Endothelial cell-specific deletion of transcription factor FoxM1 increases urethane-induced lung carcinogenesis

Abstract

Vascular endothelial cells provide essential support to the tumor microenvironment, but little is known about the transcriptional control of endothelial functions during tumorigenesis. Here we define a critical role for the Forkhead transcription factor FoxM1 in modulating the development of tumor-associated endothelial cells. Pulmonary tumorigenesis induced by urethane administration was compared in mice genetically deleted for FoxM1 in endothelial cells (enFoxm1(-/-) mice). Notably, lung tumor number and size were increased in enFoxm1(-/-) mice. Increased tumorigenesis was associated with increased proliferation of tumor cells and increased expression of c-Myc and cyclin D1. Furthermore, perivascular infiltration by inflammatory cells was elevated and inflammatory cells in BAL fluid were increased. Expression of Flk-1 (vascular endothelial growth factor receptor 2) and FoxF1, known regulators of pulmonary inflammation, was decreased in enFoxm1(-/-) mice. siRNA-mediated knockdown of FoxM1 in endothelial cells reduced Flk-1 and FoxF1 expression, which was driven by direct transcriptional induction by FoxM1 as target genes. Endothelial specific deletion of FoxM1 in vivo or in vitro also decreased expression of Sfrp1 (secreted frizzled-related protein 1), a known inhibitor of canonical Wnt signaling, in a manner that was associated with increased Wnt signaling. Taken together, our results suggest that endothelial-specific expression of FoxM1 limits lung inflammation and canonical Wnt signaling in lung epithelial cells, thereby restricting lung tumorigenesis.

Figure 1. Deletion of Foxm1 from lung endothelial cells increases tumor formation

A. qRT-PCR analysis of Foxm1 mRNA expression in control Foxm1^fl/fl and enFoxm1^−/− lungs. RNA was isolated from the total lung. β-actin mRNA was used for normalization. B. Increase in the total number of urethane-induced tumors in enFoxm1^−/− mice. enFoxm1^−/− and control Foxm1^fl/fl mice were administered 6 weekly urethane injections and lungs were harvested 30 weeks after initial urethane injection. C. Increased diameter of tumors in enFoxm1^−/− mice. Mean number of tumors per lung (±SE) and mean tumor diameter (±SE) were calculated from n = 12 mouse lungs per group. D. H&E staining demonstrates an increase in the size of lung tumors (Tu) in enFoxm1^−/− mice. Tumors in both enFoxm1^−/− and control mice are TTF1- and SPC-positive, and CCSP-negative. Magnifications: panels D, 100x; insets, 400x. A p value <0.01 is shown with (**).

Figure 2. Increased proliferation of tumor cells in enFoxm1^−/− lungs

enFoxm1^−/− and control Foxm1^fl/fl lungs were harvested 30 weeks after initial urethane injection and used for immunohistochemistry with Ki-67 antibodies or to isolate total lung mRNA for qRT-PCR. A. Increased proliferation of tumor (Tu) cells in enFoxm1^−/− lungs. B. enFoxm1^−/− lungs have increased number of Ki-67-positive tumor cells compared to control mice. Ki-67-positive cells were counted in ten random microscope fields of control or enFoxm1^−/− tumors. Increased mRNA levels of c-Myc (C) and Cyclin D1 (D) in enFoxm1^−/− lungs after urethane treatment was demonstrated by qRT-PCR. β-actin mRNA was used for normalization. Magnifications: panels A, 100x. A p value <0.05 is shown with (*), a p value <0.01 is shown with(**).

Figure 3. Increased perivascular leukocyte infiltration associates with increased tumor sizes in enFoxm1^−/− mice

A. H&E staining of lungs from enFoxm1^−/− and control Foxm1^fl/fl mice 30 weeks after urethane treatment. Lung inflammation was not observed in untreated enFoxm1 ^−/− or control mice. B-C. Increased infiltration of inflammatory cells around bigger tumors (Tu) and vessels in enFoxm1^−/− lungs 30 weeks after urethane treatment. Perivascular infiltration of inflammatory cells is shown with arrows. Higher magnification of the representative vessels from control and enFoxm1^−/− lungs shown in bottom panels. V – blood vessel. Magnifications: panels A-C, 100x; bottom panels in C, 1000x. D. Percentage of vessels exhibiting leukocyte infiltration was determined in ten random microscope fields and presented as mean + SD.

Figure 4. Decreased expression of Flk-1 and Foxf1 in enFoxm1^−/− lungs and cultured endothelial cells

A. enFoxm1^−/− mice had decreased Flk1 protein levels in tumors (Tu) compared to control Foxm1^fl/fl tumors (top panels). The decrease in Flk-1 and Foxf1 protein levels in enFoxm1^−/− tumors was specific to endothelial cells (bottom panels). Immunostaining was performed using antibodies against Flk-1 (red) and either endothelial specific vWF or Sox17 (green). Endothelial specific CD34 or Sox17 (green) antibodies were used to co-localize with Foxf1 (red). The nuclei were counterstained with DAPI (blue). Arrowheads indicate endothelial cells. B. enFoxm1^−/− mice showed decreased Flk1 and Foxf1 mRNAs either prior to or after urethane treatment. qRT-PCR was performed using total lung RNA from either untreated mice or mice harvested 30 weeks after urethane treatment. Mouse β-actin mRNA was used for normalization. C. Foxm1 depletion in HMVEC-L cells reduced Flk-1 and Foxf1 mRNA expression. HMVEC-L human endothelial cells were mock transfected (MOCK) or transfected with short interfering RNA (siRNA) duplex specific for Foxm1 mRNA (siFoxm1). Human β-actin mRNA was used for normalization. D. Flk-1 and Foxf1 are direct transcriptional targets of Foxm1. A schematic drawing of promoter regions of the mouse Flk-1 and Foxf1 genes. Locations of potential Foxm1 DNA binding sites are indicated (grey boxes). ChIP assay demonstrated that Foxm1 protein binds to promoter regions of Flk-1 and Foxf1 genes. Foxm1 binding to genomic DNA was normalized to IgG control antibodies. Diminished binding of Foxm1 to the endogenous mouse promoter regions of the Flk-1 and Foxf1 genes was observed after siFoxm1 transfection in MFLM-91U endothelial cells. Luciferase assay demonstrated that Foxm1 induced the transcriptional activity of Flk-1 and Foxf1 promoters. MFLM-91U cells were transfected with CMV-Foxm1b expression vector and luciferase (LUC) reporter driven by either -1.5kb mouse Flk-1 or -2.7kb mouse Foxf1 promoter regions. CMV-empty plasmid was used as a negative control. Cells were harvested at 24 hr after transfection and processed for dual LUC assays to determine LUC activity. Transcriptional activity of the mouse Flk-1 and Foxf1 promoters was increased by CMV-Foxm1b transfection. Magnifications: top panels in A, 100x; middle panels, 400x; bottom panels, 1000x. A p value < 0.05 is shown with asterisk (*).

Figure 5. Canonical Wnt signaling is activated in enFoxm1^−/− lung epithelial cells after urethane treatment

A. Sfrp1 mRNA is decreased in enFoxm1^−/− lungs. B. A schematic drawing of the – 1.0Kb promoter regions of the mouse Sfrp1 gene. Locations of two potential Foxm1 DNA binding sites are indicated (grey boxes). ChIP assay demonstrated that Foxm1 protein binds to promoter of mouse Sfrp1 gene. Foxm1 binding to genomic DNA was normalized to IgG control antibodies. Diminished binding of Foxm1 to the Sfrp1 promoter region was observed after siFoxm1 transfection in MFLM-91U. C. enFoxm1^−/−/TOPGAL mice had increased β-gal activity in epithelial cells compared to Foxm1^fl/fl/TOPGAL mice (top panels). Immunohistochemistry with Ki-67 antibodies onβ-gal stained lung sections shows the presence of Ki-67 protein (arrows) in β-gal-positive cells in enFoxm1^−/− mice (middle panels). Immunohistochemistry with pro-SPC antibodies on β-gal stained lung sections shows that pro-SPC co-localized with β-gal in subset of epithelial cells (arrows in bottom panels:). D. Increased nuclear localization of β-catenin in enFoxm1^−/− tumors. Membrane β-catenin staining in control Foxm1^fl/fl tumors shown with arrows. Magnifications: top panels in C, 100x; inserts, 400x; middle and bottom panels in C and D, 1000x. A p value < 0.05 is shown with asterisk (*), a p value < 0.01 is shown with asterisk (**).

Figure 6. Role of Foxm1 in the cross-talk between endothelial and epithelial cells during lung tumorigenesis

After Foxm1 deletion from endothelial cells, expression of Flk-1, Foxf1 and Sfrp1 is decreased. These endothelial genes are direct Foxm1 targets. Decreased Flk-1 and Foxf1 expression promotes chronic lung inflammation in enFoxm1^−/− lungs. Decreased Sfrp1 and Foxf1 expression causes activation of canonical Wnt signaling in respiratory epithelium. Chronic lung inflammation and Wnt-induced epithelial proliferation promote lung tumorigenesis in enFoxm1^−/− mice.