KYNU, a novel potential target that underpins CD44‐promoted breast tumour cell invasion

Abstract Using a validated tetracycline‐off‐inducible CD44 expression system in mouse model, we have previously demonstrated that the hyaluronan (HA) receptor CD44 promotes breast cancer (BC) metastasis to the liver. To unravel the mechanisms that underpin CD44‐promoted BC cell invasion, RNA samples were isolated from two cell models: (a) a tetracycline (Tet)‐Off‐regulated expression system of the CD44s in MCF‐7 cells and; (b) as a complementary approach, the highly metastatic BC cells, MDA‐MB‐231, were cultured in the presence and absence of 50 µg/mL of HA. Kynureninase (KYNU), identified by Microarray analysis, was up‐regulated by 3‐fold upon induction and activation of CD44 by HA; this finding suggests that KYNU is a potential novel transcriptional target of CD44‐downtstream signalling. KYNU is a pyridoxal phosphate (PLP) dependent enzyme involved in the biosynthesis of NAD cofactors from tryptophan that has been associated with the onset and development of BC. This review will attempt to identify and discuss the findings supporting this hypothesis and the mechanisms linking KYNU cell invasion via CD44.


Abstract
Using a validated tetracycline-off-inducible CD44 expression system in mouse model, we have previously demonstrated that the hyaluronan (HA) receptor CD44 promotes breast cancer (BC) metastasis to the liver. To unravel the mechanisms that underpin CD44-promoted BC cell invasion, RNA samples were isolated from two cell models: as target genes that underpin CD44, along with their downstream signalling pathways. 7-9 Among these 200 genes, we have selected Kynureninase (KYNU) in order to provide and discuss lines of evidence from the literature, supporting our hypothesis that KYNU might be a novel transcriptional target of CD44-downstream signalling.
KYNU is a hydrolase involved in Tryptophan metabolism, contributing to the synthesis of NAD + cofactors via the Kynurenine pathway; a vital pathway of L-tryptophan catabolism in both bacteria as well as eukaryotes. 10 In the pathway, KYNU catalyses L-kynurenine (bacteria) and 3-hydroxy-L-kynurenine (3HK) (eukaryotes) through a pyridoxal-5′-phosphate (PLP) dependent mechanism, to produce anthranilic acid and 3-hydroxyanthranilic acid (3-HAA), respectively. 10 KYNU is expressed in almost all body organs, and in higher levels in the liver, the urinary bladder and the appendix. 11 KYNU is involved in various inflammatory and cardiovascular diseases, in addition to several types of cancers, acting via different pathways ( Figure 1). [12][13][14][15] Here we discuss the findings, from the literature, supporting the hypothesis that KYNU is a transcriptional target of CD44 as well as known signalling pathways linking the activation of CD44 by HA to the transactivation of KYNU in promoting breast tumour cell invasion.  16 KYNU, normally confined within the cytoplasm of the cells of various body tissues, requires the cofactor pyridoxal-5′-phosphate for its activity. 17 KYNU protein consists of 465 amino acids and exists as a homodimer structurally homologous to other members of the PLPdependent aspartate aminotransferase family. 18 Each monomer is composed of two regions: a small and a large domain, with a sizeable opening, containing the active site, formed at the junction between these domains in the dimerized form. 18 Like other members of this family, KYNU's active site features a conserved lysine, which forms the PLP-enzyme Schiff base, with a variety of nearby amino acids, maintaining the cofactor's proper orientation through hydrogen bonding. 18 Similarly, a conserved arginine appears to be critical to binding and orienting the substrate within the active site. 16 This binding results in a conformational change that puts strain on the substrate's bonds, which would be released upon hydrolysis. 16

| FUN C TI ON S OF K YNU
KYNU is involved in the biosynthesis of NAD + from tryptophan via the kynurenine pathway. 19 Specifically, it degrades kynurenine, a catabolite in tryptophan metabolism, into anthranilic acid.

| Physiological functions of KYNU in normal cells
In most mammalian cells, the KYNU pathway is the primary path of tryptophan metabolism, producing metabolites, such as kynurenic  20 Of these three, 3-HAA is the main product of this pathway and is eventually converted to NAD+ (21), while KYNA and XA appear to only be produced when KYNU is fully saturated. 21

| Functions of KYNU in vertebral, cardiac, renal and limb defects syndrome 2 (VCRL2)
KYNU is linked with tryptophan utilization and metabolic diseases, including vertebral, cardiac, renal and limb defects syndrome 2 (VCRL2), 19 19 It was found that the elevated levels of niacin in mice were plausibly transferred from their mothers, thus, providing a protective effect on genetic-based NAD paucity. 19 To sum up, the kynurenine pathway synthesis of NAD + is essential and mutations in KYNU leads to congenital malfunctions and inviable embryos. 19

| Functions of KYNU in breast cancer and its association to CD44-signalling
Although KYNU is often associated with metabolic diseases, its role in cancer lies nascent and only a few studies have investigated the link between KYNU and CD44, and its association with cancer. One of the key pathways dysregulated in cancer is the PI3K/AKT pathway; this pathway regulates various physiological functions, including cellular migration, invasion and cell survival. 22 Another study analysing inflammation-associated mechanisms, which are regulating and blocking the TNF-induced signal transduction in primary human monocytes, identified KYNU as one of the proteins linked to nuclear factor κB (NF-κB) pathway. 25 Moreover, NF-κB plays a crucial role in tumour invasion and metastasis, 26 thus suggesting a role of KYNU in promoting tumour cell invasion. The study also reported that long-term incubation of cells in TNF correlated with increased expression of KYNU and increased phosphorylation of CD44. 25 Similarly, to the dual controversial role of many other genes (eg p53), while, some studies support its oncogenic role, 27-33 a few studies have also shown its role as a tumour suppressor. 14,34 The first study by Rose et al, (1967) reported increased activity of the kynurenine pathway, along with increased KYNU, KMO and kynurenine aminotransferase-II activity in BC patients, 33 indicating an oncogenic role of KYNU. Further, microarray data from invasive BC patients showed differential expression of KYNU in the BC subtypes. 28,30 While, no change in KYNU expression was observed in the luminal subtypes, KYNU expression was enhanced in the HER2-positive, claudin low and aggressive basal BC subtypes, 28

| P OTENTIAL INHIB ITOR S OF K YNU
KYNU may play a role in underlying mechanisms resulting in the production of the excitotoxin moiety quinolinic acid (QUIN), which is a metabolite of tryptophan that has been shown to be a neurotoxin. 37 Several studies have been carried out to develop suitable inhibitors targeting KYNU in order to guide the design of appropriate therapeutic strategies in bacteria as well as mammals.
In fact, few of the bacterial KYNU inhibitors that mimic the mo-

| CON CLUS ION
KYNU appears to play a major role in the development and dissemination of breast tumours, but its underlying mechanisms are still poorly understood. KYNU interacts with several signalling pathways that promote breast tumour cell invasion and metastasis. In particular, findings from our own work and others support our hypothesis that CD44-HA interaction might activate NF-kB, which in turn transactivate KYNU, ultimately leading to BC cell invasion ( Figure 2).
On the other hand, another finding from our own previous study identified the PI3K pathway as a potential molecular link between HA/CD44 activation and survivin transcription, 42

ACK N OWLED G EM ENTS
This research was funded by Qatar University Internal grant number: QUST-1-CAS2019-22, Qatar Foundation grant number: UREP24-117-1-027 and APC. Open Access funding was provided by the Qatar National Library.

CO N FLI C T O F I NTE R E S T
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Data sharing is not applicable to this article as no new data or datasets were created, generated or analysed in this study.