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

Abstract

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.

Figure 1.. FoxM1DD/DD PYMT+ mouse mammary tumors are deficient in metastasis.

(A) FoxM1 protein expression in MEFs. (B) FoxM1 interaction with CBP and Rb in FoxM1+/DD and FoxM1DD/DD MEFs. IP was performed using FoxM1 antibody. (C) Number of tumor nodules at the end-point of the experiment. (D) Percentage of mice that did not reach the end-point at indicated time point. (E) Quantification showing percentage of mice with visible lung metastatic nodules at the end-point of the experiment. (F) Lungs of FoxM1+/+ PYMT+ and FoxM1DD/DD PYMT+ female mice. Arrows are pointing at metastatic nodules. (G) Metastatic area in the lungs. p value was calculated using Student’s unpaired t test (*p < 0.05).

Figure 2.. Lack of metastasis in FoxM1DD/DD PYMT-driven tumors is tumor cell-intrinsic.

(A) Images of orthotopic tumors and corresponding lungs. (B) Table summarizing incidences of mammary tumors and lung metastasis after orthotopic transplantation. (C-D) Images of lungs (C) and quantification (D) after tail vein injection of FoxM1+/+ or FoxM1DD/DD PYMT+ cells into c57/bl6 recipient female mice. Arrows are pointing at metastatic nodules (E). CD90+CD24+CD45- cancer stem cells were analyzed by FACS. Quantification is shown in the bottom panel. p value was calculated using Student’s unpaired t test.

Figure 3.. Analysis of FoxM1+/+ PYMT+ and FoxM1DD/DD PYMT+ primary mammary tumors using single-cell RNAseq.

(A) UMAP plot of pooled FoxM1+/+ and FoxM1DD/DD cells isolated from primary mammary tumors. (B-C) Dot plots showing expression of epithelial, stromal, immune (B) and metastatic (C) marker genes. (D) UMAP plot of total cells separated by genotype. (E) Pie charts showing percentage of each cell populations in FoxM1+/+ and FoxM1DD/DD PYMT+ tumors.

Figure 4.. FoxM1/Rb interaction is critical for maintenance of poorly-differentiated state of breast cancer cells.

(A) Dot plot showing expression of mammary epithelial lineage markers. (B) Frequency of identified tumor cell lineages. (C) Violin plot showing expression of kappa-casein (Csn3). (D) Validation of differential expression of milk protein mRNAs by RT-PCR. (E) Feature plot showing Gata3 and Elf5 expression. (F) Validation of differential expression of luminal differentiation factors Gata3 and FoxA1 by RT-PCR. p value was calculated using Student’s unpaired t test. ns=nonsignificant, p>0.05, *p<0.05, **p<0.01, ***p<0.001.

Figure 5.. FoxM1/Rb complex is critical for Pten transcriptional repression in mouse and human breast cancers.

(A) Feature plot showing Pten expression. (B) Pten mRNA expression in 3 different pairs of FoxM1+/+ and FoxM1DD/DD PYMT+ primary tumors. (C) Western blot showing protein levels of Pten, Akt and pAktS308 in mammary gland tumors. (D) Quantification of phosphorylated Akt(S308) relative to total pan-Akt in tumor extracts. (E) Pten mRNA expression after FoxM1 silencing in MCF7 cells. (F) Pten mRNA expression after GFP-FoxM1 or GFP-FoxM1DD transient overexpression measured by RT-PCR. (G and H) ChIP assay performed on chromatin extract isolated from MCF7 cells transiently transfected with GFP, GFP-FoxM1 and GFP-FoxM1DD. Pull down was performed with GFP (G) or Rb (H) antibody and was normalized to the value from GFP transfected samples. (I) FoxM1 and Pten inverse correlation in human breast cancer samples. Analysis was performed using cBioportal (http://cbioportal.org). Pearson and Spearman correlations are indicated. p value was calculated using Student’s unpaired t test. **p<0.01, ***p<0.001.

Figure 6.. FoxM1DD/DD PYMT+ mammary gland tumors are deficient in TAMs, neutrophils and endothelial cells.

Immunohistochemical staining of FoxM1+/+ and FoxM1DD/DD PYMT-driven endogenous mammary gland tumors was performed with CD68 (A), CD206 (B), Ly6G (C) or CD31 antibody (D). IHC staining of mammary gland orthotopic tumors was carried out with CD68 (E), CD206 (F), Ly6G (G) or CD31 antibody (H). p value was calculated using Student’s unpaired t test. *p<0.05, **p<0.01.

Figure 7.. FoxM1DD/DD PYMT+ tumors are deficient in tumor cells involved in recruitment of tumor-infiltrating cells.

(A) Dot plot showing expression of Csf1, Ccl2, Mif, Il1β, Vegfα, Pdgfα and Ptges across epithelial cell clusters. (B) Feature plots showing expression of Mfge8, Il1β, Ccl5 and Ccl4 in FoxM1+/+ and FoxM1DD/DD epithelial cells separately. (C-D) Validation of single-cell RNAseq data by RT-PCR. p value was calculated using Student’s unpaired t test. *p<0.05, ***p<0.001.

Figure 8.. Schematic diagram depicting the role of FoxM1/Rb in breast cancer progression and metastasis.

In the breast tumor cells, high FoxM1 expression and intact retinoblastoma protein generate FoxM1/Rb repression complex that plays critical role in accumulation of the poorly differentiated cells, cancer stem-like and pro-metastatic cells. These cells in turn secrete recruitment factors required for establishment of pro-metastatic tumor microenvironment (TME) and subsequent metastasis. If FoxM1/Rb interaction is disrupted, tumors fail to accumulate poorly differentiated and pro-metastatic cells, resulting in reduced expression of chemokines and cytokines required for recruitment of tumor-infiltrating cells, altered TME and decreased metastatic potential.