MMP-14 (MT1-MMP) Is a Biomarker of Surgical Outcome and a Potential Mediator of Hearing Loss in Patients With Vestibular Schwannomas

Yin Ren, Hiroshi Hyakusoku, Jessica E Sagers, Lukas D Landegger, D Bradley Welling, Konstantina M Stankovic, Yin Ren, Hiroshi Hyakusoku, Jessica E Sagers, Lukas D Landegger, D Bradley Welling, Konstantina M Stankovic

Abstract

Improved biomarkers are needed for vestibular schwannoma (VS), the most common tumor of the cerebellopontine angle, as existing clinical biomarkers have poor predictive value. Factors such as tumor size or growth rate do not shed light on the pathophysiology of associated sensorineural hearing loss (SNHL) and suffer from low specificity and sensitivity, whereas histological markers only sample a fraction of the tumor and are difficult to ascertain before tumor treatment or surgical intervention. Proteases play diverse and critical roles in tumorigenesis and could be leveraged as a new class of VS biomarkers. Using a combination of in silico, in vitro, and ex vivo approaches, we identified matrixmetalloprotease 14 (MMP-14; also known as MT1-MMP), from a panel of candidate proteases that were differentially expressed through the largest meta-analysis of human VS transcriptomes. The abundance and proteolytic activity of MMP-14 in the plasma and tumor secretions from VS patients correlated with clinical parameters and the degree of SNHL. Further, MMP-14 plasma levels correlated with surgical outcomes such as the extent of resection. Finally, the application of MMP-14 at physiologic concentrations to cochlear explant cultures led to damage to spiral ganglion neuronal fibers and synapses, thereby providing mechanistic insight into VS-associated SNHL. Taken together, MMP-14 represents a novel molecular biomarker that merits further validation in both diagnostic and prognostic applications for VS.

Keywords: MMP-14; biomarker; hearing loss; surgical outcome; vestibular schwannoma.

Copyright © 2020 Ren, Hyakusoku, Sagers, Landegger, Welling and Stankovic.

Figures

Figure 1
Figure 1
Identification of protease candidates differentially expressed in vestibular schwannoma (VS). (A) Schematic of workflow utilizing the Human Degradome and meta-analysis of genome-wide expression in VS, yielding 16 differentially expressed proteases with 10 being significantly upregulated after Bonferroni correction (P < 0.05). (B) Matrixmetalloprotease 14 (MMP-14) levels are significantly elevated in VS secretions (n = 16) when compared to great auricular nerve (GAN) controls (n = 6). The mean ± SEM is denoted by a bar and error bars; two-tailed Student’s t-test, *P < 0.05. (C) Gene expression of several members of the MMP family in HEI-193 human schwannoma cell line as determined by qRT-PCR. N = 4 independent experiments. Error bars represent SEM, ns, not significant by one-way ANOVA with Tukey’s post hoc multiple comparison tests between MMP-14 gene expression and expression of other proteases. (D) MMP gene expression in primary VS cultures (n = 5). Error bars represent SEM, ***P < 0.001 for all comparisons between the expression of and other proteases, by one-way ANOVA with Tukey’s post hoc multiple comparison tests.
Figure 2
Figure 2
Expression of MMP-14 protein in human VS. (A–D) Immunohistochemical staining (IHC) of MMP-14 protein in archived VS specimens from patients with sporadic, unilateral VS. Staining is graded ranging from mild (0+, A) to mildly moderate (1+, B), moderate (2+, C), and intense (3+, D). Representative IHC from 10–12 histological sections per specimen is shown. A total of 21 different VS and 6 GAN specimens were analyzed. Scale bar, 100 μm. (E) Corresponding MMP-14 IHC staining scores for VS (n = 21) vs. GAN controls (n = 6).
Figure 3
Figure 3
Correlation of MMP-14 with preoperative sensorineural hearing loss (SNHL) in VS patients. (A) Plasma MMP-14 levels positively and significantly correlate with tumor secreted MMP-14 from the same patients (n = 19). (B) MMP-14 plasma levels correlate positively and significantly with the degree of ipsilateral SNHL as measured by pure tone average (PTA; dB). (C) MMP-14 plasma levels correlate negatively with word recognition (WR) scores (%). P-values are shown and rho represents Spearman’s rank correlation coefficient.
Figure 4
Figure 4
Development of a functional assay to sense plasma MMP-14 activity in VS. (A) Fluorescence de-quenching measurements of protease-mediated cleavage of MMP-14 specific fluorescent resonance energy transfer (FRET) substrate, using plasma samples obtained from VS patients. Controls indicate no added specimen. (B) Fluorescence de-quenching is MMP-14 specific and is inhibited by both Marimastat (blue), a broad-spectrum protease inhibitor, and NSC 405020 (green), an MMP-14 specific inhibitor. Controls (orange) indicate no added specimen. Error bars represent SEM from n = 6 independent experiments. (C) Relative fluorescence measurements of MMP-14 cleavage in the presence of protease inhibitors. Controls (orange) indicate no added specimen. Error bars represent SEM from n = 6 independent experiments. (D) Dose-dependent inhibition of MMP-14 activity by NSC 405020. Error bars represent SEM from four to six independent experiments. **P < 0.01, ***P < 0.001 by one-way ANOVA.
Figure 5
Figure 5
Elevated MMP-14 is associated with subtotal tumor resection (STR). (A) Comparison of the level of MMP-14 enzyme found in tumor secretions obtained from GTR (n = 7) vs. STR (n = 11) cohorts. Mean ± SEM is denoted by a horizontal line and error bars; two-tailed unpaired Student’s t-test, **P < 0.01. (B) Comparison of the total proteolytic activity of MMP-14, as determined by the plasma level of MMP-14 and its proteolytic activity, between patients who underwent GTR (n = 7) or STR (n = 9). Mean ± SEM is denoted by a horizontal line and error bars; two-tailed unpaired Student’s t-test, *P < 0.05. A.u., arbitrary units. (C) Receiver operating curves (ROC) and calculated area under curve (AUC) values for MMP-14 proteolytic activity, secreted MMP-14, plasma MMP-14, tumor volume, and growth rate. The gray line represents a predictor with no discriminatory power (AUC = 0.50).
Figure 6
Figure 6
Application of MMP-14 to murine cochlear explants leads to loss of spiral ganglion neurites and cochlear synapses. (A) Representative images of cochlear explants receiving no treatment (Ctrl, n = 10 different explants), incubated with 10 ng/ml of murine MMP-14 (mMMP-14) for 72 h (n = 15 different explants), or with 30 ng/ml of murine MMP-14 for 72 h (n = 16 different explants). Myo7a (green) marks hair cells and NF-H (red) marks spiral ganglion neurites. Scale bar, 20 μm. (B) Representative images of untreated cochlear synapses (Ctrl, left column, n = 10 explants), synapses after incubation with 10 ng/ml of mMMP-14 (middle column, n = 15 explants), or 30 ng/ml of mMMP-14 (right column, n = 16 explants). CtBP2 (red) marks pre-synaptic ribbons and PSD95 (green) marks post-synaptic densities. Scale bar, 10 μm. (C–E) Quantification of the number of IHCs per 100 μm (C), spiral ganglion neuron (SGN) fiber bundles per IHC measured 10 μm below the basolateral poles of hair cells, normalized to no-treatment controls (D), and CtBP2-PSD95 juxtapositions per IHC normalized to no-treatment controls (E). Error bars represent SEM. **P < 0.01, ***P < 0.001 by one-way ANOVA.

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