Poster S19 - MMP-9 Mediated N-Cadherin Shedding: Unraveling Its Role in IL-6 Signaling and Vestibular Schwannoma Progression

Introduction: Vestibular Schwannoma (VS) is the most common brain tumor in the cerebellopontine angle that causes significant morbidities including deafness and brainstem compression. VS management remains a significant clinical challenge, largely due to its unpredictable growth,poor prognosis in advanced stages, and lack of available medical therapies. Emerging evidence emphasizes the crucial role of the tumor microenvironment (TME) in promoting the growth of VS. Previously, we and others have identified matrix metalloproteinase-9 (MMP-9) as a key mediator of VS progression through extracellular matrix (ECM) remodeling which facilitates tumor migration and invasion1. MMP substrates include cell adhesion molecules abundantly expressed in the normal healthy brain and tumors like gliomas. These substrates, such as N-cadherin, is upregulated and is linked to aggressive tumor characteristics and reduced survival rate. N-cadherin can influence glioma cell migration and interaction with the ECM to facilitate invasion into brain tissue. However, its role in VS progression remains unclear. Furthermore, IL-6 is a cytokine secreted by Schwannoma cells to recruit tumor-infiltrating macrophages and may contribute to tumor progression 2-4. Here, we investigate the impact of MMP9 on N-cadherin expression and whether soluble N-cadherin could influence IL-6 production and VS proliferation in patient tumor samples and schwannoma cells.

Materials and

Methods: Patients with VS undergoing microsurgical resection were included in the study, and tumors were collected intraoperatively. Wild-type murine Schwann cells (WT-MSC) from mouse sciatic nerves and syngeneic mouse Schwann cell line deficient in merlin (genetic deletion of the NF2 gene using Cre-Lox)5 (MD-MSC) were used to validate the expression of adhesion molecules. The NF2-/- human schwannoma cell line (HEI-193) was used to validate the N-cadherin expression. Human VS samples were analyzed for spatial expression of N-cadherin using immunofluorescence. The cleavage of N-cadherin by matrix metalloproteinases was examined using an in vitro digest assay. To observe the impact of N-cadherin in homotypic cell-cell adhesion interaction, adhesion assays were conducted using GC-4 as an N-cadherin neutralizing antibody and ADH-1, a small molecule N-cadherin inhibitor. To measure the pro-inflammatory response to the soluble N-cadherin, IL-6 secretion was determined by ELISA. We characterized the activity of NF-kB using western blot and immunofluorescence to assess p65 and IkB-α translocation.

Results, Conclusions, and Discussions: To investigate whether N-cadherin is upregulated during VS growth, we found N-cadherin was highly expressed on schwannoma cell surface and significantly enriched in the parenchyma of tumors >3 cm compared to tumors < 1.5 cm (Fig 1A, B). HEI-193 cells strongly expressed N-cadherin on the surface which colocalized with MMP9 (Fig 1C). We found that MMP9 and several cell-surface adhesion molecules were overexpressed in MD-MSC cells compared to WT-MSC (Fig 1D-G). We then assessed whether N-cadherin is necessary for homotypic schwannoma cell-cell adhesion. Treatment with ADH-1 or GC-4 resulted in a 30-70% decrease in adhesion (Fig 1H-I). MMP-9 has been shown to increase N-cadherin shedding6. We then examined the in vitro cleavage of full-length N-cadherin by recombinant MMPs. MMP-9 exhibited the greatest efficacy in N-cadherin cleavage (Fig 2A). N-cadherin has been shown to promote tumor growth via IL-6/STAT3 signaling7. Primary tumor culture from large VS secreted higher levels of IL-6 than small VS (tumor diameter 3.4 vs 1.0 cm, Fig 2B). In MD-MSC, IL-6 expression was 25-fold higher than in WT-MSC (Fig 2C), and IL-6 secretion was further stimulated by exposure to soluble N-cadherin (Fig 2D). To understand the mechanism by which N-cadherin activates IL-6 secretion, we found an increase in cytoplasmic NF-kB p65 and a decrease in IkB-α after N-cadherin treatment. Immunofluorescence staining confirmed the nuclear translocation of p65, substantiating the mechanism by which soluble N-cadherin activates NF-kB signaling (Fig 2E-F).

Conclusions: This study demonstrates the role of MMP-9 in the progression of vestibular schwannoma through its interaction with N-cadherin, showing that MMP-9-mediated cleavage of N-cadherin disrupts cell adhesion and promotes inflammation and tumor growth via IL-6 secretion and NF-kB activation. These insights suggest that targeting MMP-9 and N-cadherin could offer new therapeutic for managing this challenging tumor, potentially reducing reliance on high-risk surgeries. In the future, developing therapeutics against N-cadherin VS could represent a breakthrough in managing these challenging tumors.

Acknowledgements (Optional): National Institute on Deafness and Other Communication Disorders (NIDCD) grant 1K08DC020761-01 (YR) and the American Neurotology Society Research Grant (YR)

References

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