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The role of Popeye domain-containing protein 1 (POPDC1) in the progression of the malignant phenotype

The Popeye domain-containing protein 1 (POPDC1), a tight junction-associated transmembrane protein with a unique binding site for cAMP, has been shown to act as a tumour suppressor in cancer cells. Through interaction with many downstream effectors and signalling pathways, POPDC1 promotes cell adhesion and inhibits uncontrolled cell proliferation, epithelial-to-mesenchymal transition and metastasis. However, POPDC1 expression is down-regulated in many types of cancer, thereby reducing its tumour-suppressive actions. This review discusses the role of POPDC1 in the progression of the malignant phenotype and highlights the broad range of benefits POPDC1 stabilisation may achieve therapeutically. Cancer stem cells (CSCs) are a key hallmark of malignancies and commonly promote treatment resistance. This article provides a comprehensive overview of CSC signalling mechanisms, many of which have been shown to be regulated by POPDC1 in other cell types, thus suggesting an additional therapeutic benefit for POPDC1-stabilising anti-cancer drugs. This interaction has mainly been shown in cardiac and skeletal muscle cells; however, an increasing body of evidence is emerging that demonstrates POPDC1 interaction with these in cancer cells.


| INTRODUCTION
The Popeye domain-containing protein 1 (POPDC1) was discovered in 1999, and research over the last 20 years has shown it to be involved in the progression of malignancies. Initially discovered in cardiac tissue, POPDC1 (also known as blood vessel epicardial substance [BVES]) has a wide tissue distribution and is found in smooth and skeletal muscle, the brain, gastrointestinal (GI) tract, and liver (Han et al., 2019). It is also present in many cancers, such as breast cancer (BC), gastric cancer (GC), colorectal cancer (CRC), non-small cell lung carcinoma (NSCLC), hepatocellular carcinoma (HCC), and uveal melanoma (UM) (Han et al., 2019). Although POPDC1 proteins regulate signalling pathways in cardiac and skeletal muscle tissue (Brand, 2018), the focus of this review will be their role as tumour suppressors. Dysregulation of POPDC1 expression in cancer cells may offer an interesting and novel therapeutic target in the treatment of cancer and will be discussed in the following article.
1.1 | The structure of the POPDC1 protein Popeye domain-containing proteins exist in three isoforms: POPDC1, POPDC2, and POPDC3. Structurally, these isoforms differ in their amino acid chain length: 360, 364, and 291 residues, respectively (Brand & Schindler, 2017). It has been suggested that these three subtypes and their splice variants exist to facilitate tissue-specific target interactions, as a result of truncated binding regions (Benesh et al., 2013). In addition to the variations in amino acid chain length, the POPDC2 and POPDC3 isoforms differ in their intracellular phosphorylation sites (Brand & Schindler, 2017) and have only one extracellular glycosylation site at aa4 (UniProt Consortium, 2019, available at: https://www.uniprot.org/uniprot/Q9HBU9 and https://www. uniprot.org/uniprot/Q9HBV1). Interestingly, whereas the POPDC2 gene is located on 3q13.33, the POPDC1 and POPDC3 genes are found on the same chromosome (6q21), which suggests that the POPDC3 gene may have arisen as a result of a tandem duplication of the POPDC1 gene (Schindler et al., 2012).
POPDC1 has been extensively studied over the last 20 years since its discovery, while less work has been done on elucidating the role(s) POPDC2 and POPDC3 may play in health and disease.
POPDC2 and POPDC3 proteins are highly expressed in both skeletal muscle and cardiac tissue (Brand & Schindler, 2017), and various mutations in these isoforms have been identified that are associated with tissue dysfunction at these locations (Table 1) (Amunjela et al., 2019;Vissing et al., 2019). The data in Table 1 suggest that the action of POPDC2 seems more confined to striated muscle tissues (Brand, 2019), whereas POPDC3 is dysregulated across a range of malignant phenotypes. In line with their similar genetic origin outlined above, the role of POPDC3 resembles that of POPDC1 in cancer cells.
For example, down-regulation occurs in a similar manner (e.g., promoter hypermethylation), and POPDC3 has a tumoursuppressive role in GC (Kim et al., 2010). However, while POPDC1 shows tumour-suppressive properties across most cancers, POPDC3 seems to have more restricted oncogenic roles in BC, head and neck squamous cell carcinoma (HNSCC), oesophageal cancer, and lung cancer.
A summary of the data gathered on POPDC2 and POPDC3 to date is presented in Table 1. Due to the greater body of evidence and understanding of the POPDC1 protein, this review will focus on the role of POPDC1 in the progression of the malignant phenotype.
POPDC1 is a transmembrane protein ( Figure 1) and is present in all muscle types, various parts of the GI tract, and, most importantly for this review, many subtypes of cancer. POPDC1 has a short extracellular N-terminal domain (42 amino acids long) with two asparagine residues that are subject to N-glycosylation (residues 2 and 30) (UniProt Consortium, 2019, available at: https://www.uniprot.org/ uniprot/Q8NE79). Variation in the location of N-glycosylation sites (e.g., aa20 and aa27) (Amunjela et al., 2019;Knight et al., 2003), may occur in different splice variants of the POPDC1 protein. The level of glycosylation seems to differ between tissue types, with the extent of glycosylation shown to be greater in skeletal muscle and brain tissue than cardiac tissue, which may be of functional relevance (Brand, 2019;Vasavada et al., 2004). The extent of POPDC1 glycosylation in cancer cells and the functional relevance of this has yet to be investigated. (aa114-aa360). The intracellular CTD contains the highly conserved POPEYE domain (aa172-aa266), which contains the binding sites for cAMP and other downstream effectors, such as TREK-1, a potassium ion channel, caveolin-3 (CAV-3), a plasma membrane protein, the guanine exchange factors GEFT and GEFH, the PR61α subunit of protein phosphatase 2, and ZO-1, a tight junction (TJ)-associated protein (Brand, 2018;Parang et al., 2018) (Table 2).
The cAMP binding domain, also referred to as the phosphate binding cassette (PBC), consists of two highly conserved tetrapeptide sequences (DSPE and FQVT) that are linked by a sequence of variable length (Brand, 2018). The three-dimensional structure of the PBC is very similar to that of other cAMP-activated proteins such as PKA; however, there is a large difference in the amino acid sequence encoding this domain (Schindler et al., 2012), which may offer an avenue for therapeutic exploitation.
POPDC1 forms a homodimer in the cell membrane mediated by a disulfide bond established between intracellular cysteine residues. This is facilitated by two highly conserved lysine (K) residues at positions 272 and 273 (Kawaguchi et al., 2008;Russ et al., 2011).
Dimerisation seems to be essential for protein trafficking to the membrane as well as protein function whilst in the membrane leaflet. This is evidenced by loss or mutations of KK residues leading to reduced membrane localisation and loss of cell adhesion (Brand, 2018;Kawaguchi et al., 2008). However, some evidence has shown that POPDC1 can still form a homodimer in the absence of the KK motif (Schindler & Brand, 2016).
In vitro studies by Osler et al. (2005) using epicardial mesothelial cells showed that before cell-cell contact was established, POPDC1 was localised in the cytoplasm. Once contact between neighbouring cells formed, POPDC1 inserted itself into the cell membrane and colocalised with other junctional proteins, resulting in cell adhesion (Osler et al., 2005). Furthermore, POPDC1 proteins that are no longer inserted into the cell membrane (e.g., in BC or HCC cells) have been shown to accumulate intracellularly (Amunjela et al., 2019;Han et al., 2015;Russ et al., 2011), but it remains unclear whether this accumulation is a pathway to POPDC1 degradation or a progressive storage mechanism. The potential effect of nuclear POPDC1 on gene transcription has also yet to be elucidated (Amunjela et al., 2019).

| Hallmarks of cancer
The structure of Popeye domaincontaining protein 1 (POPDC1). POPDC1 is a transmembrane protein highly expressed in cardiac myocytes, skeletal muscle, and some cancer cells. The short extracellular N-terminal domain contains two glycosylation sites (asparagine residues at positions aa2 and aa30). This is followed by three membrane-spanning domains and a long intracellular C-terminal domain (CTD). The CTD contains the highly conserved POPEYE domain (aa172-aa276) with a unique cAMP binding site. Other binding sites exist for TREK1, VAMP3, Bnip3, LRP6, ZO-1, GEFH, CAV-3 (aa242-aa246), GEFT (aa250-aa300), and PR61α (aa330-aa345). A disulfide bridge forms between intracellular cysteine residues with lysine residues (aa272/aa273) critically required for this dimerisation, which promotes membrane stabilisation. Image created with BioRender.com nutrient supply allowing tumour growth), and tissue invasion and metastasis (Hanahan & Weinberg, 2000 Note: The POPDC1 protein interacts with many downstream including TREK1, CAV-3, VAMP3, GEFT, GEFH, ZO-1, occludin, Bnip3, LRP6, and PR61α. This interaction has mainly been shown in cardiac and skeletal muscle cells; however, an increasing body of evidence is emerging that demonstrates POPDC1 interaction with these targets in cancer cells.  Table 2). Although the interactions of POPDC1 with TREK1, CAV-3 and Bcl-2 and adenovirus E1B 19-kDa interacting protein 3 (Bnip3) has thus far mainly been demonstrated in cardiac and skeletal muscle function (Brand, 2019), recent studies have found that TREK1 is overexpressed in prostate cancer (PC) cells, CAV-3 is up-regulated in anaplastic thyroid carcinoma (Han et al., 2019), and Bnip3 levels are increased in BC and NSCLC (Lee & Paik, 2006). However, the presence of POPDC1 and interaction with these proteins in cancer cells has yet to be established. The known effects of POPDC1 on downstream targets in cancer cells will be discussed in this review.

| Down-regulation of POPDC1 in cancer cells
The down-regulation of POPDC1 proteins has been demonstrated in a large range of cancer cell types. Loss of POPDC1 expression has been proposed to be initiated by four main mechanisms. Firstly, hypermethylation of the cytosine-phosphate-guanine islands of the POPDC1 gene promoter has been demonstrated in a wide range of cancers (Table 3) and has hence been suggested as a biomarker for early detection of cancer (Parang et al., 2018). Secondly, the underexpression of microRNA (miRNA)-122 (Wang et al., 2014) and, thirdly, overexpression of netrin-1 (Han et al., 2015) are known to suppress POPDC1 expression. Finally, increased EGFR stimulation leads to a significant suppression of POPDC1 activity, most likely as a result of phosphorylation of the POPDC1 CTD (Amunjela & Tucker, 2017a).
The mechanisms implicated in different cancers are shown in Table 3.
The loss of POPDC1 expression and its membrane integration means it is no longer able to influence cell adhesion and interact with the various signalling pathways and proteins outlined above (Table 2). This article will discuss the effects this has on cancer cell proliferation, transformation, and migration.
F I G U R E 2 Regulatory roles of POPDC1 in cancer. POPDC1 acts as a tumour suppressor by influencing three main processes involved in cancer progression. Its interaction with tight junction-associated proteins (e.g., ZO-1, occludin, and ZONAB) and VAMP3 ensures maintenance of cell adhesion. Furthermore, POPDC1 inhibits uncontrolled cell proliferation through its interactions with guanine nucleotide exchange factors (GEFT/GEFH) and the c-Myc and Wnt signalling pathways. POPDC1 also plays an important role in suppressing cancer cell migration and metastasis. Loss of these regulatory roles exerted by POPDC1 can occur as a result of intracellular phosphorylation (through EGFR) or reduced gene expression due to promoter hypermethylation, reduced miRNA-122 expression, or increased netrin-1 activity. Image created with BioRender.com

| THE ROLE OF POPDC1 IN CANCER CELL PROLIFERATION
Uncontrolled cell proliferation is a key hallmark of cancer progression (Hanahan & Weinberg, 2000) and is often a result of dysregulated growth factor signalling. Not only do cancer cells increase their sensitivity to exogenous growth-stimulating factors, but they also acquire the ability to generate their own growth signals that act in an autocrine manner to promote proliferation. Moreover, signalling pathways in cancer cells often become dysregulated, resulting in reduced transcription of growth-inhibiting genes and increased transcription of growth-promoting genes (Hanahan & Weinberg, 2011). POPDC1 has been implicated in the regulation of numerous signalling pathways involved in cell proliferation. Previous studies have shown that, at least in BC cells, increased cAMP levels lead to higher POPDC1 expression and protein stabilisation, resulting in reduced cell proliferation and migration (Amunjela & Tucker, 2017b). The mechanisms of POPDC1-mediated control of cancer cell proliferation will be discussed in the following section.

| Interaction with ZO-1 and ZONAB
POPDC1 interacts with ZO-1 (aka TJ protein 1, TJP1) to maintain cell adhesion and TJ-associated signalling and to prevent migration . This interaction allows ZO-1 to recruit ZONAB/DbpA to the TJ and prevent its nuclear localisation (Amunjela et al., 2019). If

| Interaction with GEFT and GEFH
Rac/Cdc42-specific exchange factor, a guanine nucleotide exchange factor more commonly referred to as GEFT, can interact (via aa300-aa450) with POPDC1 via its C-terminal residues aa250-aa300 (Smith et al., 2008). GEFT preferentially binds to and increases Rac1 and Cdc42 (cell division control protein 42) activity, which promote cell proliferation. In addition, activation of Rac1 and Cdc42 disrupts cell adhesion and promotes cell migration, which is discussed later in this review. POPDC1 negatively regulates GEFT (Smith et al., 2008) and thereby reduces Rac1 and Cdc42 activity, supporting its role as a tumour suppressor. High GEFT activity has been demonstrated in brain tumours (Guo et al., 2003), but the GEFT/POPDC1 interaction has so far only been demonstrated in fibroblasts (NIH T3T cells) (Smith et al., 2008) and human corneal epithelia, where POPDC1 interacting with two different GEFs, GEFT and GEFH, seems to promote TJ formation by reducing RhoA activity (Russ et al., 2010;Russ et al., 2011). When epithelial cell confluence has been established through formation of desmosomal, tight and adherens junctions, the activation of RhoA signalling that stimulates further cell proliferation is inhibited by GEFH and cingulin (TJ-associated protein) (Parang et al., 2018;Russ et al., 2011). Hence, loss of TJ formation in cancer cells that under-express POPDC1 could re-establish RhoA signalling and induce cancer cell proliferation.

| c-Myc pathway
c-Myc is a pro-oncogenic transcription factor commonly over-  structural, and catalytic subunit. Once the S62 residue has been dephosphorylated, c-Myc is ubiquitinated and targeted for proteasomal degradation (Seshacharyulu et al., 2013;Yeh et al., 2004). The c-Myc pathway has long been recognised as a promising anti-cancer drug target, especially as the Myc oncogene is involved in driving many hallmarks of cancer (proliferation, suppressed apoptosis, and altered cell metabolism) and that it is dysregulated in over 50% of all cancers . However, targeting c-Myc specifically has proven difficult due to its nuclear localisation as a transcription factor. Consequently, other parts of the c-Myc pathway must be investigated for drug development . As described above, PP2A is crucial for c-Myc ubiquitination and subsequent degradation, and various approaches to increase PP2A activity are currently being investigated for therapeutic use (Remmerie & Janssens, 2019).
First data have suggested that POPDC1 interacts with the PR61α subunit of PP2A to increase c-Myc inactivation. Therefore, we hypothesise that stabilising POPDC1/PP2A interactions in cancer cells may be a novel therapeutic approach for promoting c-Myc degradation and reducing its effects on malignant progression.

| Wnt/β-catenin pathway
The Wnt/β-catenin pathway is a major cell signalling pathway where It also triggers transcription of genes involved in metastasis, which will be discussed later. The activation of the non-canonical pathway and its involvement in cancer cell metastasis will also be examined within this review.

| Bnip3
Bnip3 is a mitochondrial, membrane-bound protein that is part of the Bcl-2 family. The Bcl-2 protein family balances cell proliferation and apoptosis, a balance often dysregulated in cancer cells (Hanahan & Weinberg, 2011 (Lee & Paik, 2006). POPDC1 suppressed Bnip3 activity in cardiac myocytes, and loss of POPDC1 resulted in Bnip3 up-regulation and increased apoptosis (Kliminski et al., 2017). Further, reduced POPDC1 expression also reduced expression of Bnip3. However, the role of the POPDC1/Bnip3 interaction in cancer cells remains to be investigated.

| Outlook-Inhibiting cancer cell proliferation by stabilising POPDC1
From the data explored above, it is evident that increasing expression

| ROLE OF POPDC1 IN CELL ADHESION AND METASTASIS
Another important hallmark of cancer is the loss of cell adhesion, which allows cells to gain motility and spread to distant sites in the body (metastasise). Cancer cell metastasis presents one of the greatest hurdles in successful anti-cancer treatment, especially as secondary tumours preferentially arise in tissues that are very difficult to target with traditional therapy (e.g., brain and bone). It is estimated that metastasis is the cause of around 90% of cancer fatalities (Guan, 2015). This highlights why early intervention and prevention of metastasis is key to therapeutic success.
Metastasis occurs in four main stages: loss of cell adhesion; cytoskeletal remodelling and gain of cell motility through EMT; intravasation and entry into the vascular or lymphatic system; and finally, extravasation (re-entry into surrounding tissue at a distal site).
POPDC1 proteins have been shown to play an important role in cell adhesion and cancer cell metastasis. The following will review the various signalling mechanisms and downstream targets of POPDC1 implicated in these processes.

| Cell adhesion
As previously discussed, POPDC1 has been shown to play an impor-    (Aiello et al., 2018) to reach a quasi-mesenchymal state (Dongre & Weinberg, 2019). Instead of epithelial protein expression being completely suppressed, these cells express proteins associated with both the epithelial and mesenchymal state. The resulting quasimesenchymal phenotype seems to confer greater cell motility to allow metastasis than the fully mesenchymal phenotype (Aiello et al., 2018), and of course, these cells also display sufficient plasticity to return to an epithelial state when the secondary tissue is penetrated. Formation and acquisition of a mesenchymal phenotype in POPDC1 knockout cells were also observed by Kawaguchi et al. (2008). This is in accordance with the expression pattern associated with EMT induction described by Dongre and Weinberg (2019). Restoration of POPDC1 expression in CRC cell lines re-introduced an epithelial-like morphology , further supporting the findings that POPDC1 seems to play a key role in EMT switching.
POPDC1 proteins interact with VAMP3 via their CTD, which experiments have mapped to a region after aa118 (Hager et al., 2010). As previously discussed, high levels of β1-integrin proteins in the cell membrane are associated with the mesenchymal cell type (Dongre & Weinberg, 2019). The POPDC1/VAMP3 interaction creates equilibrium between β1-integrin integration and recycling in the cell membrane to maintain cell function. If POPDC1 expression is suppressed in cancer cells, this interaction is lost, and VAMP3-mediated recycling of β1 integrin is impaired. A disruption in this equilibrium leads to higher concentrations of β1 integrin in the cell membrane, which promotes migration. Preventing the loss of POPDC1 proteins in cancer cells to maintain its interaction with VAMP3 may potentially provide a means to reduce cancer cell migration.

| GEFT/Rac1/Cdc42
Furthermore, as described previously, POPDC1 has been shown to negatively regulate GEFT (Smith et al., 2008), which in turn leads to reduced activation of Rac1 and Cdc42. These are members of the Rho family of GTPases and have been shown to induce membrane ruffling and lamellipodia, as well as filopodia and formation of actin microspikes (Guo et al., 2003). These processes increase cell motility and promote cancer metastasis. Moreover, Rac1 and Cdc42 increase the transcriptional activities of serum response elements (SREs) (e.g., Elk1 and SAP1) of the c-fos proto-oncogene, which promote proliferation and induce EMT. As increased GEFT activity has been shown to induce lamellipodia, filopodia, and actin cytoskeleton rearrangement through Rac1 and Cdc42 activity (Guo et al., 2003), preventing this activity by inhibiting the POPDC1/GEFT interaction may be beneficial to reduce cancer cell metastasis.
Despite Guo et al. (2003) showing that GEFT preferentially activates Rac1 and Cdc42 over RhoA, RhoA can be activated via other mechanisms, such as TJ downstream signalling pathways and the TJassociated GEFH . RhoA activates ROCK, which in turn stimulates myosin light chain kinase (MLCK), causing cytoskeletal rearrangement and allowing cell migration .
GEFH is usually sequestered to the TJ (via POPDC1 interaction) where it cannot activate RhoA (Han et al., 2015;Parang et al., 2018).
Down-regulation of POPDC1 and loss of TJ formation in cancer cells results in increased RhoA activation through the mechanisms outlined above, thereby triggering migration.
Studies by Wang et al. (2014) have identified that miRNA-122 is under-expressed in HCC cells. Besides leading to down-regulation of POPDC1 (discussed previously), their studies also showed miRNA-122 to increase RhoA signalling and subsequently induce EMT.
Hence, inhibiting the loss of miRNA-122 expression in cancer cells could therefore prevent metastasis not only by stabilising POPDC1 and maintaining the integrity of TJs but also by dampening down RhoA-induced EMT. The activation of the non-canonical Wnt signalling pathway has also been shown to activate both Rac1 and RhoA as a result of ROR-Fzd receptor complex activation (see Section 2.4).
This switches on RhoA and Rac1 signalling, which activates the ROCK signalling cascade, whilst the latter activation of the G q -coupled Wnt5a receptor triggers an increase in intracellular Ca 2+ concentration (Zhan et al., 2017). All of the above trigger changes in actin polymerisation and cytoskeletal rearrangement, facilitating cell motility and metastasis.
In summary, combined activation of Wnt signalling, RhoA, Rac1, and Cdc42 promotes EMT, migration, invasion, angiogenesis, and cell proliferation (discussed previously). Hence, inhibiting GEFT, Cdc42, Rac1, RhoA, and Wnt signal activation is a promising approach in the treatment of cancer and will be outlined in the outlook below.

| NDRG4
Studies by Benesh et al. (2013) have shown that n-Myc downstream-regulated gene 4 (NDRG4) binds to the CTD of POPDC1 (outside the Popeye domain in the region of aa300-aa357). This binding site is unique to the POPDC1 protein and is not found in the other cAMP binding proteins. NDRG4 is a known tumour suppressor (Amunjela et al., 2019), which represses cell proliferation, angiogenesis, metastasis, process extension, and invasion, and, like POPDC1, has been shown to also be down-regulated due to hypermethylation in cancer cells (Benesh et al., 2013). The interaction between POPDC1 and NDRG4 has previously been identified in epicardial cells. A loss of this interaction resulted in impaired fibronectin trafficking, prevention of VAMP3 docking to the cell membrane, and uncoordinated, non-directional cellular migration (Benesh et al., 2013). While evidence for this interaction in cancer cells has yet to be established, stabilising the POPDC1 protein to maintain POPDC1-NDRG4 interaction could be a useful approach to prevent the spread of cancer cells.

| Netrin-1
Netrin-1 is considered an oncogene, which inhibits apoptosis and promotes proliferation, differentiation, EMT, and metastasis (Han et al., 2015). Netrin-1 binds to four different receptors: deleted in CRC (DCC) and UNC5A, UNC5B, and UNC5C. Netrin-1 is upregulated in many cancer types, especially when the tumours are high grade or metastatic, such as BC, LC, CRC, PAC, GC, and glioblastoma (Kefeli et al., 2017). Of the four netrin-1 receptors, DCC and UNC5B have been shown to function as tumour suppressors in the absence of their ligand (Kefeli et al., 2017).
The absence of netrin-1 allows DCC-mediated apoptosis through caspase activation, and the UNC5B receptor contains a binding site for p53 and enables p53-induced apoptosis. However, along with p53, the DCC and UNC5B receptors have been shown to be downregulated in CRC and GC through gene methylation (Kefeli et al., 2017). Despite metastasis allowing the receptors to increase their distance from the netrin-1 ligand, their down-regulation stops them from inducing apoptosis to prevent this migratory step (Arakawa, 2004).
Netrin-1 also promotes other features of cancer, for example, angiogenesis, by exerting a similar effect on the endothelium as VEGF (Kefeli et al., 2017). It also influences proliferation and invasion, which have been shown to be mediated through DCC binding (Arakawa, 2004) and activation of the RhoA, Rac1, Cdc42, and PI3K signalling pathways (Ylivinkka et al., 2016). Clearly, while netrin-1 promotes activation of these pathways, POPDC1 mediates inhibition. Therefore, netrin-1 up-regulation and corresponding POPDC1 down-regulation are associated with a progressively more malignant phenotype, with both linked to downstream increases in RhoA, Rac1, and Cdc42 signalling in cancer cells.
Further evidence for netrin-1 promoting metastasis, invasion, and angiogenesis via PI3K/Akt and ERK signalling was demonstrated by Zhang et al. (2018) in NSLCC, whilst Han et al. (2015) showed this to occur in HCC and correlate with POPDC1 down-regulation. Subsequent re-expression of POPDC1 reduced the metastatic potential.
Netrin-1 also up-regulates EGF expression. The EGFR signalling pathway has been separately shown to decrease POPDC1 expression (Amunjela & Tucker, 2017a). It is therefore a reasonable assumption that restoring POPDC1 expression and/or preventing its downregulation by netrin-1 could provide a means of preventing cancer metastasis.

| Outlook-Maintaining cell adhesion and inhibiting metastasis by stabilising POPDC1
From the data outlined above, limiting POPDC1 loss could prevent disassembly of TJs and loss of cell adhesion, metastasis, and cell spreading, as well as decreasing the EMT induced by POPDC1 loss.
This could be achieved in three different ways: stopping POPDC1 down-regulation through miRNA-122 loss or increased netrin-1 signalling, increasing POPDC1 expression, or directly stabilising existing POPDC1 proteins. The latter may, for example, reduce changes in cell migration if its interaction with NDRG4 and VAMP3 to promote recycling of β1 integrin were maintained.
The important role played by the RhoGTPase family in cancer has been discussed in detail above and highlights that this protein family presents itself as an ideal candidate for targeted cancer therapy. As the globular structure of the RhoGTPases has been deemed "undruggable," alternative approaches need to be investigated (del  (Clayton & Ridley, 2020;Lin & Zheng, 2015;Tajiri et al., 2017). To summarise, stabilising the POPDC1 protein in cancer cells could prevent metastasis, one of the main causes of cancer-associated deaths. First, however, more evidence for an interaction between PODPC1 and these proteins in cancer cells is required before these approaches can be further explored.

| POPDC1 AND CSCs-RELATIONSHIP BY ASSOCIATION
A malignant neoplasm is a heterogeneous tissue consisting of many different cell types, including cancer-associated fibroblasts, cancer cells, epithelial cells, pericytes, and inflammatory immune cells (Hanahan & Weinberg, 2011), which are involved in different aspects driving tumour progression. One of these cell types can self-renew and differentiate into all types of cancer cells (pluripotency) (Du et al., 2019). These cells, known as CSCs, are implicated in the main difficulties of therapeutic cancer treatment. The formation of CSCs and quasi-mesenchymal cells somewhere along the EMT axis has already been discussed in detail, and this process is key to understanding anti-cancer drug resistance. CSCs are resistant to traditional cytotoxic chemotherapy agents due to their high expression of drug extrusion pumps, such as P-glycoprotein (Shibue & Weinberg, 2017;Zhang et al., 2017) and, similar to quasi-mesenchymal cells, possess mechanisms to avoid immune system detection. Furthermore, CSCs can also gain motility and undergo the process of metastasis outlined above and lie quiescent at local or distal sites for many years, before eventually re-entering the cell cycle leading to the development of a secondary tumour. It is important to note that some questions have been raised recently about the existence of CSCs in solid tumours, where, instead, tumour-initiating cells (TICs) have been suggested as a preferred term. This debate is beyond the scope of this article, and the reader is referred to other reviews (Machida, 2017;Magee et al., 2012;Suraneni & Badeaux, 2013) for further discussion of this point. Anti-cancer drugs that specifically target CSCs could potentially solve these key issues driving disease progression (Zhang et al., 2017), which perhaps makes CSCs the most promising target of all for developing new anti-cancer treatments (Yang et al., 2020).

| Signalling proteins in CSCs
CSCs use the same signalling pathways as normal stem cells (Yang et al., 2020), including the hedgehog, NF-κB, JAK-STAT and PI3K/ Akt/mTOR pathways, and the Wnt pathway (Yang et al., 2020). The latter is a critical signalling pathway in CSCs, and aberrant Wnt signalling has been demonstrated in various leukaemias, BC, CRC, lung cancer, PC, and ovarian cancer (Zhong & Virshup, 2020 (Yu et al., 2012;Zhan et al., 2017). Furthermore, in APC-deficient cancers (e.g., colon adenoma or CRC), the Wnt signalling pathway is constitutively active, because loss of the APC gene leads to an increase in GEF-mediated activation of Rac1, which in turn stimulates NF-κB (Myant et al., 2013). This co-activation, which increases Wnt signalling, not only promotes cell proliferation but has also been shown to initiate de-differentiation of normal intestinal cells into CSCs .

| Transcriptional regulation in CSCs
The most important transcription factors in CSCs include Sox2, Nanog, Oct4, KLF4, and Myc (Yang et al., 2020), of which the latter is involved in regulating cell metabolism, self-renewal, and proliferation.
Its expression is often dysregulated in CSCs , with high levels of c-Myc being achieved by reducing the amount of c-Myc ubiquitination and subsequent degradation, as well as increased Wnt signalling. c-Myc also promotes the hallmarks listed above and the formation of invasive CSCs.
As previously discussed, POPDC1 interacts with the regulatory

| EMT and differentiation
Two important steps in the metastatic cascade have been discussed in this review: the loss of cell adhesion, which induces EMT, and the emergence of CSCs along the EMT axis, contributing to anti-cancer drug resistance and the formation of secondary tumours previously mentioned (Dongre & Weinberg, 2019).
POPDC1 down-regulation has been shown to result in loss of cell adhesion and EMT , and EMT-mediated induction of stemness has been evidenced in mammary carcinoma cells (Mani et al., 2008) and intestinal stem cells (ISCs) (Reddy et al., 2016).
Although a link has yet to be established, the loss of POPDC1-inducing TJ disassembly and EMT could be driving the generation of CSCs. are key to ISC proliferation. Subsequent rapid expansion of the ISC population was also observed, leading to the conclusion that POPDC1 down-regulation and ensuing stem cell proliferation, in response to radiation therapy may be an underlying cause of resistance to radiotherapy treatment. The same phenomenon has also been observed as a result of POPDC3 down-regulation in other cancers such as HNSCC, lung cancer, and oesophageal cancer (Table 1) (He et al., 2019).
Stabilising these two isoforms of POPDC1 in the respective cancers may therefore reduce CSC formation and resistance to radiotherapy.
In addition, NF-κB-induced Wnt signalling in intestinal epithelial cells has also shown to promote dedifferentiation of normal epithelial cells into tumour-initiating stem cells . One could hypothesise that the POPDC1 protein could be involved in this Rac1/NF-κB co-activation of the Wnt signalling pathway as described above and that preventing POPDC1 loss in intestinal epithelia could result in reduced cell dedifferentiation into tumour-initiating CSCs.
The presence of POPDC1 proteins in CSCs and possible interaction with CSC signalling remains a relationship by association and still needs to be demonstrated experimentally. However, evidence shows that POPDC1 proteins play a crucial role in regulating c-Myc and Wnt signalling pathways in normal cancer cells, and it is also known that CSCs use exactly the same signalling mechanisms (Dongre & Weinberg, 2019;Yang et al., 2020). If POPDC1 protein expression is also down-regulated in CSCs, then stabilising the POPDC1 protein could be a useful therapeutic approach to directly inhibit cell proliferation and metastasis in CSCs.

| CONCLUDING REMARKS
The many ways in which PODPC1 acts as a tumour suppressor in cancer cells by mediating cell adhesion and suppressing proliferation and metastasis through numerous signalling pathways have been discussed ( Figure 2). However, its tumour-suppressive actions are often lost due to down-regulation in various types of cancer, including HCC, CRC, PC, BC, NSCLC, HNSCC, GC, and glioma (Table 3).
Many downstream targets of POPDC1, such as ZO-1, GEFT/ GEFH, Wnt signalling, c-Myc, VAMP3, and netrin-1, are already established targets for drug development. However, their ubiquitous expression may produce wide-ranging side effects. Hence, specifically stabilising POPDC1 in cancer cells to maintain interaction with these effectors may present itself as a novel, targeted approach to developing anti-cancer drugs. In line with the data presented above, POPDC1 stabilisation could have a broad effect by making subtle changes to many different signalling pathways to promote cell adhesion, as well as reduce cancer cell proliferation and metastasis.
Future studies should aim to develop a low MW compound that could stabilise POPDC1 and determine whether this would have the desired anti-tumour effects suggested above. Complete mapping of the interaction between POPDC1 and its target proteins to specific amino acid sequences will also be a vital step towards therapeutically targeting all POPDC1 downstream effectors. Further exploration of how POPDC1/PR61α interaction may have a dual effect on both Wnt and c-Myc signalling may also be promising. Finally, investigation of whether POPDC1 is expressed in CSCs is crucial for confirming the possibilities of CSC targeting outlined in this review. The potential roles that POPDC1 may play in other hallmarks of cancer, such as angiogenesis, have also been touched upon and highlight the importance of this protein across a broad spectrum of characteristics of this disease. Further study may therefore unveil additional roles of POPDC1 in the progression of the malignant phenotype.

| Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 Alexander, Fabbro, et al., 2019a, b;.