The ability to adapt to changing environmental conditions is essential for cellular survival. A central feature of the eukaryotic stress response is the inhibition of bulk mRNA nuclear export, while stress-induced transcripts are specifically exported. However, the molecular mechanisms that simultaneously inhibit bulk mRNA export while mediating selective export of specific transcripts remain poorly understood. Here, we performed comparative phosphoproteomic analyses of S. cerevisiae under different stress conditions. We identified a heat shock-induced increase in phosphorylation within the N-terminal domain of the mRNA export adaptor Yra1. Preventing this phosphorylation significantly reduces nuclear accumulation of poly(A)+ RNA during heat stress and concomitantly enhances the export of heat-induced transcripts. Mechanistically, Yra1 phosphorylation appears to weaken its interaction with the export receptor Mex67, thereby contributing to nuclear accumulation of bulk poly(A)+ RNA under heat stress. Together, our findings establish Yra1 phosphorylation as a previously unrecognized regulatory mechanism that promotes nuclear mRNA accumulation during heat stress and contributes to selective mRNA export.

Background Obesity-related cardiometabolic disease is linked to impaired adipose tissue function, but the underlying molecular programs are difficult to assign to specific adipose-resident cell types, to mechanistically connect to the activation state of macrophages, and to distinguish from alterations that may normalize with weight loss. Methods We integrated a layered design combining untargeted proteomics and lipidomics to define obesity-associated, cell-type-resolved molecular phenotypes across adipocytes and adipose microvascular endothelial cells, explore whether an inflammatory milieu reproduces adipose-resident cell dysfunction, and identify features that show evidence of recovery after weight loss. Next, we validated in adipose tissue transcriptomes the elements that exhibit the strongest association with dyslipidaemia, hypertriglyceridemia, and/or hyperglycaemia to identify gene signatures of cardiometabolic relevance. Results Adipocytes from subjects with obesity show suppression of mitochondrial energy metabolism together with impaired lipid plasticity, as reflected by triglyceride remodelling. By mimicking an inflammatory milieu with macrophage-conditioned media, we reproduced many of these changes in adipocyte cultures. Endothelial cells exhibited yet another, opposite trajectory in obesity, with reduced cell-cycle signalling and increased mitochondrial activation, which were recapitulated in vitro when these cells were exposed, respectively, to the secretions of inflamed macrophages and adipocytes. Bulk adipose tissue proteomes and lipidomes showed evidence of metabolic improvement after weight loss, including restoration of mitochondrial and substrate-handling pathways alongside reciprocal triglyceride remodelling. Together with inflammation-responsive adipocyte mitochondrial and lipid-handling dysfunction, our cell-type-informed framework probes macrophage and adipocyte-to-endothelial activation in obesity, and delineates cross-context cellular programs associated with weight loss. Notably, when integrated with transcriptomic resources, these layers of information prioritized determinants linked to impaired metabolism, and were used to generate models that can assess cardiometabolic vulnerability in subjects with obesity (AUROC values between 0.88 and 0.99). Conclusions Our study reveals adipocyte and endothelial cell-specific elements acting as molecular signatures of adipose tissue inflammation with cardiometabolic implications.

BackgroundThe effects of menstrual blood-derived mesenchymal stromal cell secretome (S-MenSC) on macrophage polarization remain unclear. This study studied the impact of secretomes from basal MenSCs (S-bMenSCs) and those preconditioned with IFNγ and TNFα (S-pMenSCs) on human monocytes and macrophages in vitro.MethodsS-MenSCs were used to assess their effects on three stages of monocyte-derived cell maturation: i. monocyte differentiation; ii. polarization of monocyte-derived macrophages (MDMs) toward M1-like or M2-like phenotypes; and iii. reprogramming of pre-polarized M1 or M2 macrophages. Surface markers were analyzed by flow cytometry and cytokine gene expression by RT-qPCR. In addition, a proteomic profiling was performed to identify proteins involved in the observed effects.ResultsOur results confirmed the capacity of S-MenSCs of modulating innate immune response and in particular macrophage polarization. More concretely, the in vitro experiments showed that: i. both secretomes partly promoted monocyte differentiation into an M1-like phenotype; ii. during macrophage polarization, S-bMenSCs partially limited the shift to an M1 phenotype, whereas treatment with S-pMenSCs boosted it; and, iii. in the pre-polarized macrophages, S-bMenSCs reinforced M1 traits, whereas S-pMenSCs promote partial phenotype switching. Finally, proteomic analysis revealed significant differences in the composition of both secretomes, comprising key proteins associated with macrophage polarization.ConclusionThese findings extend the knowledge on the immunomodulatory capacity of the S-MenSC, supporting that MenSCs, particularly when preconditioned, may play a significant role in regulating macrophage polarization, and, thus, modulating the inflammatory response.

Accurate quantification of circulating proteins is critical for assessing biological variation and integrating proteomics with other omics to understand biological processes and disease mechanisms. Protein measurements, however, can be substantially influenced by preanalytical variability arising from differences in sample collection, handling, and storage, whereas technical variation introduced by the assay and workflow is typically well controlled through established validation procedures. Identifying proteins that capture these systematic influences enables their incorporation into downstream analyzes, thereby improving statistical power. In this study, we applied highly multiplexed aptamer-based affinity proteomics to plasma samples from three independent cohorts─German, Arab-Asian and Qatari to evaluate how adjusting for all measured proteins influences protein quantitative trait loci (pQTLs) associations. Using the p-gain statistic as an indicator of improved association strength, we identified clusters of proteins whose covariation patterns suggested potential preanalytical effects. One cluster contained HSP90 (Heat Shock Protein 90), a marker linked to white blood cell lysis, while others were enriched for proteins involved in complement and coagulation cascades or platelet activation. Our work presents a data-driven framework for detecting latent sources of variation in large-scale proteomic data sets and lay the groundwork for future efforts to quantify the impact of hidden confounding factors.

AIMS:A subset of endothelial cells referred to as immunomodulatory endothelial cells (IMEC) has been proposed to regulate T-cell responses in atherosclerosis and after myocardial infarction. Here, we studied the inflammation-induced emergence of IMEC and characterized their crosstalk with T cells. METHODS AND RESULTS:An in vitro model to study IMEC was characterized using flow cytometry and proteomics. Endothelial cell-specific translatome and single-cell transcriptome data from a murine atherogenesis model and single-cell transcriptome data from human atherosclerotic arteries were used to determine pathophysiological relevance. Immunopeptidomics was performed to detect antigen presentation. T-cell chemotaxis, adhesion, and activation were assessed through flow cytometry and microscopy. IMEC were induced by treating human endothelial cells with interleukin-1β, interferon-γ, and transforming growth factor-β2 and expressed lower levels of classical endothelial cell markers and disrupted VE-cadherin expression accompanied by impaired barrier function. IMEC expressed major histocompatibility complex (MHC) class II, proteins involved in antigen processing and presentation (CD83, CD80, and CD86) and pro-inflammatory cytokines as well as chemokines, including CXCL9. An IMEC-like subpopulation was identified in the lumen of carotid arteries in a mouse model of accelerated atherogenesis as well as in human atheromas. Conditioned medium from IMEC enhanced the migration of peripheral blood mononuclear cells and induced T-cell chemotaxis, which was partially inhibited by antagonizing CXCL9. IMEC exhibited a significant down-regulation of proteins related to glycosaminoglycan degradation, consistent with the key role of the glycocalyx in the establishment of chemokine gradients. Indeed, the accumulation of heparan sulphates in IMEC contributed to the adhesion of T cells. Notably, IMEC that had been exposed to monocyte lysates presented 627 peptide antigens on MHC class II and induced T-cell expansion. CONCLUSION:Our data highlight the role of IMEC as non-professional antigen-presenting cells that potentially contribute to T cell-mediated immune responses in cardiovascular disease.

Ovarian cancer (OC) progression and metastasis are promoted by ascites, which constitutes a central part of the tumor microenvironment (TME). In this fluid, tumor-associated macrophages (TAMs) represent a prominent immune cell type. In addition to tumor and other host cells such as TAMs, ascites is highly enriched in soluble factors as well as extracellular vesicles (EVs). How TAMs contribute to the EV compartment of the OC TME remains, however, underexplored. In this work peripheral blood monocytes from healthy donors were differentiated into monocyte-derived macrophages (MDMs) and polarized into classically activated (M1-like), alternatively activated (M2-like) and TAM-like (by ascites incubation). For all subtypes, serum-free conditioned medium was collected for 24 h and EVs were isolated and characterized by nano-flow cytometry (nFC), label-free mass spectrometry-based proteomics and electron microscopy, among others. Our results demonstrated distinct traits for EV release and cargo across the different macrophage subtypes. Specifically, TAM-like macrophages exhibited impaired release of small EVs and reduced frequency of tetraspanin-positive particles. These EV subpopulations displayed sizing profiles closer to M1-like than to M2-like samples. Also, the low EV release in TAM-like MDMs was accompanied by altered expression of biogenesis-related markers like flotillin-1 (FLOT1) and a decreased N-glycosylation of CD63 protein, which was validated in patient-derived samples. Remarkably, the EV-associated proteome of TAMs displayed significant enrichment in both pro- and anti-inflammatory molecules with clinical value. Markers significantly enriched in the ascites TAM-EV signature were mostly associated with poor prognosis, whereas M1-like EV-related markers (pro-inflammatory) were mostly associated with longer survival. Our results confirmed previous data for proteins like CD163 and MRC1 to be associated to TAM-EVs, while also describing novel candidates with diagnostic (i.e., COLEC12) and/or prognostic (i.e., MSR1) value in plasma. Taken together, our data support a unique secretory profile of TAMs in OC and provide new EV-associated biomarkers with translational impact. Our results pave the way for a better understanding of the mechanisms behind TAM-EV cargo loading and function, and how these cells participate in the TME landscape.

Cancer cells dynamically reprogram their metabolism to adapt to changing microenvironmental conditions during tumor growth and metastatic dissemination. Metastasis of solid tumors-the principal cause of cancer-related mortality-is often driven through activation of epithelial-mesenchymal transition (EMT), regulated by the transcription factor ZEB1, which is frequently upregulated during tumor progression. To investigate the role of metabolic plasticity in metastasis, we employed murine pancreatic ductal adenocarcinoma (PDAC) cell lines with distinct EMT states, ZEB1 expression and lung colonization capacities. Highly plastic epithelial-type cancer cells (KPCepi) efficiently colonize the lung, whereas Zeb1-deficient cancer cells (KPCZ) with compromised metabolic plasticity show markedly reduced colonization, correlated with absent glycolytic reserve, mitochondrial dysfunction, and reduced anti-oxidant metabolite levels. Interestingly, mesenchymal-type cancer cells (KPCmes) also exhibit poor lung colonization despite retaining normal glycolytic capacity and a high proportion of functional mitochondria; however, similar to KPCZ cells, they display diminished levels of detoxifying metabolites. Low metastatic capacity correlates with increased susceptibility to ferroptosis even in epithelial-type KPCZ cells, indicating a limited ability to counteract reactive oxygen species under stress. Together, these findings demonstrate that metabolic plasticity and redox homeostasis are essential prerequisites for efficient lung colonization. Thus, concurrent targeting of metabolic adaptability and redox buffering may represent a promising strategy to prevent metastasis in aggressive PDAC tumors.

The biological activity of extracellular vesicles (EVs) is largely defined by their molecular cargo, yet the impact of isolation workflows on EV proteomes and function remains incompletely understood. Here, we compared four isolation strategies for EVs derived from malignant ascites and ES-2 ovarian cancer cell culture supernatants, assessing yield, particle size, protein cargo, and EV-associated enzymatic activity. Proteomic analyses of particle-normalized preparations were performed according to MISEV2023 guidelines, and vesicle-associated protease activity was profiled using a FRET-based assay with inhibitor panels. Principal component and overlap analyses identified a common EV proteome signature for ascites and ES-2 EVs, which was complemented by workflow-dependent detection of additional proteins. Ultracentrifugation/density gradient (UC-DG) and tangential flow filtration/size exclusion chromatography (TFF-SEC) achieved the highest enrichment of canonical EV markers, whereas TFF/ultrafiltration (TFF-UF) was enriched in lipoproteins and secreted proteins. Functionally, UC-DG and TFF-SEC samples exhibited strong ADAM10-associated activity, while TFF-UF retained residual non-metalloprotease activity. These results reveal to what extent EV purification methods impact both, EV composition and function. This methodological awareness is critical for advancing EV-based biomarker discovery, diagnostics, and therapeutic platforms.

Abstract Background Endothelial cells express numerous microproteins (miPs) encoded by small open reading frames (smORFs), yet the biological function of most remains unknown. This study set out to characterize a novel 69 amino acid miP encoded within the FERM domain containing kindlin-3 transcript (miP-FERMT3), which is upregulated under inflammatory conditions. Methods Confocal microscopy was used to determine miP-FERMT3 localization, and its interaction partners were determined by mass spectrometry and immunoblotting. RNA sequencing and quantitative mass spectrometry were performed to assess transcriptional and proteomic alterations. Cell proliferation and cell cycle progression were examined by live cell imaging, EdU incorporation and flow cytometry, while senescence was determined by β-galactosidase staining, live cell imaging and RT-qPCR-based analysis of telomere length. Results In endothelial cells, miP-FERMT3 localized mainly to centriole subdistal appendages, where it colocalized with ninein and CEP170 and induced centrosome amplification. The expression of miP-FERMT3 caused cell cycle arrest and DNA damage, evidenced by γ-H2AX foci and nuclear p53 accumulation. Consistent with this, miP-FERMT3-expressing endothelial cells exhibited downregulation of genes required for cell-cycle progression and upregulation of genes involved in cell cycle inhibition and senescence. However, canonical p53 target genes were not induced and cell cycle arrest occurred independently of p53. Mechanistically, miP-FERMT3 interacted with proteins involved in ubiquitin/proteasome-dependent protein catabolism, including PSMD9, CUL2 and TRIM8, and its expression increased protein ubiquitination, centrosomal neddylation and proteasomal activity. Notably, enhanced proteasomal turnover of p21 in miP-FERMT3-expressing endothelial cells resulted in replication stress, as evidenced by increased CHK1 phosphorylation. These alterations culminated in rapid induction of cellular senescence, characterized by enlarged cell size, β-galactosidase activity, telomere shortening and a paracrine pro-inflammatory activation of naïve endothelial cells. Analyses of independent murine and human transcriptomic and proteomic aging datasets further revealed that FERMT3 expression and protein abundance increase with age. Conclusions miP-FERMT3 is a novel regulator of protein catabolism that promotes p21 degradation, replication stress and p53-independent cell cycle arrest and senescence in endothelial cells. Given the aging-associated upregulation of FERMT3 in mouse and human endothelial cells, increased miP-FERMT3 expression may contribute to the onset of vascular senescence as a hallmark of aging.

AIMS:Microproteins (miPs) translated from small open reading frames (smORFs) are crucial regulators of cell function. However, the expression and function of miPs in endothelial cells and alterations in miP expression linked with inflammation and cardiovascular disease, remain largely unexplored. METHODS AND RESULTS:An optimized proteogenomic approach combining RiboTag RNA-sequencing and mass spectrometry of the small molecular mass proteome was utilized to identify endothelial cell-specific miPs. Heart, lung, and blood vessels from endothelial cell-specific RiboTag mice and human endothelial cells were studied under homeostatic and inflammatory conditions. We identified 2739 murine as well as 1365 intracellular and 607 extracellular human endothelial cell miPs encoded from previously non-canonical (unannotated) smORFs. Vascular inflammation induced in vitro by interleukin-1β (IL-1β) and in vivo through PCSK9 overexpression, high-fat diet, and partial carotid artery ligation significantly altered smORF expression. An additional 347 miPs were detected in human serum, 23 decreasing and 31 increasing, after cardiac damage. The expression of an inflammation-induced miP encoded by an internal smORF within the proline-serine-threonine phosphatase interacting protein 2 (PSTPIP2) transcript, that is, miP-PSTPIP2, was assessed using a custom antibody. miP-PSTPIP2 expression was upregulated in IL-1β-treated human endothelial cells, in pre-atherosclerotic murine carotid arteries and detected in carotid arteries from patients with atherosclerosis. The relevance of 250 miPs for endothelial cell growth and viability was demonstrated using a high-throughput clustered regularly interspaced Short palindromic Repeats (CRISPR)/Cas9 screen. CONCLUSION:Taken together, we document the existence of a large number of human and murine miPs encoded by non-canonical smORFs and their altered expression in inflammatory conditions. The identification of secreted miPs suggests that they may also exert autocrine or paracrine functions. These novel small peptides modulate cell proliferation and survival in endothelial cells and may play a significant role in human cardiovascular disease.

Obesity-related metabolic disease is linked to impaired adipose tissue function, but the underlying molecular programs are difficult to assign to specific adipose-resident cell types, to mechanistically connect to inflammation, and to distinguish from alterations that normalize with weight loss. We integrated here a layered design combining untargeted proteomics and lipidomics to define obesity-associated, cell-type-resolved molecular phenotypes across isolated adipocytes and adipose microvascular endothelial cells, explore whether an obesity-like inflammatory milieu reproduces adipose-resident cell dysfunction, and identify molecular features that show evidence of recovery after surgery-induced weight loss. As expected, adipocytes from people with obesity show suppression of mitochondrial energy metabolism together with impaired lipid plasticity, as reflected by triglyceride remodelling. By mimicking an obesity-like inflammatory milieu with macrophage-conditioned media, we reproduced most of these changes in adipocyte cultures. Endothelial cells exhibited yet another, opposite trajectory in obesity, with reduced cell-cycle signalling and increased mitochondrial activation, which were recapitulated in vitro when these cells were exposed, respectively, to the secretions of inflamed macrophages and adipocytes. Bulk adipose tissue proteomes and lipidomes showed evidence of metabolic improvement after weight loss, with broad restoration of mitochondrial and substrate-handling pathways and reciprocal triglyceride remodelling. Alongside the inflammation-responsive adipocyte mitochondrial and lipid-handling dysfunction, our cell-type-informed framework probes macrophage and adipocyte-to-endothelial activation in obesity and delineates cross-context cellular programs that recover with weight loss. Additionally, we identified the elements that exhibit the strongest association with dyslipidaemia, hypertriglyceridemia and hyperglycaemia in individuals with obesity, confirming molecular signatures relevant to metabolic obesity in two cross-sectional samples.

Aim: Extracellular vesicles (EVs) play a pivotal role in tumor progression, influencing the tumor microenvironment. Despite significant research, the targeted analysis of EVs directly derived from primary tumors remains limited, particularly in ovarian cancer. The majority of existing studies have focused on EVs derived from peritoneal fluid (ascites), which encompasses contributions from different cell types. This study aims to isolate and characterize EVs secreted specifically by ovarian cancer spheroids derived from primary patient ascites. Methods: A three-dimensional cell culture model was employed to cultivate tumor spheroids in a defined medium, with EVs purified via differential ultracentrifugation and size-exclusion chromatography. Purified EVs were characterized by nanoparticle tracking analysis, nanoflow cytometry, and electron microscopy prior to performing high-resolution mass spectrometry. Results: This approach allowed the identification of known cancer-associated proteins, including danger molecules, which are linked to poor prognosis. Moreover, enzyme-linked immunosorbent assay (ELISA) analysis demonstrated that the ascites abundance levels of novel candidates [RAB14 (Ras-related protein Rab-14), SCAMP3 (secretory carrier membrane protein 3), and FAM3C (FAM3 metabolism regulating signaling molecule C)] correlated with patients’ progression-free survival, further validating their clinical relevance. Finally, we used the Gene Expression Profiling Interactive Analysis 2 (GEPIA2) database to compare our dataset with The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) data. Thereby, we revealed a signature of three TOP genes encoding proteins within our dataset (CORO1B, LAMP2, MSLN), which were differentially expressed in ovarian cancer patients compared to healthy individuals. Conclusion: This study provides the first proteomic profile of EVs derived directly from primary tumor spheroids, and paves the way for a better mechanistic understanding of EV-associated proteins and for the development of biomarkers or therapeutic strategies.

Despite the high prevalence of type 2 diabetes (T2D), the mechanisms driving pathology in pancreatic islet β cells remain poorly understood. We utilized a multiomics approach to evaluate the transcriptional and biochemical makeup of islets from human organ donors with T2D and nondiabetic controls. Transcriptomic (N = 10), proteomic (N = 6), and untargeted high-resolution metabolomic (N = 10) data were analyzed individually and then integrated using sparse partial least-squares regression, and differential network analysis was performed. In individual data sets, 25 transcripts, 30 proteins, and 30 metabolites were differentially abundant between T2D and nondiabetic islets, representing some pathways not previously characterized in T2D islets including purine and pyrimidine, branched-chain amino acid, and histidine metabolism. Network analysis of integrated data sets highlighted disrupted relationships among features in T2D islets compared to those from nondiabetic individuals. Fatty and amino acid metabolism and immune activity were identified as prominent drivers of the distinctions in biochemical interactions in T2D networks. Our findings also suggested greater abundance and influence of industrial chemicals, including polychlorinated and polybrominated biphenyls, in T2D islets. This pilot study demonstrates that multiomics profiling can identify candidate molecules and mechanisms impacting islet cell activity in T2D, which could represent targets for therapeutic intervention.

BACKGROUND:High expression of basal cell adhesion molecule (BCAM) is a hallmark of ovarian cancer (OC) progression. BCAM facilitates transcoelomic dissemination by promoting mesothelial cell clearance at peritoneal attachment sites of tumor cell spheroids. We investigated how BCAM mediates this effect and potentially drives other pro-metastatic functions. METHODS:The impact of BCAM on the tumor cell secretome and the mesothelial cell phenotype was analyzed by affinity proteomics, bulk and single-cell RNA sequencing, life-cell and multiphoton microscopy, biochemical and functional in vitro assays as well as a murine tumor model. BCAM manipulation involved ectopic overexpression, inducible expression and treatment with soluble BCAM. RESULTS:All forms of BCAM enhanced the secretion of cytokines that impact cell motility, mesenchymal differentiation and angiogenesis, including AREG, CXCL family members, FGF2, TGFB2, and VEGF. Notably, their levels in OC ascites were correlated with BCAM expression, and recombinant BCAM-induced cytokines triggered mesothelial-mesenchymal transition (MMT). Mesothelial cells undergoing MMT exhibited enhanced motility away from attaching tumor spheroids, leading to mesothelial clearance at spheroid attachment sites. BCAM-mediated MMT-associated transcriptional changes were also observed in subpopulations of omental mesothelial cells from OC patients, and were associated with poor survival. Consistent with the secretome data, BCAM induced endothelial tube formation in vitro and markedly promoted tumor angiogenesis in a mouse model. CONCLUSION:We have identified previously unknown functions of the BCAM-induced secretome potentially impacting distinct stages of OC metastasis. While BCAM's impact on MMT may facilitate initiation of micrometastases, neo-angiogenesis is essential for tumor growth. Taken together with the observed clinical adverse association, our findings underscore the potential of BCAM as a therapeutic target.

Olfactomedin-2 (OLFM2) is a pleiotropic glycoprotein emerging as a regulator of energy homeostasis. We here show the expression of OLFM2 to be adipocyte-specific and inversely associated with obesity. OLFM2 levels increase during adipogenesis and are suppressed in inflamed adipocytes. Functionally, OLFM2 deficiency impairs adipocyte differentiation, while its over-production enhances the adipogenic transformation of fat cell progenitors. Loss and gain of function experiments revealed that OLFM2 modulates key metabolic and structural pathways, including PPAR signaling, citrate cycle, fatty acid degradation, axon guidance and focal adhesion in 3T3 cell lines and primary human adipocytes. On the molecular level, OLFM2 deficiency in differentiated adipocytes predominantly downregulates genes involved in cell cycle. Extending these findings in vivo, both whole-body Olfm2 knockout and adipose-specific Olfm2 depletion in mice resulted in impaired adipose cell cycle gene expression, with the latter also displaying fat mass accretion and metabolic dysfunction. Collectively, our results underscore a critical role for OLFM2 in adipocyte biology, and support a causative link between reduced adipose OLFM2 and the pathophysiology of obesity.

Non-small cell lung cancer (NSCLC) is the most frequent lung cancer (LC). While erlotinib is an epidermal growth factor (EGFR) tyrosine kinase inhibitor (TKI) used in the treatment of NSCLC, it remains unclear how this FDA-approved drug affects the genome. We performed integrative multi-omics studies in human pulmonary carcinoma cells to elucidate the epigenetic mechanisms induced by erlotinib. We identified 746 genes (including 34 tumor suppressor genes, TSG) that were upregulated after treatment with erlotinib or gefitinib (another EGFR-TKI). Interestingly, 45% of the upregulated genes (including 24 TSG) were in broad domains of the euchromatin histone mark H3K4me3, and 63% (including 26 TSG) exhibited reduced levels of the heterochromatin histone mark H3K27me3 after erlotinib treament. Further, H3K27ac-specific chromosome conformation capture-based methods revealed that erlotinib significantly increased number and length of chromatin loops between promoters of upregulated genes and active enhancers. We also detected augmented chromatin accessibility after erlotinib treatment at the promoters of upregulated genes, which correlated with binding of the transcription activator FOXA2. Remarkably, we identified gene clusters that seem to be upregulated by promoters with enhancer activity (Epromoters) enriched with FOXA2. The clinical relevance of our findings was confirmed by data from The Cancer Genome Atlas, showing significantly improved survival outcomes in LC patients with high levels of FOXA2 and/or the 34 TSG found upregulated by erlotinib. Our results establish 3D genome rearrangements as molecular mechanism mediating EGFR-TKI effects in NSCLC cells, supporting the design of more specific therapies for NSCLC targeting different chromatin features. ### Competing Interest Statement The authors have declared no competing interest.

Rationale: Autophagy is a dynamic intracellular catabolic process and a quality control mechanism that maintains cellular homeostasis by degrading long-lived proteins or damaged organelles. It is facilitated by a concerted action of several autophagy-related (ATG) proteins that convert the cytosolic form of Microtubule-associated protein 1A/1B-light chain 3 (LC3BI) to the membrane-bound LC3BII. The interaction of LC3B with various proteins, like p62, ULK1, ATG4, and ATG7 has proven to be crucial in orchestrating the multiple stages of autophagy. Our current work focuses on deciphering this intricate network of protein interactions in the autophagy pathway. We previously demonstrated the localization of LC3B to lamellar bodies of the alveolar epithelial type II cells (AECII) in healthy and fibrotic (IPF) lungs & its interaction with Cathepsin A, a lysosomal protective protein. Shot-gun proteomic data for endogenous LC3B in AECII suggested a potential interacting partner, Syndecan-4 (SDC4). It is a transmembrane heparan sulfate proteoglycan that acts as a modulator of cellular signaling by interacting with a multitude of signaling and structural proteins in both the ECM & cytoplasm. Methods: Shot gun proteomics was performed for endogenous LC3B, mouse lung epithelial cells 12 (MLE12) were cultured to investigate the interaction of LC3B with SDC4 via immunoprecipitations (IP) followed by western blotting. Site-directed mutagenesis (SDM) was performed to generate mutations in the LC3 interacting region (LIR) of SDC4. In addition, we also performed immunofluorescence for co-localization studies. Results: In silico analysis revealed the presence of LIR within the transmembrane domain of SDC4. The LC3B-SDC4 interaction was established in MLE12 cells via IP and reverse IPs as well as by SDM constructs. Contrarily, other SDC family members (SDC 1-3) did not interact with LC3B. Elevated levels of SDC4 and its cytosolic and membrane-bound localization in pro-SPC positive AECII were observed in IPF as well as in LC3B-/- mice. In addition, SDC4 protein significantly increased upon autophagy inhibition, but overexpression of SDC4 did not influence the autophagy pathway. Conclusion: SDC4 is the only member of the SDC family that interacts with LC3B in a sequence-specific manner. The localization of SDC4 to fibrotic AECII as seen in IPF lungs or in LC3B-/- mice and its fluctuation upon influencing autophagy flux indicates autophagy-dependent regulation of SDC4 that may play a significant pathophysiological role in the development of lung fibrosis.

Lung adenocarcinoma (LUAD) is a biologically and clinically heterogeneous disease that poses a major challenge for prognosis and treatment. In this study, we performed proteomic profiling in a cohort of 88 LUAD patients to identify molecular subgroups and investigate their clinical relevance. Unsupervised clustering of the proteomic data allowed us to identify two distinct patient groups with different demographic, clinical, and molecular characteristics. Cluster 1 consisted predominantly of older patients and showed increased expression of immune and inflammatory pathways, including significant enrichment of Tumor Necrosis Factor (TNF) and Toll-like receptor signaling. This suggests a stronger innate immune response that may be associated with better disease control. In contrast, Cluster 2 was characterized by younger demographics, a higher proportion of female patients, and a greater frequency of smoking. This cluster showed reduced activation of immune-related pathways and a significantly shorter time to disease recurrence, suggesting a more aggressive clinical course and poorer prognosis. The differential expression of immune pathways between clusters underscores the role of the tumor microenvironment in disease progression and response to treatment. Our results demonstrate the value of integrating proteomic and clinical data to identify biologically distinct LUAD subtypes. This molecular stratification can improve the understanding of tumor behavior and inform personalized treatment strategies. Thus, proteomic profiling is a promising tool to guide biomarker-directed treatment of LUAD.