Targeting matrix metalloproteinase-14 disrupts DNA repair and reduces viability in adrenocortical carcinoma
Changxian Shen1, Liudmila V. Popova1, Daniel M. Chopyk1, Lindsey Hartshorn1, Zhongguang Li2, Yaoling Shu’, Caleb Araujo”, Shivam Priya3, Varsha Thakur4, John E. Phay’, Barbra S. Miller, Barbara Bedogni4, Haichang Li2,6, Priya H. Dedhia1, 6,7, *
1Division of Surgical Oncology, The Ohio State University and Arthur G. James Comprehensive Cancer Center; Columbus, Ohio, USA.
2Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio, USA
3Department of Cancer Biology and Genetics, College of Medicine, The Ohio State University; Columbus, Ohio, USA.
4Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Sylvester Comprehensive Cancer Center, Miami, Florida, USA.
21 ‘Division of Surgical Oncology, University of Arizona College of Medicine, Tucson, AZ
Translational Therapeutics Program, The Ohio State University and Arthur G. James Comprehensive Cancer Center; Columbus, Ohio, USA.
7Center for Cancer Engineering, The Ohio State University; Columbus, Ohio, USA.
*Correspondence
Priya Dedhia, MD, PhD Division of Surgical Oncology
The Ohio State University and Arthur G. James Comprehensive Cancer Center Department of Surgery Division of Surgical Oncology N924 Doan Hall, 410 W. 10th Avenue Columbus, OH 43210
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bioRxiv preprint doi: https://doi.org/10.64898/2026.01.06.697992; this version posted January 7, 2026. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.
ABSTRACT
Background: Adrenocortical carcinoma (ACC) is a rare and aggressive endocrine cancer with limited treatment options and poor prognosis. Identifying novel therapeutic targets requires understanding the molecular drivers of ACC progression and establishing translational models for preclinical validation.
Results: Matrix metalloproteinase-14 (MMP-14) is the most highly expressed MMP in ACC, and high MMP-14 expression is associated with worse overall and disease-free survival. We demonstrate that MMP-14 is essential for ACC cell survival and serves an unexpected role in maintaining genome stability. Genetic silencing or pharmacologic inhibition of MMP-14 significantly reduced viability in both NCI-H295R cells and patient-derived tumor organoids (PTOs). MMP-14 knockdown induced CHK1 activation and S-phase checkpoint arrest. Mechanistically, MMP-14 translocates to the nucleus and binds to chromatin following DNA damage induced by ionizing radiation or cisplatin. Loss of MMP-14 resulted in accumulation of DNA double-strand breaks, as evidenced by increased yH2AX foci, and impaired non- homologous end joining (NHEJ)-mediated repair.
Conclusions: These findings reveal a novel nuclear function for MMP-14 in DNA repair and identify MMP-14 as a promising therapeutic target in ACC. Targeting MMP-14 may sensitize ACC tumors to DNA-damaging chemotherapy by impairing the repair of therapy-induced
lesions.
KEYWORDS: Adrenocortical carcinoma, matrix metalloproteinase-14, hemopexin domain,
DNA replication stress, DNA repair, patient derived tumor organoid
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BACKGROUND
Adrenocortical carcinoma (ACC) is a rare yet highly aggressive malignancy of the adrenal cortex. The majority of patients present with advanced stage disease, for which effective therapeutic options remain limited[1]. Poor survival outcomes underscore the urgent need for novel therapeutic targets. Current first-line treatment-mitotane combined with etoposide, doxorubicin, and cisplatin-extends progression-free survival by only 5 months with response rates of approximately 23%[2]. Targeted therapies-including IGF-1R and multikinase inhibitors- and immunotherapy have shown limited efficacy[3-6]. These therapeutic failures highlight the critical need to identify novel molecular targets in ACC.
Matrix metalloproteinases (MMPs) remodel the extracellular matrix (ECM) through proteolytic degradation of collagen and other ECM components, and can contribute to cancer progression through cell migration, invasion, and angiogenesis[7]. MMP-14, also known as membrane type matrix metalloproteinase 1 (MT-MMP1), is distinct from secreted MMPs in that it is anchored to the plasma membrane, enabling spatially restricted proteolysis at the cell surface[3]. This membrane localization allows MMP-14 to proteolytically activate other pro- MMPs and pro-proteins[7, 8], thereby altering the tumor microenvironment through release of embedded growth factors, cytokines, and their receptors into soluble, active signals that influence nearby cells[9-11].
MMP-14 promotes cell migration and invasion via multiple mechanisms. Specifically, MMP- 14 localizes to invadopodia-actin-rich membrane protrusions that mediate focused ECM degradation-where the protein directs matrix breakdown to enable cell migration[7]. Beyond ECM remodeling, MMP-14 can induce an epithelial-to-mesenchymal transition (EMT)-like phenotype and facilitate cell motility through cleavage of cell-cell adhesion molecules such as E- cadherin and indirect upregulation of mesenchymal markers (e.g., vimentin). Additionally,
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MMP-14 can be packaged into exosomes and transferred to neighboring cells, promoting migration and conferring chemoresistance in recipient cells[12]. MMP-14 also promotes angiogenesis through two mechanisms: 1) degradation of the vascular basement membrane to guide endothelial cell migration and sprouting, and 2) activation of pro-angiogenic factors such as (VEGF) and Heparin-Binding EGF-like Growth Factor (HB-EGF) through proteolytic cleavage[7, 13].
The diverse functions of MMP-14 are mediated by its distinct structural domains. MMP- 14 contains a catalytic domain, a hemopexin domain, a transmembrane domain, and a cytoplasmic tail[14]. Beyond proteolysis by the catalytic domain, the MMP-14 cytoplasmic tail 96 engages in non-proteolytic signaling, which can activate intracellular signaling pathways in tumor cells such as ERK/MAPK, PI3K/AKT, Src, and RhoA/Rac1[9, 15-18]. While canonical ECM-related functions are well established, emerging evidence suggests MMP-14 may have non-canonical roles in DNA replication[19]. However, the precise underlying molecular mechanisms and the biological context of these functions remain largely unknown[20].
Consistent with pro-tumorigenic functions, MMP-14 overexpression occurs frequently across multiple cancer types and correlates with tumor progression and poor prognosis[21-24]. In ACC specifically, MMP-14 expression is elevated, and patients with higher MMP-14 levels demonstrate worse survival outcomes[24]. Despite this clinical association, the therapeutic potential of MMP-14 inhibition in ACC has not been evaluated.
Given the elevated expression of MMP-14 in ACC and its poorly understood non-canonical functions, we investigated the therapeutic potential of targeting MMP-14 in ACC. Using genetic silencing and pharmacologic inhibition of the hemopexin domain, we demonstrate that MMP-14 targeting reduces viability in both NCI-H295R ACC cells and patient-derived organoids (PTOs).
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Further mechanistic investigation revealed that MMP-14 is a critical regulator of cell proliferation and DNA repair in ACC.
RESULTS
Elevated MMP-14 expression correlates with poor survival in ACC
We profiled the expression of 24 human MMP genes in ACC using the TCGA bulk RNA sequencing dataset. Of these, MMP14 was the highest expressed MMP in ACC (Fig. 1A). Furthermore, We found that MMP14 expression increased with tumor stage (p=0.02, Fig. 1B), in contrast to most other cancers (Fig. S1), prompting further investigation into its functional role in ACC. Importantly, high MMP-14 expression was significantly associated with worse overall survival (OS) (p = 0.0065, HR 5.01, 9% CI 1.56-16.14) and disease-free survival (DFS) (p=0.00035, HR 5.78, 95% CI 2.23-15.01) of ACC patients (Figs. 1C - 1D). These findings demonstrate that MMP-14 is associated with ACC progression and represents a negative prognostic factor in ACC.
Inhibition of MMP-14 reduces ACC cell viability
NCI-H295R cell line
Because MMP14 is highly expressed in ACC and associated with poor outcomes, we investigated the functional role of MMP-14 using the ACC cell line, NCI-H295R. We first examined MMP-14 localization by immunofluorescence analysis, and revealed MMP-14 expression throughout cellular compartments, including strong perinuclear staining (Figs. 2A and S2A). This localization pattern suggests potential functions of MMP-14 beyond its canonical extracellular membrane-associated activities.
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We assessed MMP-14’s role in ACC cell survival using lentiviral short hairpin RNA (shRNA) to silence MMP-14 expression. Genetic knockdown of MMP-14 significantly reduced NCI-H295R cell viability (Fig. 2B), demonstrating that MMP-14 is essential for ACC cell survival. To examine its noncatalytic contributions[16, 25, 26] to this effect, we treated cells with NSC405020, a small-molecule inhibitor that specifically targets the hemopexin domain of MMP- 14 without affecting catalytic activity[27]. Unlike traditional MMP inhibitors that target the catalytic domain, NSC405020 acts allosterically by binding the hemopexin domain and preventing homodimerization of MMP-14[27]. NSC405020 treatment reduced NCI-H295R cell viability in a dose-dependent manner (Fig. 2C). Together, these findings demonstrate that MMP- 14 is essential for ACC cell growth and survival, in part due to its noncatalytic functions.
ACC patient-derived organoids (PTOs)
Patient-derived tumor organoids (PTOs) are 3D cultures that recapitulate key aspects of tumor biology in vitro and have emerged as powerful tools for preclinical therapeutic screening[28-31]. We previously established PTOs from ACC tumors that maintain adrenal cortex characteristics, including expression of the adrenal marker, SF-1 and steroid secretion[32]. To validate the role of MMP-14 in this physiologically relevant model system, we examined MMP-14 expression in three ACC PTO cultures (OSU1, OSU2, and OSU3). Immunofluorescence staining revealed MMP-14 expression across all three ACC PTOs, with relatively higher levels in OSU2 (Figs. 3 and S2B). We next tested whether MMP-14 inhibition affects PTO viability. Genetic silencing of MMP-14 significantly reduced ACC PTO viability (Fig. 4A), consistent with our observations in NCI-H295R cells. Similarly, NSC405020 treatment inhibited ACC PTO viability (Fig. 4B). These findings in both cell line and patient-
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derived organoid models demonstrate that MMP-14 represents a promising therapeutic target in
ACC.
MMP-14 downregulation leads to DNA replication checkpoint activation
To elucidate the mechanism by which MMP-14 maintains ACC cell viability, we investigated DNA damage response pathways. Prior studies in breast cancer cells demonstrated that MMP-14 modulates DNA damage response through stimulation of integrinß1, which activates ERK and AKT signaling cascades[19, 33]. However, MMP-14 silencing in NCI-H295R cells did not alter AKT or ERK1/2 phosphorylation. Instead, we observed robust activation of CHK1 (Fig. 5A), a key kinase in the replication stress checkpoint pathway. CHK1 activation triggers downstream signaling that arrests cells in S-phase, providing time for DNA repair before replication continues[30]. To determine whether MMP-14 loss activates this checkpoint, we performed cell cycle analysis. Flow cytometry revealed that MMP-14 knockdown resulted in marked S-phase arrest in NCI-H295R cells (Fig. 5B), consistent with CHK1-mediated checkpoint activation.
DNA damaging agents induce MMP-14 nuclear accumulation and chromatin binding
Another principal role of DNA damage response is to promote DNA damage repair. To investigate whether MMP-14 is functionally lined to DNA damage repair in ACC, we analyzed the TCGA dataset and found that higher MMP-14 expression correlated with elevated hallmark DNA repair signatures (Fig. 6A). Because nuclear translocation following DNA damage indicates recruitment to DNA repair pathways, we examined MMP-14 subcellular localization in response to DNA damage. We treated NCI-H295R cells with 4 Gy ionizing radiation (IR) and
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performed immunofluorescence analysis of MMP-14 at 1, 6, and 24 hours post-treatment. While MMP-14 was distributed throughout all cellular compartments under basal conditions, IR progressively induced MMP-14 nuclear accumulation over time (Fig. 6B and Fig. S3). Treatment with the DNA-damaging agent cisplatin produced similar results. Cisplatin (10 uM, 2 hours) increased nuclear accumulation of MMP-14 in NCI-H295R cells (Fig. 6C and Fig. S4). To further characterize MMP-14 redistribution following DNA damage, we performed subcellular fractionation after cisplatin treatment (10 µM, 2 and 6 hours), separating cytoplasmic, nuclear soluble, and chromatin-bound fractions. Cisplatin induced chromatin binding of the DNA damage response proteins TOPBP1 and RAD51, as expected. Notably, cisplatin also induced MMP-14 binding to chromatin (Fig. 6D), demonstrating that MMP-14 redistributes to chromatin in response to DNA damage.
MMP-14 inhibition induces DNA damage and impairs DNA double strand break (DSBs) repair
The nuclear and chromatin localization of MMP-14 following DNA damage suggested a functional role in DNA repair. Consistent with this hypothesis, MMP-14 knockdown in NCI- H295R cells induced DNA damage, as demonstrated by increased yH2AX levels (Fig. 7A). Pharmacologic inhibition with NSC405020 produced the same effect (Fig. 7B). Similarly, both genetic and pharmacologic MMP-14 inhibition increased yH2AX levels in ACC PTOs (Fig. 7C- 7D). The accumulation of yH2AX following MMP-14 inhibition indicates impaired repair of DNA double-strand breaks (DSBs). To identify which DSB repair pathway requires MMP-14 function, we employed homologous recombination (HR) and non-homologous end joining (NHEJ) reporter assays[34]. MMP-14 knockdown did not affect HR capacity, whereas
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NSC405020 treatment significantly reduced HR activity (Fig. 7E). In contrast, both genetic and pharmacologic MMP-14 inhibition substantially impaired NHEJ-mediated DSB repair (Fig. 7F).
These data demonstrate that MMP-14 contributes to DSB repair primarily through the NHEJ pathway.
Collectively, these data demonstrate that MMP-14 maintains genome integrity during DNA replication through dual mechanisms: direct nuclear/chromatin association following DNA damage and CHK1-dependent checkpoint signaling, a pathway distinct from the integrinß1/AKT/ERK signaling reported in breast cancer.
DISCUSSION
Our study revealed that MMP-14 function extends beyond ECM remodeling to include non- canonical functions roles that contribute to ACC cell survival. In TCGA analysis, high expression of MMP-14, the most highly expressed MMP in ACC, was associated with worse overall and disease-free survival. Consistent with this clinical association, genetic and pharmacologic inhibition of MMP-14 reduced viability in NCI-H295R cells and PTOs. To elucidate the underlying mechanism, we investigated MMP-14’s intracellular functions and uncovered an unexpected role in genome maintenance: MMP-14 translocates to the nucleus following DNA damage and participates in NHEJ-mediated DNA repair.
The mechanism by which MMP-14 supports NHEJ remains to be elucidated. MMP-14 may interact with core NHEJ machinery, either through direct protein-protein interactions or by modulating chromatin accessibility at break sites. Alternatively, MMP-14 may regulate the activity or localization of NHEJ factors through proteolysis. Our data suggest that MMP-14 coordinates multiple aspects of genome maintenance through distinct structural elements. MMP-
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14 knockdown induced robust CHK1 activation and S-phase checkpoint arrest. This response likely reflects the accumulation of unrepaired DNA damage and stalled replication forks when NHEJ is compromised. Interestingly, NSC405020 treatment, which targets the hemopexin domain without affecting catalytic activity, induced DNA damage but did not activate the CHK1/S-phase checkpoint (Fig. S5). These differential effects suggest that the ATR-CHK1 replication stress response is regulated by the MMP-14 catalytic domain, while NHEJ-mediated repair is mediated by the hemopexin domain.
Our findings reveal an intriguing paradox: while MMP-14 appears to maintain genome stability in ACC cells, prior studies in other cancer models demonstrate that MMP-14 promotes genomic instability[35]. MMP-14 localizes to centrosomes and cleaves pericentrin, a protein essential for mitotic spindle formation[36]. In mammary epithelial cells, MMP-14 overexpression disrupts centrosome function, resulting in abnormal spindle formation, aneuploidy, and chromosome instability[37], hallmarks of malignant transformation. Wali et al. further demonstrated that MMP-14 cleaves centrosomal BRCA2, regulating its removal from centrosomes during metaphase and disrupting centrosome duplication[38]. These destabilizing effects contrast with our observation that MMP-14 inhibition in ACC cells induces DNA damage, CHK1 activation, and impaired NHEJ repair.
Several explanations may reconcile this apparent contradiction. MMP-14 may exert opposing effects at different subcellular locations, with the dominant effect depending on cellular context. At centrosomes, MMP-14 cleavage of pericentrin and BRCA2 disrupts mitotic fidelity and promotes chromosomal instability, while in the nucleus, MMP-14 supports NHEJ-mediated repair. Cancer cells with high replication stress may depend on MMP-14’s nuclear repair function to tolerate ongoing DNA damage. In this context, loss of MMP-14 may trigger catastrophic DNA
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damage accumulation and checkpoint arrest. The tissue-specific nature of MMP-14 function is further evident when comparing ACC to breast cancer. In breast cancer cells, MMP-14 modulates DNA damage response through extracellular integrinß1 signaling[19], whereas MMP- 14 knockdown in ACC cells did not affect integrinß1 downstream targets, as evidenced by unchanged AKT and ERK phosphorylation. The selective pressure in ACC-an aggressive cancer with limited treatment options-may have driven particular reliance on the direct DNA repair functions of MMP-14.
These considerations suggest that MMP-14 occupies a complex role in cancer biology, acting as both a driver of genomic instability during early transformation and a support for genome maintenance in established tumors. The observation that both genetic and pharmacologic MMP-14 inhibition reduces ACC PTO viability suggests therapeutic potential. NSC405020, which targets the hemopexin domain, impairs NHEJ and reduces cell viability, offering a more selective therapeutic approach than broad-spectrum MMP inhibitors. Importantly, targeting MMP-14 could sensitize ACC cells to DNA-damaging agents like cisplatin and ionizing radiation by impairing the repair of therapy-induced DNA damage.
Elucidating the molecular mechanisms by which MMP-14 contributes to NHEJ will be critical for developing targeted therapeutic strategies. Equally important will be understanding the relative contributions of centrosomal versus nuclear MMP-14 dysfunction to tumor cell lethality, which will inform whether inhibitors should target specific subcellular pools or global MMP-14 activity. Beyond these mechanistic insights, determining whether ACC’s dependency on MMP-14 represents a general feature of aggressive cancers or a tumor type-specific vulnerability will be essential for predicting which patients are most likely to benefit from MMP- 14-targeted therapies.
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This study establishes MMP-14 as a critical regulator of genome maintenance in ACC, extending well beyond its canonical role in ECM remodeling. Our finding that ACC cells depend on MMP14 for survival provides a mechanistic basis for the association between high MMP-14 expression and poor clinical outcomes. Targeting this novel dependency represents a promising therapeutic strategy that could sensitize ACC tumors to DNA-damaging chemotherapy and improve outcomes in this aggressive malignancy.
MATERIALS AND METHODS
NCI-H295R cell culture, chemicals and antibodies
The NCI-H295R ACC cell line was originally purchased from American Type Culture Collection (ATCC, Manassas, VA). Cells were maintained in pheno-red free DMEM/F12 with L-glutamine and 15 mM HEPES (Thermo Fisher Scientific, Waltham, MA) supplemented with 2.5% FBS (VWR, Radnor, PA), 1x ITS Premix Universal Cell Culture Supplement (Corning), and 1% penicillin-streptomycin (Millipore-Sigma, Burlington, MA). Cells were confirmed to be free of mycoplasma prior to experimental procedures. All cells utilized for experiments were under 25 passages in culture. MMP-14 inhibitor NSC405020 (cat.444295) was obtained from EMD Millipore (Burlington, MA). Anti-pERK1/2 (Thr202/Tyr204), ERK1/2, pAKT-S473, AKT, pCHK1-S345, CHK1, phospho-H2AX (Ser139), and GAPDH antibodies were purchased from Cell Signaling Technology (Danvers, MA).
ACC tumor collection and PTO generation
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ACC PTOs were generated as previously described[39]. Briefly, patients undergoing adrenalectomy for histology-confirmed ACC were consented for collection of specimens and clinical information using a protocol approved by The Ohio State University Institutional review board. A diagnosis of Long-lived ACC PTO cultures were generated from tissue specimens from three ACC patients with biochemical evidence of autonomous cortisol production (OSU1) or overt hypercortisolism (OSU2, OSU3). All PTO cultures were maintained in DMEM/F12 supplemented with 10 nM HEPES (Cytiva, Marlborough, MA), 1x Glutamax, 1x B27 Supplement (both Thermo Fisher Scientific), 1.25 mM n-acetylcysteine, 10 mM nicotinamide, 10 nM [Leu15]-Gastrin, 0.5 mM A-83-01, 1 mM PGE2 (all Millipore-Sigma), 0.5 mg/mL R- Spondin-1, 50 ng/ml EGF (both R&D Systems, Minneapolis, MN), 0.1 mg/ml FGF-10, 30 ng/ml recombinant murine Wnt3a, and 0.1 mg/mL recombinant human Noggin (all Thermo Fisher Scientific).
Immunofluorescence
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NCI-H295R cells were seeded on poly-L-lysine coated glass coverslips (Electron Microscopy Sciences, Hatfield, PA), fixed, and stained using the following antibodies: rabbit anti-human MMP-14 at 1:200 (Abcam, Cambridge, UK; ab51074), phalloidin-Alexa Fluor® 594 at 1:1000 (Abcam ab176757), and goat anti-rabbit Alexa Fluor® 488 at 1:1000 (Abcam ab150081) or donkey anti-rabbit Alexa Fluor® 647 at 1:1000 (Abcam ab150063). NCI-H295R cells incubated in the absence of primary antibodies were used as negative controls. ACC PTOs were processed and stained for immunofluorescence microscopy using the following antibodies: rabbit anti- human MMP-14 at 1:200 (Abcam ab51074), goat anti-rabbit Alexa Fluor® 488 at 1:1000 (Abcam ab150081), and phalloidin-Alexa Fluor® 647 1:400 (Thermo Fisher Scientific
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A2287). Negative control samples were stained with rabbit IgG isotype control at an equivalent concentration (Abcam ab172730). All images were obtained by a Nikon Ti-2 Eclipse Confocal Fluorescence Microscope (Nikon, Tokyo, Japan).
HR and NHEJ reporter assays
U2OS cells stably expressing fluorescent NHEJ (EJ5-GFP) and HR (DR-GFP) reporter assays were used as described previously[40, 41], and cultured in DMEM medium without sodium pyruvate (10-017-CV, Corning) supplemented with 10% FBS and 1% penicillin/streptomycin. Briefly, cells were transiently transfected with plasmids expressing I-SceI restriction enzyme for 24 hrs, followed by transduction with lentivirus expressing shCtrl or shMMP-14, or treated with different concentrations of NSC405020. After 48 hrs, GFP-positive cells were measured on a Cytek NL-3000 (CYTEK) flow cytometry.
Cytotoxicity assays
NCI-H295R cytotoxicity assays
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NCI-H295R cells were seeded at 1x104 cells per well in a 96-well plate and allowed to adhere for 24 hours. Media was then replaced with fresh media containing specified drugs or equivalent DMSO vehicle control (Corning). Cell viability was assessed by water-soluble tertozolium-8 (WST-8) based Cell Counting Kit-8 assay (Dojindo, Mashiki, Kumamoto, Japan) per the manufacturer’s instructions at 4 days. Absorbance readings were conducted using a SpectraMax® iD5 plate reader and SoftMax® Pro Software (Molecular Devices, San Jose, CA). All assays were performed in biologic triplicates with 3-5 replicates per condition. The following drugs were used at concentrations ranging from 1 to 500000 nM: NSC405020. Mean viability of each drug
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treatment group was measured as a percentage of baseline DMSO vehicle treated cell absorbance. The half-maximal inhibitory concentration (IC50) for each drug was calculated by performing a four-parameter nonlinear best fit analysis of log concentration by percent viability on GraphPad Prism® 7.0a software (GraphPad Software, La Jolla, CA). Differences in viability across each concentration of a particular drug were compared using one-factor ANOVA with post-hoc multiple comparison analysis by Dunnett’s test in GraphPad Prism® 7.0a software (GraphPad Software). All data are reported as means ± standard error of the mean (SEM) with statistical significance set as p <0.05.
ACC PTO cytotoxicity assays
Single-cell suspensions were generated by incubating well-established ACC organoid domes with 1 mg/mL dispase II (Thermo Fisher Scientific). 1.5x104 cells per well were seeded in 96- well plates in 80% Matrigel® as described above. Three days after seeding, media was replaced with media containing specified drugs or DMSO vehicle control. Organoid viability was assessed at 4 days of treatment by WST-8 assay as described above using the cell free Matrigel® domes for background absorbance readings. Differences in viability across each concentration of a drug were compared using one-factor ANOVA with post-hoc multiple comparison analysis by Dunnett’s test in GraphPad Prism® 7.0a software (GraphPad Software, La Jolla, CA). Each drug was evaluated in 5 replicates per condition across three or more independent experimental replicates.
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Gene silencing by lentivirus delivered shRNA
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The plasmids of shRNAs of non-target control (SHC016VN) and targeting human MMP-14 (TRCN0000050855) were purchased from Sigma-Aldrich. Lentiviral particles for shRNA were produced in HEK293T cells transfected with shRNA plasmids, packaging (psPAX2) and envelope (pMD2G) plasmids (both from Addgene) using Lipofectamine 3000 (Invitrogen) according to manufacturer’s protocol. The lentiviral supernatants were harvested twice at 24- and 48-hour time points after transfection and passed through a membrane filter with 0.45 um pore size. NCI-H295R cells were transduced with the lentiviral particles in the presence of 8 ug/mL polybrene (Sigma-Aldrich, TR-1003-G). ACC PTOs were transduced in a similar manner, with minor modifications: ACC PTOs were dissociated with dispase prior to lentiviral transduction, and were plated in Matrigel 6 hours after transduction.
Cell lysates preparation and immunoblotting
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For NCI-H295R cells, cells cultured in plates were washed twice with ice-cold PBS, and lysed in RIPA lysis buffer (ThermoFisher) supplemented with 1x protease inhibitor (cOmplete, Roche) and phosphatase inhibitors (PhosSTOP, Roche) for 15 min on ice. Organoids cultured in Matrigel were mixed with 1 ug/mL dispase for 2 hours to completely resolve the Matrigel, followed by washing twice with ice-cold PBS, and lysed in RIPA lysis buffer supplemented with 1x protease inhibitor for 30 min on ice. The cell lysate was centrifuged at 150,000 rpm for 15 min at 4℃ and the supernatant was collected. Protein quantification was determined with Dc protein assay kit (Bio-Rad, Hercules, CA). For immunoblotting, equal amounts of protein were resolved by SDS/PAGE (Bio-Rad) and transferred onto nitrocellulose membranes (cat. 1620115, Bio-Rad). Primary antibodies at a dilution of 1:1,000-5,000 were allowed to bind overnight at 4℃. After washing in Tris-buffered saline with 0.1% Tween 20 (TBS-Tween), membranes were
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incubated with a secondary antibody (1:5,000-10,000) conjugated to a near-infrared (NIR) fluorophore that binds to the primary antibody at room temperature for 1 hour in the dark. Membranes were washed with TBS-Tween and scanned using an Azure imaging system (Azure Biosystem) to detect the fluorescence signal. Secondary antibodies of donkey anti-rabbit IRDye 680RD (926-68073), donkey anti-rabbit IRDye 800CW (926-32212), and donkey anti-mouse IRDye 680RD (926-68072) were purchased from LI-COR.
Isolation of subcellular fractions and chromatin-bound proteins
To isolate cytoplasmic, soluble nuclear, and chromatin-binding proteins, the cells were lysed in 500 ul ice-cold Buffer A [50 mM HEPES-KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA (pH 8.0), 10% glycerol, 0.5% Nonidet P-40, 0.25% Triton X-100, 1 mM DTT, 1×protease inhibitor cocktail]. After centrifugation (700×g, 10 min), the supernatant was collected as the cytoplasm fraction and pellets were washed with Buffer A and resuspended in ice-cold Buffer B [10 mM Tris-HC1 (pH 8.0), 200 mM NaCl, 1mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0)], 1×protease inhibitor cocktail]. After extraction and centrifugation (20,000×g, 10 min), the soluble nuclear extract (NE) was collected. The pellet was washed with Buffer B and resuspended in Buffer C [500 mM Tris-HCl (pH 6.8), 500 mM NaCl, 1×protease inhibitor cocktail]. The suspension was sonicated for 2×10s and centrifuged (20,000×g, 10 min), and the supernatant was collected for chromatin binding fraction.
Flow cytometric analysis for cell cycle phases
Around 0.2-5 x 106 NCI-H295R cells were seeded per well of a 12-well plate and incubated for 24 hours. Cells were treated with the specified compounds for the specified duration. All sample
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wells were harvested by trypsinization. Cell pellets were washed with PBS and were then then fixed with 70% ethanol at 4℃ overnight. All samples were then incubated with 10 µg/mL propidium iodide and 200 µg/mL RNAse A for 1-2 hours at 37 °℃. Samples were then stored at 4 ℃ until analyzed on a Cytek NL-3000 flow cytometry within 1-2 days.
TCGA data analysis
TCGA RNA sequencing dataset containing log2(TPM+1) normalized data was downloaded from the UCSC Treehouse Tumor Compendium v11 Public PolyA cohort. The dataset was filtered to include adult ACC patients only (age at diagnosis >= 18 years old); as a result, gene expression data for 74 ACC patients were analyzed. ggplot2 v3.5 for R v4.1 [42] was used to compare log2(TPM+1) expression values of all human MMP genes in ACC. ANOVA trend analysis was performed in R v.4.1. For the pan-cancer ANOVA trend analysis, 21 cancer types were included. Hematological cancers, glioma, glioblastoma, and cancers with fewer than 10 patients were excluded from analysis. To perform survival analysis, packages survival v. 3.2 and survminer v. 0.4 were used[43] (https://rdrr.io/cran/survminer/). For the overall and the disease-free survival analyses, ACC patients with MMP14 expression in the upper quartile (n = 19) were considered to have high MMP14 expression, whereas ACC patients with MMP14 expression in the lower quartile (n = 19) were considered to have low MMP14 expression. The Hallmark DNA repair signature was downloaded from the Molecular Signatures Database (MSigDB)[44].
Statistical analysis
Graphs for cytotoxicity assays were constructed using GraphPad Prism (GraphPad Software, San Diego, CA). All data are presented as mean ± SEM. Log-rank tests were used to calculate P- values in Kaplan-Meier analysis. Wilcoxon mean rank-sum tests were used for two-group
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comparison of continuous variables. Statistical significance was determined by unpaired two- tailed t-test or two-way ANOVAs. P< 0.05 was considered statistically significant.
Acknowledgements: We acknowledge resources from the Campus Microscopy and Imaging Facility, Biospecimen Services Shared Resource, Flow Cytometry Shared Resource, Immune Monitoring and Discovery Platform, and the Microscopy Shared Resource. These shared resources are supported in part by the Cancer Center Support Grant P30 CA016058, National Cancer Institute, Bethesda, MD. We thank Dr. Jeremy Stark (City of Hope, Duarte, CA) for providing NHEJ (EJ5-GFP) and HR (DR-GFP) reporter system. We also acknowledge Matthew Ringel, MD, Wayne Miles, PhD, Abby Green, MD, PhD, Matthew Summers, PhD, and Monica Venere, PhD for insightful discussions.
Funding: This project was supported by Ohio Cancer Research, The Victory Over Cancer Foundation, DOD W81XWH-22-PRCRP-CDA-SO, NIH R01CA279997, and NIH R21CA277083 (PHD). DMC is supported by training grant PF-23-1036284-01-CDP via the American Cancer Society (https://doi.org/10.53354/ACS.PF-23-1036284-01-CDP.pc.gr.168151). LVP is supported by the Pelotonia Scholars Program. Any opinions, findings, and conclusions expressed in this material are those of the author(s) and do not necessarily reflect those of the NIH, Pelotonia Scholars Program, OSU, or the American Cancer Society.
Author contributions: Conceptualization: P.H.D. Methodology: C.S. L.V.P. and P.H.D. Investigation: C.S., D.M.C, L.V.P., Y.S., C.A., Z.L., S.P., V.T., J.E.P, B.S.M., B.B. and H.L. Specimen Acquisition: P.H.D., J.E.P., and B.S.M. Formal analysis: C.S., L.V.P. and D.M.C.
430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452
bioRxiv preprint doi: https://doi.org/10.64898/2026.01.06.697992; this version posted January 7, 2026. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.
Funding acquisition: P.H.D. Project administration, supervision, and validation: P.H.D. Writing- original draft: C.S., L.V.P., D.M.C. Writing-review and editing: C.S., L.V.P., and P.H.D.
Competing interests: The authors declare no competing interests.
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617 FIGURES
A
12
B
10
10
Normalized Expression
8
Normalized Expression
8
6
6
4
$
2
2
p=0.02
0
0
MMP3
MMP7
MMP8
MMP13
MMP20
MMP26
MMP27
MMP12
MMP21
MMP10
MMP25
MMP24
MMP1
MMP16
MMP23A
MMP9
MMP19
MMP17
MMP23B
MMP28
MMP11
MMP2
MMP15
MMP14
Stage
Stage II
Stage III
Stage IV
AJCC Tumor Stage
Gene Name
C
D
1.00
Disease-Free Survival Prbability
1.00
Overall Survival Prbability
0.75
MMP14-low
MMP14-low
0.75
0.50
MMP14-high
0.50
0.25
MMP14-high
0.25
p=0.0065
p=0.00035
0.00
0
1000
2000
Time (Days)
3000
4000
0.00
0
1000
2000
Time (Days)
3000
4000
618 619 620 621 622 623 624 625 626
DAPI
MMP-14
Phalloidin
Merged
A
B
150
H295R
C
NSC405020
NSC405020
shMMP14
Relative cell viability
175
150-
150
IC50 = 66 38 LLM
100
% viability
125
shCtrl
% viability
100
100
—
—
75-
50
50-
50
MMP-14
25
0
GAPDH
DMSO
1 µM
5 AM
10 MM
25 MM
50 AM
100 MM
250 UM
500 KM
-1
0
1
2
3
0
Concentration (log [.M)
shCtrl shMMP14
627 628 629 630 631 632 633 634 635 636 637 638 639 640
DAPI
MMP-14
PhalloldIn
Merged
OSU1
60 um
60 pm
60 µm
60 pm
OSU2
50 um
50 pm
50 pm
50 pm
OSU3
50 um
50 pm
50 um
50 pm
Figure 3. MMP-14 is expressed in ACC PTOs. Immunofluorescence demonstrating nuclei (DAPI, blue), MMP-14 (green), and actin (Phalloidin, red) in ACC PTO OSU1, OSU2 and OSU3.
641 642 643 644 645 646 647 648 649 650
651
A
150
B
OSU1
OSU1
200
150
shCtrl
shMMP14
Relative cell viability
175
% viability
IC
% viability
50
= 154.3 µM
100
150
125
100-
100
75
50-
MVP14
50
50
25
GÅPDH
0
DMSO
250 UM
500 μΜ
1
0
1
2
3
OSU1
I’M S
16M
25M
5PM
25 UM
0
100 g
Concentration (log uM)
shCtrl stMMP14
150
OSU2
OSU2
Relative cell viability
150-
200
shCtrl
shMMP14
% viability
175
% viability
IC50 = 129.6 AM
100
150
125
100
100
75
50
MVP14
50
50
25
GÅPDH
0
OSU2
DMSO
UM
5 UM
10 UN
25 UM
105MM
100 1
250 UM
500 PM
-1
0
1
2
3
0
Concentration (log uM)
shCtrl shMMP14
150
OSL3
OSU3
Relative cell viability
200
150
shMMP14
**
% viability
175
IC50 = 147.41 µM
100
150
125
100
I
shCtrl
% viability
100
75
50-
MMP14
50
50
25
GAPDH
0
OSU3
DMSO
7 0
UM
5 UM
10 LA
25 m
UM
100M
250 UM
500 µM
-1
0
1
2
3
0
Concentration (log uM)
shCtrl shMMP14
652 653 654 655 656 657 658 659 660 661 662
663
A
shCtrl
shMMP14
B
shCtrl 48 hr
shCt-1 72 hr
shCtrl 96 hr
G1
S
G2M
G1
S G2M
G1
S G2M
G1: 65.37%
G1: 59.13%
Counts
S: 14.99%
pAKT-S473
Counts
G1: 62.25% 15 S: 15.29% G2M: 22.06% DNA content
Counts
LA DNA Content
S: 11.20%
G2M: 18.87%
G2M: 29.16%
pERK1/2
DNA content
pCHK1-S345
shMMP14 48 hr
shMMP14 72 hr
shMMP14 96 hr
G1
S
G2M
G1
S G2M
G1
S G2M
CHK1
G1: 58.99%
Counts
S: 25.54%
AF Counts DNA Content
G1: 57.77%
WE Counts DNA content
G1: 65.54%
S: 28.95%
S: 22.89%
GAPDH
G2M: 11.81%
G2M: 11.08%
G2M: 5.39%
DNA content
664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679
A
B
MMP-14
YH2AX
DAPI
Merged
Hallmark DNA Repair Signature
Untreated
1000
R=0.63, p= 2e-09
750
IR 1hr
500
250
IR 6hr
0
0
2.5
5
7.5
10
MMP14
IR 24hr
C
D
Cytoplasm
Nuclear soluble
Chromatin binding
MMP-14
YH2AX
DAPI
Merged
DMSO
DMSO
CDDP 2hr
CDDP 6hr
DMSO
CDDP 2hr
CDDP 6hr
DMSO
CDDP 2hr
CDDP 6hr
MMP -* 4
TOPBP1
CDDP
RAD5*
H3
Raptor
680 681 682 683 684 685 686 687 688 689 690 691
A
B
C
D
shMMP14
shCtrl
shMMP14
shCtrl
DMSO
NSC
DMSO
MMP14
NSC
MMP14
YH2AX
7H2AX
H2AX
YHZAX
GAPDH
GAPDH
GAPDH
GAPDH
H295R
H295R
OSU1
OSU1
E
Percentage of GFP positive cells
HR-GFP *
HR-GFP
F
0.8
Percentage of GFP positive cells
Percentage of GFP positive cells
NHEJ-GFP **
NHEJ-GFP *
1.5
ns
4
Percentage of GFP positive cells
5
ns
ns
0.6
3
4
1.0
0.4
2
3
0.2
0.5
2
1
1
T
0.0
0.0
D
shCtrl shMMP14
DMSO
NSC 50 UM
shCtrl shMMP14
0
NSC 100 UM
DMSO
NSC 50 UM
NSC 100 UM
692 693 694 695 696 697 698 699 700 701 702 703
bioRxiv preprint doi: https://doi.org/10.64898/2026.01.06.697992; this version posted January 7, 2026. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.
transduction of control or MMP14 shRNA for 48 hours (left panel) or treated with vehicle (DMSO) or MMP-14 inhibitor NSC405020 for 48 hours (right panel). F. NHEJ reporter assays of U2OS cells (stable reporter cell lines) after transduction of control or MMP14 shRNA for 48 hours (left panel) or treated with vehicle (DMSO) or MMP-14 inhibitor NSC405020 for 48 hours (right panel). ns - not significant, * - p<0.05, ** - p<0.01.
704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726
A
B
| MMP14 Median Expression | ||||||
|---|---|---|---|---|---|---|
| Disease | Stage I | Stage II | Stage III | Stage IV | Stage I - IV+ | p-value |
| THCA | 5.45 | 5.19 | 5.66 | 5.83 | 0.38 | 0.008 |
| KICH | 4.33 | 3.81 | 3.83 | 3.74 | -0.59 | 0.012 |
| TGCT | 5.15 | 5.7 | 6.44 | NA | 1.29 | 0.015 |
| ACC | 3.22 | 3.71 | 1.87 | 5.19 | 1.97 | 0.019 |
| KIRC | 6.39 | 6.19 | 6.28 | 6.43 | 0.04 | 0.023 |
| READ | 5.92 | 6.24 | 6.51 | 6.46 | N/A | 0.09 |
| COAD | 6.09 | 6.22 | 6.86 | 6.22 | N/A | 0.109 |
| CHOL | 6.99 | 6.96 | 3.75 | 7.15 | N/A | 0.149 |
| SKCM | 7.93 | 8.17 | 8.01 | 8.4 | N/A | 0.21 |
| STAD | 6.47 | 6.78 | 6.66 | 6.81 | N/A | 0.222 |
| PAAD | 6.97 | 7.89 | 7.76 | 8.21 | N/A | 0.338 |
| HNSC | 7.76 | 7.65 | 7.34 | 7.53 | N/A | 0.478 |
| ESCA | 6.13 | 6.89 | 7.12 | 6.77 | N/A | 0.548 |
| LUSC | 7.04 | 7.19 | 6.92 | 7.85 | N/A | 0.553 |
| BRCA | 7.33 | 7.04 | 7.21 | 7.38 | N/A | 0.565 |
| LIHC | 3.75 | 4.04 | 4.13 | 4.25 | N/A | 0.624 |
| UVM | NA | 7.27 | 7.52 | 73 | N/A | 0.626 |
| BLCA | 6.96 | 6.71 | 7.07 | 7.06 | N/A | 0.63 |
| KIRP | 6.35 | 6.78 | 6.74 | 6.32 | N/A | 0.633 |
| MESO | 8.33 | 7.51 | 7.02 | 8.44 | N/A | C.GG |
| LUAD | 6.34 | 6.43 | 6.65 | 6.23 | N/A | 0.888 |
| *For TGCT, Stage I - III | ||||||
10
..
MMP14 Expression
8
A.JCC Pathologie Tumor Stage
6
Stage
Stage I
Stage II
4
Stage IV
2
KICH
KIAC
THCA
TGCT
ACC
Disease
727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742
A
DAPI
Goat anti-rabbit 488
PhalloldIn
Merged
H295R
50 um
50 μη
50 um
50 um
B
Rabbit Isotype lgG/Goat anti-Rabbit 488
DAPI
Phalloidin
Merged
OSU1
50 um
50 um
50 um
50 um
OSU2
50 um
50 um
50 um
50 um
OSU3
50 um
50 pm
50 um
50 um
743
744 745 746
MMP-14
YH2AX
DAPI
Merged
Untreated
1
1 hr post 4Gy IR
cosS
09 88
6 hr post 4Gy IR
24 hr post 4Gy IR
=
=
747
749 750 751 752 753
DAPI
MMP-14
YH2AX
Merged
DMSO
Cisplatin
754 755 756 757 758 759 760 761
762
763 764 765
766 767 768
24 hrs DMSO
48 hrs DMSO
G1 | S | G2M
G1 |S| G2M
G1: 64.02%
G1: 66.96%
Counts
1ª DNA content
S: 10.47%
Counts
Não DNA ocntont
S: 10.21%
G2M: 23.79%
G2M: 20.49%
T
24 hrs 100 μM NSC405020
48 hrs 100 UM NSC405020
G1 | S| G2M
G1 |S |G2M
G1: 58.55%
Counts
G1: 57.10%
S: 12.58%
Counts
S: 14.94%
G2M: 23.34%
G2M: 23.24%
DNA content
DNA contont
769 770 771 772