Search

The transcription factor Forkhead box M1 (FoxM1) is overexpressed in breast cancers and correlates with poor prognosis. Mechanistically, FoxM1 associates with CBP to activate transcription and with Rb to repress transcription. Although the activating function of FoxM1 in breast cancer has been well documented, the significance of its repressive activity is poorly understood. Using CRISPR–Cas9 engineering, we generated a mouse model that expresses FoxM1-harboring point mutations that block binding to Rb while retaining its ability to bind CBP. Unlike FoxM1-null mice, mice harboring Rb-binding mutant FoxM1 did not exhibit significant developmental defects. The mutant mouse line developed PyMT-driven mammary tumors that were deficient in lung metastasis, which was tumor cell-intrinsic. Single-cell RNA-seq of the tumors revealed a deficiency in prometastatic tumor cells and an expansion of differentiated alveolar type tumor cells, and further investigation identified that loss of the FoxM1/Rb interaction caused enhancement of the mammary alveolar differentiation program. The FoxM1 mutant tumors also showed increased Pten expression, and FoxM1/Rb was found to activate Akt signaling by repressing Pten. In human breast cancers, expression of FoxM1 negatively correlated with Pten mRNA. Furthermore, the lack of tumor-infiltrating cells in FoxM1 mutant tumors appeared related to decreases in pro-metastatic tumor cells that express factors required for infiltration. These observations demonstrate that the FoxM1/Rb-regulated transcriptome is critical for the plasticity of breast cancer cells that drive metastasis, identifying a prometastatic role of Rb when bound to FoxM1. Significance: This work provides new insights into how the interaction between FoxM1 and Rb facilitates the evolution of metastatic breast cancer cells by altering the transcriptome.

PDF file - 4340K, Differentially expressed genes of enriched GO functional categories.

Supplementary Data from Transcriptional Repression by FoxM1 Suppresses Tumor Differentiation and Promotes Metastasis of Breast Cancer

XLS file - 5815K, Significant DE genes between "L & M" and "H & EH" groups.

PDF file - 1006K, CAF gene signature does not identify prognostic outcome in stage I-II patients.

PDF file - 1180K,CAF sub-culture passages. Comparison between CAF- and NF-derived paracrine pro-migratory effects on colon cancer cells. Analysis of CAFs' pluripotency by osteoblast differentiation.

PDF file - 176K, Supplementary Table S1: Sequence of the primer and conditions for the amplification of each gene. Supplementary Table S2: mRNA expression data of IGFBP3, OAS2, MX1 and ROBO2 in a series of 142 colon cancer patients.

XLS file - 271K, CAF signature genes.

Purpose: Cancer-associated fibroblasts (CAF) actively participate in reciprocal communication with tumor cells and with other cell types in the microenvironment, contributing to a tumor-permissive neighborhood and promoting tumor progression. The aim of this study is the characterization of how CAFs from primary human colon tumors promote migration of colon cancer cells.Experimental design: Primary CAF cultures from 15 primary human colon tumors were established. Their enrichment in CAFs was evaluated by the expression of various epithelial and myofibroblast specific markers. Coculture assays of primary CAFs with different colon tumor cells were performed to evaluate promigratory CAF-derived effects on cancer cells. Gene expression profiles were developed to further investigate CAF characteristics.Results: Coculture assays showed significant differences in fibroblast-derived paracrine promigratory effects on cancer cells. Moreover, the association between CAFs' promigratory effects on cancer cells and classic fibroblast activation or stemness markers was observed. CAF gene expression profiles were analyzed by microarray to identify deregulated genes in different promigratory CAFs. The gene expression signature, derived from the most protumorogenic CAFs, was identified. Interestingly, this “CAF signature” showed a remarkable prognostic value for the clinical outcome of patients with colon cancer. Moreover, this prognostic value was validated in an independent series of 142 patients with colon cancer, by quantitative real-time PCR (qRT-PCR), with a set of four genes included in the “CAF signature.”Conclusions: In summary, these studies show for the first time the heterogeneity of primary CAFs' effect on colon cancer cell migration. A CAF gene expression signature able to classify patients with colon cancer into high- and low-risk groups was identified. Clin Cancer Res; 19(21); 5914–26. ©2013 AACR.

The transcription factor Forkhead box M1 (FoxM1) is overexpressed in breast cancers and correlates with poor prognosis. Mechanistically, FoxM1 associates with CBP to activate transcription and with Rb to repress transcription. Although the activating function of FoxM1 in breast cancer has been well documented, the significance of its repressive activity is poorly understood. Using CRISPR–Cas9 engineering, we generated a mouse model that expresses FoxM1-harboring point mutations that block binding to Rb while retaining its ability to bind CBP. Unlike FoxM1-null mice, mice harboring Rb-binding mutant FoxM1 did not exhibit significant developmental defects. The mutant mouse line developed PyMT-driven mammary tumors that were deficient in lung metastasis, which was tumor cell-intrinsic. Single-cell RNA-seq of the tumors revealed a deficiency in prometastatic tumor cells and an expansion of differentiated alveolar type tumor cells, and further investigation identified that loss of the FoxM1/Rb interaction caused enhancement of the mammary alveolar differentiation program. The FoxM1 mutant tumors also showed increased Pten expression, and FoxM1/Rb was found to activate Akt signaling by repressing Pten. In human breast cancers, expression of FoxM1 negatively correlated with Pten mRNA. Furthermore, the lack of tumor-infiltrating cells in FoxM1 mutant tumors appeared related to decreases in pro-metastatic tumor cells that express factors required for infiltration. These observations demonstrate that the FoxM1/Rb-regulated transcriptome is critical for the plasticity of breast cancer cells that drive metastasis, identifying a prometastatic role of Rb when bound to FoxM1.Significance:This work provides new insights into how the interaction between FoxM1 and Rb facilitates the evolution of metastatic breast cancer cells by altering the transcriptome.

XLS file - 3976K, Statistical detail of enrichment analysis of Gene Ontology Biological Process (GOBP) categories.

PDF file - 5471K, Golgi-related gene module expression in colon cancer patients and its prognostic effect.

PDF file - 2087K, Prognostic value confirmation of "CAF signature" included genes.

The canonical role of the transcription factor E2F is to control the expression of cell cycle genes by binding to the E2F sites in their promoters. However, the list of putative E2F target genes is extensive and includes many metabolic genes, yet the significance of E2F in controlling expression of these genes remains largely unknown. Here, we used the CRISPR/Cas9 technology to introduce point mutations in the E2F sites upstream of five endogenous metabolic genes inDrosophila. We found that the impact of these mutations on both the recruitment of E2F and the expression of the target genes varied, with the glycolytic gene,Phosphoglycerate kinase(Pgk), being mostly affected. The loss of E2F regulation onPgkgene led to a decrease in glycolytic flux, TCA cycle intermediates levels, ATP content and an abnormal mitochondrial morphology. Remarkably, chromatin accessibility was significantly reduced at multiple genomic regions inPgkΔE2Fmutants. These regions contained hundreds of genes, including metabolic genes that were downregulated inPgkΔE2Fmutants. Moreover,PgkΔE2Fanimals had shortened life span and exhibited defects in high-energy consuming organs, such as ovaries and muscles. Collectively, our results illustrate how the pleiotropic effects on metabolism, gene expression and development in thePgkΔE2Fanimals underscore the importance of E2F regulation on a single E2F target,Pgk.