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28 February 2026

15 Pages

Prolonged Mitogen-Activated Protein Kinase Kinase (MEK) Inhibition Induces Increase in Proteolysis and Compensatory Phosphorylation of MEK and Protein Kinase B (AKT) in Plexiform Neurofibroma Cells

,
,
and
1
Department of Neurology, Henry Ford Health System, Detroit, MI 48202, USA
2
Department of Pharmacology, Wayne State University, Detroit, MI 48201, USA
3
Department of Pharmacology and Toxicology, Brody Medical School, East Carolina University, Greenville, NC 27834, USA
*
Authors to whom correspondence should be addressed.
Cells2026, 15(5), 434;https://doi.org/10.3390/cells15050434
This article belongs to the Section Cell Microenvironment
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Abstract

Plexiform neurofibromas associated with neurofibromatosis type I (pNF1s) are benign tumors caused by the complete loss of function of the NF1 gene, which encodes a negative regulator of the RAS/mitogen-activated protein kinase (MAPK) pathway. pNF1s carry a significant risk of progression to malignant peripheral nerve sheath tumors (MPNSTs), which are highly aggressive and largely incurable. FDA-approved mitogen-activated protein kinase kinase (MEK) inhibitors, selumetinib and mirdametinib, have shown ~30% tumor shrinkage in 70% and 42% pNF1 patients, respectively. However, not all pNF1s respond to MEK inhibition, and treatment is often associated with adverse effects such as dermatologic and gastrointestinal toxicities, underscoring the need for improved therapeutic strategies with minimal side effects. Here, we demonstrate that prolonged MEK inhibition increases proteolytic activity in 3D pNF1 tumor structures, consistent with enhanced extracellular matrix degradation. Prolonged treatment with four mechanistically and chemically distinct MEK inhibitors consistently reduced ERK phosphorylation, a downstream effector of the RAS/MAPK pathway, yet induced adaptive phosphorylation of MEK and AKT in pNF1 tumor cells. Phosphorylation of MEK is required for its catalytic activation and subsequent phosphorylation of ERK. Increased MEK phosphorylation in the presence of MEK inhibitors reflects upstream pathway reactivation but does not lead to ERK phosphorylation and activation because of the presence of the inhibitor. This response was also observed in MPNST cell lines treated with MEK inhibitors. These findings suggest that adaptive activation of upstream and parallel survival pathways may counteract the intended effects of MEK inhibition and support the rationale for combination strategies to improve therapeutic outcomes in NF1-associated tumors.

1. Introduction

Plexiform neurofibromas associated with neurofibromatosis type I (pNF1s) are benign peripheral nerve sheath tumors that arise from loss-of-function mutations in the NF1 gene [1]. NF1 is one of the most common inherited tumor predisposition syndromes, affecting approximately 1 in 2500–3500 individuals worldwide [2,3]. Among individuals with NF1, approximately 30–50% are born with pNF1s, which arise during early development and may enlarge and become clinically significant over time [4,5]. The NF1-encoded protein, neurofibromin, functions as a Ras GTPase–activating protein and thereby negatively regulates Ras/mitogen-activated protein kinase (MAPK) signaling [2,6]. Loss of neurofibromin leads to constitutive Ras/MAPK pathway activation and uncontrolled tumor cell proliferation [2,6].
Clinically, pNF1s cause substantial morbidity through disfigurement, pain, and compression of vital organs [7,8]. Histologically, pNF1s are highly collagenous, extracellular matrix (ECM)–rich tumors composed of Schwann cells, fibroblasts, mast cells, and abundant fibrillar collagen, reflecting extensive tumor–matrix interactions within peripheral nerve environments [9,10,11]. This ECM–rich architecture contributes to their infiltrative growth pattern along nerves and surrounding tissues [11,12,13]. Moreover, 8–13% of pNF1 patients develop malignant peripheral nerve sheath tumors (MPNSTs), highly aggressive sarcomas with limited treatment options and poor prognosis [14], that arise due to progression from pNF1s. While pNF1s are benign, ECM–rich, infiltrative tumors primarily driven by NF1 loss, MPNSTs arise through additional genetic alterations that confer malignant growth and invasive behavior [15,16]. Despite these differences, both tumor types retain dependence on Ras/MAPK signaling as a consequence of NF1 deficiency [16]. The only FDA-approved therapies for pNF1s are MEK inhibitors, selumetinib (AZD6244) [17,18] and mirdametinib (PD0325901) [19,20], while several other approaches, including additional MEK inhibitors, are under clinical investigation [21]. FDA-approved treatments induce tumor shrinkage in approximately 30% of pNF1 patients [22,23]; however, clinical responses are often incomplete, and tumor regrowth after treatment discontinuation is common [24,25,26]. The biological mechanisms underlying this limited efficacy remain poorly understood. Moreover, the long-term durability of MEK inhibition remains uncertain [25], and adverse effects, including dermatologic, gastrointestinal, and cardiac toxicities, pose challenges for chronic administration [27]. Thus, despite these recent advances, MEK inhibitor–based strategies provide only partial and often transient benefit, highlighting the urgent need for improved therapeutic options for pNF1s.
MEK inhibitors reduce tumor volume in a subset of pNF1 patients, and most studies have focused on tumor shrinkage and signaling inhibition, with limited attention to how MEK inhibition affects tumor–microenvironment interactions [28,29]. In many solid tumors, tumor cell–mediated ECM degradation is a prerequisite for tissue infiltration and tumor progression, as it allows cells to locally modify and traverse their surrounding matrix environment [30,31,32]. However, whether MEK inhibition influences this process in pNF1 tumors has not been examined. Specifically, whether prolonged MEK inhibition increases ECM proteolytic activity in pNF1 tumors beyond acute cytotoxic responses remains unknown. This knowledge gap is notable given the infiltrative growth pattern of pNF1s along nerves and surrounding tissues [33,34], where ECM degradation is likely to influence tumor behavior and treatment durability.
In this study, we investigated the long-term effects of MEK inhibition on proteolysis and signaling in pNF1 and MPNST cell lines using four chemically and pharmacologically distinct MEK inhibitors: selumetinib [17,18], binimetinib (MEK162) [35], CI-1040 (PD184352) [36], and U0126 [36]. We found two clinically relevant findings. First, prolonged selumetinib or binimetinib treatment (3–4 days) significantly increased proteolytic activity in 3D cultured pNF1 tumor structures, revealing that MEK inhibition is associated with increased ECM degradation. Second, prolonged (2-day) MEK inhibitor treatment with U0126, CI-1040, or selumetinib reduced ERK phosphorylation, as expected, but induced adaptive phosphorylation of MEK and AKT in both pNF1s tumor cells and MPNST cells, indicating activation of compensatory signaling pathways. Together, these results highlight a previously underappreciated consequence of MEK inhibition in pNF1s: enhanced proteolysis and activation of compensatory pathways that may limit therapeutic efficacy and contribute to tumor growth or side effects. Our findings suggest that therapeutic strategies combining MEK inhibition with approaches targeting compensatory survival pathways, such as MEK/AKT signaling, may offer improved efficacy and underscore the importance of evaluating the long-term consequences of MEK inhibition in the context of NF1.

2. Materials and Methods

2.1. Reagents and Antibodies

Reconstituted basement membrane (rBM; CultrexTM reduced growth factor basement membrane extract) and type I Collagen (rat tail, high concentration) were purchased from Bio-Techne (Minneapolis, MN, USA) and Corning (Corning, NY, USA), respectively. Phenol red-free Dulbecco’s Modified Eagle Medium (DMEM) and MycoZapTM Plus-CL were purchased from Lonza (Basel, Switzerland). Fetal bovine serum (FBS) was obtained from Cytiva (Marlborough, MA, USA). Selumetinib was purchased from Selleckchem (Houston, TX, USA). L-glutamine, U0126, CI-1040 (PD184352), selumetinib, and binimetinib (MEK162), and all other chemicals, unless otherwise specified, were purchased from Sigma-Aldrich (St. Louis, MO, USA). LIVE/DEADTM Viability/Cytotoxicity kit, dye-quenched (DQTM) collagens I and IV, Hoechst33342 and 4′-6-Diamidino-2-phenylindole (DAPI) were purchased from ThermoFisher Scientific (Waltham, MA, USA). Antibody against phospho-ERK (pERK) was purchased from Sigma-Aldrich, and antibodies against phospho-MEK (pMEK), phospho-AKT (pAKT), and total ERK, MEK, AKT, and GAPDH antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

2.2. Cell Lines and Cell Maintenance

Two immortalized human plexiform neurofibroma cell lines (NF1−/−; hereafter called pNF1 tumor cells) were used: ipNF95.11b C and ipNF05.5 obtained from ATCC or provided by Dr. Margaret Wallace, University of Florida (Gainesville, FL, USA). The human MPNST cell line TRS462TY, derived from a xenograft of S462 cells in immune-deficient mice, was provided by Dr. Nancy Ratner (University of Cincinnati College of Medicine, Cincinnati, OH, USA) [37]. All cell lines were maintained as monolayers at 37 °C, 5% CO2 in DMEM/high glucose supplemented with 10% FBS. Routine screening confirmed the absence of mycoplasma contamination.

2.3. Three-Dimensional (3D)/4D (3D in Real-Time) Culture Models

3D cultures were established either in patented microfluidic culture devices called TAME (tissue architecture and microenvironment engineering; Patent#, US10227556B2) chips [38] or on glass coverslips for live-cell proteolysis imaging, as previously published [28,39]. Fabrication of TAME chips, including separate and linked well designs [38], and protocols for 3D monocultures have been described previously [40]. Briefly, cells were seeded on a reconstituted basement membrane (rBM; Cultrex™ and collagen I mixture) containing intramolecularly quenched fluorescent protein substrates (DQTM-collagen I and DQTM-collagen IV) in the glass-bottom wells of TAME chips or standard culture plates [41]. For 3D cultures, the rBM/DQTM-collagen mixture was polymerized at 37 °C for 30 min, then cells were seeded on the top of the matrix at a density of 6000–8000 cells per well. Cultures were overlaid with DMEM supplemented with 2.5% FBS and 2% rBM and maintained for 3–4 days. MEK inhibitors were added 24 h after seeding to allow formation of early 3D tumor structures and were maintained in the culture media for the indicated culture periods. Prior to live-cell 3D imaging, nuclei were stained with Hoechst 33342 to assess cell integrity and morphology, and total live-cell numbers were quantified using viability markers such as Calcein-AM.

2.4. Image Acquisition for Quantitative Analysis in 3D

Optical sections spanning the full depth of the 3D structures were acquired from 4 contiguous fields (2 × 2) using either an upright Zeiss LSM 780 confocal microscope (Carl Zeiss Microscopy, Jena, Germany) within a temperature- and CO2-controlled environmental chamber, or an inverted Leica Stellaris 5 system (Leica Microsystems, Deerfield, IL, USA) equipped with a Tokai Hit stage-top incubator to maintain physiological conditions. Image stacks were processed and reconstructed in 3D using Volocity software (Version 7.0.0; PerkinElmer, Waltham, MA, USA) to visualize and quantify live/dead cell distributions. Spatial orientation within reconstructed volumes is indicated by x (green), y (red), and z (blue) arrows in the lower corner of each image. DQTM-collagen fluorescence was imaged using identical acquisition settings across all conditions, and proteolytic activity was quantified as total fluorescence intensity within each 3D structure using Volocity software.

2.5. Immunoblotting

pNF1 cell line, ipNF95.11bC, and MPNST cell line, TRS462TY, were grown for 24 h before treatment with DMSO (vehicle control), CI-1040, U0126, or PMA (a positive control to activate the MAPK pathway) for various times (30 min or 48 h). Whole-cell lysates were collected in boiling Laemmli sample buffer modified for cell lysis (75 mM Tris-HCl, pH 6.8, 1.5% (w/v) sodium dodecyl sulfate (SDS), 7.5% (w/v) glycerol, 200 mM β-mercaptoethanol, 0.03% (w/v) bromophenol blue, 0.003% (w/v) pyronin-Y) [42]. Whole-cell lysates were resolved on an SDS-PAGE gel and transferred to a nitrocellulose membrane. After blocking for 1 h with 5% dry milk in TBST buffer (20 mmol/L Tris-HCl pH 7.4, 150 mmol/L NaCl, 0.1% Tween-20), blots were probed overnight at 4 °C with the following primary antibodies: pERK, pMEK, pAKT, ERK, MEK, AKT, and GAPDH. Blots were reacted with peroxidase-conjugated antibody for 1 h, followed by visualization of the proteins using ECL detection reagents. Band intensities normalized to their respective total proteins, or GAPDH, were quantified using ImageJ 1.54g software (National Institutes of Health, Bethesda, MD, USA).

2.6. Statistical Analysis

Data are presented as bar graphs, representing the mean ± standard deviation (SD). The number of replicates is provided in each corresponding figure legend. Statistical significance was assessed using either one-way ANOVA followed by Tukey’s post hoc test for multiple group comparisons, or Student’s t-test for comparisons between two groups. A p-value ≤ 0.05 was considered statistically significant throughout the study.

3. Results

3.1. MEK Inhibition Increases Proteolytic Activity in 3D pNF1 Tumor Structures

To investigate whether MEK inhibition increases ECM proteolytic activity in pNF1 tumor cells, we generated 3D tumor structures by embedding ipNF95.11bC or ipNF05.5 cells in reconstituted basement membrane (rBM) containing intramolecularly quenched fluorescent collagen substrates, DQ™-collagen I and DQ™-collagen IV, which fluoresce upon enzymatic cleavage by ECM–degrading proteases, on glass-bottom TAME chips (Figure 1). Cells were seeded as a 3D overlay culture on polymerized rBM/DQTM-collagen matrix and allowed to form multicellular tumor structures for 24 h prior to drug treatment. Tumor structures were treated with or without the FDA-approved MEK inhibitor, selumetinib, for 4 days and cell viability was assessed by Calcein-AM. In untreated cultures, viable pNF1 tumor structures were readily observed (Figure 1A(a),B(a)), and basal proteolytic activity was evident as green fluorescence from degradation of DQTM-collagens in the surrounding matrix (Figure 1A(b,c),B(b,c)). Selumetinib treatment reduced the number of viable tumor structures (Figure 1A(d),B(d)), consistent with its growth-inhibitory effects as shown in our prior study [39]. Strikingly, however, the remaining live tumor structures exhibited a marked increase in proteolytic activity compared to untreated controls (Figure 1A(e,f),B(e,f)). DQTM-collagen fluorescence and cell viability were imaged under identical acquisition settings, and proteolytic activity was quantified as total fluorescence intensity per 3D structure using Volocity software. Quantification confirmed a significant elevation of DQTM-collagen degradation in both ipNF95.11bC and ipNF05.5 tumor structures following selumetinib treatment (Figure 1C). This increase was consistently observed across multiple fields and independent experiments.
Figure 1. Four-day selumetinib treatment increases proteolysis in pNF1 tumor structures in 3D culture. (A,B) pNF1 tumor structures derived from ipNF95.11bC or ipNF05.5 cells were embedded in rBM containing quenched fluorescent derivatives of types I and IV collagen (DQTM-collagens; green) and treated with or without selumetinib for 4 days. Tumor structures were stained with Calcein AM, a live-cell marker (green; (a,d)) and Hoechst 33342 (nuclei; pseudo-white; (b,e)). Panels (c,f) show fluorescence from DQTM-collagen cleavage products (green), indicating sites of proteolysis. Images are tiled from four contiguous fields; A grid represents 174 μm. (C) Total proteolytic activity was quantified as normalized integrated fluorescence intensity of DQTM-collagen cleavage products across all optical sections of each tumor structure, normalized to cell number, using Volocity software. Data represent mean ± SD (n = 3).
To determine whether this effect was specific to selumetinib, we repeated the assay with another clinically relevant MEK inhibitor, binimetinib (Figure 2). Similar to selumetinib, binimetinib treatment reduced the number and size of viable tumor structures but substantially increased proteolytic activity in pNF1 tumor structures. These findings demonstrate that MEK inhibition is associated with enhanced ECM proteolysis, despite reducing overall tumor viability in 3D pNF1 tumor structures.
Figure 2. Bimimetinib treatment increases proteolysis in pNF1 tumor structures in 3D culture. pNF1 tumor structures derived from ipNF95.11bC cells were embedded in rBM containing quenched fluorescent type IV collagen (DQTM-collagen; green) and treated with (lower) or without binimetinib (upper) for 3 days (n = 2). Green fluorescence indicates cleavage products of DQTM-collagen, reflecting proteolytic activity. Nuclei were counterstained with Hoechst 33342 (blue). Grid = 42 μm.

3.2. Prolonged MEK Inhibition Suppresses ERK Phosphorylation While Increasing MEK and AKT Phosphorylation in pNF1 and MPNST Cells

To define signaling changes induced by prolonged MEK inhibition, we examined MAPK and PI3K/AKT pathway activity in both pNF1 and MPNST tumor cells following extended MEK inhibitor treatment (Figure 3). The pNF1 tumor cell line, ipNF95.11bC (Figure 3A,B), and the MPNST cell line, TRS462TY (Figure 3C,D), were treated with the MEK inhibitors, U0126 or selumetinib, for 48 h, and phosphorylation of ERK and MEK was assessed by immunoblotting. In ipNF95.11bC cells, prolonged treatment with either U0126 or selumetinib markedly reduced ERK phosphorylation compared with DMSO-treated controls, indicating sustained suppression of downstream MAPK signaling (Figure 3A,B, and Supplementary Figure S1A). In contrast, phosphorylation of MEK was increased following prolonged exposure to both inhibitors. Selumetinib induced a dose-dependent increase in MEK phosphorylation across the tested concentration range (0.05–5 μM). Densitometric analysis was performed from three independent experiments and normalized to total protein or GAPDH as indicated. Given the well-established cross-talk between the MAPK and PI3K/AKT pathways [43,44], we also examined AKT phosphorylation following prolonged MEK inhibition. AKT phosphorylation was increased after prolonged treatment with either U0126 or selumetinib, with selumetinib producing a robust, dose-dependent effect. A similar signaling pattern was observed in TRS462TY MPNST cells (Figure 3C,D, and Supplementary Figure S1B). Prolonged MEK inhibitor treatment suppressed ERK phosphorylation while concurrently increasing MEK and AKT phosphorylation, demonstrating that sustained MEK inhibition elicits comparable compensatory signaling responses in both benign pNF1 tumor cells and malignant MPNST cells.
Figure 3. Prolonged MEK inhibitor treatment reduces ERK phosphorylation and induces increased MEK and AKT phosphorylation in pNF1 and MPNST cells. (A) Immunoblot analysis of the pNF1 cell line, ipNF95.11bC, treated with DMSO (vehicle), U0126, selumetinib (0.05, 0.5, or 5 μM), or PMA (positive control for MAPK pathway activation). Cells were harvested after 48 h (DMSO, U0126, selumetinib) or 5 min (PMA). Blots were probed for pERK, pMEK, and pAKT. GAPDH served as a loading control. (B) Densitometric quantification corresponding to panel (A) (n = 3). Phosphorylated protein levels were normalized to GAPDH. (C) Immunoblot analysis of the MPNST cell line, TRS462TY, under the same treatment conditions as in panel (A). (D) Densitometric quantification corresponding to panel (C) (n = 3). Total ERK, total MEK, and GAPDH served as loading controls. * p < 0.05 compared to the DMSO-treated group.
We next compared early (5 and/or 30 min) and prolonged (48 h) signaling responses to MEK inhibition using two other mechanistically distinct MEK inhibitors, CI-1040 and U0126, in both pNF1 tumor, ipNF95.11bC, and the MPNST cell line, TRS462TY (Figure 4). Short time points (5 and 30 min) were chosen to capture acute pathway inhibition, whereas 48 h represented prolonged exposure during which adaptive signaling was observed. In ipNF95.11bC cells, treatment with CI-1040 or U0126 suppressed ERK phosphorylation at all examined time points (5 min, 30 min, and 48 h), demonstrating sustained inhibition of ERK signaling. In contrast, treatment with CI-1040 or U0126 induced phosphorylation of MEK and AKT at 5 min and persisted through 30 min and 48 h (Figure 4A,B, and Supplementary Figure S2A). These changes occurred despite continued suppression of ERK phosphorylation. In the MPNST cell line TRS462TY, short-term treatment (30 min) with either CI-1040 or U0126 reduced phosphorylation of both ERK and MEK, indicating effective acute inhibition of MAPK signaling. With prolonged treatment (48 h), ERK phosphorylation remained suppressed, whereas MEK and AKT phosphorylation increased, consistent with compensatory pathway activation during sustained MEK inhibition (Figure 4C,D, and Supplementary Figure S2B).
Figure 4. Prolonged MEK inhibition decreases ERK phosphorylation while increasing MEK and AKT phosphorylation compared to short-term treatment in pNF1 and MPNST cells. (A) Immunoblot analysis of pNF1 cell line, ipNF95.11bC cells, treated with DMSO (vehicle), CI-1040, U0126, or PMA for 5 min, 30 min, or 48 h. Blots were probed for pERK, pMEK, and pAKT. GAPDH served as a loading control. (B) Densitometric quantification corresponding to panel (A) (n = 3). Phosphorylated protein levels were normalized to GAPDH. (C) Immunoblot analysis of the MPNST cell line, TRS462TY, treated with DMSO (vehicle) or PMA for 5 min, and with CI-1040 or U0126 for 30 min and 48 h. Blots were probed for phosphorylated ERK (pERK) and MEK (pMEK). Total ERK, total MEK, and GAPDH served as loading controls. (D) Densitometric quantification corresponding to panel (C) (n = 3). Phosphorylated protein levels were normalized to their respective total protein levels. * p < 0.05 compared to the DMSO-treated group.
Together, these findings show that prolonged MEK inhibition induces adaptive phosphorylation of MEK and AKT in both benign pNF1 tumor cells and malignant MPNST cells. This adaptive signaling response temporally coincides with the enhanced proteolytic activity observed in 3D pNF1 tumor structures, suggesting a potential mechanistic link between sustained MEK inhibition, signaling rewiring, and ECM degradation, as summarized schematically in Figure 5.
Figure 5. Schematic summary of NF1–RAS–MAPK signaling and adaptive responses observed during prolonged MEK inhibition in NF1 tumor models. Neurofibromin, encoded by NF1, functions as a Ras GTPase–activating protein (RAS-GAP) that promotes conversion of Ras-GTP to Ras-GDP under normal conditions. Loss of NF1 in pNF1 and MPNST cells results in constitutive Ras-GTP and activation of downstream RAF–MEK–ERK and PI3K–AKT signaling pathways. MEK inhibitors (CI-1040, U0126, selumetinib, and binimetinib) suppress ERK phosphorylation downstream of MEK. During prolonged MEK inhibition, sustained ERK suppression is associated with feedback release, leading to increased Ras signaling and adaptive phosphorylation of MEK and AKT. These signaling changes occur in parallel with increased tumor cell–mediated ECM proteolysis in 3D tumor structures. Black solid lines indicate established NF1–RAS–MAPK signaling relationships. Red solid line with blunt bar indicates the site of MEK inhibitor action. Red solid lines with arrows indicate concurrent adaptive phosphorylation during prolonged MEK inhibition.

4. Discussion

In this study, we identify adaptive consequences of MEK inhibition in NF1-associated tumor models. Using 3D NF1 tumor models, we found that selumetinib or binimetinib treatment significantly increased proteolytic activity despite reducing overall tumor viability. Mechanistically, prolonged MEK inhibitor exposure over 48 h led to increased phosphorylation of MEK and AKT in both pNF1 and MPNST cells, consistent with adaptive feedback signaling. Both pNF1 [45,46] and MPNST models [47,48] used here are well-established NF1-null systems in which loss of neurofibromin results in constitutive RAS pathway activation. While adaptive MEK/AKT feedback activation has been described in other tumor types following MEK inhibition, this signaling behavior has not previously been characterized in pNF1 models treated with clinically relevant MEK inhibitors. These results reveal that MEK inhibition not only suppresses tumor growth but also increases tumor cell-mediated ECM proteolysis, which may compromise therapeutic durability.
The observation that selumetinib and binimetinib enhanced ECM degradation highlights a previously underexplored effect of MEK inhibitors in pNF1s. The DQ-collagen assay used in this study represents a well-established functional method to quantify tumor cell–mediated pericellular ECM degradation in 3D environments and has been widely validated as a surrogate for invasive, matrix-degrading behavior across multiple tumor models [49,50,51,52,53]. ECM degradation is a critical step in tumor cell invasion and tissue infiltration in many solid tumors, as it enables tumor cells to locally remodel their surrounding matrix environment [54,55].
Importantly, MAPK and PI3K/AKT signaling are well-known regulators of matrix metalloproteinase (MMP) expression and protease activity that drive ECM degradation and invasion in many cancers [56,57], providing a plausible mechanistic link between adaptive signaling responses and the increased proteolysis observed in our models. In NF1 tumors specifically, MMP-1, MMP-2, MMP-9, and membrane type (MT)1-MMP have been reported in tumor regions and are capable of degrading basement membrane collagens [58,59,60]. Consistent with this framework, proteolysis is a key regulator of tumor biology, driving invasion, angiogenesis, immune evasion, and therapeutic resistance [61,62,63]. While studies focusing on the contributions of proteolysis in pNF1 progression remain limited, our findings suggest that prolonged MEK inhibition enhances this ECM degradation, potentially facilitating tumor progression once drug pressure is relieved.
Notably, proteolytic activity was assessed using DQ-collagen, a self-quenched collagen substrate that fluoresces upon cleavage by collagen-degrading proteases (e.g., MMPs and cathepsins); therefore, the increased DQ-collagen fluorescence reflects enhanced collagen/ECM proteolysis rather than apoptotic caspase activity [64,65,66,67]. This adaptive increase in proteolysis may help explain why MEK inhibitors, though effective in shrinking tumor volume in pNF1 patients, often show incomplete responses and frequent tumor regrowth after treatment discontinuation [22,23,24,25,26]. These findings align with a broader oncologic paradigm in which initial tumor shrinkage in response to therapy is followed by adaptive resistance and subsequent regrowth over time, reflecting dynamic signaling reprogramming rather than static tumor suppression [68].
Our mechanistic analyses indicate that long-term MEK inhibition disrupts feedback regulation within the Ras/MAPK network. While short-term treatment with U0126 or CI-1040 effectively suppressed ERK phosphorylation, as expected, prolonged treatment revealed divergent outcomes: U0126 and selumetinib reduced ERK phosphorylation but induced adaptive phosphorylation of MEK. In parallel, selumetinib induced robust, dose-dependent phosphorylation of MEK and AKT. These findings suggest that MAPK inhibition relieves negative feedback on upstream elements, such as receptor tyrosine kinases, leading to compensatory activation of MEK and PI3K/AKT pathways. Phosphorylation of MEK at activating residues is required for its kinase activity toward ERK; therefore, increased MEK phosphorylation reflects upstream pathway reactivation rather than direct ERK activation by unphosphorylated MEK. While such compensatory MEK and AKT activation has been reported in MPNST [69] and other cancers [43,44], it has not been characterized in pNF1 tumor cells. Our results show similar activation in pNF1s, along with a previously unrecognized increase in proteolytic activity. In both pNF1 and MPNST cells, this signaling rewiring may contribute to enhanced proteolysis and potentially limit therapeutic durability.
A more detailed temporal analysis and drug withdrawal studies would further clarify the kinetics and persistence of this adaptive response and help distinguish transient feedback signaling from more sustained cellular adaptation. This adaptive signaling pattern is consistent with an ERK-dependent negative feedback relief mechanism in which suppression of ERK activity reduces expression of feedback regulators such as DUSP6 and SPRY2. This, in turn, can lead to receptor tyrosine kinase (RTK) reactivation and increased upstream signaling that can drive RAS reactivation and parallel MEK and PI3K/AKT pathway activity [70,71,72,73,74]. While detailed dissection of RTK activation and feedback regulator dynamics was beyond the scope of this study, our findings in pNF1 and MPNST models align closely with this established MAPK feedback circuitry.
In addition to kinase-driven feedback reactivation, phosphatase-mediated mechanisms may further modulate adaptive signaling. Protein phosphatase 2A (PP2A), a known mediator of MEK dephosphorylation, has emerged as a therapeutic target in several tumor types [75]. Pharmacologic activation of PP2A, including with agents such as perphenazine that have been shown to reactivate PP2A and suppress tumor growth in glioblastoma models [76], may represent a potential strategy to attenuate compensatory MEK phosphorylation. Evaluation of phosphatase modulation in NF1 tumor models warrants future investigation. Future studies will include profiling RTK activation and ERK-dependent feedback regulators, testing dual MEK–PI3K/AKT inhibition, and evaluating whether protease activity contributes functionally to the observed ECM proteolysis.
Clinically, these results suggest that MEK inhibitor monotherapy may inadvertently foster a more proteolytically active and potentially aggressive tumor microenvironment. This dual effect, tumor shrinkage accompanied by enhanced ECM remodeling, may complicate long-term outcomes in pNF1 patients. Combination strategies that block both MAPK and PI3K/AKT pathways, or directly target proteolytic activity, may enhance therapeutic benefit, reduce the risk of regrowth, and potentially mitigate some of the adverse effects associated with chronic MEK inhibition. Unlike for pNF1s, MEK inhibitor monotherapy has not been found effective for MPNST treatment. Consideration of these issues will be an additional factor in the development of combinatorial approaches for therapy of unresectable MPNST [77].
Our study has several limitations. First, the findings were obtained in vitro using 3D tumor structures, and in vivo validation will be essential to confirm the clinical relevance. Second, while we observed increased collagen degradation, the specific proteases responsible were not identified; future work should dissect the contributions of specific types of MMPs and other protease families. Finally, although we examined both pNF1 and MPNST cell models, further studies across diverse NF1 tumor contexts are needed to assess the generalizability of these mechanisms.

5. Conclusions

In 3D pNF1 tumor models, MEK inhibitor exposure increases ECM proteolysis and triggers an adaptive signaling response characterized by increased phosphorylation of MEK and AKT despite sustained ERK suppression. These effects were observed in both pNF1 and MPNST cell lines and represent adaptive changes that emerge during extended MEK inhibitor treatment. Our findings offer a potential explanation for the limited durability of MEK inhibitor therapy and tumor regrowth after treatment discontinuation in pNF1 patients. Given that MEK inhibitors are currently the only FDA-approved treatment for pNF1s, understanding these adaptive tumor responses is clinically important. Together, our results highlight the need for therapeutic strategies that target both adaptive survival signaling and ECM proteolysis to improve long-term treatment outcomes in NF1-associated tumors.

6. Patents

The present study is associated with the U.S. Patent US10227556B2.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/cells15050434/s1: Supplementary Figure S1: Uncropped immunoblot images corresponding to Figure 3, Supplementary Figure S2: Uncropped immunoblot images corresponding to Figure 4.

Author Contributions

Conceptualization, K.J. and R.R.M.; investigation, K.J., J.F.C. and T.J.R.; writing—original draft, K.J.; writing—review and editing, K.J., J.F.C., T.J.R. and R.R.M.; supervision, K.J. and R.R.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research work was supported by a Department of Defense Neurofibromatosis Research Program New Investigator Award (W81XWH-22-1-0564 to K.J.) and an Exploration-Hypothesis Development Award (NF220063 to R.R.M.). Work in the R.R.M. laboratory was also supported by the Neurofibromatosis Therapeutic Acceleration Program (NTAP) and NF Michigan. T.J.R. was a summer undergraduate fellow of the Cancer Biology Program at the Karmanos Cancer Institute and Wayne State University.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

Acknowledgments

We are deeply grateful to Bonnie F. Sloane, K.J.’s exceptional mentor, who sadly passed away from lung cancer. Her guidance, feedback, and encouragement were instrumental in shaping the NF1 projects and continue to inspire this work. Finally, we thank the NTAP for providing funding to support the purchase of immortalized human pNF1 cell lines used in this study.

Conflicts of Interest

The authors declare no conflicts of interest.

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