Hockla, Alexandra

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.

Selective inhibition of the more than 100 S1 family serine proteases is a long-standing challenge due to their active site similarity. Mesotrypsin, implicated in cancer progression, exemplifies these difficulties; no current inhibitors achieve selectivity over other human trypsins. We found an unexpected autoinhibited conformation of mesotrypsin via x-ray crystallography, revealing a cryptic pocket adjacent to the active site. Using high-throughput virtual screening targeting this cryptic pocket, we identified a conformationally selective small-molecule inhibitor that stabilizes the inactive state of mesotrypsin. This inhibitor demonstrates selectivity for mesotrypsin over other trypsins. Our findings challenge the accepted view of digestive trypsins as constitutively active enzymes lacking potential for allosteric regulation. Furthermore, analyses of other structures suggest that dynamic sampling of closed states with analogous allosteric cryptic pockets appears widespread among S1 serine proteases. These observations point to a potentially generalizable strategy to achieve selective inhibition, offering broad implications for drug development targeting serine proteases in cancer and other diseases.

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 significant drivers of many diseases, including cancer, and are established targets for drug development. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous MMP inhibitors and are being pursued for the development of anti-MMP therapeutics. TIMPs possess many attractive properties for drug candidates, such as complete MMP inhibition, low toxicity, low immunogenicity, and high tissue permeability. However, a major challenge with TIMPs is their rapid clearance from the bloodstream due to their small size. This study explores a method for extending the plasma half-life of the N-terminal domain of TIMP2 (N-TIMP2) by appending it with a long, intrinsically unfolded tail containing Pro, Ala, and Thr (PATylation). We designed and produced two PATylated N-TIMP2 constructs with tail lengths of 100 and 200 amino acids (N-TIMP2-PAT100 and N-TIMP2-PAT200). Both constructs demonstrated higher apparent molecular weights and retained high inhibitory activity against MMP-9. N-TIMP2-PAT200 significantly increased plasma half-life in mice compared to the non-PATylated variant, enhancing its therapeutic potential. PATylation offers distinct advantages for half-life extension, such as fully genetic encoding, monodispersion, and biodegradability. It can be easily applied to N-TIMP2 variants engineered for high affinity and selectivity toward individual MMPs, creating promising candidates for drug development against MMP-related diseases.

Ovarian cancer remains a devastating disease with 5 year survival rate in advanced stage of only 27%. Advanced stage tumors metastasize through continuous shedding of tumor cells into the peritoneal fluid, aided by the resistance of these cells to anoikis, a form of apoptosis normally triggered by loss of cell‐matrix interactions. We have previously shown that the serine protease inhibitor SPINK1 activates anoikis resistance in ovarian cancer cells via its protease inhibitory function; however, the specific relevant protease target(s) remain unknown. To find these targets, we have synthesized an activity‐based protein probe with a diphenylphosphonate warhead that covalently modifies serine proteases, with greatest specificity towards trypsin‐like proteases. We have used this probe to identify ovarian cancer cell secreted and membrane‐bound serine proteases possessing differential activity in the presence versus absence of SPINK1, using a proteomics approach. We are evaluating candidate proteases for involvement in anoikis resistance in ovarian cancer models. Our studies implicate a serine protease network in regulation of anoikis in ovarian cancer cells. By identifying critical proteases that control anchorage‐independent viability in ovarian cancer metastasis, we aim to elucidate important downstream pathways and ultimately develop therapeutic approaches that can intervene in mechanisms of anoikis resistance. Support or Funding Information Mayo Clinic Graduate School, Rochester MN

The PRSS23 gene, encoding putative serine protease 23, has been associated with several diseases by correlative gene expression studies and genetic‐based comparative models. Our group has recently developed data implicating the expression of this gene as a potential driver of ovarian cancer malignancy. However, there have been no studies defining PRSS23 protein structure or demonstrating its hypothesized proteolytic function. We aim to elucidate PRSS23 protein structure and enzymatic activity. The first step to achieve this goal is to develop a robust expression system and purification protocol for recombinant PRSS23 protein. We hypothesize that developing a methodology to produce PRSS23 recombinant protein will enable us to determine its crystal structure, evaluate its biological and enzyme function, and ultimately target the enzyme therapeutically. We strategized to maximize chances of producing PRSS23 in its native form and with high yield by testing multiple systems and host species: human, yeast, and bacterial. We anticipate, based on the protein sequence, that PRSS23 is endogenously secreted and may be activated in the secretory pathway by furin, releasing the C‐terminal catalytic domain from a smaller N‐terminal domain. The protein is also predicted to be glycosylated. We generated expression plasmids for full‐length PRSS23, codon‐optimized for expression in human and Pichia pastoris expression systems, incorporating a His‐tag for detection and purification. In the human HEK 293F and HEK 293T cell systems, despite optimizing transfection protocols and testing multiple expression constructs, secretion signals, and promoters, the expression level remained very low and was judged unlikely to produce sufficient protein for structural and functional studies. Integration of codon‐optimized PRSS23 into the genome of P. pastoris produced integrants showing a spectrum of expression levels from very low to moderate. Experiments are ongoing to optimize expression conditions of the best clone and develop a purification workflow involving ammonium sulfate precipitation, immobilized nickel ion chromatography, and additional chromatographic steps as needed. In parallel, we cloned the catalytic domain of PRSS23, appropriately codon‐optimized and also containing a C‐terminal His‐tag, into an expression vector for Escherichia coli. High levels of protein were produced as insoluble inclusion bodies, and a protocol was developed for nickel ion affinity purification under denaturing conditions. Experiments are ongoing to identify suitable conditions for in vitro refolding of the PRSS23 catalytic domain. Additionally, we are evaluating a maltose binding protein fusion with the PRSS23 catalytic domain as an alternative strategy to improve soluble protein production in E. coli. Significant quantities of pure protein will be needed to enable crystal structure determination and enzymatic activity elucidation, to unravel the biological mechanism of action and identify endogenous substrates, and potentially lead to new therapeutic strategies targeting PRSS23. The expression systems and purification workflows developed here will be a step forward toward these long‐term goals.

Abstract Large cell carcinoma (LCC) is an aggressive lung cancer subtype with poor prognosis and no targeted therapies. We previously reported that tumor-associated fibroblasts (TAFs) derived from LCC tumors exhibit premature senescence, and coculture of pulmonary fibroblasts with LCC cell lines selectively induces fibroblast senescence, which in turn drives LCC cell growth and invasion. Here we report that MMP1 is overexpressed specifically in LCC cell lines. Notably, silencing MMP1 expression in LCC cancer cell lines using shRNA revealed that MMP1 expression by LCC cells is necessary for induction of fibroblast senescence in coculture experiments with normal pulmonary fibroblasts, as revealed by the analysis of a panel of standard senescence markers, including β-galactosidase (SA-βgal) staining, permanent growth arrest and expression of senescence-associated secretory factors. Injecting control (shScr) or shMMP1 LCC cells into immunodeficient nude mice revealed that tumor growth, tumor take and cancer cell dissemination to the lung were reduced in shMMP1 H460 tumors compared to control tumors. We also observed fewer senescent fibroblasts in tumors from shMMP1 H460 cells using Sentragor staining, which allows identification of senescent cells in paraffin embedded tissues. Moreover, we found that recombinant active MMP1 in combination with TGF-β1 were sufficient to induce normal fibroblast senescence. In terms of the potential underlying mechanisms, treatment with the antioxidant n-acetyl cysteine (NAC) significantly attenuated the increase in SA-βgal+ fibroblasts elicited by co-stimulation with rMMP1 and TGF-β1, and its corresponding conditioned medium elicited a significantly lower growth and invasion in LCC cancer cells, revealing the oxidative stress implication in fibroblast senescence induction and associated pro-tumorigenic secretome. In summary, our results establish a new role for MMP1 in cancer and support that LCC cells elicit a tumor-supporting niche through the aberrant secretion of MMP1 and TGF-β1 to induce senescence in adjacent fibroblasts. Furthermore, we implicate oxidative stress in MMP1/TGF-β1-induced TAF senescence and support a novel therapeutic strategy in LCC based on targeting senescent TAFs. Citation Format: Marta Gabasa, Evette S. Radisky, Rafael Ikemori, Giulia Bertolini, Marselina Arshakyan, Alexandra Hockla, Paula Duch, Ornella Rondinone, Alejandro Llorente, Maria Maqueda, Alexandre Perera, Noemí Reguart, Luca Roz, Derek C. Radisky, Jordi Alcaraz. MMP1 and TGF-β1 cooperate to drive tumor progression in large cell carcinoma of the lung through fibroblast senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2515.

Serine protease inhibitors of the Kunitz‐bovine pancreatic trypsin inhibitor (BPTI) family, such as tissue factor pathway inhibitor‐1 Kunitz domain 1 ( KD1 TFPI1) and amyloid precursor protein inhibitor (APPI), are biological regulators of proteolysis and serve as important scaffolds for developing therapeutics. Mesotrypsin is a unique human protease with cancer‐promoting properties which rapidly cleaves and inactivates serine protease inhibitors; because of this, it is a challenging therapeutic target and an excellent model for developing therapeutic inhibitors with improved proteolytic resistance. We hypothesize that stabilization of the Kunitz inhibitor binding loop by disulfide bond anchoring to the scaffold can reduce proteolytic susceptibility and improve binding efficacy. Here we have engineered a new disulfide bond into both KD1 TFPI1 and an APPI variant previously engineered for mesotrypsin affinity. The new disulfide bond anchors the binding loop to the scaffold, resulting in reduced mesotrypsin proteolytic cleavage by 17‐fold and 3‐fold, respectively. To gain insight into the structural basis for improved proteolytic stability, we crystallized mutant KD1 TFPI1 and APPI with mesotrypsin and solved these novel structures. We then performed all‐atom molecular dynamics simulations using these mesotrypsin‐mutant inhibitor complexes compared to models lacking the engineered disulfide bond. Our simulation results support our hypothesis that the cause of decreased proteolysis rates is dampened conformational fluctuations in the binding loop and minimized motion at the enzyme‐inhibitor interface. We also performed simulations using unbound inhibitors to show that stabilization of the binding loop to states similar to the bound configuration correlates with stronger binding affinity. Our disulfide engineering approach is potentially a useful tool in biopharmaceutical development for mesotrypsin, other serine proteases, and possibly for other enzyme targets. Support or Funding Information NIH grant R01CA154387 to E.S.R., European Research Council grant 336041 to N.P., US‐Israel Binational Science Foundation grant BSF2015134 to E.S.R. and N.P. Xray diffraction data were measured at the Berkeley Center for Structural Biology at the Advanced Light Source and at the European Synchrotron Radiation Facility. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Abstract Ovarian cancer remains a challenging disease, with an average 5 year survival rate of 46%. At time of diagnosis, over 70% of patients have advanced metastatic disease, for which there are no effective treatment options. Of patients with metastatic disease, more than 70% of patients develop peritoneal carcinomatosis, making the peritoneum one of the main areas of dissemination. We have previously found that expression of the serine protease inhibitor Kazal type 1 (SPINK1) by ovarian cancer cells drives anoikis resistance, allowing the tumor cells to circumvent apoptosis protocols and facilitating tumor cell metastasis. For this new study we focused on ovarian clear cell carcinoma (OCCC), a histotype of ovarian cancer that has particularly poor prognosis if diagnosed in late stage. Using OCCC cell lines, we found that knockdown of SPINK1 reduces cell survival and stimulates apoptotic pathways in OCCC cells, when cultured under attachment-free conditions to mimic metastatic disease. We then identified Interleukin-6 (IL-6) as an upstream regulator of SPINK1 expression and a potential therapeutic target for metastatic OCCC: we showed that IL-6 silencing reduced SPINK1 mRNA expression and cell survival, while treatment with recombinant IL-6 increased SPINK1 expression and attachment-free survival. We developed a new OCCC mouse model and showed that knockdown of SPINK1 results in significant reduction of metastatic lesions, highlighting the impact of SPINK1 on OCCC metastasis. In ongoing studies we are testing intraperitoneal injections of FDA approved IL-6 inhibitors in a preclinical model to assess impact on abdominal dissemination of tumor cells. By targeting the IL-6 – SPINK1 signaling axis, we aim to develop therapeutic approaches to reduce the metastatic burden and peritoneal carcinomatosis of late stage disease, resulting in better prognosis and survival of patients with ovarian clear cell carcinoma. Citation Format: Christine Mehner, Erin Miller, Mathew A. Coban, Alexandra Hockla, Derek C. Radisky, Evette S. Radisky. Targeting ovarian cancer induced peritoneal carcinomatosis by inhibition of signaling axis between Interleukin-6 (IL-6) and serine protease inhibitor Kazal type 1 (SPINK1) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3758.

Abstract Patients diagnosed with ovarian cancer face an average 5 year survival rate of 46%; low survival is driven by early and ongoing intraperitoneal dissemination and metastasis of the tumor. Among the different histotypes of ovarian cancer, ovarian clear cell carcinoma (OCCC) has a particularly poor prognosis when diagnosed in late stage, as these tumors tend to be chemoresistant, leaving no effective therapeutic options. Intraperitoneal metastasis requires cells to become anoikis resistant, where apoptosis normally induced by loss of attachment is suppressed. We seek to better understand the mechanisms involved in anoikis resistance in OCCC, which in the future could lead to new therapeutic strategies for these patients. We have found that Serine Protease Inhibitor Kazal type 1 (SPINK1), an endogenous inhibitor of trypsin like serine proteases, is also an important regulator of anoikis resistance in some ovarian cancers. In this study, we aimed to dissect the role of SPINK1 specifically in OCCC, defining its contribution to anoikis resistance using cultured OCCC cell lines and elucidating its mechanism of action. We compared anoikis resistance of cells with endogenous SPINK1 expression versus cells in which SPINK1 was silenced using lentiviral shRNA constructs, when culturing cells on ultra-low attachment plates to mimic cell detachment from the extracellular matrix. We found that knockdown of SPINK1 reduced survival and stimulated apoptotic pathways in OCCC cells grown under detached conditions, implicating SPINK1 in anoikis resistance of OCCC cells. As a secreted protease inhibitor, SPINK1 may be expected to confer anoikis resistance by inhibiting an extracellular serine protease involved in triggering anoikis. Because many proteases represent potential targets of SPINK1, we have designed a strategy using Activity Based Protein Profiling (ABPP) to discover the targets of SPINK1 from among this large pool of candidates. In pilot studies, we have successfully used a novel activity-based probe to covalently label the active sites of serine proteases secreted from OCCC cells. In ongoing efforts, we are optimizing methods to identify labeled proteases with high sensitivity using tandem mass spectrometry. We will then identify those proteases regulated by SPINK1 through comparison of labeled proteomes from OCCC cells with and without SPINK1 treatment,, and candidate proteases will be further analyzed for their role in triggering anoikis. The identification of SPINK1 regulated proteases that are responsible for mediating anoikis in our models will give insight into important mechanisms whereby tumor cells acquire resistance to anoikis in OCCC. In-depth understanding of anoikis resistance, a critical component of ovarian cancer progression and metastasis, may lead to novel biomarkers of disease progression as well as important therapeutic targets. Citation Format: Christine Mehner, Mathew A. Coban, Alexandra Hockla, Derek C. Radisky, Evette S. Radisky. Serine protease inhibitor Kazal type 1 (SPINK1) drives anoikis resistance in ovarian clear cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 442.