Molecular Oncology

Neutrophils display marked functional plasticity in cancer; however, it remains poorly understood how soluble factors derived from hypoxic and irradiated head and neck squamous cell carcinoma (HNSCC) cells reprogram neutrophil phenotype and function. Here, we employed a well characterized and controlled in vitro model to examine how tumor-conditioned media (TCM) from HNSCC cell lines cultured under ambient (21% O2) or hypoxic (1% O2) conditions, with or without 6 Gy gamma irradiation, modulate human neutrophil phenotype, and functional and transcriptional responses. Initial analyses were performed using TCM from three different HNSCC cell lines, whereas subsequent mechanistic characterization focused on FaDu-derived TCM. We show that TCM prolongs neutrophil survival in a cell line-dependent manner. Among the tested cell lines, hypoxia-conditioned FaDu-derived TCM promoted immunomodulatory neutrophil state characterized by enhanced survival, selective priming of ROS production, and elevated TRAIL-R3/TRAIL-R2 ratio. Induction of classical activation markers (CD11b, CD62L) was not evident. Transcriptomic analysis revealed minimal effects of normoxic TCM. Hypoxia-conditioned TCM induced a pronounced transcriptional program enriched in hypoxia- and stress-associated pathways. In contrast, irradiation of tumor cells had a limited additional impact on neutrophil reprogramming. Together, these findings indicate hypoxia-conditioned tumor secretomes as important drivers of neutrophil functional adaptation in vitro, supporting a model in which soluble factors alone are sufficient to induce a persistent, immunomodulatory neutrophil phenotype. This work provides mechanistic insight into tumor-neutrophil crosstalk, highlighting hypoxia-driven signaling as a potential therapeutic target in radioresistant HNSCC and supporting a role for neutrophil reprogramming in this context.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA (lncRNA) implicated in cancer progression. In thyroid cancer, MALAT1 has been proposed as a potential biomarker, but its role in disease progression remains incompletely understood. Here, we analyzed MALAT1 RNA levels in paired tumoral and adjacent non-tumoral thyroid samples from a Chilean patient cohort. We found a positive correlation of MALAT1 levels with lymphatic infiltration that was not replicated when modeling MALAT1 expression in a larger cohort obtained from the TCGA-THCA database. An exploratory RNA-seq comparison of one matched tumor-adjacent tissue pair confirmed higher tumor abundance of MALAT1 and the epithelial-to-mesenchymal transition-marker VIM, together with lower abundance of cell-adhesion gene PCDH10. To investigate the impact of MALAT1 on thyroid cancer and cellular metabolism, we targeted MALAT1 in the papillary thyroid cancer cell line TPC1. MALAT1 knock-down reduced proliferation and migration while enhancing mitochondrial respiration with no changes in glycolysis. Notably, although MALAT1 was not localized within mitochondria, its silencing modulated the expression of transcripts associated with mitochondrial dynamics and mitophagy. Consistent with these results, transcriptomic correlation analysis in the TCGA-THCA cohort showed that MALAT1 expression was largely uncoupled from oxidative phosphorylation and glycolysis gene programs, while negatively correlating with core regulators of mitophagy and mitochondrial dynamics, pointing to a link with mitochondrial quality control rather than direct bioenergetic reprogramming. Our findings highlight MALAT1 as a contributor to thyroid cancer aggressiveness and reveal a link between MALAT1 and mitochondrial quality control independent of direct mitochondrial localization. Besides, our results support a tissue-specific mechanism and population-specific role of MALAT1 in cancer biology.

Introduction. TAC/SARIFA has been introduced as a new robust and easy-to-evaluate biomarker in several cancer entities, including colorectal cancer. It is defined by direct contact between at least five tumour cells and one adipocyte and is believed to indicate metabolic reprogramming associated with adverse outcome. However, the mechanism that leads to TAC/SARIFA positivity remains unclear. To investigate whether there is an individual component, we conducted a study on double and triple cancers, establishing a within patient design. Methods. We retrospectively analysed a total of 135 cases with 276 colorectal cancers from two academic medical centres. The TAC/SARIFA status was evaluated, as were the basic histopathological factors. The median follow-up time was 120 months. Results. Cases with any TAC/SARIFA positive tumours showed significantly reduced overall survival (62 vs. 88 months; p = 0.011). Analysing the entire cohort, the rates of concordant and discordant cases followed a random distribution. However, restricting the analysis to synchronous pT3/4 cases revealed a significant deviation from a random distribution (p = 0.016). Conclusion. This study reveals significant concordance of TAC/SARIFA status in synchronous locally advanced colorectal double/triple carcinomas, supporting the concept that tumour adipocyte interaction reflects a host related microenvironmental condition linked to metabolic reprogramming rather than a purely tumour intrinsic event.

AbstractAutophagy and Toll-like receptor (TLR) signaling are both implicated in cancer progression, but whether they act as tumor suppressors or promoters remains unclear. To explore this relationship, we investigated the role of autophagy downstream of therapeutic TLR activation in oral squamous cell carcinoma (OSCC). Using the FDA- approved TLR agonists Bacillus Calmette-Guerin (TLR2/4) and imiquimod (TLR7), we assessed their effects in vitro on SCC-4 cells and in vivo in a chemically induced OSCC hamster model. Both agents, alone or in combination with each other or with Monophosphoryl Lipid-A induced robust autophagy, as measured by LC3B staining in vitro and flow cytometry in vivo. Autophagy induction correlated with reduced tumor volume and prolonged survival, with outcomes comparable to cisplatin, the current standard chemotherapeutic, but with less treatment-associated morbidity and mortality. Autophagy was also associated with cisplatin antitumor effects. Notably, imiquimod produced the most pronounced and sustained autophagic and antitumor effects. To our knowledge, this is the first study to directly link the therapeutic efficacy of TLR agonists in OSCC to autophagy modulation, providing both mechanistic and translational insights into their potential as immunotherapeutic agents.

CD47 is a ubiquitously expressed transmembrane protein that functions as a negative immune checkpoint, marking host cells as "self" by delivering an inhibitory "dont-eat-me" signal to phagocytes. Cancer cells co-opt this mechanism, upregulating CD47 to evade immunosurveillance and phagocytosis by innate immune cells, a pattern observed across solid tumours and haematological malignancies. CD47 overexpression correlates with poor prognosis across most cancer types, including therapy-resistant disease. Despite extensive efforts to develop CD47-targeted therapies, the downstream biological consequences of aberrant CD47 expression within tumour cells remain poorly characterised beyond its established anti-phagocytic role. This study investigated non-immunological, pro-tumorigenic functions of CD47 to define the cellular effects, beyond immune evasion, that CD47-targeted therapy might disrupt. We found that CD47 exerts cancer type-specific effects: in DLBCL, CD47 loss impaired mitochondrial metabolism and sensitised cells to R-CHOP standard-of-care chemoimmunotherapy, whereas in triple-negative breast cancer (TNBC), CD47 knockdown delayed cell cycle progression, enhanced migration, and conferred resistance to specific chemotherapeutic agents. These findings indicate that CD47 has multifaceted, context-dependent roles in tumour biology that extend beyond immune checkpoint signalling. Clinically, this suggests CD47-targeted therapies may produce cancer type-specific off-target effects on tumour metabolism, proliferation, and drug sensitivity, which are considerations that should inform their rational combination with existing targeted therapies.

Integrated prognostic signatures combining ferroptosis, cuproptosis, and disulfidptosis are increasingly reported in oncology as advances in risk stratification, yet their added value over simpler pathway-specific or proliferation-related models remains unclear. Here, we developed an integrated regulated cell-death signature and evaluated it through an adversarial pan-cancer benchmark. Using the TCGA pan-cancer cohort comprising 9,808 tumours across 33 cancer types, we curated 118 genes associated with the three cell-death programmes, characterised inter-pathway crosstalk, and derived a 26-gene LASSO-Cox risk signature. The model showed reproducible prognostic performance across cancers, with a pan-cancer concordance index of 0.573 (95% CI, 0.552-0.594), and was independently validated in METABRIC and CGGA cohorts, remaining significant after adjustment for standard clinical variables. However, benchmarking revealed that the integrated signature, although superior to size-matched random gene sets (empirical p < 0.001), did not outperform a ferroptosis-only model (DeLong p = 0.81), indicating no measurable gain from pathway integration. Moreover, much of the prognostic signal reflected tumour proliferation rather than regulated cell death. After adjustment for the proliferation meta-signature (meta-PCNA), ferroptosis performance declined from 0.573 to 0.504, while the integrated model decreased to 0.554. High-risk tumours were more sensitive to anti-proliferative drugs, and the risk score was most strongly associated with E2F, MYC, and G2M target programmes. The signature stratified prognosis but did not predict immune-checkpoint blockade response in IMvigor210 (AUC {approx} 0.50). Importantly, the underlying biology was not merely a modelling artefact. Signature genes showed concordance with protein abundance in CPTAC cohorts, and the three cell-death programmes co-varied within individual malignant cells, with correlations ranging from {rho} = 0.46 to 0.66. Overall, our findings indicate that integrated multi-death signatures are reproducible and biologically grounded, yet prognostically redundant and substantially confounded by proliferation. This study provides a cautionary benchmark for the rapidly expanding use of composite regulated cell-death signatures in cancer prognosis.

Despite continuous progress in diagnosis and therapy, breast carcinoma (BC) remains a major health problem. Triple-negative (Estrogen Receptor-/Progesterone Receptor-/HER2-) breast cancer (TNBC) is the most aggressive subtype due to its high metastatic potential and resistance to chemotherapy. The Y-box binding protein 1 (YB-1) transcription factor, a protein present in both cytoplasm and nucleus, is a driver of TNBC malignancy as it stimulates its cancer stem cell phenotype and disrupts cell cycle progression. Here, we hypothesized that YB-1-containing sEVs deliver YB-1 to the nuclear compartment of recipient cancer cells and play a major role in the activation of the metastatic process. We found a selective enrichment of YB-1 in sEVs from MDA and 4T1 cells, with [~]65% and 50% of all sEVs positive for YB-1 by d-STORM. Administration of sEVs from wild-type MDA and 4T1 to their YB-1 knockout counterparts resulted in nuclear translocation of sEV-associated YB-1 and increased tumorsphere formation. Pharmacological blockade of the nuclear transport machinery based on the inhibition of the formation of the "VOR" complex (VAP-A-ORP3-Rab7) by PRR851 impaired both nuclear translocation and the YB-1-induced increase in tumorsphere formation. YB-1 phosphorylation at S102 was required for nuclear localization. In fact, loss of YB-1 phosphorylation inhibited tumorsphere growth and stemness of cancer cells and YB-1-positive sEVs restored the oncogenic behavior of cancer cells expressing phospho-mutant YB-1. Moreover, PRR851 inhibited the nuclear translocation of the phosphorylated form of YB-1 and the oncogenic behavior of the TNBC cells. These data support the conclusion that the nuclear translocation of sEV-associated phosphorylated YB-1 is an important factor in the malignant behavior of TNBC and a potential therapeutic target.

The m6A methyltransferase METTL3 functions as a critical oncogenic driver in triple-negative breast cancer (TNBC). However, its specific downstream targets and mechanistic functions in less metastatic TNBC subtypes remain poorly characterized. To address this, we evaluated METTL3 expression and the phenotypic effects of its siRNA-mediated knockdown in normal mammary epithelial (MCF10A), low-metastatic TNBC (HCC1143), and high-metastatic TNBC (MDA-MB-231) cell lines. We assessed global m6A levels, cell viability, cell cycle progression, and migration. To uncover specific downstream pathways, transcriptomic profiling was performed on HCC1143 cells, followed by RT-qPCR validation and m6A site prediction. METTL3 depletion reduced global m6A levels and cell viability across all cell lines. Notably, in low-metastatic HCC1143 cells, METTL3 knockdown induced a pronounced G2/M cell cycle arrest and dramatically impaired migratory capacity. Transcriptomic analysis of HCC1143 revealed altered expression of genes associated with the observed phenotypic changes. Specifically, critical transcripts harboring predicted m6A motifs, including LIMK1, CCNB2, and CDH1, were significantly dysregulated, pointing to potential alterations in pathways governing cytoskeletal remodeling, actin organization, and cell-cell adhesion. Taken together, we propose that METTL3 promotes cell viability and motility in low-metastatic TNBC by regulating key transcripts involved in cell cycle progression and actin dynamics. Significance StatementEpitranscriptomic studies on TNBC predominantly focus on highly metastatic models, leaving less aggressive subtypes poorly understood. This study uniquely addresses this gap by investigating the function of METTL3 in HCC1143, a low-metastatic TNBC cell line, alongside aggressive TNBC cell lines. We discovered that METTL3 depletion uniquely triggers a severe halt in cell division (G2/M arrest) in HCC1143 cells, while universally disrupting actin-associated cell motility across different backgrounds. These findings demonstrate that METTL3 acts as a context-dependent modulator of cell fate rather than a monolithic driver. Ultimately, highlighting these distinct cellular responses underscores the need to consider specific molecular backgrounds when evaluating epitranscriptomic targets in heterogeneous cancers, such as TNBC.

Human papillomavirus (HPV)-related and HPV-unrelated oropharyngeal squamous cell carcinomas (OPCs) are distinct entities with different clinical outcomes. While p16 immunohistochemistry (IHC) is widely used as a surrogate marker for HPV-driven OPC, a subset of HPV-unrelated OPCs also overexpress p16, and the biological basis of this discordance remains unclear. Here, we performed integrated clinicopathological, transcriptomic, genomic, and functional analyses of OPCs and demonstrated that dysregulation of the p16-CDK6 axis characterizes HPV-unrelated p16-positive OPCs. Although these tumors closely resembled HPV-unrelated p16-negative OPCs in their clinicopathological and transcriptomic characteristics, they exhibited a more favorable prognosis. CDK6 was recurrently upregulated in HPV-unrelated OPC regardless of p16 status and was already detectable in high-grade dysplastic leukoplakia, suggesting that CDK6 activation is an early event in HPV-unrelated tumorigenesis. In experimental models, CDK6 overexpression induced compensatory p16 upregulation, creating selective pressure for subsequent CDKN2A inactivation. Consistent with this model, homozygous CDKN2A loss predominated in p16-negative tumors. We further identified CDKN2A frameshift mutations generating p14ARF-p16 chimeric proteins that retain p16 immunoreactivity despite functional loss of wild-type p16, revealing a previously unrecognized diagnostic pitfall of p16 IHC. These findings provide a biological framework for p16 overexpression in HPV-unrelated OPC and suggest that assessment of the p16-CDK6 axis may refine molecular classification and risk stratification beyond p16 IHC alone.

Fluoropyrimidine-based chemotherapies, including 5-fluorouracil (5-FU) and floxuridine (FuDR), are widely used in cancer treatment, but their efficacy is limited by adaptive resistance driven by TYMS upregulation. The upstream mechanisms controlling TYMS expression remain poorly defined. Here, we identify INPPL1 (SHIP2) as a critical regulator of TYMS expression and fluoropyrimidine response in breast cancer cells. We show that SHIP2 enhances basal and drug-induced TYMS expression at the transcriptional level independently of its phosphatase activity. Mechanistically, SHIP2 increases SRC levels and nuclear accumulation of {beta}-catenin, driving TYMS expression. Inhibition of SRC or {beta}-catenin suppresses TYMS induction and restores sensitivity to FuDR. Importantly, SHIP2 rewires TYMS regulation from a P53-dependent program to a {beta}-catenin-driven pathway, enabling sustained TYMS expression under chemotherapeutic stress. Consistent with this model, differential sensitivity to SHIP2 depletion correlates with baseline TYMS levels across cell lines. Analysis of patient cancer datasets reveals that high INPPL1 expression correlates with increased TYMS levels and poor clinical outcomes. These findings identify SHIP2 as a non-canonical regulator of TYMS and a potential therapeutic target to overcome fluoropyrimidine resistance.

BCL2L13 is a mitochondrial BCL2 family protein linked to mitophagy and ceramide metabolism, but its role in NSCLC metastatic plasticity remains unclear. Human lung cancer Tissue Microarray and matched patient specimens showed subtype and site dependent BCL2L13 expression, with higher cytoplasmic granular staining in primary NSCLC and reduced, heterogeneous staining in lymph node metastases, most evident in adenocarcinoma and squamous cell carcinoma. Because Epithelial mesenchymal transition and anoikis resistance are central requirements for metastatic dissemination, this primary to node attenuation provided the rationale to test BCL2L13 knockdown and overexpression in metastasis relevant NSCLC models. In A549 and LLC cell lines. TGF beta 1 induced coordinated mitophagy and EMT with mitochondrial enrichment of BCL2L13. BCL2L13 knockdown impaired TGF beta 1 and carbonyl cyanide m chlorophenyl hydrazone associated mitophagy, reducing LC3 beta mitochondria colocalization, TOMM20, LAMP1 overlap and mitochondrial LC3 II, p62, TOMM20 turnover; BNIP3 and NIX redistribution did not compensate. BCL2L13 loss enhanced EMT marker switching and migration, whereas overexpression partially opposed these changes. During detachment, BCL2L13 knockdown reduced anoikis associated apoptosis despite preserved mitochondrial recruitment of BAX, BAK, BNIP3,NIX, altered BID processing, non parallel caspase activity and shifted FAK phosphorylation. Pharmacological autophagy modulation did not reverse this anoikis phenotype. Lipidomics identified adhesion state dependent ceramide synthases CerS2, CerS6 linked sphingolipid remodeling: BCL2L13 knockdown increased C24 linked sphingolipid species in attached cells but reduced C16, C24 ceramide related profiles during anoikis. These findings identify BCL2L13 downregulation as a metastasis associated mitochondrial-lipid state that limits mitophagic quality control while favoring EMT and detachment survival in NSCLC adenocarcinoma.

Long non-coding RNAs (lncRNAs) have attracted increasing interest because of their roles as modulators of tumor progression, acting either as oncogenic drivers or tumor suppressors, depending on the cellular context. LINC01133 has been implicated in regulation of multiple tumor-related mechanisms; however, its role in breast cancer, particularly in the triple-negative subtype, remains poorly characterized. In this study, we investigated the impact of LINC01133 depletion on malignant phenotypes and on the expression of migration- and invasion-associated genes using the Hs578T triple-negative breast cancer (TNBC) cell line, through comparative analyses of parental, control, and LINC01133-knockout cell lines, namely Hs578T_wt, Hs578T_ctr, and Hs578T_ko. Functional characterization included morphological analysis, growth assays, anchorage-independent colony formation, migration, invasion, and quantitative biomolecular experiments. Depletion of LINC01133 led to reduction of cell diameter, a significant increase in colony-forming capacity, and marked enhancement of migratory and invasive potential. At the molecular level, LINC01133 loss induced the expression of genes associated with extracellular matrix remodeling and cellular plasticity, including fibronectin, vimentin, integrins, FOXC1, and TWIST1, concomitant with reduced expression of ZEB1, TWIST2, and N-cadherin. Collectively, these data indicate that LINC01133 acts as a potential fine regulator of in vitro migration and invasion processes in TNBC, with its expression favoring a more asymptomatic mode of tumor progression, whereas its loss markedly enhances tumor malignancy.

Breast cancer is a heterogeneous disease whose clinical management relies heavily on accurate molecular subtyping. The murine E0771 mammary carcinoma cell line is widely used in preclinical studies, yet its molecular identity remains controversial, with reports variably classifying it as luminal B or triple-negative. In this study, we performed an integrated molecular and functional characterization of two independently sourced E0771 cell line stocks to resolve this discrepancy. Both stocks were genetically authenticated and exhibited concordant phenotypes. Immunohistochemical and molecular analyses demonstrated absence of oestrogen and progesterone receptors, classifying E0771 as triple-negative. Functionally, E0771 cells showed no transcriptional response to oestrogen and displayed resistance to endocrine therapy both in vitro and in vivo. Collectively, our results establish E0771 as an oestrogen-independent, basal-like triple-negative breast cancer model, supporting its appropriate use in studies of hormone-resistant breast cancer biology.

G protein-coupled receptor (GPCR) signaling represents a critical interface between oral bacteria and host cellular regulation in oral squamous cell carcinoma (OSCC). Here, we integrated systems biology, exploratory machine learning, and structure-based drug design to characterize potential associations between bacteria-related signaling and autophagy and to identify candidate therapeutic targets. Taste-associated signaling genes belonging to the GPCR superfamily were curated from KEGG, while OSCC- and autophagy-associated proteins were obtained from STRING, Reactome, UniProt, KEGG, and HMDB. Ten bacteria-associated host-interaction datasets were integrated using NetworkAnalyst to construct protein- protein interaction networks, and key hub nodes were identified through degree and betweenness centrality. Feature matrices derived from network topology were analyzed using exploratory dimensionality reduction (PCA), hierarchical clustering, and supervised models (SVM and Gradient Boosting) to assess whether network-derived features showed separability according to literature-informed bacterial reference categories; a Dysbiosis Index was additionally calculated. Results suggested that bacterial sensing through taste-associated GPCR signaling may converge on a MAPK1-centered axis linking calcium signaling, autophagy, and oncogenic pathways. Pathobiont-associated networks showed greater representation of inflammatory and terminal-autophagy-related signaling through MAPK1-STAT3, whereas commensal-associated networks were more closely aligned with cytoprotective autophagy through balanced MAPK1-TP53/PTEN networks. Exploratory machine learning analyses highlighted MDM2 and AKT3 as high-contribution, network-associated candidate features linked to group separability within the current dataset. A dual-target MTDL (SG101) was designed to target downstream nodes (MDM2 and JAK2), showing favorable predicted docking interactions and computationally predicted ADMET properties. In conclusion, bacteria-associated host taste signaling may be linked to differing autophagy-related network states in OSCC, and targeting downstream regulatory hubs with multi-target ligands represents a hypothesis-generating strategy that warrants experimental validation for pathway-oriented therapy.

Despite advances in screening and treatment, breast cancer remains a leading cause of cancer-related mortality. APOBEC enzymes, particularly APOBEC3B (A3B), are upregulated in many cancers, contributing to a characteristic C-to-T mutational signature found in 30-50% of breast cancers. However, the relationship between A3B mutational signatures and A3B expression across subtypes, and the resulting potential biologic consequences, have not been fully defined. Using TCGA and ICGC datasets, we analyzed DNA and RNA expression data to assess the relationship between A3B mRNA expression and APOBEC enrichment scores. Pathway enrichment analyses (KEGG, GO, Reactome) were performed to identify biological processes associated with high A3B expression, specifically stratifying by breast cancer intrinsic subtypes (HR+/HER2-, HR+/HER2+, HR-/HER2+, and TNBC). Over 64% of tumors with enriched A3B mutational genomic signatures demonstrated above-median A3B mRNA expression (p < 0.001). High A3B-expressing tumors exhibited specific alterations in drug metabolism pathways. Notably, we observed reduced expression of CYP2D6 and CYP3A isoforms which is required for the conversion of tamoxifen to its active metabolites. Conversely, genes involved in pyrimidine metabolism, including IMPDH1, NME1, TK1, and DPYS, were downregulated in high A3B tumors. Elevated A3B expression correlates with mutational signatures and may contribute to impaired tamoxifen activation and endocrine resistance, while concurrently creating metabolic vulnerabilities to pyrimidine-based chemotherapies. Targeting A3B or exploiting these metabolic dependencies may improve therapeutic response in selected patient subsets.

Aims: Serrated lesions contribute substantially to colorectal cancer (CRC), while routine management of small distal hyperplastic polyps (HPs) assumes low risk. Surveillance guidelines nevertheless incorporate uncertainty at the HP/SSL interface and recommend shortened intervals for large serrated lesions. We tested whether fibroblast activation protein-alpha; (FAPalpha) expression by stromal fibroblasts within expert-reviewed HPs stratifies risk of subsequent neoplasia. Methods and results: In a single centre historical cohort, FAPalpha; immunohistochemistry (Abcam ab53066, 1:200) was performed on FFPE colon tissues from 64 patients (normal colon n=10; HP n=39; low grade TA n=6; high-grade TA n=4; adenocarcinoma n=5). FAPalpha positive stromal fibroblasts were quantified in 20 randomly selected fields at magnification 1000 by two blinded readers (ICC 0.93). Among 39 patients with expert reviewed index HPs and colonoscopic follow up, the endpoint was metachronous adenoma occurring in the same general colonic area as the index HP, with proximal defined as ascending colon and distal as descending colon. Follow-up colonoscopies were scheduled every 2 years for up to 10 years. ROC analysis identified an optimal threshold of [&ge;]9 FAPalpha positive fibroblasts (AUC 0.8658; sensitivity 81.25%, specificity 87.93%). FAPalpha high status (44% of HPs) was associated with shortened neoplasm free survival (log-rank p=0.0012): five-year neoplasm free survival 41% versus 91% for FAPalpha; no/low. In multivariable Cox modelling, FAPalpha high status remained independently associated with metachronous adenoma (HR 4.5, 95% CI 1.2-16.8, p=0.022). Conclusion: FAPalpha+ fibroblasts in expert-reviewed colorectal HPs identify a high-risk subgroup for metachronous adenoma, supporting stromal activation markers as a feasible pathology-anchored stratification tool.

Neuroblastoma is a pediatric, sympathoadrenal tumour accounting for 7-10% of childhood malignancies. Some neuroblastomas are driven by activating mutations in ALK kinase and inhibitors show promise in clinical trials. Nevertheless, with resistance an ever-present concern, it remains important to understand better the effectors and modulators of ALK signaling. Wild type ALK promotes ERK activation, raising expression of negative regulators such as the dual-specificity phosphatase (DUSP) DUSP6. DUSP6 though can be pro- or anti-oncogenic in different cancers and its role in neuroblastoma cells remains unclear. We sought to understand its role in cells with either wild type or mutated ALK. Mutated ALK strongly promotes DUSP6 transcription, but apparently not through the ERK pathway. DUSP6 also appears to promote neuroblastoma cell proliferation without affecting ERK. Additionally, when DUSP6 is lost, the cells become more sensitive to ALK inhibitors lorlatinib and crizotinib. Phosphoproteomic analysis of such cells demonstrates that mutated ALK cooperates with DUSP6 to maximise signaling through several potential pathways, but again not through ERK or AKT. Their cooperation may also maintain optimal levels of N-Myc in MYCN-amplified neuroblastoma cells. While key substrates of DUSP6 remain to be determined in neuroblastoma cells, our study defines a novel role for this phosphatase in supporting the action of oncogenic ALK.

P2Y12 receptor (P2RY12), mainly expressed on platelets, is known for its central role in hemostasis. P2RY12 activation is also involved in cancer development through platelet adhesion to cancer cells supporting immune-evasion, promoting tumor angiogenesis and metastasis, among others. P2RY12 is known as an actionable target, and P2RY12 antagonists are in clinical use for cardiovascular diseases. However, very little data are available regarding the protein expression of P2RY12 in lung carcinomas. We performed immunohistochemical staining for P2RY12 in a cohort of non-small cell lung cancer (NSCLC) samples comprising 320 adenocarcinomas (LUAD) and 158 squamous cell carcinomas (LUSC). Results were evaluated using a dual approach combining microscopic assessment and digital image analysis (QuPath). Results were correlated with clinical-pathological data. We found significantly higher P2RY12 protein expression in LUSC compared to LUAD (p<0.001) via eyeballing (absent/low expression in 21.7% (34/158) and moderate/high expression in 78.3% (124/158) of LUSC cases versus absent/low expression in 98.4% (315/320) and moderate/high expression in 1.6% (5/320) of LUAD cases). Digital analysis yielded similar results. High P2RY12 expression was associated with a significantly better 5-year overall survival rate for the entire cohort (p=0.0048) as well as for the LUAD (p=0.015) and LUSC (p=0.05) subgroups. Furthermore, P2RY12 showed excellent discriminatory performance for classifying carcinomas as LUAD or LUSC, with an AUC of 0.916 in ROC-analysis. High P2RY12 expression is linked to a better prognosis and might serve as a promising novel prognostic biomarker for NSCLC. Its assessment could be implemented in future routine diagnostic workup. At the same time, the data suggest that P2RY12 could also serve as a diagnostic marker for LUSC.

Non-small cell lung cancer (NSCLC) is the most common form of lung cancer accounting for most cancer-related deaths worldwide. Despite substantial recent advances in targeted therapies and immunotherapy, the prognosis for advanced-stage disease remains comparably poor, which is why the identification of novel molecular biomarkers as well as therapeutic targets influencing tumor development, progression, and metastasis remain important. This study focusses on SERPINB13, a serine-protease inhibitor expressed in selected tissues that is dysregulated in several tumor entities. However, its role in NSCLC still remains largely unclear. We analyzed SERPINB13 transcription in a cohort of non-small cell lung cancer (NSCLC) cases including both lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) by transcriptome profiling. Epigenetic modifications were assessed via Methylation BeadChips. Additionally, SERPINB13 protein expression was assessed by immunohistochemistry (IHC) in an independent cohort of NSCLC comprising 126 LUSC patients. Correlation analyses were performed to associate SERPINB13 expression with key clinico-pathological parameters, including overall survival and extent of tumor-infiltrating immune cells. To functionally investigate the regulatory influence of peripheral blood mononuclear cells (PBMCs) on SERPINB13 expression in LUSC tumor cells in vitro, we utilized the SERPINB13-expressing LUSC cell line LUDLU-1. Here, gene transcription was analyzed by quantitative real-time PCR (RT-qPCR), confirmed by Western blot on the protein level. Transcriptome analysis revealed a significant upregulation of SERPINB13 in lung squamous cell carcinoma (LUSC) compared to lung adenocarcinoma (LUAD), highlighting a subtype-specific expression pattern. This differential expression was further associated with a distinct epigenetic DNA methylation signature at the SERPINB13 loci in LUSC, suggesting transcriptional regulation via hypomethylation. IHC analysis demonstrated that high SERPINB13 protein expression is significantly associated with prolonged overall survival in LUSC. Notably, SERPINB13 expression was enriched in immune-inflamed ("hot") tumors, characterized by elevated infiltrating lymphocytes and immune activation. Mechanistically, co-culture experiments with PBMCs induced SERPINB13 expression in a LUSC cell line in a dose- and time-dependent manner in the absence of direct cell contact. This suggests that soluble factors secreted by immune cells might play a key role in regulating SERPINB13 expression in the tumor microenvironment. Taken together, SERPINB13 is a novel prognostic indicator in LUSC that is modulated by Immune cells. Further studies are necessary to decipher the crosstalk of Immune cells on the Serpin B13 expressing tumor cells in depth, with regard to a possible interventional strategy. immunomodulatory potential strategies and personalized therapeutic approaches in NSCLC.

WNT11, a non-canonical WNT ligand, plays well-defined roles in development and tissue architecture; however, its function in cancer remains ambiguous. Here, we characterize WNT11 as a context-dependent suppressor of cancer stemness, invasion, and in vivo tumor formation in human epithelial cancer models. We show that WNT11 upregulation reduces the expression of stemness-promoting genes, suppresses epithelial-to-mesenchymal transition, and inhibits sphere formation and tumor growth. Conversely, downregulation of WNT11 enhances these aggressive malignant properties of cancer cells. Mechanistically, we found that the ability of WNT11 to inhibit RAC1 GTPase activation is essential for its regulation of invasion and self-renewal. In cells unresponsive to WNT11, the connectivity between WNT11 and RAC1 activity is disengaged. Direct manipulation of RAC1 activity in these cells recapitulates the phenotype and molecular signature of WNT11-responsive cells, establishing RAC1 as a critical effector of WNT11-mediated tumor suppression. Taken together, these findings identify the cellular context in which WNT11 suppresses RAC1 activation as a key determinant of its anti-tumor effects and provide a mechanistic framework for understanding the diverse, and sometimes opposing, roles of WNT11 reported in cancer.