Aitchison, Ella E.

Abstract Matrix metalloproteinases (MMPs) are key regulators of tumor progression, invasion, and metastasis; however, clinical efforts to inhibit MMPs have largely failed due to a lack of selectivity and unintended suppression of protective family members. In triple-negative breast cancer (TNBC), MMP-9 (gelatinase B) is strongly associated with tumor aggressiveness, extracellular matrix remodeling, and metastatic dissemination. In contrast, MMP-8 has been shown to exert anti-tumorigenic effects in breast cancer by limiting inflammation and suppressing metastasis. This functional divergence underscores the importance of selectively targeting MMP-9 while preserving MMP-8 activity as a rational therapeutic strategy in TNBC. To address this challenge, we engineered variants of the endogenous MMP inhibitor tissue inhibitor of metalloproteinases-1 (TIMP-1) with enhanced selectivity for MMP-9 over MMP-8. A yeast surface display library of human TIMP-1 was constructed and screened using fluorescence-activated cell sorting through iterative rounds of positive selection for recombinant MMP-9, followed by competitive counter-selection against MMP-8 to eliminate off-target binders. This strategy enabled enrichment of TIMP-1 variants with improved specificity for MMP-9 compared with wild-type TIMP-1. Several TIMP-1 variants exhibited enhanced binding and inhibitory selectivity toward MMP-9, with TIMP-1-C6.2 emerging as the most promising candidate. TIMP-1-C6.2 preferentially inhibited MMP-9 with a Ki of 0.3 ± 0.04 nM, while demonstrating substantially weaker inhibition of MMP-8 (Ki = 6.8 ± 0.27 nM), corresponding to an approximately 23-fold selectivity for MMP-9. Importantly, functional evaluation in Matrigel invasion assays using MDA-MB-231 TNBC cells revealed that TIMP-1-C6.2 significantly enhanced inhibition of tumor cell invasion compared with wild-type TIMP-1 in a dose-dependent manner (p < 0.0001). This improved anti-invasive activity reflects refined target selectivity rather than a generalized increase in inhibitory potency. Collectively, these findings demonstrate that selective engineering of TIMP-based inhibitors can overcome a central limitation of MMP-targeted therapies by suppressing pro-tumorigenic MMP-9 while sparing anti-tumorigenic MMP-8. This work provides a strong foundation for the development of selective MMP-9 inhibitors with improved safety and therapeutic potential for TNBC and other MMP-driven cancers. Citation Format: Alireza Shoari, Alexandra Hockla, Mathew A. Coban, Ella E. Aitchison, Alexandra M. Dimesa, Evette S. Radisky. Protein engineering of selective MMP-9 inhibitors reveals a therapeutic strategy for triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB233.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteolytic enzymes that are crucial for the remodeling of the extracellular matrix, a process that is often co-opted by cancers, including brain tumors, to facilitate growth, invasion, and metastasis. In gliomas, MMPs contribute to a complex interplay involving tumor proliferation, angiogenesis, and immune modulation, thereby influencing tumor progression and patient prognosis. This review provides a comprehensive analysis of the roles of various MMPs in different types of gliomas, from highly malignant gliomas to metastatic lesions. Emphasis is placed on how the dysregulation of MMPs impacts tumor behavior, the association between specific MMPs and the tumor grade, and their potential as biomarkers for diagnosis and prognosis. Additionally, the current therapeutic approaches targeting MMP activity are discussed, exploring both their challenges and future potential. By synthesizing recent findings, this paper aims to clarify the broad significance of MMPs in gliomas and propose avenues for translational research that could enhance treatment strategies and clinical outcomes.