UvA-DARE (Digital Academic Repository) Forkheads in neuroblastoma & human longevity: From the womb to the tomb Santo, E.E. Publication date 2015 Document Version Final published version Link to publication Citation for published version (APA): Santo, E. E. (2015). Forkheads in neuroblastoma & human longevity: From the womb to the tomb. General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:02 Oct 2021 Chapter 1 Introduction 1 2 Forkhead Box (FOX) Family Transcription Factors The first member of what was to become the forkhead family (fork head, Fkh) was identified in Drosophila melanogaster by a genetic screen which yielded flies whose heads exhibited abnormal involution patterns early in development (Jurgens 1988). Fkh was subsequently cloned and described as being a nuclear protein with no identifiable sequence similarities to contemporary transcription factors of the time (Weigel 1989). The DNA binding domain now termed the forkhead domain was identified when Fkh was aligned with the independently discovered HNF-3A in rats (Lai 1990 & Weigel 1990). Throughout the last 25 years additional members of this superfamily which all contain the approximately 100 amino acid forkhead domain (the only criteria for inclusion) have been identified. So far phylogenetic analyses have resulted in the identification of 19 different subfamilies containing 50 members in humans and 44 in mice (Jackson 2010 & Fig. 1). The members of these subfamilies typically have sequence features outside of the forkhead domain common with each other but these features are not found within other FOX subfamilies. This familial tree structure was annotated using a nomenclature where FOX describes the membership in the superfamily, a letter of the alphabet describes membership in a subfamily and a number describes the particular subfamily member; e.g. FOXA1, FOXA2, FOXA3 and FOXD1, FOXD2, FOXD3 and FOXD4 (Hannenhalli 2009). Forkheads also exhibit extreme conservation with orthologues identifiable in yeast (fkh1 & 2, Zhu 2000). The following is an overview of select forkhead subfamilies that have exhibited powerful roles in shaping mammalian biology. 3 Figure 1 Evolutionary tree of mouse forkhead box (Fox) genes (adapted from Hannenhalli 2009). A neighbor-joining tree is shown that is based on the protein sequence of the forkhead domain. The relationships shown in the tree are based on multiple alignment with each branch annotated with a bootstrap value that was based on 1000 permutations. The branch lengths are proportional to the mutation rate. In red boxes the forkheads which are the primary subjects of this Thesis are indicated. 4 FOXA The FOXA subfamily was first identified from rat liver extracts as a protein that bound to the promoters of the transthyretin (Ttr), α1-antitrypsin (Serpina1) and albumin (Alb1) genes that contained a hepatocyte nuclear factor 3 (HNF-3) DNA binding element and were subsequently named HNF-3A, B & C (now FOXA1, 2 & 3 respectively) (Lai 1990). Since their discovery they have been shown to be involved in organ development and are thought to function as “pioneer” chromatin modification factors (for review see Friedman 2006). In addition to being fundamental for liver development they have also been shown to be pivotal for the maintenance of glucose homeostasis (for review see Le Lay 2010). Despite the expected and previously demonstrated redundancy of function, FOXA1 knockout mice exhibit severe hypoglycemia. This may be at least in part due to a deficiency of pancreatic glucagon production as well as an attenuated transcriptional gluconeogenic response to glucagon by the liver. Liver-specific deletion of FOXA2 was euglycemic while FOXA3 knockouts only exhibit mild hypoglycemia after prolonged fasting. Surprisingly, the FOXA1 orthologue in c. elegans, PHA-4, has been demonstrated to be robustly essential for dietary restriction-induced longevity as well as longevity in response to the loss of mTORC1 signaling (Panowski 2007 & Sheaffer 2008). FOXD The FOXD subfamily consists of four members (FOXD1-4) with 2 & 4 being poorly characterized. FOXD1 is pivotal in eye and kidney formation; playing an organogenic role like that of the FOXA subfamily (Herrera 2004 & Fetting 2014). FOXD3 is of particular interest due to its emerging roles as a maintenance factor in embryonic stem cells, an important player in neural crest development and a tumor suppressor. FOXD3 knockout mice were embryonic lethal with the epiblasts depleted of their progenitor cells (Hanna 2002). It was also impossible to derive teratomas or culture embryonic stem cells from FOXD3 null embryos; demonstrating that FOXD3 was essential for ES cell survival. Using a neural crest targeted Cre knockout model FOXD3 loss caused severe defects in NC development such as impaired formation of craniofacial features, abolishment of the enteric nervous system and reduction of the peripheral nervous system (Teng 5 2008). A follow-up study demonstrated that the neural crest stem cell features of multipotency, self-renewal capacity and ability to form neuronal lineages were maintained by FOXD3 with its loss causing a strong mesenchymal differentiation (Mundell 2011). In the neural crest derived tumor neuroblastoma FOXD3 was shown to have tumor suppressive functions (Li 2013). Overexpression of FOXD3 inhibited growth and invasion in cell lines and xenografts while knockdown enhanced these tumor driving properties. A similar role was discovered for FOXD3 in hepatocellular carcinoma and gastric cancer (Cheng 2013 & Liu 2014). FOXM FOXM1 is the only member of its subfamily and is an important factor for cell cycle progression at the G2 checkpoint as well as for faithful chromosomal segregation; although it is not completely essential (Laoukili 2005). Its expression pattern is tightly linked to the cell cycle with an onset in S phase and loss of expression by the end of mitosis (Korver 1997). Unsurprisingly, FOXM1 has also become a very well documented tumor driver in numerous systems. It has been found amplified in 5.6% of breast cancers, 42% of non-Hodgkin’s lymphomas and 58% of malignant peripheral nerve sheath tumors (Katoh 2013). Its potency has been further demonstrated in neuroblastoma and nearly every other tumor type (Wang 2011 & Wierstra 2013). FOXM1 has also been found to promote resistance to the chemotherapeutic cisplatin in breast cancer (Kwok 2010). FOXO The FOXO subfamily (FOXO1, 3, 4 & 6) is the best studied and perhaps most famous subfamily of forkheads to-date. Across mammalian tissues their expression patterns differ with FOXO3 being the most ubiquitous and FOXO1, FOXO4 and FOXO6 exhibiting more restrictive profiles (for review see Salih 2008). They directly interact with DNA by binding the “DBE” (DNA Binding Element, TTGTTAC) and the more degenerate thymidine-rich IRS (Insulin Responsive Sequence) elements (Furuyama 2000 & Brent 2008). Although they have specific roles they also have many redundant functions due to their sometimes overlapping expression and DNA binding patterns. This redundancy was especially relevant in regard to their tumor suppressive capabilities. In genetic mouse models of single 6 FOXO knockouts their roles as tumor suppressors were heavily masked by this redundancy and not revealed until a conditional triple knockout mouse for FOXO1/3/4 was constructed which then developed leukemia (Paik 2007). Beyond tumor suppression they have been implicated in many processes with some of the most important being apoptosis, cell cycle regulation, metabolism, longevity, response to oxidative stress, stem cell maintenance, cell-type specification, differentiation and immune response modulation. FOXO1 (previously FKHR) was first discovered as an aveolar rhabdomyosarcoma fusion gene where the n-terminus of PAX3 and PAX7 (which contains the PAX DNA binding domains) are found fused to the c-terminus of FOXO1; this portion of FOXO1 contains the familial FOXO transactivation domain (Davis 1994 & Fredericks 1995). This fusion product is oncogenic as demonstrated by its ability to transform primary cells (Scheidler 1996). Physiologically, FOXO1 has been described as being of central importance in the liver where it acts to maintain whole-body glucose homeostasis. It is a potent activator of gluconeogenesis and VLDL cholesterol biosynthesis that in turn is tightly inhibited by the Insulin/Irs1- 2/PI3K/Akt pathway (Nakae 2002, Dong 2008, Kamagate 2008 & Lu 2012). Recently, knockout of FOXO1/3/4 in the liver has demonstrated that FOXO3 and FOXO4 act redundantly with FOXO1 in both the activation of gluconeogenesis and suppression of lipogenesis (Haeusler 2014). Initially, the importance of FOXO1 in whole-body glucose