A peptide binding to the tetraspanin CD9 reduces cancer ...

Introduction

Cancer is one of the leading causes of death globally and most cancer deaths are resulted from metastasis—the formation of primary cancer-derived secondary tumors at distant sites [1]. Cancer metastasis is a multi-step process involving a series of events that allow cancer cells to invade from the primary tumor, survive in blood vessels or the lymphatic system, extravasate, and form secondary tumors in distant organs [2]. Due to the complexities of metastasis, clinical approaches against metastatic cancers are limited, which causes poor treatment outcomes and inefficient drug discovery [3]. Thus, the development of strategies and agents to prevent metastatic cancer outgrowth is necessary [3].

The CD9 membrane protein is a member of tetraspanins or the transmembrane 4 superfamily (TM4SF) proteins, defined by similarities in size (approximately 20–30 kDa) and topology (four transmembrane domains together with two extracellular and one intracellular loops) (Scheme 1A) [4]. Through its large extracellular loop (LEL) and four transmembrane regions, CD9 interacts with specific partner proteins, including other tetraspanins, thereby forming tetraspanin webs for partner-dependent functions (Scheme 1A) [4], [5]. CD9 is expressed in various cellular components (e.g., the plasma membrane, endocytic compartment, and extracellular vesicles (EV)) and is often concentrated in highly curved membrane subdomains (e.g., microvillar-like projections and filopodia) [6]. Depending on cell types and associated molecules, CD9 plays an important role in many biological and pathological processes, such as motility, fusion, signaling, egg–sperm fusion, virus infection, glomerular disease progression, and cancer metastasis [6], [7], [8], [9], [10]. There is abundant evidence suggesting that CD9 has roles in cancer differentiation, migration, invasion, and metastasis because of its specific overexpression in metastatic cancer cell lines, cancer-derived EVs, and late-stage cancers [11], [12], [13], [14], [15]. As an organizer of biological membranes, CD9 may induce changes in tetraspanin web organization and/or cancer-specific signaling pathways (e.g., TNF-α and TGF-β) in cancer cells [6], [16], [17], [18]. During EV-based intercellular communication and metastasis, CD9 in high-curvature regions may be responsible for membrane curvature generation/fusion in EV exocytosis as well as adhesion/fusion in EV endocytosis [4], [19], [20]. Taken together, the tetraspanin CD9 is a potential therapeutic target for metastatic cancers.

The therapeutic benefit of targeting tetraspanins, nevertheless, is hindered by the adverse impact and inefficient delivery of nucleic acids as well as the issues with antibody size, susceptibility, side effects, and resistance [6], [21], [22]. Alternatively, peptides or short chains of amino acids with strong binding affinity are characterized by small size, high stability, low cytotoxicity, and possible tunability [22], [23]. Hence, peptide utilization may be useful for the therapeutic targeting of tetraspanins, including CD9. We have previously identified an eight-mer CD9-binding peptide (CD9-BP, RSHRLRLH) from the amino-acid sequence of CD9's major partner, EWI-2 protein, by the peptide array technique (Scheme 1B) [24]. CD9-BP showed a good affinity for CD9 via self-delivery to the membrane-proximal LEL domain and potentially impaired the partner association of CD9 (Scheme 1B) [24], [25]. CD9-BP was earlier used as an optional probe for targeting CD9-enriched nanoscale EVs [24]. Besides, CD9-BP preferentially suppressed the migration of cancer cells rather than normal cells [25]. However, the mechanism by which CD9-BP blocks CD9-related functions and its impact on cancer metastasis is still unknown.

In this study, we first evaluated the effect of CD9-BP on the formation of tetraspanin webs. Furthermore, the abilities of CD9-BP to reduce cancer cell migration and invasion were evaluated. The CD9-BP effects on secretion and uptake of nanoscale EVs or exosomes were also evaluated. Finally, we evaluated the ability of CD9-BP to reduce lung metastasis in a mouse model. The results of these analyses clearly demonstrated that CD9-BP is a promising biomolecule for targeted therapy aimed at preventing metastasis.

Section snippets

Impact of CD9-BP on tetraspanin web formation of melanoma cells

Tetraspanins recruit and sort partner proteins, including other tetraspanins, and form tetraspanin webs [5], [26]. To study the impact of CD9-BP on tetraspanin web formation, helical D-form CD9-BP (Fig. S1) was prepared, labeled, and applied to stain melanoma B16/BL6 cells, which show high CD9 expression and are widely used in studies of cancer metastasis and solid tumor formation [27]. As shown in Fig. 1A, similar stain patterns were observed for fluorescein isothiocyanate (FITC)-labeled

Conclusion

We demonstrated that CD9-BP is a candidate biomaterial for reducing cancer metastasis. Binding to CD9 LEL, CD9-BP had an adverse effect on the diameter and number of tetraspanin webs—networks of CD9 and its partner proteins. Consequently, CD9-BP decreased cancer cell migration and invasion as well as exosome secretion and uptake in vitro, all of which are CD9- and metastasis-related processes. CD9-BP also reduced the lung metastasis of melanoma cells in a mouse model. We believe that CD9-BP

Peptide preparation

D-Form CD9-BP (RSHRLRLH) powder was prepared following previously described methods [23], [25]. CD9-BP was synthesized on H-Rink-Amide-ChemMatrix® resin using an automated peptide synthesizer (Initiator+; Biotage, Uppsala, Sweden), according to the manufacturer's instructions with some modifications. The synthesis cycle for each amino acid was started with the deprotection of Fmoc group at the N-terminus by 20 % piperidine in N, N-dimethylformamide (DMF). DMF was later used to wash the

CRediT authorship contribution statement

Conceptualization, T.S., K.M. and M.O.; Methodology, T.S., K.I., M.T., Y.M., K.M. and M.O.; Validation, M.T., A.H., Y.M., K.M. and M.O.; Formal analysis, T.S., M.T., Y.M., K.M. and M.O.; Investigation, T.S., K.I., K.S., Y.M. and K.M.; Resources, A.H., K.M. and M.O.; Data curation, T.S., K.M. and M.O.; Writing—original draft preparation, T.S. and K.I.; Writing—review and editing, M.T., A.H., Y.M., K.M. and M.O.; Visualization, T.S. and K.I.; Supervision, A.H., K.M. and M.O.; Project

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We highly appreciate Mr. Takeshi Chiaki, Ms. Hiroko Kato and Mr. Takeshi Igarashi at Leica (Tokyo, Japan) for supporting the STED observation. The present study was funded by the Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (21H01726, 21H01725, 22K19913). Partially, this work was supported by the Moonshot Research and Development Program from the Japan Agency for Medical Research and Development (AMED) (JP21zf0127004),