Therapeutic potential of inhibitors of gelatinases MMP-2 and MMP-9 for the treatment of breast cancer

Abstract

Matrix metalloproteinases (MMPs) are secreted and cell membrane-associated enzymes that remodel the extracellular matrix (ECM) and cleave extracellular proteins to promote tumor invasion, angiogenesis, immune evasion, and many other aspects of cancer progression. Among this family, the gelatinases MMP-2 and MMP-9, specialized for cleaving collagen IV, are tightly linked to metastatic progression in breast cancer and adverse clinical outcomes. Here, we summarize gelatinase biochemistry and regulation, including zymogen activation, TIMP control, cell-surface trafficking and tethering, and receptor-mediated clearance, and explain how crosstalk between ECM and proteases amplifies invasion and metastatic seeding. We then review therapeutic strategies in two parts: direct inhibition and indirect pathway modulation. Direct approaches emphasize selective small molecules (thiirane mechanism-based inhibitors, allosteric blockers of pro-MMP-9 activation) and protein biologics (monoclonal antibodies, nanobodies, engineered TIMPs). Indirect strategies target upstream signals that drive MMP-2/−9, including MAPK/AP-1, PI3K/Akt/mTOR, NF-κB, EGFR/JAK/STAT, and nuclear receptor and Nrf2/HO-1 programs, with agents that curb invasion, angiogenesis, and metastasis in breast cancer models. Together, these advances define a maturing toolkit for precise gelatinase control and support prospective evaluation in rational combinations to restrain metastatic progression in high-risk breast cancer.

Graphical Abstract

Breast Cancer: Clinical burden and unmet metastatic need

In 2022, female breast cancer was the second most prevalent cancer globally, with approximately 2.3 million new cases, representing 11.6% of all cancer diagnoses. Additionally, it ranked as the fourth leading cause of cancer-related mortality worldwide, resulting in 666,000 deaths, which accounted for 6.9% of all cancer fatalities [1, 2]. Global incidence is still climbing by ~3 % per year, driven largely by population aging and lifestyle shifts [3]. Mortality remains unequally distributed, with rates about 90% higher in low-resource regions such as Western Africa and the Caribbean compared to high income regions including North America, Western Europe, and Australia/New Zealand) [4]. While not all cancer cases are fatal, they substantially diminish quality of life and impose considerable economic burdens [5].

In the United States, the 5-year relative survival rate for women diagnosed with localized breast cancer is 98.9%. This rate decreases to 85.7% for regional breast cancer and drops further to just 28.1% for metastatic breast cancer [6]. Worldwide as well, women diagnosed de novo with stage IV disease still face 5-year survival rates below 30 % [7]. These survival statistics highlight the drastic impact of metastasis on disease outcome and the critical need for improved metastatic inhibitors for treatment in late-stage breast cancer therapy. For localized tumors, management with curative intent combines surgery, radiotherapy and increasingly tailored systemic therapy to eradicate the primary tumor and forestall metastatic spread [8], whereas treatment of metastatic breast cancer focuses on symptom palliation and extending survival through systemic drugs plus selective local measures [9]. In nearly all cases, metastatic breast cancer remains incurable in currently affected patients [10], underscoring the urgency of interventions that prevent or blunt the metastatic cascade.

A key contributor to distant metastasis is the degradation of the extracellular matrix (ECM), which facilitates cancer cell invasion, intravasation, and eventual colonization of distant organs [11, 12]. This process is driven by proteases secreted from tumor and stromal cells; proteases from several mechanistic classes cooperatively participate in ECM degradation and remodeling [13, 14]. In normal physiological processes such as wound healing and angiogenesis, proteolysis is precisely regulated by controlled activation and endogenous inhibitors, preserving tissue architecture [15–17], but this balance is progressively subverted during tumor progression [18]. Within the extracellular tumor microenvironment, the dynamic interplay between proteases, their substrates, and effectors orchestrates key pathological processes that drive cancer progression [14]. Among these enzymes, matrix metalloproteinases (MMPs) are frequently upregulated across malignancies and correlate with poor clinical outcome [19], including advanced stage, increased tumor cell invasion, and metastatic burden in breast cancer [20]. Because gelatinases MMP-2 and MMP-9 specifically cleave native collagen IV, the principal structural component of basement membranes, and can further degrade the denatured collagen fragments (“gelatin”) produced by initial proteolysis, they are implicated as key mediators of metastatic spread in breast cancer [21–23]. We next overview gelatinase structure and biochemistry, before turning to the roles of these enzymes in breast cancer progression.

Gelatinases in context: biochemistry, activation, and regulation

The MMPs are a family of zinc-dependent endopeptidases (23 in humans) that orchestrate ECM remodeling in physiological and pathological settings [24, 25]. These enzymes are traditionally classified into 6 broad groups based on substrate specificity and domain architecture, comprising collagenases (MMP-1, −8, −13), gelatinases (MMP-2, −9), stromelysins (MMP-3, −10, −11), matrilysins (MMP-7, −26), membrane-type (MMP-14, −15, −16, −17, −24, −25), and other, non-classified members. MMPs are multidomain enzymes, containing an N-terminal prodomain that maintains latency through the “cysteine switch” P-R-C-X-X-P-D motif [26], a catalytic domain coordinating the active site Zn²⁺ ion, and in most family members, a C-terminal hemopexin domain that modulates substrate binding, receptor interactions and inhibition [27, 28]. Most MMPs are secreted as inactive zymogens and require pericellular activation by serine proteases or other MMPs, while their activity is counter-balanced by endogenous tissue inhibitors of metalloproteinases (TIMPs) [25]. Within this framework, the two gelatinases stand out for their unusual collagen IV-directed activity, an attribute central to basement membrane breach during metastasis.

MMP-2 (gelatinase A) and MMP-9 (gelatinase B) share the core MMP scaffold and additionally possess three fibronectin type II repeats inserted into the catalytic domain (Figure 1), a module that confers high-affinity binding to denatured collagens and gelatins [29]. MMP-9 alone contains, in addition, a flexible, O-glycosylated domain (~ 64 amino acids) between the catalytic and hemopexin domains (Figure 1) [30]. Although most MMPs genes cluster on chromosome 11, MMP2 maps to 16q12.2 and MMP9 to 20q11.2, reflecting early evolutionary divergence [31]. Both enzymes cleave native type IV collagen, the principal structural component of basement membranes, and efficiently process the gelatin fragments generated by initial proteolysis, thereby driving progressive matrix degradation [32, 33].

Fig. 1. Gelatinase structure and domain organization.

(A) Human MMP-2 (gelatinase A) showing modular domain organization: signal peptide (S, light gray), prodomain (Pro, pink), catalytic domain (CAT, yellow), fibronectin type II–like repeats (F, red), zinc-binding site (Z, beige), and hemopexin-like domain (PEX, green). The crystal structure of soluble MMP-2 (PDB: 1CK7) illustrates the typical gelatinase architecture, with the CAT domain in yellow, the four-bladed PEX domain in shades of green, zinc ions shown in gray, and calcium ions in green. (B) Human MMP-9 (gelatinase B) with a similar domain organization but containing an additional O-glycosylated domain (OG, gold), which serves as a flexible linker of variable length connecting the CAT and PEX domains. The crystal structure of soluble MMP-9 (PDB: 1L6J) represents its characteristic architecture. Structures were rendered in PyMOL.

Physiologically, MMP-2 and MMP-9 contribute to ECM turnover during wound healing [34] and organogenesis [35]; their tightly controlled expression in these contexts contrasts sharply with the sustained over-production observed in breast tumors [21–23]. MMP-9 transcription is readily induced by proinflammatory cytokines (IL-1β, TNF-α), growth factors (EGF), and hypoxia via AP-1, NF-κB, and HIF-1α responsive elements in its promoters [36–38]. The MMP-9 promoter also has GC-rich Sp1/Ets motifs that can drive expression; for example in breast cancer cells, Ets-1 binding was found to contribute to MMP-9 upregulation and invasive potential [39]. The GC-rich promoter of MMP-2 lacks canonical NF-κB/AP-1 sites and is controlled chiefly by Sp1/Ets motifs; stimuli such as TGF-β or hypoxia up-regulate MMP-2 more indirectly through these elements [37, 38].

Both gelatinases are secreted as latent pro-enzymes: basal pro-MMP-2 (72 kDa) is released at low levels by most mesenchymal and epithelial cells and can be further up-regulated by TGF-β or oncogenic signaling, whereas pro-MMP-9 (92 kDa) is normally restricted to neutrophils, macrophages and other inflammatory cells but is strongly induced in tumor and stromal compartments [40, 41]. Pro-MMP-2 is activated at the cell surface by the membrane-type MMP-14 (MT1-MMP) in a trimolecular complex where TIMP-2 tethers the zymogen to the activator [42, 43]. Pro-MMP-9 can be activated by serine proteases such as trypsin or other MMPs such as MMP-3 [28, 44]. Activity is further regulated by endogenous inhibitors and binding partners. TIMP-1 preferentially inhibits MMP-9, whereas TIMP-2 shows higher affinity for MMP-2; imbalance of these inhibitors is common in breast cancer [45]. MMP-9 uniquely forms a stable complex with NGAL, which shields the enzyme from autodigestion and prolongs activity; the complex can be detected in serum and urine of breast cancer patients [46]. Conversely, both active gelatinases can be internalized for lysosomal degradation through the LRP-1 scavenger receptor [47–49], a clearance route frequently down-regulated in tumors [50].

These regulatory mechanisms enable precise deployment of MMP-2 and MMP-9 to support normal tissue repair, vascular remodeling, and resolution of inflammation. During cutaneous and mucosal wound healing, transient gelatinase induction coordinates controlled ECM turnover, promotes keratinocyte migration, and facilitates angiogenic sprouting required for granulation tissue [17, 34, 51]. In parallel, gelatinases process cytokines and chemokines, for example cleaving CCL2 and CXCL9/10/11 to generate truncated forms with reduced CCR2 or CXCR3 receptor signaling, which limits ongoing leukocyte recruitment and aids resolution of inflammation [52]. In genetic models, Mmp9 deficiency delays re-epithelialization and disrupts collagen fibrillogenesis, underscoring a non-redundant role in orderly wound repair [53]. These mechanisms also support host defense. During infection, MMP-9 expression by neutrophils facilitates their migration across endothelium and basement membrane barriers to sites of infection [54], gelatinase processing of chemokine CXCL8/IL-8 amplifies signaling activity ten-fold and enhances early neutrophil recruitment [55], while cleavage of CXCL9/10/11 promotes the resolution phase [52, 56]. These homeostatic activities highlight how gelatinase activity is normally time-limited and spatially restricted, in contrast to the sustained overproduction observed in tumor settings. Collectively, specialized domain architecture and multilayered regulatory mechanisms endow MMP-2 and MMP-9 with potent, yet finely tunable, capacity to degrade collagen and cleave other critical biological substrates—properties co-opted by breast tumors to breach basement membranes and remodel the metastatic niche (Figure 2).

Fig. 2. Multifaceted roles of gelatinases (MMP-2 and MMP-9) in breast cancer (BC) progression.

Gelatinases act at multiple stages of breast cancer biology. At the tumor front, MMP-2/−9 degrade extracellular matrix (ECM) and basement membrane (BM) components, promoting local invasion enabling cancer cells to penetrate surrounding stroma and intravasate into blood vessels, where they circulate as circulating tumor cells (CTCs). In the pre-metastatic niche, gelatinase activity remodels the ECM to facilitate recruitment of tumor-associated stromal and immune cells, priming distant organs for colonization. Gelatinases also contribute to dormancy escape by releasing matrix-sequestered growth factors that drive proliferative reactivation of disseminated tumor cells. In addition, MMP-2/−9 support angiogenesis and vasculogenic mimicry by reshaping vascular architecture and enabling tumor cell incorporation into vessel-like structures. Collectively, these processes are reinforced by a gelatinase–ECM crosstalk feedback loop that sustains proteolysis-driven tumor progression and metastatic dissemination. (Illustration created with BioRender.com).

Functional roles of gelatinases MMP-2 and MMP-9 in breast cancer progression

Breach of basement membrane and invasion

Many studies have demonstrated that gelatinase expression and activation of their latent forms are critical for ECM degradation and tumor invasion in breast cancer [22]. Immunohistochemistry first showed that “type IV collagenase” activity is present at the invasive edge of breast carcinomas but absent in ductal carcinoma in situ [23]. These enzymes were later identified as the gelatinases MMP-2 and MMP-9, whose preference for type IV collagen, the main structural scaffold of basement membrane, was defined biochemically with synthetic substrate libraries [32, 33]. Gelatinases also act on other basement-membrane components; for example, MMP-2 cleaves the laminin-332 γ2 chain, exposing a cryptic sequence that stimulates breast cancer cell motility [57]. Functional relevance of gelatinase-mediated invasion to metastasis has also been demonstrated in vivo; for example, silencing tumor cell MMP-9 in human triple-negative breast cancer cells greatly reduced invasion through Matrigel artificial basement membranes and blocked spontaneous metastasis in an orthotopic model [58]. Together, these studies establish a direct link between gelatinase activity, basement-membrane degradation, and the onset of invasive behavior.

Actin-based invadopodia focus the enzymes where they are needed; these matrix-degrading protrusions leverage a protease ensemble led by MMP-14 and the gelatinases [59]. Trafficking of pro-MMP-2 and pro-MMP-9 to the invadopodial membrane requires the small GTPase Rab40b, the loss of which dramatically reduces focal matrix degradation and invasion of breast cancer cells [60]. At the cell surface, active MMP-9 is positioned for pericellular proteolysis by the receptor CD44, promoting localized ECM degradation and tumor invasion [61, 62]. Cell surface MMP-9 also cooperates with activated integrin αvβ3 to drive directed migration of breast cancer cells [63]. The tumor microenvironment amplifies this program: breast fibroblasts signal through thrombospondin-1, and macrophages through TNF-α, to enhance tumor cell MMP-9 expression, thereby promoting invasion in co-culture models [64, 65]. Notably, collective invasion of groups of cells is increasingly recognized as a major route, wherein aggressive leader cells (tumor-derived cells, cancer-associated fibroblasts, or tumor-associated macrophages) clear a path through ECM and create tracks for followers [66]. Gelatinases also play a key role here, as demonstrated by the upregulation of MMP-9 in mesenchymal leader cells at the invasive front [67]. In human breast cancers, MMP-2 was observed to be specifically expressed by fibroblasts in close contact with pre-invasive tumor clusters [68]. These converging mechanisms—subcellular membrane targeting, receptor tethering, and stromal induction—underscore the central role of gelatinases in breaching basement membrane and driving local invasion in breast cancer.

Dissemination, colonization, and escape from dormancy

Before tumor cells leave the breast, distant tissues can be primed by host-derived gelatinases to receive them. In an early seminal study, subcutaneous tumors in mice provoked the VEGFR-1–driven upregulation of MMP-9 in lung endothelial cells and macrophages; genetic deletion of either the VEGFR-1 kinase domain or Mmp9 itself significantly reduced lung infiltration and colonization of tail vein-injected tumor cells [69]. Similar lung protection was seen in the immunocompetent MMTV-PyMT mouse mammary tumor model, where germ-line Mmp9 knockout or systemic administration of a gelatinase-neutralizing antibody lowered early lung foci and circulating tumor cell seeding [70]. MMP-2 plays a complementary role: lung fibroblast-derived MMP-2 activates latent TGF-β1, driving fibroblast activation and collagen deposition; loss of stromal MMP-2 substantially curtailed proliferative expansion of breast cancer metastatic foci in an experimental metastasis model [71]. Analogously, in bone, osteoblast-secreted MMP-2 liberated active TGF-β, supporting breast cancer cell survival and osteolysis; bones from Mmp2-null mice showed greater tumor cell apoptosis and smaller lesions [72].

Beyond matrix remodeling, gelatinase activity shapes a permissive immune microenvironment that aids colonization and early outgrowth. First, gelatinase-released TGF-β is potently immunosuppressive, dampening anti-tumor immunity by promoting regulatory T cells (Tregs), suppressing CD8+ cytotoxic T-cell (CTL) effector programs, and inhibiting dendritic-cell activation. It also activates fibroblasts and desmoplasia, which can physically impede T-cell entry into metastatic niches. In breast cancer models, MMP-2 activates latent TGF-β1 in lung and bone niches [71, 72], and tumor cell surface MMP-9, tethered by CD44, can also activate latent TGF-β in the pericellular space [61]. Second, MMP-9 can reduce T-cell trafficking by cleaving and inactivating the CXCR3-binding chemokines CXCL9/10/11, which are the signals that recruit anti-tumor CD8+ cytotoxic T lymphocytes (CTLs) and their activating CD4+ Th1 helper T cells. In a HER2-driven orthotopic model, an anti-MMP-9 antibody preserved intratumoral CXCL10 and Th1-type cytokine signals, which in turn increased effector-memory CD4+ and CD8+ T cells, while combination with anti-PD-L1 further amplified these effects, inhibiting tumor growth [73]. Consistent with this trafficking mechanism, MMP-9 inhibition in PyMT tumors increased intratumoral CD8+ T-cell infiltration and activation [70].

Tumor cells themselves can reinforce this program. In an orthotopic tumor model, lentiviral knockdown of MMP-9 in triple-negative MDA-MB-231 cells eliminated spontaneous metastasis to lung, despite only a modest reduction in primary-tumor size [58]. Forced expression of MMP-2 in MDA-MB-231 cells enhanced systemic colonization of brain, bone, liver, and kidney after intracardiac injection [74]. Furthermore, functional selection of lung-tropic MDA-MB-231 sublines to identify genes responsible for lung metastasis identified MMP-2 as one of four lung-metastatic virulence genes, upregulated exclusively in the most aggressive sublines [75]. MMP-2 was also one of 54 genes in a lung metastasis gene signature that predicted poor lung metastasis-free survival in independent patient cohorts [75]. Collectively, both host- and tumor-derived gelatinases facilitate successful arrival and early outgrowth of disseminated cells by remodeling extracellular matrices, activating TGF-β, and damping antitumor immunity.

Gelatinases also govern whether dormant micrometastases remain silent or resume growth. In mouse lungs harboring quiescent breast cancer cells, chronic inflammation can induce neutrophil influx and formation of neutrophil extracellular traps (NETs). These NETs serve as a scaffolding to facilitate sequential cleavage of laminin by neutrophil elastase and MMP-9, unveiling an integrin-α3β1 binding site that forces dormant tumor cells to awaken and proliferate [76]. This study showed that blocking NET formation, inhibiting MMP-9 activity, or neutralizing the laminin neoepitope revealed by MMP-9 cleavage prevented tumor cell awakening [76]. In a three-dimensional model in which serum-withdrawal was used to induce breast cancer cell dormancy, cells survived extended dormancy by assembling a fibrillar fibronectin matrix via αvβ3 and α5β1 integrin adhesion [77]. MMP-2 and MMP-9 were highly upregulated during growth recovery, while regrowth in response to serum-stimulation was reduced by a pan-MMP inhibitor, suggesting that the observed degradation of fibronectin by gelatinases is an important step during dormant tumor cell reactivation and outgrowth [77]. These studies place gelatinases at both ends of the metastatic timeline—preparing distant niches for colonization and later dismantling dormancy barriers to permit overt relapse.

Angiogenesis and vasculogenic mimicry

In diverse models of multiple cancers, catalytically active MMP-9, especially that secreted by neutrophils in the tumor microenvironment, has been repeatedly demonstrated to be a key driver of tumor angiogenesis, by liberating sequestered VEGF and other angiogenic factors from the ECM [78–80]. In breast cancer, several orthogonal approaches converge on the same conclusion. In a transgenic model, stromal Mmp9 deficiency reduced CD31+ vessel density in primary tumors, consistent with a requirement for host-derived MMP-9 to support neovascularization [81]. Direct suppression of MMP-9 in PyMT tumors using an anti-MMP-9 DNAzyme lowered microvessel density and diminished VEGF bioavailability, linking MMP-9 proteolysis to growth factor release in vivo [82]. Tumor-cell MMP-9 also contributes knockdown in triple-negative MDA-MB-231 xenografts produced a marked reduction in endothelial-lined vessels and disrupted vascular morphology [58]. Secreted, catalytically active MMP-9 can increase angiogenic signaling, as shown by enhanced VEGF-VEGFR2 complex formation and greater capillary density when wild-type (soluble) MMP-9 was expressed in breast cancer xenografts [83]. Complementing this, cell-surface localization of MMP-9 to CD44 on mammary carcinoma cells was sufficient to restore angiogenesis in vivo and to activate latent TGF-β in co-culture and tumor lysates, demonstrating a pericellular route by which MMP-9 can promote neovascularization in breast cancer [61]. Taken together, these studies support complementary modes of action—diffusible MMP-9 from both stromal and tumor compartments that liberates matrix-bound VEGF and tethered MMP-9 that activates TGF-β in the immediate pericellular space—both of which favor angiogenesis in breast tumors.

A supporting role for MMP-2 in breast tumor angiogenesis also emerges in specific contexts. Endothelial αvβ3 integrin recruits MMP-2 via its hemopexin (PEX) domain, while a purified PEX fragment that disrupts this interaction blocked collagenolysis and suppressed angiogenesis in chick embryos and a melanoma model in vivo [84]. The relevance of MMP-2 to breast cancer angiogenesis is supported by human breast specimens, where tumor cell MMP-2 immunoreactivity correlates with a higher fraction of small caliber microvessels, consistent with a role in early capillary formation [85]. Mechanistically, inhibiting MMP-14, the principal cell-surface activator of pro-MMP-2, prevents proMMP-2 processing and diminishes VEGF-driven endothelial invasion and tumor angiogenesis, underscoring the MMP-14/MMP-2 axis as an amplifier of pericellular proteolysis during neovessel formation and endothelial invasion [86].

Vasculogenic mimicry refers to the ability of aggressive cancer cells to organize into perfusable, endothelial-like channels independent of host endothelium, providing an alternative route for blood supply to feed tumor growth. Under hypoxia, HIF-1/2 signaling upregulates VE-cadherin, EphA2, and matrix metalloproteinases—particularly MMP-14, MMP-2, and MMP-9—which cooperate to sculpt pericellular matrix into patterned conduits. In this cascade, MMP-14 activates pro-MMP-2, and active MMP-2 cleaves laminin-332 γ2 to generate promigratory fragments, while MMP-9 further remodels matrix and increases VEGF availability [87]. In triple-negative breast cancer, CD133+ stem-like subclones express VE-cadherin and high levels of MMP-2/-9 and form capillary-like networks on Matrigel, a hallmark of vasculogenic mimicry in vitro [88]. Clinically, breast tumors that exhibit vasculogenic mimicry often co-express endothelial markers with elevated gelatinases and are associated with worse outcomes and resistance to anti-angiogenic therapy [89]. These observations position MMP-2 and MMP-9 as key effectors of vasculogenic mimicry in breast cancer, complementing their established roles in endothelial cell-driven angiogenesis.

Gelatinase-ECM crosstalk in the tumor microenvironment

Matrix mechanics regulate gelatinase expression and function in breast cancer models, and gelatinase activity in turn modulates matrix deposition and characteristics. In three-dimensional collagen, high-density (stiffer) gels amplified prolactin-ERK signaling of breast cancer cells to increase MMP-2 and MMP-9 expression and the level of active MMP-2 in conditioned medium, leading to increased invasiveness that could be suppressed by MMP inhibition [90]. Similarly, MDA-MB-231 cells cultured on substrates of increasing stiffness responded by increasing gelatinase activity, while MMP inhibition reduced cell spreading, lowered FAK phosphorylation, and weakened integrin-based adhesions, consistent with a mechanotransduction pathway linking rigid extracellular matrix to integrin-FAK signaling and gelatinase-driven invasion [91]. Gelatinase activity then feeds back onto the matrix to promote desmoplasia. In metastatic lung niches, stromal fibroblast MMP-2 activated latent TGF-β1, induced myofibroblast differentiation, and increased collagen I/IV and fibronectin synthesis; reducing MMP-2 or neutralizing TGF-β1 diminished these effects, whereas exogenous TGF-β1 restored them [71]. Fibroblast-derived MMP-9 similarly increased active TGF-β1, enhanced Smad3 signaling, and drove collagen gel contraction and fibronectin expression; loss or inhibition of MMP-9 reduced these responses, and reintroduction of MMP-9 rescued them [92]. Together, these studies support a positive feedback mechanism in which matrix stiffening elevates gelatinase output, and gelatinase-dependent activation of TGF-β further increases stromal matrix deposition and tissue stiffness.

Gelatinase cleavage of basement membrane and interstitial matrices also generate bioactive fragments that reinforce malignant behavior. MMP-2 cleavage of laminin-332 at the γ2 chain exposes a cryptic site that promotes epithelial migration, with the cleaved laminin-332 detected in remodeling breast tissue and tumors [57]. During mammary tissue remodeling, MMP-dependent processing of laminin-332 releases an EGF-like γ2 fragment that binds the EGF receptor, activates MAPK signaling, increases MMP-2 expression, and enhances migration [93]. In breast cancer dormancy models, NET-associated neutrophil elastase and MMP-9 can sequentially cleave laminin-111 to reveal an integrin α3β1-binding epitope that activates FAK, Src, MYLK, and YAP signaling and induces dormant cells to resume proliferation, whereas neutralizing the neoepitope or degrading the NETs prevents reactivation [76]. In the interstitial matrix, MMP-9-derived fibronectin fragments bind integrin αvβ6, amplify FAK, Src, and ERK as well as PI3K/Akt and Smad pathways, and increase migration and invasion in breast carcinoma models [94]. Collectively, these examples illustrate reciprocal exchange: ECM stiffness and architecture regulate gelatinase production and activity, and gelatinase proteolysis reshapes matrix structure and generates signaling cues that promote invasion, fibrosis, and metastatic outgrowth.

Beyond ECM remodeling activities, gelatinases can cleave select cell-surface receptors and adhesion molecules, thereby modulating key signaling pathways. For example, MMP-2 cleaves the FGFR1 ectodomain, releasing a soluble receptor fragment that retains ligand-binding capacity and can dampen FGF-2 signaling, adding an additional dimension to protease–matrix crosstalk [95, 96]. Similar receptor-shedding events have been reported for various cadherins and integrins, indicating the potential for gelatinases to influence not only ECM turnover but also receptor availability and downstream signaling. For MMP-9, most evidence points to indirect modulation of receptor tyrosine kinase signaling, for example through crosstalk with Neu1 sialidase to activate signaling ligands of receptor tyrosine kinases such as TrkA and EGFR, linking gelatinase activity to receptor transactivation and biased signaling [97, 98]. In breast cancer, where growth-factor pathways interface tightly with the ECM, these mechanisms likely interact with the ECM processing roles of gelatinases in a context-dependent manner, although definitive data elucidating the consequences in breast cancer models remain limited.

Clinical correlates and biomarker evidence for gelatinase activity in breast cancer

Across clinical studies, elevated gelatinase expression or activity generally associates with more aggressive breast cancer biology. Expression of MMP-2 and MMP-9 in breast tumors is associated with malignant vs. benign tumors [99] and with higher stage or grade, lymphatic metastasis, and poorer overall or progression-free survival [100–102].

MMP-2 (gelatinase A)

Multiple tissue studies and meta-analyses report that higher tumor MMP-2 correlates with worse prognosis [103]. In immunohistochemical and in situ hybridization analyses, MMP-2 is detected in primary breast carcinomas and associates with adverse clinicopathologic features [68, 102]. A study on a large series of poor-prognosis hormone receptor-negative patients revealed a prognostic role for MMP-2 in breast carcinoma [104]. Other studies demonstrated a correlation between expression level of MMP-2 and poorer overall or recurrence-free survival; in one study, the first 10 years of follow-up showed that MMP-2 positivity was associated with an elevated risk of death (1.8-fold higher) [102, 105]. A systematic meta-analysis concluded that MMP-2 could be a prognostic biomarker in breast cancer, and more recent pooled analyses similarly associated tumor MMP-2 overexpression with poorer outcomes, while lower MMP-2 mRNA relates to better overall survival [21, 106].

MMP-9 (gelatinase B)

Concordant observations implicate MMP-9 as an adverse marker in breast cancer [45]. Transcriptional microarray data have shown MMP-9 gene expression to be consistently prognostic for poor survival in numerous large cohorts of breast cancer patients [107, 108]. Moreover, MMP-9 is part of the Rosetta poor-prognosis signature for breast cancer [109]. Tissue microarray and immunohistochemical studies of primary breast tumors have revealed that total MMP-9 staining and specific detection in mononuclear inflammatory cells, stromal fibroblasts, and tumor cells were each associated with poorer relapse-free survival, distant metastasis, and shorter survival [110–113]. Systemic measurements echo these patterns: serum levels of MMP-9 and its endogenous inhibitor TIMP-1 were significantly higher in breast cancer than in benign breast disease or healthy control subjects [114]. The MMP-9/NGAL complex that stabilizes and prolongs MMP-9 activity was detected in 87% of urine samples from breast cancer patients, though rare in healthy controls [46], and elevated serum levels of NGAL, MMP-9, and their complex were associated with disease severity [115]. Conversely, despite the prognostic impact of plasma MMP-9 and TIMP-1 when measured individually in other studies, the inhibited complex was not associated with prognosis in a cohort of breast cancer patients [116], consistent with the idea that catalytically active, TIMP-free MMP-9 is most relevant pathologically in breast cancer progression [78].

Assay considerations and clinical implications

Reported effect sizes and significance vary across studies with assay platform (protein or transcript), sample source (tumor/stroma or circulation), and antibody/epitope differences, which should temper cross-study comparisons. Nevertheless, the aggregate evidence supports a consistent message: higher levels of MMP-2 and MMP-9, particularly when localized to tumor or stromal compartments, associate with greater risk of recurrence and death. These clinical associations align with the mechanistic roles summarized earlier, including basement membrane breach, growth factor mobilization, priming metastatic sites, and immune evasion (Figure 2); together, they provide a rationale for evaluating gelatinase-focused diagnostics and therapeutics in breast cancer.

Direct inhibition of gelatinases: small-molecule strategies

First-generation, broad-spectrum hydroxamates: lessons from breast cancer trials

The development of small molecule inhibitors targeting MMPs began in the 1990s, driven by the recognition that MMPs, including the gelatinases MMP-2 and MMP-9, were key mediators of extracellular matrix degradation, angiogenesis, and tumor invasion (Table 1) [117]. First-generation inhibitors such as batimastat (BB-94) and marimastat (BB-2516) were designed as hydroxamate-containing compounds that chelated the catalytic zinc ion common to all MMPs, and showed initial promise in preclinical tumor models [118]. However, broad inhibition of all MMPs disrupted physiological tissue remodeling and immune regulation, leading to adverse events like musculoskeletal pain and inflammation, particularly in long-term treatments, exposing the central problem of poor selectivity [119]. Clinical experience in breast cancer confirmed the limitation: despite strong biologic rationale for suppressing tumor invasion, angiogenesis, and metastasis, broad-spectrum MMP inhibitors did not improve outcomes and were often associated with dose-limiting toxicities, including musculoskeletal side effects [120].

Table 1.

Direct gelatinase inhibitors in breast cancer: clinical-stage agents, selective chemotypes, and preclinical leads

Inhibitor	Class / chemotype	Primary gelatinase target(s) (Ki/IC50)	Selectivity notes	Breast cancer model / key finding	Clinical status	Ref.
A. Classical broad-spectrum hydroxamates (pan-MMP active-site chelators)
Batimastat (BB-94)	First-generation hydroxamate; broad-spectrum MMP inhibitor	Multiple MMPs incl. MMP-2/−9 (low-nM enzymatic)	Non-selective; intraperitoneal/poor oral bioavailability	preclinical anti-metastatic activity but limited clinical development	Not advanced in breast cancer	[117, 118]
Marimastat (BB-2516)	Oral hydroxamate; broad-spectrum MMP inhibitor	Multiple MMPs incl. MMP-2/−9	Non-selective; dose-limiting musculoskeletal toxicity (arthralgia/tendonitis)	No improvement in PFS/OS as maintenance after first-line chemotherapy in metastatic breast cancer (ECOG E2196)	Phase III negative in metastatic breast cancer	[118, 121–124]
BAY 12–9566 (tanomastat)	Oral biphenyl inhibitor; preferential for MMP-2	MMP-2 (Ki≈11 nM) ≫ MMP-9 (Ki≈301 nM)	Improved vs hydroxamates but still multi-MMP activity	Reduced regrowth and lung metastases post-resection in xenografts; no objective responses in Phase I (advanced solid tumors incl. breast)	Program terminated after negative Phase III in SCLC; no further breast cancer trials	[125–127]
Prinomastat (AG3340)	Non-peptidic hydroxamate; designed for MMP-2/−9	MMP-2/−9 (nanomolar enzymatic)	Broader MMP inhibition; musculoskeletal toxicity in other indications	Suppressed tumor growth (~50%) in MDA-MB-435 xenografts; paradoxical migration effects in short-term cell assays	No breast cancer trials; development discontinued	[80, 128, 129]
Rebimastat (BMS-275291)	Second-generation oral MMPI (reduced collagen-mimic features)	Broad MMP panel incl. MMP-2/−9	Still non-selective; class-typical musculoskeletal toxicity	Preclinical anti-angiogenic/anti-metastatic activity; adjuvant feasibility study in early breast cancer terminated early for toxicity	Phase II feasibility negative (early breast cancer)	[130, 131]
B. Gelatinase-selective, mechanism-based small molecules
SB-3CT (and analogs)	Mechanism-based thiirane; time-dependent inhibition	MMP-2/−9 (selective vs many MMPs)	Prefers gelatinases via transition-state stabilization; spares many non-gelatinase MMPs	Anti-angiogenic/anti-metastatic in non-breast models; reduced PD-L1 and enhanced checkpoint efficacy in syngeneic tumors	Preclinical; no breast cancer trials	[134, 136]
ND-322 (water-soluble prodrug)	SB-3CT-based prodrug with improved PK	MMP-2 ≈ MMP-14 ≫ MMP-9 (context-dependent)	Enhanced metabolic stability; retains gelatinase bias	Reduced tumor growth/invasion in melanoma models; rationale for breast cancer testing	Preclinical	[137, 138]
JNJ-0966	Allosteric inhibitor of pro-MMP-9 activation	MMP-9 (zymogen activation step)	Isoform-selective; does not block catalytic site or other MMPs	Efficacy in inflammation/neuro models; conceptually attractive where activated MMP-9 drives pathology	Preclinical (non-oncology)	[139]
AZD-1236	Dual MMP-9/−12 small molecule inhibitor	MMP-9 and MMP-12	Designed for airway disease; off-target sparing vs pan-MMPs	Biomarker modulation in COPD; potential repurposing where MMP-9 dominates	Clinical (non-oncology)	[140]
C. Medicinal-chemistry leads and natural-product scaffolds with preclinical potential
Arylamide series (hemopexin-domain binders)	Non-catalytic site binders to MMP-9 Hpx domain	MMP-9 (EC50 ≈125–139 μM in 4T1 cells)	Conceptually selective for MMP-9 domain; modest cellular potency	Suppressed gelatinase activity in 4T1 breast cancer cells	Preclinical (cellular)	[141]
Ugi bis-amide non-hydroxamates (e.g., 8, 11, 28)	Non-zinc-binding MMP-9 inhibitors	MMP-9 (IC50 ≈3.6–7.5 nM)	Selectivity over other MMPs reported in vitro	Potent enzymatic and MCF-7 anti-migration activity; no in vivo breast data	Preclinical (enzymatic/cellular)	[142]
Star-shaped triazine dendrimer 8a	Zinc-binding multivalent dendrimer	MMP-9 (cell-based IC50 ≈3.8 nM); ~4× vs MMP-2	Direct active-site binding; improved MMP-9 preference	Inhibited MMP-9 in MDA-MB-231; reduced VEGF expression	Preclinical (cellular)	[143]
Sanguinarine	Natural alkaloid; active-site binder (docking/MD)	MMP-9 (IC50 ≈19 μM)	Limited data vs MMP-2/other MMPs	Reduced MMP-9 activity/expression in TNBC (MDA-MB-468)	Preclinical (cellular/biochemical)	[150]
Hinokiflavone	Natural biflavonoid	MMP-9 (IC50 ≈43 μM; MCF-7)	Not fully profiled	In silico selection; reduced MMP-9 and cell viability	Preclinical (cellular/biochemical)	[151]
Green tea extract (catechin-rich)	Polyphenol mixture; zinc-chelating activity	MMP-2/−9 (~50% inhibition at 10 μg/mL; enzyme assays)	Non-selective mixture; high concentrations required	Direct enzyme inhibition; limited mechanistic specificity	Preclinical (biochemical)	[149]
In silico (Lig-1; L1)	Virtual-screen/4D-QSAR leads	Predicted high-affinity MMP-9 binders	Computational; needs empirical validation	Strong docking/MD stability; no breast cancer data	Discovery (computational)	[145, 146]

Abbreviations: MMP, matrix metalloproteinase; Hpx, hemopexin domain; PK, pharmacokinetics; SCLC, small-cell lung cancer; PFS, progression-free survival; OS, overall survival; TNBC, triple-negative breast cancer.

The most informative dataset comes from marimastat, an orally bioavailable broad-spectrum MMP inhibitor that effectively suppressed metastasis in breast and lung cancer models [121]. In a randomized Phase III trial in metastatic breast cancer (ECOG E2196), marimastat was evaluated in 179 patients with stable or responding disease following first-line chemotherapy [122]. Marimastat treatment did not extend progression-free survival (PFS) or overall survival (OS) versus placebo and was associated with a higher incidence of musculoskeletal toxicity. Notably, patients who developed grade 2–3 toxicity had significantly worse survival outcomes, and higher plasma levels of marimastat correlated with toxicity but not efficacy [122]. Earlier phase studies also highlighted a narrow therapeutic window and dose-dependent inflammatory polyarthritis [123, 124].

Experience with other early broad-spectrum MMP inhibitors was concordant. Tanomastat (BAY 12–9566) suppressed tumor growth and metastasis in breast cancer xenograft models [125]. It was generally well tolerated and reached target plasma levels in Phase I studies that included breast cancer patients, but no objective tumor responses were observed; the program was discontinued following a negative phase III study in another indication [126, 127]. Prinomastat (AG3340) suppressed growth in a breast cancer xenograft model [128], but did not reach clinical trials in breast cancer, and was ultimately discontinued due to lack of efficacy and high toxicity observed in other settings [129]. Rebimastat (BMS-275291) showed anti-tumor activity in preclinical breast cancer models [130], yet failed a Phase II feasibility trial as prolonged adjuvant therapy in early-stage breast cancer due to musculoskeletal toxicity and early discontinuations, with no survival readouts collected [131]. Collectively, these results argue that poorly selective MMP inhibitors designed to exploit potent zinc chelation moieties are unlikely to succeed in breast cancer, motivating the selective, mechanism-based strategies developed subsequently.

Gelatinase-selective, mechanism-based chemotypes

The early clinical failures with broad, zinc-chelating MMP inhibitors prompted a shift in focus toward selectivity and structural precision. The shortcomings of pan-MMP inhibition revealed the need for inhibitors that could distinguish between individual MMPs, especially those with tumor-promoting roles like MMP-2 and MMP-9, versus those with homeostatic or anti-tumorigenic functions [132]. Building on structure-guided design, fragment screening, and insights into noncatalytic MMP substrate-binding domains (including hemopexin and fibronectin repeats), second-generation chemotypes emerged that could offer therapeutic efficacy with reduced off-target effects (Table 1) [133]. For example, later-generation inhibitors SB-3CT, ND-322, JNJ-0966, and AZD-1236 represent distinct chemical scaffolds and mechanisms designed to selectively engage gelatinases or to block their activation rather than broadly chelating zinc.

Mechanism-based thiirane inhibitors represented a conceptual break from simple zinc chelation by requiring catalytic engagement to form a long-lived inhibitory complex with gelatinases. SB-3CT is a thiirane inhibitor that selectively targets gelatinases [134], and has demonstrated significant anti-metastatic and anti-angiogenic activity in vivo [135]. Notably, the combination of SB-3CT with immune checkpoint blockade enhanced antitumor immune response in murine models of several cancers, consistent with the immune-modulatory effects linked to MMP-9 activity [136]. ND-322 is an orally bioavailable derivative of SB-3CT, developed to improve metabolic stability and pharmacokinetics while retaining selective inhibition of MMP-2 and MMP-9 [137]. It has been tested in melanoma models where it showed potent anti-invasive and anti-tumor effects, supporting the translational premise of catalysis-dependent, gelatinase-selective inhibition [138]. Although direct breast cancer data remain limited for these agents, their mechanism aligns with the gelatinase-driven biology described in earlier sections.

A complementary strategy is to prevent zymogen activation rather than inhibit the catalytic site. JNJ-0966 targets the activation of pro-MMP-9 rather than inhibiting the catalytic domain, binding an allosteric pocket in the pro-domain to prevent MMP-9 activation without affecting other MMP family members, thereby minimizing liabilities of pan-MMP inhibition [139]. AZD-1236, developed primarily for inflammatory lung disease, is a small-molecule inhibitor that achieves dual selectivity for MMP-9 and MMP-12 through careful scaffold design and side-chain chemistry. It has advanced to early-phase clinical trials and demonstrates the feasibility of achieving MMP-9-biased pharmacology with an acceptable safety profile in non-oncology settings [140]. Thus, JNJ-0966 exemplifies allosteric blockade by preventing zymogen activation, while AZD-1236 illustrates how rationally designed zinc-binding scaffolds can achieve greater isoform selectivity while reducing the off-target toxicities that limited less selective inhibitors. In parallel, mechanism-based thiiranes such as SB-3CT and ND-322 demonstrate the feasibility of mechanism-dependent trapping of MMP-2 and MMP-9 in vivo. Together, these approaches define an emerging playbook for direct gelatinase inhibition, spanning catalytic trapping, activation blockade, and selective zinc-binding design, and support further testing in breast cancer contexts where MMP-9 drives angiogenesis, immune exclusion, and metastatic fitness.

Medicinal-chemistry leads and natural-product scaffolds with preclinical potential

Medicinal chemistry programs have explored alternative binding modes and non-hydroxamate scaffolds to improve selectivity for gelatinases and to avoid broad zinc chelation (Table 1). One line of work targets noncatalytic domains: a series of aryl-amide derivatives incorporating heterocyclic moieties were designed as selective inhibitors targeting the hemopexin domain of MMP-9 [141]. These molecules effectively suppressed MMP-9 activity in the 4T1 breast cancer cell line, albeit at high micromolar effective concentrations (EC₅₀ ~ 125–139 μM), underscoring an early proof-of-concept with substantial room for potency optimization [141]. A complementary approach uses non-hydroxamate chemotypes to engage the catalytic domain with greater isoform discrimination. Using an Ugi multicomponent strategy, bis-amide scaffolds were reported with dual activity against MMP-9 and AKT in the single-digit nanomolar range and excellent selectivity over closely related MMP isoforms [142]. These compounds displayed both cytotoxicity and inhibition of migration in breast cancer cell models. While attractive as non-hydroxamate leads, these bis-amides require further studies to disentangle their polypharmacology and to establish in vivo selectivity, pharmacokinetics, and efficacy.

Other chemical architectures attempt to recover selectivity while retaining zinc coordination. For example, star-shaped triazine-based dendrimers bearing terminal carboxylic acid groups as potential zinc-binding moieties were developed as direct inhibitors of MMP-9 [143]. However, the low nanomolar cytotoxicity of these compounds, far below the concentrations required for MMP inhibition, raises concern that the observed biological effects reflect off-target activities rather than specific gelatinase inhibition. Dendrimeric scaffolds may therefore be viewed as concept-generating, illustrating a design space in which classical zinc binding can be paired with topological features to improve isoform bias, rather than validated MMP-9-directed chemotypes at this stage. Additional early-stage concepts include novel potential MMP-9 inhibitors that demonstrate efficient docking scores, offering promising scaffolds for future therapeutic development [144]. For example, a virtual screen based on an MMP-9 E-pharmacophore model identified candidate inhibitors with predicted MMP-9 preference [145], while a separate 4D-QSAR approach predicted β-N-biaryl ether sulfonamide hydroxamate derivatives as potential high-affinity MMP-9 inhibitors [146]. At present, these remain rational starting points for lead optimization and in vivo evaluation rather than validated chemotypes.

Natural products and phytochemical scaffolds, with their long-standing relevance in cancer pharmacology [147, 148], provide another source of candidate gelatinase inhibitors. Among several dietary antioxidants, green tea extract showed the strongest direct inhibition by zymography of both MMP-2 and MMP-9 enzymatic activities, likely due to its potent zinc-chelating ability [149], although the concentrations required were high. Curcumin inhibited MMP-2 at high concentrations but had minimal effect on MMP-9, while resveratrol and olive extract were weaker still [149]. Several additional purified natural products demonstrate clearer mechanistic specificity: sanguinarine, a benzophenanthridine alkaloid derived from bloodroot, inhibited the activity of recombinant human MMP-9 with an IC₅₀ of 19 μM, with predicted binding at the active site, and decreased both the activity and expression of MMP-9 in triple-negative breast cancer cells [150]. Hinokiflavone, a biflavonoid derived from Juniperus communis identified through a ligand-based pharmacophore screen, inhibited MMP-9 in MCF-7 breast cancer cells with an IC₅₀ of 43 μM [151]. Although modest in potency compared to synthetic leads, these examples illustrate how natural scaffolds can serve as starting points for medicinal-chemistry optimization.

Together, current small molecule efforts reflect a pivot from broad zinc chelation toward selective engagement of MMP-2/−9. The most persuasive evidence comes from mechanism based thiiranes and prevention of pro-MMP-9 activation; in contrast, many other medicinal chemistry leads and natural product scaffolds remain early stage, with modest potency, incomplete selectivity profiling, and limited in vivo validation. Given the clinical history with nonselective hydroxamates, credible progress will require clear on-target pharmacodynamic readouts in breast cancer models, acceptable exposure and tolerability, and demonstration of benefit—likely in rational combinations. With these criteria in mind, we next consider protein-based strategies that offer inherently higher selectivity with a different risk-benefit profile.

Direct inhibition of gelatinases: protein-based strategies

Therapeutic antibodies: preclinical mechanisms and early clinical experience

Over the last three decades, therapeutic antibodies have altered the targeted cancer therapy landscape and monoclonal antibodies represent rising class of targeted anticancer agents that improve natural immune system functions to restrain cancer cell activity and eradicate cancer cells [152]. By binding a single protease with epitope-level precision, anti-gelatinase antibodies aim to neutralize MMP-9 (or MMP-2) without the cross-family zinc chelation that produced dose-limiting toxicity with early small molecules. In immunocompetent mouse models of HER2-driven breast cancer, a mouse-specific anti-MMP-9 monoclonal antibody, a preclinical surrogate to the humanized antibody andecaliximab (GS-5745) [153], achieved selective MMP-9 blockade and reduced tumor growth [73]. Mechanistically, MMP-9 inhibition improved T-cell trafficking by preserving the function of CXCR3 ligands (CXCL9, CXCL10, CXCL11), reduced fibrillar collagen, and enhanced effector/memory CD4⁺ and CD8⁺ T-cell infiltration. Notably, combination with anti-PD-L1 further augmented antitumor immunity [73], suggesting that selective MMP-9 inhibition may significantly improve the effectiveness of immune checkpoint blockade therapies. Importantly, selective MMP-9 inhibition was shown to avoid the musculoskeletal syndrome typical of pan-MMP inhibitors in preclinical safety studies [153].

Andecaliximab has been clinically evaluated across solid tumors. In a phase I trial (NCT01803282) in patients with advanced solid tumors including breast cancer, recommended dosing was established, confirming target engagement and favorable safety [154]. An expansion cohort in gastric/gastroesophageal junction adenocarcinoma demonstrated encouraging efficacy with a ~50% objective response rate [154]. A subsequent Phase III trial in gastric and gastroesophageal junction adenocarcinoma was negative for PFS/OS improvement, highlighting the importance of disease context and combinations [155]. In breast cancer, a phase I study combining andecaliximab with paclitaxel in patients with previously treated metastatic disease showed reported feasibility and acceptable safety, with signals of activity [156], although no breast cancer data from randomized trials are yet available. Considering the role of MMP-9 in immune suppression, a concept already supported preclinically and under exploration clinically in other tumor settings, MMP-9 inhibition may warrant exploration of combinations with immune checkpoint blockade therapies in breast cancer patients [73].

Compared with early broad-spectrum, zinc-chelating inhibitors, more selective gelatinase-directed agents like andecaliximab and MMP-9-biased small molecule AZD-1236 have shown more favorable tolerability, suggesting the potential for a wider therapeutic window [140, 154]. At the same time, because MMP-2 and MMP-9 contribute to wound repair, vascular remodeling, and aspects of host defense as described earlier, sustained systemic inhibition could carry on-target risks such as delayed wound closure or altered inflammatory responses [51, 53, 157], so peri-operative timing and prospective safety monitoring are important considerations in translational studies. Combination regimens raise two further considerations: potential additive effects on vascular remodeling with anti-angiogenic drugs that could heighten wound-healing risk [158], and a mechanistic rationale for complementarity with immune checkpoint blockade (preservation of CXCR3-ligand chemokines and enhanced T-cell trafficking) [56, 73, 159] that, in principle, could also intensify the immune-mediated toxicities typical of checkpoint therapy. These considerations frame the context for advancing antibody and other engineered-protein strategies discussed next.

Single-domain antibody (“nanobody”) inhibitors provide a complementary format of biologics, offering high specificity, stability, and tissue penetration, making them a promising strategy for selective gelatinase inhibition in cancer therapy [160]. A panel of highly selective nanobodies targeting human MMP-2 has been described, offering potential therapeutic or imaging agents; translation to breast cancer models remains to be shown [161]. Given their short half-life, engineering for half-life extension, such as albumin- or Fc-fusion, may be a consideration for systemic therapy. Beyond antibody-derived constructs, we next turn to compact protein and peptide scaffolds engineered for gelatinase selectivity.

Other protein and peptide scaffolds: engineered TIMPs and peptide inhibitors

Beyond antibodies, small proteins and peptides can be engineered to engage gelatinases with domain-level selectivity. Several peptide-based inhibitors have been developed targeting MMP-2 and MMP-9. For example, hydroxamate-modified peptides were designed that bind to the fibronectin type II domains of MMP-2, achieving IC₅₀ values of 10–100 μM [162]. In previous studies, we used phage display to identify the linear peptide RSH-12, which selectively binds the fibronectin domain of MMP-2/9 and inhibits gelatinolytic activity in vitro [163]. RSH-12 was further optimized by cyclization to enhance serum stability, producing a more potent inhibitor of MMP-9 activity and fibrosarcoma cell invasion [164]. An MMP-2/9-targeted cyclic peptide, CTT, was shown to possess potent gelatinase inhibitory activity, while its radiolabeled derivative was employed for in vivo tumor imaging in models with high MMP-2/9 expression [165]. However, none of these peptides have yet been tested for their anti-cancer activities in models of breast cancer.

Another recent strategy aims to engineer endogenous TIMPs, natural tight-binding protein inhibitors of active MMPs that in their native forms are broadly cross-reactive. By leveraging structure-function insight at the TIMP-MMP interface, protein engineering using yeast surface display and directed evolution can alter the inhibitory spectrum of TIMPs to become much more selective [166]. In practice, mutational tuning of the TIMP binding loops, supported by computation-guided design, enables conversion of TIMP scaffolds into isoform-selective inhibitors while preserving high potency.

The N-terminal domain of TIMP-2 (N-TIMP2) is a compact, well-behaved inhibitory scaffold that retains full MMP-binding activity and is amenable to display-based selection. Using dual-color sorting, N-TIMP2 variants with 30- to 1175-fold enhanced selectively for either MMP-9 or MMP-14 were obtained; biochemical selectivity extended to cells, where MMP-9-selective variants blocked the mobility of MCF-7 cells overexpressing MMP-9 but not their MMP-14-overexpressing counterparts [167]. A next-generation MMP-9-selective N-TIMP2 variant (REY) further improved inhibitory affinity for MMP-9 (Ki^app ~0.15 nM). and exhibited >1000-fold selectivity over off-target MMPs like MMP-3, MMP-10, and MMP-14 [168]. In functional assays with MDA-MB-231 breast cancer cells, REY potently inhibited cell invasion and proliferation in a dose-dependent manner, achieving >90% inhibition at 100 nM without cytotoxicity [168]. Complementing the MMP-9 program, computation-guided evolution yielded an ultra-high-affinity inhibitor of MMP-14 (K_i ~0.9 pM; 900-fold enhanced affinity and up to 16,000-fold higher selectively relative to other MMPs) that functionally suppressed MMP-dependent breast cancer cellular invasiveness [169]. Extending this concept to the pericellular activation machinery, a multi-specific N-TIMP2 heterodimer co-targeting MMP-14 and integrin αvβ3 displayed superior capability to suppress MMP-2 activation and curtailed endothelial invasion and tube formation [170]. This innovative engineering approach underscores a tractable route to interfere with the MMP-14/MMP-2 activation axis relevant to breast cancer stromal and tumor compartments.

Full-length TIMP-1, comprised of two domains, offers a broader epitope surface than N-TIMP2, enabling extensive fine-tuning of affinity and selectivity through directed evolution. In work using MMP-3 as a model, we showed that mutations distributed across both the N- and C-terminal domains can act synergistically to strengthen affinity and shape specificity; variants achieved low-picomolar inhibition and demonstrated that the two domains cooperate in matrix metalloproteinase recognition [171]. A counter-selective screening strategy was further developed to drive fine discrimination between closely related MMPs, illustrating how distal TIMP-1 epitopes can be leveraged to bias binding toward one among near-identical enzymes [172]. We have recently applied these principles to generate a TIMP-1 variant (TIMP-1-C15) with markedly enhanced specificity and potency for inhibiting MMP-9. TIMP-1-C15 incorporated mutations that exploit interactions not only with the MMP-9 catalytic domain but also its unique fibronectin domains, resulting in an improved equilibrium inhibition constant (K_i) for MMP-9, 0.61±0.02 nM for TIMP-1-C15 versus 3.36±0.07 nM for wild-type TIMP-1, a ~5.5-fold enhancement [173]. TIMP-1-C15 also demonstrated dose-dependent suppression of cell invasion in two triple-negative breast cancer cell lines (MDA-MB-231 and BT-549), outperforming wild-type TIMP-1 across tested concentrations [173]. These findings support the idea that full-length TIMP-1 can be programmed for single-MMP selectivity through multisite engagement while retaining a human scaffold conducive to further translational optimization.

Pharmacokinetic engineering is beginning to address a practical barrier for TIMP therapeutics. PEGylation extended the circulation half-life of TIMP-1 from about an hour to roughly a day in mice while preserving inhibitory activity and anti-invasion effects [174], while an unfolded Pro–Ala–Thr extension (“PATylation”) similarly prolonged N-TIMP2 exposure without loss of potency against MMP-9 [175]. Against this backdrop, highly selective TIMP variants that inhibit MMP-9 or MMP-14, nanobody approaches to MMP-2, and clinical-stage anti-MMP-9 antibodies such as andecaliximab define a coherent path for direct gelatinase inhibition by biologics in breast cancer. The mechanistic selectivity and early safety signals with selective MMP-9 blockade suggest a way to leverage gelatinase biology while avoiding the toxicity that limited broad small molecules. However, translation will depend on in vivo validation in breast cancer models, durable target engagement in the tumor microenvironment, and rational combinations with chemotherapy or immune checkpoint blockade. With continued optimization of selectivity, exposure, and delivery, protein inhibitors, including monoclonal antibodies, nanobodies, and engineered TIMPs, are well positioned to test the hypothesis that precise gelatinase inhibition can improve outcomes in aggressive breast cancer.

Indirect modulation of gelatinase expression and activity

Beyond direct catalytic inhibition, gelatinase activity in breast cancer is shaped by upstream signaling networks that regulate MMP-2 and MMP-9 expression. Key pathways include MAPK/AP-1, PI3K/Akt/mTOR, NF-κB, EGFR/JAK/STAT, nuclear receptors, and VEGF-linked stromal loops integrate external cues with transcriptional control, driving invasion, angiogenesis, and metastasis. Numerous pharmacological inhibitors, natural compounds, and repurposed drugs attenuate these nodes, thereby reducing gelatinase output and metastatic behavior (Table 2).

Table 2.

Indirect modulation of gelatinase expression and activity in breast cancer—therapeutic nodes and representative modulators.

Pathway / node	Mechanism and dominant readouts	Therapeutic modulators (representative refs)
Growth factor and PKC signaling: MAPK/AP-1 and NF-κB convergence	ERK/JNK/p38 drive AP-1 and NF-κB-dependent MMP-9 transcription. Inhibitors reduce gelatinase activity and invasion; some show in vivo anti-metastatic/anti-angiogenic effects.	Dihydroavenanthramide D [186]; silibinin [178–181]; berberine [182, 183]; curcumin [187]; orientin [188]; triptolide [189]; capillarisin [191]; delphinidin [192]; centchroman [193]; emodin [184]; AP-1–targeting PI polyamide [185]; ursolic acid [194]; Salvia miltiorrhiza extract [195].
PI3K/Akt/mTOR integration with NF-κB/AP-1	Agents inhibit PI3K/Akt and/or mTOR (often with ERK crosstalk), lower NF-κB/AP-1 signaling, suppress MMP-2/−9, invasion, and (for several) angiogenesis/metastasis in vivo.	DMBT [200]; CoQ0 [201]; dihydroartemisinin [202]; EGCG [203]; piceatannol [204]; sinomenine [206]; antroquinonol [207]; docetaxel/quercetin nanoparticles [197]; curcumol [198]; gypensapogenin H [199]; MPPa-PDT [208].
NF-κB-centered control of gelatinases	Direct inhibition of the Akt/IKK/NF-κB axis reduces MMP-9 transcription/activity and invasion; several agents validated with genetic/pharmacologic tools and in vivo.	DPP23 [210]; DHMEQ [212]; sulforaphane [213]; Saussurea lappa extract (ESL) [214]; celastrol [190, 215]; magnolol [211]; Frondoside A [217]; gallic acid-AuNP [218]; formononetin [219]; docosahexaenoic acid [220]; Euphorbia humifusa [221]; metformin [222]; Anisomeles indica extract (AI)/apigenin [216].
Receptor-level control: EGFR/HER2 and JAK/STAT	EGFR→JAK3/ERK signaling upregulates MMP-9; modulators inhibit EGFR phosphorylation and downstream NF-κB/AP-1, reducing MMP-9 and invasion.	Lapatinib-derivative conjugates (3a, 5b) [225]; pomolic acid [205].
Nuclear receptor and antioxidant programs: PPARγ, retinoids, and Nrf2/HO-1	PPARγ and retinoids elevate TIMPs and/or damp NF-κB/AP-1; Nrf2/HO-1 induction suppresses MMP-9 and invasion.	PPARγ agonists (pioglitazone, rosiglitazone, GW7845) and 15d-PGJ₂ [226–228]; all-trans retinoic acid [226, 227]; nicardipine [229]; letrozole [230]; tamoxifen [231]; tamoxifen/tranilast [232]; tamoxifen/orlistat nanocrystals [233].
Microenvironment and angiogenesis: VEGF-linked loops and stromal mediators	Breaking MMP-9/VEGF stromal feed-forward loops or blocking VEGF→VEGFR2→FAK signaling lowers MMP-9, curtails angiogenesis, and limits metastatic spread.	Amino-bisphosphonates (zoledronate, pamidronate) [235]; cucurbitacin B [236].

Note: “DHA” refers to dihydroartemisinin in [202]; docosahexaenoic acid is listed explicitly in [220]. Abbreviations: EGCG, epigallocatechin-3-gallate; AuNP, gold nanoparticle.

Growth factor and PKC signaling: convergence on MAPK/AP-1 and NF-κB

Mitogen-activated protein kinases link growth factor and PKC signals to transcriptional programs that drive gelatinase expression in breast cancer. The MAPK family including ERK, JNK, and p38 represents one of the most extensively studied upstream regulators of MMP-2 and MMP-9. These kinases integrate extracellular cues such as growth factors, cytokines, and stress signals, transmitting them to transcriptional activators like AP-1 and NF-κB that directly control gelatinase expression. Early pathway dissection with heregulin-β1 in SKBr3 and MCF-7 cells revealed that PKCε and p38 were essential and ERK also contributed, while PI3K/Akt was dispensable [176]. Pharmacologic blockade with RO318220 (PKC), SB203580 (p38), or PD098059 (MEK/ERK) effectively suppressed MMP-9 induction and invasion [176]. Selective inhibition of p38α also reduced basal and TGF-β1-induced MMP-9, impaired vitronectin-driven motility, and suppressed osteolytic metastasis in vivo, linking p38 signaling to bone colonization in breast cancer [177].

Multiple agents exploit this node therapeutically. For example, silibinin, a flavonoid from milk thistle, blocked MEK/ERK phosphorylation, reduced MMP-9 and COX-2 expression (phenocopied by the MEK inhibitor U0126 and reversed by constitutively active MEK, establishing pathway dependence), and inhibited invasion in MCF-7 and MDA-MB-231 cells [178–181]. Berberine, an isoquinoline alkaloid, suppressed PKCα/MEK/ERK signaling, reducing MMP-9 expression and activity in MCF-7 and MDA-MB-231 breast cancer cells and blocking migration and invasion [182, 183]. Modulators with in vivo corroboration include emodin, an anthraquinone derivative, which suppressed TNFα-induced MMP-9 expression via p38/IKK/NF-κB signaling, reducing primary tumor invasiveness and lung metastases in xenografted mice [184]. An orthogonal approach blocks AP-1 at the MMP-9 promoter: a pyrrole–imidazole polyamide targeting the AP-1 recognition site reduced MMP-9 mRNA, protein, and activity in MDA-MB-231 cells, curtailing migration and invasion and reducing liver metastasis and angiogenesis in vivo [185]. Numerous additional natural or synthetic modulators converge on the same MAPK/AP-1 and NF-κB axis and recapitulate these phenotypes in breast cancer models, illustrating how pharmacologic blockade of MAPK signaling not only prevents gelatinase expression but blunts their tumor-promoting effects [178–180, 186–195].

In sum, dysregulated MAPK signaling enhances gelatinase-driven invasion and metastasis, making this pathway a central therapeutic target. Across breast cancer models, interrupting ERK/JNK/p38 activation or preventing AP-1 promoter engagement consistently lowers MMP-9 transcription and gelatinolytic activity and curbs migration, invasion, and, in some studies, metastatic spread. Given frequent interplay with PI3K/Akt and NF-κB, rational combinations that co-target cooperating nodes may yield more durable suppression of gelatinase-driven invasion; we address these related signaling pathways in sections below.

PI3K/Akt/mTOR integration with NF-κB/AP-1

The PI3K/Akt axis, often coupled with mTOR, is a central signaling hub orchestrating cell survival, motility, metabolism, and angiogenesis, and is strongly implicated in MMP regulation [196]. Activation of Akt promotes nuclear signaling cascades that upregulate MMP-2 and MMP-9 transcription and secretion, whereas pharmacological inhibition or blockade by natural compounds suppresses gelatinase activity and breast cancer invasiveness. Given its frequent hyperactivation in aggressive tumors, PI3K/Akt/mTOR represents a critical regulatory node for targeting breast cancer metastasis.

Several synthetic agents and natural products have demonstrated anti-invasive properties by suppressing Akt- and mTOR-driven pathways. For example, co-delivery of docetaxel and the Akt inhibitor quercetin in hyaluronic acid (HA)–modified PLGA–PEI nanoparticles in 4T1 murine breast cancer cells downregulated p-Akt and MMP-9, suppressing migration (~95%) and invasion (~99%) in vitro [197]. In vivo, the HA-nanoparticles accumulated in tumors and lungs of 4T1-bearing mice, effectively curtailing both primary tumor growth and pulmonary metastasis [197]. Curcumol attenuated PI3K/Akt-dependent NF-κB activation, reduced MMP-9, and limited tumor growth and metastasis in vivo [198]. Gypensapogenin H similarly suppressed the PI3K/Akt/NF-κB/MMP-9 axis, restraining growth and migration of triple-negative breast cancer in preclinical models [199]. Additional modulators converge on this node, including agents that reduce Akt phosphorylation and downstream NF-κB/AP-1 activity with reductions in gelatinase expression and activity, and consequent reductions in endpoints such as migration, invasion, metastasis, and angiogenesis [200–208]

Collectively, these studies underscore the centrality of PI3K/Akt and mTOR in regulating MMP-2/−9 expression, with multiple agents acting at different levels of the pathway, from receptor tyrosine kinases to Akt phosphorylation and mTOR effectors. A recurring caveat is pleiotropy: many agents modulate PI3K/Akt together with MAPK and NF-κB; while this complicates mechanism assignment, it may be advantageous therapeutically when simultaneous suppression of cooperating nodes is desired. The convergence of findings across synthetic drugs, phytochemicals, and nanoparticle formulations highlights PI3K/Akt/mTOR as a versatile and druggable signaling axis. Given its broad integration with NF-κB, AP-1, and EMT-inducing transcription factors, inhibition of this pathway offers a promising therapeutic avenue to suppress gelatinase-driven invasion, angiogenesis, and metastasis in breast cancer.

NF-κB-centered control of gelatinases

NF-κB is a master transcriptional regulator of genes linked to inflammation, angiogenesis, and invasion, including MMP-2 and MMP-9. Constitutive NF-κB activation is a hallmark of many breast cancer subtypes, particularly triple-negative disease, where chronic signaling supports metastatic behavior [209]. In this context, multiple studies demonstrate that natural products, synthetic small molecules, and nanoparticle formulations suppress MMP-2/−9 expression by disrupting NF-κB activation, thereby attenuating invasion and metastasis. Together, these observations establish NF-κB as a therapeutically relevant node for indirect gelatinase control.

Therapeutic modulation has been demonstrated with mechanistic and in vivo support. The synthetic chalcone DPP23 blocked the Akt/IKK/NF-κB axis, suppressing TNFα-induced MMP-9 transcription, actin remodeling, migration, and invasion; in a 4T1 syngeneic model it attenuated liver metastasis, highlighting pathway-dependent anti-metastatic activity [210]. Magnolol inhibited IκBα phosphorylation and NF-κB nuclear translocation, potently suppressing MMP-9 while sparing MMP-2 across multiple breast cancer cell types, and reduced tumor growth and MMP-9 levels in xenograft models [211]. Additional agents converge on the same pathway and recapitulate anti-gelatinase, anti-invasive phenotypes in breast cancer models [209, 212–222].

These findings reinforce NF-κB as a critical transcriptional driver of gelatinase expression in breast cancer. Across diverse pharmacologic and natural inhibitors, a consistent theme emerges as blocking NF-κB nuclear translocation or promoter engagement reduces MMP-2/−9 expression, dampens gelatinolytic activity, and limits invasion and metastatic spread. These results not only establish NF-κB as a therapeutic target but also highlight its central role as a signaling hub integrating inputs from PI3K/Akt, MAPKs, and oxidative stress responses into MMP regulation.

Receptor-level control: EGFR/HER2 and JAK/STAT signaling

Growth factor signaling via EGFR and its downstream mediators JAK and STAT are central drivers of oncogenic programs in breast cancer, including regulation of MMP expression [223]. Mechanistically, EGF activates MMP-9 transcription through an EGFR/JAK3/ERK-dependent pathway, whereas STAT3 can act as a negative regulator [224]. In HER2-positive SKBR3 cells, pharmacological inhibition of EGFR (AG1478), JAK3 (WHI-P131), or MEK1/2 (UO126), or alternatively JAK3 siRNA or STAT3 overexpression, markedly reduced EGF-induced MMP-9. In contrast, JAK1 or JAK2 inhibition had no effect, defining JAK3/ERK as essential positive regulators and STAT3 as a negative modulator of EGF-induced gelatinase expression [224].

Therapeutically, EGFR-directed strategies reduce MMP-9 output and invasion in breast cancer models. In triple-negative disease, lapatinib-derived conjugates linked to cancer stem cell inhibitors (compounds 3a and 5b) reversed lapatinib resistance by attenuating EGFR signaling, suppressing Wnt/β-catenin, and reducing MMP-2/−9 [225]. In vivo, pretreatment with 3a reduced tumor initiation in xenografted TNBC models, highlighting the therapeutic potential to limit metastasis [225]. In EGF-stimulated MDA-MB-231 cells, the triterpenoid pomolic acid suppressed EGFR phosphorylation and downstream ERK and PI3K/Akt/mTOR signaling, reduced MMP-9 transcriptional activity and protein levels, inhibited NF-κB and AP-1 DNA binding, and decreased FAK phosphorylation, thereby limiting invasion and migration [205].

Collectively, these studies underscore EGFR/JAK/STAT signaling as a pivotal regulator of MMP-9 in breast cancer. Targeting this axis, via direct EGFR inhibition, selective JAK3 blockade, or dual pathway strategies that suppress ERK and PI3K/Akt, emerges as a coherent approach to limit gelatinase-driven invasion and metastasis in tumors with EGF-responsive phenotypes.

Nuclear receptor and antioxidant programs: PPARγ, retinoids, and Nrf2/HO-1

Nuclear receptor signaling through peroxisome proliferator-activated receptor gamma (PPARγ), retinoic acid receptors, and estrogen receptor (ER), together with the antioxidant nuclear factor erythroid 2–related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) axis, provides an additional layer of transcriptional control over MMP activity in breast cancer. These pathways frequently exert anti-inflammatory and antioxidant effects, indirectly reducing gelatinase expression by upregulating TIMPs or suppressing NF-κB and AP-1 transcriptional activity. Importantly, their modulation by natural ligands and FDA-approved drugs highlights their therapeutic potential for limiting invasion and metastasis.

PPARγ and retinoids illustrate two complementary modes of gelatinase control. In MDA-MB-231 cells, PPARγ agonists (pioglitazone, rosiglitazone, GW7845) and the endogenous ligand 15-deoxy-Δ¹²,¹⁴-prostaglandin J₂ (15d-PGJ₂), as well as all-trans retinoic acid (ATRA), suppressed invasion. While MMP-9 protein levels remained unchanged and MMP-2 was undetectable, these ligands robustly increased TIMP-1 expression, thereby elevating the TIMP-1/MMP-9 ratio and reducing gelatinolytic activity; neutralization experiments confirmed that MMP-9 was the dominant mediator of invasion in this context [226]. In ER-positive MCF-7 cells, ATRA treatment significantly decreased pro-MMP-2 activity and expression while increasing TIMP-2 and E-cadherin [227]. It also suppressed MMP-14, EMMPRIN, integrins (α5, β1), FAK, NF-κB, phosphorylated ERK, and VEGF, linking retinoid signaling to integrin/FAK and MAPK nodes that impinge on gelatinase regulation and cell-ECM adhesion [227].

Antioxidant programs intersect with these nuclear receptors via HO-1. In MCF-7 cells, 15d-PGJ₂ inhibited TPA-induced MMP-9 expression and invasion through an HO-1-dependent mechanism; PPARγ activity was required, as the antagonist GW9662 reversed the effect [228]. Mechanistically, 15d-PGJ₂ blocked nuclear translocation and DNA binding of NF-κB and AP-1, and induced HO-1 expression via Nrf2 activation; positive feedback between HO-1 and PPARγ reinforced this axis [228]. Clinically accessible modulation of this axis is exemplified by nicardipine, an FDA-approved calcium channel blocker, which suppressed MMP-9 expression and activity in triple-negative breast cancer models through Nrf2-dependent HO-1 induction, inhibiting migration, invasion, and colony formation [229]. Pharmacological or siRNA blockade of HO-1 restored MMP-9 activity, while HO-1 end products such as bilirubin downregulated MMP-9 [229]. These findings point to a drug repurposing path for Nrf2/HO-1 activation in gelatinase control.

Endocrine signaling adds context as another nuclear receptor modulating gelatinase expression and activity. In ER-positive MCF-7 cells, aromatase inhibition with letrozole reduced androgen-driven proliferation and suppressed secretion of pro-MMP-2 and pro-MMP-9, attenuating estradiol-induced gelatinase upregulation. These results highlight the dual action of aromatase inhibition on both growth and invasion pathways in ER+ breast cancer [230]. In another study, estradiol and the selective estrogen receptor modulator tamoxifen differentially regulated gelatinases in MCF-7 cells, with estradiol generally reducing MMP-2/−9 and tamoxifen increasing them, emphasizing context-dependent ER control of the protease/anti-protease balance [231]. Combination strategies can sharpen the effect: tamoxifen with tranilast decreased VEGF and MMP-9 more than either agent alone in both ER-positive and ER-negative models [232], and tamoxifen with orlistat nanocrystals likewise enhanced tumor suppression with concurrent downregulation of MMP-9 and VEGF among other tumor markers [233].

Taken together, ligands of PPARγ and retinoic acid receptors, inducers of Nrf2/HO-1, and ER-directed endocrine approaches converge on lowering MMP-2/−9 activity by enhancing TIMP expression and repressing integrin-FAK or NF-κB/AP-1 pathways. With several agents already in clinical use or repurposing range, this node offers pharmacologically tractable strategies to restrain gelatinase-driven invasion in breast cancer.

Microenvironment and angiogenesis: VEGF-linked loops and stromal mediators

Angiogenesis is a hallmark of breast cancer progression, and VEGF serves as its central driver. Matrix metalloproteinases, particularly MMP-2 and MMP-9, facilitate extracellular matrix degradation that permits endothelial sprouting and vessel remodeling [234]. This functional interdependence establishes a VEGF/MMP axis critical for tumor vascularization and metastatic dissemination.

Amino-bisphosphonates, including zoledronate and pamidronate, disrupted an MMP-9/VEGF feed-forward loop that promoted myeloid-derived suppressor cell (MDSC) expansion and stromal remodeling in a HER2/neu-driven breast cancer model [235]. Treatment reduced MMP-9 and VEGF, normalized bone marrow progenitor expansion, and curtailed MDSC infiltration, alleviating immunosuppression [235]. Direct interference with VEGF signaling also suppresses gelatinase-linked angiogenesis. Cucurbitacin B bound VEGF and blocked VEGF/VEGFR2/FAK signaling, reducing migration, invasion, and tube formation of tumor and endothelial cells [236]. In vivo, it diminished tumor growth, lung metastases, and microvessel density, while reducing FAK phosphorylation and MMP-9, establishing the VEGF/VEGFR2/FAK/MMP-9 axis as a therapeutic target [236].

Together, these findings illustrate how targeting the VEGF/MMP-9 circuitry, either by breaking stromal feed-forward loops or by blocking VEGF/VEGFR2 signaling, can restrain angiogenesis, remodel the microenvironment, and limit metastatic progression in breast cancer models. Translationally, this axis is well-suited to combination strategies with anti-angiogenic or immune-checkpoint therapies in settings where MMP-9-dependent vascular remodeling sustains metastatic outgrowth.

Conclusions

Gelatinases, particularly MMP-2 and MMP-9, are integral to breast cancer progression, enabling basement-membrane breach, angiogenesis, immune evasion, and metastatic outgrowth. Their dual nature as biomarkers and therapeutic targets has long been appreciated, yet early broad-spectrum MMP inhibitors faltered in the clinic because they indiscriminately chelated zinc across the protease family, leading to toxicity and limited benefit. These experiences underscored the need for selectivity—discriminating tumor-promoting MMP activities from homeostatic functions.

Recent advances point to a more tractable path. Direct inhibitors, including mechanism-based thiiranes, allosteric blockers of pro-MMP-9 activation, and more selective zinc-binding scaffolds, demonstrate that potency can be combined with isoform bias. In parallel, biologics, including monoclonal antibodies, nanobodies, and engineered TIMP variants, offer epitope-level selectivity and encouraging signs of tolerability, with pharmacokinetic engineering beginning to address exposure. These approaches enable precise targeting of gelatinase activity in relevant tumor and stromal compartments.

Complementing direct strategies, indirect targeting of gelatinases through upstream regulatory pathways, including MAPK/AP-1, PI3K/Akt/mTOR, NF-κB, EGFR/JAK/STAT, and nuclear receptor networks, consistently lowers MMP-2/-9 expression and activity, curbing invasion, angiogenesis, and metastasis in preclinical models. Because these pathways frequently intersect, rational combinations and sequencing with chemotherapy, endocrine therapy, or immune checkpoint blockade may be particularly effective in subtypes such as triple-negative disease.

Translationally, progress will hinge on clear pharmacodynamic readouts of on-target gelatinase inhibition, adequate tumor exposure, and biomarker-guided trial designs that align mechanism with patient selection. With these elements in place, selective gelatinase inhibition, achieved by refined small molecules or protein-based agents, has the potential to move from concept to clinic and improve outcomes for patients with aggressive, treatment-resistant breast cancers.

Abbreviations

AKT

Protein kinase B

AP-1

Nuclear translocations of activator protein-1

COX-2

Cyclooxygenase-2

CXCR

CXC chemokine receptors

CXCL

Chemokine (CXC motif) ligand

DHAvD

Dihydroavenanthramide D

DHMEQ

Dehydroxymethylepoxyquinomicin

DMBT

6, 6’-bis (2, 3-dimethoxybenzoyl)-a,a-D-trehalose

DPP23

E-3-(3,5-dimethoxyphenyl)-1-(2-methoxyphenyl) prop-2-en-1-one

ECM

Extracellular matrix

EGFR

Epidermal growth factor receptor

ERK

Extracellular signal regulated kinase

FAK

Focal adhesion kinase

FGF-2

Fibroblast growth factor 2

FGFR1

Fibroblast growth factor receptor 1

HER2

Human epidermal growth factor receptor 2

HO-1

Heme oxygenase-1

IL-1β

Interleukin-1 beta

JAK

Janus kinase 3

JNK

c-Jun N-terminal kinase

MAPK

Mitogen-activated protein kinase

MMPs

Matrix metalloproteases

mTOR

Mammalian target of rapamycin

NF-κB

Nuclear factor kappa B

NGAL

Neutrophil gelatinase-associated lipocalin

PD-L1

Programmed death-ligand 1

PI3K

Phosphoinositide 3-kinase

PKC-α

Protein kinase C alpha

PPARγ

Peroxisome proliferator-activated receptor γ

STAT

Signal transducer and activation of transcription

TGF

Transforming growth factor

TIMP

Tissue inhibitor of metalloproteinase

TNF

Tumor necrosis factor

TPA

12-O-tetradecanoylphorbol-13- acetate

VEGF

Vascular endothelial growth factor

15d-PGJ2

15-Deoxy-Delta-12,14-prostaglandin J2