Exploring Retrograde Trafficking: Mechanisms and Consequences in Cancer and Disease

Retrograde trafficking (RT) orchestrates the intracellular movement of cargo from the plasma membrane, endosomes, Golgi or endoplasmic reticulum (ER)–Golgi intermediate compartment (ERGIC) in an inward/ER‐directed manner. RT works as the opposing movement to anterograde trafficking (outward secretion), and the two work together to maintain cellular homeostasis. This is achieved through maintaining cell polarity, retrieving proteins responsible for anterograde trafficking and redirecting proteins that become mis‐localised. However, aberrant RT can alter the correct location of key proteins, and thus inhibit or indeed change their canonical function, potentially causing disease. This review highlights the recent advances in the understanding of how upregulation, downregulation or hijacking of RT impacts the localisation of key proteins in cancer and disease to drive progression. Cargoes impacted by aberrant RT are varied amongst maladies including neurodegenerative diseases, autoimmune diseases, bacterial and viral infections (including SARS‐CoV‐2), and cancer. As we explore the intricacies of RT, it becomes increasingly apparent that it holds significant potential as a target for future therapies to offer more effective interventions in a wide range of pathological conditions.


| Introduction
Understanding the machinery and roles of retrograde trafficking (RT) is critical for deciphering cellular processes and maintaining cellular homeostasis.In this review, we will delve into how dysregulation of RT contributes to disease and cancer progression through disruption of immune response, proteostasis and growth signalling.Investigating RT provides insights into the intricate mechanisms of intracellular transport and sheds light on the importance of maintaining proper cargo localisation within the cell.

| Key Machinery in Intracellular Trafficking
Intracellular trafficking plays a crucial role in maintaining cellular homeostasis and proper function.The process of anterograde trafficking describes the movement of cargo, primarily proteins and lipids, within the secretory pathway.New proteins are synthesised at ribosomes in the endoplasmic reticulum (ER) and translocated to the Golgi for post-translational modifications.The cargo is then packaged into secretory vesicles and transported within the cell where it can perform its function, or to the plasma membrane where the vesicle will fuse and exocytose its contents into the extracellular space [1].Coat protein complexes help to collate and assemble cargo into vesicles for intracellular transport [2].The coat protein complex II (COPII) mediates cargo export from the ER, facilitating anterograde transport [3].
In contrast to anterograde trafficking, RT is the reverse process, whereby cargo is trafficked in an inbound, or ER direction from the plasma membrane, endosomes, Golgi or ER-Golgi intermediate complex (ERGIC) [4].Following cargo endocytosis from the plasma membrane, an early endosome is formed, a focal point of intracellular trafficking, from which three main routes of trafficking can occur (Figure 1) [5,6].The first is the degradation route, whereby the early endosome matures into a late endosome, which subsequently binds to existing lysosomes.Cargo proteins are then degraded due to an increase in acidification of the lysosome lumen [7].The second route is the recycling pathway.Recycling of cargo can occur via fast recycling, direct recycling from the early endosome to the plasma membrane or slow recycling, where the early endosome matures into a recycling endosome from which the cargo can be transported to the plasma membrane [8].The final route of endosomal trafficking from the early endosome is onward RT to the Golgi [9]; however, cargo can also be rescued from the recycling or late endosome and retrograde-trafficked to the Golgi [10] (readers are directed to a review by Elkin et al. [11] for more detailed information on endosome maturation and trafficking).RT may also take place downstream of the Golgi, with cargo trafficked from the Golgi to the ER and ERGIC to the ER, and, in some cases, may further embark on nuclear trafficking from the ER to the nucleus [12].To mediate retrograde transport of cargo, three coated transport vesicles exist within mammalian cells (Figure 2): coat protein complex I (COPI), retromer and clathrin, with aid from other RT components.

| Clathrin Vesicles
Within its role in RT, clathrin mediates the transport of cargo between the plasma membrane to endosomes and the trans-Golgi network (TGN) [13].Clathrin machinery may also require accessory proteins specific to the cargo it is transporting, to ensure efficient and selective transport of cargo.This includes epsinR and adaptor protein 1 (AP-1) in the transport of the bacterial toxin Shiga toxin B-subunit (STxB) [14] and mannose 6-phosphate receptors (MPRs), respectively [15,16].Clathrin and retromer act in a sequential manner in two successive steps, whereby cargo transported by clathrin is next transported by retromer for further RT [16].Receptor-mediated endocytosis 8 (RME-8) protein binds to sorting nexin 1 (SNX1) of retromer, colocalising with clathrin-coated early endosomes.This recruits the ATPase heat-shock cognate 70 (HSC70), which is responsible for the uncoating of clathrin, thereby allowing the retromer to process and further traffic the cargo [17,18].Alternatively, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), a clathrin-binding protein, may bind to SNX1 and also recruit a retromer [17].

| Retromer
Retromer facilitates RT from early and late endosomes to the TGN in addition to transporting cargo from the endosomes in the recycling pathway [19,20].It consists of two subcomplexes: first, the core retromer consisting of a heterotrimer of VPS26, VPS29 and VPS35, of which VPS35 is the largest subunit and is responsible for selecting and binding cargo for transportation [21].Of the other subunits, VPS26 binds to the N-terminal of VPS35, and VPS29 binds to the C-terminal [22].The second subcomplex is the SNX dimer consisting typically of a combination of SNX1, SNX2, SNX5 or SNX6, which aids in the binding of retromer to the endosome membrane and inducing membrane curvature [23,24].Retromer is responsible for the RT of a plethora of cargo including Wntless [25] and MPRs [20].

| Sorting Nexins
SNXs are a family of membrane-associated proteins involved in intracellular trafficking, including the retrograde route, and are characterised by the presence of a phosphoinositide-binding phox homology (PX) domain [26,27].Thirty-three sorting nexins have been identified in mammals, where they function to identify, organise and guide cargo through the endosomal trafficking system [28].Many SNX proteins have been shown to interact with retromer, facilitating the trafficking of a variety of cargoes to the plasma membrane or TGN, most notably, SNX1, SNX2, SNX3, SNX5, SNX6, SNX27 and SNX32 [28].Furthermore, some SNX proteins can facilitate retromer-independent RT [29].

| COPI Vesicles
COPI primarily acts within the early secretory pathway to mediate RT between the cis-Golgi and the ER [30].It is composed of two components: ADP-ribosylation factor 1 (Arf1) and a Golgi coat protomer, known as coatomer [31].The first step in COPI vesicle formation is the binding of the guanine nucleotide exchange factor (GEF), Golgi Brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1), to activate the exchange of guanosine diphosphate (GDP) to guanosine triphosphate (GTP) in the small GTPase, Arf1 [32].Arf1-GTP can then recruit the coatomer to the Golgi membrane [33].The coatomer is made of two functional subunits: the 'cage-like' B subcomplex, composed of COP proteins α/β′/ε [34] and the F subcomplex, composed of COP proteins β/δ/ γ/ζ, together forming a heptameric complex [35].Cargo is selected for RT based on sorting signals present on the cargo [30].The best characterised signals are the KKxx or KxKxx motifs found on the cytoplasmic C-terminal domain of transmembrane proteins that interact with the αor β′-COP subunits [36].Concurrently, the COPI vesicle assembly process involves interaction with vesicle SNARE (v-SNARE) proteins, which are seen to prime COPI coat assembly [37][38][39].SNARE proteins indeed play a crucial role in facilitating RT, and readers are directed to a review covering their role in trafficking by Wang et al. [40].Additional proteins are then recruited to aid in the formation and scission of the COPI vesicle from the Golgi membrane [41].Once the COPI vesicle has reached the ER, the hydrolysis of Arf1-GTP to Arf1-GDP, mediated through Arf1 GTPase-activating proteins (ArfGAPs), will trigger the uncoating and release of the cargo [42,43].For further information on the COPI complex, we refer readers to the recent review by Taylor et al. [43].In addition to the COPI-mediated Golgi to ER pathway, there exists COPI-independent Golgi to ER trafficking.This is primarily mediated through the small GTPase Rab6, recently reviewed by Dornan and Simpson [44]; however, Bottanelli et al. also report an Arf1-dependent, COPI-independent Golgi to ER trafficking [45].

| Examples of Key Rab GTPases
Approximately 70 members exist within the Rab GTPase family, of which almost three-quarters are involved in regulating endosomal trafficking [46].In their inactive state, Rab proteins are GDP-bound and, however, are activated upon switching of GDP to GTP via a GEF protein [47].After the Rab protein has performed its activity, a GAP protein will hydrolyse GTP to GDP, terminating its activity [47].Key Rab proteins that mediate RT include Rab2, which promotes the formation of retrograde COPI vesicles [48]; Rab6A, which mediates COPIindependent Golgi to ER trafficking [10]; Rab6A′, an alternative splice variant that differs from Rab6A by three amino acid residues due to an exon duplication [49], which is also shown to mediate Golgi-ER trafficking in addition to its involvement in cargo docking at the TGN [50,51]; Rab9, which interacts with retromer or tail-interacting protein (TIP47) to facilitate late endosome to TGN trafficking [52,53]; and the Rab11 family, which functions primarily in recycling endosomes and can help facilitate recycling endosome to TGN trafficking [54].A more comprehensive overview of the function of Rab proteins can be found in Galea et al. [55].

| Regulating Cell Polarity
The roles of RT are diverse and essential for cellular functions.RT is required to regulate cell polarity, which is important for continued cell homeostasis, and to avoid a diseased phenotype [56].Pocha et al. have shown Crumbs (Crb), a transmembrane protein responsible for defining the apical edge of the cell and maintaining apical-basal polarity [57], to be a cargo of retromer, and that RT is essential to maintaining Crb localisation and, hence, epithelial cell polarity [58].The polarity protein Scribble (Scrb) is needed to maintain planar cell polarity in vertebrate epithelial cells but has also been implicated in controlling the trafficking of other polarity proteins [59].Scrb blocks the association of E-cadherin to retromer, inhibiting it from being retrograde-trafficked and re-localised from the cell membrane, thus maintaining the cell polarity [60].However, when cell polarity needs to change, RT is also involved.A migratory phenotype requires integrins to be at the leading edge of cells, and Shafaq-Zadah et al. have shown that β1 integrin undergoes RT from the plasma membrane to the TGN, where it is subsequently re-secreted specifically to the leading edge of migratory cells [61].This contrasts with the recycling of β1 integrin through recycling endosomes and Rab11, which maintains β1 integrin at the leading edge of migratory cells, in a random fashion [62].

| Retrieval of Resident Proteins
Proteins that are required to mediate anterograde trafficking from the ER to the Golgi or Golgi to the plasma membrane need to be recycled back to their original locations to facilitate continuous rounds of anterograde trafficking.For example, the transmembrane sorting receptor, Wntless (Wls), is essential for the anterograde trafficking of Wnt signalling proteins from the TGN to the plasma membrane and consequently needs to be recycled back to the TGN to facilitate multiple rounds of trafficking [25,63].Recycling of Wls relies on clathrin and retromer to be translocated to the TGN [64].Proteins involved in anterograde trafficking from the ER to Golgi also need to be retrieved back to the ER to facilitate a new round of secretion.This includes v-SNARE proteins, including Sec22p, involved in the tethering of cargo at the Golgi [65], and Sortilin, a recycling receptor that works alongside MPRs to transport hydrolases from the TGN to endosomes in anterograde trafficking [66].However, Sortilin and MPRs need to be recycled back to the TGN for another round of trafficking [66].This is mediated through RT in SNX1-positive vesicles from the early endosome to the TGN [66].

| Mis-Localised Proteins
Proteins that have escaped controls to enter the wrong compartment during anterograde trafficking need to be identified and retrograde-trafficked back to their correct location.ERresident chaperone proteins can mistakenly stay resident in the Golgi following ER to cis-Golgi trafficking and need to be retrieved back to ER via COPI-mediated RT [67].To be recognised as being mis-localised, ER-resident proteins contain a C-terminal Lys-Asp-Glu-Leu (KDEL) sequence, which are recognised at the cis-Golgi by KDEL receptors (KDELR) in a pH-dependent manner [68].The KDELR strongly binds to the protein KDEL motif at the cis-Golgi, which has a luminal pH of ~6.The binding of the protein to the receptor triggers its association with the COPI vesicles, allowing the mis-localised protein to be returned to the ER [69].The increase in pH ~7 in the ER lumen triggers the dissociation of the protein from the KDELR.Once the mis-localised protein is unloaded at the ER, the KDELR is then transported back to the cis-Golgi via COPII anterograde trafficking [69,70].For example, immuneglobulin heavy chain binding protein (BiP) is an ER chaperone protein that helps in protein folding, sensing ER stress and signalling to activate the unfolded protein response (UPR) [71].If BiP leaves the ER attached to misfolded proteins and traffics to the cis-Golgi, the KDEL motif will bind to KDEL receptors within the cis-Golgi triggering the association with COPI vesicles allowing BiP to be shuttled back to the ER via retrograde transport [30].

| Dysregulation of Key RT Machinery and Their Function in Disease
Having outlined the key machinery and role of RT, we now examine how dysregulation of RT contributes to disease through hijacking, upregulation and downregulation.The mechanisms through which key RT machinery is dysregulated are many and consequently disrupt normal RT, causing disease.These diseases caused by mutations or expression alterations to subunits of coated transport vesicles are collectively named 'coatopathies'.In some cases, high expression of coat subunits is associated with a poorer outcome, such as for α-COP in cervical cancer [72], and β′-COP (COPB2) and ζ1-COP (COPZ1) in breast cancer [73,74].In contrast, for some subunits, low expression is associated with a poor outcome, such as ε-COP (COPE) in pancreatic adenocarcinoma [75] and ζ2-COP (COPZ2) in breast cancer [76].In addition, point mutations in subunits, such as the VPS35 subunit of retromer, have been shown to have links to Parkinson disease (PD) [77,78], whereas mutations within the δ subunit of the COPI complex encoded by the ARCN1 gene result in craniofacial defects [79].For a more extensive breakdown of mutations to coat proteins, we direct the readers to a review by Dell'Angelica et al. [80].Besides key subunits of coated vesicles, alterations to additional RT machinery also have links to disease.For example, mutations in the conserved oligomeric Golgi (COG) complex, which coordinate intra-Golgi RT, alter the localisation of key glycosylation enzymes, giving rise to congenital disorders of glycosylation (CDGs) [81].Patients present with a variety of symptoms, including microcephaly, hypotonia and failure to thrive, amongst others, dependent on the COG mutation [81,82].

| Hijacked: Insights From Pathogens
In this section, we explore how RT is hijacked by pathogens to infiltrate human cells and cause disease.It is well established that RT is utilised by bacterial toxins including the Shiga toxin, cholera toxin and ricin toxin to infiltrate and infect cells [83,84].Indeed, RT can be targeted to treat infection from these toxins [18].Recent advances have now harnessed the RT properties of these toxins to provide newly targeted therapies in cancer [85] and beyond (reviewed by Lingwood [86]).Furthermore, bacterial toxins have been used to better understand the RT route, including the use of labelled toxins to quantitatively measure RT [87].New pathogens that exploit host RT machinery are emerging, navigating cellular environments and manipulating critical immune surveillance mechanisms.Understanding these mechanisms provides valuable insights into novel therapeutic strategies for combating these diseases.

| The Human Immunodeficiency Virus
The human immunodeficiency virus (HIV) belongs to the family of retroviruses, which upon invasion into the host cells will make use of the host cytoskeleton and RT machinery to transport its genetic material to the nucleus as part of a preintegration complex before the viral DNA can be converted into dsDNA by reverse transcription [88,89].The HIV genome is packaged into a capsid protein (CA), which is released into the cytoplasm following fusion with a target cell membrane [90].The CA containing the viral RNA and proteins required for infection then binds to bicaudal D2 (BICD2), a dynein adaptor that facilitates the binding of HIV to the dyneindynactin complex and traffics along microtubules towards the nucleus, in retrograde fashion [88,91].HIV primarily invades CD4+ cells and utilises not only RT to facilitate its entry and infection of host cells but also to evade immune detection once established [92].This is achieved by sequestering major histocompatibility complex 1 (MHC I) and cluster of differentiation 4 (CD4) from the cell surface to the early endosome and either onwards to the late endosome and eventually degradation in lysosomes, or to the TGN [93][94][95][96].Reduced MHC-I expression on the surface of infected cells hampers the presentation of HIV antigens to the immune system, particularly cytotoxic T lymphocytes, impairing their ability to recognise and eliminate infected cells [94].The HIV accessory protein negative regulatory factor (Nef) is essential for modulating the expression of cell surface proteins, mediating this immune evasion [97].Downstream pathways of Nef ultimately increase ARF6 activation, which increases the rate of MHC-I endocytosis to the TGN, downregulating its expression at the cell surface [98,99].Cells expressing endogenous Nef and infected with HIV showed a significant reduction in MHC-I cell surface molecules compared with non-infected cells [96].Groppelli et al. established in 2014 the association between retromer and HIV.Specifically, the HIV glycoprotein, Env, binds to the VPS35 subunit of retromer [100].Retromer facilitates the RT of HIV from the early endosomes to the TGN, which is essential for its recycling to the plasma membrane, or indeed further RT within the cell [100].Recently, in response to this, Hoffman et al. demonstrated minimal co-localisation of the Env protein with Golgi or TGN markers [101].This suggests that interactions between retromer and Env may not be a result of direct endosome-to-TGN/Golgi RT, but could involve alternative endosomal pathways, potentially including Rab14 [101].These interactions may play a role in the retromer's involvement in anterograde trafficking between the early endosome and the plasma membrane.However, they do acknowledge that the absence of accumulation may stem from the limited presence of Env at these compartments, likely due to rapid recycling [101].Notably, RT of the HIV Env from the plasma membrane to early/recycling endosomes is essential to facilitate its subsequent anterograde movement back to the plasma membrane-the designated assembly site for HIV virions [102].HIV further hijacks the RT pathway in dendritic cells, whereby the inward trafficking of HIV is essential for its onward anterograde trafficking to the plasma membrane, where it can engage with trans-infection with nearby T cells [103].Bayliss et al. have shown that a reduction in retromerassociated genes reduces trans-infection [103].

| SARS-CoV-2
The SARS-CoV-2 respiratory virus responsible for the COVID-19 pandemic was first discovered in 2019, and since then, much effort has been applied to understand the molecular pathway undertaken by the virus to replicate [104].The virus is composed of a nucleoprotein (N)-encapsidated RNA genome enveloped by three proteins, the spike (S) protein, responsible for binding to target cell membranes; membrane protein (M), responsible for the recruitment of the viral genome within the virion; and the E protein (E), a minor envelope protein [105].The latest research points to RT as the SARS-CoV-2 mode of entry into the cell and onwards to its site of replication, sharing similar trafficking mechanisms of SARS-CoV, responsible for the SARS epidemic of 2002 [106].The S protein first binds to the cell surface receptor, angiotensin-converting enzyme 2 (ACE2), through its receptor-binding domain [107].Upon successful entry into the target cell, the RNA genome is released and uncoated, undergoing translation of key viral replication machinery, and replication and translation of the viral genome.Translation then occurs of the key structural proteins, S, M and E, at the ER.The S protein will traffic to the Golgi (via COPII anterograde trafficking) to be posttranslationally modified (e.g., glycosylation and palmitoylation) [108,109].The S protein then needs to be trafficked to the ERGIC, the site of progeny assembly.Dey et al. employed mutagenesis and binding assays to demonstrate how the α-COP selectively binds to the S protein of SARS-CoV-2 to mediate RT from the Golgi to the ERGIC [110].The S protein contains the canonical K-x-H dibasic sequence needed for binding to COPI and consequent RT to the ERGIC [110].Newly formed virions are subsequently exocytosed to infect nearby cells [111].

| Human Papillomaviruses
Human papillomaviruses (HPVs) are a common group of viruses responsible for 5% of human cancers worldwide, including but not limited to cervical, anogenital and oropharyngeal cancers [112].HPV has recently been established to hijack the retrograde route to gain entry to intracellular compartments for virus replication in the nucleus and evade the fate of lysosomal degradation [113,114].This includes the use of the RT coat complexes, retromer and COPI, alongside other RT accessory proteins, through interaction with the L2 CA to facilitate trafficking [114][115][116][117][118]. By utilising endogenous RT compartments, HPV can evade innate immune surveillance mediated by cyclic GMP-AMP (cGAS)/stimulator of interferon genes (STING) [119].

| Downregulated: Autophagy, ER Stress and the UPR
In this section, we explore how RT is downregulated to cause an imbalance of proteostasis, ultimately inducing autophagy, ER stress and the UPR.These three processes heavily rely on intracellular trafficking mechanisms.Autophagy, an intracellular self-degradation process, facilitates the removal of damaged cytoplasmic components, including organelles and proteins, through the lysosomal system [120].It is essential for maintaining cellular homeostasis under normal and stressful conditions.Autophagy involves the formation of autophagosomes, membrane-enclosed vesicles containing materials for degradation.These autophagosomes fuse with lysosomes to form autolysosomes, where degradation occurs through hydrolases [121].Autophagy was first described as a protective mechanism, ensuring cell survival; however, later evidence points to its role in mediating cell death [122].Consequently, the relationship between autophagy and cancer has been established as a 'double-edged sword' with autophagy exhibiting tumour-suppressing capabilities during early tumorigenesis, while it can promote tumour progression in later stages [123].Retrograde machinery plays a crucial role in facilitating autophagy, especially in neurons where autophagic vacuoles must traffic long distances along axons to reach lysosomes [124].Disruption of RT can result in defective clearance of cytotoxic proteins through the endolysosomal system, which is heavily dependent on RT components such as retromer and Traffic, 2024 Rab7 [125,126].Such disruptions may contribute to the onset of classical neurodegenerative diseases such as Alzheimer disease (AD) and PD.
ER stress results from an accumulation of misfolded proteins in the lumen of the ER.To restore homeostasis, the UPR, a signal transduction pathway, is activated by stress sensors including inositol-requiring protein 1 (IRE1), protein kinase RNA-like ER kinase (PERK) and activating transcription factor 6 (ATF6) [127].These can go on to regulate responses including ER-associated degradation (ERAD), whereby misfolded proteins are retrotranslocated to the cytosol to undergo ubiquitination and degradation by the proteasome [128].Given the close relationship with the ER, associations between ER stress and RT are primarily through defective COPI trafficking [129][130][131].In particular, the α-COP subunit, which when altered, can lead to defective RT and resulting disorders [129][130][131].

| Neurodegenerative Diseases
Links between impaired autophagy and disease are evident in neurodegenerative diseases.RT is essential for the trafficking of autophagic vacuoles from distal locations in neurons to the soma, where most lysosomes reside for autophagic clearance [124,132].Disrupted RT was shown by Tammineni et al. to promote autophagic stress in the context of AD [124].The accumulation of autophagosomes is seen in AD models due to RT failure and defective clearance of the cytotoxic Aβ protein that may directly contribute to the pathogenesis of AD [133].The resulting accumulation of Aβ protein in autophagosomes inhibits their fusion to lysosomes for protein degradation [133].Mechanistically, this has been proposed to be linked to the failure of activation of the Rab7 protein [125].Rab7 is essential in axonal RT and regulation of the biogenesis of lysosomes [125].Cai et al. show that overexpression of the CCZ1-MON1A complex, a GTP exchange factor for Rab7, enhances the clearance of Aβ and phosphorylated Tau (both implicated in AD pathogenies) leading to improved memory [125].This represents a potential target for future AD-alleviating therapies.
Mutations in the retromer component, VPS35, have been linked to late-onset, autosomal dominant familial PD through autophagy and could represent a novel target for PD treatment [126].It is characterised by the accumulation of the αsynuclein protein that contributes to the formation of deposits called Lewy bodies in the brain stem, in addition to the loss of dopaminergic neurons in the substantia nigra pars compacta [134].A missense mutation in VPS35 (p.D620N) has been identified and reported in both familial and sporadic forms of PD [77,78].The D620N mutation in VPS35 impairs the association of retromer to the WASH complex at endosomes, resulting in defective autophagy [135,136].The WASH complex was found to be necessary for autophagosome formation, and therefore, D620N may contribute to the pathology of PD through autophagy dysfunction.Furthermore, impaired trafficking of autophagy-related gene 9A (ATG9A) in cells expressing VPS35 D620N mutations is required in early autophagosome formation, and therefore likely contributes to the decreased autophagy phenotype [135].More recently, Rahman et al. further built upon this, revealing that disrupted signalling of the brain ECM component, hyaluronan (HA), may further contribute to PD pathogenesis through defective autophagy [137].Knockdown of hyaluronan receptor, HAmediated motility receptor (HMMR), rescued autophagy in VPS35 D620N cells, suggesting the importance of ECM signalling in driving autophagy inhibition, further contributing to our knowledge of defective autophagy in PD pathology [137].

| COPA Syndrome
Recent publications have outlined a molecular mechanism behind the newly described autoimmune disease, COPA syndrome, arising from missense mutations in the WD40 region of α-COP and characterised by aberrant STING activation and subsequent interferon signalling due to defects in RT [129,[138][139][140]. Patients typically present in infancy with interstitial lung disease and arthritis [129].Understanding the molecular mechanism of this disease has linked it to ER stress and the UPR [129,130,141].Owing to mutations in the α-COP subunit of the COPI complex, cargo binding is reduced, leading to defective RT between the Golgi and ER [129].It is hypothesised that the resulting ER stress may be twofold.First, defective RT leads to a lack of ER proteins needed for anterograde trafficking; consequently, the cell drastically increases its protein production rate, resulting in ER stress [130].This subsequently leads to the activation of the UPR to mitigate stress.Second, ER stress may arise due to defects in autophagy, which COPA patients also display, as autophagy is another mechanism to reduce ER stress [129].

| Cancer
The COPI complex has been shown to be an inhibitor of autophagy in cancer cells, as depletion of COPI components, including α-COP, β-COP (COPB1), β′-COP (COPB2), γI-COP (COPG1), δ-COP (COPD) and ζ1-COP (COPZ1), induces autophagy [142].Furthermore, the induction of autophagy also led to other well-linked processes including ER stress and activation of the UPR due to protein accumulation in the ER because of disrupted trafficking [142].Di Marco et al. further suggest that ER stress due to COPI depletion can lead to an interferon response, and hence an immunogenic cell death in the context of thyroid cancer [143].
To better define the role of faulty COPI in cancer, much interest has been paid to endogenous RNA editing of the α-COP subunit, which has been found to be upregulated in several instances.Adenosine deaminase acting on RNA 1 (ADAR1), which is highly expressed in some cancer tissues, catalyses A-to-I RNA editing.When acting upon α-COP, COPA I164V is formed and is a non-functional version of α-COP that results in defective RT, and hence ER stress.Wang et al. have recently shown that overexpression of COPA I164V drives metastasis through ER stress [131].In colorectal cancer cells, RNA editing to COPA I164V induced ER stress, followed by the UPR, which in turn activated transcription factors including MALAT1, MET and ZEB1, which upregulated metastasis-related genes [131].In contrast, in non-tumour cells expressing COPA I164V , there was evidence of ER stress followed by the UPR, which triggered apoptosis [131], clearly demonstrating the fine balance of ER stress that a cell can manage.
Brefeldin A (BFA) is a fungal lactone with antibiotic and anti-tumour activity, inhibiting trafficking between the ER and Golgi [144].BFA targets the short-lived ARF-GDP-Sec7domain reaction intermediate, stabilising it and preventing COPI vesicle formation and thus disrupting cargo trafficking [145].BFA is seen to stimulate ER stress in colorectal cancer cells, promoting autophagy and consequently cell death [146].Zhou et al. show how increased ER stress, resulting from BFA treatment, increases BiP expression, which interacts with Akt and decreases Akt phosphorylation, hence activating autophagy [146].

| Upregulated: Dysregulated Growth Factor Signalling
Dysregulated growth factor signalling, particularly involving receptor tyrosine kinases (RTKs), plays a critical role in cancer development and treatment resistance.RTKs, such as cMET, fibroblast growth factor receptors (FGFRs), epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2), are typically found on the cell surface, regulating cellular processes such as proliferation and migration.However, emerging evidence suggests that internalised and trafficked RTKs can promote tumorigenesis and impact therapeutic outcomes.In this section, we delve into the intriguing phenomenon of upregulated RT of RTKs, focusing on cMET, FGFR, EGFR and HER2, and their intricate intracellular trafficking pathways that contribute to cancer progression and resistance to treatment.

| cMET
The hepatocyte growth factor receptor (cMET) is an RTK activated by its ligand, hepatocyte growth factor, which leads to downstream effects on proliferation, migration, wound healing and organogenesis [147].Aberrant activation of cMET has well-established links to poorer prognosis in cancers including glioblastoma, head and neck, breast, and gastrointestinal [148][149][150][151]. Recently, Chen et al. have published a proposed mechanism outlining the RT of cMET from the plasma membrane to the nucleus in breast cancer cells [151].RT was achieved through microtubule-dependent but dynein-independent plasma membrane to Golgi trafficking; COPI-mediated Golgi to ER trafficking; and 'integral trafficking from the ER to the nuclear envelope transport' (INTERNET) ER to nucleus trafficking [151].The authors demonstrated how H 2 O 2 , a reactive oxygen species generated from cell metabolism and chemotherapy agents, induced nuclear accumulation of cMET in a dose-dependent manner through RT [151].Nuclear accumulation of cMET is required for DNA damage repair through interaction with Ku proteins, suggesting RT of cMET can be harnessed as a resistance mechanism by breast cancer cells [151].The authors went on to show that nuclear accumulation of cMET contributes to resistance to PARP inhibitors in triplenegative breast cancer (TNBC) and pancreatic cancer [152,153].A cMET inhibitor alongside a PARP inhibitor or ROSinducing chemotherapy increased DNA damage and, therefore, cell death [153].

| FGFR
The FGFRs are activated by their ligands, fibroblast growth factors, with the aid of heparin [154].Four receptors, FGFR1, FGFR2, FGFR3 and FGFR4, exist within the FGFR family, but in the context of cancer, FGFR1 is the most frequently overexpressed [155].Like many RTKs, FGFRs are primarily cell surface-bound, but can be trafficked intracellularly, to either be recycled back to the plasma membrane to maintain consistent signalling, degraded in lysosomes or retrogradetrafficked towards the nucleus [155].Trafficking of full length or cleaved FGFR1 to the nucleus can occur via the Golgi and ER, via integrative nuclear FGFR1 signalling (INFS), where the FGFR1 is released from the ER to the cytoplasm, before translocation to the nucleus mediated by importin-β [156,157].Nuclear localisation of FGFRs is correlated with a poor prognosis in a variety of cancers including breast, cervical and pancreatic [158][159][160][161]. Within the nucleus, FGFR1 can act as a regulatory factor, working alongside nuclear proteins to activate the expression of genes involved in cell migration including KRTAP5-6, SFN and PRSS27 and repressing GRINA and EBI3, promoting a more aggressive phenotype [159].Recently, FGFR1 overexpression and nuclear localisation have been associated with endocrine resistance in oestrogen receptor-positive breast cancer [162].This is due to FGFR1 regulating gene transcription, which promotes resistance to antioestrogens [162].Furthermore, the pan-FGFR1 inhibitor, erdafitinib, which inhibits the enzymatic activity of FGFR1, does not decrease nuclear FGFR1 levels nor decrease the growth of breast cancer cells overexpressing FGFR1, suggesting that an alternate mechanism to target nuclear FGFR may be required to overcome therapeutic resistance [162].One such mechanism has recently been proposed by Gregorczyk et al. who showed N-glycosylation to be a reversible switch that can control FGFR1 localisation [163].N-glycosylated FGFR1 is seen to localise to the plasma membrane, whereas FGFR1 mutated to not undergo N-glycosylation localises to the nuclear envelope [163].

| EGFR
The EGFR belongs to the ErbB family of RTKs, which are involved in key cellular processes including proliferation, growth and survival [164].Specifically, canonical EGFR signalling leads to the activation of the PI3K/Akt/PTEN/mTOR and RAS/RAF/MEK/ERK pathways, playing a crucial role in cell division and survival [165][166][167].Mutations and overexpression of EGFR have frequently been observed in a variety of cancers including lung, breast and gallbladder [168][169][170].However, a non-canonical oncogenic role for EGFR that is found intracellularly has been proposed.EGFR is endocytosed in a clathrin or non-clathrin-mediated fashion [171] with the endocytic vesicle fusing to an early endosome, where it is RT to the Golgi by the interaction with SNARE protein syntaxin 6, which regulates fusion with the Golgi [172].EGFR is transported along microtubules by motor protein dynein [172].From the Golgi, EGFR translocates to the ER via COPImediated RT [173].From the ER, EGFR may be targeted for translocation to the nucleus, in INTERNET transport [174], regulated by importin-β and Sec61b [175].Once in the nucleus, nuclear EGFR (nEGFR) is involved in gene transcription regulation as a co-transcription factor, DNA repair, resistance to chemotherapy and radiotherapy and DNA replication by phosphorylating proliferating cell nuclear antigen (PCNA) to increase the rate of cell proliferation [168,169,[176][177][178][179].Maisel et al. have indirectly modulated EGFR localisation through targeting Mucin-1 (MUC1).The authors show how, upon EGF binding to EGFR and being internalised, MUC1 promotes the retention of EGFR in EEA1-positive vesicles.This prevents the trafficking of EGFR to lysosomes and the degradation pathway, extending the availability of intracellular EGFR within endosomes.This reduces the therapeutic effectiveness of cetuximab and impacts key cancer phenotypes, such as increasing migration speed [170].

| HER2
The HER2 or ErbB2 is another member of the ErbB family of RTKs and has similarly been shown to traffic from its canonical location at the plasma membrane to the nucleus, where it can elicit cancer-promoting effects.HER2 is retrograde-trafficked to the Golgi and onwards to the ER [180].From the ER, it undergoes INTERNET nuclear trafficking mediated by importin-β, which interacts with its nuclear localisation signal [175].Nuclear HER2 has been well documented in cancers and diseases including liver disease [181], but its role in breast cancer has been particularly well studied [182].Within HER2+ breast cancer, a high incidence of nuclear HER2 has been reported and is correlated with a poorer prognosis.Moreover, in in vitro studies, nuclear HER2 has been shown to contribute to resistance to chemotherapeutic agents including paclitaxel [183], and conversely, cells expressing mutant forms of HER2 unable to translocate to the nucleus had increased sensitivity to HER2-targeted therapies including trastuzumab [184].Furthermore, it has recently been elucidated that in some instances of TNBC, HER2 is localised to the nucleus, where it can drive cancer growth due to its role as a transcription factor [185].However, Madera et al. show that the RT inhibitor, Retro-2, induces an accumulation of HER2 at the plasma membrane or Golgi, depending on the HER2 isoform, resulting in a significant reduction in cell growth [186].

| Conclusion and Future Perspectives
RT is a complex process that plays a critical role in maintaining cellular homeostasis and mediating various cellular functions.Understanding the machinery and roles of RT in the context of cancer and disease is complex as RT has been shown to be upregulated, downregulated or indeed hijacked.Investigating these mechanisms provides insights into the intricate processes of intracellular transport and highlights the importance of maintaining proper cargo localisation within the cell.Further research in this field will enhance our understanding of cellular processes and potentially lead to the development of targeted therapies to combat diseases associated with RT dysfunction.
The well-established RT inhibitor, BFA, is known to have poor bioavailability in vivo, which has hindered its clinical use [187].However, derivatives of BFA have shown anti-cancer activity against a plethora of cancer types, with most recent work targeting cervical, breast and hepatocellular cancers, with improved bioavailability [188][189][190][191][192]. Other methods to circumvent this issue are centred on improvement to drug delivery, such as that by Zhang et al. who are using nanomicelles to improve the delivery of BFA [193][194][195][196]. Future work could combine these two strategies to bring BFA into the clinic.
Retro-2 has emerged as a compound for therapeutically targeting a broad spectrum of RT cargo including bacterial toxins, viruses and RTKs [83,170,186,197,198]. Furthermore, the Retro-2 derivative, Retro-2.1, which is more potent than the parent form, is also being investigated for treatment against herpes simplex virus and polyomavirus in vitro, and more recently in vivo [197][198][199].Similar to BFA, recent efforts by Vinck et al. have aimed to improve the delivery of Retro-2.1 using hydrogels and if coupled with recent evidence by Madera et al.Retro-2.1 could prove to have promising applications in cancer therapeutics [186,199].
Other RT machinery have been targeted in therapeutics.This includes SNX1, which, in the context of cancer, usually promotes the RT of EGFR to the nucleus where it can have oncogenic functions.Atwell et al. have designed a novel peptide-based therapeutic that mimics the EGFR-binding domain of SNX1, cSNX1.3, which binds to EGFR, thus inhibiting its nuclear localisation [200].Phenotypically, this resulted in a reduction in migration and tumour regression in vivo, suggesting that targeting RT in EGFR-driven cancers may be a therapeutic option [200].
Furthermore, the RT of HPV has proved a potential therapeutic vulnerability, using a cell-penetrating peptide containing the HPV-retromer binding site [116].The peptide competes for retromer binding, sequestering it from the virion, therefore preventing HPV from retromer-mediated trafficking [116].In doing so, HPV cannot traffic to its site of replication, thus limiting the persistence of the disease [116].
Finally, recent work by McHugh et al. has uncovered the therapeutic potential of targeting COPI vesicle formation in senescent cells, as the COPI pathway is seen to be important for their survival [201].Senescent cells contribute to tissue dysfunction through the generation of a senescence-associated secretory phenotype (SASP) as well as fostering a pro-inflammatory environment [202].Senescent cells rely on COPI due to their need to recycle proteins essential to the maintenance of the SASP [201].Halting intracellular trafficking led to an accumulation of SASP-related proteins, activating the UPR, leading to apoptosis [201].The COPI complex can, therefore, be seen as a senolytic target, expanding the scope of targeting RT to senescent cells, with the potential of treating age-related diseases and cancer.
In sum, the broad nature of how RT is associated with disease makes it an attractive target for new therapies.Future applications, particularly for cancer therapeutics, would need to ensure the specificity of RT-targeted therapy to minimise off-target side effects, due to the essential nature of RT to normal functioning cells.