Unpacking the Tumor Protein D52-like Family: Roles in Intracellular Trafficking and Cancer Progression.

1. Introduction
The intricate interactions that take place to maintain cellular homeostasis are regulated by vesicle trafficking of newly synthesized proteins and cellular secretion mechanisms [1]. These intracellular trafficking pathways are fundamental to many cellular processes that precisely control the direction of proteins within the intracellular space as well as protein expression on, and secretion from, the cell surface [2]. Proteins synthesized within a cell must first undergo sorting to the correct destination to ensure an appropriate function. Therefore, if dysregulation of such pathways occurs, it can be catastrophic to the normal cellular function and can lead to the development of disease, such as Alzheimer’s disease, diabetes, or cancer [3]. When hijacked during cancer progression, changes to proteins expressed at the cell surface or secreted can aid and accelerate the hallmarks of cancer (e.g., sustained proliferative signaling, resistance to cell death, angiogenesis, metastasis, anti-tumor immunity, metabolic reprogramming), contributing significantly to disease progression [4]. Thus, identifying regulators of intracellular trafficking pathways presents a unique opportunity in cancer research, as disruption of these highly regulated mechanisms could potentially lead to a multi-targeted anti-cancer approach.
Over the years, many proteins have been identified to contribute to dysregulated intracellular trafficking during cancer progression, e.g., Rab GTPases and their effector proteins [5]. More recently, the Tumor Protein D52 (TPD52)-like family have been identified as a group of small lipid-binding proteins (140 to 224 amino acids long) with emerging roles in vesicle-mediated trafficking (e.g., exocytosis, Golgi transport, granule secretion, etc.) [6]. The TPD52 family consists of four members (TPD52, TPD53, TPD54, and TPD55), each characterized by highly conserved coiled-coil motifs. In 1995, Byrne and colleagues were the first to document that TPD52 is overexpressed in approximately 40% of breast carcinomas [7]. Since this discovery, there has been increasing interest in the other family members and their mechanistic roles in cancer progression. As detailed below, elevated protein expression of TPD52 and TPD54 has been associated with more aggressive cancer phenotypes. In addition, these proteins have shown both tissue-specific and isoform-specific roles, which may elucidate different underlying mechanisms in various disease contexts [8,9]. A better understanding of the TPD52 family may identify cancer-specific mechanisms that could guide therapeutic intervention. Importantly, intracellular trafficking directly affects cancer hallmarks, as trafficking determines the expression levels of signaling receptors (e.g., receptor tyrosine kinases and integrins), immune checkpoint proteins, and metabolic regulators at the cell surface. Therefore, abnormal vesicle trafficking or recycling can impact proliferative signaling, invasion, the immune microenvironment, and cell metabolism. Hence, trafficking regulators, including the TPD52-like family, affect multiple oncogenic processes at the same time. In this timely article, we review the reported roles of the TPD52-like family in trafficking mechanisms and cancer progression.

2. The TPD52-like Family
The TPD52-like family consists of four proteins with canonical sequences ranging from 140 to 224 residues in length and sharing 41–59% sequence identity: TPD52, TPD53, TPD54, and TPD55 (Table 1). Each protein has several isoforms, featuring insertions, deletions, and/or mutations compared to the canonical sequence. TPD52 was the first member of this family to be identified with elevated expression in breast carcinomas [7].
TPD52-like proteins are conserved at least across vertebrates, with orthologues found from fish to mammals, as determined by orthology searches using OMA (https://omabrowser.org (accessed on 19 January 2026)). The presence of paralogues across vertebrate species suggests that gene duplication contributed to gene expansion of the family early during vertebrate evolution, followed by divergence of the four human family members. More distant invertebrate homologues may exist, although they are likely to be less readily identifiable. Consistent with this gene divergence, a paralogue similarity tree of the four human TPD52-like family members shows clear sequence separation among the paralogues (Figure 1). This is consistent with functional diversification within the gene family.
To date, an experimental protein structure has not been determined for any member of the family. These proteins are characterized by coiled-coil motifs ranging from 29 to 52 amino acids (determined with UniProt Align), with TPD55 having the shortest motif. Structural predictions by AlphaFold3 [10] show high confidence in the formation of a coiled-coil domain (pLDDT > 90) in dimeric TPD52-like family models (Figure 2). Outside of this domain, alpha helices and unstructured regions are predicted, with more unstructured regions and reduced confidence in the model (down to pLDDT < 50) nearer to the N- and C-terminals.
The coiled-coil motif was documented to be essential for dimer formation between TPD52-like family proteins, with some contribution from C-terminal regions in facilitating and/or stabilizing the dimers [11,12]. All possible dimeric interactions between these proteins were observed in a yeast two-hybrid system; however, the homodimeric interactions of TPD53 were preferred over heterodimeric reactions, while the opposite was true for TPD52 and TPD54 [11]. Whether the ratio of homodimers and heterodimers formed by TPD52-like family members influences their cellular function remains to be explored.
The structural significance of two other motifs in the family is less clear. TPD52, TPD53, and TPD54 share a consensus sequence known as the D52 motif, namely, (V,M)(T,Q)X(T,S) XAY(v,K)KTXETL [13,14], but it does not play a role in interactions between the TPD52-like proteins [14]. PEST motifs overlap with the N-terminal end of the TPD52-like family’s coiled-coil motifs (Table 2 and Figure 3). These motifs are hydrophilic sequences rich in proline, aspartate or glutamate, serine, and threonine residues, with a PEST score (determined by the EMBOSS epestfind algorithm [15]) indicating they are likely biologically relevant proteolytic cleavage sites [16]. C-terminal PEST motifs have also been identified for the TPD52-like family of proteins, but their PEST scores are below the threshold for biological relevance.
Finally, the TPD52-like family proteins contain an amphipathic lipid-packing sensor (ALPS) motif. First identified in TPD54 [17], the highly conserved ALPS motif is also present in TPD52, TPD53, and TPD55, downstream of the coiled-coil motif (Figure 3). Reynaud et al. [18] report that beyond the coiled-coil region, TPD54 is intrinsically unstructured, with the ALPS motif contained in one of several amphipathic helices (Figure 2). These amphipathic helices are expected to fold upon interaction with lipid membranes. Indeed, Reynaud et al. [18] show that there is an increase in alpha helical structure in residues’ C-terminal to the coiled-coil motif upon binding of TPD54 to highly curved, unsaturated lipid membranes. The coiled-coil motif itself is not involved in membrane binding, and monomeric TPD54 can bind to membranes [19]. The protein–membrane interaction is instead facilitated by two amphipathic helices at residues 101–120 and 141–158, the latter of which contains the ALPS motif [18]. Via these interactions of amphipathic helices with lipid membranes, TPD54, TPD53, and TPD52 are able to bind a class of small intracellular transport vesicles. Dubbed intracellular nanovesicles (INVs), these transport vesicles are defined by their association with at least one of the TPD52-like proteins [19].
The TPD52-like proteins have been associated with proteins outside of the family. In the context of INVs, the TPD52-like proteins have been associated with a wide variety of Rab GTPases [19]. Direct interactions have also been reported, and these appear to be mediated through a number of different regions of the TPD52-like proteins [12,20,21,22]. The binding of TPD52 to the integral membrane protein PLP2 and the membrane-associated protein Rab5c was found not to require the coiled-coil motif, but a region that includes the ALPS motif [12]. The formation of a stable TPD52-Activated Protein Kinase (AMPK) 1 complex involves interactions between the AMPK1 α1 and α2 subunits and the N-terminal residues 1–61 of TPD52 [21]. TPD52 interacts with Peroxiredoxin 1 (PRDX1) in prostate cancer cells via C-terminal residues to promote peroxidase activity [22].
Thus, the TPD52-like proteins are versatile in their ability to interact with each other, with heterologous binding partners and with lipid membranes. The TPD52-like protein structures are highly dynamic, with some regions becoming more structured as they interact with other proteins and lipid membranes. The different preferences for forming homo- and heterodimers within the TPD52-like protein family, and the variety of associations with heterologous proteins suggest that some of the functions of TPD52-like proteins are mediated and controlled through an array of protein–protein interactions along their length. Understanding the structure and function of the TPD52-like proteins is further complicated by the existence of multiple isoforms of each TPD52-like protein.
Alternative splicing is believed to be a key feature of the TPD52-like family, with the presence or absence of cDNA inserts possibly determining the protein function and achieving isoform-specific roles (Table 2 and Figure 3) [13]. Of note, the addition or removal of a consensus 14-3-3 binding site via alternative exon splicing has been identified as a novel mode of regulating 14-3-3 binding in TPD52-like isoforms. TPD53, unlike other family members, retains a 14-3-3 binding motif identifying this as a unique protein interaction and exhibiting an integral role in TPD53 protein function [23]. Although, it has been suggested that TPD52 and TPD54 may exhibit neural-specific functions of 14-3-3 binding (due to the presence of a shorter serine-rich exon expressed in neural tissues prior to the 14-3-3 encoding exon), and as such, regulation of 14-3-3 binding with a subset of TPD52-like proteins may be altered in neural tissues [23]. In contrast, sequences designated as insert 2 are restricted to TPD54 isoforms (Figure 3) [13]. Although the function of this insert is not clear, TPD54 isoforms showed stronger interactions with TPD54+ins2 isoforms compared to TPD54-ins2 isoforms, indicating a role in protein interactions and, consequently, protein function [11]. In addition to insert 2, other inserts (i.e., ins1, ins3 (containing a 14-3-3 binding motif), ins4) are subject to alternative splicing across TPD52, TPD53, and TPD54 isoforms [13]. This diversity of alternative splicing events across the TPD52-like family suggests that various isoforms may modulate TPD52-like protein function. Interestingly, in addition to splicing events, TPD52 was found to be more sensitive to prominent post-transcriptional regulation (particularly by T-cell intercellular antigen 1 (TIA-1) and TIA-related protein binding in the 3′ untranslated region), which affected mRNA stability when compared to other family members [24].
Since their initial characterization, frequent upregulation of the TPD52-like proteins at both gene and protein levels in cancer has been associated with tumor progression and tumorigenesis. For example, Buffart et al. [28] observed a high TPD52 copy number ratio in liver metastases relative to primary colorectal tumors, supporting a contributory role in cancer progression. In addition to copy number and transcript-level alterations, TPD52-like family proteins have been reported to be increased in absolute protein copy numbers [13,29,30,31]. This high baseline abundance may exacerbate the functional consequences of the gene dosage and overexpression during tumor progression.
Of the four family members, TPD52 and TPD54 are most consistently dysregulated in cancer, and their co-expression has been proposed as a marker for acute myeloid leukemia and acute lymphoblastic leukemia [32].
Building on these observations, the functional relevance of the TPD52-like proteins in cancer is further underscored by their long-recognized involvement in intracellular trafficking pathways. The first heterologous partner identified for TPD52-like proteins was MAL2 (Myelin and lymphocyte protein 2), a proteolipid essential for apically directed protein transport, thereby implicating the family in vesicle trafficking [6]. To better understand the roles of TPD52-like proteins in cellular transport, the following section outlines the key principles of intracellular vesicle trafficking.

3. A Brief Overview of Intracellular Vesicle Trafficking
Intracellular vesicle trafficking pathways are integral to a broad range of normal cellular functions, such as protein internalization, secretion, intercellular and interorganelle communication, and signal transduction [33]. These pathways are highly complex and have been the focus of extensive, in-depth reviews in recent years [33,34,35,36,37,38,39]; here, they are discussed briefly.
During the life cycle of a plasma membrane protein, synthesis begins in the endoplasmic reticulum (ER), after which the newly translated protein is transported to the Golgi apparatus for post-translational modifications. The Golgi, a cytoplasmic organelle located near the nucleus, is organized into cis, medial, and trans compartments [40]. Proteins arrive at the cis-Golgi network and move sequentially through the medial compartment before reaching the trans-Golgi network (TGN). At the TGN, fully modified proteins, lipids, and polysaccharides are packaged into intracellular transport vesicles and directed to either the plasma membrane (via exocytosis), early endosomes, or late endosomes [2].
Endosomes are highly dynamic organelles with functionally distinct regions that enable sorting of cargo while simultaneously undergoing protein turnover [41]. They serve as the central trafficking hubs of the cell and are categorized into early endosomes, late endosomes, and recycling endosomes. Increasing evidence suggests that the early endosome and recycling endosome exist in a unified state, often referred to as sorting endosomes, with distinct microdomains enriched in specific proteins or lipids [42]. As a central hub for protein organization, the sorting endosome receives cargo from both the Golgi and the plasma membrane following endocytosis, and subsequently directs it along one of three pathways: a recycling pathway that returns it to the plasma membrane, a retrograde pathway that transports it to the TGN, or degradation via late endosomes [43]. Late endosomes maintain a slightly acidic environment (pH 5–5.5) to prepare cargo for degradation through fusion with lysosomes to form endolysosomes [44]. Lysosomes contain enzymes responsible for digesting unwanted cellular material to facilitate protein degradation.
Autophagy provides an additional degradation pathway targeting cellular debris, damaged organelles, and protein aggregates for recycling through the fusion of autophagosomes and lysosomes, late endosome invagination, or chaperone-mediated pathways [45]. The resulting degradation products (e.g., amino acids) are recycled to support cellular energy production and protein synthesis.
Intracellular vesicles carrying cargo are drawn to the target membrane in part by Rab GTPases. These small proteins serve as master regulators of intracellular trafficking pathways by cycling between an active GTP-bound state and an inactive GDP-bound state [46]. Additional Rab-dependent regulation is exerted through the recruitment of Rab GTPase effector molecules, which include adaptors, phosphatases, motor proteins, kinases, tethers, and regulators of membrane fusion [47]. To date, 66 distinct Rab GTPases have been identified, each associated with specific organelles or trafficking steps to ensure correct cargo delivery [48]. For example, Rab10 is localized to the TGN to regulate TGN-to-plasma membrane trafficking, whereas Rab12 is localized to recycling endosomes and lysosomes to facilitate lysosomal trafficking [46].
Upon arrival at the target membrane, vesicle docking and membrane fusion are mediated by soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) complexes [3]. These complexes are responsible for driving vesicle fusion and exocytosis, and they are necessary for cargo delivery, although they do not provide transport specificity like Rab proteins [49].
Proteins that are delivered to the plasma membrane via Rab GTPases and SNARE complexes can then participate in signaling cascades, function as adherence molecules, or undergo secretion. When proteins are no longer required at the cell surface, they are internalized via endocytosis to the sorting endosome, where they are routed to late endosomes or lysosomes for degradation, or towards the plasma membrane or Golgi apparatus for recycling [50]. A schematic overview of these trafficking pathways is shown in Figure 4.
Equally important to the pathways discussed, intracellular transport vesicles are defined by their protein coats. Coat proteins, including coat protein complex (COP) I, COPII, and clathrin, play essential roles in vesicle budding, formation, and functional specificity to ensure correct vesicular-mediated transport. COPII facilitates the formation of vesicles that drive anterograde transport (Figure 4n), carrying newly synthesized proteins destined for secretion or other organelles [51]. In contrast, COPI-coated vesicles mediate retrograde transport (Figure 4o) and support intra-Golgi transport (Figure 4c) [51]. Clathrin-coated vesicles participate in endocytosis and transport of lysosomal proteins to the lysosome (Figure 4h,i,k) [43,52]. Collectively, these coat proteins influence vesicle formation, directionality, and, in some cases, cargo selection, thereby shaping the overall architecture of intracellular transport.
Of particular interest, TPD54 has recently been identified by Larocque et al. [29] to bind to the membrane of INVs, a population of highly maneuverable, small transport carriers proposed to function as “express” trafficking routes within the cell [2]. Although the underlying mechanisms of INV biogenesis remain unsolved, TPD54 is required for their function. This raises the possibility that TPD52-like proteins may influence vesicle-mediated transport in a comparable manner to classical coat proteins. Supporting this idea, TPD52 and TPD53 have been observed to partially localize to intracellular vesicle structures, suggesting similar roles shared throughout the protein family [6,53]. Among these, TPD54-vesicle interactions are the best characterized, and the involvement of TPD52-like proteins in INV biology is discussed further in Section 6.
Beyond vesicle association, members of the TPD52-like family have been implicated in multiple intracellular trafficking pathways. Briefly, TPD52 localizes to the Golgi apparatus and to several endosome compartments, including sorting endosomes and late endosomes [54,55]. TPD53 also partially localizes to the early endosomes but primarily has a role in SNARE complex assembly through the coiled-coil domain to participate in membrane fusion events [53]. Similar to TPD52, TPD54 associates with the Golgi apparatus, and it has been reported to maintain Golgi integrity [29]. The protein also interacts with Rab GTPases to support trafficking between the ER, Golgi, and endosomes (anterograde transport), as well as participating in endocytosis and recycling pathways [29]. In contrast, the involvement of TPD55 in intracellular trafficking remains undefined. The trafficking pathways involving TPD52-like proteins are summarized in Figure 4, while the specific functions of each family member are elaborated in their respective sections below.

Targeting Intracellular Trafficking in Cancer
Dysregulated membrane trafficking is a characteristic of various disease states, such as cancer, where transport pathways are leveraged to execute multiple strategies (e.g., inducing proliferative signaling, halting autophagy, promoting migration and invasion, and secreting enzymes that aid tumorigenesis and contribute to drug resistance) [56]. Although the adaptation of membrane trafficking itself is not classified as a traditional hallmark of cancer [4], it significantly affects many established hallmarks and thus plays a vital role in cancer progression.
At a cellular level, the function of pro-tumorigenic receptors and proteins is intricately linked to their synthesis, processing, and trafficking within the cell. For these receptors to exert their effects, they must first be synthesized in the ER, undergo post-translational modifications in the Golgi apparatus, and be trafficked to the cell surface via vesicular transport as discussed above. Once at the cell surface, they can interact with their ligands or other signaling molecules [57], contributing to processes such as immune escape, tumor angiogenesis, and metastasis. Given that many key pro-tumorigenic proteins rely on this trafficking pathway to function, targeting the machinery that controls their movement to the cell surface presents a potentially transformative approach. By inhibiting key components of this trafficking machinery, it may be possible to prevent the surface expression of multiple pro-tumorigenic proteins simultaneously. This could not only diminish the tumor’s ability to evade immune surveillance but also block other pathways involved in tumor progression, such as angiogenesis, cell survival, and migration. The advantage of such a strategy lies in its potential to target multiple tumor-driving mechanisms at once, reducing the likelihood of resistance compared to single-target therapies.
Recent work increasingly implicates trafficking regulators as active contributors to tumorigenesis rather than passive cellular components, including Apolipoprotein L4 (APOL4) in glioblastoma [58], dysregulated Rab GTPases (e.g., Rab11 in breast carcinomas) [5], and Golgi phosphoprotein 3 (GOLPH3) in breast cancer [59]. This growing recognition, coupled with increasing interest in the therapeutic potential of targeting intracellular transport pathways, places renewed emphasis on vesicle trafficking-associated proteins. Among these, the TPD52-like proteins represent a particularly relevant family, as their trafficking-related functions and dysregulation, both at gene and protein levels, have been increasingly implicated in cancer [29,53,60].

4. Mechanistic Functions of TPD52
TPD52 has been strongly implicated in membrane trafficking functions, with protein localization to major trafficking compartments including the Golgi apparatus, early endosomes, late endosomes, and recycling endosomes [54,55]. It has also been reported to reside in close proximity to exocrine secretory granules (i.e., large membrane-bound vesicles), supporting roles in exocytotic pathways [6]. Moreover, TPD52 expression has been found in both the endocytic and exocytic compartments of pancreatic acinar cells, with roles in direct regulation of endolysosomal secretion (i.e., secretion of proteins via the endolysosomal pathway), indirect regulation of secretory granules containing digestive enzymes, and sensitivity to secretagogue stimulation (i.e., molecules that promote secretion by cells) [54,61,62]. Many of these intracellular trafficking roles have been identified via TPD52 binding partners, such as Ca2+-dependent binding to annexin VI (a protein implicated in endocytosis), MAL2 (a regulator of intracellular vesicle transport) [54,63], Lysosome-associated membrane protein 1 (LAMP1) (a major component lysosomal membranes that is Ca2+-sensitive) [60,64], and Rab5c (localized to early endocytosis and sorting endosomes) [12]. Additionally, through these Ca2+-dependent mechanisms, TPD52 expression plays a key role in membrane trafficking during cytokinesis, suggesting a role in cell division [64]. Taken together, these binding partners implicate TPD52 in major protein trafficking pathways both within the cell and for secretion.
Another major role commonly described for TPD52 is its involvement in intracellular lipid storage through lipid droplet biogenesis [55,65,66]. Co-localization with Golgi markers suggests a potential role in transporting lipid droplets directly from the Golgi apparatus, which is consistent with other reported trafficking roles discussed above [25,55]. Through overexpression studies in zebrafish, it has been reported that the AMPK pathway is a primary target of TPD52, leading to lipid accumulation, adipocyte differentiation, and adipose tissue expansion [67]. In addition, TPD52 was found to bind to the alpha subunits of AMPK to form stable complexes (TPD52-AMPK), thereby inhibiting kinase activity and supporting roles in metabolism [21].

Implications of TPD52 in Cancer
TPD52 is widely reported to be aberrantly expressed in numerous types of cancers, including pancreatic [68,69], breast [70,71,72,73,74,75,76], lung [77], cervical [78,79], ovarian [80], brain [81], bladder [82,83], colorectal [84], prostate [85,86,87,88,89], testicular germ cell tumors [90], and blood cancers [91,92]. This overexpression is primarily due to the increased copy number caused by amplification of the chromosome band 8q21 in which TPD52 resides [93,94]. When overexpressed in non-malignant mouse fibroblasts (3T3), Tpd52 induced transformation to a malignant phenotype that promoted tumorigenesis and metastasis [95]. Consistent with this oncogenic activity, Zeng et al. [96] identified TPD52 as a novel deubiquitinating target for Ubiquitin-specific Peptidase 10 (USP10) in gastric cancer cells, enabling TPD52 to evade protein degradation and providing an additional mechanism contributing to dysregulated expression. However, decreased expression of TPD52 has also been reported in renal cell carcinoma [97] and primary hepatocellular carcinoma [98], suggesting that this protein may act as a potential tumor suppressor in these malignancies. These contradictory findings indicate tissue-dependent functions of TPD52 in the progression of cancer, as summarized in Table 3.
Dysregulated TPD52 expression has been implicated in cancer cell proliferation, migration, and invasion in breast cancer [70,107,112,113], cervical cancer [78,79,121,123], melanoma [135], pancreatic cancer [69], oral squamous cell carcinoma [139], and lung squamous cell carcinoma [77]. Many cellular mechanisms have been reported to underlie these pro-oncogenic roles, including PAX-3 (paired box gene 3)-mediated regulation [135], regulation of mitotic checkpoint genes (e.g., Cyclin B1/2, TTK protein kinase, Minichromosome maintenance complex component 4 (MCM4)) [77], involvement in cell signaling axes such as NEAT1/miR-218-5p/TPD52 [112], promotion of epithelial-to-mesenchymal transition (EMT) [107], links to the proliferation-related gene CD58 [91], and suggested sensitivities to hypoxia [139]. In renal cell carcinoma, TPD52 has additionally been reported to act through the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway to modulate cell proliferation, migration, invasion, and EMT phenotypes [97].
Similar to the tissue-specific function described above, TPD52 has also been reported to exhibit isoform-specific functions. Most notably, TPD52 amplification is well-documented in prostate cancer, with prostate-specific isoforms (commonly referred to as PrLZ or PC-1) identified as androgen-responsive [86,88]. TPD52 has also been reported to interact directly with the androgen receptor via androgen-response elements in the 5′ untranslated region, contributing to disease progression from androgen dependence to androgen independence (i.e., castration-resistant prostate cancer) [9,149,155]. As such, TPD52 is considered a putative pro-oncogene in prostate cancer progression due to its roles in promoting cell growth, survival, migration, and invasion (regulated through interactions with 14-3-3 proteins and suppression of kinase activity by the tumor suppressor Liver Kinase V1 (LKV1)) [85,87,157,160,166].
TPD52 has been linked to the modulation of various cell signaling pathways involved in cell growth, survival, and metabolism. Multiple studies implicate TPD52 in Nuclear Factor kappa-light-chain-enhancer of activated B-cells (NF-κB), Signal Transducer and Activator of Transcription (STAT) 3, and Akt signaling in cancer cells, all of which are involved in crucial pathways for cellular processes such as cell growth, survival, immune response, and metabolism [102,133,176]. TPD52-mediated activation of the Janus Kinase (JAK)/STAT, PI3K/Akt, and Raf/MEK/ extracellular signal-regulated kinase (ERK) signaling pathways has been reported in neuroblastoma cells to modulate cell differentiation post retinoic acid treatment [138]. This role in cell differentiation has also been reported by other studies, including involvement in the maintenance of neuroendocrine, EMT, and hematopoietic stem cell phenotypes [91,174,180], and B-cell differentiation into plasma cells [101]. This supports early investigations demonstrating that TPD52 expression is involved in the development and maintenance of epithelial cells and embryogenesis [145,184]. Some studies suggest that TPD52 is a downstream target of receptor tyrosine kinase (RTK) signaling, with ERBB2 (also known as HER2) being shown to be co-expressed and have complementary cellular functions with TPD52 to promote breast cancer cell survival [73,75]. Additionally, TPD52 was demonstrated to promote genomic instability through direct interactions with ATM (ataxia telangiectasia mutated) to reduce signaling and, consequently, impair DNA repair capabilities of SK-BR-3 (breast cancer) cells [115]. Genome-wide gene expression analysis of lung squamous cell carcinoma cells supported this finding through the identification of genomic instability as a downstream effect of TPD52 [77]. Taken together, TPD52 appears to promote cell growth and survival through multiple signaling pathways and increases the mutational burden through promoting genomic instability in some cancer cells. Interestingly, there is a reported role for TPD52 in cell stress, with TPD52 driving activating transcription factor 6 (ATF6) activation during ER stress to subsequentially activate the unfolded protein response in liver cancer cells, to promote cell death and ultimately act as a tumor suppressor [82]. Precise mechanisms of TPD52 in these pathways require further elucidation, but the varying reports of a cellular function of the TPD52 protein emphasize its likely context-dependent functions. Additionally, TPD52 has been identified as a downstream target of many different microRNAs to modulate oncogenic mechanisms, as summarized in Table 3 (miRNAs in bold).
As discussed above, TPD52 is primarily reported to be a cell-trafficking protein, a role that extends to its implied roles in cancer progression. It has been reported to bind to annexin VI in a Ca2+-dependent manner in both pancreatic acinar cells [63] and myeloma plasma cells [101] to regulate cellular secretory pathways. Through calcium binding, annexin VI plays an important role in membrane interactions and endocytosis, implying that TPD52 may function in intracellular trafficking during oncogenesis [185]. Additionally, TPD52 has been reported to form a complex with Heat Shock Protein Family A Member 8 (HSPA8; a molecular chaperone involved in protein folding, stabilization, and degradation) to activate chaperone-mediated autophagy in prostate cancer cells [163], which serves to aid in protein translocation for cellular recycling and lysosomal degradation. Consistent with this role in trafficking, TPD52 regulates the ER size in response to stress, suggesting potential roles in maintaining ER integrity [82].
In addition to its functional roles, TPD52 has also been reported to be involved in immune cell infiltration in cancer [71]. Through in silico analysis, TPD52 has been associated with the modulation of immune cell infiltration, tumor immune landscape, and immune checkpoints [83,113,183]. There are also a number of reports on TPD52 roles in radio- and chemotherapy sensitivity in triple-negative breast cancer [72] and prostate cancer [158,162,164,175].
In summary, TPD52 is the most well-characterized family member in cancer progression, and although there are varying reports describing either oncogenic or tumor suppressor-like activity, it has well-characterized mechanisms in pro-tumorigenic functions. These functions are likely carried out through intracellular trafficking (including endocytosis, secretion, recycling endosomes, and late endosomes), lipid storage and biogenesis, and cell division.

5. Mechanistic Functions of TPD53
Like the other family members, TPD53 has homo- and heterologous binding partners and has been reported to have roles in membrane trafficking, cell cycle regulation, and apoptosis. TPD53 has been shown to partially localize to early endosomes and intracellular vesicle structures [53]. Additionally, TPD53 is involved in the assembly of endosomal SNARE complexes to participate in homotypic or heterotypic membrane fusion as well as the recycling of synaptic vesicles. TPD53 can bind directly to Synaptobrevin 2 (Sb2) via the coiled-coil domain, and Syntaxin (Stx1), to form SNARE-like complexes (Sb2/Stx1/TPD53), which then can go on to participate in membrane fusion [53]. As the coiled-coil domain is a feature shared by all members of the TPD52-like family, it is possible that, if TPD53 functions as a SNARE, the other family members may also possess similar capabilities; however, this is yet to be reported.
TPD53 has been identified as a cell cycle-regulated protein. Protein expression is upregulated at the G2-M transition phase in parallel with cyclin B1 (a known regulator of transition from G2 to M phase) and remains highly expressed into early mitosis (i.e., prophase) before rapidly decreasing at metaphase. As such, dysregulated TPD53 expression in breast cancer cells adversely affects mitosis, leading to the accumulation of multinucleated cells and cell death [186]. Microarray analysis showed that TPD53 expression fluctuates according to the cell cycle stage in human fibroblasts [187] and HeLa cells [188], as well as being a circadian clock cycling gene in the mouse liver (i.e., involved in the regulation of the daily rhythm of metabolic processes (e.g., lipid metabolism, bile acid synthesis, etc.)) [189]. Moreover, overexpression of TPD53 also occurs during replicative senescence in fibroblasts [190]. Taken together, TPD53 appears to have a role in cell cycle regulation, with dysregulated expression possibly affecting normal cellular division. Supporting this, TPD53 has been reported to undergo alternative splicing events (as discussed above), which allow it to directly bind with 14-3-3, a negative regulator of the cell cycle progression [23]. Other TPD52-like proteins lose this binding motif during alternative splicing (Figure 2 and Figure 3), making this interaction with 14-3-3 unique to TPD53. Following increased TPD53 expression during G2-M phase transition, increased protein binding with 14-3-3 was also observed, suggesting a combined effort to regulate the cell cycle [186]. As 14-3-3 proteins are involved in many cellular processes in both healthy and disease states, this interaction suggests that additional cellular roles for TPD53 are yet to be discovered. Roles for TPD53 in apoptosis have also been demonstrated, with protein expression maintaining apoptosis signal-regulating kinase 1 (ASK1) in a moderately active state to promote the accumulation of pro-apoptotic factors such as caspase [191].

Implications of TPD53 in Cancer
Unlike TPD52, TPD53 is not overtly implicated in cancer (Table 4). However, a number of reports have described upregulation and involvement in the disease progression of breast cancer [23,186,192], lung adenocarcinoma [193], oral squamous cell carcinoma [194], and colorectal cancer [195]. The limited studies of TPD53 in these cancers include overexpression leading to the modulation of cell viability, migration, and invasion, matrix metalloprotease (MMP) activity, and cell cycle regulation [194,195]. Although the cellular mechanisms are currently unknown, TPD53 has been identified as having prognostic significance in thyroid cancer [196], colorectal cancers [195,197], hepatocellular carcinoma [198], and nasopharyngeal cancer [199]. Supporting oncogenic functions in these cancers, TPD53 protein expression in patients with ovarian tumors decreased following chemotherapy treatment [200], alluding to some importance in cancer cell survival. Additionally, there is evidence suggesting that TPD53 may have a role in acquired chemotherapy resistance through novel TPD53-ROS1 fusion rearrangements (fusion of TPD53 exons 1-3 to ROS1 exons 33-43) in lung adenosquamous cell carcinoma [201]. Overall, while there is limited evidence that TPD53 functions as a pro-tumorigenic protein in some cancers, the precise mechanisms remain to be elucidated.

6. Mechanistic Functions of TPD54
The recent literature has characterized TPD54 as a vesicle-trafficking protein that exhibits partial localization to the Golgi apparatus as well as to cytoplasmic compartments [29]. TPD54 exhibits strong associations with Rab14 and Rab2a, and shows a more modest association with Rab5c [29]. Rab14 is important in membrane trafficking between the Golgi apparatus and endosomes, Rab2a is localized to the ER-Golgi pathway, and Rab5c is involved in the endocytic pathway [203,204,205]. These associations implicate TPD54 in multiple cell-trafficking pathways, including anterograde trafficking, endosomal recycling, and the maintenance of Golgi integrity [29]. Among these roles in cell trafficking pathways, TPD54 has recently been identified to be bound to INVs (Figure 5) [29]. As outlined above, INVs are small, highly maneuverable transport vesicles that are hypothesized to provide an efficient transport pathway within the cell [2]. Multiple copies of TPD54 (monomer) can directly bind to the INV membrane via positively charged residues located within the C-terminal region of the TPD54 protein to regulate the trafficking of ligands carried within the INV [2,8]. Residues 83-125, located in the C-terminal domain of TPD54, are responsible for direct membrane binding, while cellular assays showed that positive residues (including K154, R159, K175, and K177) in this region are required for specific binding to INVs in the cell [19]. Interestingly, TPD52—and, to a lesser extent, TPD53—are also associated with INVs and bind independently of TPD54. Larocque et al. [19] reported four INV populations defined by the presence of at least one member of the TPD52-like family (as summarized in Table 5) as well as intermediate populations, but there is evidence that TPD54 is required for correct INV function.
Intracellular vesicles commonly range from 40 nm to 1000 nm, whereas INVs are only 30 nm in size, possibly allowing for fast intracellular transport to rapidly adapt to extracellular cues and precisely control protein flow to and from the plasma membrane [29,206]. INVs have been documented to transport integrins, notably α5β1, with depletion of TPD54 leading to the retention of integrins within intracellular compartments [2,8]. Integrin trafficking has a central role in cell migration and tumorigenesis, suggesting that the TPD54/INV pathway may be altered during cancer progression [2]. Further supporting this, gene enrichment analysis of TPD54 and co-expression genes revealed an enrichment of cell migration pathways and ER-to-Golgi vesicle-mediated transport [207]. Currently, the proteome of INVs is unknown, and it is not fully understood what other cargo they carry. However, TPD54 may act as a major regulator in the intracellular transport of a variety of proteins. Thus, it is tempting to suggest that when TPD54 expression is altered, it may contribute to cancer progression.

Implications of TPD54 in Cancer
Similar to TPD53, there is limited literature outlining the contribution of TPD54 in cancer (Table 6). However, TPD54 overexpression is linked to pro-oncogenic mechanisms and disease progression in breast cancer [8], clear cell renal cell carcinoma [208], colon cancer [209], glioma [210,211], head and neck squamous carcinoma [207], lung adenocarcinoma [212], pancreatic cancer [213], and prostate cancer [214,215]. In contrast, in oral squamous cell carcinoma [140,216], low TPD54 expression correlates with poor patient outcomes and contributes to oncogenesis. Qiang et al. [217] reported that TPD54 is heterogeneously expressed in glioblastoma with conflicting functions due to its modulation of the Wnt pathway to regulate the EMT status of cancer cells. These findings suggest possible tissue-dependent functions similar to that noted for TPD52.
Dysregulated TPD54 expression in solid cancers has been reported to modulate cell proliferation, migration, invasion, and colony formation. However, the specific mechanisms driving TPD54 involvement in these oncogenic processes are not well understood. In vitro studies demonstrated that TPD54 knockdown inhibited PIK3CA/Akt signaling in pancreatic cancer cells, a pathway that enhances cell survival, growth, and cell cycle regulation [213]. TPD54 inhibition induced cell cycle arrest in the G0/G1 phase, further strengthening its role in cell cycle control [210]. Additionally, TPD54 was shown to reverse tumor-suppressing microRNA activity to promote pro-tumorigenic processes in glioma [211]. Several other studies have also identified novel microRNA molecules that exert tumor-suppressing effects by targeting TPD54 in cancer, further alluding to its importance in solid cancer progression [213,215].
A role for TPD54-driven integrin activation and trafficking during cell attachment and migration in both breast cancer and oral squamous cell carcinoma cells has been reported [8,216], further supporting its involvement in protein trafficking. Further gene enrichment results also found that pathways such as focal adhesion and ER-to-Golgi vesicle-mediated transport were significantly enriched in head and neck squamous carcinoma [207]. This provides preliminary insight into how the TPD54 trafficking pathway may be hijacked during cancer progression to promote cancer cell survival. Further investigation into the role of TPD54 trafficking of other known pro-tumorigenic proteins or growth factors would support this hypothesis.
In silico analysis provided insights into the ability of TPD54 to modulate immune-cell infiltration (i.e., TAMs and Tregs) in the TME to promote cancer progression [207,212]. These pro-tumor immune cells upregulate the secretion of inflammatory cytokines and chemokines for immune escape. TPD54 was reported to have associations with key immunosuppressive genes (e.g., CD274 (PD-L1), EGFR, CD44, TGF-β1, and TGF-βR1) to further modulate the TME and alter the patient response to treatment in head and neck squamous cell carcinoma and lung adenocarcinoma, highlighting its involvement in tumor immunity [207,212]. Multiple studies have shown a correlation between TPD54 expression and patient sensitivity to traditional treatments (i.e., radiation and chemotherapy) in breast cancers. However, reports of the mechanism of TPD54 in cancer treatment are conflicting. Zhang et al. [8] report that breast cancer patients with high TPD54 tumor expression had significantly shorter survival times after treatment with radiation therapy or targeted molecular therapy, whereas Zhuang et al. [218] reported that TPD54 expression enhanced breast cancer cell sensitivity to metformin treatment via modulation of pyruvate dehydrogenase (PDH) enzyme activity. TPD54 has also been reported to have prognostic significance in pancreatic cancer patients with early tumor reoccurrence post-surgery [219]. Preliminary studies describe a role for TPD54 in modulating tumor immunity and roles in the treatment response, both of which are of clinical interest and warrant further elucidation.
The current literature provides limited insights into the mechanistic actions of TPD54 in tumorigenic processes; however, it is tempting to speculate that its role in intracellular vesicle trafficking may be as a courier system for cancer cells to hijack and facilitate pro-tumorigenic protein transport to the cell surface. In theory, this would enable the cancer cell to control protein flow and protein recycling, thus aiding cell survival, and ultimately, disease progression. The current evidence for the role of TPD54 in cancer warrants further investigation.

7. Mechanistic Functions of TPD55
The least researched member of the TPD52-like protein family, TPD55, was described by Cao et al. [30] to be exclusively expressed in the normal testis, with fertile men having higher expression than infertile men [220]. Interestingly, the adult testis show a 5.6-fold higher expression level of TPD55 than the fetal testis, supporting a potential involvement in testis development and spermatogenesis [30]. TPD55 was also identified as a novel human sperm tail protein and identified to have roles in sperm capacitation (i.e., physical changes that sperm undergoes in the female reproductive track in order to fertilize an egg) [221]. Although limited, the literature convincingly reports a significant role for TPD55 in the testis and in spermatogenesis.
Interestingly, one of the three known isoforms (isoform 3) was detected in placenta, heart, liver, and adult testis tissue [30]. This aligns with the potential isoform-specific functions identified within this protein family and suggests that isoform- or tissue-dependent functions may be characteristic of the TPD52-like members. TPD55 was also shown to successfully interact with itself and other TPD52-like family members in a yeast two-hybrid system and in vitro [30], likely through the coiled-coil domains characteristic of its family. Thus far, only one heterologous interaction partner has been identified, namely Nuclear Factor I-C (NFIC), which plays roles in cellular differentiation, DNA replication, and gene regulation [222]. Further investigations into this protein are required to fully elucidate its mechanistic actions and significance in cell function.
Utilizing next-generation RNA sequencing, Takematsu et al. [223] reported that TPD55 was the most downregulated gene in skin samples from type 2 diabetes mellitus patients when compared to samples from non-diabetic patients. To note, additional studies to validate the role of TPD55 in type 2 diabetes are yet to be published, and as such, TPD55 remains widely accepted as a testis-specific protein.

Implications of the TPD55 in Cancer
Although there is no current literature implicating TPD55 in cancer, it is tempting to speculate a potential role for TPD55 in testicular cancer. Clearly, more research is required.

8. Summary and Conclusions
In summary, the TPD52-like family members appear to share several mechanistic aspects in common: coiled-coil domain-mediated homo- and heterodimerization, which regulates vesicle trafficking between the Golgi, endosomes, and plasma membrane. The level and direction of misexpression in cancer, as well as its cancer relevance, typically differ by tissue-of-origin, isoform, and binding context. However, each family member has been associated with the modulation of protein trafficking. This suggests that the cell type and isoform-specific expression patterns determine whether TPD52-like proteins promote or suppress tumorigenesis.
There is increasing evidence that members of the TPD52-like family play critical roles in intracellular trafficking, protein interactions, and cancer progression. Although the precise mechanistic actions are yet to be fully elucidated, the isoform-specific functions driven by alternative splicing may be key to a better understanding of their roles in cancer. The roles that TPD52-like family homo- and heterodimers play in healthy and cancerous cell functions also remain to be answered. However, TPD52 and TPD54 (and, to a lesser extent, TPD53) present themselves as fascinating proteins with roles in intracellular trafficking pathways (e.g., exocytosis, endosome transport, Golgi integrity) and well-documented evidence of overexpression in many cancers. Targeting the TPD52 family as key components of intracellular trafficking pathways offers a single approach to simultaneously prevent the expression of pro-tumorigenic molecules (e.g., integrins) at the cell surface. To this end, studies using cancer vaccines to target tumor self-antigens such as TPD52 have reported improved immune responses against breast and prostate tumors [105,151,224]. In addition, an anti-TPD52 antibody has been identified as a tool for the diagnosis of B-cell malignancies [101]. Moreover, exosomes designed to bind HER2-positive breast cancer cells successfully delivered TPD52-targeting siRNA [117], providing a possible targeted approach to gene therapy. These reports provide confidence in the potential therapeutic benefits of targeting the TPD52-like family to combat cancer.
In conclusion, this review presents evidence that the TPD52-like family has essential roles in cancer cell pathophysiology. Disrupting these intracellular trafficking mechanisms by targeting TPD52/TPD54 in cancer could offer a single approach to interfere with a suite of pro-cancerous cellular mechanisms that underpin cancer progression. Herein, we also identify major gaps in our collective knowledge of the mechanistic actions of these family members that would support TPD52-like based therapies in achieving success in the clinical setting. An important next step will be to elucidate the isoform-specific roles in different tissues and cancer types because alternative splicing may affect family member interactions, trafficking, and downstream signaling. Thus, isoform-specific proteomics and interaction studies will be key for rationally developing therapies that target TPD52-like proteins.