NAR Cancer

Ovarian cancer is the most lethal gynecological malignancy, largely because of late diagnosis and marked genomic instability, with high-grade serous ovarian cancer (HGSOC) representing its most common and aggressive subtype. Amplification of chromosome 8q24.3 is a recurrent event in HGSOC, yet the regulation and clinical relevance of the non-coding RNA output from this locus remain poorly defined. Here, we performed an integrative analysis of 8q24.3-encoded miRNAs in ovarian cancer using copy-number, transcriptomic, isoform-resolved, and clinical data from TCGA and NCBI datasets. We identified pronounced heterogeneity in miRNA abundance and strand usage across this locus. Copy-number gain broadly associated with increased miRNA expression, although this effect was not uniform across all candidates. Intronic miRNAs showed variable coupling with their host genes, indicating that mature miRNA output is shaped by both genomic dosage and post-transcriptional regulation. Isoform-level analysis revealed marked strand asymmetry and regulatory complexity, but did not strengthen copy-number or histotype associations compared with total miRNA measurements. Clinically, higher expression of miR-937, miR-4664, and miR-6849 was associated with improved overall survival in HGSOC. Functional enrichment of validated targets highlighted pathways related to cellular stress responses, senescence, p53 signaling, endocytosis, and metabolic adaptation. Together, these findings define 8q24.3 as a heterogeneous non-coding regulatory hub in ovarian cancer and provide a basis for future mechanistic and biomarker studies.

Long noncoding RNAs (lncRNAs) are important regulators of gene expression and are frequently dysregulated in cancer. The mitotically associated lncRNA MANCR is highly expressed in aggressive cancers and contributes to genomic instability in triple-negative breast cancer (TNBC), but the molecular mechanisms underlying its activity remain poorly defined. Here we integrate computational and experimental approaches to examine the structure and regulatory interactions of MANCR isoforms. Analysis of transcriptomic datasets revealed tumor-type-specific expression patterns for seven MANCR isoforms in breast cancer cell lines. Computational prediction of RNA secondary structures identified conserved structural features across isoforms, suggesting potential functional specialization. We identify p53 as a MANCR-interacting protein through computational docking and RNA immunoprecipitation sequencing (RIP-seq) and demonstrate that MANCR depletion reduces p53-dependent transcriptional activity. Chromatin isolation by RNA purification sequencing (ChIRP-seq) revealed 1, 250 genomic regions associated with MANCR, including enrichment of p53 consensus motifs and GC-rich sequence elements. Motif analysis further identified candidate sequence features associated with MANCR-occupied chromatin regions. Computational prediction of RNA-miRNA interactions identified multiple potential miRNA binding sites across MANCR isoforms, including miR-6756-5p, which targets the androgen receptor (AR). Consistent with this prediction, AR expression decreased following MANCR knockdown in TNBC cells. Together, these results suggest that MANCR isoforms may contribute to transcriptional regulation in TNBC through interactions with chromatin, p53 signaling pathways, and potential miRNA regulatory networks. One Sentence SummaryMitotically-associated lncRNA (MANCR) is prevalent in aggressive cancers interacting with DNA, P53, and miRNAs, to mediate multiple levels of epigenetic transcriptional control in triple negative breast cancer.

The TGIF1 transcription factor gene is present on chromosome 18, which is subject to whole chromosome copy number reduction in colon cancer. Despite this, TGIF1 expression is significantly higher in cancer than in normal. In mice complete deletion of Tgif1 reduced tumor burden in an Apc mutant model of intestinal cancer. Here we show that reducing TGIF1 expression in a human colon cancer cell line slows proliferation and reduces growth of orthotopic xenografts. To ask if additional genes with copy number loss are more highly expressed in tumors we identified chromosomal regions subject to copy number reductions from ten TCGA cancer datasets. Within these regions a small proportion of genes, generally less than 10%, are expressed at higher levels in the tumor than in corresponding normal samples. Enrichment analysis using a set of 435 genes that have copy number reduction and increased expression identified mitosis as the most enriched gene set and FOXM1 and E2F family transcription factors as potential regulators. For mitotic genes, the average expression increase in tumor compared to normal is independent of copy number. In contrast, while DepMap common essential genes are generally more highly expressed in cancer than normal tissue, the relative increase in expression tracks well with copy number. Similarly, expression differences for gene sets such as S-phase, rRNA processing and DNA repair show increased expression in cancer versus normal, but changes also track with copy number. Thus, genes with increased expression despite copy number reduction may represent the output of key pro-tumorigenic transcriptional programs and could be potential therapeutic targets.

BackgroundTumors exhibit substantial cellular and molecular diversity driven by genetic and epigenetic mechanisms. Large-scale profiling efforts have established aberrant DNA methylation as a universal hallmark of cancer. Beyond changes in mean methylation levels, tumor tissues exhibit elevated DNA methylation variability at specific genomic regions within and across tumors. This constitutes a fundamental dimension of cancer epigenomes, reflecting disrupted maintenance of epigenomic states and stochastic drift, which may enable adaptation to the microenvironment, phenotypic plasticity, invasion, disease progression, and treatment resistance. However, the genome-wide organization and functional consequences of DNA methylation variability across cancer types remain incompletely understood. MethodsWe analyzed paired tumor-normal DNA methylation profiles across 16 cancer types to systematically quantify DNA methylation variability. Pan-cancer DNA methylation variability was consistently observed using complementary statistical approaches and multiple modes of data representation. We identified cancer-specific and pan-cancer differentially variable regions and evaluated their associations with genomic features, transcriptional and chromatin regulators, and biological processes. Variability was quantified using three measures per sample: the proportion of intermediately methylated sites (PIM), genome-wide Shannon entropy, and a DNA methylation-based stemness index. Associations with genomic instability, tumor biological features, and clinical outcomes were subsequently assessed. ResultsTumor samples consistently exhibited higher DNA methylation variability than matched normal tissues, reflected by increased dispersion and wider interquartile ranges. Pan-cancer variably methylated regions were depleted in promoters and enriched in open sea regions, in heterochromatic H3K27me3-decorated PRC2-repressed domains, and at enhancers. They preferentially contained motifs for transcription factors involved in developmental regulation. Elevated DNA methylation variability, captured by higher PIM, entropy, and stemness scores, was associated with increased genomic instability manifested by higher aneuploidy, increased DNA break points, a greater fraction of the genome altered, and increased tumor mutational burden, as well as with aggressive tumor features such as lymph node involvement, post-therapy neoplasm events, and elevated hypoxia scores. Importantly, tumors with high DNA methylation variability exhibited significantly worse overall, progression-free, and disease-free survival. ConclusionsDNA methylation variability is a pervasive and clinically relevant feature of tumor epigenomes, reflecting epigenetic and genetic instability, expanded regulatory plasticity, and tumor aggressiveness.

Anti-hormonal therapies such as selective estrogen receptor modulators like tamoxifen or aromatase inhibitors like letrozole represent a cornerstone for breast cancer prevention and therapy of estrogen receptor-positive breast cancer. Therapeutic monitoring can include blood tests and imaging; however, genetically-based approaches are not yet in practice. Ideally, a test would be able to detect a positive molecular response across different estrogen pathway-suppressive approaches. Circular RNAs are a species of non-coding RNAs detectable in plasma that have been proposed as non-invasive therapeutic biomarkers. To determine whether a set of specific circular RNAs is altered across estrogen-suppressive pathway approaches, we analyzed mammary gland-specific total RNA sequencing data from two individual genetically engineered mouse models (GEMMs) of estrogen pathway-induced breast cancer, with or without exposure to tamoxifen or letrozole. The nf-core/circrna pipeline was used to identify circRNAs that were differentially expressed in response to either tamoxifen or letrozole. We then screened for circRNAs that were differentially regulated by both anti-hormonals. Four up-regulated and 31 down-regulated circRNAs with host genes known to be expressed in human breast epithelial cells were identified as showing reproducible differential regulation in response to anti-hormonal treatment.

BackgroundRetrotransposable elements (RE) comprise approximately 45% of the human genome and are typically repressed by DNA methylation to preserve genomic integrity. In cancer, global DNA hypomethylation can lead to RE derepression, resulting in genomic instability and activation of innate immune pathways through viral mimicry. While individual RE classes have been examined in clear cell renal cell carcinoma (ccRCC), the integrated epigenetic landscape of multiple RE families and their clinical relevance remain incompletely characterized. MethodsWe performed a genome-wide prediction of DNA methylation across three major RE classes (Alu, LINE-1, and LTR elements) using a validated computational framework applied to Illumina methylation array data from two independent ccRCC tumor cohorts. Integrated unsupervised clustering of RE methylation profiles was used to define the epigenetic subtypes. Associations with clinicopathologic variables, tumor immune microenvironment composition (DNA Methylation-derived), hypoxia signaling, innate immune activation, and overall survival were evaluated. Prognostic relevance was assessed using multivariable Cox regression models adjusting for age, sex, AJCC stage or AUA risk group, and immune and angiogenic tumor microenvironment features. Key findings were then externally validated in CPTAC-ccRCC and independently replicated in an institutional Dartmouth Cancer Center (DCC) cohort with matched methylation and RNA-sequencing data. ResultsIntegrated clustering identified three reproducible RE methylation subtypes, Repressed, Transient, and Active. In the discovery cohort, the Active subtype showed significantly worse overall survival than the Repressed subtype, with a graded survival pattern across RE methylation states that persisted after multivariable adjustment. RE hypomethylation was associated with reduced EPAS1 (HIF2A) expression, increased immune infiltration, elevated PD-1 expression, and heightened cGAS-STING and interferon signaling, consistent with an immune-inflamed yet immunosuppressed tumor state. In the external CPTAC validation cohort, RE methylation subtypes recapitulated key molecular features and showed supportive survival trends. In the independent DCC replication cohort, an Active RE state was again associated with poorer survival, lower EPAS1 expression, increased PD-1 expression, greater CD8 T-cell and Treg infiltration, and elevated T-cell exhaustion signatures, supporting the reproducibility of the prognostic and immune-exhausted phenotype across cohorts. ConclusionsWe identified RE methylation subtypes with distinct molecular, immunologic, and prognostic features in ccRCC. External validation in CPTAC and independent replication in DCC support the robustness of this RE methylation framework across large-scale and institutional cohorts. These findings highlight the prognostic potential of RE methylation profiles and support their integration into molecular classification strategies to improve risk stratification in ccRCC.

Network biology traditionally identifies gene correlations that reflect biological pathways. While LIONESS enables individualized gene networks, the influence of replication timing on these correlations remains unexplored. Replication timing reflects the temporal order of DNA synthesis and is tightly linked to chromatin state, methylation, and transcriptional stability, all of which affect tumor behavior. Integrating replication-timing proxies derived from methylation data therefore offers a bridge between epigenetic state and functional gene coordination, while morphology provides an additional route for inferring gene expression. This is the first study to integrate replication-timing proxies and morphological embeddings into individualized LIONESS gene networks. The aim is to determine how replication timing and morphology derived from bulk methylation and image embeddings influence gene coexpression in pancreatic cancer. Patient-specific networks were generated for basal and classical pancreatic ductal adenocarcinoma subtypes using TCGA data. Results show an 80% AUC for RNA-replication-timing-based subtype prediction modules and a 75% AUC for morphology-based networks. Incorporating replication timing and morphology increased network robustness while maintaining classification performance. Notably, the 80% AUC was achieved using only 17 of the 50 Moffitt genes, with 16 overlapping the PURIST gene set, indicating that replication timing captures clinically relevant regulatory structure. These findings suggest that replication-timing proxies can act as epigenetic indicators of mechanistic gene coordination and may help identify patients with distinct replication stress or chromatin accessibility profiles relevant to therapeutic response.

Y-box binding protein 1 (YB-1; YBX1) is a multifunctional DNA- and RNA-binding protein involved in cell cycle regulation, DNA repair, stress adaptation, and therapy resistance. Elevated YBX1 mRNA expression is associated with aggressive disease across multiple cancers, yet its pan-cancer genomic and clinical correlates remain unclear. Here, we performed a comprehensive pan-cancer analysis across 53 datasets spanning 33 tumour types, integrating RNA expression, somatic mutations, copy number, hypoxia, and clinical outcomes. YBX1 was rarely mutated or amplified, indicating that oncogenic relevance is primarily driven by its expression. Tumours with high YBX1 mRNA exhibited a conserved transcriptional program enriched for cell cycle, DNA repair, and chromatin regulation pathways, and were preferentially mutated in genes involved in maintaining genomic stability, including TP53. These tumours were associated with increased mutation burden, fraction of genome altered, homologous recombination deficiency, and elevated hypoxia. Clinically, high YBX1 mRNA associated with advanced stage, higher grade, shorter progression-free survival, and reduced overall survival. Collectively, high YBX1 mRNA expression defines a conserved, genomically unstable, and clinically aggressive tumour state across multiple cancer types.

TP53 mutations occur in 80-90% of triple-negative breast cancers (TNBCs) and drive genomic instability and metastatic progression. Poly (ADP-ribose) polymerase (PARP) is critical for DNA repair and replication fork stability. How oncogenic signaling influences PARP function to sustain proliferation during replication stress remains unclear. Mutant p53 (mtp53) R273H associates tightly with chromatin, forms complexes with PARP, and enhances PARP recruitment to replication forks [1-3]. The C-terminal region of mtp53 mediates mtp53-PARP and mtp53-Poly (ADP-ribose) (PAR) interactions that facilitate S phase progression [4, 5]. The PARP inhibitor talazoparib (TAL) combined with the alkylating agent temozolomide (TMZ) produces synergistic cytotoxicity selectively in mtp53, but not wild-type p53 (wtp53), breast cancer cells and organoids. Herein we evaluated the mechanism of mtp53-associated cell death and tested if this could translate to a preclinical xenograft model. We found that TMZ+TAL treatment induced elevated cleaved PARP and {gamma}H2AX and reduced the metastasis-promoting oncoprotein MDMX. In orthotopic xenografts expressing mtp53 R273H, but not wtp53, combination therapy significantly decreased circulating tumor cells (CTCs) and lung metastases. Transcriptomic profiling of tumors from combination treated animals demonstrated downregulation of MDMX, VEGF, and NF-{kappa}B, consistent with the observed suppression of CTCs and lung metastasis, and increased {gamma}H2AX, indicative of replication stress in mtp53 xenografts. Inhibition of metastasis was also observed in mtp53 R273H WHIM25 and p53-undetectable WHIM6 TNBC patient-derived xenografts (PDX). The mtp53 C-terminal domain (347-393) demonstrated a critical tumor promoting function, as CRISPR-mediated deletion impaired replication fork progression, tumor growth, and metastatic dissemination. DNA fiber combing showed that expression of full-length mtp53 R273H, but not C-terminal deleted {Delta}347-393, supported sustained single-stranded DNA gaps (ssGAPs) following Poly (ADP-ribose) glycohydrolase (PARG) inhibition. These findings support that mtp53 uses C-terminal amino acids to exploit PARP to enable replication stress adaptation and that mtp53 is a predictive biomarker for combined PARP inhibitor and DNA damaging therapies targeting TNBC. Significance statementTP53 mutations are the most common genetic alterations in TNBC and a major driver of replication stress and metastasis. This study shows that missense mutant p53 uses C-terminal amino acids to reprogram PARP activity to maintain tumor cell survival under replication stress. We demonstrate that p53 status governs the response to combined PARP inhibitor (PARPi) and DNA-damaging chemotherapy, establishing an additional molecular basis beyond BRCA1 mutations for treating TNBC with PARPi therapy. These findings reveal a previously unrecognized mechanism by which the mutant p53-PARP axis enables replication stress tolerance and drives cancer metastasis. We show mutation of p53 in TNBC provides an additional biomarker-guided framework to improve PARPi therapeutic outcomes.

The microRNAs miR-15a and miR-16 are key regulators of the anti-apoptotic oncogene BCL2, playing a significant role in tumorigenesis. These miRNAs function as tumor suppressors by directly targeting BCL2, whose overexpression contributes to cell survival and resistance to therapy in multiple malignancies, including chronic lymphocytic leukemia (CLL). The downregulation or deletion miR-15a/miR-16-1 cluster located on chromosome 13q occurs in about 50% of CLL patients and leads to the overexpression of the oncogenic BCL2, contributing to the survival and proliferation of cancer cells. In this confirmatory study, we provide additional evidence supporting the mechanism by which these miRNAs mediate the inhibition of BCL2 translation, leading to reduced levels of BCL2 protein with no significant effect on BCL2 mRNA degradation. This mechanism has been previously established as a critical pathway in the regulation of apoptosis, particularly in cancer cells where BCL2 overexpression is often associated with resistance to cell death. Our findings reinforce the notion that miRNAs, such as miR-15 and miR-16, bind to the 3-UTR of BCL2 messenger RNA (mRNA), specifically repressing its translation without inducing mRNA degradation. The results from our study align with previous research, confirming that the miRNA-mediated inhibition of BCL2 translation serves as a precise regulatory mechanism that targets protein synthesis rather than mRNA stability. These findings highlight the role of miRNAs in fine-tuning post-transcriptional gene regulation, offering a targeted approach to downregulate oncogenic proteins like BCL2 without disrupting the underlying mRNA, which could be leveraged for more refined therapeutic strategies.

Extrachromosomal DNA (ecDNA) has emerged as a critical mediator of oncogene amplification and transcriptional dynamics in aggressive cancers, yet its contribution to chemotherapy resistance in vivo remains incompletely understood. This study investigates the contribution of ecDNA-associated molecular features to predictive chemotherapy resistance in TNBC. We analyzed RNA-seq data from 4T1 TNBC cells and 4T1 bulk tumors at different growth stages (1-, 3-, and 6-week) to identify differentially expressed ecDNA alterations. We then utilized molecular docking tools to predict ecDNA protein-drug interactions and employed machine learning (ML) models to predict ecDNA-associated therapeutic resistance. Our results revealed changes in global gene expression, including expression of ecDNA-associated genes, that continued over time, with significant molecular remodeling observed at six weeks. Additionally, we found gradual accumulation of mutations in ecDNA genes, which may have contributed to reduced drug binding affinity, indicating potential resistance. ML models generated stable, high-confidence classifications of resistant phenotypes, consistently identifying ecDNA burden and prevalence as dominant predictive features of drug resistance. Drug specific predictions further highlighted elevated resistance probabilities for paclitaxel and doxorubicin, whereas hydroxyurea, which depletes ecDNA, showed reduced resistance probabilities, indicating potential roles of ecDNA in chemoresistance. This study provides new insights into temporal remodeling of ecDNA within TNBC tumors over time and their potential association with drug resistance.

Gastric cancer is driven by aberrant kinase signaling that supports uncontrolled proliferation and metabolic adaptation. Calcium/calmodulin dependent protein kinase kinase 2 (CAMKK2) is overexpressed in gastric cancer; however, its role in regulating metabolic programs that sustain tumor growth remains incompletely understood. In this study, we employed an integrated multi-omics approach with a primary focus on untargeted metabolomics to investigate the consequences of CAMKK2 inhibition in gastric cancer cells. Pharmacological inhibition of CAMKK2 using STO-609 in AGS cells resulted in significant suppression of proliferation, clonogenic growth, migration, and invasion, accompanied by pronounced nuclear abnormalities and multinucleation indicative of mitotic defects. Global metabolomic profiling revealed extensive and time-dependent metabolic reprogramming following CAMKK2 inhibition, characterized by a marked depletion of nucleotide intermediates, including purine and pyrimidine metabolites required for DNA synthesis. Pathway enrichment analysis highlighted suppression of nucleotide metabolism, lipid metabolism, and central carbon metabolic pathways, indicating a broad impairment of biosynthetic capacity. Integration with proteomic and phosphoproteomic datasets demonstrated that metabolic alterations were accompanied by downregulation of DNA replication machinery and attenuation of kinase signaling pathways governing cell cycle progression. Protein metabolite interaction and docking analyses further supported functional coupling between nucleotide metabolites and key replication-associated enzymes, revealing disruption of metabolite enzyme interactions upon CAMKK2 inhibition. Collectively, these findings identify CAMKK2 as a critical regulator of metabolic programs that support DNA replication and cell cycle progression. Its inhibition induces replication stress through coordinated depletion of nucleotide pools and disruption of replication-associated signaling, leading to impaired proliferation and mitotic failure. These results highlight CAMKK2 as a potential therapeutic target for exploiting metabolic vulnerabilities in gastric cancer.

Hypoxia is a defining feature of the solid tumour microenvironment and a major determinant of therapeutic response. Hypoxia-inducible factors (HIFs) are central regulators of transcriptional reprogramming under hypoxic stress. Hypoxia can paradoxically elicit both tumour-promoting and tumour-suppressive outcomes, suggesting regulatory mechanisms beyond canonical HIF-dependent pathways. Emerging evidence indicates that hypoxia-responsive RNAs (HRRs) may also be regulated independently of HIFs, with posttranscriptional stabilization playing a critical determinant of hypoxic adaptation. Cytoplasmic mRNA recapping mediated by the cytoplasmic capping enzyme (cCE) has recently emerged as an important post-transcriptional regulatory process, yet its role in hypoxia-driven RNA regulation remains poorly understood. Here, we aimed to identify novel HRRs that modulate cellular adaptability to hypoxia and to determine whether these transcripts are regulated by cCE. Using CoCl2-induced hypoxia, we observed a significant reduction in osteosarcoma cell aggressiveness, characterized by decreased proliferation, clonogenic survival, and migratory capacity. Transcriptomic profiling of hypoxic osteosarcoma cells identified RORA and KCTD16 as significantly upregulated and function as suppressors of tumour cell aggressiveness. Integrative in-silico CAGE tag analysis followed by cap-specific biochemical assays confirmed that both transcripts are post-transcriptionally stabilized by cCE. Mechanistically, hypoxia-induced stabilization of HIF1 transcriptionally elevated RORA and KCTD16 expression, while cCE further reinforced their stability post-transcriptionally. Stabilization of these cCE-targeted HRRs resulted in suppression of the oncogenic proliferation driver c-Myc, thereby attenuating the aggressive phenotype of hypoxic osteosarcoma cells. Collectively, our findings identify cCE as a previously unrecognized post-transcriptional regulator in hypoxia biology and reveal a RNA-centric mechanism by which hypoxia can restrain tumour aggressiveness.

The human genome consists of nearly 50% repetitive DNA, referred to for decades as "junk DNA". These repetitive sequences, usually under the strict control of epigenetic silencing, have been observed to be aberrantly expressed in cancer. Some of these expressed sequences, e.g., transposable elements (TEs), can induce innate immune responses when de-repressed following treatment with epigenetic therapies. As a result, epigenetic therapy has been suggested to augment cancer therapies. TEs are traditionally ignored in most RNA-seq studies and their expression is often excluded from publicly available data sources. Thus, the vast amount of publicly available RNA-seq data is an untapped resource for exploring the role of TE expression in cancer and cancer treatment. Here, we present a uniform re-analysis of over 7,000 RNA-seq samples, encompassing more than 2,000 differential expression experiments across 220 cancer cell lines and 700 drug treatments. We observed that TE expression is more prone to batch effects than gene expression alone, necessitating the use of meta-analysis techniques to probe the dataset for global trends. We confirm that DNMTi and HDACis are powerful inducers of TEs. We also show that non-epigenetic compounds such as CDK and topoisomerase inhibitors can also induce robust up-regulation of transposable elements and confirm that this TE induction is consistent with viral mimicry response. We make all of the reprocessed data, web application, and database publicly available at: https://dataexplorer.coriell.org/TEDEdb/

BackgroundLong non-coding RNAs (lncRNAs) have emerged as key regulators of tumor biology, however, thus far none have translated to cancer therapies. The lncRNA MALAT1 is overexpressed in more than 20 cancers, including breast cancer and has been shown to function via various mechanisms in a context-dependent manner, in 2D cell lines and mouse models. However, its functional role and therapeutic potential have not been evaluated in clinically relevant patient-derived models. MethodsWe investigated the therapeutic potential of a MALAT1-targeting antisense oligonucleotide (ASO) for breast cancer, using clinically relevant 3D human patient-derived organoids (PDOs) and PDO-xenograft (PDO-X) models. We systematically evaluated the efficiency of MALAT1-targeting ASOs using a biobank of 28 PDO models. Using three independent PDO-X models of triple negative breast cancer (TNBC), we targeted MALAT1 in vivo to study its impact on transcription, alternative splicing, stromal remodeling and metastasis. ResultsAcross PDO-X models, MALAT1 depletion reproducibly drove widespread alternative splicing changes across all event types, particularly intron retention events, accompanied by modest gene expression alterations. Differentially spliced transcripts were enriched for targets of shared cancer-associated transcription factors, and MALAT1 knockdown altered the relative abundance of previously unannotated splicing isoforms. Beyond tumor-intrinsic effects, tumor-specific MALAT1 depletion induced a consistent reduction in macrophage-associated gene signatures and reduced lung metastatic burden. ConclusionsOur data define MALAT1s multifaceted role in TNBC, coordinating alternative splicing, transcriptional fine-tuning, tumor-stroma crosstalk, and metastatic progression. Our study provides strong preclinical evidence supporting MALAT1-targeted ASO therapy and establishes PDO-X models as a clinically relevant platform for functional interrogation of TNBC therapies.

We previously reported that the murine lncRNA Epr is essential for maintaining colon mucosal integrity and permeability. Mice lacking Epr in the colon are more susceptible to colitis and tumor development. Additionally, we demonstrated that human EPR expression is reduced in ulcerative colitis and in a small cohort of colon adenocarcinoma patients. Here, we present evidence that human and mouse EPR share several key physiological features: preferential binding to the KH1 domain of their interacting protein, KSRP; specific expression in canonical and immature goblet cells of the large intestine; and a functional role in intestinal goblet cell development. The correlation between EPR levels and survival in large cohorts of metastatic colon adenocarcinoma patients, together with the capacity of human EPR to inhibit cell proliferation and induce apoptosis in two distinct human colon adenocarcinoma cell lines, suggests that EPR may serve as both a valuable prognostic marker for goblet cell-derived adenocarcinomas and a potential therapeutic target.

Hypertranscription and transcription-replication conflicts (TRCs) are frequent features of cancer cells. RAS oncogenes promote hypertranscription to allow cell growth and proliferation, which can the lead to TRCs. Here, we report that hyperactivation of the PI3K-AKT signalling pathway is required for TRCs induced by RAS oncogenes. Oncogenic HRAS causes more TRCs than oncogenic KRAS or BRAF, because HRAS hyperactivates PI3K. PI3K hyperactivation is associated with in glycogen synthase kinase-3{beta} (GSK3{beta}) inhibition, increased E2F and MYC transcription programmes, increased nascent transcription of ribosome biogenesis genes and small nucleolar RNAs (snoRNA) expression. Small molecule inhibition of PI3K signalling prevents RAS-induced replication stress, and small molecule PI3K activation promotes replication stress. RAS-induced TRCs require a cooperation of MAPK and Pi3K signalling, S phase entry and hypertranscription. Our findings suggest a mechanistic explanation for replication stress variability between RAS activation models and identify PI3K pathway activation as a potential new determinant of TRCs in cancer.

The RNA polymerase-associated factor 1 complex (PAF1C) is an evolutionarily conserved transcription elongation complex that regulates RNA polymerase II-mediated transcription and chromatin modification. LEO1, a core subunit of PAF1C, has been implicated in developmental gene regulation, WNT signaling, and leukemogenesis; however, its role in solid tumor progression remains poorly understood. In this study, we found that although LEO1 expression is generally elevated in colorectal cancer (CRC), its expression is reduced in stage IV tumors and is associated with poor clinical outcomes. To investigate its function, we established LEO1 -deficient HCT116 cell line and performed transcriptomic analyses. Loss of LEO1 suppressed epithelial differentiation and developmental gene programs while inducing cell cycle delay. Despite these changes, LEO1-deficient cells exhibited aggressive phenotypes, including enlarged nuclei and increased expression of migration-associated genes, which were further enhanced under glucose deprivation. Motif analysis identified FOXM1 as a key regulator of these migration-related genes. Mechanistically, LEO1 deficiency promoted accelerated transcriptional activation of GRP78, a central regulator of endoplasmic reticulum (ER) stress adaptation. GRP78 was required for survival under ER stress conditions, and its inhibition suppressed both migration and migration-associated gene expression. In addition, transcriptomic analyses revealed upregulation of cholesterol metabolism-related genes in LEO1-deficient cells. Consistently, treatment with the HMG-CoA reductase inhibitor atorvastatin selectively impaired their survival, indicating cholesterol metabolic dependency. Collectively, these findings demonstrate that LEO1 loss promotes ER stress-adapted migration and cholesterol metabolic dependency in CRC, suggesting that these pathways may represent therapeutic vulnerabilities in metastatic LEO1-low CRC.

Increased matrix metalloproteinase (MMP) expression has long been recognized as a common feature of colorectal cancers (CRCs), yet less is known about how these enzymes interact to impact cancer progression. Taking advantage of single-cell and spatial transcriptomic data, we analyzed the cell-type-specific and spatial expression of MMPs in CRCs. Distinct colon cancer-associated fibroblast (CAF) subtypes were found to express different MMP combinations, including MMP1/3-expressing and MMP11-expressing CAFs. Conversely, myeloid cells (monocytes, macrophages, and dendritic cells) expressed varying levels of the "myeloid MMPs" 9, 12, and 14, which correlated closely with secretory gene expression. Finally, a small population of cancer cells expressed high levels of MMP7. The MMP7-expressing cancer cells frequently co-expressed MMP1, MMP14, and several Wnt-related genes, consistent with a cancer cell type at high risk of malignancy and metastasis. Spatial transcriptomic data showed MMP expression in discernible clusters driven in part by cell-type localization, including fibroblast-heavy stromal regions and inflammatory cell hubs. Epithelial-rich areas showed subregions of MMP7-expressing cancer cells, including areas where cancer cell and myeloid MMP expression overlap. Tumors showed a wide variation in MMP1-expressing CAFs, a variation reflected in primary CAF cell lines. In vitro, MMP1 expression was a stable phenotype that persisted through multiple rounds of division. MMP1-expressing CAFs were frequently positioned at the stromal interface, suggesting a role in facilitating cell movement across the tumor boundary. Our analysis indicates that cell-type and positional MMP expression varies between tumors and may play a role in determining lesion progression and cancer spread.

Aberrant expression of Long Interspersed Element-1 (LINE-1/L1) retrotransposons is increasingly linked to genomic instability in cancer, particularly in the context of compromised p53 function. The endonuclease (EN) domain of the LINE-1 ORF2 protein (ORF2p) generates DNA strand breaks, yet its role in shaping downstream chromatin and transcriptional responses remains poorly defined. Here, we investigate the impact of ORF2p-EN activity on DNA damage response signalling and epigenomic remodeling in isogenic wild-type and p53-deficient A375 melanoma cells using a L1 expression system. ORF2p-EN expression induced activation of ATM dependent DNA damage signalling, as evidenced by increased {gamma}H2AX accumulation. This response was accompanied by altered levels of the DNA repair factor XRCC5, consistent with engagement of non-homologous end joining pathways. Notably, EN-associated DNA damage correlated with increased p300 activity and enhanced H3K27ac enrichment at regulatory regions, suggesting coupling between DNA damage signalling and chromatin acetylation. Pharmacological inhibition of ATM attenuated both {gamma}H2AX accumulation and H3K27ac levels, supporting a model in which chromatin remodeling occurs downstream of DNA damage signalling. Collectively, our findings position ORF2p endonuclease activity as an initiating source of DNA damage that is functionally linked to chromatin remodeling and transcriptional reprogramming, with p53 acting as a critical modulator of this axis. These results provide mechanistic insight into how LINE-1 activation may contribute to oncogenic gene expression programs in cancer.