Cell Death & Differentiation

Disseminated cancer cells (DCC) in non-metastatic carcinoma patient bone marrow (BM) are predictive of metastasis. Those detected by epithelial cytokeratin or EpCAM expression have poorly-characterized transcription profiles due to their extreme rarity: 1~2 cells per two million BM cells in every third non-metastatic patient. We here characterize the transcriptomes of DCCs. Single-cell RNA-sequencing (scRNA-seq) of 864 EpCAM-positive cells (from 1,151 cancer patients) in BM or lymph nodes (LN) revealed plasma, immune, myeloid, erythroid progenitor cells and two candidate DCC populations, termed M0-DCC and M1-DCC. M0-DCC, mostly from non-metastatic M0-stage patients, displayed the highest known adult stemness scores, and were transcriptomically reminiscent of human cleavage-stage, preimplantation embryos. M1-DCC represented cancer cells undergoing the epithelial-mesenchymal transition (EMT), corresponding to later, implanting and gastrulating embryos. Detection of early-embryo-like DCC categorised patients at highest risk for metastatic progression. Furthermore, high M0-DCC scores predicted the metastatic potential of human cell lines from the Cancer Cell Line Encyclopedia. M0-DCC gene expression profiles can be reversibly induced from M1-DCC-like cells in vitro. The close correspondence between gene expression profiles in immediate early embryonic development and metastatic founder cell candidates provides strong evidence that the onset of cancer and metastasis recruits mechanisms employed in fertilization.

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.

High-grade serous ovarian carcinoma (HGSC) responds poorly to immune checkpoint blockade, partly due to a macrophage-dominated immunosuppressive microenvironment. We integrated single-cell spatial proteomics and spatial transcriptomics across 50 HGSC tumors and applied SPACEstat to resolve higher-order immune communities and their transcriptional programs. We identified six immune community types, with macrophage-dominated Myelonets representing the predominant spatial pattern of immune organisation. In chemotherapy-exposed tumors, Myelonets showed coordinated lipid metabolism-immunosuppression and inflammation-MHC-II macrophage transcriptional programs, with SPP1, C1Q, VEGF, MMPs, and CCL18 linked to immunosuppressive states and fibroblasts emerging as key mediators of macrophage communication. Chemotherapy contracted large Myelonets while increasing CD8+ T-cell organization into Lymphonets. Persistent macrophage dominance within Myelonets was associated with adverse outcomes among patients who achieved a complete response to treatment. Together, we identify Myelonets as clinically relevant, multicellular immunoregulatory niches sustained by spatiotemporally coordinated macrophage programs and stromal crosstalk.

Pancreatic ductal adenocarcinoma (PDAC) exhibits profound metabolic rewiring and strong resistance to DNA-damaging therapies, yet how metabolic pathways regulate genome maintenance remains poorly understood. The hexosamine biosynthetic pathway (HBP) integrates nutrient availability with protein glycosylation through production of UDP-GlcNAc, but its role in DNA damage response (DDR) regulation is unclear. Here we show that inhibition of the HBP enzyme phosphoglucomutase-3 (PGM3) reduces DNA repair capacity in pancreatic cancer cells. Transcriptomic and functional analyses reveal that the selective PGM3 inhibitor FR054 amplifies gemcitabine-induced replication stress, disrupts ATR-CHK1 and ATM-CHK2 checkpoint signaling, and selectively impairs homologous recombination. Glycoproteomic profiling identifies the AAA+ ATPase RUVBL2 as a key metabolic-DDR node. Gemcitabine increases RUVBL2 O-GlcNAcylation, with Thr81 identified as a modified residue within the Walker A nucleotide-binding motif. Structural modelling predicts that Thr81 O-GlcNAcylation stabilizes the RUVBL1-RUVBL2 complex without compromising ATP-Mg engagement. PGM3 inhibition and Thr81 mutation similarly reduced ATR and ATM abundance and promoted persistent DNA damage, supporting a role for RUVBL2 Thr81 O-GlcNAcylation in sustaining checkpoint signalling and genome stability. Consequently, PGM3 inhibition induces a BRCAness-like state that sensitizes pancreatic cancer cells to PARP inhibition, both in vitro and in vivo, as well as to ionizing radiation. These findings reveal a nutrient-sensitive mechanism linking protein glycosylation to genome maintenance and identify HBP-dependent DNA repair as a potentially actionable vulnerability in pancreatic cancer.

Hirschsprung disease (HSCR) is a severe birth defect where ganglia of the enteric nervous system (ENS) are missing from distal bowel. The aganglionic segment is also characterized by increased epithelial permeability and pro-inflammatory immune activation. These problems may sequentially lead to translocation of gut microbes into the colon wall and systemic circulation, resulting in enterocolitis and sepsis. Current HSCR treatment via surgical resection of the aganglionic segment is lifesaving but not curative, often leaving patients with persistent gastrointestinal complications including recurrent risk of enterocolitis. As alternative, we are developing a regenerative medicine strategy based on in situ stimulation of tissue-resident ENS progenitors via rectal administration of the neurotrophic factor GDNF. Here, we report that GDNF-based therapy has pleiotropic gastrointestinal effects in a mouse model of short-segment HSCR, beyond its role in ENS regeneration. Interestingly, we found that these protective effects are not restricted to the aganglionic distal colon, also positively impacting the ENS-containing proximal colon. GDNF treatment reduces bacterial translocation both locally and in peripheral organs, and this is associated with recovery of the key epithelial junction proteins CLDN3, ZO1 and DSG2. Furthermore, multiparameter flow cytometry-based analysis of 55 lymphoid and 17 myeloid cell subtypes revealed that GDNF treatment has global anti-inflammatory effects, preferentially affecting innate over adaptive immunity. Overall, these findings highlight a critical role for GDNF treatment in reestablishing proper epithelial and immune cell homeostasis, offering promising therapeutic avenues not only for HSCR but also potentially for other intestinal disorders with overlapping pathophysiology.

The cell-intrinsic complement system has emerged as an important orchestrator of a variety of cell-physiological processes, with complement components interacting with intracellular effector systems to regulate cellular responses to pathogens or noxious stimuli. For instance, intracellular C5 signaling through a mitochondrial C5a receptor (C5aR1) controls IL-1{beta} production in human monocytes and macrophages. Here, we investigated whether cell-intrinsic C3 similarly regulates inflammatory responses in macrophages. In LPS-stimulated C3 knockout THP-1-derived macrophages, interferon (IFN)-{beta} production was increased, accompanied by elevated expression of interferon-stimulated genes and enhanced secretion of IFN-induced cytokines and chemokines. C3-deficient cells showed increased phosphorylation of IRF3 at Ser396 and a stabilization of the interaction between IRF3 and TBK1, along with enhanced IRF3 dimerization and nuclear translocation. TBK1 phosphorylation was unaffected, indicating that C3 limits IRF3-TBK1 complex formation rather than upstream TBK1 activation. Small-molecule inhibitors of complement factors B and D restored full-length C3 abundance in LPS-stimulated primary human macrophages, consistent with inhibition of the C3 convertase. It also reduced LPS-induced IFN-{beta} production in primary human macrophages and THP-1 cells, suggesting that full-length, uncleaved C3 suppresses IFN-{beta} production. Collectively, these findings identify cell-intrinsic C3 as a suppressor of IFN-{beta} production in human macrophages, highlighting the importance of the cell-intrinsic complement system in fine-tuning inflammatory responses to pathogens.

Brown adipose tissue (BAT) is a mitochondria-rich thermogenic organ whose function depends on high oxidative capacity, yet how primary mitochondrial dysfunction remodels BAT identity and metabolism remains poorly defined. Using the Deletor mouse model of progressive mtDNA deletion disease, we identify a pseudohypoxiairon-NAD+ axis as a central organiser of BAT pathology. Deletor BAT underwent profound structural, transcriptional and metabolic remodelling, characterised by mitochondrial ultrastructural damage, loss of thermogenic identity, PHD3/HIF associated pseudohypoxic signalling, iron dysregulation and NAD+/NADH redox imbalance. Indirect calorimetry confirmed that this molecular disease program translates to functional thermogenic failure under physiological demand. Deletor mice showed significantly reduced heat production under acute cold challenge and failed to switch to fatty acid oxidation Metabolomic profiling revealed altered TCA cycle intermediates, glycolytic rewiring and selective amino acid accumulation. Pharmacological perturbation showed that the PHD inhibitor roxadustat worsened disease-associated features, whereas HIF-1 suppression with PX-478 attenuated the integrated stress response, indicating that pseudohypoxic signalling is maladaptive in this setting. Nicotinamide riboside broadly attenuated the disease metabolome and transcriptome, restoring NAD+/NADH balance, suppressing ISRmt, iron-stress and pseudohypoxic gene programs, and correcting selective carnitine and acylcarnitine abnormalities consistent with impaired fatty-acid handling. These findings define a therapeutically tractable pseudohypoxia-iron-NAD+ axis as a core determinant of BAT dysfunction in mitochondrial disease.

Antral CCK2R+ stem cells are regulated by gastrin, but how endocrine and neural cues integrate under chronic injury remains unclear. Here we show that inducible hypogastrinemia shifts from asymmetric renewal to symmetric expansion of CCK2R+ stem cells. With carcinogenic stress, these cells acquire a cycling, injury responsive progenitor state revealed by single-cell RNA profiling. Acute gastrin loss activates a CCK2R+ nodose DMV vagal reflex that increases acetylcholine release, NGF production, cholinergic innervation, and Chrm3 expression, driving ERK and YAP signaling in CCK2R+ stem cells. Vagotomy, Trk inhibition, or Chrm3 deletion each suppressed stem cell expansion. In H. pylori and MNU injury models, hypogastrinemia amplified inflammation, dysplasia, and CCK2R+ clone expansion, whereas gastrin suppressed these responses. Human scRNA seq and spatial profiling confirmed G cell depletion and progenitor state enrichment. These findings define an endocrine neural epithelial axis in which gastrin loss boosts vagal M3R signaling to initiate antral preneoplasia, highlighting this pathway for early interception.

The role of UBE2N in myeloid cell-mediated immune suppression in cancer remains undefined. Here, we examined the function of UBE2N in myeloid cell-mediated tumor progression using a temporally inducible myeloid-specific knockout model (LysMCreERUbe2nfl/fl). Temporally induced deletion of Ube2n in myeloid cells (Ube2nMyeKO) significantly hindered growth of YUMM1.7 melanoma. This was accompanied by reduced myeloid cell burden within the tumor microenvironment. We observed altered abundance of PD-1, PD-L1, and SPP1 in the Ube2nMyeKO tumor microenvironment at the tissue level. In vitro analysis showed that knock-in expression of a catalytically deficient UBE2NC87S mutant in bone marrow-derived macrophages (BMDMs) markedly decreased expression of Spp1. We observed decreased SPP1 secretion in Ube2nMyeKO BMDM-conditioned media (CM). Treatment with Ube2nMyeKO BMDM-CM decreased co-expression of PD-1, TIM-3, and LAG-3 on chronically stimulated T cells. Antibody-mediated neutralization of SPP1 in Ube2nWT BMDM-CM decreased PD-1 expression on CD8+ T cells. Together, these findings suggest a role for myeloid UBE2N in YUMM1.7 progression.

Apoptosis-inducing factor is a mitochondrial flavoprotein that links redox metabolism to mitochondrial homeostasis through its interaction with the disulfide relay protein CHCHD4. Although NADH-dependent AIF dimerization has been proposed as the activated state mediating CHCHD4 engagement, whether it is strictly required for productive AIF-CHCHD4 function remains unclear. Here, combining cellular, biochemical and biophysical approaches, we show that disruption of the AIF dimer interface compromises oxidative phosphorylation, respiratory-chain organization and CHCHD4-dependent mitochondrial homeostasis, yet preserves partial AIF function. Our data reveal that the AIF-CHCHD4 system operates as a conformational dynamic redox module in which distinct AIF oligomeric and redox states sustain CHCHD4 activity with different efficiencies. Mechanistically, dimerization is coupled to NADH-dependent conformational changes that regulate coenzyme binding, charge-transfer complex stabilization and catalytic efficiency. In turn, CHCHD4 binding remodels AIF conformational and redox properties, partially compensating for defects in dimer stabilization or redox coupling. Consistently, a peptide derived from the CHCHD4 N-terminus partially restores redox function in a pathogenic AIF variant defective in dimer stabilization, supporting partner-assisted allosteric regulation as a potential therapeutic strategy.

Hematopoietic stem cell (HSC) aging is associated with epigenetic remodeling, yet the molecular mechanisms driving these changes, their overlap with stress-induced alterations, and whether this course can be durably reset remain incompletely understood. Here, we show that transient induction of the Yamanaka factors OCT4, SOX2, KLF4, and MYC in young mice durably delays and partially reverses physiological and LPS-driven HSC aging in mice. Transient reprogramming improved hematopoietic reconstitution, reduced myeloid bias, and limited DNA damage. Multi-omic analyses revealed reduced chromatin accessibility at AP-1-enriched regulatory regions, attenuated age-associated AP-1 transcriptional programs, and repression of transposable elements (TEs). Pharmacological AP-1 inhibition prevented LPS-induced TE activation and loss of HSC clonogenicity. Reverse transcriptase inhibition in aged mice reduced DNA damage and improved HSC function, demonstrating a functional contribution of TE activity to HSC decline. Together, these findings identify AP-1-associated chromatin remodeling as a candidate mechanism linking inflammatory stress, TE activation and HSC aging.

MYCN amplification drives replication stress in high-risk neuroblastoma, yet how MYCN-amplified tumour cells tolerate this stress to sustain proliferation remains poorly understood. Here we show that FBXL12, an SCF ubiquitin ligase substrate receptor that targets the Fanconi anaemia protein FANCD2 for degradation at replication forks, as well as the broader Fanconi anaemia and replication stress transcriptional program are elevated in high-risk and MYCN-amplified neuroblastoma. High FBXL12 expression independently predicts poor survival across neuroblastoma patient cohorts. FBXL12 loss stabilizes FANCD2 on chromatin, elevates ATR-dependent replication stress signalling and DNA damage during S phase, and impairs proliferation of MYCN-amplified neuroblastoma cells in vitro and in vivo. Mechanistically, MYCN directly engages the FBXL12-FANCD2 complex and antagonises FBXL12-mediated degradation of FANCD2 at replication forks, revealing that the oncogenic driver of replication stress also actively preserves the chromatin-bound FANCD2 pool required to tolerate it. Beyond S phase, FBXL12 loss disrupts FANCD2-dependent mitotic DNA synthesis and transmits unresolved replication intermediates into daughter cells. FBXL12-deficient cells consequently show transcriptional activation of MYC target gene, ATR, and mTOR signalling programs, and this pathway-concordant state confers differential sensitivity to ATR, and mTOR-targeting compounds, nominating candidate therapeutic strategies for this disease subset. Together, these findings define a MYCN-FBXL12-FANCD2 axis as a clinically relevant vulnerability in high-risk neuroblastoma.

Nuclear bile acid (BA) signaling plays a central role in liver homeostasis and represents a major therapeutic axis in fibrotic liver diseases. The farnesoid X receptor (FXR), a master nuclear effector of BA signaling, is expressed in several liver-resident cell types, suggesting that it may regulate distinct biological programs beyond the hepatocyte (HC) compartment. Using complementary pharmacological, genetic, and computational approaches across in vitro, ex vivo, and in vivo models of mouse and human origin, we investigated the role of hepatic stellate cell (HSC) FXR (FXRHSC) in both unchallenged and injured livers, which has remained controversial. FXR is robustly expressed in both HCs and HSCs with distinct isoform distributions, and these isoforms exhibited differential capacities to activate gene expression in an HSC context. We found that the potent selective FXR agonist tropifexor triggers a transcriptional program reminiscent of that observed after partial hepatectomy and associated with HC proliferation. This cell cycle-related response was also observed in HSCs and did not require intestinal FXR expression. An HSC-specific response to tropifexor was observed for several genes, including members of the glutathione-S-transferase (GST) family or Scube1. FXRHSC was sufficient to observe the anti-fibrotic effects of tropifexor in precision-cut liver slices, an ex-vivo model of fibrosis. Finally, we identified the regulation of the chemerin-encoding gene Rarres2 as a relevant example of FXRHSC-dependent control of hepatic intercellular communication. Together, these findings identify FXRHSC as an important contributor to hepatic adaptation and therapeutic response to BA analogs and confirmed HSCs as a significant site of nuclear bile acid signaling in liver biology.

KRAS is one of the most frequently mutated oncoproteins in cancer. Its ability to induce malignant transformation relies on metabolic reprogramming that causes cells to become dependent on aerobic glycolysis as a primary source of energy and for generating biological building blocks. Thus far, the signaling mechanism used by oncogenic KRAS to promote these changes in cancer cell metabolism has not been fully elucidated. However, through studies in pancreatic ductal adenocarcinoma (PDAC) cell lines and patient-derived organoids, we now demonstrate how oncogenic KRAS triggers an increase in glycolytic activity and identify Survivin as a newly discovered and critical KRAS-signaling partner essential for promoting these metabolic changes. We show that oncogenic KRAS potently upregulates the expression of Survivin in PDAC cells and patient-derived organoids undergoing increased glycolysis, whereas depleting Survivin expression inhibits their glycolytic activity and growth. Through a combination of cellular, biochemical, and imaging approaches, we further show that Survivin promotes the formation of unique microtubule-based structures that resemble invadosome rosettes, allowing for the recruitment of the glycolytic enzymes triose phosphate isomerase (TPI) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to these super-structures which drives the increases in glycolysis. These findings demonstrate that by directing the assembly of a microtubule-based complex of metabolic enzymes, Survivin serves as a vital link in a KRAS signaling pathway responsible for promoting the metabolic changes necessary for the accelerated growth of PDAC cells, and thus potentially highlight new therapeutic strategies for treating KRAS-dependent cancers.

Solid tumors remain refractory to conventional treatments, yet cell surface proteins, by virtue of their extracellular accessibility and critical roles in tumor signaling, represent an attractive class of targets for precision-targeted therapy. Here, we report that TMEM132A is an essential and previously unrecognized pan-cancer target. TMEM132A interacts directly with EGFR and stabilizes its expression, thereby tethering EGFR at the plasma membrane and sustaining constitutive activation of lipid synthesis. Mechanistically, the TMEM132A-EGFR axis promotes lipogenesis by facilitating SREBP nuclear translocation, which in turn upregulates ACLY and ACSS2 expression to drive acetyl-CoA production and downstream lipid biosynthesis, ultimately disrupting lipid droplet homeostasis. To therapeutically target this axis, we developed a nanobody, LFNanoT132A#3, which effectively blocks the TMEM132A-EGFR interaction, abrogates downstream signaling activation, and potently inhibits proliferation across multiple solid tumor types. Notably, LFNanoT132A also exerts robust antitumor activity against H1975 xenografts, a model resistant to first- and second- generation EGFR inhibitors, underscoring its potential to overcome conventional drug resistance. Our findings establish TMEM132A#3 as a critical node in membrane-tethered oncogenic signaling and metabolic rewiring, and position LFNanoT132A#3 as a promising therapeutic candidate for precision cancer therapy.

Skin, the bodys largest mechanosensitive organ, relies on a tension gradient across epidermal layers to maintain structure and function, but how mechanical force contributes to epidermal development and disease pathogenesis is poorly understood. By anchoring intermediate filaments (IF) to the plasma membrane, desmosomes, the most abundant intercellular junctions in the epidermis, help create a supracellular scaffolding that provides mechanical resilience to the tissue. However, the contribution of the desmosome-IF network to the epidermal response to mechanical strain remains unknown. Here we show that the desmosome-IF connection is not only required to induce a proper cellular mechano-response but is actively strengthened in response to stretch through the PP2A-mediated phospho-regulation of the cytoskeletal linker protein desmoplakin (DP). Additionally, we show in human skin dephosphorylated DP localizes to high tension layers, suggesting this mechano-response mechanism is coordinated with the epidermal tension gradient. Furthermore, in models of Carvajal syndrome, a cardio-cutaneous disorder caused by truncating DP mutations, cells lose mechano-responsive behavior and exhibit abnormal morphology in high-tension epidermal layers. Together, these findings identify the DP-IF network as a key component of the response to mechanical strain and show that its disruption compromises epidermal homeostasis and contributes to disease pathogenesis.

Keratin gene mutations are often associated with inflammatory skin disorders, in which the ensuing immunopathology is generally attributed to disrupted barrier integrity and consequent microbial invasion. Here, we challenge this paradigm by demonstrating a role for keratin 76 (Krt76) in thymic central tolerance to skin-targeting autoimmune responses. We show that transfer of Krt76-/- thymic lobes under the kidney capsules of athymic recipients is sufficient to induce expansion of effector T cells in the secondary lymphoid organs, T cell skin infiltration, and autoantibody reactivity to both skin and oral mucosa tissue. Mechanistically, we demonstrate that loss of thymic Krt76 expression disrupts canonical differentiation of the thymic medulla and impacts the development of post-AIRE-expressing keratinocyte-like mimetic medullary epithelial cells (termed CorneoTECs). Notably, Krt76-expressing CorneoTECs differentially express a specific skin and oral mucosa-associated gene signature, including skin-specific tissue self-antigens (TSAs). Importantly, in the absence of Krt76 this skin and oral mucosa TSA signature is almost entirely lost, and T cell negative selection is affected. Collectively, these data highlight a heretofore unanticipated role for Krt76 in thymic central tolerance to skin and oral mucosatargeting T cells and suggest that loss-of-keratin-associated skin disorders could also include autoimmune pathologies.

Keloids are benign fibroproliferative disorders majorly characterized by excessive extracellular matrix deposition, with recurrence rates exceeding 80% following conventional therapy. Although epigenetic dysregulation has been implicated in keloid pathogenesis, whether genome-wide DNA methylation actively drives pathological cellular reprogramming, and whether this state is therapeutically reversible, remains unclear. We performed genome-wide DNA methylation profiling on keloid tissues, matched primary keloid fibroblasts, and normal controls. Our analysis revealed a shared DNA hypermethylation pattern between keloid tissues and fibroblasts, which was validated by three independent public cohorts. By integrating DNA methylome and transcriptome, we demonstrated that DNA methylation-regulated genes were enriched in osteochondrogenesis-related pathways, such as cartilage and bone development pathways. Furthermore, pharmacologic inhibition of DNA hypermethylation by DNA demethylating agent decitabine reduced the expression of osteochondrogenic markers and inhibited collagen deposition and keloid growth in primary keloid fibroblasts and patient-derived xenograft (PDX) model, offering a potential therapeutic strategy of keloid.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by high metastatic dissemination, therapy resistance, and poor clinical outcome. Inhibitor of differentiation 1 or ID1, is frequently overexpressed in PDAC and is associated with tumour progression and adverse clinical outcome. However, the mechanisms governing its post-transcriptional regulation remain insufficiently characterized. Here, we identify tumour-suppressive miR-615-5p as a regulator of ID1 expression in PDAC. Integrative in-silico target prediction prioritized miR-615-5p based on seed complementarity and thermodynamic stability with the ID1 3' -UTR. Expression analysis of available PDAC clinical datasets revealed reduced miR-615-5p expression associated with increased ID1 expression. Direct association was validated using luciferase reporter assays, where miR-615-5p suppressed 3' -UTR reporter activity of ID1 in a sequence dependent manner, while mutation of the predicted binding site attenuated this effect. Further biotinylated-RIP and AGO2-RIP assays demonstrated the co-enrichment of ID1 transcripts and miR-615-5p with AGO2 associated RISC complexes, while AntimiR mediated inhibition of miR-615-5p perturbs association between miR/ID1 to AGO2, supporting interaction specificity. Functionally, modulation of miR-615-5p altered ID1 expression and impacted PDAC cell migration in vitro. Mechanistic analyses further indicated that the miR-615-5p/ID1 axis influences autophagic flux where miR-615-5p mediated inhibition of autophagy suppresses ID1 dependent cellular migration. Collectively, these findings define a previously uncharacterized miRNA-dependent regulation of ID1 expression and link this axis to autophagy-associated migratory responses in PDAC cells. The study expands the post-transcriptional regulatory landscape of ID1 and provides a possible mechanism where suppression of miR-615-5p leads to ID1 overexpression and subsequent poor clinical outcome in PDAC cells.

ZNF217 is an established oncogenic transcription factor that promotes cancer progression and therapeutic resistance; however, the mechanisms regulating ZNF217 protein abundance remain poorly understood. Here, we identify ubiquitin-specific peptidase 15 (USP15) as a critical regulator of ZNF217 stability and define a reciprocal USP15-ZNF217 signaling loop that sustains malignant phenotypes in ovarian cancer. Stable overexpression of ZNF217 in OVCA420 ovarian cancer cells enhanced proliferation, epithelial-mesenchymal transition, migration, invasion, and extracellular matrix adhesion. Notably, ZNF217 overexpression increased USP15 protein abundance without altering USP15 mRNA levels, whereas ZNF217 depletion reduced USP15 protein levels, suggesting post-transcriptional regulation. Conversely, USP15 depletion markedly reduced ZNF217 protein abundance while increasing ZNF217 mRNA levels, indicating that USP15 regulates ZNF217 predominantly at the post-transcriptional level. Proteasome inhibition restored ZNF217 protein levels following USP15 depletion, further demonstrating that USP15 promotes ZNF217 protein stability. Functionally, USP15 depletion in ZNF217-overexpressing ovarian cancer cells suppressed proliferation and multiple metastatic phenotypes, including migration, invasion, extracellular matrix adhesion, anoikis resistance, and multicellular aggregate formation. In vivo, USP15 depletion significantly reduced tumor progression and metastatic burden and prolonged survival in mice bearing ZNF217-driven ovarian tumors. Furthermore, USP15 depletion enhanced the sensitivity of ZNF217-overexpressing cells to carboplatin, paclitaxel, and doxorubicin. Collectively, these findings identify USP15 as an upstream regulator of ZNF217 protein stability and reveal a positive-feedback loop between USP15 and ZNF217 that reinforces oncogenic signaling. Targeting USP15 may therefore represent an indirect therapeutic strategy for suppressing ZNF217-driven ovarian cancer, particularly given the challenges associated with directly targeting oncogenic transcription factors.