Foxf1 Expression Research Articles

MiR-182 promotes cell growth and invasion by targeting forkhead box F2 transcription factor in colorectal cancer.

Forkhead box F2 transcription factor (FoxF2) has been described to promote organ development, extracellular matrix (ECM) synthesis and epithelial-mesenchymal interaction. Although recent studies reported decreased FoxF2 expression in several types of cancers, indicating its potential role in carcinogenesis, the mechanistic role of FoxF2 is yet to be explored. MicroRNAs (miRNAs) are strongly implicated in carcinogenesis. The oncogenetic properties of miR-182 have been described in multiple cancers. In the present study, we aimed to investigate the role of miR-182 in colorectal cancer (CRC) and identify the regulation of FoxF2 by miR-182. Bioinformatic analyses on gene expression profiling datasets showed decreased FoxF2 expression in colorectal adenomas, primary tumors compared to normal colon epithelial and a negative association between FoxF2 and β-catenin expression. Restoration of FoxF2 in CRC cells suppressed β-catenin expression and simultaneously inhibited cell growth and invasion. Furthermore, we observed that miR-182 was aberrantly upregulated in CRC. Knockdown of miR-182 in CRC cells impeded cell growth and invasion. The direct binding of miR-182 to the 3' untranslated region (3'UTR) of FoxF2 mRNA was confirmed using a luciferase reporter gene assay. Importantly, elevated FoxF2 expression was observed in miR-182-knockdown cells with a simultaneous reduction in β-catenin. In conclusion, the present study describes a potential mechanism underlying an miR-182/FoxF2 link contributing to CRC development. miR-182-induced downregulation of FoxF2 partly accounts for increased activity of β-catenin signaling. Inhibition of miR-182 represents a potential strategy against CRC.

Forkhead Box F2 Regulation of Platelet-derived Growth Factor and Myocardin/Serum Response Factor Signaling Is Essential for Intestinal Development

Alterations in the forkhead box F2 gene expression have been reported in numerous pathologies, and Foxf2(-/-) mice are perinatal lethal with multiple malformations; however, molecular mechanisms pertaining to Foxf2 signaling are severely lacking. In this study, Foxf2 requirements in murine smooth muscle cells were examined using a conditional knock-out approach. We generated novel Foxf2-floxed mice, which we bred to smMHC-Cre-eGFP mice to generate a mouse line with Foxf2 deleted specifically from smooth muscle. These mice exhibited growth retardation due to reduced intestinal length as well as inflammation and remodeling of the small intestine. Colons of Tg(smMHC-Cre-eGFP(+/-));Foxf2(-/-) mice had expansion of the myenteric nerve plexus and increased proliferation of smooth muscle cells leading to thickening of the longitudinal smooth muscle layer. Foxf2 deficiency in colonic smooth muscle was associated with increased expression of Foxf1, PDGFa, PDGFb, PDGF receptor α, and myocardin. FOXF2 bound to promoter regions of these genes indicating direct transcriptional regulation. Foxf2 repressed Foxf1 promoter activity in co-transfection experiments. We also show that knockdown of Foxf2 in colonic smooth muscle cells in vitro and in transgenic mice increased myocardin/serum response factor signaling and increased expression of contractile proteins. Foxf2 attenuated myocardin/serum response factor signaling in smooth muscle cells through direct binding to the N-terminal region of myocardin. Our results indicate that Foxf2 signaling in smooth muscle cells is essential for intestinal development and serum response factor signaling.

FOXF1 inhibits hematopoietic lineage commitment during early mesoderm specification.

The molecular mechanisms orchestrating early mesoderm specification are still poorly understood. In particular, how alternate cell fate decisions are regulated in nascent mesoderm remains mostly unknown. In the present study, we investigated both in vitro in differentiating embryonic stem cells, and in vivo in gastrulating embryos, the lineage specification of early mesodermal precursors expressing or not the Forkhead transcription factor FOXF1. Our data revealed that FOXF1-expressing mesoderm is derived from FLK1(+) progenitors and that in vitro this transcription factor is expressed in smooth muscle and transiently in endothelial lineages, but not in hematopoietic cells. In gastrulating embryos, FOXF1 marks most extra-embryonic mesoderm derivatives including the chorion, the allantois, the amnion and a subset of endothelial cells. Similarly to the in vitro situation, FOXF1 expression is excluded from the blood islands and blood cells. Further analysis revealed an inverse correlation between hematopoietic potential and FOXF1 expression in vivo with increased commitment toward primitive erythropoiesis in Foxf1-deficient embryos, whereas FOXF1-enforced expression in vitro was shown to repress hematopoiesis. Altogether, our data establish that during gastrulation, FOXF1 marks all posterior primitive streak extra-embryonic mesoderm derivatives with the remarkable exception of the blood lineage. Our study further suggests that this transcription factor is implicated in actively restraining the specification of mesodermal progenitors to hematopoiesis.

Abstract 598: Forkhead transcription factor FOXF1 is a novel p53 target gene and regulates cancer cell migration and invasion

Abstract p53 is an established tumor suppressor that can activate the transcription of multiple target genes. Recent evidence suggests that p53 may contribute to the regulation of cell invasion and migration. In this study, we show that the forkhead box transcription factor FOXF1 is a novel target of p53 because FOXF1 is upregulated by p53. We show that FOXF1 is induced upon DNA damage in a p53-dependent manner. Furthermore, we identified a response element located within the FOXF1 gene that is responsive to wild-type p53, TAp73 as well as TAp63. The ectopic expression of FOXF1 inhibited cancer cell invasion and migration, whereas the inactivation of FOXF1 stimulated cell invasion and migration. We also show that FOXF1 regulates the transcriptional activity of E-cadherin (CDH1) by acting on its FOXF1 consensus binding site located upstream of the E-cadherin gene. Collectively, our results show that FOXF1 is a p53 family target gene, and our data suggest that FOXF1 and p53 form a portion of a regulatory transcriptional network that appears to play an important role in cancer cell invasion and migration. Citation Format: Takashi Tokino, Miyuki Tamura, Masashi Idogawa, Yasushi Sasaki. Forkhead transcription factor FOXF1 is a novel p53 target gene and regulates cancer cell migration and invasion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 598. doi:10.1158/1538-7445.AM2014-598

Exogenous Sonic hedgehog protein does not rescue cultured intestine from atresia formation

BackgroundThe mechanism of intestinal atresia formation remains undefined. Atresia in fibroblast growth factor receptor 2IIIb (Fgfr2IIIb−/−) mutant mouse embryos is preceded by endodermal apoptosis and involution of the surrounding mesoderm. We have observed that involution of the atretic segment is preceded by the downregulation of Sonic hedgehog (SHH) in the endoderm, which is a critical organizer of the intestinal mesoderm. We hypothesized that supplementation of Fgfr2IIIb−/− intestinal tracts with exogenous SHH protein before atresia formation would prevent involution of the mesoderm and rescue normal intestinal development. MethodsIn situ hybridization was performed on control and Fgfr2IIIb−/− intestinal tracts for Shh or forkhead box protein F1 (FoxF1) between embryonic (E) day 11.5 and E12.0. Control and Fgfr2IIIb−/− intestinal tracts were harvested at E10.5 and cultured in media supplemented with fibroblast growth factor (FGF) 10 + SHH, or FGF10 with a SHH-coated bead. In situ hybridization was performed at E12.5 for Foxf1. ResultsSHH and Foxf1 expression were downregulated during intestinal atresia formation. Media containing exogenous FGF10 + SHH did not prevent colonic atresia formation (involution). A SHH protein point source bead did induce Foxf1 expression in controls and mutants. ConclusionsShh and Foxf1 expression are disrupted in atresia formation of distal colon, thereby serving as potential markers of atretic events. Application of exogenous SHH (in media supplement or as a point source bead) is sufficient to induce Foxf1 expression, but insufficient to rescue development of distal colonic mesoderm in Fgfr2IIIb−/− mutant embryos. Shh signal disruption is not the critical mechanism by which loss of Fgfr2IIIb function results in atresia formation.

Noncoding RNAs in cardiovascular biology and disease.

The genome continues to fascinate the enthusiasts, and the captivation seems unabating because of the continuous stream of new discoveries. What was once considered a junk DNA has now emerged to contain a large number of important regulatory elements.1 As an example of such discoveries is the recent finding that the genome contains ≈6200 enhancer elements that are operational in the human fetal and adult hearts.2 Genes occupy only ≈1.5% and coding exons only ≈1% of the genome, which make up only ≈60 million nucleotides of the 6.4 billion nucleotides (3.2 billion base pairs) in the genome.3 Yet, ≈5% of the human genome has undergone purifying selection and hence are likely functional.4 It is intriguing that about two third of the evolutionary constrained genomic elements are located in introns and the intergenic regions, suggestive of their regulatory roles in the genome.4 These conserved regions are enriched in loci that have been found to be associated with clinical phenotypes in the genome-wide association studies.4 The initial findings of the ENCODE (Encyclopedia of DNA Elements) project, although preliminary, illustrate the presence of numerous regulatory elements in the genome, including the enhancers.1 The discoveries largely made possible by the recent advances in the high-throughput RNA sequencing point to enormous RNA splicing diversity and the plethora of alternative splicing variants. Approximately 95% of the multiexon genes undergo alternative splicing, resulting in ≈100 000 abundant splice variants in various tissues.5 Moreover, it seems that almost all genomic regions in 1 form or shape are transcribed, which seems perplexing as only ≈1% of the genome codes for proteins, as has been understood to date. Only recently we have started to appreciate the diverse biological functions of these nonprotein coding transcripts, which are referred to as noncoding RNAs (ncRNAs). …

Forkhead transcription factor FOXF1 is a novel target gene of the p53 family and regulates cancer cell migration and invasiveness

p53 is an established tumor suppressor that can activate the transcription of multiple target genes. Recent evidence suggests that p53 may contribute to the regulation of cell invasion and migration. In this study, we show that the forkhead box transcription factor FOXF1 is a novel target of the p53 family because FOXF1 is upregulated by p53, TAp73 and TAp63. We show that FOXF1 is induced upon DNA damage in a p53-dependent manner. Furthermore, we identified a response element located within the FOXF1 gene that is responsive to wild-type p53, TAp73β and TAp63γ. The ectopic expression of FOXF1 inhibited cancer cell invasion and migration, whereas the inactivation of FOXF1 stimulated cell invasion and migration. We also show that FOXF1 regulates the transcriptional activity of E-cadherin (CDH1) by acting on its FOXF1 consensus binding site located upstream of the E-cadherin gene. Collectively, our results show that FOXF1 is a p53 family target gene, and our data suggest that FOXF1 and p53 form a portion of a regulatory transcriptional network that appears to have an important role in cancer cell invasion and migration.

Expression of Bmi1, FoxF1, Nanog, and γ-Catenin in Relation to Hedgehog Signaling Pathway in Human Non-small-Cell Lung Cancer

Hedgehog signaling is known to be involved in both lung organogenesis and lung carcinogenesis. The aim of this study was to examine potential downstream targets of the hedgehog signaling pathway in non-small-cell lung cancer. Protein expression of Bmi1, FoxF1, Nanog, and γ-catenin was examined by immunohistochemistry in 80 non-small-cell lung cancer samples. Correlations with the previously immunohistochemically recovered results for sonic hedgehog, Ptch1, Smo, Gli1, and Gli2 in the same cohort of tumors as well as the clinicopathological characteristics of the tumors were also evaluated. Bmi1 was expressed in 78/80 (97.5%) cases of non-small-cell lung cancer and correlated with male gender and expression of Gli1. Positive expression of FoxF1 was found in 62/80 (77.5%) cases. Expression of FoxF1 correlated with lymph node metastases, Bmi1, and hedgehog pathway activation. Overexpression of Nanog was also noted in 74/80 (92.5%) tumors and correlated with Bmi1. Cytoplasmic accumulation of γ-catenin was observed in 85% (68/80) of the tumors and correlated with the expression of Bmi1, FoxF1, and Nanog. Several developmental pathways seem to be implicated in non-small-cell lung cancer. It is also suggested that Bmi1 and FoxF1 may cooperate with hedgehog signaling in non-small-cell lung carcinogenesis.

Impaired hepatocyte maturation, abnormal expression of biliary transcription factors and liver fibrosis in C/EBPα(Cebpa)-knockout mice.

Inactivation of the C/EBPα gene (Cebpa) in the mouse not only causes impaired hepatocyte maturation, but also induces pseudoglandular structures in the liver parenchyma. The present study was undertaken to determine how the expression of other transcription factors controlling differentiation into hepatocytes and biliary epithelial cells is affected, and how the hepatic architecture, including the bile and vascular systems, is disordered in the fetal knockout liver. Histochemical analyses demonstrated that the expression of HNF1α and HNF4α was heterogeneous in the knockout liver, and that not all parenchymal cells (pseudoglandular) expressed these transcription factors, whereas parenchymal cells in the wild-type liver homogeneously expressed these transcription factors. SOX9, which was expressed only in biliary cells in the wild-type liver, was detectable in many pseudoglandular cells of the knockout liver. Although the pseudoglandular cells often coexpressed SOX9 and HNF1α/HNF4α, cells expressing SOX9 but not expressing HNF1α/HNF4α (biliary cells) were sometimes detectable in the parenchyma. Periportal biliary structures were abnormal in their segregation from the parenchyma and in their expression of the transcription factors and Ep-CAM, a biliary adhesion molecule. These results suggest that the inactivation of the Cebpa gene causes unstable expression of liver-enriched transcription factors or biliary transcription factors and elevated expression of Ep-CAM, which may lead to abnormal biliary morphogenesis in the knockout liver. The impaired maturation of the parenchyma caused elevated expression of PECAM-1, desmin and Foxf1, suggesting that the maturation of the parenchyma plays an important role in the normal histogenesis of nonparenchymal cells (stellate cells and sinusoidal endothelial cells).

Decreased FOXF2 mRNA Expression Indicates Early-Onset Metastasis and Poor Prognosis for Breast Cancer Patients with Histological Grade II Tumor

The transcription factor, FOXF2, plays an important role in tissue development, extracellular matrix synthesis, and epithelial-mesenchymal interactions, implying that it may be associated with the metastatic capabilities of cancer cells. However, the relationship between FOXF2 expression and breast cancer progression, metastasis, and prognosis, remains to be elucidated. In this study, FOXF2 mRNA levels in 305 primary breast cancer tissues were examined using RT-QPCR. Results showed that FOXF2 mRNA levels in primary breast cancer were negatively associated with tumor progression, including tumor size, number of metastatic lymph nodes, and clinical stage. Patients with low FOXF2 mRNA levels had a high risk of relapse and metastasis within three years. Low FOXF2 mRNA levels could predict shorter disease-free survival for those patients with histological grade II and triple-negative breast cancer. Taken together, we conclude that decreased FOXF2 expression indicates the early-onset metastasis and poor prognosis for patients with histological grade II and triple-negative breast cancer.

Abstract A60: Foxf1 transcription factor drives progression of benign lung adenomas to malignant adenocarcinomas

Abstract Lung cancer is the leading cause of cancer-related deaths in the United States with current 5-year survival only 8—12%. Patients with non-small cell lung cancer (NSCLC) develop metastases prior to clinical symptoms, which reduces successful treatment outcomes. The new therapeutic approaches targeting progression and metastasis of NSCLC are needed. Foxf1 transcription factor is expressed in mesenhyme-derived cells, such as fibroblasts, smooth muscle cells and endothelial cells, but absent from epithelial-derived cells, such as alveolar and airway epithelial cells, and benign lung adenoma cells. In present study, we used immunohistochemistry to demonstrate that Foxf1 expression is up-regulated in the invasive front of human lung adenocarcinomas. Foxf1 expression is also induced in mouse lung adenocarcinomas, caused by either activation of K-Ras G12D transgene or transplantation of Lewis Lung Carcinoma (LLC) cells. In vitro, shRNA-mediated depletion of Foxf1 from LLC cells decreased migration, invasion and colony formation of LLC cells on soft agar. Depletion of Foxf1 reduced mRNA and protein levels of Twist1, and prevented TGFbeta-induced EMT. In vivo, Foxf1-depleted LLC cells failed to form lung metastasis in the transplantation model of lung cancer. Furthermore, we generated transgenic mice with Doxycycline-inducible expression of Foxf1 under control of epithelial-specific SPC-promoter (epFoxf1 mice). Lung adenomas in these mice were induced by urethane. Activation of the Foxf1 transgene in pre-existing lung adenomas promoted their progression towards adenocarcinomas that invaded adjacent bronchiolar lumens and pleural surfaces. Transgenic expression of Foxf1 in tumor cells resulted in the loss of cell polarity, decreased E-cadherin, and increased expression of aSMA, Twist1 and other EMT associated genes. Foxf1 directly bound to promoter region of Twist1, implicating the Foxf1 in the regulation of the Twist1 gene. The present study provides the first genetic evidence that aberrant expression of Foxf1 induces EMT in lung tumors and promotes the progression of lung adenomas to invasive adenocarcinomas, suggesting that Foxf1 is a promising target for anti-cancer therapy. Citation Format: Xinjian Wang, David Balli, Tien Le, Vladimir V. Kalinichenko, Tatiana V. Kalin. Foxf1 transcription factor drives progression of benign lung adenomas to malignant adenocarcinomas. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A60.

Foxm1 transcription factor is required for lung fibrosis and epithelial-to-mesenchymal transition

Alveolar epithelial cells (AECs) participate in the pathogenesis of pulmonary fibrosis, producing pro-inflammatory mediators and undergoing epithelial-to-mesenchymal transition (EMT). Herein, we demonstrated the critical role of Forkhead Box M1 (Foxm1) transcription factor in radiation-induced pulmonary fibrosis. Foxm1 was induced in AECs following lung irradiation. Transgenic expression of an activated Foxm1 transcript in AECs enhanced radiation-induced pneumonitis and pulmonary fibrosis, and increased the expression of IL-1β, Ccl2, Cxcl5, Snail1, Zeb1, Zeb2 and Foxf1. Conditional deletion of Foxm1 from respiratory epithelial cells decreased radiation-induced pulmonary fibrosis and prevented the increase in EMT-associated gene expression. siRNA-mediated inhibition of Foxm1 prevented TGF-β-induced EMT in vitro. Foxm1 bound to and increased promoter activity of the Snail1 gene, a critical transcriptional regulator of EMT. Expression of Snail1 restored TGF-β-induced loss of E-cadherin in Foxm1-deficient cells in vitro. Lineage-tracing studies demonstrated that Foxm1 increased EMT during radiation-induced pulmonary fibrosis in vivo. Foxm1 is required for radiation-induced pulmonary fibrosis by enhancing the expression of genes critical for lung inflammation and EMT.

Cytoplasmic mislocalization of overexpressed FOXF1 is associated with the malignancy and metastasis of colorectal adenocarcinomas

Our previous studies have revealed that the human FOXF1 gene, encoding a transcription factor member of the forkhead box (FOX) family, functions as a tumor suppressor and its expression is frequently silenced in breast cancer via DNA hypermethylation. Moreover, we recently reported that FOXF1 expression is preferentially silenced in colorectal cancer cell lines with inactive p53 and knockdown of FOXF1 caused genomic instability in FOXF1-expressing colorectal cancer cells with a defect in the p53-p21WAF1 checkpoint, suggesting that FOXF1 plays a key role in colorectal tumorigenesis. Given that the in vivo role of FOXF1 in colorectal cancer remains unknown, the study here was aimed at delineating the clinical relevance of FOXF1 in colorectal adenocarcinomas. To characterize FOXF1 protein expression in colorectal cancer, designed tissue microarrays, comprising 50 cases of primary colorectal adenocarcinoma paired with matched adjacent normal tissue, were utilized in the immunohistochemistry (IHC) study. The IHC results showed that for adjacent normal colorectal tissue, the FOXF1 protein was only detected in stroma, not in epithelium, with either cytoplasmic staining (70% of total cases) or a mix of cytoplasmic and nuclear staining (6%). In contrast, for colorectal adenocarcinomas, FOXF1 staining was predominately identified in the cytoplasm of tumor epithelial cells (40% of total cases) and tumor-associated stromal cells of some cases (10%) also exhibited FOXF1 positivity in their cytoplasm. Cytoplasmic FOXF1 protein expression in tumor epithelial cells positively correlated with the histologic grade, depth of invasion, stage and lymphatic metastasis of colorectal adenocarcinomas (p<0.05). Moreover, in silico meta-analysis of Oncomine's cancer microarray database indicates that FOXF1 mRNA is overexpressed in a significant subset of colorectal adenocarcinoma tumors compared with normal colorectal tissue and other types of cancers. Our findings for the first time have revealed that the FOXF1 protein is overexpressed as well as mislocalized in cancerous epithelial cells and underexpressed/lost in tumor-associated stromal fibroblasts of colorectal adenocarcinomas, and suggest that FOXF1 is a potential prognostic marker due to its association with the malignancy and metastasis of colorectal cancer.

The p53–p21WAF1 checkpoint pathway plays a protective role in preventing DNA rereplication induced by abrogation of FOXF1 function

We previously identified FOXF1 as a potential tumor suppressor gene with an essential role in preventing DNA rereplication to maintain genomic stability, which is frequently inactivated in breast cancer through the epigenetic mechanism. Here we further addressed the role of the p53–p21WAF1 checkpoint pathway in DNA rereplication induced by silencing of FOXF1. Knockdown of FOXF1 by small interference RNA (siRNA) rendered colorectal p53-null and p21WAF1-null HCT116 cancer cells more susceptible to rereplication and apoptosis than the wild-type parental cells. In parental HCT116 cells with a functional p53 checkpoint, the p53–p21WAF1 checkpoint pathway was activated upon FOXF1 knockdown, which was concurrent with suppression of the CDK2-Rb cascade and induction of G1 arrest. In contrast, these events were not observed in FOXF1-depleted HCT116-p53−/− and HCT116-p21−/− cells, indicating that the p53-dependent checkpoint function is vital for inhibiting CDK2 to induce G1 arrest and protect cells from rereplication. The pharmacologic inhibitor (caffeine) of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) protein kinases abolished activation of the p53–p21WAF1 pathway upon FOXF1 knockdown, suggesting that suppression of FOXF1 function triggered the ATM/ATR-mediated DNA damage response. Cosilencing of p53 by siRNA synergistically enhanced the effect of FOXF1 depletion on the stimulation of DNA rereplication and apoptosis in wild-type HCT116. Finally, we show that FOXF1 expression is predominantly silenced in breast and colorectal cancer cell lines with inactive p53. Our study demonstrated that the p53–p21WAF1 checkpoint pathway is an intrinsically protective mechanism to prevent DNA rereplication induced by silencing of FOXF1.

Endothelial Cell–Specific Deletion of Transcription Factor FoxM1 Increases Urethane-Induced Lung Carcinogenesis

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.

Epigenetic Inactivation of the Potential Tumor Suppressor Gene FOXF1 in Breast Cancer

The expression of several members of the FOX gene family is known to be altered in a variety of cancers. We show that in breast cancer, FOXF1 gene is a target of epigenetic inactivation and that its gene product exhibits tumor-suppressive properties. Loss or downregulation of FOXF1 expression is associated with FOXF1 promoter hypermethylation in breast cancer cell lines and in invasive ductal carcinomas. Methylation of FOXF1 in invasive ductal carcinoma (37.6% of 117 cases) correlated with high tumor grade. Pharmacologic unmasking of epigenetic silencing in breast cancer cells restored FOXF1 expression. Re-expression of FOXF1 in breast cancer cells with epigenetically silenced FOXF1 genes led to G(1) arrest concurrent with or without apoptosis to suppress both in vitro cell growth and in vivo tumor formation. FOXF1-induced G(1) arrest resulted from a blockage at G(1)-S transition of the cell cycle through inhibition of the CDK2-RB-E2F cascade. Small interfering RNA-mediated depletion of FOXF1 in breast cancer cells led to increased DNA re-replication, suggesting that FOXF1 is required for maintaining the stringency of DNA replication and genomic stability. Furthermore, expression profiling of cell cycle regulatory genes showed that abrogation of FOXF1 function resulted in increased expression of E2F-induced genes involved in promoting the progression of S and G(2) phases. Therefore, our studies have identified FOXF1 as a potential tumor suppressor gene that is epigenetically silenced in breast cancer, which plays an essential role in regulating cell cycle progression to maintain genomic stability.

Nuclear Janus-Activated Kinase 2/Nuclear Factor 1-C2 Suppresses Tumorigenesis and Epithelial-to-Mesenchymal Transition by Repressing Forkhead Box F1

Progression to metastasis is the proximal cause of most cancer-related mortality. Yet much remains to be understood about what determines the spread of tumor cells. This paper describes a novel pathway in breast cancer that regulates epithelial-to-mesenchymal transition (EMT), motility, and invasiveness. We identify two transcription factors, nuclear factor 1-C2 (NF1-C2) and Forkhead box F1 (FoxF1), downstream of prolactin/nuclear Janus-activated kinase 2, with opposite effects on these processes. We show that NF1-C2 is lost during mammary tumor progression and is almost invariably absent from lymph node metastases. NF1-C2 levels in primary tumors correlate with better patient survival. Manipulation of NF1-C2 levels by expression of a stabilized version or using small interfering RNA showed that NF1-C2 counteracts EMT, motility, invasiveness, and tumor growth. FoxF1 was found to be a direct repressed target of NF1-C2. We provide the first evidence for a role of FoxF1 in cancer and in the regulation of EMT in cells of epithelial origin. Overexpression of FoxF1 was associated with a mesenchymal phenotype, increased invasiveness in vitro, and enhanced growth of breast carcinoma xenografts in nude mice. The relevance of these findings is strengthened by the correlation between FoxF1 expression and a mesenchymal phenoype in breast cancer cell isolates, consistent with the interpretation that FoxF1 promotes invasion and metastasis.

Identification and gastrointestinal expression of Xenopus laevis FoxF2

FoxF genes are essential for visceral mesoderm development from Drosophila to human. However, part of the difficulty of studying the visceral mesoderm is its relative inaccessibility during early development. Owing to its external development Xenopus laevis presents considerable advantages for the study of visceral mesoderm formation, yet FoxF2 has not been identified in this system. Here, we describe the cloning and expression pattern of XFoxF2 during embryonic development, metamorphosis and adulthood, and compare and contrast it to the expression of FoxF1 in Xenopus laevis and FoxF2 in mouse.

Overexpression of Foxf2 in adipose tissue is associated with lower levels of IRS1 and decreased glucose uptake in vivo

Many members of the forkhead genes family of transcription factors have been implicated as important regulators of metabolism, in particular, glucose homeostasis, e.g., Foxo1, Foxa3, and Foxc2. The purpose of this study was to exploit the possibility that yet unknown members of this gene family play a role in regulating glucose tolerance in adipocytes. We identified Foxf2 in a screen for adipose-expressed forkhead genes. In vivo overexpression of Foxf2 in an adipose tissue-restricted fashion demonstrated that such mice display a significantly induced insulin secretion in response to an intravenous glucose load compared with wild-type littermates. In response to increased Foxf2 expression, insulin receptor substrate 1 (IRS1) mRNA and protein levels are significantly downregulated in adipocytes; however, the ratio of serine vs. tyrosine phosphorylation of IRS1 seems to remain unaffected. Furthermore, adipocytes overexpressing Foxf2 have a significantly lower insulin-mediated glucose uptake compared with wild-type adipocytes. These findings argue that Foxf2 is a previously unrecognized regulator of cellular and systemic whole body glucose tolerance, at least in part, due to lower levels of IRS1. Foxf2 and its downstream target genes can provide new insights with regard to identification of novel therapeutic targets.

The FOXF2 pathway in the human prostate stroma

Forkhead box 2 (FOXF2) is a member of the large family of forkhead transcription factors and its expression pattern suggests a role in prostate cancer development. FOXF2 expression is stroma-specific and higher expressed in the prostate transition zone than the prostate peripheral zone. Moreover, expression of FOXF2 is decreased in prostate cancer. To identify the genes and pathways regulated by FOXF2, we compared microarray expression profiles of primary prostate stromal cells (PrSC) treated with control or small interfering RNA (siRNA) directed against FOXF2. From our microarray analyses, we selected 190 differentially expressed genes, of which 104 genes were higher expressed in PrSC cells treated with FOXF2 siRNA and 86 were higher expressed in PrSC cells treated with negative control siRNA. Eight of the strongest differentially expressed genes were validated by RT-PCR. Genes down-regulated by FOXF2 included MT1E, MT1F, PDGFA, ITGB1, and PSG7 and genes up-regulated by FOXF2 included WASF2, BAMBI, and CXCL12. Ingenuity pathway analysis showed several pathways significantly regulated by FOXF2, including PPAR signaling, PDGF signaling, and extracellular matrix (ECM) signaling. GSEA analysis revealed that FOXF2 up-regulated genes were down-regulated in the same PrSC cells treated with transforming growth factor 3 (TGFbeta3). The distinct expression pattern of FOXF2 in the prostate, its effect on expression of ECM signaling, and its opposing role in the TGFbeta3 pathway, suggests a role for FOXF2 in prostate homeostasis and stroma-epithelial interactions.