Tetraspanins, also known as the transmembrane 4 superfamily (TM4SF), comprise 33 family members. They regulate biological processes such as cell adhesion, proliferation, tissue differentiation, and immune response by forming dimers or heterodimers or interacting with other protein molecules such as integrins, adhesion molecules, major histocompatibility complex class II (MHC II), and T cell receptors. Increasing evidence suggests that some TM4SF molecules are closely associated with the occurrence and development of tumors, participating in multiple stages such as migration, epithelial-mesenchymal transition, thrombus formation, tumor stem cells, and exosome signaling transduction. A deep understanding of the functions and regulatory mechanisms of TM4SF that can promote or inhibit the occurrence and development of tumors will provide new strategies for future targeted interventions.
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Introduction of TM4SF
The TM4SF proteins, part of the transmembrane 4 superfamily, are situated within the plasma membrane of eukaryotic cells. In mammals, there exist 33 highly conserved subtypes.
TM4SF proteins were first reported gradually until 1990, including differentiation-related proteins 9 (CD9), CD37, CD53, CD81, CD82, CD151, and TSPAN8 (tetraspanin-8). These molecules are increasingly recognized for their involvement across a spectrum of biological processes, including tumor metastasis, fertilization, membrane dynamics, infection, synaptogenesis, platelet aggregation, and immune responses. Beyond the cell membrane, TM4SF is abundant in endocytic organelles and exosomes, serving pivotal roles in intercellular signaling and receptor-cell selection.
Structurally, TM4SF features a four-transmembrane arrangement, comprising short amino and carboxyl termini, four transmembrane regions (TM), and two extracellular loops (ECL). Polar residues within the TM region contribute to stabilizing its tertiary structure. ECL2, with a conserved and variable region, is implicated in dimerization and interactions with other proteins. Some TM4SF proteins, identified by a tyrosine-based motif, are directed to specific intracellular compartments or internalized through protein associations.
The highly conserved cysteine residues within the ECL2 domain are distinctive, with palmitoylation of cysteine residues initiating the TM4SF network formation, shielding it from lysosomal degradation, and facilitating binding to cholesterol and glycosphingolipids. Palmitoylation of specific integrins aids in TM4SF complex formation. TM4SF, upon binding with cholesterol and glycosphingolipids, forms complexes known as TEMs, enabling protein-protein interactions and signal transduction regulation. While TEMs share some features with lipid rafts, they exhibit independent structural compositions, distinguished by their disruption by Triton X-100 at 4°C and the absence of classical lipid raft molecules and functions.
TM4SF in Tumor Development
Tumor metastasis involves a cascade reaction process where cancer cells escape from the primary site, infiltrate blood vessels, circulate, and eventually settle in distant locations. Existing evidence suggests that TM4SF regulates tumor cell proliferation, migration, and adhesion by integrating signals from integrins. TM4SF can also interact with peptidases, disintegrin, metalloproteases (particularly ADAM10) matrix metalloproteinases (MMPs), and urokinase-type plasminogen activator receptor (uPAR) to regulate cellular invasiveness. Although TM4SF members exhibit high structural conservation and no fundamental differences in the assembly of transmembrane and signaling molecules within TEMs, their regulatory roles in tumor development vary, either promoting or inhibiting tumor occurrence and progression.
Fig. 1 Structure of tetraspanins (Yang Y. G., et al. 2016).
TSPAN8
TSPAN8, initially deemed a tumor-associated antigen, shows heightened expression in various human tumors, particularly linked to liver metastasis. The exact mechanism driving its upregulation in tumor progression remains unclear. In breast cancer, the activation of the EGF/EGFR pathway triggers TSPAN8 transcription via SOX9. Its expression further escalates in metastatic tumors, facilitating tumor progression by promoting cellular migration, increasing cell motility, and fostering liver metastases. Moreover, TSPAN8 induces ADAM12 expression, contributing to cancer cell migration, invasion, and metastasis regulation. Notably, elevated TSPAN8 expression in breast cancer stem cells correlates with treatment resistance and adverse prognosis, mediated by its interaction with PTCH1 to enhance stemness. TSPAN8, conventionally believed to function on the cell membrane, is also present in the cytoplasm and nucleus, affecting chromatin remodeling and transcription of metastasis-related genes. This aberrant EGFR-AKT-TSPAN8 axis activation is implicated in various cancers, correlating with aggressive phenotypes and poor prognosis.
CD81
CD81 belongs to the tetraspanin protein family, and the crystal structure of the complete CD81 protein has been resolved. Within the four transmembrane domains of the CD81 molecule, there are tight cholesterol binding sites, and cholesterol binding contributes to conformational changes in the CD81 extracellular loops (ECL). CD81 regulates the progression of breast cancer and colorectal cancer through epithelial-mesenchymal transition. Inhibition of CD81 expression can lead to weakened cancer cell migration and reduced lung metastasis. Additionally, increased expression of CD81 in human melanoma, osteosarcoma, and colon cancer cells also promotes tumor progression and metastasis.
CD151
CD151 overexpression is observed in various tumor types such as breast cancer, pancreatic cancer, colorectal cancer, and non-small cell lung cancer, correlating with poor prognosis. However, in prostate cancer, high CD151 expression is associated with well-differentiated histological grades. Knocking out CD151 in tumor cells reduces the expression of MMP2, MMP7, and MMP9. CD151 promotes MMP7 activation and extracellular matrix (ECM) degradation by binding to proMMP7, and anti-CD151 antibodies inhibit MMP expression. CD151 plays a crucial role in integrin trafficking, regulating cell migration by interacting with α3β1, α6β4, and MMPs. While CD151 does not affect the steady-state expression of integrins α3 and α6, it regulates the localization of α3β1 and α6β4 in tumor-stromal cells. Mutation or knockout of CD151 significantly impairs the internalization of α3β1, α5β1, and α6β1 and their accumulation in intracellular vesicles, leading to impaired cell motility and enhanced adhesion.
CD82
CD82, with six cysteine residues in ECL2 and an internalization motif, is found in endosomes, lysosomes, and exosomes. Its downregulation in advanced cancers correlates with poor prognosis and metastasis in various cancers. CD82 inhibits cancer cell migration and invasion by interacting with integrin α6 and EGFR, regulating laminin adhesion and promoting uPAR binding to α5β1. It interferes with HGFR signaling, activating Rac and CDC42 to inhibit invasion. Ganglioside GD1a influences CD82's regulation of EGFR activity. While CD82 mutations in tumors are lacking, high CD82 CpG island methylation is observed in multiple myeloma. NF-κB p50 promotes CD82 transcription, and HTATIP downregulation is linked to metastatic cells. Selective splicing may contribute to CD82 downregulation.
CD9
CD9 was initially thought to suppress tumor metastasis by inhibiting integrin-mediated motility. In ovarian cancer, CD9 levels correlate with integrins β1, α2, α3, α5, and α6, and reduced CD9 leads to weakened adhesion and diffuse growth. Gangliosides promote CD9-integrin complex formation; high GM3 expression inhibits SRC and Rac via GM3-CD9-integrin α3 complexes. CD9 influences cell migration through the EGF-EGFR pathway, promoting EGFR internalization and inhibiting TGFα cleavage, thus suppressing autocrine growth. CD9 regulates Wnt-related molecules associated with invasive growth and binds to podoplanin, inhibiting metastasis. Inconsistent findings exist about CD9's role in tumors; it may mark pancreatic cancer-initiating cells, enhancing organoid formation. CD9 also localizes at tumor edges and increases MMP2 expression in melanoma cells, promoting migration.
CD63
CD63 expression is generally associated with tumor metastasis inhibition, but contradictory reports exist. Inhibition of metastasis formation by CD63 may depend on integrin internalization, MMP14 lysosomal degradation, and recruitment of tissue inhibitors of metalloproteinases 1 (TIMP1). CD81 reshapes the cytoskeleton of liver cancer cells by interacting with α-actinin 4 to inhibit cell motility. In melanoma, the G protein-coupled receptor GPR56 forms a complex with Gαq and CD81 and binds to tissue transglutaminase 2 (TGM2), the main crosslinking enzyme in the extracellular matrix. The GPR56-Gαq-CD81 complex binding to TGM2 promotes cell adhesion, interfering with tumor cell migration. Further research is needed to elucidate the potential role of CD63 in tumor progression.
While an increasing number of studies have revealed the significance of TM4SF family members in the occurrence and development of tumor cells, the complexity of their biological functions necessitates further exploration into the specific molecular mechanisms and regulatory diversity within this family.
References
- Yang Y. G., et al. Tetraspanins: Spanning from solid tumors to hematologic malignancies. Experimental Hematology. 2016, 44(5): 322-8.
- Hemler M.E. Tetraspanin proteins promote multiple cancer stages. Nature Reviews Cancer. 2014, 14(1): 49-60.
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