = 0.004). Of 7 phosphatidylinositol 3-kinase/AKT pathway phosphorylation sites, 6 were more highly phosphorylated in MBCs than in other breast tumor subtypes. The majority of MBCs displayed mRNA profiles different from those of the most common, including basal-like cancers. By transcriptional profiling, MBCs and the recently identified claudin-low breast cancer subset constitute related receptor-negative subgroups characterized by low expression of GATA3-regulated genes and of genes responsible for cell-cell adhesion with enrichment for markers linked to stem cell function and epithelial-to-mesenchymal transition (EMT). In contrast to other breast cancers, claudin-low tumors and most MBCs showed a significant similarity to a "tumorigenic" signature defined using CD44(+)/CD24(-) breast tumor-initiating stem cell-like cells. MBCs and claudin-low tumors are thus enriched in EMT and stem cell-like features, and may arise from an earlier, more chemoresistant breast epithelial precursor than basal-like or luminal cancers. PIK3CA mutations, EMT, and stem cell-like characteristics likely contribute to the poor outcomes of MBC and suggest novel therapeutic targets. Comparison of reference samples against treatment

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MMTV-cre;Ccn6 knockout mice develop tumors recapitulating human metaplastic breast carcinomas.

Publications

MMTV-cre;Ccn6 knockout mice develop tumors recapitulating human metaplastic breast carcinomas.

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