Lung cancer is the leading cause of deaths in cancer patients in the United States. Identification of new molecular targets is clearly needed to improve therapeutic outcomes of this devastating human disease. Activating mutations in K-Ras oncogene and increased expression of FOXM1 protein are associated with poor prognosis in patients with non-small-cell lung cancer. Transgenic expression of activated KrasG12D in mouse respiratory epithelium is sufficient to induce lung adenocarcinomas; however, transcriptional mechanisms regulated by K-Ras during the initiation of lung cancer remain poorly understood. Foxm1 transcription factor, a downstream target of K-Ras, stimulates cellular proliferation during embryogenesis, organ repair and tumor growth, but its role in tumor initiation is unknown. In the present study, we used transgenic mice expressing KrasG12D under control of Sftpc promoter to demonstrate that Foxm1 was induced in type II epithelial cells before the formation of lung tumors. Conditional deletion of Foxm1 from KrasG12D-expressing respiratory epithelium prevented the initiation of lung tumors in vivo. The loss of Foxm1 inhibited expression of K-Ras target genes critical for the nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) pathways, including Ikbkb, Nfkb1, Nfkb2, Rela, Jnk1, N-Myc, Pttg1 and Cdkn2a. Transgenic overexpression of activated FOXM1 mutant was sufficient to induce expression of these genes in alveolar type II cells. FOXM1 directly bound to promoter regions of Ikbkb, Nfkb2, N-Myc, Pttg1 and Cdkn2a, indicating that these genes are direct FOXM1 targets. FOXM1 is required for K-Ras-mediated lung tumorigenesis by activating genes critical for the NF-κB and JNK pathways.
Lung cancer is the leading cause of deaths in cancer patients worldwide with the current 5-year survival of 8–12%. Existing treatments for lung cancers have not significantly improved patient survival, leading to a critical need for new therapeutic approaches.1 Lung adenocarcinoma, the most common type of non-small-cell lung cancer (NSCLC), exhibits metastases before clinical symptoms become apparent, thus reducing successful treatment options.2 NSCLC arise from pulmonary epithelial cells and are often associated with inactivation of tumor-suppressor genes and activating mutations in K-Ras oncogene. Identification of new molecular targets is clearly needed to improve therapeutic outcomes of this devastating human disease.
Expression levels of FOXM1 protein (also known as HFH-11B, Trident, Win or MPP2) are dramatically increased in NSCLC cancers from mice and humans.3 FOXM1 is a nuclear protein from the Forkhead Box (FOX) family of transcription factors. FOXM1 stimulates cellular proliferation during embryogenesis, organ repair and tumor growth by activating transcription of multiple cell cycle regulatory genes, including Cdc25A, cyclins B1 and A2, Cdc25B, Polo-like and Aurora B kinases.4, 5 FOXM1 is induced before the onset of cellular proliferation and deletion of the Foxm1 gene in transgenic mice inhibits the progression of quiescent cells into the cell cycle (ref). Positive correlation was shown between increased FOXM1 and poor prognosis in NSCLC patients.6 In mice, overexpression of FOXM1 in Rosa26-FOXM1 transgenic mice accelerated proliferation of tumor cells and increased the number and size of lung tumors after treatment with 3-methylcholanthrene/butylated hydroxytoluene.7 Likewise, deletion of Foxm1 caused a significant reduction in numbers and sizes of lung adenomas induced by either 3-methylcholanthrene/butylated hydroxytoluene or urethane.3, 8 Activated ERK and p38, both of which are known targets of the Kras signaling pathway, directly activate the Foxm1b protein in cultured tumor cells and in vivo (ref).
Although these studies demonstrated a critical role of FOXM1 in proliferation of neoplastic cells during lung tumor growth, the role of FOXM1 in tumor initiation is unknown. In the present study, we used transgenic mice expressing KrasG12D under control of Sftpc promoter to examine the kinetic of Foxm1 expression after activation of K-Ras/Raf/ERK signaling pathway in vivo. We demonstrated that Foxm1 is induced in alveolar type II cells before the formation of lung tumors. Conditional deletion of Foxm1 from type II cells prevented the initiation of lung tumors by oncogenic K-RasG12D through inhibition of genes critical for the nuclear factor-κB (NF-κB) and JNK pathways.
Results and Discussion
KrasG12D induces Foxm1 protein in lung tumor cells and alveolar type II cells
Previous studies demonstrated that transgenic overexpression of activated KrasG12D in mouse respiratory epithelium is sufficient to induce lung adenocarcinomas.9, 10, 11 Oncogenic FOXM1 protein is a downstream target of Ras/ERK and Ras/p38 pathways in cultured tumor cells and mouse embryos12, 13, 14, 15 but its role in K-Ras-mediated lung carcinogenesis is unknown. To examine Foxm1 expression during K-Ras-mediated lung tumorigenesis in vivo, transgenic mice that express a mutant KrasG12D transcript under control of Sftpc promoter (SPC-rtTA/TetO-KrasG12D mice,9) were used. Consistent with previous studies,9 doxycycline (Dox)-mediated activation of KrasG12D transgene induced lung adenocarcinomas in a time-dependent manner (Figure 1a). Activation of KrasG12D-induced phosphorylated extracellular signal-regulated kinase (pERK) and Foxm1 proteins in lung tumor and alveolar regions (Figure 1a). Neither pERK nor Foxm1 were found in lungs of mice without Dox (Figure 1a). All lung tumors in SPC-rtTA/TetO-KrasG12D mice contained proSP-C (Supplementary Figure 1), a specific marker for type II alveolar epithelial cells,16 indicating that these tumors were derived from a distal epithelial lineage. Interestingly, both pERK and Foxm1 were increased in type II cells before the formation of lung tumors (Figures 1b and c), implicating Foxm1 in tumor initiation.
Conditional deletion of Foxm1 in respiratory epithelial cells prevents K-Ras-mediated lung tumorigenesis
To determine whether Foxm1 is required for KrasG12D-driven lung tumorigenesis, we generated quadruple transgenic mice containing SPC-rtTA, TetO-KrasG12D and TetO-Cre transgenes as well as the Foxm1-floxed allele (SPC–rtTAtg/−/TetO-Krastg/−/TetO-Cretg/−/Foxm1fl/fl or epKrasG12D/epFoxm1−/− mice). In these mice, Dox induces simultaneous expression of KrasG12D and Cre proteins in alveolar type II cells and airway Clara cells, resulting in Cre-mediated excision of exons 4–7 of the Foxm1 gene that encode DNA binding and transcriptional activation domains of the Foxm1 protein (Figure 2a). Efficient deletion of the Foxm1-floxed allele by the SPC–Cre transgene was shown in our previous studies.8, 16 Mice without Cre (SPC–rtTAtg/−/TetO-Krastg/−/Foxm1fl/fl or epKrasG12D/Foxm1fl/fl mice) were used as controls. After Dox treatment, microCT scan imaging and computer-based three-dimensional reconstruction of whole mouse lungs were performed to determine the number and volume of lung tumors. Activation of KrasG12D-induced lung adenocarcinomas in control epKrasG12D/Foxm1fl/fl mice (Figure 2b), which was confirmed by histological evaluation of these tumors (Figure 2c). Increased expression of pERK and pAKT was observed in tumor cells (Supplementary Figure 3) and was consistent with activation Raf/ERK and PI3K/AKT signaling pathways in KrasG12D-induced tumors.17, 18 In contrast, deletion of Foxm1 from KrasG12D-expressing respiratory epithelium dramatically reduced the number and size of lung tumors (Figures 2b–d). Although approximately 25% of Kras/Foxm1−/− mice still developed single lung tumors (Figure 2d), these tumors were positive for Foxm1 after histological examination (data not shown), indicating that they developed because of incomplete deletion of Foxm1.
Interestingly, deletion of Foxm1 did not protect from KrasG12D-mediated hyperplasia in airway epithelium and terminal bronchioles of Dox-treated epKrasG12D/epFoxm1−/− mice (Figure 2c and Supplementary Figure 2). pERK and pAKT were detected in hyperplastic respiratory epithelium of epKrasG12D/epFoxm1−/− mice (Supplementary Figure 3), a finding consistent with increased K-Ras signaling. Proliferating epithelial cells were found in Foxm1-deficient epithelium as demonstrated by immunostaining for proliferation-specific markers Ki-67 and phosphorylated histone H3 (Supplementary Figure 4). Thus, deletion of Foxm1 does not prevent cellular proliferation in hyperplastic epithelial cells. Altogether, our results suggest that Foxm1 is required for progression of epithelial hyperplasia into the lung tumor by acting downstream of the K-Ras/pERK signaling pathway.
Deletion of Foxm1 prevents K-Ras-mediated activation of genes critical for the JNK and NF-κB signaling pathways
To determine Foxm1 requirements in oncogenic K-Ras signaling, we focused on early events of tumor initiation. epKrasG12D/epFoxm1−/− mice were treated with Dox for 7 days to activate KrasG12D and Cre in the adult lung (Figure 3a). Dox-treated epKrasG12D/Foxm1fl/fl mice and mice without Dox were used as controls. This short Dox exposure induced pERK, pAKT and Foxm1 proteins in type II cells, but was insufficient to induce epithelial hyperplasia or lung tumors in all groups of mice (data not shown). Dox-treated epKrasG12D/epFoxm1−/− lungs displayed an increase in Cre mRNA and a decrease in mRNAs of Foxm1 and its target genes, Cdc25b and Plk1 (Figure 3b), findings consistent with deletion of Foxm1 by the Cre recombinase. To identify K-Ras target genes that are dependent on Foxm1, expression of 25 downstream targets of K-Ras17, 18 was examined by real-time reverse transcription PCR. Consistent with previous studies,17, 19, 20, 21, 22 activation of K-Ras decreased mRNA levels of Cxcr2, IL1b, Saa1, Pparg and Cdkn1a, whereas c-Myc and Zeb2 mRNAs were increased (Figure 3c). Deletion of Foxm1 did not influence mRNA levels of these genes in epKrasG12D/epFoxm1−/− lungs (Figure 3c), indicating that expression of these K-Ras targets is independent of Foxm1. In contrast, deletion of Foxm1 prevented K-Ras-mediated changes in expression of Ttf1, Sftpb, Gpr65, Plunc and Angptl3 (Figure 3d), all of which are critical for proper homeostasis, metabolism and function of lung epithelial and tumor cells.23, 24, 25 Mutations in TTF1 were found in patients with squamous cell carcinoma,26 whereas mutations in Sftpb are associated with chronic interstitial lung disease.27 Gpr65, Plunc and Angptl3 were implicated in tumor growth and lipid metabolism.24, 25, 28 Thus, Foxm1 regulates a subset of K-Ras target genes during tumor initiation.
Consistent with previous studies,29, 30 activation of KrasG12D increased mRNA levels of genes critical for JNK signaling (Figure 3d). Deletion of Foxm1 prevented upregulation of JNK1 in epKrasG12D/epFoxm1−/− lungs (Figure 3d), a result consistent with transcriptional regulation of the Jnk1 promoter by Foxm1.31 mRNAs of JNK1 target genes, including cJun, N-Myc, Atf2, Pttg1, Cdkn2a and Cdh1, were decreased in Foxm1-deficient lungs (Figure 3d), indicating decreased JNK1 signaling. Furthermore, KrasG12D increased mRNA levels of genes critical for NF-κB signaling (Figure 3d), a finding consistent with activation of cell cycle. Deletion of Foxm1 prevented K-Ras-mediated changes in expression of NF-κB-associated genes, such as Ikbkb, Nfkb1, Nfkb2, Rela, IL1rn and Sqstm1 (Figure 3d). As both JNK and NF-κB pathways are required for the initiation of lung tumorigenesis by activated KrasG12D in vivo,30, 32 inhibition of these signaling pathways may contribute to tumor resistance in epKrasG12D/epFoxm1−/− mice. Altogether, our data demonstrate that initiation of lung tumorigenesis by oncogenic KrasG12D requires the Foxm1 transcription factor, which activates genes critical for the JNK and NF-κB signaling pathways.
Overexpression of FOXM1 in alveolar type II cells increases expression of genes critical for the JNK and NF-κB signaling pathways
To determine whether FOXM1 is sufficient to induce genes associated with the JNK and NF-κB pathways, we used double transgenic mice expressing an activated human FOXM1 mutant protein (hFOXM1) under control of Dox-inducible Sftpc promoter (SPC-rtTA/TetO-hFOXM1 mice33). After activation of hFOXM1, alveolar type II cells were purified from transgenic lungs and used to prepare total RNA. Type II cells from Dox-treated TetO-hFOXM1 mice were used as controls. The purity of type II cell populations was 90–95% (data not shown). Transgenic hFOXM1 mRNA was induced in type II cells from SPC-rtTA/TetO-hFOXM1 mice but was absent in controls (Figure 4a), indicating an efficient activation of the hFOXM1 transgene. Plk1 mRNA was increased, whereas mRNAs of endogenous Foxm1 and Cdc25B were not changed (Figure 4a). Consistent with the loss-of-function studies (Figure 3d), activation of hFOXM1 induced expression of genes associated with the JNK and NF-κB pathways, including Jnk1, N-Myc, Ccnd1, Pttg1, Cdkn2a, IL1rn, Ikbkb, Nfkb2 and Rela (Figures 4b and c). Chromatin immunoprecipitation assay demonstrated that the FOXM1 protein directly bound to promoter regions of N-Myc, Pttg1, Cdkn2a, Ikbkb and Nfkb2 genes (Figure 4d), indicating a direct transcriptional regulation. FOXM1 did not bind to the Nfkb1 promoter region (Figure 4d). Thus, FOXM1 overexpression in respiratory epithelial cells is sufficient to induce genes associated with the JNK and NF-κB signaling pathways.
In summary, our data demonstrate that FOXM1 is induced in alveolar type II cells before the formation of lung tumors. Deletion of Foxm1 prevented the initiation of lung tumors by oncogenic K-RasG12D. FOXM1 is critical for K-RasG12D-mediated activation of the NF-κB and JNK signaling pathways in alveolar type II cells. Ikbkb, Nfkb2, Pttg1, N-Myc and Cdkn2a are novel transcriptional targets of FOXM1.
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We thank Ron Pratt for technical support with microCT imaging and Craig Bolte for helpful comments. This work was supported by NIH grants HL84151 (VVK) and CA142724 (TVK), the Career Development Award from National Lung Cancer Partnership (I-CW), research grants from National Science Council of Taiwan 102B0023V8 (I-CW) and Toward World-Class University Project of National Tsing Hua University 102N2052E1 (I-CW).
The authors declare no conflict of interest.
Supplementary Information accompanies this paper on the Oncogene website
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Wang, IC., Ustiyan, V., Zhang, Y. et al. Foxm1 transcription factor is required for the initiation of lung tumorigenesis by oncogenic KrasG12D. Oncogene 33, 5391–5396 (2014). https://doi.org/10.1038/onc.2013.475
- non-small-cell lung cancer
- transgenic mice
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