Abstract
Tetraspanins are a highly conserved family of transmembrane proteins that play a pivotal role in cellular signaling by forming tetraspanin-enriched microdomains (TEMs) at the plasma membrane. These microdomains interact with various signal transduction-associated proteins, thereby regulating critical processes such as cell migration, protein trafficking, and multiple signaling pathways. Dysregulation of tetraspanins at transcriptional and translational levels is implicated in several pathological conditions, including cancer. While extensive research has focused on the regulation of tetraspanins at the protein level, their transcriptional and post-transcriptional regulation remains poorly understood. This review provides a comprehensive and up-to-date synthesis of the regulatory mechanisms governed by transcription factors, epigenetic modifiers, microRNAs (miRNAs), and long noncoding RNAs on all tetraspanin family members. We have also examined the mutational landscape of tetraspanins and their pathological consequences across various diseases. Furthermore, we highlight critical gaps in current knowledge and propose future directions for research to advance our understanding of tetraspanin regulation and its therapeutic potential in disease biology.
Introduction
Tetraspanins are members of a well-conserved family of integral membrane proteins that play crucial roles in cellular communication. The tetraspanin family consists of numerous members across different species. There are 33 tetraspanins in Homo sapiens, 37 in Drosophila melanogaster, 20 in Caenorhabditis elegans, and 17 in Arabidopsis thaliana. 1 The human tetraspanin family comprises TSPAN1, TSPAN2, TSPAN3, TSPAN4, TSPAN5, TSPAN6, TSPAN7, TSPAN8, TSPAN9, TSPAN10, TSPAN11, TSPAN12, TSPAN13, TSPAN14, TSPAN15, TSPAN16, TSPAN17, TSPAN18, TSPAN19, UPK1B, UPK1A, PRPH2, ROM1, CD151, CD53, CD37, CD82, CD81, CD9, CD63, TSPAN31, TSPAN32, and TSPAN33. This review emphasizes and explores the regulation of human tetraspanin family members. The gene names of all 33 human tetraspanin family members with their aliases, chromosomal location and cellular localization have been summarized in Table 1.
Human tetraspanin family members with their gene names and aliases, chromosomal location, and cellular localization
NCBI database (https://www.ncbi.nlm.nih.gov/).
Uniprot database (https://www.uniprot.org/).
As the name suggests, tetraspanins consist of four transmembrane domains (TM1, TM2, TM3, and TM4), which span the plasma membrane four times. Other than four transmembrane domains, there are two extracellular loops (EC1/SEL and EC2/LEL) and one small inner loop (IL) (Fig. 1a-1c). The EC1 is small, while the EC2 is relatively large, consisting of one conserved and variable domain. The variable domain is unique to each tetraspanin member, and N and C termini of the protein lie inside the cytoplasmic region. 1 Several amino acid residues in these domains are conserved across all the tetraspanin members. 1 The EC2 domain contains conserved cysteine residues, a characteristic feature of tetraspanins. These conserved cysteine residues form 2–4 disulfide bonds, and a cysteine-cysteine-glycine (CCG) motif, which helps in the proper folding of ectodomain. 2 The domain structures of all the human tetraspanins are illustrated in Fig. 2. A multiple sequence alignment of protein sequences of all 33 human tetraspanins is performed using the Constraint-based Multiple Alignment Tool (COBALT) (Fig. 3). 3 The analysis shows the conserved (marked red) and nonconserved (marked blue) regions across all the members of the tetraspanin superfamily, which emphasize similar as well as unique features of tetraspanin proteins (Fig. 3). Each tetraspanin expresses at approximately 30,000–100,000 copies per cell and is expressed in almost every tissue in the body. 4 The ubiquitous nature of tetraspanins suggest their crucial role in physiology of organisms. 4 The full crystal structures for following human tetraspanins are resolved and available in the Protein Data Bank (PDB) database (https://www.rcsb.org/): CD53 (PDB ID: 6WVG), CD9 (PDB ID: 6K4J), and CD81 (PDB ID: 5TCX) 5 (Fig. 1c). In addition, the partial crystal structure (extracellular loop of CD9, TSPAN15, and CD81) and receptor complexes (PRPH2-ROM1, CD19-CD81, and TSPAN15-ADAM10) of various tetraspanins are also solved using X-ray diffraction studies. 5
Schematic representation of tetraspanins. (a) Basic structure of tetraspanin protein spanning the plasma membrane. (b) General domain structure. (c) The crystal structures of human CD9 (PDB ID: 6K4J), CD81 (PDB ID: 5TCX), and CD53 (PDB ID: 6WVG).
Domain structures of all the 33 human tetraspanins. Abbreviations: N, N-terminus; I, Transmembrane I; II, Transmembrane II; III, Transmembrane III; IV, Transmembrane IV; EC1, Small extracellular loop; EC2, Large extracellular loop; IL, Inner loop; and, C, C terminus. The amino acid locations were retrieved from the Uniprot database (https://www.uniprot.org/).
The multiple sequence alignment of protein sequences for all the 33 human tetraspanins. Query 10001 to Query 10033 represent the input sequences from TSPAN1 to TSPAN33. Red color indicates highly conserved positions, and blue color indicates lower conservation, whereas the gray color represents gaps. Numbers in the End column represent the final length of the proteins. The multiple sequence alignment was performed, using the COBALT tool (https://www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi).
Although the structural properties of tetraspanins are well studied, 5 their gene regulation has remained unexplored. This literature review aims to comprehensively present findings on the transcriptional and post-transcriptional regulation of human tetraspanins. To our knowledge, no other review has been published that summarizes this specific area. This review highlights the crucial discoveries and lacunae in the field that might drive the tetraspanin research further toward the open questions regarding their transcriptional and post-transcriptional regulation.
Tetraspanin Functions
All 33 human tetraspanins share a similar core structure (Fig. 2), however their evolution has assigned them specific functions. For example, due to the lack of ligand binding domain and catalytic activity, tetraspanins act through specialized structures known as tetraspanin-enriched microdomains (TEMs). 4 TEMs extensively interact with other membrane receptors such as integrins, G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs), and even intracellular signaling molecules including protein kinase C (PKC), phosphatidylinositol 4-kinase, GTPases, and β-catenin to regulate their functions. 1
Tetraspanins and signal transduction
The TEMs and membrane receptors form multi-molecular signaling platform affecting physiological processes such as development, movement, and growth. 6 Tetraspanins regulate adhesion-mediated signaling by colocalizing with focal adhesion proteins, vinculin and talin, and perform PKC-mediated signaling. 1 The tetraspanins CD9, CD63, CD81, CD82, and CD151 interact with phospho-focal adhesion kinase (FAK). 1 The downregulation of these tetraspanins hampers FAK phosphorylation and integrin signaling. 1 CD9 and CD81 regulate the GPCR signaling via interacting with GPR56, while CD82 and CD9 influence the RTK signaling by modulating EGFR phosphorylation and activation. 1
Tetraspanins as exosomal markers
Tetraspanin CD81, CD63, and CD9 are major exosomal markers. 7 TEMs at the plasma membrane interact with several receptors and play a crucial role in exosomal biogenesis, miRNA and protein cargo sorting, exosomal uptake by target cells, and antigen presentation to immune cells. 8
Role of tetraspanins in infections
Being surface proteins, tetraspanins are crucial for entry of many viral and nonviral pathogens. CD81 has been shown to mediate entries of influenza virus and malarial parasite in liver cells. 6 Furthermore, TSPAN9 regulates alphavirus entry, while CD151 is known to regulate the entry of human papillomavirus 16 (HPV16) and human cytomegaloviruses with the help of integrins and other receptors. 9 CD63 and TSPAN9 are regulators in viral trafficking of human immunodeficiency virus, influenza A, HPV, and Lujo virus. 9 Interestingly, tetraspanins influence both viral entry and viral exit. 9
Regulation of Tetraspanins
The gene expression is a complex yet orchestrated and tissue-specific process, which ensures that the right protein is produced in the right amount at the right time in the cell. In general, gene expression can be regulated at the following levels: 1) transcriptional, 2) RNA processing, 3) RNA transport and localization, 4) mRNA degradation, 5) translation, and 6) protein activity. 10 Transcriptional regulation is defined as regulation at the transcriptional level, whereas post-transcriptional regulation involves RNA processing, transport, localization, and mRNA degradation. 10
Given the extensive research articles and reviews published on the regulation of tetraspanins at the protein level, 1 this review focuses on the regulation of tetraspanins at the transcriptional and post-transcriptional levels. In the section on transcriptional regulation, we review transcription factors, epigenetic regulators, and alternative splicing (AS) events reported in the tetraspanin family, whereas, in the section on post-transcriptional regulation, we have emphasized the regulation of tetraspanin genes by microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) (Fig. 4).
A schematic diagram showing the transcriptional and post-transcriptional regulators of tetraspanins and their implications on tetraspanin functions.
Transcriptional regulation of tetraspanins
Regulation at the transcriptional level is carried out by binding of various transcription factors, such as activators, repressors, and co-regulators to DNA regulatory elements. 11 The binding of these factors can be either directly onto the DNA regulatory elements or indirectly to other regulatory DNA binding proteins. The direct and indirect transcriptional regulation can cause either positive or negative alteration in gene expression. 11 In addition to transcription factors, genes can be regulated via epigenetic mechanisms. The main epigenetic mechanisms of gene regulation are DNA methylation, histone modifications, chromatin remodeling, and regulation by noncoding RNAs. 12 The DNA methylation at CpG sites often causes downregulation of genes, whereas histone modifications can be active or inactive gene expression signatures depending on the modification type. Several proteins or epigenetic modifiers which carry out these functions are classified according to their role. DNA methyl transferases, ten-eleven translocation methyl cytosine dioxygenases, histone methyl transferases, histone acetyltransferases (HATs), histone deacetylases (HDACs), and histone demethylases are different classes of epigenetic modifiers. 13
In addition to the regulation by transcription factors and epigenetic regulators, the nascent transcripts also undergo splicing to remove introns during transcription. 14 The spliceosome complex with accessory proteins binds to the exon-intron boundary and removes introns, followed by the joining of coding exons. Many transcripts also undergo AS and express in multiple isoforms, adding another layer of gene regulation. 14 The transcription factors, epigenetic regulators, and AS events regulating transcription in the tetraspanin family are summarized in Table 2.
Transcriptional and post-transcriptional regulators of human tetraspanin family members
HCV, hepatitis C virus.
Regulation of tetraspanins by transcription and epigenetic factors
A common network of transcription factors binds to multiple tetraspanin genes due to sequence similarity across the tetraspanin family. One such example is the binding of Specificity protein 1 (Sp1) transcription factor to multiple tetraspanins and modulating their gene expressions. In liver cancer cells, Sp1 binds to the promoter region of CD151 and is required for its basal activation. 15 Sp1 also binds to the UPK1B gene promoter, and its binding site is lost due to CpG methylation in urothelial tumor cells. 16 In another report, multiple Sp1 binding sites are present on CD53 regulatory sequences, and they are utilized differentially in different cell lines. 17 CD63 also contains potential binding sites for Sp1 in its 5′ flanking regulatory region. 18 Another tetraspanin, CD82 (also known as KAI1), has 9 Sp1 binding sites in its promoter element. 19
Similar to Sp1, Activator protein 1 (AP-1), Activator protein 2 (AP-2), and TEA domain transcription factor 2 (ETF) transcription factors also bind directly to promoters of several tetraspanin genes. The CD82 gene has five AP-2 binding sites in its promoter region. 19 In addition to AP-2, CD82 promoter activity is also regulated by binding of p53 and junB. 20 The loss of p53, junB, and AP-2 reduces CD82 expression in prostate cancer. 20 Along with Sp1, the CD63 gene has binding sites for AP-1, AP-2, and ETF transcription factors. 18
In a study by Zhou et al., 21 the canonical WNT-CTNNB1 pathway upregulates TSPAN1. It has been shown that the CTNNB1-TCF4 transcription complex binds directly to the TSPAN1 promoter and increases its expression. In addition, the mRNA level of TSPAN1 is upregulated by FAM83A through facilitating the CTNNB1-TCF4 complex recruitment to its promoter region. These results indicated that TSPAN1 is a direct target of the WNT-CTNNB1 signaling pathway. 21 In B cell lymphoma, Interferon regulatory factor 8 (IRF8) binds to the promoter of CD37 and transcriptionally activates its expression. 22 TSPAN8 is regulated by transcription factor SOX9 in pancreatic cancer. 23
The tetraspanin family is also regulated by various epigenetic mechanisms such as DNA methylation, histone modifications and regulation by noncoding RNAs (noncoding RNAs are discussed in the next section). The CD82 gene is a well-studied tumor suppressor, and its downregulation is reported in many cancers.24,25 Apart from its regulation by many transcription factors such as AP-2, p53, and junB, some reports suggest CD82 regulation via epigenetic mechanisms, although it has remained controversial.24,26 Three independent research groups have reported that the downregulation of CD82 is not due to its promoter methylation in prostate cancer, oral cancer, and melanoma cell lines.24–26 These groups have shown that inhibiting methylation by 5-aza-2’-deoxycytidine does not revert the CD82 expression.24–26 In contrast, another research study in 2017 showed that promoter methylation at CpG islands plays a vital role in regulating CD82 expression in prostate cancer. 27 The alternative suggested mechanism for CD82 regulation includes loss of heterozygosity instead of promoter methylation in melanoma cells. 26 Moreover, the promoter methylation is also observed to be the reason behind the downregulation of CD82, CD81 and CD9 genes in multiple myeloma. 28 In addition to the controversial promoter methylation, CD82 expression is also reported to be regulated by histone modifiers. PLU1 (an H3 demethylase and an oncogene) represses the expression of KAT5 (a HAT), which positively regulates CD82.25,29 Hence, by inhibiting KAT5, PLU1 epigenetically represses the expression of tumor suppressor CD82. 29 Similar to CD82, CD81 is also a tumor suppressor, and its promoter hypermethylation has been reported in glioblastoma, multiple myeloma, and gastric cancer cells.28,30,31 In addition to promoter hypermethylation, CD81 expression also reverts back due to HDACs inhibition in glioblastoma. 32 A similar mechanism has been reported for the CD9 gene in multiple myeloma and B cell lymphoma, where CD9 expression is downregulated because of deacetylation by HDAC activity.33,34 Supporting HDAC-mediated CD9 inhibition, another report in 2016 observed the repression of CD9 by HDAC5 in neuroblastoma. 35 Furthermore, eugenol, a phenolic compound, is reported to increase H3K27 acetylation, and thus increasing the expression of CD151 in cardiomyocytes. 36
As all the members of the tetraspanin family have a similar domain structure, the other tetraspanins may also be regulated via similar epigenetic mechanisms.
AS of tetraspanins
AS is a regulatory process in which different exons (sometimes parts of introns) join in multiple combinations, generating many splice isoforms from a single transcript. These multiple isoforms are being translated into different protein isoforms, which perform separate functions. According to an estimate, more than 90% of genes undergo AS, increasing the gene products by many folds. 37 The tetraspanin family of genes are also reported to go through AS events and express as multiple isoforms with varied functions. 37
A comprehensive search led by Lang and group (2019) has reported the expression of 31 nonconventional splice isoforms transcribed from 18 tetraspanin genes. 37 Nonconventional tetraspanin isoforms are defined as the translated isoforms other than the full-length tetraspanins. The structure and function of nonconventional isoforms differ from their conventional counterpart. Interestingly, the AS variants of tetraspanins form monospanins (have only one TM domain), dispanins (two TM domains), trispanins (three TM domains), and nonconventional tetraspanins, which span the membrane four times but are different from conventional 33 tetraspanins. 37 Examples of monospanins occur in AS variants of TSPAN6 and TSPAN14 isoforms. The dispanins AS events are more frequent and occur in CD53, TSPAN4, TSPAN6, CD37, CD81, CD9, CD63, TSPAN31, TSPAN17, TSPAN3, and TSPAN15 isoforms. The AS events forming trispanins include TSPAN2, CD82, TSPAN17, TSPAN16, UPK1A, TSPAN3, and CD63 isoforms. Finally, the AS events making nonconventional tetraspanins are of TSPAN10, TSPAN11, TSPAN17, CD53, and CD82 isoforms. 37 Furthermore, the functional studies on different isoforms suggest that the absence of TM domains causes the retention of some isoforms inside the endoplasmic reticulum (e.g., TSPAN15 and TSPAN20). 37 Some variants, such as the CD81 isoform, translocate to the plasma membrane like conventional CD81 but inhibit viral uptake, resulting in aberrant cellular signaling. 37 The AS events also play a crucial role during disease conditions. For example, tumor suppressor CD82 undergoes aberrant AS in breast cancer, leading to the expression of a CD82 isoform (KAI1-SP), which shows tumor-promoting characteristics. 38
The expression of numerous tetraspanin AS variants indicates their functional relevance at an evolutionary level. Overall, the AS events for structural proteins are still unexplored and further investigation is required to understand the function of these nonconventional tetraspanin isoforms.
Post-Transcriptional Regulation of Tetraspanins
Post-transcriptional regulation can occur at any stage after transcription. After transcription, splicing and AS events, the premRNA transcript undergoes further processing to become a mature mRNA transcript by adding a 7-methylguanosine cap at the 5′ end and polyadenylation at the 3′ end. The mRNA processing stabilizes the nascent transcript so it can go outside the nucleus for further translation. However, the cellular machinery quite elegantly adds another level of regulation in the cytoplasm to decide which transcript will be chosen to be translated into a protein and which one will be degraded by regulatory RNAs. The gene regulation by noncoding RNAs is another example of epigenetic gene regulation. The most studied class of regulatory RNAs are miRNAs and lncRNAs. Regulation by miRNA and lncRNA primarily depends on cell type, developmental stage, and spatial and temporal state of the cell, and this regulation is immensely dysregulated in disease conditions such as cancer. Similar to other mRNA transcripts, tetraspanin transcripts are also regulated by miRNAs and lncRNAs in a context-specific manner (as described later). Here, we have comprehensively reviewed the upstream miRNAs and lncRNAs regulation on different tetraspanin members in various cancers (Table 2).
MiRNA-mediated regulation of tetraspanins
Recent research has established that not all RNAs in the cell code for functional proteins, but many RNAs also perform other regulatory functions (GeneCards, https://www.genecards.org/). MiRNAs are a class of small noncoding RNAs of approximately 18–25 nucleotides in length. Similar to protein-coding genes, miRNA genes are transcribed within the nucleus by RNA Pol II/III, forming a primary miRNA transcript (pri-miRNA). 39 The pri-miRNA is processed by Drosha/DGCR8 into a premature miRNA (pre-miRNA) in the nucleus. The pre-miRNA translocates to the cytoplasm and is further processed by Dicer/TRBP into a mature miRNA. Finally, the mature miRNA is loaded onto the accessory proteins (Argonaute being the most important one) and makes a functional unit known as the RNA-induced silencing complex (RISC). 39 Due to miRNA sequence specificity, the RISC is highly specific to its target mRNAs. In general, miRNA binds to the 3′UTR of its cognate target mRNA in a sequence-specific manner and negatively regulates its expression. A perfect base-pair complementarity between a miRNA and its target mRNA leads to the degradation of the target mRNA, while a moderate complementarity results in translational repression.40,41 However, in both ways, gene expression goes down. A single miRNA can have hundreds of target mRNAs, and this regulation is highly dependent on tissue type, developmental stage, and disease condition. In addition to the 3′UTR, the miRNAs can have binding sites in the promoter region, 5′UTRs, and coding regions of their target mRNAs. 42 Interestingly, recent evidence suggests that miRNA binding to the promoter region often results in gene activation rather than repression. 42 Therefore, miRNAs can function as both gene activators and suppressors. Overall, miRNA-mediated gene regulation is crucial in development, cellular growth, proliferation, and other physiological processes. 40 According to the miRbase database (https://www.mirbase.org/), at the time of writing, there have been 2,654 mature miRNAs documented for humans, having tens of thousands of targets in the cell. 43
Numerous research groups have reported the miRNA-mediated regulation of members of the tetraspanin family. There are many examples when one tetraspanin exerts an opposing phenotype due to regulation by different miRNAs in the context of different tissues (as described later). This is a complicated yet thought-provoking observation. It seems like the function of tetraspanin is overruled by the upstream miRNA regulation. However, this information is scattered and does not infer any therapeutic potential. Here, we have summarized all the miRNAs targeting all the tetraspanins across various cancers, which will clarify the current understanding in the field (Table 2).
TSPAN1- TSPAN17
The first member of the tetraspanin family, TSPAN1 has been explored across various cancers. TSPAN1 expression is upregulated in multiple cancers and it acts as an oncogene in most cancers. 44 In 2022, a review article summarizing the role of TSPAN1 in carcinogenesis has been published. 44 In colorectal cancer, TSPAN1 is targeted by miR-638, leading to reduced cell proliferation and invasion. 45 Similar to colorectal cancer, TSPAN1 acts as an oncogene in pancreatic cancer and is directly targeted by three different miRNAs: MiR-454, miR-573, and miR-216a.21,46,47 The tumor suppressor miR-454 targets TSPAN1 and inhibits cell proliferation. 21 Another tumor suppressor, miR-573 inhibits cell proliferation, migration and invasion of pancreatic cancer cells while targeting TSPAN1. 46 Furthermore, miR-216a also negatively regulates TSPAN1 and reduces pancreatic cancer progression. 47 The tight regulation of TSPAN1 by three different miRNAs suggests its strong oncogenic potential in pancreatic carcinogenesis.21,46,47
TSPAN1 also acts as an oncogene in gastric cancer and is targeted by miR-573, similar to pancreatic cancer, affecting cell proliferation, cell cycle progression and invasion. 48 Furthermore, TSPAN1 increases epithelial-to-mesenchymal transition (EMT) and metastasis by increasing the PI3K-AKT signaling and is targeted by miR-194-5p in cholangiocarcinoma. 49 Another tumor suppressor miRNA, miR-491-3p targets TSPAN1 and suppresses growth and invasion in osteosarcoma. 50 In cervical cancer, miR-361-3p targets TSPAN1, modulating cell viability, migration, and invasion. 51 Interestingly, in nonsmall cell lung cancer, oncogenic miR-200a is reported to target TSPAN1 in its promoter region and increase its expression. 52 As previously mentioned, miRNA binding to the promoter region enhances the expression of its target mRNA. Thus, miR-200a-mediated activation of TSPAN1 is an example of RNA activation by miRNA. 52 The suppression of TSPAN1 by numerous miRNAs reflects its tumor promoting potential, and it can be a useful target in cancer diagnostics and therapeutics.
Aberrant elevated expression of TSPAN3 has been reported in acute myeloid leukemia (AML). The expression of TSPAN3 is negatively regulated by four different tumor suppressor miRNAs (i.e., miR-570-3p, miR-370-3p, miR-193a-3p, and miR-139-5p) in the context of AML.53–56 Moreover, the miRNA mentioned above is negatively regulated by their upstream oncogenic lncRNAs, leading to increased AML progression (discussed in the next section).53–56
In triple-negative breast cancer (TNBC), miR-155 negatively regulates TSPAN5, increasing stemness and chemoresistance. 57 TSPAN5 is also regulated by miR-322-5p in vascular dementia. 58 For TSPAN6, only one miRNA regulator is known so far. miR-199a-5p negatively regulates TSPAN6 in cardiac fibroblast cells. 59 TSPAN8 is negatively regulated by miR-324-5p in gastric cancer cells. The miR-324-5p reduces viability and induces apoptosis via down-regulating TSPAN8; hence, TSPAN8 behaves oncogenic in gastric cancer. 60 In diabetic neuropathy, miR-543 downregulates TSPAN8 and regulates high glucose-induced fibrosis and autophagy. 61 During hepatocellular carcinoma, TSPAN9 is downregulated due to inhibition by miR-9-5p, an upstream regulator of TSPAN9, resulting in poor survival of hepatocellular carcinoma (HCC) patients. 62
In non-small cell lung cancer (NSCLC), TSPAN12 acts as a tumor suppressor. 63 An oncogenic miRNA, miR-196b-5p directly targets TSPAN12, lowering its expression and promoting tumorigenesis in vitro and in vivo. 63 In contrast, TSPAN12 increases chemoresistance in small-cell lung cancer (SCLC) and is regulated by miR-495. 64 Similar to TSPAN12, TSPAN13 also acts as both tumor suppressor and oncogene. In breast cancer, TSPAN13 is a tumor suppressor and gets negatively regulated by miR-4732-5p. 65 However, TSPAN13 is oncogenic in papillary thyroid cancer and is targeted by miR-369-3p. 66 In osteosarcoma, TSPAN15 is targeted by miR-16-5p, leading to increased cell proliferation, migration, and invasion of bone cancer cells. 67 Similar to TSPAN15, TSPAN17 also increases cell proliferation and migration in glioblastoma and is targeted by miR-378a-3p. 68
TSPAN18-TSPAN33
CD151, CD82, and CD81 are the most well-studied tetraspanins. CD151 is reported to be oncogenic and gets negatively regulated by numerous miRNAs. The miR-199a-3p is one such miRNA reported to negatively regulate CD151 expression across different cancers and other disorders.69–71 As described previously, tetraspanins play a vital role in cell signaling. The activation of GPCR in TNBC elevates tumor suppressor miR-199a-3p expression, decreasing oncogene CD151 expression and suppressing proliferation, invasion, and EMT of TNBC. 70 Similarly, in hepatocellular carcinoma, miR-199a-3p shows antitumor properties via targeting CD151. 69 Moreover, miR-199a-3p shows antitumor behavior in mesenchymal stromal cells via targeting CD151. 71 The miR-199a-3p/CD151 axis is also reported in other diseases like hypertrophic cardiomyopathy, 72 hypertensive diabetic nephropathy, 73 and cardiomyocyte proliferation. 74 Another well-studied miRNA regulator for CD151 is miR-124. In oestradiol-treated breast cancer cells, the expression miR-124 decreases, and its target CD151 expression increases, eventually increasing tumorigenesis. 75 In another study, miR-124 suppresses cell growth by targeting CD151 in breast cancer. 76 Interestingly, to increase CD151 expression, two upstream mRNAs are reported, which inhibit binding between miR-124 and CD151 in hepatocellular carcinoma. LAMC1 mRNA competitively binds to miR-124, elevating CD151 expression. 77 Another mRNA, PIK3C2A mRNA sponges miR-124 and does not allow its binding to CD151. 78 In heart failure, miR-124 aggravates the condition by inhibiting the CD151-induced angiogenesis. 79 Moreover, CD151 is negatively regulated by miR-152 80 and miR-22 81 in gastric cancer and by miR-506 in breast cancer. 82
Like CD151, CD82 also has a lot of upstream regulatory miRNAs. CD82 acts as a tumor suppressor in many cancers, so the upstream-acting miRNAs of CD82 are oncogenic.83,84 In gastric cancer, an increased miR-197 expression downregulates its target CD82, activating the EGFR-ERK1/2-MMP7 signaling pathway and promoting invasion and metastasis. 83 The oncogenic miRNA miR-633 targets CD82 in melanoma 85 and in gastric cancer 84 in order to promote carcinogenesis. The CD82 level is also being kept low by transforming growth factor (TGF)-β signaling. Due to TGF-β release, SMAD2/3 binds to the promoter of miR-362-3p, increasing its expression. 86 The elevated level of miR-362-3p downregulates CD82 by binding to its 3′UTR. 86 Some other oncogenic miRNAs that target CD82 to aggravate tumorigenesis are miR-338-5p in melanoma, 87 miR-203 in lung cancer, 88 miR-362-3p in gastric cancer, 89 and miR-197 in liver cancer. 90
As noted earlier, CD81, CD63 and CD9 are the most common exosomal markers. 7 The miR-483-3p is overexpressed in exosomes released during influenza viral infection and is pro-inflammatory. 91 Interestingly, miR-483-3p negatively targets the exosomal marker CD81, which itself is a negative regulator of inflammation. 91 Another exciting scenario between CD81 and miRNA has been shown during hepatitis C viral (HCV) infection. The treatment with epigallocatechin gallate (an antioxidant present in green tea) increases the expression of miR-548m, which reduces the expression of its target CD81. 92 The reduction in CD81 level attenuates HCV entry. 92 Another exosomal marker, CD9 is negatively regulated by miR-518f-5p in prostate and breast cancers.93,94 In another study, the inhibition of LSD1 (histone lysine-specific demethylase 1) reduces carcinogenesis in gastric cancer by decreasing miR-142-5p and increasing CD9 expression. 95 Finally, TSPAN31 is a critical positive regulator of hepatocellular carcinoma, where it is targeted by miR-135b. 96
The multiple reports of CD151 being an oncogene and CD82, a tumor suppressor make them another attractive targets for cancer therapeutics. Further research on these molecules might place the tetraspanins as potential cancer biomarkers. There are no upstream miRNAs known so far for TSPAN2, TSPAN4, TSPAN7, TSPAN10, TSPAN11, TSPAN14, TSPAN16, TSPAN18, TSPAN19, UPK1B, PRPH2, ROM1, CD53, CD37, CD63, TSPAN32, and TSPAN33 genes.
LncRNA-mediated regulation of tetraspanins
LncRNAs) are transcripts that are more than 200 bp in length with no or significantly less coding potential. 97 Similar to mRNAs, most lncRNAs are also transcribed by RNA polymerase II. Moreover, many of the lncRNAs undergo capping, splicing and polyadenylation. According to the LNCipedia database (https://lncipedia.org/), there are 56,946 lncRNA genes present in the human genome, which code for 1,27,802 lncRNA transcripts. 98 Although the number of discovered lncRNA genes is large, very few are functionally characterized. Unlike miRNAs, there is no single mechanism by which lncRNAs exert their action. One well-defined function of lncRNA is to act as a sponge for miRNAs and sequester them so that miRNAs cannot bind to their target mRNAs. Hence, lncRNA indirectly increases the expression of miRNA target genes. LncRNA can also stabilize mRNA and increase its expression. Moreover, lncRNA can positively and negatively regulate transcription via interaction with transcription factors. LncRNA can also regulate the splicing and translation process. Another critical function of lncRNA is interacting with epigenetic modifiers and regulating gene expression. 99
LncRNAs regulate the various members of the tetraspanin family.51,100 Although the literature on lncRNA-mediated regulation of tetraspanins is not very wide, we have summarized the available research on the topic in Table 2.
In cervical cancer, the oncogenic lncRNA LINC01123 sponges the miR-361-3p, increasing the expression of its target TSPAN1 and aggravating the cancer phenotype. 51 As previously mentioned, TSPAN3 is regulated by four different miRNAs (i.e., miR-570-3p, miR-370-3p, miR-193a-3p, and miR-139-5p) in AML.53–56 Because TSPAN3 is an oncogene and regulation by these miRNAs inhibits its expression, there is further negative regulation by noncoding RNAs upstream to these miRNAs. Circ_0004136 (a circular RNA) inhibits miR-570-3p, lncRNA GAS6-AS1 sponges miR-370-3p, and KCNQ1OT1 sponges miR-193a-3p and hence upregulating the oncogene TSPAN3 expression.53–56 Another circular RNA, circ_103809 modulates TSPAN3 expression by downregulating its upstream miRNA, miR-197-3p, hence increasing the expression of TSPAN3. 101
In lung cancer, lncRNA SOX21-AS1 regulates the expression of TSPAN8 by binding to transcription factor GATA6. 100 GATA6 binds to the TSPAN8 promoter, and the binding of SOX21-AS1 to GATA6 increases the TSPAN8 expression. 100 Another circular RNA, circNFIC decreases breast cancer progression by sponging miR-658, increasing the expression of its target UPK1A. 102
CD151 is regulated by many upstream lncRNAs/miRNA axes. In esophageal squamous cell carcinoma, lncSUMO1P3 sponges miR-486-5p and increases the CD151 expression to elevate malignancy. 103 Another lncRNA, MT1JP negatively regulates CD151 expression in breast cancer. 104 LncRNA SNHG3 sponges miR-128 and elevates the CD151 expression in hepatocellular carcinoma. 105 In gastric cancer, lncRNA PVT1 sponges miR-152 so that they cannot bind to their downstream target CD151, hence increasing the expression of oncogene CD151. 106
Genetic Mutations in Tetraspanin Family Genes Across Genetic Disorders and Cancer
According to the human gene mutation database, there are several mutations in tetraspanin family genes, resulting in various diseases such as autism, intellectual disability, schizophrenia, ocular disorders, and even cancer (Table 3). Although all tetraspanin members share a similar core protein structure, significant differences exist across different tetraspanin proteins, which govern their unique functions in cellular physiology. The mutations in tetraspanin family members cause a broad spectrum of disease phenotypes, from ocular disorders and neurological defects to cancer. Different types of mutations occur in the tetraspanin family genes, such as missense, nonsense, substitutions, insertions, deletions, and translocations. 107 Most mutations in TSPAN12, PRPH2, and ROM1 cause ocular disorders such as retinitis pigmentosa, pattern dystrophy, and macular dystrophy (Table 3). The high number of mutations in tetraspanins in eye-related disorders suggests the crucial role of tetraspanins in visual signal transduction and its potential as a biomarker and therapeutic target. Extensive research for correcting these mutations or tetraspanin therapy might provide us with new treatments for ocular disorders.
Genetic mutations in human tetraspanin family genes and their associated disorders
According to the Human gene mutation database (HGMD), https://www.hgmd.cf.ac.uk/ac/search.php.
Mutations in TSPAN4, TSPAN5, TSPAN6, TSPAN7, TSPAN8, TSPAN10, TSPAN17, TSPAN18, and CD151 cause autism and other neurological disorders (Table 3). 107 Furthermore, some mutations in the tetraspanin family are also associated with cancer. The mutation in the TSPAN1 gene increases the risk of gastric cancer, whereas the gross deletions in the TSPAN5 gene cause colorectal cancer and nonpolyposis colon cancer. 107 Moreover, the missense/nonsense mutations in the CD9 gene increase the risk of breast cancer (Table 3). 107 The wide variety of mutations in the tetraspanin family makes them a potential target for diagnosis and therapeutics.
Conclusions and Prospects
Tetraspanins play a crucial role in cell signaling across the plasma membrane by forming TEMs. By delving into the intricate regulatory networks governing tetraspanin gene expression, researchers aim to uncover potential therapeutic targets for various diseases, including cancer. Here, we have reviewed the regulation of 33 members of the human tetraspanin family and their roles across different cancers. We have summarized the actions of transcription factors, epigenetic regulators, miRNAs, and lncRNAs in regulating the human tetraspanin members. The tight regulation of tetraspanins by numerous transcription and post-transcription factors suggests the crucial nature of tetraspanins in biological processes.
As reported across many studies, cancer cells have evolved to elevate multiple oncogenic miRNAs to reduce the expression of tumor suppressor tetraspanins (e.g., CD82). In return, tumor suppressor tetraspanins have also evolved ways to express themselves and protect the cells. In contrast, cancer cells have developed mechanisms to decrease the expression of tumor suppressor miRNAs (like sponging by upstream lncRNAs) so that the downstream oncogenic tetraspanin (e.g., TSPAN1 and CD151) can flourish and aggravate tumorigenesis. This tug-of-war between oncogenes and tumor suppressors has been the core of cancer progression. Interestingly, the same tetraspanin gene can act as both tumor suppressor and oncogene (e.g., TSPAN12 and TSPAN13) when regulated by different miRNAs in different cancers (Table 2). Tetraspanins such as TSPAN1, CD151, and CD82 are well-explored and have potential for becoming the target for cancer diagnosis and therapeutics.
Nevertheless, despite several studies,1,2,6 there are still many tetraspanins such as TSPAN2, TSPAN4, TSPAN7, TSPAN10, TSPAN11, and TSPAN14 for which no upstream regulators have been identified. Understanding the transcriptional and post-transcriptional mechanisms that fine-tune tetraspanin gene expression is essential for developing targeted interventions to modulate these crucial protein functions in health and diseases. As tetraspanins are membrane proteins and are present on the cellular surface, there is a possibility that tetraspanins can be detected much easier than soluble proteins. Utilizing oncogenic tetraspanins such as TSPAN1 and CD151 as biomarkers for diagnosis can be a step forward for early detection of cancer and other diseases.
We have also summarized the mutational landscape of human tetraspanins and their associated phenotype. The wide spectrum of disease-causing mutations in the tetraspanin family genes further suggests their vital roles in neurological and ocular disorders. The correction of these mutations by gene therapy might improve the current treatments.
Footnotes
Authors’ Contributions
S.G.: Conceptualization, writing—original draft, writing—review and editing. A.K.: Supervision, writing—review and editing.
Author Disclosure Statement
The authors declare no conflicts of interest.
Funding Information
As this is a review article, no funding was required.
Acknowledgments
The authors thank the anonymous reviewer and the editor for valuable suggestions and comments to improve the quality of the manuscript.