FOXC1 Research Articles

Abstract B127: Evaluating PRKACA as a therapeutic target for Fibrolamellar Carcinoma

Abstract Introduction: Fibrolamellar Carcinoma (FLC) is a rare primary liver malignancy, affecting children and young adults without chronic liver disease. FLC tumors are largely resistant to chemotherapy, making the identification of effective treatment options urgently needed. Recent genomic data strongly suggest that DNAJB1-PRKACA kinase fusions are the drivers of the vast majority of FLC cases. However, it has not been assessed whether FLC tumors remain dependent on DNAJB1-PRKACA expression and whether PRKACA inhibition could be a therapeutic approach for FLC. Here we summarize the preclinical evaluation of PRKACA as a potential therapeutic target for FLC. Methods: We established a xenograft model from a FLC- patient and then developed inducible PRKACA shRNA cell lines from this model. We also designed potent tool compounds that selectively inhibit the PRKACA protein to assess PRKACA as a potential therapeutic target for FLC. Results: We characterized a patient-derived xenograft (PDX) model of FLC (LI5132) and confirmed DNAJB1-PRKACA fusion expression and constitutive PRKACA pathway activation measured by phospho-VASP. The model also shows fibrolamellar type histology by H&E staining and expression of typical FLC markers like cytokeratin 7 and CD68 by IHC. Using inducible PRKACA-specific shRNA cell lines from this PDX model we demonstrated that the FLC transcriptional gene signature correlates strongly with expression of the DNAJB1-PRKACAfusion protein. Importantly, we demonstrated for three inducible PRKACA shRNA cell-line-derived xenograft models that the in vivo tumor growth remained dependent on DNAJB1-PRKACA fusion expression (TGI-72%-78%, day 22). PRKACA knockdown tumors displayed reduced Ki67 index (6.4 %) when compared to non-induced controls (37.1 %) further confirming that proliferation of the tumors depends on the fusion expression. To investigate the PRKACA catalytic dependency of the FLC model, we designed potent and selective PRKACA inhibitors based on starting points from our proprietary kinase inhibitor library. These investigational compounds are the first selective and potent PRKACA inhibitors and provide excellent tools to assess in vitro and in vivo PRKACA dependency. These compounds achieved potent PRKACA pathway inhibition and dose-dependent inhibition of FLC-specific gene expression, including genes such as carbamoyl phosphate synthetase (CPS1) and forkhead box C1 (FoxC1). We established a pharmacokinetic/ pharmacodynamic relationship and demonstrated in vivo PRKACA pathway inhibition in PDX tumors, as measured by phospho-VASP. Importantly, oral delivery of a potent and selective PRKACA inhibitor achieved up to 80% PRKACA kinase inhibition and led to statistically significant FLC tumor growth inhibition (54%, day 34) on a tolerated schedule. These data demonstrate that FLC depends on PRKACA kinase activity. Conclusion: This study is the first evaluation of PRKACA kinase inhibition as a therapeutic approach for FLC. The results from these preclinical experiments provide strong evidence that FLC depends on PRKACA catalytic activity and that novel PRKACA inhibitors may significantly decrease tumor growth in vivo. Citation Format: Stefanie S Schalm, Erin O’Hearn, Kevin Wilson, Timothy LaBranche, Grace Silva, Lucian DiPietro, Neil Bifulco, Richard Woessner, Nicolas Stransky, Darshan Sappal, Adam Shutes, Robert Campbell, Riadh Lobbardi, Michael Palmer, Joseph Kim, Stephen Miller, Marion Dorsch, Christoph Lengauer, Timothy Guzi, Vivek Kadambi, Andrew Garner, Klaus P Hoeflich. Evaluating PRKACA as a therapeutic target for Fibrolamellar Carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B127. doi:10.1158/1535-7163.TARG-19-B127

Candidate gene analysis of germline variants in Finnish patients with juvenile open‐angle glaucoma

AbstractPurposeJuvenile open‐angle glaucoma (JOAG) is a rare type of glaucoma diagnosed in children older than 3 years and adults less than 40 years of age. Predisposition to JOAG is generally inherited in an autosomal dominant pattern. Pathogenic MYOC germline variants are reported in 8‐36% of patients. Rarer pathogenic variants in other genes could also predispose to JOAG. Our aim was to detect disease‐associated germline variants in glaucoma candidate genes in Finnish patients with JOAG.MethodsWe studied 46 unrelated patients with JOAG treated between 2010 and 2018 at the Department of Ophthalmology, Helsinki University Hospital, Finland. We first sequenced all exonic regions and flanking splice sites of MYOC using Sanger sequencing. In 42 patients without a pathogenic MYOC germline variant, we then performed whole exome sequencing (WES) to investigate variants in 17 glaucoma‐related genes.ResultsFour probands had a likely pathogenic variant in MYOC: c.1109C>T, p.(Pro370Leu), 1130C>T p.(Thr377Met), 1132G>A p.(Asp378Asn), and c.1204G>A, p.(Val402Ile). Three of these patients had a family history of dominantly inherited JOAG. One patient and his mother with JOAG had a loss‐of‐function variant in the FOXC1 gene, c.G366A, p.(Trp122Ter). One patient with sporadic glaucoma had a homozygous likely pathogenic variant in the LTBP2 gene, c.G3938A, p.(Cys1313Tyr). The frequency of the variant carriers in Finns is 0.03% (the gnomAD database). The frequency of MYOC variants was 9% (4/46; 95% CI, 2‐21). The detected genetic variants explained 13% (6/46; 95% CI 5‐26%) of JOAG in our patient series.ConclusionsGenetic variants in known JOAG‐associated genes seem to be less frequent in Finnish patients, compared to other reported populations. Because of the specific population history of Finland, the genetic background of Finns differs from other European populations. Therefore, it might be possible to identify novel glaucoma genes through our JOAG series in the future.

P38-regulated FOXC1 stability is required for colorectal cancer metastasis.

Aberrant expression of forkhead box C1 (FOXC1) promotes tumor metastasis in multiple human malignant tumors. However, the upstream modulating mode and downstream molecular mechanism of FOXC1 in metastasis of colorectal cancer (CRC) remain unclear. Herein we describe a systematic analysis of FOXC1 expression and prognosis in CRC performed on our clinical data and public databases, which indicated that FOXC1 upregulation in CRC samples was significantly associated with poor prognosis. FOXC1 knockdown inhibited migration and invasion, whereas FOXC1 overexpression caused the opposite phenotype in vitro and in vivo. Furthermore, MMP10, SOX4 and SOX13 were verified as the target genes of FOXC1 for promoting CRC metastasis. MMP10 was demonstrated as the direct target and mediator of FOXC1. Interestingly, Ser241 and Ser272 of FOXC1 were identified as the key sites to interact with p38 and phosphorylation, which were critically required for maintaining the stability of FOXC1 protein. Moreover, FOXC1 was dephosphorylated by protein phosphatase 2A and phosphorylated by p38, which maintained FOXC1 protein stability through inhibiting ubiquitination. Expression of p38 was correlated with FOXC1 and MMP10 expression, indirectly indicating that FOXC1 was regulated by p38 MAPK. Therefore, FOXC1 is strongly suggested as a pro-metastatic gene in CRC by transcriptionally activating MMP10, SOX4 and SOX13; p38 interacts with and phosphorylates the Ser241 and ser272 sites of FOXC1 to maintain its stability by inhibiting ubiquitination and degradation. In conclusion, the protein stability of FOXC1 mediated by p38 contributes to the metastatic effect in CRC. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Forkhead box C1 promotes metastasis and invasion of non-small cell lung cancer by binding directly to the lysyl oxidase promoter.

Increasing evidence indicates that human forkhead box C1 (FOXC1) plays important roles in tumor development and metastasis. However, the underlying molecular mechanism of FOXC1 in non–small cell lung cancer (NSCLC) metastasis remains unclear. Here, we identified FOXC1 as an independent prognostic factor in NSCLC and showed clear biological implications in invasion and metastasis. FOXC1 overexpression enhanced the proliferation, migration and invasion of NSCLC cells, whereas FOXC1 silencing impaired the effects both in vitro and in vivo. Importantly, we found a positive correlation between FOXC1 expression and lysyl oxidase (LOX) expression in NSCLC cells and patient samples. Downregulation of LOX or LOX activity inhibition in NSCLC cells inhibited the FOXC1‐driven effects on cellular migration and invasion. Xenograft models showed that inhibition of LOX activity by β‐aminopropionitrile monofumarate decreased the number of lung metastases. Mechanistically, we demonstrated a novel FOXC1‐LOX mechanism that was involved in the invasion and metastasis of NSCLC. Dual‐luciferase assay and ChIP identified that FOXC1 bound directly in the LOX promoter region and activated its transcription. Collectively, the present study offered new insight into FOXC1 in the mediation of NSCLC metastasis through interaction with the LOX promoter and further revealed that targeted inhibition of LOX protein activity could prevent lung metastasis in murine xenograft models. These data implicated FOXC1 as a potential therapeutic strategy for the treatment of NSCLC metastasis.

FOXC1 Regulation of miR-31-5p Confers Oxaliplatin Resistance by Targeting LATS2 in Colorectal Cancer.

Colorectal cancer (CRC) is the second leading cause of cancer-related illness worldwide and one of the most common malignancies. Therefore, colorectal cancer research and cases have gained increasing attention. Oxaliplatin (OXA) is currently used in first-line chemotherapy to treat stage III and stage IV metastatic CRC. However, patients undergoing chemotherapy often develop resistance to chemo drugs being used. Evidence has confirmed that microRNAs regulate downstream genes in cancer biology and thereby have roles related to tumor growth, proliferation, invasion, angiogenesis, and multi-drug resistance. The aim of our study is to establish whether miR-31-5p is an oncogene in human colorectal cancers that are resistant to OXA and further confirm its malignant phenotype-associated target molecule. From the results of miRNA microarray assay, we establish that miR-31-5p expression was upregulated in oxaliplatin-resistant (OR)-LoVo cells compared with parental LoVo cells. Moreover, through in vitro and in vivo experiments, we demonstrate that miR-31-5p and large tumor suppressor kinase 2 (LATS2) were inversely related and that miR-31-5p and Forkhead box C1 (FOXC1) were positively correlated in the same LoVo or OR-LoVo cells. Importantly, we reveal a novel drug-resistance mechanism in which the transcription factor FOXC1 binds to the miR-31 promoter to increase the expression of miR31-5p and regulate LATS2 expression, resulting in cancer cell resistance to OXA. These results suggest that miR-31-5p may be a novel biomarker involved in drug resistance progression in CRC patients. Moreover, the FOXC1/miR31-5p/LATS2 drug-resistance mechanism provides new treatment strategies for CRC in clinical trials.

FOXC1 up-regulates the expression of toll-like receptors in myocardial ischaemia.

Myocardial ischaemia (MI) remains a major cause of death and disability worldwide. Accumulating evidence suggests a significant role for innate immunity, in which the family of toll‐like receptors (TLRs) acts as an essential player. We previously reported and reviewed the changes of Tlr expression in models of MI. However, the underlying mechanisms regulating Tlr expression in MI remain unclear. The present study first screened transcription factors (TFs) that potentially regulate Tlr gene transcription based on in silico analyses followed by experimental verification, using both in vivo and in vitro models. Forkhead box C1 (FOXC1) was identified as a putative TF, which was highly responsive to MI. Next, by focusing on two representative TLR subtypes, an intracellular subtype TLR3 and a cell‐surface subtype TLR4, the regulation of FOXC1 on Tlr expression was investigated. The overexpression or knockdown of FoxC1 was observed to up‐ or down‐regulate Tlr3/4 mRNA and protein levels, respectively. A dual‐luciferase assay showed that FOXC1 trans‐activated Tlr3/4 promoter, and a ChIP assay showed direct binding of FOXC1 to Tlr3/4 promoter. Last, a functional study of FOXC1 was performed, which revealed the pro‐inflammatory effects of FOXC1 and its destructive effects on infarct size and heart function in a mouse model of MI. The present study for the first time identified FOXC1 as a novel regulator of Tlr expression and described its function in MI.

Prognostic Significance of FOXC1 in Various Cancers: A Systematic Review and Meta-Analysis.

Forkhead box C1 (FOXC1), a member of the Forkhead box (Fox) transcription factor family, plays an essential role in lymphatic vessel formation, angiogenesis and metastasis. Observational studies examining the relationship between the protein biomarker FOXC1 and breast cancer prognosis have reported conflicting findings. This systematic review and meta-analysis evaluates the prognostic value of the FOXC1 expression in association with patient survival in breast cancer and other types of cancers in order to identify the overall prognostic effectiveness of FOXC1. This study followed the guidelines established in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). We conducted a broad search on the online bibliographic databases EMBASE, PubMed, Science Direct and Scopus, limiting search to publications from 2010 to 2018. The prognostic value was demonstrated by a random effects model meta-analysis using the hazard ratio (HR) with 95% confidence interval (CI) for overall survival (OS) in various cancer patients. The heterogeneity was measured by the I2 statistic. Publication bias and quality assessment for the selected articles was performed. Subgroup analysis was conducted based on the data available from the selected articles. A total of 16 studies met the predefined selection criteria established for our systematic review and meta-analysis, with multiple studies using diverse methodologies and reported on differing clinical outcomes, falling under a common banner of FOXC1 expression and survival in cancer. Overall, we observed a statistically non-significant association between FOXC1 protein expression and patients survival (HR: 1.186 and 95% CI 1.122-1.255, p=0.000, I2=88.83%). In summary, FOXC1 protein expression indicated poor survival outcome in various carcinomas, especially in patients with breast cancer, suggesting it as a possible biomarker for the prognosis in multiple carcinomas. Further clinical evaluation and large-scale cohort studies are required to accurately identify its possible clinical utility.

A novel mutation of FOXC1 in a Chinese family with Axenfeld-Rieger syndrome.

Axenfeld-Rieger syndrome (ARS) is a disorder affecting the anterior segment of the eye and causing systemic malformations, and follows an autosomal-dominant inheritance pattern. The aim of the present study was to identify the underlying cause of ARS in a Chinese family. Genomic DNA was extracted from the peripheral blood of the subjects from a family with ARS. The pathogenic variant was identified by targeted next-generation sequencing and confirmed by Sanger sequencing. A novel heterozygous mutation of the forkhead box (FOX)C1 gene (c.1494delG, p.G499Afs*20) was detected in all affected members of the family, while no mutation was identified in the unaffected members or in the 150 normal controls. The affected members exhibited typical ocular and craniofacial anomalies. The results of the present study demonstrated that a novel deletion in exon 1 of the FOXC1 gene caused ARS in this Chinese family.

Abstract 2617: FOXC2-associated regulation of melanoma cell adhesion to the extracellular matrix and lymphatic endothelial cells

Abstract The FOXC2 transcription factor has emerged as a tumor-promoting oncogene in many cancer types. We have recently investigated the impact of Foxc2 gene disruption and FOXC2 overexpression in melanoma on tumor cell adhesion to extracellular matrix (ECM) proteins and lymphatic endothelial cells. We show that melanoma-associated FOXC2 is a critical regulator of tumor cell adhesion to the ECM proteins fibrinogen and fibronectin as well as to lymphatic endothelial cells. These effects on tumor cell adhesion correlate with FOXC2-mediated regulation of the integrins ITGA5 and ITGA9. As changes in cellular adhesion frequently accompany tumor cell transformation and progression, our data suggest that FOXC2 and genes regulated by this transcription factor may play key roles in promoting melanoma invasion and metastasis. Citation Format: Coleman E. Johnson, Corey J. Williams, Kristian M. Hargadon. FOXC2-associated regulation of melanoma cell adhesion to the extracellular matrix and lymphatic endothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2617.

A novel frameshift mutation in the PITX2 gene in a family with Axenfeld-Rieger syndrome using targeted exome sequencing

BackgroundAxenfeld-Rieger syndrome (ARS) is an autosomal dominant genetic disorder that is characterized by specific abnormalities of the anterior segment of the eye. Heterozygous mutations in two developmental transcription factor genes PITX2 and FOXC1 have been identified within ARS patients, accounting for 40 to 70% of cases. Our purpose is to describe clinical and genetic findings in a Chinese family with ARS.MethodsAn ARS family with three affected members was recruited. The patients underwent a series of complete ophthalmologic examinations, general physical examination and dental radiography. DNA samples of proband II-1 were used for targeted exome sequencing of the FOXC1 and PITX2 genes. Sanger sequencing was used to validate the variation in PITX2. Quantitative real-time PCR was carried out to detect the expression of PITX2 in patients and normal controls.ResultsAll affected members showed iris atrophy, corectopia, shallow anterior chamber, complete or partial angle closure, and advanced glaucoma. In addition, they revealed systemic anomalies, including microdontia, hypodontia, and redundant periumbilical skin. A novel heterozygous frameshift variation, c.515delA, in PITX2 was found in the proband, which might lead to a truncated PITX2 protein (p.Gln172ArgfsX36). Sanger sequencing validated that the variation completely cosegregated with the ARS phenotype among this family, but was absent in 100 unrelated controls. Quantitative real-time PCR analysis revealed that the mRNA expression of PITX2 was significantly decreased in patients compared with that in unrelated normal controls.ConclusionsPITX2 c.515delA (p.Gln172ArgfsX36) was the genetic etiology of our pedigree. The mutation led to decreased PITX2 gene expression and a truncated mRNA transcript.

Progress on the function of forkhead box C2

FOXC2, a transcription factor of the forkhead/winged-helix family, possesses a classical forkhead domain, which is required for embryonic and prenatal development.FOXC2 acts as a crucial modulator during tumor cell proliferation, metastasis, angiogenesis, lymphangiogenesis, renal disease and metabolism, but very little is known about the precise underlying mechanisms.Therefore, an in-depth study of FOXC2 is helpful to further understand the pathogenesis and developmental mechanism of FOXC2-related diseases.The purpose of this review is to summarize the current understanding of FOXC2 and its function. Key words: FOXC2; Epithelial-to-mesenchymal transition; Tumor; Kidney

Satb2 expression in Foxc1-promoted osteogenic differentiation of MC3T3-E1 cells is negatively regulated by microRNA-103-3p.

The forkhead transcription factor C1 (Foxc1) is a cell-fate-determining factor that controls cranial bone development and osteogenic differentiation. Previously, it was demonstrated that various microRNAs (miRNAs) play important roles in osteogenesis and regulate the complex process of osteogenic differentiation. However, it remains unclear how miRNA expression changes during Foxc1-promoted osteogenic differentiation. In this study, we successfully overexpressed the Foxc1 gene in MC3T3-E1 cells and investigated the alterations in the miRNA expression profile on day 3 after osteogenic induction by using a miRNA microarray. Nine downregulated miRNAs and eight upregulated miRNAs were found to be differentially expressed. Among these miRNAs, miR-103-3p was consistently downregulated in the Foxc1-overexpressing MC3T3-E1 cells and was identified as a negative regulator of osteogenic differentiation by using a gain- and lose-of-function assay. The special AT-rich sequence-binding protein 2 (Satb2), a pivotal osteogenic transcription factor, was identified as the miR-103-3p targeting gene and was verified by real-time polymerase chain reaction, western blot analysis, and luciferase assay. Overexpression of miR-103-3p markedly inhibited the expression of Satb2 and attenuated Foxc1-promoted osteogenic differentiation. Taken together, our results elucidated the miRNA expression profiles of MC3T3-E1 cells in the early stage of Foxc1-promoted osteogenic differentiation and suggested that miR-103-3p acts as a negative regulator of the osteogenic differentiation of MC3T3-E1 cells by directly targeting Satb2.

Unexpected phenotype in a patient with two chromosomal deletions involving 6pter and 22q11

The 6p terminal deletions are rare and usually early diagnosed because of their association with eye and cranio-facial anomalies, particularly as part of Axenfeld-Rieger syndrome in relation with the haploinsufficiency of FOXC1 gene. Deletions in the 22q11 region are frequent, highly correlated with DiGeorge syndrome also named CATCH22, and may be associated with many clinical features of various severities. We report a 31-year-old man with an unbalanced 45,XY,der(6)t(6;22)(p25;q11.2),-22 karyotype leading to monosomies in both 6p25 and 22q11 regions, confirmed by FISH and array-CGH. The length of the deletions was respectively 770 Kb for 6pter and 2.9 Mb for 22q11. This karyotype was discovered at adult age following problems of fertility. The chromosomal formula was unexpected, regarding the patient's medical history and clinical features. This case makes a great example of the difficulties to correlate genotype and phenotype, and furthermore demonstrates the complexity of genetic counselling even in a case with two different chromosomal unbalances.

Integrated analysis of gene expression profiles identifies transcription factors potentially involved in psoriasis pathogenesis.

Psoriasis is a common inflammatory skin disease mediated by cells and molecules in both the innate and adaptive immune systems. Recently, gene expression profile analysis revealed a large set of immune-related differentially expressed genes (DEGs) in psoriasis. However, the associations between these DEGs and their transcriptional regulation mechanisms have not been completely elucidated. In this study, several psoriasis Gene Expression Omnibus data sets were systematically analyzed using bioinformatics tools to uncover important transcription factors (TFs) that regulate the expression of immune-related DEGs and further enhance our understanding of psoriasis pathogenesis. Common DEGs encoding chemokines, cytokines, antimicrobial peptides, and keratins were identified in psoriasis, and extensive correlations existed among these DEGs. Several common TFs that bind the promoters of the DEGs, including the well-known signal transducer and activator of transcription 1 (STAT1), STAT3, and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) as well as ETS homologous factor (EHF), Fos-like antigen 1 (FOSL1), and Forkhead box C1 (FOXC1), which are rarely studied in psoriasis, were also identified. STAT1, EHF, FOSL1, STAT3, and NFKB1 were positively correlated with these DEGs in psoriasis lesions, whereas FOXC1 was negatively correlated with most DEGs. The decreased expression of the DEGs was accompanied by the downregulation of STAT1, EHF, FOSL1, STAT3, and NFKB1 and the upregulation of FOXC1 upon blocking interleukin 17 (IL-17) or tumor necrosis factor α signaling in psoriasis. Additionally, the downregulation of IL37 in psoriasis was negatively correlated with STAT1 and CXCL10, which are associated with Th1 responses. These results suggest that TFs play an important role in the pathogenesis of psoriasis, and interfering with the activity of key TFs may be a promising therapeutic strategy for psoriasis.

Time-dependent Pax3-mediated chromatin remodeling and cooperation with Six4 and Tead2 specify the skeletal myogenic lineage in developing mesoderm.

The transcriptional mechanisms driving lineage specification during development are still largely unknown, as the interplay of multiple transcription factors makes it difficult to dissect these molecular events. Using a cell-based differentiation platform to probe transcription function, we investigated the role of the key paraxial mesoderm and skeletal myogenic commitment factors—mesogenin 1 (Msgn1), T-box 6 (Tbx6), forkhead box C1 (Foxc1), paired box 3 (Pax3), Paraxis, mesenchyme homeobox 1 (Meox1), sine oculis-related homeobox 1 (Six1), and myogenic factor 5 (Myf5)—in paraxial mesoderm and skeletal myogenesis. From this study, we define a genetic hierarchy, with Pax3 emerging as the gatekeeper between the presomitic mesoderm and the myogenic lineage. By assaying chromatin accessibility, genomic binding and transcription profiling in mesodermal cells from mouse and human Pax3-induced embryonic stem cells and Pax3-null embryonic day (E)9.5 mouse embryos, we identified conserved Pax3 functions in the activation of the skeletal myogenic lineage through modulation of Hedgehog, Notch, and bone morphogenetic protein (BMP) signaling pathways. In addition, we demonstrate that Pax3 molecular function involves chromatin remodeling of its bound elements through an increase in chromatin accessibility and cooperation with sine oculis-related homeobox 4 (Six4) and TEA domain family member 2 (Tead2) factors. To our knowledge, these data provide the first integrated analysis of Pax3 function, demonstrating its ability to remodel chromatin in mesodermal cells from developing embryos and proving a mechanistic footing for the transcriptional hierarchy driving myogenesis.

The Diverse Consequences of FOXC1 Deregulation in Cancer.

Forkhead box C1 (FOXC1) is a transcription factor with essential roles in mesenchymal lineage specification and organ development during normal embryogenesis. In keeping with these developmental properties, mutations that impair the activity of FOXC1 result in the heritable Axenfeld-Rieger Syndrome and other congenital disorders. Crucially, gain of FOXC1 function is emerging as a recurrent feature of malignancy; FOXC1 overexpression is now documented in more than 16 cancer types, often in association with an unfavorable prognosis. This review explores current evidence for FOXC1 deregulation in cancer and the putative mechanisms by which FOXC1 confers its oncogenic effects.

Familial arachnoid cysts: a review of 35 families

Arachnoid cysts are commonly considered congenital lesions, but this has not been proven. With the development of neuroimaging and DNA testing technology, more cases of familial arachnoid cysts have been reported. Herein, we review such cases. The PubMed, Embase, and Web of Science databases were searched for case reports of arachnoid cysts published through April 2018. Case reports were included only if two or more related patients were diagnosed with an arachnoid cyst by neuroimaging or intraoperatively. For each report, the following data were extracted: first author name, date of publication, number of families, number of patients, location of the arachnoid cysts, patient age, patient sex, and genetic mutations and associated disease. Our searches identified 33 case reports involving 35 families and 115 patients. The locations of arachnoid cysts were similar in 25 of the 35 families. Spinal extradural arachnoid cysts were reported most often, followed by arachnoid cysts in the middle fossa and posterior fossa. A left-sided predominance was noticed for arachnoid cysts of the middle fossa. Mutation of the FOXC2 gene was reported most often, and arachnoid cysts may be associated with mutations on chromosome 16. Although the origin of arachnoid cysts is believed to have a genetic component by some researchers, the genes associated with arachnoid cysts remain unknown. Unfortunately, the evidence remains insufficient.

Prevalence of FOXC1 Variants in Individuals With a Suspected Diagnosis of Primary Congenital Glaucoma

Both primary and secondary forms of childhood glaucoma have many distinct causative mechanisms, and in many cases a cause is not immediately clear. The broad phenotypic spectrum of secondary glaucoma, particularly in individuals with variants in FOXC1 or PITX2 genes associated with Axenfeld-Rieger syndrome, makes it more difficult to diagnose patients with milder phenotypes. These cases are occasionally classified and managed as primary congenital glaucoma. To investigate the prevalence of FOXC1 variants in participants with a suspected diagnosis of primary congenital glaucoma. Australian and Italian cohorts were recruited from January 1, 2007, through March 1, 2016. Australian individuals were recruited through the Australian and New Zealand Registry of Advanced Glaucoma and Italian individuals through the Genetic and Ophthalmology Unit of l'Azienda Socio-Sanitaria Territoriale Grande Ospedale Metropolitano Niguarda in Milan, Italy. We performed exome sequencing, in combination with Sanger sequencing and multiplex ligation-dependent probe amplification, to detect variants of FOXC1 in individuals with a suspected diagnosis of primary congenital glaucoma established by their treating specialist. Data analysis was completed from June 2015 to November 2017. Identification of single-nucleotide and copy number variants in FOXC1, along with phenotypic characterization of the individuals who carried them. A total of 131 individuals with a suspected diagnosis of primary congenital glaucoma were included. The mean (SD) age at recruitment in the Australian cohort was 24.3 (18.1) years; 37 of 84 Australian participants (44.0%) were female, and 71 of 84 (84.5%) were of European ancestry. The mean (SD) age at recruitment was 22.5 (18.4) years in the Italian cohort; 21 of 47 Italian participants (44.7%) were female, and 45 of 47 (95.7%) were of European ancestry. We observed rare, predicted deleterious FOXC1 variants in 8 of 131 participants (6.1%), or 8 of 166 participants (4.8%) when including those explained by variants in CYP1B1. On reexamination or reinvestigation, all of these individuals had at least 1 detectable ocular and/or systemic feature associated with Axenfeld-Rieger syndrome. These data highlight the genetic and phenotypic heterogeneity of childhood glaucoma and support the use of gene panels incorporating FOXC1 as a diagnostic aid, especially because clinical features of Axenfeld-Rieger syndrome can be subtle. Further replication of these results will be needed to support the future use of such panels.

MicroRNA-regulated pathways of flow-stimulated angiogenesis and vascular remodeling in vivo

BackgroundVascular shear stress promotes endothelial cell sprouting in vitro. The impact of hemodynamic forces on microRNA (miRNA) and gene expression within growing vascular networks in vivo, however, remain poorly investigated. Arteriovenous (AV) shunts are an established model for induction of neoangiogenesis in vivo and can serve as a tool for analysis of hemodynamic effects on miRNA and gene expression profiles over time.MethodsAV shunts were microsurgically created in rats and explanted on postoperative days 5, 10 and 15. Neoangiogenesis was confirmed by histologic analysis and micro-computed tomography. MiRNA and gene expression profiles were determined in tissue specimens from AV shunts by microarray analysis and quantitative real-time polymerase chain reaction and compared with sham-operated veins by bioinformatics analysis. Changes in protein expression within AV shunt endothelial cells were determined by immunohistochemistry.ResultsSamples from AV shunts exhibited a strong overexpression of proangiogenic cytokines, oxygenation-associated genes (HIF1A, HMOX1), and angiopoetic growth factors. Significant inverse correlations of the expressions of miR-223-3p, miR-130b-3p, miR-19b-3p, miR-449a-5p, and miR-511-3p which were up-regulated in AV shunts, and miR-27b-3p, miR-10b-5p, let-7b-5p, and let-7c-5p, which were down-regulated in AV shunts, with their predicted interacting targets C–X–C chemokine receptor 2 (CXCR2), interleukin-1 alpha (IL1A), ephrin receptor kinase 2 (EPHA2), synaptojanin-2 binding protein (SYNJ2BP), forkhead box C1 (FOXC1) were present. CXCL2 and IL1A overexpression in AV shunt endothelium was confirmed at the protein level by immunohistochemistry.ConclusionsOur data indicate that flow-stimulated angiogenesis is determined by an upregulation of cytokines, oxygenation associated genes and miRNA-dependent regulation of FOXC1, EPHA2 and SYNJ2BP.

The FOXC2 Transcription Factor Promotes Melanoma Outgrowth and Regulates Expression of Genes Associated With Drug Resistance and Interferon Responsiveness.

The FOXC2 transcription factor promotes the progression of several cancer types, but has not been investigated in the context of melanoma cells. To study FOXC2's influence on melanoma progression, we generated a FOXC2-deficient murine melanoma cell line and evaluated The Cancer Genome Atlas (TCGA) patient datasets. We compared tumor growth kinetics and RNA-seq/qRT-PCR gene expression profiles from wild-type versus FOXC2-deficient murine melanomas. We also performed Kaplan-Meier survival analysis of TCGA data to assess the influence of FOXC2 gene expression on melanoma patients' response to chemotherapy and immunotherapy. FOXC2 promotes melanoma progression and regulates the expression of genes associated with multiple oncogenic pathways, including the oxidative stress response, xenobiotic metabolism, and interferon responsiveness. FOXC2 expression in melanoma correlates negatively with patient response to chemotherapy and immunotherapy. FOXC2 drives a tumor-promoting gene expression program in melanoma and is a prognostic indicator of patient response to multiple cancer therapies.