Influence of FOX genes on aging and aging-associated diseases

H2S impairs the insulin/IGF-1 pathway in neuroblastoma (Liu et al., 2013) and blocks nutrient-sensing mTOR pathway and protein aggregations. (Talaei et al., 2013) H2S inhibits FOXO1 and FOXO3 phosphorylation and is an endogenous regulator of PTEN, the main antagonist of the PI3K-Akt axis in the insulin/IGF-1 signal pathway. The insulin/IGF1 signalling pathway and the FOXO transcription factors modulate the expression of pro-longevity genes (Lee et al., 2003 Oh et al., 2006 Murphy et al., 2003) Marjolein et al., 2017 also demonstrated Foxa2- FOXO4-p53 interaction and its role in cell aging. Its overexpression has been reported in metastases of prostate cancer (Mirosevich et al., 2006) and in colorectal carcinoma (Lehner et al., 2007), while in thyroid cancer cells Foxa2 suppression has been observed. (Akag et al., 2008)

Other FOX family genes are involved in apoptosis, cell invasion and migration, cellular development, metabolism, cell proliferation and development. (Myatt et al., 2007)

Their mutations can lead to cellular cancerization. (Kalin et al., 2006), (Paik et al., 2007)

Several other publications also confirmed the relationship between FoxP expression and cell proliferative, oncological and inflammatory processes as well as the relationship between other FOX genes and vitiligo (FOXD3), ptosis (FOXL2), adipose disease (FoxY2) (Zhou et al. 2018) and lymphoedema-associated autosomal dominant lymphedema-distichiasis syndrome (Fang et al., 2000).

FOXG1 plays a role in apoptosis but is also expressed in the placenta. (Entrez Gene: FOXG1B forkhead box G1B FoxF2).

Among other, FOXJ1 regulates the immune system. (Lin et al., 2005)

FOXA2 influences aging relevant changes in nuclear lamina and increases the lamin B1- and aberrant prelamin A isoform (called Progerin)-level (Bochkis et al., 2014; Scaffidi et al., 2006).

FOXM1B, the „fountain of youth gene“, stimulates tissue growth. (Laoukili et al., 2005; Wierstra and Alves, 2007) and caloric restriction effect on aging can happen via FOXL2 and Foxa2 (hepatocyte nuclear factor 3-beta, HNF3b).

Foxa2 among others effects dopaminergic neurons (its mutation contributes to locomotor deficits in animals that remember the human Parkinson's disease (Kittappa et al., 2007; Arenas, 2008; Lin et al., 2009). It also influences sleep through melanin-concentrating hormone (MCH) and orexin (which also plays a role in alcohol dependence).

FOXP3 importance for carcinogenesis The tumor suppressor gene p53 increases Foxp3 expression by binding directly to the its promoter region of the conserved non-coding DNA sequence CNS-2. (Kawashima et al., 2013) P53 may not only play its immunosuppressive but also its anticarcinogenic role through FOXP3 activation. As already mentioned, protooncogene p53 plays an important role in aging and aging relevant diseases.(Reinhardt And Schumacher, 2012; Carrasco‐Garcia et al., 2017; Nicolai et al., 2015) p53 controls ROS in connection with expression of GLS2 via Tap63. p53 plays a role in apoptosis (Li et al., 2014) especially with the help of mitochondrial pathway of PUMA (Westphal et al., 2014; Yu et al., 2003) and with the help of pro-apoptotic proteins.(Zahran et al., 2014) e.g., via NOXA (Cuadrado Cuadrado et al.,2007) p53 also activates tumor metastasis via SNAI2 (Wu et al., 2005). Further ATP-dependent chromatin remodeler p53 interacts with CSB, which is important for DNA repair.(Lake et al., 2011) It would be interesting to investigate whether and to what extent FOXP3 is involved in a variety of carcinogenic diseases with T inflammatory response.

According to the 28th annual meeting of the „German Association for the Study of the Liver“ (GASL) almost every 5th cancer is caused by chronic inflammation. For example, H. pylori produces the toxic ammonia and vacuolating cytotoxin A, they are demaging gastric tissue. This activates cytokines, which in turn activate neutrophilic granulocytes, T cells and macrophages. H. Pylori attempts to induce apoptosis of macrophages and T cells. This dysregulation of tumor-protective immune processes leads to the development of gastric carcinoma. (Liliane Sygulla, 1998) Human herpesvirus-8 (HHV-8) also involves vascular endothelial growth factor interleukin-6 and VEGF-like genes, which promotes tumorigenesis. (Masood et al., 2002) Human T-lymphotropic virus 1 (HTLV-1) can induce tropical spastic paraparesis or adult T-cell leukaemia (ATL) in which T cells respond independently of the external growth signals. (Liesz et al., 2012)

Gerontologic medicine also tries to prevent the inflammations on a cellular level. For example: many natural anti-aging agents protect against inflammatory process (e.g. curcumin and cayenne pepper) (Aggarwal, 2010) or to influence growth processes (polyphenols, e.g. epigallocatechin EGCG from green tea or procyanidin from apples). (Lombardo et al, 2015)

Posselt's et al. working group investigated 2016 in their work „Spatial distribution of FoxP3 + and CD8 + tumor infiltrating T cells reflects their functional activity“ FoxP + and CD8 + distribution in regulatory and cytotoxic T cells. According to their results the cytotoxic T cells play the key role in the immunological cancer response and offer promising new ways in the fight against cancer. Further results were reported by Schmidt et al. (2011), Lin et al. (2015), Gerber et al. (2014), Li et al. (2016) and Balbaco et al. (2014). These studies showed that T-cell activity were decreased by the PD-L1 and increased by the CTLA-4 antibodies. Both molecules are influenced by the FOXP3 gene. Jim Allison took advantage of this feature in the development of the cancer immunotherapeutic Ipilipumab 2008. FOXP3 interaction with other chemotherapeutic agents has been reported by Takada et al. (2018), Tang et al. (2008) and Landoire et al. (2016). Interestingly, Programmed Cell Death Ligand 1 correlated with FOXP3 expression. (Enkhbat et al., 2018)

The role of l n RNA in aging

Long noncoding RNAs are also aging relevant (Kim et al., 2016) e.g. via phosphatase and tensin homolog PTEN. ln RNA represses expression of the age related tumor-suppressor gene via its interaction with DNMT3a. lncRNA TARID induces promoter demethylation of TCF21. lncRNAs 7SL and MEG3, which is of importance in carcinogen processes (Li et al., 2018), decreased cell proliferation via MDM2 and the tumor suppressor p53 inhibition (Zhou et al, 2007) is decreased in liver, bladder cancer and in pituitary cancer (Braconi et al. 2011; Zhang et al. 2003; Ying et al., 2013) It influences autophagy inversely in age related gastric cancer. LncRNA HULC triggers autophagy direct via stabilizing Sirt1 and attenuates the chemosensitivity of HCC cells (Xiong et al., 2017) As others lncRNAs (Reviewed Yanget al., 2014) HULC influences tumorigenesis in liver and colorectal cancer. (Panzitet al., 2007; Wang et al., 2010)

ncRNAs also modulate protein transcription indirectly via microRNAs. E.g., lncRNAs such as linc-MD1 and lincRNA-RoR change the level of another mRNAs. Grammatikakis

et al., 2014; Cesanaet al., 2011; Wanget al., 2013) In the same time Uchl1 lncRNA may influence senescence directly via p14ARF and p53 and its repressor MDM2 (via proteasome). NF90, required for hypoxia-induced cancer cell invasion (Petrovics et al, 2004; Yang et al., 2013) also effects lnc RNA decrease and is also cancer and apoptosis relevant , because it reduces p21, P53, caspase 7, PARP and PCGEM1 level. (Fu et al., 2006) Cancer relevant lncRNA PVT1 is downregulated via MYC, which is also associated with proliferation and apoptosis of breast and ovarian cancer (Carramusaet al., 2007).

According to Kim et al. „Long noncoding RNAs in Diseases of Aging“, 2016 MALAT1 interact with TP53 and decreases with aging, based on changing serine/arginine (SR) splicing factors level. (Abdelmohsen al. 2013 Tripathi et al.; Tripathi et al., 2010; Melzeret al., 2013) Its level is increased lung and liver carcinoma (Qi et al., 2013; Luo et al., 2006) and influences muscle aging.

ANRIL influences cell cycle as well senescence via upregulation of the tumor suppressor gene p15 (INK4B) (Latrese et al, 2000; Kotake et al., 2011; Peters 2008) and plays a role in breast and another carcinoma, e.g. in gastric, prostate cancer cells and non-small cell lung cancer cell via KLF2 and p21 downregulation (Yap et al., 2010; Nie et al., 2014;1:268–277; Zhang et al., 2014; Hannou et al., 2015; Pasmant et al., 2011) and disturbs miR-99 and miR-449a. LncRNA PINT interacts with PRC2 complex via SUZ12, with plays a role in histone modification and cancer. (Marin-Bejar et al., 2013)

HOTAIR also influences senescence and cancer, e.g., breast tumors as well as endometrial, colorectal, cervical carcinomas and metastasis via H3K27me3 changes and chromatin modifiers, e.g. PRC2. (Kogo et al., 2011; Zhang et al., 2013; Heet al, 2014; Huanget al., 2014; Gupta et al., 2010)

The lncRNA XIST is decreased in breast cancer (Vincent-Salomon et al., 2007) and correlates with Taxol sensitivity. (Huang et al., 2002)

a 200-nt-long ncRNA BCYRN1 is associated with AD b and aging (Mus et al., 2007) but also with breast (Iacoangeli et al. 2004), parotid, tongue, oesophagus, lung, cervix and ovary cancer (Chen et al, 1997).

GAS5 lncRNA suppressed GR and induced apoptosis (Mourtada-Maarabouni et al., 2009) and is downregulated in human HCC and breast cancer (Tu and al., 2014).

lncRNA-17A BACE1, repressed GABABR2 variant A, promoted variant B, and enhanced accumulation of peptides Aβ42 and Aβ40 (Massone et al., 2011), BCYRN1 (Mus et al.,2007) and GOMAFU (MIAT)BC089918 GAS5 RMSTAS are associated with neurodegeneration. (Wapinski et al.; 2011Yu et al., 2013; Ng et al., 2013; Kim et al., 2013; Meier et al., 2010) and cancer (Mourtada-Maarabouni et al., 2009)

BACE1 protein levels and activity increase with brain aging and AD (Modarresi et al., 2011; Fukumoto et al., 2002)

7 SL interacts with the TP53 mRNA and suppresses p53 translation, during HuR can displace 7SL and increase p53 translation. lncRNA 7SLsuppresses TP53 (Abdelmohsen et al., 2014) and influences autophagy, senescence and cancer (Grammatikakis et al., 2014; Chen et al., 1997).

FOXP2 and mRNA in aging

But other RNAs are also an important component of aging, e.g., extracellular RNA play an important role in this process.(Douglas et al. 2016) 24–31nts piRNAs are responsible for proper genome in germ-line cells. 142.22 nts microRNAs cooperate with the 3′ UTR of target mRNAs binding RISC. This complex leads to mRNA degradation and disturbs protein translation. This affects components of the RNA spliceosome, 100–300 nucleotides Small nuclear RNAs (snRNAs), which process mRNAs in the nucleus. (Carthew et al. 2009) Aging relevant oxidative stress effects 5′ tRNA and 3′ tRNA fragments (Fu et al., 2009), which are necessary for cellular proliferation and interact with Argonauts proteins like extracellular miRNAs. (O'Brien et al., 2018) AGO2 is known to influence tumorigenesis through miRNAs-dependent or independent ways. (ZhenLong et al., 2015) Inflammatory miR-223 regulate the help of HDL ICAM-1 46 protein expression and snoRNAs, responsible for chromatin remodelling and rRNA modification. (Vickers and Remaley, 2017; Tabet et al., 2014) 200 nts non-coding lncRNAs regulate gene expression via chromatin association, Y-RNA 80–112 nts, coded in 4 genes, initiate DNA replication (Kheir & Krude, 2017; Yeri at al.2017) and are important for apoptosis. (Chakrabortty et al. 2015; Fritz et al., 2016). Circular RNAs, created from spliced exons and introns, influence tissue-specific development via miRNA. (Chen et al., 2015 Salzman et al., 2016). In endothelial cells miRNAs influence senescence, inflammation, cell differentiation and angiogenesis with the help of HDL Cockerill et al., 1995; Tabet et al., 1995, 2009; Schroen et al, 2012; Sun X, et al.,2012; Kane et al., 2012; Sumi et al. 2007;Pu and Liu L., 2008) Mi 223 also corelates with Hepatitis C healing. (Hyrina et al., 2017)

73 miRNAs e.g. miR-24-3p, miR-371a-5p, miR-3175, miR-3162-5p, miR-671-5p, miR-4667-5p, 146a-5p, 342-5p and miRs 5107-5p, which influence Gpx3 and thyroids, have significantly increased level in serum of old mouse. Level of 47 miRNAs e.g. miR-195a-5p and miR-34c-5p, also decrease with age. (Machida et al., 2015) This process can be influenced by caloric restriction. Mi RNAs also effects axon growth, melanogenesis, MARK, adherent and gap junctions (Victoria et al. 2015) and neuronal activity is affected by miRs 5107-5p, 146a-5p, and 342-5p. miR-5107-5 protects thyroids via Gpx3 and miR-195a-5p affect the aging relevant PI3K-AKT signalling pathway. miRNAs can also influence HTLV-1 infection, pluripotent stem cells, bone microarchitecture, osteoarthritis and cancer. (Hafez et al, 2015; Rodriguez-Fontenla et al. 2014; Wu et al., 2014) as well es aging relevant Bmpr1a,Vegfa, Bcl-2, Map2k1 Wnt4. MiR-34-5p influences GABAergic neurons, vascular aging response and cancer relevant Notch1, Notch4, Ccne2, E2f3, Gabra3 and Cylcin E2 through a Notch-dependent mechanism and Sirt1 (Li et al. 2011) Prop1 leads to decreased level of GH, Igf-1 and TSH. (Bartkeet al. 2001; Victoria et al., 2015) Lysine-, Valine-, Arginine-, Cysteine-, Glycine tRNA were decreased and Histidine- and Aspartic- acid derived 5′ tRNA increased in age. These changes were also CR sensitive (Dhahbi et al. 2013)

Aging is also associated with increased mRNAs level of CSF2, which is necessary for monocytes activation (Croxfordet al. 2015), granulocyte and macrophage maturation (Hamilton, 2002), energy homeostasis and metabolism. mRNA of subunit of pyruvate dehydrogenase enzyme DLD, which is necessary for exocytosis is also increased. RAB3 GTPase activating Protein Subunit 2 (Bem et al., 2011), an alternative splicing nuclear protein, which is involved in metastasis suppression with the help of splicing factor SRSF1 RRP1B (Lee et al. 2014), CSF2RA, SLC35B4, LAMB2 (Schaeffer, 2012), LAMB2 and LC35B4 showed increased mRNA level. Some lncRNAs were also hyperexpessed in ageing. At the same time 12 snoRNAs and 20 piRNAs were decreased. Decreased rRNA, which interacts with TTF1, may play a role in age related Alzheimer disease. (Freedman et al.,2016) miR-31 decrease is associated with aging relevant chronic myeloid leukaemia and ovarian carcinoma (Mitamura et al., 2013; Rokah et al.,2012; Korner et al., 2013; Zhong et al., 2013) miR-485-5p expression also alternates in aging. (Faghihi et al.,2008; 2010;Lee et al. 2014)

FOXP2 and mi RNA in oncological processes

Like the other genes in the FOXP family, the Fox P2 gene can be inactivated in cancer cells through miRNAs and alter its action this way. The recent study by Herrero and Gitton (2018) showed that FOXP2 can be blocked by TWIST activated miRs 199a-214, miR-762, miR-1915, let-7b and miR-34a and miR-3666. let-7a-d, miRs, miR-26a, miR-10,1miR-200b (which leads to increased MT1-MMPu und decreased PTEN- expression) (Soubani et al., 2012) and decreases prostaglandin, NF-kappaB,PEG2, VEGF activity ( Ali et al., 2010; Bao et al., 2011), EpCAM and EZH2 level and inhibits NOTCH-1 in pancreatic cancer (Bao et al., 2012). Wen-Zhuo et al. (2016) investigated the regulation of the FOXP2 gene by the microRNA-190 in gastric cancer. Valencia-Sanchez et al. (2006) showed that miRNAs destroy FOXP2-mRNA through its interaction with 3 'UTR, prevent its translation and this way negatively regulate the FOXP2 target genes. Many other studies also indicated that miRNA dysregulation may play an important role in the initiation and progression of oncological processes. (Barbrotto et al., 2008; Kasinski and Slack, 2011) Two years earlier the group of Wen-Zhuo et al. used the dual luciferase enzyme assays and confirmed that the miR-190 interacts with the FOXP2. RT-PCR and Western blot verified that miR-190 overexpression suppresses expression of FOXP2. Decrease of miRNA-190 expression leads in turn to FOXP2-mRNA and -protein increase. Because FoxP2 plays an important role in many oncological processes, the miR-190 could serve as a potential gastric cancer marker. Zhang et al. (2009) investigated various miRNAs and identified eight of them, which levels were increased in pancreatic cancer, e.g. the miR-190. This miRNA correlated with the progression of gliomas (Almog et al., 2012; Cuiffo et al.,2014) demonstrated suppression of FOXP2 expression by miR-199a in breast cancer mesenchymal cells. In lung, breast, bladder, colerectal and liver cancers MiRNA expression was also increased. (Ichimi et al., 2009; Lowery et al., 2009; Navon et al., 2009; Ura et al., 2009; Ng et al., 2009)

The results indicate that miRNAs play a different role in different tumor types at different stages. The influence of various miRNAs on pancreatic cancer (Zhang et al., 2009), lymphoma (Musilova and Mraz, 2014; Jones et al., 2014) and breast cancer (Wu et al., 2009) was also shown. It would be useful to study whether this miRNAs dysregulation also influences these and other cancers in connection with FOXP2?

Regulation of other FOXP genes by various factors on the example of carcinogenic processes In oncological tissue FoxP genes as well as other FOX genes can be modulated by microRNA . (Zheng et al., 2015), (Shao et al., 2013), (Kong et al., 2013), (Zhang et al., 2015), (Kundu et al., 2016), (Mei et al., 2015), (Yu et al., 2017). This modulation determines whether FOXP2 acts as an oncogene or as a tumor suppressor gene. In these processes the FoxP genes can also be deactivated by micro RNAs. (Choi et, 2016) According to Chang et al. (2007), He et al. (2010) and Raver-Shapira et al. (2007) the transcription factor p53 induces the transcription of the microRNA miR-34s, which in turn suppresses anti-apoptotic genes, which are required for cell development and growth. According to Choi et al. (2016), Isken et al. (2008), Mraz et al. (2009), Zenz et al. (2009), miR-34a plays an important role in both chronic lymphatic and acute myeloid leukaemia and the Foxp1 is regulated by miR-34a. The miR-34a also builds a direct link between the tumor suppressor p53 and the oncoprotein Bcl-2. Rao et al. (2010) pointed out that in B lymphocytes too miR-34 modulates Foxp1 via the tumor suppressor gene p53. The tumor suppressor p53 could also be a direct FOXP2 target gene. (Herrero and Gitton, 2018)There are also further indications of androgen influences on the FoxP1 expression and associated carcinogenic processes. (Banham et al, 2007; Bates et al., 2008; Fox et al., 2004; Giatromanolaki et al., 2006; Takayama et al., 2008) Further FOXP1 appears to affect follicular lymphoma as well as in aging dependent ovarian and colorectal carcinoma. (Brown et al., 2004; Choi et al., 2016; De Smedt et al., 2015) In B cell lymphomas or hepatocellular carcinomas FOXP-1 serves as an oncogene. (Zhang et al., 2012; Jiang et al., 2012; Xia et al., 2014) In lungs, prostate and endometrial tumors FOXP1 is a potential predictor of disease progression. (Feng et al., 2012; Toma et al., 2011; Giatromanolaki et al., 2006; Takayama et al., 2008; Takayama et al., 2014)

FOXP2 and neuroplasticity

FOXP2 plays an extremely important role in neuroplasticity (Haesler et al., 2006; Ebisu et al., 2017; Konopka et al., 2009; Boeckx et al., 2014; Dodson et al., 2015; Garcia-Calero et al., 2016) and review of Wohlgemuth et al. (2014) showed that FOXP2 also provided the NMDAR-mediated neuronal plasticity affecting MAPKK and tyrosine phosphatase. The MARK is a RAS downstream effector. RAS is a a GTPase that indirectly interacts with oncological relevant ant p21 and p53 indirectly. RAS, in turn, is activated by growth factor signalling and interacts with FOXO-inhibiting PIK3. Further interaction with neuro and oncoactive Trk is also discussed. (Santos, 2011), (Mengeselt et al., 2008) It influences cell proliferation and this plays a role in the development of cancer. A direct FOXO4-p53 interaction and its importance in cell aging has been demonstrated too. (Baar et al., 2017) It would be interesting to look more closely at the role of FOXP in these processes. Possibly Fisetin, related to Qis (Zhu et al., 2017), but also FOXO4-related peptide that inhibits the PI3K/AKT/p53/p21/Serpine SCAP (Zhu et al., 2015b), recently noted to be senolytic (Baar et al., 2017) could have positive effect on cancer and aging.

Guibinga et al. showed in "Neuropathogenesis of LNS. Striatal Neurodevelopment is Dysregulated in Purine Metabolism Deficiency and Impacts DARPP-32, BDNF / TrkB Expression and Signaling: New Insights on the Molecular and Cellular Basis of Lesch-Nyhan Syndrome" (2014) that downregulating Bcl11b in HPRT-deficient cells elicited a change in DARPP-32 level in the neural stem cells. This phosphoprotein is regulated by dopamine and cAMP. Its altered level in turn led to striatal B-cell leukaemia 11b (Bcl11b). The activator of the DARPP-32 striatal BDNF / TrkB signalling pathway was highly increased in HPRT-deficient cells as well as in the striatum of the HPRT knockout mice. This protected the cells from reactive oxygen species (ROS)-mediated cell death.

There are also FOXP2 interactions with members of the histone family (H2AFX; H3f3B), two heat shock proteins (Hsp25; Hsp90a) and Calcycline-binding proteins ARHGEF9, CtBP1 and effectors Gli1, Gli2 and Gli3, EGR1, IEGs, D1R and DARPP32. In addition, FOXP2 affects NMDAR-mediated neuronal plasticity, MAPKK and tyrosine phosphatase. (Mc Auley et al., 2017)

γH2AX foci in aged HSCs is increased (Rossi et al., 2007; Beerman et al., 2014; Moehrle et al., 2015; Flach et al., 2014)