Role of the complement protein C1q in the regulation of hyaluronic acid cleavage in malignant pleural mesothelioma

Background: The component of the classical pathway C1q and hyaluronic acid (HA) play a pivotal role in malignant pleural mesothelioma (MPM) tumour microenvironment and their interaction has been demonstrated to favour pro-tumorigenic behaviours of MPM cells by enhancing adhesion, migration and proliferation (Agostinis et al., 2017), as well as by upregulating the hyaluronan synthase HAS3 (Vidergar et al., 2021), increasing the production of short pro-invasive and pro-metastatic HA fragments. Here, we aimed to determine whether HA-bound C1q can exert its modulation also on hyaluronidase (HYAL1, HYAL2, HYAL3) expression, the enzymes responsible for HA degradation, and which receptors may be involved in this process. Methods: Real-time qPCR, flow cytometry and Western blot analysis were performed on primary MPM cells to evaluate HYAL expression, upon treatment with C1q-HA matrix. Enzyme distribution was inquired by immunofluorescence, surface biotinylation assay and proximity ligation assay (PLA). The bioinformatics tool GEPIA was used for TCGA-based survival analysis. Results: Real-time qPCR in MPM cells highlighted a downregulation of HYAL1 and an upregulation of HYAL2 expression, upon treatment with C1q-HA matrix, as compared to HA alone, then confirmed at protein level by Western blot and flow cytometry. No significant modulation was observed on HYAL3. Since only HYAL2 mRNA expression levels found a correlation with MPM patient survival by bioinformatics analysis, being associated with poorer outcome, we proceeded with the characterization of HYAL2 distribution and potential interaction with HA or C1q receptors to better understand their interplay: we detected a striking overlap between HYAL2 and globular C1q receptor (gC1qR) localization both at the cell membrane and intracellularly, by immunofluorescence colabelling, surface biotinylation assay and PLA. PLA confirmed also HYAL2-CD44 interaction.Conclusion: C1q-HA interaction can act as a signaling complex by enhancing HYAL2 expression, suggesting a consequent higher rate of HA catabolism and the release of shorter HA fragments, confirming an overall pro-tumorigenic effect promoted by C1q interaction with HA. The co-localization and interaction between HYAL2 and gC1qR, being a receptor of both C1q globular head and HA, led us to hypothesize a potential involvement of gC1qR within this macromolecular complex, most likely requiring the presence of the preferential HA receptor CD44. References Agostinis, C., Vidergar, R., Belmonte, B., et al., 2017. Complement protein C1q binds to hyaluronic acid in the malignant pleural mesothelioma microenvironment and promotes tumor growth. Front. Immunol. 8, 1559, doi: 10.3389/fimmu.2017.01559. Vidergar, R., Balduit, A., Zacchi, P., et al., 2021. C1q-HA matrix regulates the local synthesis of hyaluronan in malignant pleural mesothelioma by modulating HAS3 expression. Cancers (Basel) 13 (3), 416, doi:10.3390/cancers13030416.