Dimesa, Alexandra M.

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.

Liver cancer, also known as hepatocellular carcinoma (HCC), remains a major global health concern, with high mortality driven by late-stage diagnosis, limited treatment efficacy, and frequent therapeutic resistance. Matrix metalloproteinases (MMPs), a large family of zinc-dependent endopeptidases, are central to the biological processes that drive liver tumor initiation and progression. By degrading and reorganizing extracellular matrix components, MMPs facilitate tumor expansion, tissue invasion, and metastatic dissemination. In addition, these enzymes regulate the availability of growth factors, cytokines, and chemokines, thereby influencing angiogenesis, inflammation, immune cell recruitment, and the development of an immunosuppressive tumor microenvironment. Aberrant expression or activity of multiple MMP family members is consistently associated with aggressive clinicopathologic features, including vascular invasion, increased metastatic potential, and reduced patient survival, highlighting their promise as prognostic markers and actionable therapeutic targets. Past attempts to modulate MMP activity were hindered by broad inhibition profiles and dose-limiting toxicities, underscoring the need for improved specificity and delivery strategies. Recent advances in molecular design, biologics engineering, and nanotechnology have revitalized interest in MMP targeting by enabling more selective, context-dependent modulation of proteolytic activity. Preclinical studies demonstrate that carefully tuned MMP inhibition can limit tumor invasion, enhance anti-angiogenic responses, and potentially improve the efficacy of existing systemic therapies, including immuno-oncology agents. This review synthesizes current knowledge on the multifaceted roles of MMPs in HCC pathobiology and evaluates emerging therapeutic strategies that may finally unlock the clinical potential of targeting these proteases.

Abstract Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors that regulate the function and activity of matrix metalloproteinases (MMPs), enzymes that are known for their involvement in cancer progression. MMP-9, also known as gelatinase B, plays a crucial role in tumorigenesis of several cancers including triple negative breast cancer (TNBC); tumor cell-produced MMP-9 promotes malignant progression and metastasis of basal-like TNBC. Here, we have used full-length TIMP-1 as a scaffold for engineering inhibitors that bind with higher selectivity to MMP-9, for potential therapeutic applications. We previously developed a yeast surface display library of human TIMP-1, with diversity incorporated at 17 residues within five loops; mutation burden was tuned to 3-5 mutations per clone. Here, we have screened the TIMP-1 library for affinity toward recombinant MMP-9 by using fluorescence-activated cell sorting (FACS). Yeast cells were incubated with an incrementally decreasing concentration of biotinylated MMP-9 (400 nM to 25 nM) in successive rounds of sorting. Samples were analyzed using flow cytometry and highest affinity populations were selected using FACS. Among the TIMP-1 variants with highest enrichment, we identified the TIMP-1-C15 variant that showed 12-fold enhanced binding with improved selectivity toward MMP-9 compared with wild type TIMP-1 (WT TIMP-1). We next expressed and purified soluble recombinant TIMP-1-C15 and conducted inhibition studies using a fluorogenic substrate to evaluate inhibitory potency towards MMP-9 activity. These experiments showed that TIMP-1-C15 inhibits MMP-9 activity with an inhibition constant (Ki) of 0.61 nM, more than 5-fold stronger than WT TIMP-1; these studies also corroborated the improved selectivity towards MMP-9 over other MMPs. Additionally, we further evaluated the functional impact of the TIMP-1-C15 variant on cancer cell invasiveness using TNBC cell lines in cellular invasion assays. The TIMP-1-C15 variant markedly reduced the invasive capabilities of TNBC cells to a greater extent than WT TIMP-1. Currently, we are working to isolate additional TIMP-1 variants from our library that exhibit enhanced selectivity for MMP-9, with MMP-8 included in the sorting process as a competitive target to ensure specificity. Selective inhibition of MMP-9 over MMP-8 is particularly important in breast cancer therapy, as MMP-8 has been shown to have anti-tumorigenic properties, and its inhibition could counteract therapeutic efforts aimed at reducing cancer invasiveness. Our findings suggest that engineered TIMP-1 variants could serve as potent inhibitors of MMP-9, offering a novel approach to reducing the invasiveness of breast cancer cells and potentially limiting the progression of metastatic disease. Citation Format: Alireza Shoari, Alexandra Hockla, Mathew A. Coban, Alexandra M. Dimesa, Derek C. Radisky, Evette S. Radisky. Engineered TIMP-1 for selective MMP-9 inhibition: A novel strategy to suppress breast cancer invasiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5115.

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.

Colorectal cancer (CRC) remains one of the most prevalent and lethal cancers worldwide, prompting ongoing research into innovative therapeutic strategies. This review aims to systematically evaluate the role of gelatinases, specifically MMP-2 and MMP-9, as therapeutic targets in CRC, providing a critical analysis of their potential to improve patient outcomes. Gelatinases, specifically MMP-2 and MMP-9, play critical roles in the processes of tumor growth, invasion, and metastasis. Their expression and activity are significantly elevated in CRC, correlating with poor prognosis and lower survival rates. This review provides a comprehensive overview of the pathophysiological roles of gelatinases in CRC, highlighting their contribution to tumor microenvironment modulation, angiogenesis, and the metastatic cascade. We also critically evaluate recent advancements in the development of gelatinase inhibitors, including small molecule inhibitors, natural compounds, and novel therapeutic approaches like gene silencing techniques. Challenges such as nonspecificity, adverse side effects, and resistance mechanisms are discussed. We explore the potential of gelatinase inhibition in combination therapies, particularly with conventional chemotherapy and emerging targeted treatments, to enhance therapeutic efficacy and overcome resistance. The novelty of this review lies in its integration of recent findings on diverse inhibition strategies with insights into their clinical relevance, offering a roadmap for future research. By addressing the limitations of current approaches and proposing novel strategies, this review underscores the potential of gelatinase inhibitors in CRC prevention and therapy, inspiring further exploration in this promising area of oncological treatment.