Machine learning-assisted elucidation of CD81–CD44 interactions in promoting cancer stemness and extracellular vesicle integrity

Abstract

Tumor-initiating cells with reprogramming plasticity or stem-progenitor cell properties (stemness) are thought to be essential for cancer development and metastatic regeneration in many cancers; however, elucidation of the underlying molecular network and pathways remains demanding. Combining machine learning and experimental investigation, here we report CD81, a tetraspanin transmembrane protein known to be enriched in extracellular vesicles (EVs), as a newly identified driver of breast cancer stemness and metastasis. Using protein structure modeling and interface prediction-guided mutagenesis, we demonstrate that membrane CD81 interacts with CD44 through their extracellular regions in promoting tumor cell cluster formation and lung metastasis of triple negative breast cancer (TNBC) in human and mouse models. In-depth global and phosphoproteomic analyses of tumor cells deficient with CD81 or CD44 unveils endocytosis-related pathway alterations, leading to further identification of a quality-keeping role of CD44 and CD81 in EV secretion as well as in EV-associated stemness-promoting function. CD81 is co-expressed along with CD44 in human circulating tumor cells (CTCs) and enriched in clustered CTCs that promote cancer stemness and metastasis, supporting the clinical significance of CD81 in association with patient outcomes. Our study highlights machine learning as a powerful tool in facilitating the molecular understanding of new molecular targets in regulating stemness and metastasis of TNBC.

The versatile roles of testrapanins in cancer from intracellular signaling to cell–cell communication: cell membrane proteins without ligands

“…Furthermore, CD151–α3β1/α6β4 integrin complexes can recruit and activate small G proteins (RAS, RAC1, and CDC42) in melanoma cell lines [ 8 ]. Other partners of TSPANs include growth factor receptors (EGFR [ 9 ], mtTGF-β [ 10 ]), transporters (ASCT2 [ 11 ], FATP1 [ 12 ], MDR1 [ 13 ]), membrane-linked kinases (BTRC [ 14 ], SOCSS3 [ 15 ], ATXN3 [ 16 ]), other transmembrane proteins (ADAM10 [ 17 ], CD44 [ 18 ], p120 [ 19 ]) or some nonproteins such as cholesterol [ 20 ]. For example, TSPAN6 could bind with EGFR and inhibit its downstream KRAS-ERK1/2 signaling to suppress KRAS-driven cancer initiation and metastasis [ 20 ](Fig.…”

“…Integrins are the most prominent partner of TSPANs. Other partners of TSPANs include growth factor receptors (EGFR [ 9 ], mtTGF-β [ 10 ]), transporters (ASCT2 [ 11 ], FATP1 [ 12 ], MDR1 [ 13 ]), membrane-linked kinases (BTRC [ 14 ], SOCSS3 [ 15 ], ATXN3 [ 16 ]), other transmembrane proteins (ADAM10[ 17 ], CD44 [ 18 ], p120 [ 19 ]). Thus, TSPANs can affect several signaling pathways, including PI3K/AKT, Wnt/β-catenin, ERK1/2, STAT3/5, Src, Notch pathways …”

“…CD82 was up-regulated in CD34 + /CD38 + acute myelocytic leukemia stem cells and increased the phosphorylation of transcription factor STAT5 to transactivate IL-10 transcription [ 92 ]. More recently, CD81 has been revealed to interact with CD44 to enhance the stemness of triple-negative breast cancer cells, and high CD81 expression can be found in circulating tumor cells [ 18 ]. Furthermore, TSPAN1 is found to be elevated in CSCs from head and neck squamous cell carcinoma cells and lead to drug resistance [ 93 ].…”

“…CD151 facilitates secretion of ribosomal proteins while reducing complement proteins to promote the migration and invasion of triple-negative breast cancer cells [ 111 ]. CD81 ensures the membrane integrity of exosomes, which are capable of inducing stemness in triple-negative breast cancer [ 18 ]. CD9 on the surface of EVs facilitates the uptake of EVs from cancer-associated fibroblasts by pancreatic cancer cells [ 117 ] …”

“…TSPAN1 was found to be upregulated in plasma EVs from colon cancer patients compared to those from healthy controls [ 116 ]. In addition, CD81 has been reported to ensure the membrane integrity of exosomes, which are capable of inducing stemness in triple-negative breast cancer [ 18 ]. Recently, Jérémy Nigri and colleagues showed that CD9 on the surface of EVs facilitates the uptake of EVs from cancer-associated fibroblasts by pancreatic cancer cells [ 117 ].…”