Foxf1 Expression Research Articles

Abstract 2559: FoxF1 is a potential oncogene in prostate cancer

Abstract Background: FoxF1 belongs to the family of forkhead box transcription factors. Many forkhead box proteins are linked to cancer development and progression. However, the regulation and exact function of FoxF1 remains unclear. The aim of this study was to elucidate how the transcriptional activity of FoxF1 is controlled and to assess the role of FoxF1 within prostate cancer. Design: For assessment of FoxF1 functions tumor cell lines were stably transfected with wild type FoxF1 or mutant FoxF1 constructs lacking putative phosphorylation sites. Protein expression and localization of FoxF1 were analyzed by immunprecipitation and Western Blot. Using tissue microarrays of prostate cancer cohorts we analyzed the protein expression and intracellular localization of FoxF1 in tumors compared to benign tissue. Results: Primary tumors and distant metastases exhibited a significantly higher FoxF1 expression compared to benign prostate tissue. In tumors and metastases nuclear localized FoxF1 was more abundant compared to benign tissue. Transfection of tumor cell lines with FoxF1 followed by immunprecipitation and Western Blot showed that nuclear FoxF1 but not cytoplasmic FoxF1 is phosphorylated in tyrosine residues, conceivably tyrosine-39, as suggested by literature data. We further transfected tumor cell lines with different amino acid exchange mutants of putative phosphorylation sites and analyzed the transcription of the known FoxF1 target gene Pecam-1, revealing a complex pattern of regulation, dependent on the site of phosphorylation. Overexpression of FoxF1 in tumor cell lines resulted in epithelial-mesenchymal transition, as observed by loss of E-cadherin expression, gain of Vimentin expression, the up-regulation of Snail and Twist and changes to a spindle-like morphology. Furthermore, overexpression of FoxF1 led to anchorage-independent growth in soft agar as well as increased migration rates in vitro without affecting the proliferation rate. Transfection of tumor cell lines with FoxF1 followed by immunprecipitation and Western Blot showed that nuclear FoxF1 but not cytoplasmic FoxF1 is phosphorylated in tyrosine residues, conceivably tyrosine-39, as suggested by literature data. We further transfected tumor cell lines with different amino acid exchange mutants of putative phosphorylation sites and analyzed the transcription of the known FoxF1 target gene Pecam-1, revealing a complex pattern of regulation. Conclusion: In summary, our results point to a role of FoxF1 as a potential oncogene in prostate cancer whose nuclear localization and activity is regulated by phosphorylation. Citation Format: Carolin Bossmann, Constanze Merz, Yuri Tolkach, Jessica Carlsson, Sven Perner, Ove Andrén, Glen Kristiansen, Michael Nowak. FoxF1 is a potential oncogene in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2559. doi:10.1158/1538-7445.AM2017-2559

Notochordal and nucleus pulposus marker expression is maintained by sub-populations of adult human nucleus pulposus cells through aging and degeneration

The nucleus pulposus (NP) of the intervertebral disc (IVD) demonstrates substantial changes in cell and matrix composition with both ageing and degeneration. While recent transcriptomic profiling studies have helped define human NP cell phenotype, it remains unclear how expression of these markers is influenced by ageing or degeneration. Furthermore, cells of the NP are thought to derive from the notochord, although adult NP lacks identifiable notochordal (NC) cells. This study aimed to confirm expression of previously identified NP and NC marker genes in adult human NP cells from a range of ages and degenerate states. Importantly, using gene expression analysis (N = 60) and immunohistochemistry (N = 56) the study demonstrates expression of NP markers FoxF1, Pax-1, keratin-8/18, carbonic anhydrase-12, and NC markers brachyury, galectin-3 and CD24 in cells of the NP irrespective of age or degeneration. Our immunohistochemical data, combined with flow cytometry (N = 5) which identified a small number of CA12+Gal3+T+CD24+ cells, suggests the possible presence of a sub-population of cells with an NC-like phenotype in adult NP tissue. These findings suggest that the NP contains a heterogeneous population of cells, which may possess varied phenotypic and functional profiles and thus warrant further investigation to improve our understanding of IVD homeostasis and repair.

CRISPR/Cas9-mediated deletion of lncRNA Gm26878 in the distant Foxf1 enhancer region.

Recent genome editing techniques, including CRISPR mutagenesis screens, offer unparalleled opportunities to study the regulatory non-coding genomic regions, enhancers, promoters, and functional non-coding RNAs. Heterozygous point mutations in FOXF1 and genomic deletion copy-number variants at chromosomal region 16q24.1 involving FOXF1 or its regulatory region mapping ~300kb upstream of FOXF1 and leaving it intact have been identified in the vast majority of patients with a lethal neonatal lung disease, alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Homozygous Foxf1 -/- mice have been shown to die by embryonic day 8.5 because of defects in the development of extraembryonic and lateral mesoderm-derived tissues, whereas heterozygous Foxf1 +/- mice exhibit features resembling ACDMPV. We have previously defined a human lung-specific enhancer region encoding two long non-coding RNAs, LINC01081 and LINC01082, expressed in the lungs. To investigate the biological significance of lncRNAs in the Foxf1 enhancer region, we have generated a CRISPR/Cas9-mediated ~2.4kb deletion involving the entire lncRNA-encoding gene Gm26878, located in the mouse region syntenic with the human Foxf1 upstream enhancer. Very recently, this mouse genomic region has been shown to function as a Foxf1 enhancer. Our results indicate that homozygous loss of Gm26878 is neonatal lethal with low penetrance. No changes in Foxf1 expression were observed, suggesting that the regulation of Foxf1 expression differs between mouse and human.

Human umbilical cord derivatives regenerate intervertebral disc.

Intervertebral disc (IVD) degeneration is characterized by the loss of nucleus pulposus (NP), which is a common cause for lower back pain. Although, currently, there is no cure for the degenerative disc disease, stem cell therapy is increasingly being considered for its treatment. In this study, we investigated the feasibility and efficacy of human umbilical cord mesenchymal stem cells (MSCs) and chondroprogenitor cells (CPCs) derived from those cells to regenerate damaged IVD in a rabbit model. Transplanted cells survived, engrafted and dispersed into NP in situ. Significant improvement in the histology, cellularity, extracellular matrix proteins, and water and glycosaminoglycan contents in IVD recipients of CPCs was observed compared to MSCs. In addition, IVDs receiving CPCs exhibited higher expression of NP-specific human markers, SOX9, aggrecan, collagen 2, FOXF1 and KRT19. The novelty of the study is that in vitro differentiated CPCs derived from umbilical cord MSCs, demonstrated far greater capacity to regenerate damaged IVDs, which provides basis and impetus for stem cell based clinical studies to treat degenerative disc disease. Copyright © 2016 John Wiley & Sons, Ltd.

Sonic hedgehog regulation of Foxf2 promotes cranial neural crest mesenchyme proliferation and is disrupted in cleft lip morphogenesis.

Cleft lip is one of the most common human birth defects, yet our understanding of the mechanisms that regulate lip morphogenesis is limited. Here, we show in mice that sonic hedgehog (Shh)-induced proliferation of cranial neural crest cell (cNCC) mesenchyme is required for upper lip closure. Gene expression profiling revealed a subset of Forkhead box (Fox) genes that are regulated by Shh signaling during lip morphogenesis. During cleft pathogenesis, reduced proliferation in the medial nasal process mesenchyme paralleled the domain of reduced Foxf2 and Gli1 expression. SHH ligand induction of Foxf2 expression was dependent upon Shh pathway effectors in cNCCs, while a functional GLI-binding site was identified downstream of Foxf2 Consistent with the cellular mechanism demonstrated for cleft lip pathogenesis, we found that either SHH ligand addition or FOXF2 overexpression is sufficient to induce cNCC proliferation. Finally, analysis of a large multi-ethnic human population with cleft lip identified clusters of single-nucleotide polymorphisms in FOXF2 These data suggest that direct targeting of Foxf2 by Shh signaling drives cNCC mesenchyme proliferation during upper lip morphogenesis, and that disruption of this sequence results in cleft lip.

MiR-200c inhibits metastasis of breast tumor via the downregulation of Foxf2.

The forkhead box F2 (Foxf2) gene suppresses epithelial-mesenchymal transition via the modulation of transcription of zinc finger E-box-binding homeobox 1 (Zeb1) and epithelial (E)-cadherin, thereby inhibiting tumor metastasis. Additionally, the specific binding of microRNA (miR)-200c to Foxf2 mRNA impedes metastatic pulmonary cancer. However, the role of miR-200c in breast cancer is still unknown. Therefore, in this study, miR-200c mimics were transfected into the highly metastatic breast cancer cell line MDA-MB-231. Their invasion and migration abilities were observed by scratch and transwell migration assays. Real-time PCR was used to detect mRNA levels of Foxf2, Zeb1, and E-cadherin, whereas Foxf2 protein level was determined by western blot analysis. Our results showed that, compared to the control group, miR-200c inhibited the migration or invasion of MDA-MB-231 cells. Real-time PCR and western blot analysis exhibited significant decreases in Foxf2 expression in the presence of miR-200c, along with a decrease in Zeb1 and increase in E-cadherin mRNA expressions. Thus, our preliminary data demonstrated that miR-200c could inhibit the metastasis of breast cancer cells by downregulating Foxf2 expression, providing leads for the discovery of a novel breast cancer treatment.

Antisense lncRNA FOXF1-AS1 Promotes Migration and Invasion of Osteosarcoma Cells Through the FOXF1/MMP-2/-9 Pathway.

Osteosarcoma (OS) is the most common primary malignant bone cancer in children and adolescents. Long non-coding RNAs (lncRNAs) have been shown to play significant role in various cancers, including OS. In a previous study, we have reported that a novel antisense lncRNA FOXF1-AS1, also known as FENDRR, could sensitize doxorubicin-resistance of OS cells through down-regulating ABCB1 and ABCC1. Here in, the critical role of FOXF1-AS1 in regulating OS progression was further investigated. Firstly, we found that FOXF1-AS1 and its antisense transcript FOXF1 expression were positively up-regulated in OS tissues and cell lines and correlated with poor prognosis of OS patients. Besides, FOXF1-AS1 as well as FOXF1 silencing significantly inhibited cell proliferation, migration, invasion of OS cells and tumor growth both in vitro and vivo through decreasing the expression of MMP2 and MMP9, whereas enhanced expression of FOXF1-AS1 had the opposite effects. In addition, mechanistically, both of FOXF1-AS1 and FOXF1 could regulate the expression of MMP2 and MMP9 at mRNA and protein levels, whereas FOXF1-AS1 could influence the FOXF1expression but FOXF1 did not have the same effect on FOXF1-AS1. Rescue assay further showed that FOXF1-AS1 overexpression efficiently reversed the knockdown of MMP2 and MMP9 expression induced by si-FOXF1. Thus, we concluded that FOXF1-AS1 may promote migration and invasion of OS cells through the FOXF1/MMP-2/-9 pathway. Taken together, these findings demonstrated the underlying mechanism of FOXF1-AS1 in the regulation of OS progression and provide a novel potential target in the OS therapy.

Foxf1 and SP-C contains a Cell-Type-Specific Transcriptional Activation Domain and is Expressed in Mouse Lungs

Lung development in the mammal carries series of steps starting from simple lung buds to the elaborately branched organ that fulfil the vital function of gaseous exchange. Strict transcriptional control is essential for lung development. To screen the genes responsible for the regional growth and patterning we used RNA Insitu hybridization to identify genes that show restricted expression patterns in the embryonic lung. During organogenesis, the expression of Foxf1 becomes restricted to the splanchnic mesoderm, which provides the mesenchymal cells of the intestinal tract and gut derivates such as lungs and liver. Surfactant is a lipoprotein substance that is synthesized and secreted by alveolar type II epithelial cells and acts to reduce surface tension at the air-alveolar interface. SP-C a 5 kDa, hydro-phobic, surfactant-associated protein. We used the technique of in situ hybridization to localize SP-C mRNA in mice lung tissue. SP-C mRNA was present in all of the epithelial cells of the prealveolar region of day 13 gestational age mice lung tissue. Our data are suggestive that SP-C is present in fetal lung prealveolar epithelial cells much earlier in gestation than are differentiated, surfactant-producing alveolar type II cells.

Downregulation of Forkhead box F1 gene expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia.

High mortality and morbidity in infants born with congenital diaphragmatic hernia (CDH) are attributed to pulmonary hypoplasia and pulmonary hypertension (PH). Forkhead box (Fox) transcription factors are known to be crucial for cell proliferation and homeostasis. FoxF1 is essential for lung morphogenesis, vascular development, and endothelial proliferation. Mutations in FoxF1 and also the Fox family member FoxC2 have been identified in neonates with PH. In human and experimental models of arterial PH, the Fox protein FoxO1 was found to be downregulated. We hypothesized that Fox expression is altered in the lungs of the nitrofen-induced CDH rat model and investigated the expression of FoxF1, FoxC2, and FoxO1. Following ethical approval (Rec 913b), time-pregnant Sprague-Dawley rats received nitrofen or vehicle on gestational day (D9). Fetuses were sacrificed on D21, inspected for CDH and divided into CDH (n=11) and control group (n=11). Gene expression of FoxF1, FoxC2, and FoxO1 was evaluated with qRT-PCR. Detected alterations of mRNA levels were subsequently assessed on the protein level by performing western blot analysis and laser scanning confocal microscopy. The relative mRNA level of FoxF1 was significantly downregulated in CDH lungs compared to controls (FoxF1 CDH 1.047±0.108, FoxF1 Ctrl 1.419±0.01, p=0.014). Relative mRNA levels of FoxC2 and FoxO1 were not found to be altered between the experimental groups (FoxC2 CDH 30.74±8.925, FoxC2 Ctrl 27.408±7.487, p=0.776; FoxO1 CDH 0.011±0.002, FoxO1 Ctrl 0.011±0.001, p=0.809). On the protein level, western blotting demonstrated a reduced pulmonary protein expression of FoxF1 in CDH lungs. Confocal microscopy showed a markedly diminished expression of FoxF1 in the pulmonary vasculature of CDH lungs compared to controls. Our study demonstrates a strikingly reduced expression of FoxF1 in the pulmonary vasculature of nitrofen-induced CDH. Altered FoxF1 gene expression during embryogenesis may participate in vascular maldevelopment resulting in PH in this animal model.

FoxF1 and FoxF2 transcription factors synergistically promote rhabdomyosarcoma carcinogenesis by repressing transcription of p21Cip1 CDK inhibitor.

The role of Forkhead Box F1 (FoxF1) transcription factor in carcinogenesis is not well characterized. Depending on tissue and histological type of cancer, FoxF1 was shown to be either oncogene or tumor suppressor. Alveolar rhabdomyosarcoma (RMS) is the most aggressive pediatric soft tissue sarcoma. While FoxF1 is highly expressed in alveolar RMS, the functional role of FoxF1 in RMS is unknown. The present study demonstrates that expression of FoxF1 and its closely related transcription factor FoxF2 are essential for rhabdomyosarcoma tumor growth. Depletion of FoxF1 or FoxF2 in rhabdomyosarcoma cells decreased tumor growth in orthotopic mouse models of RMS. The decreased tumorigenesis was associated with the reduced tumor cell proliferation. Cell cycle regulatory proteins Cdk2, Cdk4/6, Cyclin D1 and Cyclin E2 were decreased in FoxF1- and FoxF2-deficient RMS tumors. Depletion of either FoxF1 or FoxF2 delayed G1-S cell cycle progression, decreased levels of phosphorylated Rb and increased protein levels of the CDK inhibitors, p21Cip1 and p27Kip1. Depletion of both FoxF1 and FoxF2 in tumor cells completely abrogated RMS tumor growth in mice. Overexpression of either FoxF1 or FoxF2 in tumor cells was sufficient to increase carcinogenesis in orthotopic RMS mouse model. FoxF1 and FoxF2 directly bound to and repressed transcriptional activity of p21Cip1 promoter through −556/−545 bp region, but did not affect p27Kip1 transcription. Knockdown of p21Cip1 restored cell cycle progression in the FoxF1- or FoxF2-deficient tumor cells. Altogether, FoxF1 and FoxF2 promoted RMS tumorigenesis by inducing tumor cell proliferation via transcriptional repression of p21Cip1 gene promoter. Due to robust oncogenic activity in RMS tumors, FoxF1 and FoxF2 may represent promising targets for anti-tumor therapy.

Abstract 976: Long non-coding RNA Fendrr as biomarker for lung cancer

Abstract Background. Carcinogenesis has been extensively studied at a molecular level however, only since some years ago this research field has been fed by the discovery of a new type of long non-coding RNAs that are able to regulate gene expression and control various cellular mechanisms: Long non-coding RNA (LncRNA). In this study we have focused on the LncRNA Fendrr, which is located on chromosome 16 (16q24.1). This gene produces a long non-coding RNA transcribed bidirectionally with the Forkhead box-F1 (Foxf1) transcription factor on the opposite strand. The distance between Fendrr and FoxF1 is around 1700 bp and it seems to contain the promoter of both genes. In addition, the function of LncRNA Fendrr is currently unknown. There is controversial data about the possible correlation between Fendrr and FoxF1. Szafranski et al (Genome Res. 2013) showed that Fendrr downregulates FoxF1 (by using siRNA against Fendrr). On the other hand, Cabili et al (Genome Biol. 2015) demonstrated that the mRNA of Fendrr and Foxf1 are in the same proportion and localization (nucleus and cytoplasm) and Grote et al (RNA Biol. 2013) indicated that Fendrr interacts with PRC2 complex, which is able to inhibit FoxF1 expression. However, Xu et al (J Hematol Oncol. 2014) showed that, regardless of an overexpression or knockdown of Fenderr, any change in FoxF1 expression was found. Objective. One of the principal aims of this study is to investigate how Fendrr and FoxF1 are silenced in lung cancer and which are their functions. Results. Our preliminary results show that 17 out of 20 (85%) human lung cancer cell lines tested, didn′t express neither Fendrr nor Foxf1. However, to date, it has not been identified any mutation in Fendrr or FoxF1 in these cell lines. In addition, it has been seen that methylation of the promoter and the response of 5-aza is dependent of the p53 status. Moreover, we have observed that ectopic overexpression of LncRNA Fendrr increases the expression of FoxF1 in some lung cancer cell lines and, interestingly, inhibition of FoxF1 gene increases expression of Fendrr. In contrast, inhibition of Fendrr does not seem to produce any change in FoxF1 expression. This could mean that Fendrr regulates FoxF1 expression but requires the help of other components. Our main goal is to investigate whether Fendrr and FoxF1 are silenced in lung cancer cell lines and how demethylating agents can re-express Foxf1 and Fendrr genes. In addition, we will explore the epigenetic patterns of lncRNAs in lung cancer cell lines using sequencing-based methylome data. Conclusion. We demonstrate that Fendrr and FoxF1 are silenced by methylation in p53 mutated human lung cancer cells lines and we also have observed that Fendrr regulates the expression of FoxF1. All there results could support the hypothesis the role of Fendrr in regulating FoxF1 expression, a gene that could be a putative tumor suppressor. Citation Format: Antonio Herrera-Merchan, Marta Cuadros, Sandra Rodriguez, Raul Torres, Esther Farez, Pedro Medina. Long non-coding RNA Fendrr as biomarker for lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 976.

Long non-coding RNA stabilizes the Y-box-binding protein 1 and regulates the epidermal growth factor receptor to promote lung carcinogenesis.

Indoor and outdoor air pollution has been classified as group I carcinogen in humans, but the underlying tumorigenesis remains unclear. Here, we screened for abnormal long noncoding RNAs (lncRNAs) in lung cancers from patients living in Xuanwei city which has the highest lung cancer incidence in China due to smoky coal combustion-generated air pollution. We reported that Xuanwei patients had much more dysregulated lncRNAs than patients from control regions where smoky coal was not used. The lncRNA CAR intergenic 10 (CAR10) was up-regulated in 39/62 (62.9%) of the Xuanwei patients, which was much higher than in patients from control regions (32/86, 37.2%; p=0.002). A multivariate regression analysis showed an association between CAR10 overexpression and air pollution, and a smoky coal combustion-generated carcinogen dibenz[a,h]anthracene up-regulated CAR10 by increasing transcription factor FoxF2 expression. CAR10 bound and stabilized transcription factor Y-box-binding protein 1 (YB-1), leading to up-regulation of the epidermal growth factor receptor (EGFR) and proliferation of lung cancer cells. Knockdown of CAR10 inhibited cell growth in vitro and tumor growth in vivo. These results demonstrate the role of lncRNAs in environmental lung carcinogenesis, and CAR10-YB-1 represents a potential therapeutic target.

A Retinoic Acid-Hedgehog Cascade Coordinates Mesoderm-Inducing Signals and Endoderm Competence during Lung Specification

Organogenesis of the trachea and lungs requires a complex series of mesoderm-endoderm interactions mediated by WNT, BMP, retinoic acid (RA), and hedgehog (Hh), but how these pathways interact ina gene regulatory network is less clear. Using Xenopus embryology, mouse genetics, and human ES cell cultures, we identified a conserved signaling cascade that initiates respiratory lineage specification. We show that RA has multiple roles; first RA pre-patterns the lateral plate mesoderm and then itpromotes Hh ligand expression in the foregut endoderm. Hh subsequently signals back to the pre-patterned mesoderm to promote expression of the lung-inducing ligands Wnt2/2b and Bmp4. Finally, RA regulates the competence of the endoderm to activate the Nkx2-1+ respiratory program in response to these mesodermal WNT and BMP signals. These data provide insights into early lung development and a paradigm for how mesenchymal signals are coordinated with epithelial competence during organogenesis.

Pathogenetics of alveolar capillary dysplasia with misalignment of pulmonary veins.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal lung developmental disorder caused by heterozygous point mutations or genomic deletion copy-number variants (CNVs) of FOXF1 or its upstream enhancer involving fetal lung-expressed long noncoding RNA genes LINC01081 and LINC01082. Using custom-designed array comparative genomic hybridization, Sanger sequencing, whole exome sequencing (WES), and bioinformatic analyses, we studied 22 new unrelated families (20 postnatal and two prenatal) with clinically diagnosed ACDMPV. We describe novel deletion CNVs at the FOXF1 locus in 13 unrelated ACDMPV patients. Together with the previously reported cases, all 31 genomic deletions in 16q24.1, pathogenic for ACDMPV, for which parental origin was determined, arose de novo with 30 of them occurring on the maternally inherited chromosome 16, strongly implicating genomic imprinting of the FOXF1 locus in human lungs. Surprisingly, we have also identified four ACDMPV families with the pathogenic variants in the FOXF1 locus that arose on paternal chromosome 16. Interestingly, a combination of the severe cardiac defects, including hypoplastic left heart, and single umbilical artery were observed only in children with deletion CNVs involving FOXF1 and its upstream enhancer. Our data demonstrate that genomic imprinting at 16q24.1 plays an important role in variable ACDMPV manifestation likely through long-range regulation of FOXF1 expression, and may be also responsible for key phenotypic features of maternal uniparental disomy 16. Moreover, in one family, WES revealed a de novo missense variant in ESRP1, potentially implicating FGF signaling in the etiology of ACDMPV.

Forkhead Box F1 promotes breast cancer cell migration by upregulating lysyl oxidase and suppressing Smad2/3 signaling.

BackgroundEpithelial-mesenchymal transition (EMT) increases cell migration and is implicated in cancer cell invasion and metastasis. We have previously described the involvement of the transcription factors, nuclear factor I-C2 (NFI-C2) and Forkhead box F1 (FoxF1), in the regulation of EMT and invasion during breast tumor progression. NFI-C2 counteracts these processes and FoxF1 is a directly repressed target of NFI-C2. FoxF1 induces EMT and invasiveness and enhances xenograft tumorigenicity in nude mice. Here we identify oppositely regulated targets of NFI-C2 and FoxF1 involved in these processes and further study a possible role for FoxF1 in tumorigenesis.MethodsWe used Affymetrix microarray to detect changes in the transcriptome of a mouse mammary epithelial cell line upon overexpression of NFI-C2 or FoxF1. To elucidate the effects and signaling events following FoxF1 overexpression we investigated in vitro invasion capacity and changes in transcription and protein expression resulting from RNAi and inhibitor treatment.ResultsThe extracellular matrix enzyme lysyl oxidase (LOX) was negatively regulated by NFI-C2 and positively regulated by FoxF1, and upregulation of LOX following FoxF1 overexpression in mouse mammary epithelial cells increased in vitro cell invasion. In the nuclei of FoxF1-overexpressing cells, the phosphorylation of Smad2 decreased, while that of p38 increased. Depletion of LOX by RNAi enhanced phosphorylation of Smad2 by a focal adhesion kinase (FAK)-dependent mechanism. In addition, induced expression of FoxF1 in a non-malignant human mammary epithelial cell line showed that the increase in LOX transcription and the suppression of Smad2 activity are early effects of FoxF1.ConclusionThese data show that FoxF1 enhances invasion in a LOX-dependent manner, is involved in the regulation of Smad2 signaling, and that FoxF1 overexpression ultimately leads to activation of p38 MAPK signaling. These findings provide new insights into the regulation of signaling pathways known to be important during breast tumor progression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2196-2) contains supplementary material, which is available to authorized users.

Differential expression of FOXF1 in bladder cancer metastases and association with established bladder cancer subtypes.

464 Background: FOXF1 is a transcription factor that may act as a tumor suppressor by interaction with p53. We identified FOXF1 to be highly differentially expressed in a discovery set of primary bladder cancers and matched nodal metastases. Here we study FOXF1 in preclinical models and define the association of FOXF1 with clinical bladder cancer phenotypes in several independent cohorts. Methods: Whole genome mRNA gene expression profiling (GEP) using Illumina HT12v3-4 chips was performed on paired primary tumors and nodal metastases from a cystectomy cohort. Among the top differentially expressed genes, FOXF1 was identified as a candidate regulator of cancer cell invasion. Murine orthotopic xenografts were established from human bladder cancer cell lines (UC-3, UC-14), and mRNA expression was assessed by real-time PCR. Four independent clinical cohorts with GEP data were subtyped as luminal, p53-like, or basal according to a validated gene expression signature (Choi et al., Cancer Cell2014). Associations between FOXF1 expression with survival after cystectomy (adjusted Cox models) and bladder cancer subtype (ANOVA) were assessed. High gene expression was defined as expression > median. Results: In the initial discovery set, FOXF1 expression was 3.6-fold lower in nodal metastases than paired primary tumors (n = 33, p < 0.001). Among xenografts with nodal metastases, FOXF1 expression was 1.4-fold lower in the metastases (n = 9, p = 0.0886). In two clinical cohorts (MDA Discovery n = 73 and GSE13507 n = 55), high FOXF1 expression was associated with improved cancer specific survival (HR = 0.35, p = 0.046) and overall survival (HR = 0.45, p = 0.006), respectively. In two other cohorts (MDA Validation n = 57 and GSE32894 n = 93), FOXF1 was not associated with survival after treatment. In all four cohorts, FOXF1 was most highly expressed in tumors subtyped as p53-like (p ≤ 0.0024 for all cohorts). Conclusions: FOXF1 is differentially expressed between primary and metastatic bladder cancer lesions. Across multiple independent cohorts, FOXF1 is more highly expressed among p53-like tumors known to be resistant to neoadjuvant chemotherapy, suggesting that this may be a novel predictive biomarker.

Targeted Resequencing of 29 Candidate Genes and Mouse Expression Studies Implicate ZIC3 and FOXF1 in Human VATER/VACTERL Association.

The VATER/VACTERL association describes the combination of congenital anomalies including vertebral defects, anorectal malformations, cardiac defects, tracheoesophageal fistula with or without esophageal atresia, renal malformations, and limb defects. As mutations in ciliary genes were observed in diseases related to VATER/VACTERL, we performed targeted resequencing of 25 ciliary candidate genes as well as disease-associated genes (FOXF1, HOXD13, PTEN, ZIC3) in 123 patients with VATER/VACTERL or VATER/VACTERL-like phenotype. We detected no biallelic mutation in any of the 25 ciliary candidate genes; however, identified an identical, probably disease-causing ZIC3 missense mutation (p.Gly17Cys) in four patients and a FOXF1 de novo mutation (p.Gly220Cys) in a further patient. In situ hybridization analyses in mouse embryos between E9.5 and E14.5 revealed Zic3 expression in limb and prevertebral structures, and Foxf1 expression in esophageal, tracheal, vertebral, anal, and genital tubercle tissues, hence VATER/VACTERL organ systems. These data provide strong evidence that mutations in ZIC3 or FOXF1 contribute to VATER/VACTERL.

Whole transcriptome expression profiling of mouse limb tendon development by using RNA-seq.

Tendons are fibrous connective tissues that transmit force between muscle and bone. Whereas the molecular and cellular mechanisms of bone and muscle development have been well studied, that of tendon development is poorly understood. Using the Scx-GFP transgenic mice, we isolated GFP(+) cells from the developing mouse limbs at E11.5, E13.5, and E15.5, respectively, and carried out whole transcriptome RNA-seq analysis. Comparing the gene expression profiles of GFP(+) and GFP(-) cells in the E13.5 limb isolated over 1,500 genes that exhibited enrichment of mRNA expression by at least 1.5-fold in the GFP(+) cells. Of these, 778 genes showed expression up-regulated by more than 1.5-fold from E11.5 to E13.5 and 516 genes showed expression up-regulated by more than 1.5-fold from E13.5 to E15.5 in the GFP(+) cell population. Interestingly, over 30 genes encoding transcription factors are among the early-activated genes in the GFP(+) cells. Whole mount and section in situ hybridization analyses showed that many of these transcription factor genes have distinct patterns of expression during limb development and identified Foxf2 expression as a specific marker for differentiated dorsal limb tendon cells. Together, these data provide a valuable resource for further investigation of the molecular mechanisms regulating tendon development.

Loss of FOXF2 Expression Predicts Poor Prognosis in Hepatocellular Carcinoma Patients.

FOXF2 is a member of the forkhead box (FOX) family of transcription factors. FOXF2 plays an important role in several tumors but its expression and role in hepatocellular carcinoma (HCC) remains unknown. Using immunohistochemistry, western blot, and real-time polymerase chain reaction, we analyzed FOXF2 expression in 295 clinicopathologically characterized HCC cases. Using RNA interference (RNAi), we investigated the effects of FOXF2 depletion on tumor cell behavior in vitro. Statistical analyses were used to determine associations between FOXF2 levels, tumor features, and patient outcomes. FOXF2 downregulation was observed in HCC tissues (p < 0.001) compared with peritumorous tissues, and its expression levels were closely correlated with overall survival and recurrence-free survival (p = 0.023 and 0.006, respectively) in patients with HCC. RNAi-mediated silencing of the FOXF2 gene in the MHCC-97H cell line significantly promoted proliferation and anti-apoptosis. The results of the present study indicate that FOXF2 may serve as a prognostic biomarker for HCC and may be a promising target in the treatment of patients with HCC.

The miR-200 family and the miR-183~96~182 cluster target Foxf2 to inhibit invasion and metastasis in lung cancers.

Metastatic lung cancer is one of the most lethal forms of cancer and molecular pathways driving metastasis are still not clearly elucidated. Metastatic cancer cells undergo an epithelial-mesenchymal transition (EMT) where they lose their epithelial properties and acquire a migratory and invasive phenotype. Here we identify that expression of microRNAs from the miR-200 family and the miR-183~96~182 cluster are significantly co-repressed in non-small cell lung cancer (NSCLC) cell lines and primary tumors from multiple TCGA data sets with high EMT scores. Ectopic expression of the miR-183~96~182 cluster inhibited cancer cell migration and invasion, while its expression was tightly modulated by miR-200. We identified Foxf2 as a common, novel and direct target of both these microRNA families. Foxf2 expression tightly correlates with the transcription factor Zeb1 and is elevated in mesenchymal-like metastatic lung cancer cells. Foxf2 expression induced robust EMT, migration, invasion and metastasis in lung cancer cells, whereas Foxf2 inhibition significantly repressed these phenotypes. We also demonstrated that Foxf2 transcriptionally represses E-Cadherin and miR-200, independent of Zeb1, to form a double negative feedback loop. We therefore identified a novel mechanism whereby the miR-200 family and the miR-183~96~182 cluster inhibit lung cancer invasion and metastasis by targeting Foxf2.