ULK1-driven autophagy modulation alters tumor-promoting pathways in triple-negative breast cancer.

Introduction
Triple-negative breast cancer (TNBC) is an aggressive and molecularly heterogeneous subtype of breast cancer that lacks expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Due to the absence of these therapeutic targets, patients with TNBC often experience poor prognosis, higher recurrence rates, and limited treatment options, with chemotherapy being the mainstay despite frequent resistance and toxicity issues [1–3].
Autophagy, a conserved lysosomal degradation pathway, plays a critical role in cellular homeostasis by recycling damaged organelles and misfolded proteins [4]. The process is tightly regulated by nutrient-sensing signaling pathways involving PI3K, AKT, AMPK, mTOR, and Unc-51-like kinase 1 (ULK1). Among these, the ULK1 complex (comprising ULK1, ATG13, FIP200, and ATG101) plays a pivotal role in initiating autophagosome formation [5]. While autophagy has been shown to support cancer cell survival under metabolic and therapeutic stress, it can also lead to cell death under specific conditions. The dual role of autophagy in cancer remains highly dependent on disease stage and tumor biology. In particular, autophagy can promote tumor growth or contribute to tumor suppression depending on the tumor profile and molecular subtype [6–9].
ULK1 has recently emerged as a key therapeutic target in cancer biology. Its dual involvement in autophagy and tumor signaling pathways makes it an attractive candidate for pharmacological intervention [10]. Small-molecule modulators such as LYN-1604 (a ULK1 activator) and MRT68921 (a ULK1 inhibitor) offer valuable tools for investigating the context-dependent role of ULK1 in cancer [11–14]. However, despite their established effects on autophagic flux, the downstream molecular consequences of ULK1 modulation at the proteomic level remain poorly defined, particularly in TNBC.
In this study, we aimed to systematically investigate the proteomic alterations induced by pharmacological modulation of ULK1 in TNBC cells using label-free quantitative LC-MS/MS. We hypothesized that ULK1 activation and inhibition may converge on distinct oncogenic or immune-related pathways, potentially offering novel therapeutic avenues.

Materials and methods

Cell culture
The human triple-negative breast cancer cell line MDA-MB-231 was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were maintained at 37 °C in a humidified incubator with 5% CO₂.
LYN-1604 and MRT68921 (MedChemExpress, USA) were dissolved in sterile water to prepare 5 mM stock solutions, which were diluted in culture medium to the required working concentrations.

Phosphoprotein isolation and ULK1 activity analysis
To determine ULK1 activity, MDA-MB-231 cells were treated with increasing concentrations of LYN-1604 (4, 8, 16, 32 µM) or MRT68921 (1, 2.5, 5, 7.5, 10 nM) for 24 h. For phosphoprotein enrichment, cells were incubated in serum-free DMEM supplemented with 10 nM Calyculin A (Thermo Fisher Scientific), a serine/threonine phosphatase inhibitor, for 15 min prior to harvesting. Cells were then lysed in a buffer containing 30 mM Tris, 7 M urea, and 4% CHAPS, and the resulting lysates were used for downstream phospho-ULK1 analysis.
Western blotting was performed using an anti-phospho-ULK1 antibody (4634T; Cell Signaling Technology, USA), with β-actin as the loading control. EC₅₀ values were calculated using the AAT Bioquest EC₅₀ Calculator [15].

Western blotting
Phosphoprotein extracts, along with total protein lysates from MDA-MB-231 cells treated with LYN-1604 (EC₅₀), MRT68921 (EC₅₀), or vehicle control, were subjected to Western blot analysis. For total protein extraction, cells were washed with 250 mM sucrose/Tris-HCl buffer (pH 7.2) and lysed in a buffer containing 8 M urea, 2 M thiourea, 4% CHAPS, and protease inhibitors, using mechanical disruption (Bullet Blender; Next Advance, NY, USA) [16]. Protein concentrations were determined by the Bradford assay (Bio-Rad, USA). Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes, and blocked in 5% non-fat dry milk. Membranes were incubated overnight at 4 °C with primary antibodies against LC3B (ab192890; Abcam, UK), phospho-ULK1 (4634T; Cell Signaling Technology), and β-actin (ab8226; Abcam). After incubation with secondary antibodies, protein bands were visualized using enhanced chemiluminescence (ECL) and quantified via ImageJ, normalized to β-actin.

Autophagy detection via fluorescence microscopy
Autophagic vesicle formation was assessed using the Autophagy Detection Kit (ab139484; Abcam) following the manufacturer’s protocol. Cells were incubated with Green Detection Reagent (1:1000) and Hoechst (1:1000) in phenol-red-free DMEM for 30 min in the dark, then visualized using fluorescence microscopy (DP71; Olympus, Japan). Autophagic flux was not directly assessed using lysosomal inhibition-based turnover assays.

Enzymatic digestion and LC-MS/MS analysis
Equal amounts of protein from control and treated MDA-MB-231 cells were digested using a filter-aided sample preparation (FASP) kit (Expedeon, Cambridge, UK), according to the manufacturer’s protocol. The resulting peptides were concentrated using a SpeedVac concentrator (Eppendorf, Germany) and reconstituted in 0.1% formic acid. Peptide concentrations were quantified using a Qubit Fluorometer (Invitrogen, USA) and normalized across all experimental groups.
Peptides were analyzed using an Ultimate 3000 RSLC nano-liquid chromatography system coupled to a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, USA). Peptide separation was performed on a reverse-phase C18 column using a linear gradient ranging from 6% to 90% solvent B (acetonitrile with 0.1% formic acid) at a flow rate of 300 nL/min. A data-dependent acquisition mode was used to select the top ten most intense precursor ions within a mass-to-charge (m/z) range of 400–2000 for subsequent fragmentation at a resolution of 70,000.
Raw data were processed using the SEQUEST algorithm within Proteome Discoverer software (version 2.2; Thermo Fisher Scientific). Search parameters included a precursor mass tolerance of 10 ppm and a fragment mass tolerance of 0.2 Da. Carbamidomethylation of cysteine was set as a fixed modification, while oxidation of methionine and deamidation of asparagine were considered variable modifications. Only peptides with > 95% confidence and proteins identified by at least two unique peptides with > 99% confidence were retained. Proteins demonstrating ≥ 2-fold changes in abundance relative to control were classified as differentially regulated proteins (DRPs) [17].

Bioinformatics and statistical analysis
Protein–protein interaction (PPI) networks were constructed using STRING (https://string-db.org) based on candidate regulated proteins prioritized using a ≥ 2-fold change screening threshold following LYN-1604 or MRT68921 treatment. To retain high-confidence interactions, STRING-derived interactions were filtered using a false discovery rate (FDR) cutoff of 1e-05. The resulting interaction networks were visualized in Cytoscape (v3.10.3). Commonly regulated proteins shared between both treatment groups were identified, and hub proteins were determined using the maximal clique centrality (MCC) algorithm implemented in the CytoHubba plugin. Reactome pathway enrichment analysis of the shared protein set was performed using the EnrichR web tool (https://maayanlab.cloud/Enrichr/), and the top 10 enriched pathways were visualized according to − log₁₀ p-value significance [18].
Statistical analyses for quantitative experiments (e.g., Western blot densitometry and vesicle quantification) were performed in GraphPad Prism (v9.1.0). Comparisons be-tween groups were conducted using one-way ANOVA followed by Tukey’s post hoc test. A p-value < 0.05 was considered statistically significant.

Results

ULK1 modulation alters phosphorylation status in TNBC cells
To assess the effects of ULK1 modulators on kinase activity, MDA-MB-231 cells were treated with increasing concentrations of LYN-1604 (4–32 µM) and MRT68921 (1–10 nM) for 24 h. Western blot analysis of phospho-ULK1 revealed a dose-dependent increase upon LYN-1604 treatment and a corresponding decrease with MRT68921. Both effects were statistically significant (p < 0.001), confirming pharmacodynamic activity of the compounds (Fig. 1). EC₅₀ values were calculated as 29.8 µM for LYN-1604 and 2.6 nM for MRT68921, and these concentrations were selected for subsequent experiments.

Pharmacological modulation of ULK1 induces autophagic responses
To evaluate autophagic activity following ULK1 modulation, MDA-MB-231 cells were treated with either LYN-1604 (ULK1 activator) or MRT68921 (ULK1 inhibitor) at their respective EC₅₀ concentrations. Autophagy-related vesicular structures were visualized by fluorescence microscopy using a FITC-conjugated autophagy detection dye. As shown in Fig. 2a, both treatments increased the appearance of green punctate vesicles compared with control cells, consistent with an autophagy-associated vesicular phenotype. The vesicular FITC signal was more prominent in the LYN-1604 group, whereas MRT68921 produced a detectable but comparatively weaker increase.

Quantitative analysis revealed that the percentage of autophagic vesicle–positive cells was significantly higher in both LYN-1604- and MRT68921-treated groups compared to the negative control (Fig. 2b, p < 0.05). Additionally, mean FITC fluorescence intensity per cell was also significantly increased in both treatment groups relative to control, with LYN-1604 inducing the strongest signal (Fig. 2c, p < 0.05). These findings suggest that both pharmacological activation and inhibition of ULK1 can increased accumulation of dye-positive vesicular structures, though with differing magnitudes.
To further corroborate these microscopy-based findings at the protein level, LC3B was assessed by Western blotting. As shown in Fig. 2d, the LC3B signal was higher in LYN-1604–treated cells than in control and MRT68921-treated cells. Densitometric analysis indicated that the LC3B/β-actin ratio increased from 0.0126 in control cells to 3.0877 in the LYN-1604 group and to 0.3067 in the MRT68921 group (Fig. 2e, p < 0.05), with a substantially stronger increase under LYN-1604 than MRT68921. Densitometric values represent normalized LC3B/β-actin ratios (arbitrary units) derived from the same analysis settings.
Collectively, these results support an autophagy-associated phenotype characterized by increased vesicle-associated fluorescence and LC3B signal accumulation following ULK1 modulation. However, because lysosomal inhibitor–based turnover assays were not performed, these readouts cannot distinguish enhanced autophagic flux from altered autophagosome maturation and/or impaired lysosomal clearance.

Quantitative proteomic profiling of ULK1 modulation in TNBC cells
Label-free quantitative proteomic analysis identified 2,519 proteins in LYN-1604-treated cells and 2,588 proteins in MRT68921-treated cells. Differential expression analysis, based on a ≥ 2-fold change relative to control, revealed 182 proteins altered in response to LYN-1604 (13 upregulated, 169 downregulated) and 196 proteins altered following MRT68921 treatment (54 upregulated, 142 downregulated) (Online Resource 1–2).
Functional enrichment analysis of these DRPs revealed distinct pathway-specific signatures associated with ULK1 activation and inhibition. In the LYN-1604 group, upregulated proteins were primarily associated with RNA polymerase II transcription termination, mRNA 3′-end processing, and RNA metabolic processes (Fig. 3a). Downregulated proteins were enriched in translational pathways, including peptide chain elongation, translation initiation, and nonsense-mediated decay (Fig. 3b).
In contrast, MRT68921 treatment resulted in an upregulation of proteins involved in immune-associated pathways, including complement activation, platelet degranulation, and vesicle-mediated transport (Fig. 3c). Downregulated proteins were predominantly associated with RNA metabolism, cap-dependent translation, and ribosomal function (Fig. 3d).
A set of commonly regulated proteins was identified between the two treatment groups. PPI networks constructed using Cytoscape revealed these shared nodes, and the top five hub proteins -PSIP1, MORF4L1, SETD2, ACO2, and HNRNPC- were identified using the MCC algorithm via the CytoHubba plugin to reflect network-based prioritization. Subsequent Reactome pathway enrichment analysis of the shared protein set, performed with EnrichR, identified the top 10 enriched pathways, including Mitochondrial Calcium Ion Transport, MET-activated PTK2 signaling, and ECM proteo-glycans (Fig. 4).

Discussion
This study presents the first proteome-wide comparison of pharmacological ULK1 activation and inhibition in TNBC cells. Our findings underscore the multifaceted role of ULK1 in regulating autophagy, transcription, immune signaling, and protein homeostasis, offering valuable insights into potential therapeutic strategies for this aggressive breast cancer subtype.
We observed that both ULK1 activation via LYN-1604 and inhibition via MRT68921 led to increased autophagic vesicle formation and LC3B accumulation. These results may initially appear paradoxical, as ULK1 is canonically required for autophagy initiation. However, prior studies have shown that ULK1 inhibition does not uniformly suppress autophagic flux; rather, it can activate compensatory or alternative pathways that maintain autophagosome formation under stress conditions [19, 20]. For instance, AMPK, a major energy sensor, can become hyperactivated upon ULK1 inhibition, potentially driving autophagy through ULK1-independent mechanisms such as Beclin 1 phosphorylation or activation of ULK2 [12, 21]. Our results support this model, suggesting that ULK1 inhibition may uncouple autophagosome initiation from flux, thereby altering the qualitative nature of the autophagic response.
Proteomic profiling revealed distinct functional outcomes depending on the direction of ULK1 modulation. ULK1 activation led to upregulation of RNA polymerase II transcription termination and mRNA 3′-end processing pathways, coupled with downregulation of translational machinery. This dichotomy suggests that ULK1 activation promotes transcriptional remodeling while conserving energy by attenuating protein synthesis -features commonly associated with integrated stress responses and autophagy-mediated survival mechanisms [22, 23]. These findings align with prior evidence linking ULK1 to nuclear functions beyond its canonical cytoplasmic role, including regulation of mRNA dynamics and epigenetic signaling [11, 22, 23].
Conversely, ULK1 inhibition reprogrammed the proteome toward immunological engagement. We observed increased abundance of proteins annotated to complement-related terms, vesicle-mediated transport, and platelet degranulation, a processes frequently associated with immune signaling and antigen presentation. Although ULK1 is a canonical initiator of autophagy, our findings demonstrate that its inhibition with MRT68921 does not fully suppress autophagic activity; instead, it appears to induce compensatory autophagy through ULK1-independent mechanisms. This autophagy-associated phenotype (LC3B/vesicle signal accumulation) may reflect compensatory stress responses and/or altered autophagosome maturation/clearance; although increased autophagic flux was not directly demonstrated in this study, autophagy-related processes could still intersect with immune signaling by facilitating intracellular antigen presentation via MHC class I, promoting DAMP release, and modulating inflammatory responses [24]. Supporting this, Li Jin et al. revealed that LPS-induced activation of the MAPK p38/ULK1 pathway inhibits autophagy and induces IL-1β expression in hepatic stellate cells, highlighting the interplay between autophagy inhibition and immune activation [25]. Similarly, He Y et al. found that p38 MAPK inhibits autophagy and promotes microglial inflammatory responses by phosphorylating ULK1, suggesting that ULK1 activity modulates immune responses through autophagy regulation [26]. Notably, MRT68921 has been shown to increase immunoproteasome function and reverse immune evasion in LKB1-deficient cancers, suggesting a broader application of ULK1 inhibitors in immunotherapy [27]. Thus, rather than resulting from autophagy blockade, the immune-related enrichment signature observed in the proteomic data may reflect a distinct stress phenotype in which compensatory autophagy and disrupted proteostasis collectively enhance tumor immunogenicity.
Importantly, a subset of proteins was consistently regulated under both ULK1 activation and inhibition, suggesting that specific cellular functions are highly sensitive to ULK1 perturbation regardless of directionality. Protein–protein interaction network analysis revealed five common hub proteins: PSIP1 (LEDGF/p75), AGO2, MORF4L1, HNRNPC, and SETD2. Functional enrichment analysis indicated their involvement in autophagy-associated pathways such as mitochondrial calcium ion transport, MET–PTK2 (c-Met–FAK) signaling, extracellular matrix (ECM) remodeling, FasL/CD95 death receptor signaling, and cholesterol metabolism.
These pathways have been implicated in autophagy regulation and TNBC biology in prior studies; however, our proteomic data provide association-level evidence rather than functional confirmation. For example, autophagy can promote lysosomal degradation of the MET receptor, dampening downstream MET–FAK signaling and limiting invasive behavior [28]. Similarly, the ULK1-FIP200 complex facilitates turnover of focal adhesion components, thereby regulating cell adhesion and motility through FAK modulation [29]. Mitochondrial Ca²⁺ flux, a central node in autophagy signaling, governs cross-talk with apoptotic programs [30], while autophagy has also been shown to modulate death receptor pathways (e.g., Fas/CD95) and promote lipid droplet degradation and cholesterol efflux via HDL [31].
Our findings extend this knowledge by suggest that ULK1 modulation is associated with coordinated changes in hub proteins and pathway signatures implicated in TNBC biology; the functional consequences remain to be tested. We propose that these signatures represent testable hypotheses; whether they translate into tumor-suppressive phenotypes requires proliferation/migration/invasion assays.
Indeed, previous studies support this rationale. PSIP1 (LEDGF/p75), a chromatin-bound transcription coactivator, has been implicated in chemoresistance and metastasis in TNBC. Its silencing significantly impaired tumorigenicity and metastasis in vivo, highlighting its role in cancer aggressiveness [32]. AGO2, an essential component of the RNA-induced silencing complex (RISC), facilitates microRNA-mediated post-transcriptional repression. Overexpression of AGO2 has been associated with enhanced proliferation, therapy resistance, and epithelial-to-mesenchymal transition (EMT) in breast cancer, including TNBC [33]. MORF4L1, a chromatin remodeling factor involved in histone acetylation, has been linked to stemness, metastatic potential, and altered transcriptional landscapes in breast cancer. Its role in epigenetic regulation suggests potential as a therapeutic target in aggressive subtypes [34]. HNRNPC, a heterogeneous nuclear ribonucleoprotein, governs alternative splicing and mRNA stability. In basal-like and triple-negative breast cancer, its overexpression promotes tumor growth, and correlates with poor survival outcomes [35, 36]. SETD2, the sole H3K36 trimethyltransferase, plays a critical role in maintaining genomic integrity, DNA repair, and transcription fidelity. Mutations or reduced expression of SETD2 contribute to tumorigenesis and therapy sensitivity in TNBC, particularly in relation to PARP inhibitor response [37, 38].
These findings support the hypothesis that ULK1 modulation may exert anti-tumor effects in TNBC via coordinated regulation of these hub proteins. Targeting PSIP1, AGO2, MORF4L1, HNRNPC, and SETD2 offers a novel strategy to disrupt interconnected networks of transcriptional control, RNA processing, and chromatin remodeling—all of which are critical to the aggressive phenotype and therapy resistance of TNBC.
Collectively, our findings highlight the potential of modulating autophagy-associated networks in TNBC as a therapeutic strategy. While ULK1 remains a central regulator of autophagy, the identification of downstream targets -particularly PSIP1 (LEDGF/p75), AGO2, MORF4L1, HNRNPC, and SETD2- opens the door to more precise interventions. These proteins, which may act as intrinsic barriers to autophagy-linked tumor suppression, represent promising molecular entry points for future combination therapies. Ultimately, therapeutically targeting this specific set of hub proteins may provide a means to replicate the multi-faceted anti-tumor benefits of autophagy, while circumventing the challenges of direct ULK1 modulation.
This study has several limitations that should be considered when interpreting the findings. First, our autophagy readouts were based on an autophagy detection dye and LC3B immunoblotting, and we did not perform lysosomal inhibitor–based turnover assays to directly assess autophagic flux; therefore, the observed increases in vesicle-associated fluorescence and LC3B signal may reflect enhanced autophagosome formation and/or altered autophagosome maturation with impaired lysosomal clearance rather than confirmed increases in flux. Second, the experiments were conducted in a single TNBC cell line (MDA-MB-231), without additional TNBC models or non-malignant breast epithelial controls, which limits the generalizability of the proteomic signatures across TNBC subtypes and normal tissues. Third, while ULK1 was pharmacologically modulated using LYN-1604 and MRT68921, we did not perform genetic perturbation or orthogonal compound validation to confirm ULK1 dependency and to exclude potential off-target contributions; thus, conclusions should be interpreted as reflecting pharmacological ULK1 pathway modulation under the tested conditions. Fourth, the proteomic analyses are primarily hypothesis-generating and were not systematically corroborated by independent assays for key candidates, and no functional phenotypic assays (proliferation, migration, invasion) or in vivo validation were included. In addition, candidate proteins were prioritized using a ≥ 2-fold screening threshold; therefore, these candidate lists should be interpreted as hypothesis-generating and may include false positives in the absence of formal differential-abundance hypothesis testing with multiple-testing correction. Accordingly, enrichment results should be interpreted as pathway-level associations rather than direct evidence of functional phenotypes, and the highlighted pathways and hub proteins should be viewed as prioritized candidates for future mechanistic and translational studies.

Conclusion
This study provides the first comparative proteomic analysis of pharmacological ULK1 activation and inhibition in TNBC cells, offering novel insights into the autophagy landscape of this aggressive cancer subtype. While previous studies have established ULK1 as a central autophagy regulator, our findings go further by demonstrating that ULK1 modulation elicits distinct yet converging proteomic signatures, particularly in-volving immune signaling and protein synthesis pathways. Most notably, we identify a shared set of downregulated hub proteins - PSIP1, AGO2, MORF4L1, HNRNPC, and SETD2- implicated in autophagy-linked pro-oncogenic signaling, including pathway terms related to MET–FAK signaling, mitochondrial Ca²⁺ handling, ECM-associated processes, and immune-related signatures. Our findings suggest that ULK1 modulation is associated with converging proteomic signatures; functional and mechanistic validation is required to determine phenotypic relevance. Importantly, these hub proteins represent candidate targets; therapeutic implications remain hypothesis-generating pending functional and in vivo validation. These insights lay the foundation for novel, more selective treatment strategies that harness the benefits of autophagy modulation in aggressive breast cancer subtypes like TNBC.

Supplementary Information
Below is the link to the electronic supplementary material.