Breast cancer (BC) is the most common cancer worldwide and ranks fifth among cancer-related deaths (Zhang et al., 2023). Projections indicate a significant increase in the global burden, with cases expected to rise by nearly 40% and deaths by 68% by 2050 if current trends continue (Liao, 2025). Brain and central nervous system cancer ranks approximately 12th in incidence and 8^th in mortality in Europe based on age-standardized rates (DeCordova et al., 2020). Glioblastoma (GBM), classified as a WHO Grade 4 CNS tumor according to the 2021 WHO Classification of Tumors of the Central Nervous System, represents the most aggressive form of brain cancer (Louis et al., 2021). The 5- and 10-year survival rates still remain at 5% and 2.6%, respectively (Ma et al., 2022; Alcantara Llaguno et al., 2009). The current standard of care for GBM follows the Stupp protocol, established in 2005, which includes maximal safe surgical resection, concurrent chemoradiotherapy, and adjuvant temozolomide (TMZ) (Jezierzański et al., 2024).

The poor treatment prognosis, notably GBM, has been linked to its diverse molecular profiles, resulting in distinct phenotypes also associated with TMZ-resistance (DeCordova et al., 2020). Several fundamental mechanisms contribute to GBM’s treatment resistance and incurability. The blood-brain barrier represents a critical obstacle, preventing most systemic chemotherapeutic agents from reaching therapeutic concentrations in brain tissue (Zuccarini et al., 2018). Glioblastoma stem cells (GSCs) constitute another major resistance mechanism, exhibiting enhanced DNA repair capacity, resistance to apoptosis, metabolic reprogramming, and self-renewal capacity that maintains the tumor cell population (Nowak et al., 2021; Guan et al., 2020). Methylation of the O6-methylguanine-DNA methyltransferase (MGMT) promoter plays a critical role in TMZ-resistance. Tumors with an unmethylated MGMT promoter are significantly more resistant to TMZ, and prolonged treatment may induce loss of MGMT methylation, further contributing to acquired resistance (Li et al., 2021).

Recently, Weighted Gene Co-Expression Network Analysis (WGCNA) was used to capture the molecular heterogeneity of GBM patients on the molecular level. Authors constructed an immune-related prognostic model to predict patient sensitivity to checkpoint inhibitor blockade therapy (Ma et al., 2022). The high-risk group (non-survival) was associated with epithelial-mesenchymal transition (EMT), high immune cell infiltration, immune activation, a low mutation number, and high methylation, while the low-risk group had an adverse status (Ma et al., 2022).

BC is also extremely variable in morphology and at the molecular level, necessitating combinatorial therapy modalities depending on the molecular subtype, which is defined by hormone receptor (HR) status and human epidermal growth factor receptor-2 (HER2) expression (Li et al., 2017; Kashyap et al., 2022). These include HR-positive/HER2-negative (HR+/HER2-), HR+/HER2+, HR-/HER2+, and triple-negative breast cancer (TNBC), which lacks estrogen receptor, progesterone receptor, and HER2 expression (Li et al., 2017). TNBC is particularly aggressive, accounting for approximately 10%–15% of all breast cancers but contributing to a disproportionately high percentage of breast cancer-related deaths globally, estimated at around 40% (Rosińska et al., 2024). The 5-year relative survival rate for TNBC, combining all stages, is approximately 77%, with significantly lower rates for distant metastatic disease (Baranova et al., 2022).

The current standard of care in breast cancer varies by subtype and stage. Early-stage breast cancer typically involves surgical resection, adjuvant or neoadjuvant chemotherapy, radiation therapy, hormone therapy for hormone receptor-positive tumors, and targeted therapy for HER2-positive tumors (Bravo et al., 2023; Grant et al., 2024).

The Wnt pathway is implicated in both BC and GBM, significantly contributing to treatment resistance (Zhong and Virshup, 2020), even though precise regulatory mechanisms remain unclear (Kashyap et al., 2022). Crosstalk between Wnt signaling and other pathways contributes to cancer development and spread, including resistance to pathway inhibitors (Zhong and Virshup, 2020). This understanding drives the development of novel combination therapies to minimize toxicity and resistance (Zhong and Virshup, 2020). Therefore, exploring unexamined plant extracts or phytocompounds contributes to discovering safe, effective, and novel combination therapies (Ulrich-Merzenich et al., 2017).

TMZ and paclitaxel (PTX), key chemotherapeutics for GBM and BC, respectively, are under investigation for combination therapies (Tan et al., 2020). TMZ, a DNA alkylating agent, induces cell death by causing base mismatches and DNA strand breaks (Tan et al., 2020). Ongoing 583 clinical trials worldwide explore treatments for GBM (https://clinicaltrials.gov/), including immune checkpoint inhibitors paired with CNS-penetrant or potent inhibitors (Gueble et al., 2022). Despite multimodal treatments, GBM patients face a low median survival of 12.1–16.6 months (Neff et al., 2022; Minea et al., 2024; Fekete et al., 2023), emphasizing the urgency for novel treatment strategies (Tan et al., 2020).

Taxanes, like PTX, used in BC treatment, were derived from the Pacific yew’s bark (Wani et al., 1971). PTX induces cell death by stabilizing microtubules, causing G2/M arrest and initiating apoptosis (Manthey et al., 1992). Resistance to PTX and other anti-cancer drugs (Das et al., 2021; Bukowsk et al., 2020) is common for BC, just as resistance in GBM to TMZ (Lee, 2016). The resistance of BC to PTX is thought to be a consequence of a disequilibrium in various signaling pathways, mutations in certain genes, and epigenetic deregulations (Abu et al., 2019). In particular, the genes of the ATP-binding cassette (ABC) superfamily of drug efflux, including P-glycoprotein (P-gp), are involved in the resistance to PTX by leading to an overexpression of P-gp in BC-cells (Abu et al., 2019). In MCF7 cells, aberrantly regulated expression of FOXM1 and KIF20A was associated with PTX-resistance (Abu et al., 2019; Khongkow et al., 2016). Further, the overexpression of miR-200c-3p contributed to the resistance of BC cells to PTX by an aberrant regulation of SOX2 (Abu et al., 2019).

This diversity of resistance mechanisms promotes a search for natural compounds as adjuvants (Persano et al., 2022). Therapies triggering multiple pathways (and specifically addressing crucial survival pathways) may be more promising (Sestito et al., 2018).

Lichens have been utilized in traditional medicine for ages (Crawford and Ranković, 2015). They are symbiotic organisms consisting of a fungus (mycobiont) and either algae or cyanobacteria (photobiont) (Schwendener, 2011). They are a promising source for novel organic small molecules and synergistic therapeutic strategies. Evernia prunastri L. and Cladonia arbuscula (Wallr.) Fot. were selected based on their published bioactivity. Evernic acid (EA), the main metabolite of E. prunastri L., has shown antimicrobial, cytotoxic, neuroprotective, and anti-inflammatory properties in prior studies (Fernández-Moriano et al., 2017; Lee et al., 2021; Gökalsın and Sesal, 2016; Shcherbakova et al., 2019). However, its role in cancer therapy remains largely unexplored. Usnic acid (UA), a prominent secondary metabolite isolated from C. arbuscula (Wallr.) Fot., has shown potent antiproliferative effects in several cancer types. Notably, UA exhibited promising cytotoxicity against T-47D breast cancer cells, Capan-2 pancreatic cancer cells (Einarsdottir et al., 2010), and MCF7 breast cancer cells as well (Bačkorová et al., 2011; Galanty et al., 2017; Kiliç et al., 2019; Brisdelli et al., 2013).

We investigated extracts from E. prunastri L. (Parmeliaceae) and C. arbuscula (Wallr.) Fot. (Cladoniaceae) along with their major metabolites, evernic acid (EA) and usnic acid (UA), for their potential to reduce metabolic activity in U-87 glioma and MCF7 breast cancer cells.

Both cell lines were selected as representative in vitro models for glioblastoma and breast cancer, respectively, due to their widespread use, well-characterized molecular profiles, and relevance to the mechanisms under investigation. U-87 cells are among the most commonly used GBM models and exhibit key features of primary glioblastoma, such as rapid proliferation, resistance to temozolomide (TMZ) (Xie et al., 2015). Similarly, MCF7 cells are commonly used for BC, characterized by estrogen receptor positivity and moderate sensitivity to chemotherapeutics like paclitaxel (PTX), making them a standard model for studying HR+ BC and mechanisms of taxane resistance (Neve et al., 2006).

U-87 and MCF7 remain widely accepted platforms for early-stage anticancer research. In this study, they were used to evaluate not only the ability of the lichen extracts and their metabolites to influence cancer cell metabolic activity, but also their potential to synergize with standard chemotherapeutics (TMZ or PTX). Extracts and combinations showing efficacy were further assessed for possible mechanisms of action. This included chemographic prediction tools, cytokine response profiling, and evaluation of their potential to modulate cellular pathways involved in drug sensitivity, forming a foundation for future translational investigations.

The effectiveness of standard chemotherapeutics such as temozolomide (TMZ) in glioblastoma (GBM) and paclitaxel (PTX) in breast cancer (BC) is often limited by developing resistance and eventual treatment failure (Das et al., 2021; Lee, 2016). Therefore, we investigated the adjuvant potential of characterized extracts of E. prunastri and C. arbuscula and their metabolites evernic acid (EA) and usnic acid (UA) alone or in combination with TMZ and PTX for their immunomodulatory and chemosensitivity-increasing potential. HSKF served to evaluate general cytotoxicity and selectivity, even though these cells do not fully replicate the physiological environment or cellular characteristics of normal brain or mammary tissue. U-87 and MCF7 cells have their limitation, too. U-87 cells differ genetically from primary GBM tumors and do not fully recapitulate the intratumoral heterogeneity, stem cell populations, or invasive behavior observed in patient-derived GBM (Xie et al., 2015; Allen et al., 2016). Likewise, MCF7 cells, while representative of HR+ BC, do not model the full spectrum of BC subtypes, particularly TNBC or HER2+ disease, and lack the tumor microenvironment components that influence drug response in vivo (Neve et al., 2006; Holliday and Speirs, 2011). Both models are grown in two-dimensional monolayers, which do not capture the complexity of tumor-stroma interactions, hypoxia, or immune modulation present in human tumors. Despite these limitations, the use of U-87 and MCF7 in this study provides a well-established platform for assessing the antiproliferative and resistance-modulating effects of lichen-derived metabolites with findings that can inform subsequent validation in more complex models such as patient-derived cells, organoids, or in vivo systems.

The initial experiments revealed a reduction in cellular metabolic activity in U-87 and MCF7 cells after treated with the lichen extracts and their metabolites. However, they were less effective against MCF7 cells compared to PTX. Therefore, experiments with MCF7 cells were discontinued, even though UA had exhibited activity against MCF7 cells in earlier studies (Bačkorová et al., 2011; Hawrył et al., 2020).

For extracts of E. prunastri, IC₅₀ values of 90.1 μg/mL and 81.8 μg/mL were reported (Bézivin et al., 2004), which is in the range of our results. Effects of EA against MCF7 and U-87 have not been reported earlier. However, previous reports suggest its activity against A-172 and T98G glioblastoma cell lines (Studzińska-Sroka et al., 2021), supporting our findings.

The extracts of C. arbuscula were not further investigated due to their non-selective effect. Further experiments focused on the TMZ-resistant U-87 cells (IC₅₀ > 500 µM) (Lee, 2016) and the combination of TMZ and EA, since this combination yielded the most promising reduction in cellular metabolic activity, while showing minimal effect on HSKF.

To substantiate the antiproliferative effects, we performed Western blot (WB) analyses of members of the central MAPK families. They play a central role by regulating the cell cycle engine and other proliferation-related proteins (Bézivin et al., 2004; Studzińska-Sroka et al., 2021; Zhang and Liu, 2002). In addition, we measured AKT. In the context of cancer, Akt signaling promotes tumor cell survival, proliferation, growth, and metabolism by activating its downstream effectors. Both the PI3K/Akt and the MEK/ERK pathways cooperate in tumor growth and are involved in the development of therapeutic resistance in GBM cells (Singh et al., 2023).

WB analyses revealed that EprACN and EA reduced the expression and phosphorylation of ERK1/2 over time, explaining the observed decrease in the metabolic activity in U-87 cells and revealing an antiproliferative activity, as in most cells, sustained ERK activation is required to induce cell cycle entry. In Glioma, an aberrant activity of the RAS/MAPK/ERK pathway appears to play a crucial role in the development of gliomas (Stachyra and Grzybowska-Szatkowska, 2025). Even a large proportion of resistance mechanisms are associated with reactivation of the RTK/Raf/Ras/MEK/ERK pathway. Co-treatment with inhibitors targeting these pathways is meanwhile regarded as a compelling strategy to overcome resistance mechanisms in GBM (Yakubov et al., 2025).

Another member of the MAPK-pathway, the so-called proto-oncogene c-Jun, is also central to cancer-altered signalling: an upregulated c-Jun was described for variable tumor cells, specifically in brain tumors, contributing to its malignancy (Blau et al., 2012). Blau et al. demonstrated that the accumulation of c-Jun in tumors is regulated more translationally than transcriptionally (Blau et al., 2012) corresponding to our data with the regulation of c-Jun at the protein but not at the mRNA level.

EprACN and EA downregulated pAkt in a time- and dose-dependent manner corresponding to the reduction in metabolic activity in U-87 cells. This is particularly relevant, given that overexpression and high phosphorylation of Akt correlate with a poor prognosis in glioblastoma patients (Shahcheraghi et al., 2020). Increased pAkt activates transcription factors by phosphorylating GSK3β, leading to its inactivation and subsequent translocation of β-catenin into the nucleus. Both pathways, when activated independently, may contribute to resistance (Manoranjan et al., 2020). Conversely, β-catenin also induces the expression of Akt1 and Akt2 and the phosphorylation of Akt (Zhong and Virshup, 2020). Transcription of WNT3 was contradictorily upregulated by EA, indicating a limitation as a single treatment at higher concentrations. Nevertheless, the downregulation of Akt by EprACN and EA may contribute to the enhanced sensitivity of U-87 cells to TMZ.

In our combinatory study, GE-profiling of U-87 cells revealed the modulation of multiple components within the Wnt/β-catenin pathway. Combination EA35TMZ320 reduced the transcription of WNT5A, an upstream intracellular member (Yu et al., 2007; Chen et al., 2021) known to play a pro-tumor role in glioma (Zuccarini et al., 2018; Chen et al., 2021) to induce the migration of GBM cells (Lee, 2016) to increase cell proliferation (Yu et al., 2007; Chen et al., 2021) and to correlate with higher WHO histological glioma classification grades (Alkailani et al., 2022). A Wnt5a knockdown inhibited the activity of the GSK3β/β-catenin pathway related to glioma-derived endothelial cell angiogenesis (Chen et al., 2021).

Frizzleds (FZDs) are transmembrane receptors (Alkailani et al., 2022) inhibiting the β-catenin degradation complex (Shahcheraghi et al., 2020). In glioma, FZD7 is upregulated, correlating with poor patient outcomes (Zuccarini et al., 2018). The significant downregulation of FZD7 GE by all combinations is promising. Alike EA20TMZ380 significantly upregulated the GE of CTNNBIP1, which prevents the interaction of β-catenin and TCF (transcription factors) family members. Negative regulation of CTNNBIP1 correlates with higher grades of glioma (Tong et al., 2015).

To support the transcriptional findings, we measured the release of Wnt inhibitory factor (WIF1) protein via ELISA. WIF1 binds to Wnt-proteins, thereby inhibiting Wnt pathway signalling. Both single metabolites and combinations increased the release of WIF1 protein. In contrast, the combination of high concentrations of EA (40 µM) and TMZ (≥380 µM) reduced WIF1 releases, supporting the dose-dependent effects observed in the GE analyses, where higher concentrations induced an upregulation of WNT3. This inverse dose-dependent WIF1 regulation underscores the importance of optimized dosing to balance pathway modulation.

Cross-talks between signaling pathways are known to play a role in resistance development f. e. Wnt/ß-catenin signaling activates NF-kB in the cytoplasm, whereas Dvl inhibits NF-kB signaling in the nucleus (Guo et al., 2024). Even more in breast cancer, NF-kB has been confirmed to be a crucial link between resistance signaling pathways (Zhao et al., 2021). Activated NF-kB promotes the production of Wnt, ß-catenin, and ß-TrCP, which can lead to cytokine storms up to death (Guo et al., 2024; Jang et al., 2021). We did not measure NF-kB, but the gene expression of IL6, a product of NF-kB, which can activate, via STAT3, cell survival, proliferation, and inflammation (Guo et al., 2024). The resolution of inflammation is regarded as a novel host-focused option to complement existing therapies for glioma (Bazan et al., 2021). IL6 is frequently upregulated in GBM, where it activates JAK/STAT3 signaling to promote tumor cell survival, proliferation, and therapy resistance, and contributes to an immunosuppressive milieu (West et al., 2018). IL10 primarily exerts immunosuppressive effects in the GBM microenvironment by inhibiting effective anti-tumor immune responses and, in some contexts, directly enhancing glioma proliferation via JAK–STAT3 activation (Widodo et al., 2021). EprACN and TMZ increased the transcription of IL6 (RT-PCR), and IL10 transcripts were detectable. However, we previously observed that plant ingredients and acetylsalicylic acid stimulated the GE of IL6 and IL10 under non-stress conditions, which turned into an anti-inflammatory response under inflammatory conditions. Such cytokine regulations may keep the immune-regulatory system active and influence the cytokine release dynamics (Ulrich-Merzenich et al., 2017). This hypothesis aligns with Ahmad et al.‘s proposition that the simultaneous expression of IL6 and IL10 in tumor tissues improves the survival of breast cancer patients, although underlying mechanisms remain unclear (Ahmad et al., 2018).

We did not observe changes in the GE of IL4 and IL8. However, both exert their most critical actions via crosstalk with immune and endothelial cells. In a tumor-cell-only model, the roles of IL4 and IL8 would likely appear less central compared to their significance in the complex in vivo GBM microenvironment (Brat et al., 2005; Losur et al., 2024).

Furthermore, the IL-17B/ IL-17RB pathway has been implicated in tumorigenesis and resistance to anticancer therapies (Briukhovetska et al., 2021). The IL-17A/F, binding to the IL17RC receptor, demonstrated pro-tumoral effects (Briukhovetska et al., 2021). Although its precise role in tumor resistance remains unclear. Our RNAseq data revealed exclusive expression of RNA encoding IL17RC in resistant U-87 cells treated with high TMZ concentrations, suggesting that the IL-17 pathway may contribute to the development of resistance in U-87 cells, a finding warranting further study.

Brain-derived neurotrophic factor (BDNF), an endogenous signaling molecule, is involved in the carcinogenesis of glioma (Zheng and Chen, 2020), especially in tumor growth and metastasis in neuroblastoma (Chen et al., 2016), whereas a precursor of BDNF (proBDNF) plays a role in the modulation of cell apoptosis (Xiong et al., 2013). In our study, EA35TMZ320 and EprACN30 induced significant over-expression of BDNF without activating GE of the PI3K and MAPK pathway members. We hypothesize that such an effect does not induce cell growth or counteract the reduction in metabolic activity seen in U-87 cells upon different treatments.

GABAA forms a heterotetrameric complex (Huang et al., 2022). The expression of subunits α, β, and γ-subfamilies correlates with the malignancy grade of gliomas (Smits et al., 2012). Patients with high GABAA receptor-associated protein (GABARAPL1) expression levels were reported to have a lower risk for metastasis (Le Grand et al., 2011). In MCF7 cells, GABARAPL was demonstrated to inhibit Dvl2 (disheveled segment polarity protein 2), an inhibitor of the β-catenin degradation complex (Boyer-Guittaut et al., 2014). While TMZ, EA, and TMZ-EA did not affect the GE of Dvl2, TMZ and EA upregulated the GE of GABARAPL1. Thus, we suggest that the downregulation of the Wnt signaling cascade observed in our study is likely a direct effect on Wnt signaling members rather than through GABARAPL1.

This study demonstrates the potential of lichen-derived metabolites, particularly evernic acid (EA), to modulate key pathways associated with TMZ resistance in U-87 cells, suggesting a promising multitarget mechanism for future investigation. EA reduced the metabolic activity of TMZ-resistant U-87 cells, synergizing with TMZ to reduce viability by 75% at optimized ratios. The prediction of ChemGPS that EA acts on tubulin activity was supported by deep sequencing. Mechanistically, EA suppressed GE of oncogenic Wnt/β-catenin signaling while upregulating the protein expression of WIF1 as a central inhibitor of Wnt-signaling. Combinatorial EA-TMZ treatment further modulated MAPK/PI3K pathways, inhibiting ERK1/2, c-Jun, and Akt phosphorylation, which are critical for glioblastoma survival and resistance.

The discovery of IL17RC overexpression in resistant cells underscores a novel pathway implicated in TMZ resistance, warranting further exploration.

Even though present studies lack an in vivo validation, findings form a base for subsequent validation in more complex models. Future studies should clarify EA’s direct role in tubulin dynamics, its influence on the IL-17 pathway, on established mechanisms of drug resistance, such as MGMT promoter methylation status, DNA repair pathways, or efflux transporter activity in primary patient-derived cells, as well as in vivo, for example, in genetically engineered glioma models (GEGMs) or orthotopic animal models including pharmacodynamic and pharmacokinetic evaluations to explore clinical translation of EA-TMZ combinations. Integrating computational tools like ChemGPS-NP with multi-omics approaches will accelerate the development of natural product-based therapies to address refractory cancers. This work advances the paradigm of combinatorial, mechanism-driven strategies to disrupt resistance-associated pathways and enhance chemosensitivity in oncology.