AJUBA: A regulator of epidermal homeostasis and cancer

The epidermis, outermost layer of the skin, is constantly renewing itself through proliferative and differentiation processes. These processes are vital to maintain proper epidermal integrity during skin development and homeostasis and for preventing skin diseases and cancers. The biological mechanisms that permit this balancing act are vast, where individual pathway regulators are known, but the exact regulatory control and cross‐talk between simultaneously turning one biological pathway on and an opposing one off remain elusive. This review explores the diverse roles the scaffolding protein AJUBA plays during epidermal homeostasis and cancer. Initially identified for its role in promoting meiotic progression in oocytes through Grb2 and MAP kinase activity, AJUBA also maintains cytoskeletal tension permitting epidermal tissue development and responds to retinoic acid committing cells to initiate development of surface epidermal layer. AJUBA regulates proliferation of skin stem cells through Hippo and Wnt signalling and encourages mitotic commitment through Aurora‐A, Aurora‐B and CDK1. In addition, AJUBA also induces epidermal differentiation to maintain appropriate epidermal thickness and barrier function by activating Notch signalling and stabilizing catenins and actin during cellular remodelling. AJUBA also plays an imperative context‐dependent tumor‐promoting and tumor‐suppressive role within epithelial cancers. AJUBA’s abundant roles within the epidermis signify its importance as a molecular switchboard, vetting multiple signalling pathways to control epidermal biology.


| INTRODUC TI ON
The epidermis is the outermost layer of the skin and is comprised of a basal and suprabasal layers. 1,2 The entire epidermis renews every 2 weeks, and epidermal stem cells play a critical role in maintaining tissue homeostasis by ensuring replacement of cells that are lost during tissue turnover or following wounding. 2 Differentiation is an integral process of renewal, whereby cells transition through four keratinocyte layers: basal, spinous, granular and cornified -from stemlike to most differentiated cellular state. 3 Onset of differentiation is triggered by basal cell detachment from the underlying basement membrane, followed by intensive cell remodelling and stop of proliferation, culminating in terminal cell differentiation into highly crosslinked epidermal scales that are exfoliated from the surface of the skin. 4 While functionally mimicking human skin, murine skin has several structural differences such as epidermal and dermal thickness and hair follicle density. [5][6][7] Maintaining the epidermal primary function as a barrier is of uttermost importance to protect against mechanical impacts and pressure, variations in temperature, micro-organisms, radiation and chemicals and to prevent dehydration. As such, maintaining a properly functioning stem cell pool while at the same time ensuring highly balanced differentiation is regulated by a plethora of signalling pathways and cellular mechanisms. For example, Notch signalling plays a critical role in inducing epidermal differentiation and Notch pathway components are differentially expressed within the different keratinocyte layers. 8 It is well-documented that basal keratinocytes express Delta1(Dll1), while Jagged1 and 2 are expressed in suprabasal cell layers within murine epidermis. 1,9 Jagged1 and 2 are critical NOTCH receptor ligands, stimulating differentiation and stratification of suprabasal keratinocytes, where most studies find expression of NOTCH receptors. 1,[9][10][11][12] On the other hand, Dll1 participates in cis-inhibition of Notch signalling preventing differentiation in the basal layer. 9,[13][14][15] Thus, NOTCH ligands orchestrates cellular differentiation of the spinous cells by binding to NOTCH receptors, which triggers NOTCH cleavage and release of the NOTCH intracellular domain (NICD), which translocates to the nucleus and binds the CSL/RBPJ transcription factor complex to activate NOTCH target gene expression required for keratinocyte differentiation. 11 The preferential expression of NOTCH pathway proteins has recently been validated via single cell RNA sequencing (online interface for scRNAseq within stratified murine epidermis: https://kaspe rlab.org/ mouse skin). 9 Regeneration and controlled proliferation of epidermal stem cells within the basal layer is required to consistently renew the epidermis. Regeneration is governed by several signalling pathways, with Hippo signalling taking a centre stage. Localization of YAP/TAZ, the transcriptional co-activators of the Hippo signalling pathway, is regulated by LATS1/2 kinase-induced phosphorylation of YAP/TAZ. 4,[16][17][18][19] LATS-induced phosphorylation inhibits the transcription factors by triggering their cytoplasmic retention and prohibiting nuclear translocation. In addition, YAP/TAZ are also regulated by expression patterns of α-catenin, adherens junctions and actin, respectively. 4,[16][17][18][19] Cytoskeletal components relay extracellular matrix stiffness and cell-cell interactions and regulate YAP/TAZ nuclear translocation by several mechanisms such as binding and sequestering Hippo components to cell-cell or cell-matrix attachment sites, actin-stress induced JNK/c-Jun activation to promote LIMD1-LATS1 binding or direct inhibitory phosphorylation of LATS1 by Src in response to integrin-and FAKactivation. [20][21][22][23][24] The core Hippo cascade proteins are expressed throughout the keratinocyte layers. Within the single layer of basal keratinocytes, YAP is almost exclusively localized within the nucleus indicative of proliferating cells, 19 while nuclear accumulation of YAP decreases with increasing differentiation with YAP being exclusively cytoplasmic in granular layers. 19 Nuclear localization of YAP triggers expression of Hippo target genes, promoting both symmetric (=self-renewal) and asymmetric cell division of basal keratinocytes. [25][26][27] As such, Hippo and Notch signalling appear to have opposing functions -the YIN and YANG of skin biology.
Overall, having a balance of basal cell proliferation and suprabasal cell stratification is not only important for normal function of the skin as protective barrier, but also to prevent tumor initiation. This review summarizes AJUBA as a key gene linking the multitude of signalling pathways such as Hippo and Notch signalling within this balancing act. Since its discovery, AJUBA was shown to function as a scaffolding protein participating in the assembly of a multitude of signalling pathways. As such, it acts as a signalling hub assembling diverse signalling pathways, strategically regulating selectivity and forming cross-talks between signalling pathways. While AJUBA's precise role in signalling cascades has been recently reviewed elsewhere, 28 we here concentrate on AJUBA's physiological role during epidermal stem cell biology and carcinogenesis.

| A J UBA' S DISCOVERY
AJUBA ("curiosity" in Urdu, an Indian dialect) was first discovered in a study looking for interactors of erythropoietin receptor (EPO-R) using yeast two-hybrid screening. 29 This study identified the first functional role of AJUBA, demonstrating that it associates with Grb2 to enhance MAP kinase signalling and that it correlates with meiotic progression within mature Xenopus oocytes. 29 AJUBA, is a 58 kDa protein and belongs to the group 3 LIM-domain containing proteins alongside zyxin, LPP, Trip6, and LIMD1. Group 3 proteins contain 3-4 LIM domains at the C terminus that are rich in cysteine and histidine residues and are known to mediate protein-protein interaction (PPI) as well as distinct N-terminal domains (preLIM domain). 29 Specifically, AJUBA contains 3 tandem C-terminal LIMdomains. LIM-domains are relatively promiscuous in the sense that they tend to physically interact with a large number of proteins such as enzymes, receptors, cytoskeletal proteins thereby participating in several signalling pathways, making it difficult to mechanistically unravel their function. 29 AJUBA's preLIM domain is abundant in glycine and proline residues, a unique feature distinguishing AJUBA from other group 3 members. Specifically, the N-terminus contains two stretches of proline-rich SH3 recognition motifs, another PPI domain, and a nuclear export signal. 29 Within mammals, AJUBA LIM family proteins include the related AJUBA (JUB), LIMD1, and WTIP. Drosophila has a single homolog, djub, with greatest sequence similarity to mammalian AJUBA. 30 These homologs are tightly conserved. AJUBA resides on chromosome 14 in humans and mice and on the X chromosome in drosophila.

| A J UBA' S ROLE DURING DE VELOPMENT
In development, AJUBA is present in all embryonic germ layers and within foetal components of the developing placenta. 31 Expression of AJUBA dramatically decreases at post-embryonic day 12.5 being limited to skin epidermis, oral mucosa, nervous system and the genitourinary tract. 31 AJUBA-deficient mice are viable with no phenotypic effect on a mixed C57BL/6 J-129 X1/SvJ background. 32 However, AJUBA-deficiency C57BL/6 J mice are embryonic lethal, 33 indicating that murine background influences the phenotype of AJUBA loss. 34 Similarly, AJUBA-deficiency Drosophila melanogaster flies are embryonic lethal. 35 During early murine development, cross-talk between dermis and epidermis lead to development of the basement membrane. 4 The ectoderm undergoes FGF signalling by BMP activated of SMAD pathway to commit the ectoderm to epidermal fate, differentiating into keratin-expressing cells, forming the basal layer of the epidermis. 4,36,37 During mid-gestation, typically at embryonic day (E) 9.5 in a mouse embryo, the epidermis forms as a single-layer surface ectoderm ( Figure 1A). [38][39][40][41] A periderm develops in early stratification to protect the single layer from constant exposure to the amniotic fluid during E10-E12 ( Figure 1A). 42,43 Murine stratification is developed through asymmetric division ending with terminally differentiated, cornified cells; a phenomenon not prominent in human epidermis. 43,44 AJUBA has been shown to be involved in several signalling cascades and aspects of epidermal development ( Figure 1A).

| AJUBA communicates cytoskeletal tension into a Hippo Signalling response during development
During embryogenesis and tissue morphogenesis, mechanical forces as well as extrinsic mechanical stress induce changes of cell shape, size, position and gene expression. During these dynamic processes, adhesive complexes between cells are constantly resolve and establish anew. 45 Adherens junctions are one of the main molecular complexes responsible for cell-cell recognition and interaction, force transmission, force sensing and force generation. Adherens junctions are composed of cadherins, which are transmembrane proteins that operate as homo-or heterophilic cell-cell adhesion receptors and link to the actin cytoskeleton through p120-, α-and β-catenins. 45 Adherens junctions have several cadherin-catenin clusters regulating cell-cell intercalation and invagination during tissue remodelling. 45 These processes determine cellular fate, define embryonic compartments, and initiate epithelial development. During tissue morphogenesis, adhesion junctions have been reported to interact with microtubules in Drosophila (whereby microtubules are reported F I G U R E 1 AJUBA during epidermal development. A, Stratification of the epidermis begins at E9.5 in murine tissues, whereby direct signalling from the dermal tissue promotes initiation of stratification. AJUBA is expressed throughout keratinocyte layers, predominantly localizing to the cytoplasm and membrane and enriched in the basal layer compared to suprabasal epidermal layers. The localization patterns of AJUBA are speculated to correspond to its varying roles and gene expression profiles of the stratified epidermis. B, AJUBA is required for epidermal tissue development. Specifically, AJUBA is found at cellular compartment boundaries during drosophila wing development and binds cytoskeletal components and Wts increasing tension, driving wing growth. C, During ectoderm development, AJUBA localizes to the nucleus in response to increased retinoic acid (RA). Nuclear AJUBA was correlated with an increase in c-Jun and JNK proteins that permit differentiation into the epidermis to strengthen these cell-cell junctions specifically by regulating myosin II and β-catenin 46 ), myosin and several regulatory proteins to diversify cellular outcomes. 45 The Drosophila AJUBA homolog, Jub, plays a crucial role in the developing embryo by interacting with adherens junctions (and complexed proteins) to regulate epithelial closure and wing development ( Figure 1B).
During Drosophila development, Jub is recruited to adherens junctions in response to high actin-myosin contractility. 47 This recruitment regulates the Hippo pathway, for example during the morphogenesis of the Drosophila wing, a tissue composed of epidermal cells. 48 Wing development is dependent on the phosphorylation of Myosin II by Rho-associated kinase and the increased Myosin activity triggers recruitment of Jub to E-cadherin via α-catenin. 49

| Retinoic Acid promotes nuclear accumulation of AJUBA during ectoderm differentiation
AJUBA has also been shown to be regulated by Retinoic acid (RA) and to mediate RA-induced differentiation ( Figure 1C). RA functions as a ligand for the retinoic acid receptor (RAR) and retinoid X receptors (RXR), which upon RA binding heterodimerize in the nucleus, altering the transcriptional activity of these transcription factors from repressors to activators of transcription. 54

| A J UBA MAINTAINS ADULT EPIDERMAL HOMEOS TA S IS
During adulthood, AJUBA is broadly expressed across human tissues (although completely absent in bone marrow), but is most abundantly expressed within the skin ( Figure 1A). 31,60

| Hippo signalling
AJUBA's best characterized role is its negative regulation of the Hippo signalling pathway (Figure 2A). 61 The Hippo pathway is highly conserved and best known for regulating tissue and organ size.
Hippo signalling is also required in many other processes, such as epithelial to mesenchymal transition and cellular polarity. 14,20,[62][63][64][65][66] The Hippo pathway was initially characterized in Drosophila utilizing larval wing and eye imaginal disc epithelium. 67 The core of this path- to Aurora-A's C-and N-terminus, respectively ( Figure 2B). 73 In 293 T cells, the binding of AJUBA to Aurora-A prevents auto-inhibition of Aurora-A, thus AJUBA binding is required for phosphorylation of Aurora-A and mitotic commitment ( Figure 2B). 74 Corroborating these F I G U R E 2 Basal keratinocyte proliferation involves AJUBA's participation in several signalling pathways. A, Nuclear YAP localization and subsequent cellular proliferation. This can occur in a hippo-dependent manner by AJUBA binding WW145 and Lats1/2 or hippo-independent manner through EGFR signal transduction to permit nuclear YAP/ TAZ. B, AJUBA binds to Aurora-kinases to commit cells to mitosis to replenish the basal layer, promoting AURORA-A self-phosphorylation. C, AJUBA facilitates the binding of GSK and β-catenin, leading to β-catenin phosphorylation and subsequent degradation. Absence of AJUBA leads to cellular proliferation through sustained WNT signalling pathway findings, in Drosophila neuroblasts, Ajuba was shown to localize at mitotic poles with Aurora-A. 35 While AJUBA was clearly shown to be involved in mitotic checkpoint, chromosome dynamics, and cytokinesis within epithelial cells, this was not yet explored specifically within the epidermis. 70 In HeLa and COS-1 cells, AJUBA also forms a complex with Aurora-B and BUBR1 kinases at kinetochores. 75 Specifically, AJUBA binds via its PreLIM domain to microtubules and kinetochores during metaphase and anaphase respectively, aiding in metaphase to anaphase transition. 75 In addition, AJUBA was shown to be regulated by the cyclin-dependent kinase CDK1. CDK1 bound to cyclin B governs cellular mitosis, but CDK1 can compensate for other CDK proteins by binding with all cyclins. [76][77][78] AJUBA was found to be phosphorylated and regulated by CDK1 during prometaphase/metaphase of mitosis. 79 Interestingly, binding of AJUBA with LATS1 occurs regardless of the phosphorylation status of AJUBA and thus Hippo pathway activation seems to be independent of AJUBA phosphorylation status. This also suggests that mitotic phosphorylation of AJUBA controls cell cycle independent of Hippo, albeit one has to mention that AJUBA itself had no effect on Hippo pathway activation in the examined cells (HPNE and RCA) and might thus be context-specific. 79 Mitotic phosphorylation of AJUBA is essential for AJUBA to promote cellular proliferation, anchorage intendent growth and tumor growth. Together, these data establish AJUBA as an oncogene promoting cell survival by regulating cell cycle. While this phenomenon has yet to be explored in keratinocytes (murine nor human), these data show that AJUBA is tightly involved in cell cycle control especially during various aspects of mitosis.

| Wnt signalling
Canonical WNT signalling involves Wnt-ligands binding to Frizzled receptors and Wnt-co-receptors such as LRP5/6 to initiate a signal cascade that result in accumulation of β-catenin and its nuclear translocation, where β-catenin acts as co-activator of TCF/LEF family transcription factors ( Figure 2C). 43  Linking these processes acts like a fail-safe mechanism. 82 AJUBA acts as a negative regulator of Wnt signalling pathway ( Figure 2C). GSK-3β is responsible for phosphorylating β-catenin, tagging it for degradation. AJUBA binds to both β-catenin and GSK-3β, thus promoting β-catenin phosphorylation through proximity. 83 Thus, AJUBA leads to the degradation of β-catenin. Further studies show that upon stimulation of Wnt signalling pathway AJUBA undergoes proteasomal degradation. 83 Suggesting that AJUBA acts as a molecular switch between Wnt-β-catenin and Hippo-YAP induced proliferation. However, the exact relationship between AJUBA-Wnt-Hippo remains elusive.

| Notch signalling
Notch signalling is responsible for differentiation and maintaining the differentiated squamous layers. NOTCH receptors are was shown to directly bind NOTCH1/2, NICD1/2, and NUMB, a negative regulator of Notch signalling, 33 in primary mouse keratinocytes ( Figure 3A). AJUBA promotes differentiation by selectively sequestering NUMB, thereby blocking NUMBs association with NOTCH1/NICD and NUMB-mediated recruitment of the E3 ubiquitin-protein ligase ITCH to NOTCH/NICD, which is required for NOTCH/NICD's proteasomal degradation 84 (Figure 3A). Upon ligand-induced NOTCH activation, AJUBA knockout keratinocytes showed impaired nuclear translocation of NICD1/2 and transcription of canonical NOTCH downstream targets. Together, these data suggest that AJUBA permits cells to undergo differentiation by sequestering NUMB and permitting NOTCH transcriptional targets to be upregulated. 33 The depiction of NOTCH, Hippo and WNT signalling not being correlated (section 3.1.3) may also connect to AJUBA regulation and it would be interesting to explore cross-talk between AJUBA, NOTCH, Hippo and WNT, especially in cellular decision-making during epidermal stratification.

| Cell adhesion mediated epidermal remodelling
Adherens junctions are cadherin-catenin complexes that link/connect F-actin cytoskeleton to the plasma membrane and form cell-cell linkages throughout the stratified epidermis. 16,45,47,[85][86][87] E-cadherin, the most abundant cadherin within the epidermis, forms a complex between β-catenin and p120-catenin to α-catenin, which holds F-actin in place. 87 Desmosomes are other cell junction complexes that link desmosomal cadherins (desmocollin and desmoglein) with the catenin paralogs, plakoglobin and plakophilin, to intermediate filaments of keratins to strengthen the epidermis and have been implicated in oral lesions, thickened skin, and severe blistering when expression is altered. 16,[88][89][90] Alterations in adhesion and cytoskeletal components within the epidermis leads to specific stratification phenotypes. 47 Specifically, loss of E-cadherin within the epidermis leads to hyperproliferation of the basal layer, loss of p120-catenin leads to altered epidermal inflammatory responses, loss of β-catenin leads to hair follicle defects (while plakoglobin replaces β-catenin within the epidermis to maintain stratification) and loss of α-catenin is linked to loss of epidermal cell polarity and leads to the dissociation of cells. 16,85,86,91 Little overlap in phenotypes suggests important independent roles for maintaining keratinocyte differentiation.
In primary mouse keratinocyte, AJUBA was shown to interact with α-catenin and is recruited to cadherin-dependent cell-cell adhesion complexes ( Figure 3B). 50 AJUBA also interacts directly with F-actin through its PreLIM domain, while simultaneously binding to α-catenin ( Figure 3B). 50 Keratinocytes from AJUBA-null mice exhibit abnormal cell-cell junction formation and/or stability and function. 50 In addition, AJUBA has been shown to dynamically interact with several cellular adhesion molecules during remodelling and migration to control Rac activation. In actin remodelling, AJUBA induces Rac activation and maintains E-cadherin adhesion in human keratinocytes. 92 AJUBA is required to stabilize pre-formed adherens junctions by recruiting F-actin ( Figure 3C). F-actin-AJUBA complex binds to activated Rac via the PreLIM domain on AJUBA ( Figure 3C).

| A J UBA' S ROLE IN C AN CER S TEM CELL S OF EPIDERMAL C AN CER S
Cancer stem cells are a small subpopulation of cells within tumors with self-renewal as well as differentiation capabilities and can initiate a tumor when transplanted into a recipient animal. This section will explore AJUBA as a double-edged sword in epidermal cancer stem cell biology, with tumor-suppressive and tumor-promoting functions ( Figure 4 and Table 1). 94  Together, these data indicate that alterations of AJUBA alterations might contribute to ~18% of HNSCC and cSCC by blocking proper Notch signalling and that NOTCH inactivation is a hallmark of HNSCC and cSCC initiation. F I G U R E 4 AJUBA functions as a tumor suppressor or promoter in cancers throughout the human body. Brief illustrations summarizing AJUBA's involvement in different organ's during tumor initiation, growth and metastasis. This schematic highlights the context-dependent manner in which AJUBA influences molecular signalling pathways throughout the human body. Whereby, green boxes represent tumorpromoting roles and red boxes represent tumor-suppressive roles of AJUBA. AJUBA functions within signalling pathways to promote cancer. In epidermal squamous cell carcinoma (ESCC), AJUBA stabilizes Grb2 promoting RAS/ERK-dependent MMP10/13 expression, increasing migration and invasion. In colon cancer, AJUBA promotes migration by upregulating N-cadherin via Smad1/SNAIL. AJUBA promotes cancer within the nucleus through its role as a co-activator. In breast cancer, AJUBA acts as a co-activator of estrogen receptor (ER) to promote cellular growth. In pancreatic cancer, AJUBA generates a positive feedback loop between SP1/GC transcription of EGFR to increase cellular proliferation. AJUBA was shown to be influenced by miRNA in lung cancer leading to increased invasive and migratory cellular properties. AJUBA has also been linked to patient drug resistance in cervical cancer. As a tumor suppressor, loss of AJUBA in head and neck squamous cell carcinoma (HNSCC) affects the Notch signalling pathway, loss of AJUBA increases growth and invasive properties of cells in hepatocyte cancer by linking E-cadherin and Hippo signalling, and lastly in prostate cancer, androgen receptor causes miRNA-dependent depletion of AJUBA leading to increased migration and metastasis

| AJUBA's functions in oesophageal squamous cell carcinoma
Oesophageal cancer is the 8 th most frequently diagnosed cancer worldwide and oesophageal squamous cell carcinoma (ESCC) is the most common subtype, accounting for over 90% of cases. [97][98][99] Smoking and alcohol consumption is associated with increased risk. 99 The only familial link is a genetic abnormality at chromosome 17q25. 99

| AJUBA's function in other cancers
In addition to squamous cell carcinomas of the skin and the upper aerodigestive tract, AJUBA has been implicated in several epithelial cancers, working through a multitude of mechanisms summarized in Table 1. AJUBA can function as an oncogene to promote cell proliferation and migration. In colorectal cancer AJUBA was found to be a to induce expression of TFF1, Greb1 and SGK3 (ERα target genes), 109 respectively. Similar promotion of migration is seen in lung cancer.
Precisely, miR-193b-3p and −5p inhibition led to increased metastatic potential and both miRNA's decreased AJUBA expression resulting in a decrease in migration, proliferation and colony-forming  activity of lung cancer cell lines. 110 Lastly, cervical cancer patients displaying high AJUBA expression positively correlated with resistance to cisplatin treatment via YAP/TAZ upregulation. 111 AJUBA can also functions as a tumor suppressor in some cancers inhibiting cell growth and migration by modulating several cellular components and proteins. AJUBA overexpression was shown to inhibit hepatocellular carcinoma cell proliferation by diminishing β-catenin and YAP levels, preventing their translocation and expression of Cyclin D1 and CYR61, respectively. 112 Loss of AJUBA also enhanced migration and is correlated to prostate cancer metastasis. 113 In prostate cancer, overexpression of miR-193a-3p, an androgen receptor binding miRNA, induces migration of LNCaP and C4-2B cells by downregulating its target AJUBA. 113 These studies highlight the complexity in pinpointing the exact mechanism of AJUBA in a cancer context ( Figure 4) and lead to more questions -does AJUBA function through multiple signalling pathways and mechanisms and is its function dependent on the genetic make-up of a given tumor?

| CON CLUS ION
The adaptor protein AJUBA functions as a key molecular switch fine-

ACK N OWLED G M ENTS
This work was supported by a project grant from the Ontario Research Fund Research Excellence Round 8 (RE08-065).

CO N FLI C T S O F I NTE R E S T
The authors declare no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
KS wrote the manuscript under the supervision and guidance of DS.
All authors who contributed to the article have read and approved the final manuscript.