Neoplasia

Background and AimsThe tumor microenvironment in colorectal cancer (CRC) is richly innervated, yet the contribution of the enteric nervous system (ENS) to CRC biology remains poorly defined. ENS neurons express proenkephalin (PENK), which can be processed by proprotein convertase 1/3 (PCSK1) to generate Methionine-enkephalin (M-ENK), a bioactive peptide with growth-regulatory potential. We hypothesized that an ENS-derived PCSK1-M-ENK axis restrains CRC proliferation through opioid growth factor receptor (OGFr) signaling and is modulated by stress-associated glucocorticoid receptor (GR) signaling and GLP1 receptor (GLP1R) activity. MethodsPublicly available human CRC single-cell RNA-sequencing datasets were analyzed for OGFr expression. PCSK1 and M-ENK expression in murine ENS and tumor-associated tissue was assessed by immunofluorescence. Functional studies were performed using murine CRC organoids, and primary murine ENS neurons in mono- and co-culture. CRC proliferation was quantified by EdU incorporation following treatment with recombinant M-ENK, recombinant PCSK1, OGFr synthetic ligand naloxone, or PCSK1 inhibitors. Effects of dexamethasone and liraglutide on PCSK1 expression in ENS-containing murine tissue were evaluated. ResultsOGFr was enriched in CRC cells and positively associated with KRAS gene expression. A subset of adult murine colonic myenteric neurons expressed PCSK1 and M-ENK. M-ENK dose-dependently suppressed proliferation of CRC organoid cells. ENS neurons also suppressed CRC proliferation in a PCSK1-dependent manner. Dexamethasone reduced, whereas liraglutide increased, PCSK1 expression. ConclusionsThese findings define a previously unrecognized ENS-derived neuro-oncologic pathway that is associated with reduced CRC cell proliferation and identify the GR/GLP1R-PCSK1-M-ENK axis as a potentially actionable therapeutic node. SummaryThis study identifies a neuronal PCSK1 - M-ENK pathway in the ENS that directly suppresses colorectal cancer growth through local OGFr activation, revealing a previously unrecognized neuropeptidergic mechanism of tumor control within the intestinal microenvironment.

Temozolomide (TMZ) resistance remains a major obstacle in glioblastoma (GBM) therapy, yet the metabolic adaptations underlying this phenotype are incompletely understood. Here, we performed integrative lipidomic, ultrastructural, and pathway analyses to define lipid metabolic reprogramming associated with TMZ resistance and failure of statin-mediated sensitization. Targeted LC-MS lipidomics quantified 322 lipid species across 25 lipid classes in TMZ-sensitive and TMZ-resistant U251 cells under basal conditions and following TMZ, simvastatin, or combination treatment. Multivariate analyses (PCA, PLS-DA, and volcano plots) revealed a robust and treatment-resilient lipidomic signature in resistant cells characterized by enrichment of lysophospholipids, sphingolipids, and cholesteryl esters, alongside depletion of glycerolipid and phospholipid pools. Complementary univariate analysis confirmed these changes at the species level, demonstrating consistent elevation of lysophosphatidylcholine/ethanolamine, glycosphingolipid subclasses, and cholesteryl esters, together with reductions in phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and diacylglycerol intermediates across multiple treatment conditions. In contrast, sensitive cells displayed dynamic lipid remodeling, including phosphatidylinositol and phosphatidylethanolamine enrichment associated with autophagic membrane expansion. KEGG pathway analysis linked the resistant phenotype to Rap1, PI3K-Akt, and phospholipase D signaling networks regulating vesicle trafficking and membrane homeostasis. Transmission electron microscopy confirmed a vesicle-rich intracellular architecture consistent with persistent autophagy flux blockade in resistant cells. Collectively, these findings define a stable lipid metabolic program characterized by lysophospholipid expansion and cholesteryl ester accumulation that supports membrane integrity and therapeutic resistance. Targeting lipid buffering and cholesterol storage pathways may represent a promising strategy to overcome chemoresistance in glioblastoma. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=134 HEIGHT=200 SRC="FIGDIR/small/712341v1_ufig1.gif" ALT="Figure 1"> View larger version (78K): org.highwire.dtl.DTLVardef@178acd7org.highwire.dtl.DTLVardef@19b6a79org.highwire.dtl.DTLVardef@6b3904org.highwire.dtl.DTLVardef@16c3d01_HPS_FORMAT_FIGEXP M_FIG C_FIG Lipidomic and autophagy differences between non-resistant (NR) and temozolomide-resistant (R) glioblastoma cells. NR cells show dynamic lipid remodeling and treatment-dependent autophagy responses, whereas R cells maintain blocked autophagy flux and persistent enrichment of LPC, SM, and cholesteryl esters across treatments.

BackgroundPatient-derived tumor organoids are widely used in cancer research, yet the biological impact of tissue processing during model generation remains unclear. Fragment-based and dissociation-based approaches are commonly assumed to trade fidelity for uniformity, but their molecular consequences remain incompletely defined. MethodsWe performed a proteome-wide comparison of fragment-based (CUT) and dissociation-based (DIS) glioblastoma organoid protocols using quantitative mass spectrometry. Organoids from multiple patient tumors were cultured under growth factor-free or growth factor-supplemented conditions and compared with matched primary tissue. ResultsBoth protocols produced technically robust glioblastoma organoids when maintained in their native media. However, CUT organoids matched the reproducibility of DIS cultures while preserving a broader extracellular matrix repertoire and networks linked to collagen assembly, vascular support, and cell-matrix signaling. DIS cultures were biased toward exogenous basement membrane components and proliferative, growth factor-responsive states. Across tumors, CUT organoids consistently showed greater proteomic similarity to matched primary tissue, retaining neural, glial, stromal, and extracellular features largely absent from DIS models. ConclusionsFragment-based glioblastoma organoids can be both reproducible and biologically faithful. Tissue dissociation acts as a major perturbation that reshapes extracellular matrix organization, cellular states, and tumor identity, making protocol choice a critical determinant of model fidelity and translational relevance.

Boys experience an overall increased incidence of several childhood cancers, including medulloblastoma, a clinically heterogeneous cerebellar tumor. In subtypes of Group 3 and Group 4 medulloblastoma, males are three times more prevalent than females. As medulloblastoma is suspected to initiate during fetal development, we hypothesized that this sex bias reflects a combination of prenatal, sex-specific developmental processes and somatic alterations. To test these hypotheses, we compiled a large multi-omics dataset from children with medulloblastoma, which revealed sex-specific alterations, including frequent loss of the inactive X chromosome in females with Group 4. Generation of a sex-matched single-cell transcriptome atlas of the developing murine cerebellum enabled investigation of putative developmental factors underlying sex bias. Progenitors giving rise to Group 3/4 subgroups were more abundant, more proliferative, and harbored more open chromatin for recruitment of LMX1A and OTX2, master transcription factors defining Group 3/4 identity. Advanced genetically engineered mouse models and human cerebellar organoids were leveraged to determine whether sexual dimorphism arises from intrinsic or extrinsic factors. These models showed that the XY genotype contributed to the phenotype, but the predominant effect was driven by presence of the male gonadal hormone testosterone. Our findings provide a sex-specific genetic and neurodevelopmental explanation for male bias in an aggressive pediatric brain tumor. Outcomes from this study may inform novel treatment strategies delivered according to sex and are likely to be broadly applicable to other sex-biased malignancies arising in early life. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/714163v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@3a06faorg.highwire.dtl.DTLVardef@1a01bb7org.highwire.dtl.DTLVardef@7bc9c2org.highwire.dtl.DTLVardef@fb206d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Prostate cancer is a common cancer in males and there is an urgent unmet clinical need to identify new therapies for advanced disease. Aberrant glycosylation is common in prostate cancer and plays a functional role in disease progression. The sialyl-Tn antigen (sTn) has been widely studied in cancer, yet its involvement in prostate cancer remains relatively unexplored. Here, we utilise a novel anti-sTn antibody (L2A5) to comprehensively monitor sTn expression levels in clinical prostate cancer tissues encompassing normal, benign, primary, metastatic castrate-resistant prostate cancer (CRPC), and patient-derived xenografts (PDXs). We show that while sTn is detected at low or negligible levels in normal prostate tissues, it is expressed in 44% of prostate tumours, and prostate cancer patients with high sTn levels have significantly poorer survival times. Analysis of metastatic therapy resistant prostate-derived tumours growing in liver and bone, shows sTn is expressed in 37.5% of cases. Furthermore, we show sTn is expressed in nearly half of PDXs tested, supporting the use of PDX models as tools for testing anti-sTn therapeutic strategies. These findings identify sTn as potential prognostic biomarker and therapeutic target in prostate cancer and lay the groundwork for the development of sTn-targeted precision therapies for advanced disease.

Glioblastoma (GBM) is the most aggressive primary brain malignancy, characterized by hypoxia-driven proliferation, therapeutic resistance, and poor prognosis. While hypoxia-induced transcriptional changes are well documented, the temporal regulation of cell cycle genes under sustained hypoxia remains unclear. This study profiled transcriptomic alterations in U87MG cells cultured under normoxia and graded hypoxia for one to three days. Differentially expressed genes (DEGs) were identified and analyzed using STRING, Cytoscape, MCODE, and CytoHubba to construct protein-protein interaction (PPI) networks and extract hub genes. Functional enrichment was assessed through DAVID, ClueGO, and KEGG, while prognostic relevance was evaluated using GlioVis and ONCOMINE datasets. qRT-PCR validated expression of selected hub genes. A total of 294 DEGs were identified, forming two main functional modules enriched in cell cycle regulation and chemokine signaling pathways. Eighteen hub genes (KIF20A, CCNB1, AURKA, EGR1, CDCA3, CENPF, CDCA2, ASPM, KIF11, CCL2, CCNA2, DLGAP5, RACGAP1, TPX2, PTGS2, CTGF, and KIFC1) were significantly associated with mitotic processes and GBM progression. Survival analysis demonstrated that 17 of these genes correlated with poor overall survival (p < 0.05). qRT-PCR confirmed that hub gene expression peaked during early hypoxia and declined with prolonged exposure, indicating dynamic regulatory adaptation. These findings identify key hypoxia-responsive genes governing cell cycle progression and highlight their prognostic and therapeutic potential in glioblastoma.

Diffuse hemispheric gliomas (DHGs) are highly aggressive and infiltrative CNS tumors that are refringent to treatment, and with a 5-year overall survival of around 20%. A fraction of DHGs is driven by mutations in the histones H3.1 and H3.3. In this study, we demonstrate that the expression of histone H3.3 glycine 34 to arginine mutations (H3.3-G34R) result in the epigenetic and transcriptional activation of the NF-{kappa}B signaling pathway in DHG. To target this vulnerability, we designed high density lipoprotein (HDL) nanoparticles loaded with unmethylated CpG dinucleotides, which mimic the immune stimulatory activity of bacterial DNA. CpG are recognized by Toll-like receptor 9 (TLR9), activating the NF-{kappa}B signaling. The CpG-mediated NF-{kappa}B activation results in the release of immuno-stimulating cytokines that promote an antitumoral response. As we previously established that G34-mutant DHGs are characterized by DNA repair impairment, we combined CpG dinucleotides with a PARP (poly (ADP-ribose) polymerase) inhibitor, olaparib, in the HDL nanoparticles.

BackgroundGlioblastoma is an aggressive and incurable brain tumor. Clinical trials of immune checkpoint inhibitors showed no clinical benefit in glioblastoma when given after surgery. However, a clinical trial in which PD1 inhibition was given prior to second surgery did show pharmacodynamic evidence for activity. This suggests the possibility that immune checkpoint inhibitors may be more effective in a setting where large tumors are present. Here we have studied immune responses to large tumors in an autochthonous mouse model of glioblastoma. MethodsGlioblastoma was induced by transfection with oncogenic plasmids injected directly into the lateral ventricle of neonatal mice. Immune responses were assessed using a combination of spectral flow cytometry and immunohistochemistry. ResultsThere was a marked immune response to large tumors, with significant increases in CD4 T cells and dendritic cells. T cell changes occurred primarily at leptomeningeal/perivascular border sites. A large proportion of CD4 T cells expressed PD1 and half of these were regulatory T cells. NK cells were also increased in mice with large tumors, but were predominantly in immature states. The mouse model accurately recapitulates the formation of palisading necroses. These contain apoptotic cells and avidly recruit myeloid cells that are induced to express large amounts of TGF{beta}. ConclusionsLarge glioblastoma tumors generate a border site population of PD1 positive T cells that may explain the pharmacodynamic response in neoadjuvant trials, and a palisading necrosis-driven immunosuppressive mechanism that may explain why responses are insufficient to provide a significant clinical benefit. KEY POINTSThe SB mouse model accurately recapitulates immune features of human glioblastoma Large tumors induce a significant border site immune response Palisading necroses in large tumors counter this with a strong immunosuppressive response IMPORTANCE OF STUDYImmune checkpoint inhibitors have not shown efficacy in glioblastoma when used post-surgery, but do show pharmacodynamic activity when used in patients prior to second surgery (i.e. neoadjuvant). This suggest the possibility that immune checkpoint inhibition is more effective when large tumors are present. Using a clinically-relevant autochthonous mouse model, we show here that large tumors induce an immune response that is evident in leptomeningeal border sites. Large tumors in this mouse model also generate palisading necroses, a well-known diagnostic feature in glioblastoma tumors. These palisading necroses generate large amounts of TGF{beta}, providing a mechanism by which large tumors can suppress border site immune responses. This further supports the concept that palisading necroses are drivers of glioblastoma malignancy and suggests novel strategies to enhance responses to immune checkpoint inhibition in this cancer.

Engineering macrophages with chimeric antigen receptors is emerging as a promising cancer therapeutic. Chimeric antigen receptor-expressing macrophages (CAR-Ms) engineered to recognize tumor-specific antigens have been shown to inhibit tumor growth and activate adaptive immune responses, leading to robust tumor control in animal studies. Based on this work, clinical trials have been initiated. While the trials have shown promise, challenges remain. The dynamic interactions between CAR-Ms and cancer cells and the exact mechanisms driving anti-tumor effects remain poorly defined. Defining the dynamic interactions between CAR-Ms and cancer cells will provide critical insights for optimizing future CAR-M design and improving therapeutic efficacy. We sought to directly visualize CAR-M interactions with glioblastoma cells at high-resolution and in real-time using CAR-Ms engineered to recognize Neural-Glial Antigen 2 (NG2), an antigen expressed on glioblastoma cells. Using patient-derived glioblastoma cells, we formed glioblastoma spheroids and embedded them in a 3D matrix together with CAR-Ms. Using time-lapse microscopy, as expected, we found that NG2-targeting CAR-Ms engulfed glioblastoma cells. However, excitingly, we found that NG2-targeting CAR-Ms blocked >85% of glioblastoma cell invasion in 3D. This inhibition of glioblastoma invasion was not due to a significant change in CAR-M polarization states. Together, these data suggest that NG2-targeting CAR-Ms both engulf glioblastoma cells and block glioblastoma invasive behavior.

Intratumor heterogeneity (ITH) is one of the main reasons for the lack of effective targeted therapies for glioblastoma (GBM). Imaging-guided surgical navigation allows for tumor-wide sampling to account for variation across distant regions of the tumor, but typical drug screening is performed on cell lines derived from a single biopsy and does not account for GBM heterogeneity. Here we profiled matching MRI-guided multi-region primary tumor biopsies from 6 GBM cases (n=40 biopsies) and corresponding neurosphere cultures (n=30) derived from these spatially distinct tumor samples. We found that in vitro cultures derived from distinct regions of the same tumor display divergent phenotypes, proliferative capacity and ability to accumulate 5-aminolevulinic acid, used to visualize cancer cells during surgery. The differential drug response of the multi-region neurospheres remains linked to the gene expression of the original tumor biopsies. Thus, studies with multiregion-derived neurospheres are essential to faithfully model GBM ITH for therapeutic testing. KEY POINTSO_LIMulti-region biopsy-derived neurospheres represent distinct spatial locations in the GBM tumor. C_LIO_LICultures derived from different regions of the tumor retain phenotypic diversity. C_LIO_LIParental biopsy phenotype predicts drug response better than to in vitro phenotype. C_LI IMPORTANCE OF THE STUDYCell lines developed from spatially distinct regions of glioblastoma capture its intratumor heterogeneity. We show that while the transcriptional output of these cell lines is not connected to their spatial origin, their drug response can be linked to it. Thus, spatial heterogeneity reflected in our neurosphere collection provides a new paradigm for drug screening in these highly heterogenous and difficult to treat tumors.

BackgroundGroup 3 (MYC-driven) medulloblastoma (MB) is a highly aggressive brain tumor with poor-prognosis and limited treatment options. We previously identified protein-arginine methyltransferase-5 (PRMT5) as a promising target in Group 3 MB with its control on MYC protein stability. In this follow up study, we further mechanistically investigated PRMT5 control on MYC transcription and targeted it pharmacologically for therapeutic proof-of-concept. MethodsUsing pharmacogenetic inhibition approaches against PRMT5 in MYC-amplified (Group 3) MB cell line and neurosphere models in vitro and in vivo, we investigated molecular mechanism(s) and anti-cancer efficacy of PRMT5 inhibition. ResultsOur experiments demonstrated that PRMT5 epigenetically regulates MYC transcription in MYC-amplified MB cells by binding to the proximal-promoter region of the MYC gene and contributing to the enriched symmetric-dimethylation of histone H4R3 in the same region. We further showed that PRMT5 is recruited to the MYC promoter by its interaction with BRD4, the major BET-protein responsible for MYC transcription. PRMT5 inhibition caused the suppression of MYC-induced transcriptional programs and target genes, with widespread disruption of splicing across the transcriptome, particularly affecting metabolism-related gene products. Pharmacologic inhibition of PRMT5 using a panel of selective small-molecule inhibitors demonstrates suppression of cell growth/survival in a MYC-dependent manner in MB cells. Moreover, our in vivo analyses of PRMT5 inhibition, in mice treated with one of the potent pharmacologic inhibitors, particularly a lipid-decorated form of it, demonstrated reduced cerebellar tumor growth with suppressed MYC expression and prolonged survival of mice with MYC-amplified MB xenografts. ConclusionsOur findings establish a functional link between PRMT5 and MYC-mediated transcriptional regulation, suggesting a promising therapeutic approach targeting the PRMT5-MYC axis for MYC-driven MB. Key PointsO_LIPRMT5 acts as an epigenetic regulator of MYC transcription, RNA splicing and associated energy metabolism in MYC-driven MB. C_LIO_LIPRMT5 inhibition selectively suppresses cell growth/survival in MYC-driven MB. C_LIO_LIPRMT5 inhibition reduces tumor burden and prolongs survival in a MYC-driven MB mouse model. C_LI Importance of the StudyGroup 3 medulloblastoma is a highly aggressive pediatric brain tumor marked by MYC amplification, malignant clinical behavior, and poor survival outcomes despite intensive multimodal therapy. Because MYC remains largely undruggable, there is an urgent need for effective and less toxic treatment options for affected children. This study identifies protein arginine methyltransferase 5 (PRMT5) as a key epigenetic regulator of MYC transcription and MYC-dependent oncogenic programs in Group 3 MB. We show that PRMT5 is recruited to the MYC promoter via BRD4, sustains MYC-driven transcription and RNA splicing networks associated with metabolism, and supports MB tumor growth. Importantly, pharmacologic inhibition of PRMT5 using a selective brain-penetrant inhibitor suppresses MYC expression, reduces cerebellar tumor burden, and prolongs survival in MYC-amplified MB models. These findings provide a strong translational rationale for PRMT5 inhibition as a targeted therapeutic strategy for high-risk MB, with the potential to improve outcomes while reducing treatment-related toxicity.

Background & AimsHepatocellular carcinoma (HCC) frequently exhibits resistance to immune checkpoint inhibitors (ICIs), particularly in {beta} -catenin-driven tumors characterized by immune exclusion. While the Unfolded Protein Response (UPR) and the Integrated Stress Responses (ISR) enable tumor adaptation to metabolic stress their role in shaping tumor immunogenicity remains incompletely understood. We investigated whether ATF4, a central effector of the integrated stress response, couples metabolic reprogramming to suppression of anti-tumor immunity in HCC. MethodsWe combined transcriptomic analyses across three independent human HCC cohorts with mechanistic studies using an immunotherapy-resistant MYC/{beta}-catenin-driven murine HCC model. We integrated CRISPR/Cas9-mediated deletion of Atf4 with RNA-sequencing and targeted metabolomics. The impact of tumor-derived metabolites on macrophage differentiation and polarization was evaluated using primary bone marrow-derived cells. Therapeutic responses were evaluated in orthotopic and subcutaneous models treated with anti-PD-1 and anti-VEGFA. ResultsATF4 and XBP1 transcriptional signatures are selectively enriched in human HCC and associate with poor prognosis, vascular invasion, and an immunosuppressive myeloid-enriched tumor microenvironment. Genetic ablation of Atf4 markedly suppressed tumor growth in immunocompetent but not immunodeficient hosts, establishing a requirement for immune-mediated tumor control. Mechanistically, Atf4 loss downregulated Aldh18a1 and disrupted proline biosynthesis, resulting in extracellular proline depletion. This proline-deficient environment abrogated monocyte-to-macrophage differentiation and decreased M2 polarization, thereby reshaping the tumor microenvironment toward enhanced T cell infiltration and activation. Functionally, Atf4-deficient tumors exhibited restored sensitivity to anti-PD-1 monotherapy and showed pronounced responses to combined anti-PD-1/anti-VEGFA treatment in aggressive orthotopic models. ConclusionATF4 programs a proline-dependent metabolic axis that sustains macrophage-mediated immunosuppression and immune evasion in {beta}-catenin-driven HCC. Disruption of this pathway converts immune-excluded tumors into T cell-inflamed states and restores responsiveness to immunotherapy. By governing proline homeostasis and macrophage-mediated immunosuppression, ATF4 is a key metabolic checkpoint for immune evasion, linking stress adaptation to immune escape and a candidate therapeutic target in HCC. Impact and implicationsWe identify ATF4 as a crucial metabolic-immune orchestrator that sustains myeloid-driven immune evasion in {beta}-catenin-dependent HCC through proline-dependent circuitry. Disrupting the ATF4-proline axis converts immune-desert tumors into T cell-inflamed lesions by blocking macrophage differentiation, thereby sensitizing tumors to immune checkpoint therapy. This work positions ATF4 as a tractable therapeutic target to overcome immunotherapy resistance in HCC. Graphical abstract Highlights- ATF4 orchestrates an immunosuppressive tumor microenvironment in HCC by coupling metabolic stress adaptation to immune evasion. - Ablation of ATF4 disrupts proline biosynthesis, leading to a marked depletion of extracellular proline. - Cancer cell-derived proline availability contributes to macrophage differentiation and M2 polarization; its loss restores T cell-mediated anti-tumor surveillance and sensitizes beta-catenin-driven HCC to immune checkpoint blockade.

Colorectal cancer (CRC) poses a severe global health threat with high incidence, mortality, and poor 5-year survival rates for advanced cases despite existing treatments. This study aims to explore the role of STRIP2 in CRC progression and its underlying mechanisms. Impact of STRIP2 on CRC in vitro was investigated via CRC cell proliferation, migration, invasion, and apoptosis. The in vivo impact was investigated via nude mice models. The role of STRIP2 in CRC was investigated via transcriptomic analysis, Western blot, Co-immunoprecipitation assays and ferroptosis validations. STRIP2 is overexpressed in CRC, driving malignant phenotypes in vitro and in vivo. Mechanically, STRIP2 stabilizes the IL17 downstream effector LCN2 by blocking its K48-linked ubiquitination and degradation, enhances anti-ferroptosis of CRC cells. Oe-STRIP2 suppresses ferroptosis, boosting proliferation and reducing oxidative stress; while si-STRIP2 induces the opposite effect. This study suggests STRIP2-mediated stabilization of LCN2 and enhances CRC cells ferroptosis resistance, thus promoting CRC cell survival and mediates malignant progression in CRC, which provides a novel link between STRIP2 and ferroptosis regulation in CRC. HighlightO_LISTRIP2 is overexpressed in CRC tissues and cells C_LIO_LISTRIP2 blocks LCN2 Ubiquitination and stabilizes LCN2 C_LIO_LISTRIP2 suppresses CRC ferroptosis C_LIO_LISTRIP2 drives CRC malignant phenotypes both in vitro & in vivo C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/725308v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1baf7baorg.highwire.dtl.DTLVardef@1de15d9org.highwire.dtl.DTLVardef@16c8078org.highwire.dtl.DTLVardef@667840_HPS_FORMAT_FIGEXP M_FIG C_FIG

Atypical teratoid rhabdoid tumor (ATRT) is a malignant brain tumor of children that has an overall survival of less than 40 percent even with aggressive therapy. We identified upregulation of the mitogen activated protein (MAP) kinase pathway in ATRT. The novel, brain-penetrant MEK inhibitor mirdametinib inhibited the growth of ATRT cell lines in culture at nanomolar concentrations. Mirdametinib suppressed proliferation as measured by BrdU incorporation and induced apoptosis as measured by cPARP and Annexin V staining. Monotherapy with mirdametinib extended the life of mice bearing orthotopic xenografts. Combination therapy with the brain-penetrant cyclin dependent kinase 4/6 inhibitor abemaciclib further suppressed growth and BrdU incorporation in ATRT cell lines representing all molecular subgroups. Mirdametinib and abemaciclib combined to extend survival of mice bearing orthotopic ATRT xenografts. In conclusion, mirdametinib has single agent activity against ATRT and combines with abemaciclib to decrease proliferation and extend survival in orthotopic xenograft models of ATRT.

Ionizing radiation can be an effective therapy for prostate cancer. Unfortunately, however, more aggressive prostate cancers such as neuroendocrine prostate cancer (NEPC) are often radiation resistant, which contributes to their high degree of morbidity and mortality. In this study, we used an unbiased approach to identify novel mechanisms that contribute to resistance to radiation and that are associated with neuroendocrine differentiation. Specifically, we compared the expression of cell surface proteins by mass spectrometry in prostate cancer cell lines that had been either untreated or treated with radiation to induce resistance, a process that also promotes neuroendocrine differentiation. Among the proteins identified by this screen, we focused on folate receptor (FR) because of its known biological functions and the fact that it is a validated therapeutic target. Our data reveal that FR has a causal role in enabling prostate cancer cells to resist radiation. Importantly, we also demonstrate that the expression of FR is regulated by HIF-1, which also has a causal role in radiation resistance and neuroendocrine differentiation. Given that the ability of cells to resist damage and death in response to ionizing radiation depends largely on their ability to buffer the substantial increase in reactive oxygen species (ROS) that is generated by radiation, we also demonstrate that the folate-FR axis promotes radiation resistance by sustaining intracellular glutathione levels that buffer this increase in ROS. In summary, the data reported here highlight a novel role for FR in resistance to ionizing radiation that is intimately associated with the hypoxic microenvironment of NEPC and the ability of the folate-FRa axis to maintain redox homeostasis.

Chromophobe renal cell carcinoma (ChRCC) accounts for 5% of all renal cancer cases. Despite its generally indolent behavior and low mutational burden, there is no targeted therapy for metastatic ChRCC. Profilin-1 (Pfn1), a cytoskeletal regulator of actin and tubulin dynamics, has emerged as a potential oncogenic driver in several cancers including RCC, but its role in ChRCC, remains undefined. We observed elevated Pfn1 expression in stage IV ChRCC patients, implicating Pfn1 in advanced disease progression. To investigate this, we manipulated Pfn1 expressions in two ChRCC cell lines UOK276 and RCJ41M. Pfn1 knockdown (KD) significantly reduced proliferation, invasion, and colony formation, whereas Pfn1 overexpression (OE) in UOK276 enhanced ChRCC aggressive phenotypes. Pharmacological inhibition of Pfn1 significantly suppressed proliferation and clonogenic growth in both cell lines. Additionally, Pfn1 KD increased intracellular ROS accumulation, while overexpressed reduced ROS levels, linking cytoskeletal regulation to oxidative stress control. Together, these findings position Pfn1 as a critical mediator of ChRCC progression, linking cytoskeletal remodeling to aggressive tumor behavior. This work highlights Pfn1 as a potential therapeutic target and establishes a framework for cytoskeletal-focused strategies in advanced ChRCC.

BackgroundGlioblastoma (GB) is a highly aggressive brain tumor with a median survival of approximately 14 months, primarily due to its ability to infiltrate healthy brain tissue both as single cells and in collectives. A deeper understanding of GB cell motility, both individual and collective, is crucial for developing patient-specific therapies. We aimed to characterize migration in patient-derived GB cells using advanced modeling to identify stratification markers and therapeutic vulnerabilities. MethodsWe developed Single-Cell Behavior Live Imaging (ScBLI), an approach integrating live imaging with computational analysis, applied to 30 GB primary cell cultures. Trajectories and morphological features were tracked and analyzed. Diffusion Entropy Analysis (DEA) was applied to classify trajectories based on the Delta Scaling parameter ({delta} scaling). We evaluated functional responses correlating all findings with clinical outcomes and transcriptomic profiles. ResultsWe analyzed 4,279 cell trajectories. Based on {delta} scaling (range 0.28-0.837), we defined three distinct motility groups: Low (L, {delta} scaling [&le;]0.5), Medium (M, 0.5 < {delta} scaling [&le;] 0.7), and High (H, {delta} scaling >0.7). Functional assays demonstrated that Group H cells are more performant in both positive and negative chemotaxis. Clinically, the three groups showed a clear linear progression with patient survival: High {delta} scaling correlated with the shortest survival (poorer prognosis), while Low {delta} correlated with the longest survival, suggesting that structured motility drives invasiveness. Integrative multi-omic analysis, encompassing both exome and transcriptome profiling, demonstrated that these groups are defined by distinct molecular landscapes rather than poor behavioral traits. Moreover, exome data revealed that Group H is significantly enriched in PTEN alterations (75% vs. 8% in Group L), with PTEN gain-of-function (GoF) mutations exclusively restricted to this group (100% vs 0% in Group L). Notably, within our extended cohort (n=51) currently characterized by whole-exome sequencing, we observed that specific PTEN GoF mutations were associated with a significantly shorter survival compared to PTEN wild-type cases (median OS 6.4 vs 16.6 months; p=0.02), which typically harbor the canonical loss of chromosome 10q. A similar clinical trend was observed when comparing directly GoF carriers to patients with truncating (Ter) alterations (median OS 6.4 vs 14.3 months; p=0.09). Conversely, no survival difference was found between truncating (Ter) mutations and wild-type cases. ConclusionOur findings demonstrate for the first time that migratory efficiency, quantified through DEA, represents a powerful predictor of glioblastoma aggressiveness. Tumor cells adopting highly efficient exploration strategies are strongly associated with poor clinical outcomes and are characterized by distinct molecular signatures, notably PTEN gain-of-function alterations. Statement of significanceOur multi-scale computational framework elucidates emergent behavioral phenotypes as pivotal drivers of glioblastoma progression. By demonstrating a correlation between enhanced migratory efficiency, PTEN gain-of-function, and significantly reduced overall survival, we establish a foundational paradigm for deciphering the emergent complexity governing tumor invasiveness.

PurposeProgastrin, aberrantly expressed in colorectal cancer (CRC), is an established trophic factor for tumour epithelial cells. Whether it also promotes CRC progression by reprogramming stromal fibroblasts remains unclear. We investigated progastrin-induced colonic fibroblast activation and its functional consequences on CRC cell migration. MethodsFibroblast activation was assessed in the colonic mucosa of hGAS mice and in the human normal colonic fibroblast line CCD18Co exposed to synthetic progastrin. The impact of tumour-derived progastrin on epithelial cell motility was analysed using HCT116 cells expressing a control shRNA (shLuc) or a progastrin-targeting shRNA (shPG) in transwell migration assays performed with or without fibroblasts. Candidate paracrine mediators were evaluated by RT-qPCR, ELISA and neutralization experiments, and signalling was interrogated using the PI3K inhibitor LY294002. ResultsColonic fibroblasts from hGAS mice displayed stromal FAP and SMA expression, indicating fibroblast activation in vivo. In CCD18Co cells, progastrin increased FAP and SMA protein levels. Fibroblasts enhanced HCT116 cell migration. This effect was stronger when tumour cells expressed progastrin or when fibroblasts were preconditioned by progastrin-producing HCT116 cells. Progastrin induced CXCL12/SDF-1 and CXCL8/IL-8 expression and secretion by fibroblasts, and neutralization of either chemokine abrogated the additional migratory effect conferred by progastrin-activated fibroblasts. Progastrin triggered sustained Akt phosphorylation in fibroblasts, while PI3K inhibition suppressed CXCL12 and CXCL8 secretion and abolished fibroblast-dependent tumour cell migration. ConclusionThese data identify a stromal dimension of progastrin signalling in CRC and support a model in which tumour-derived progastrin activates colonic fibroblasts and elicits a PI3K/Akt-dependent paracrine programme that enhances CRC cell migration.

BackgroundThe Netrin-1 dependence receptor pathway plays critical roles in neural development, but its expression landscape and prognostic significance in glioblastoma (GBM) remain poorly characterized. MethodsSingle-cell RNA-seq data from 148,019 cells across 34 tumors (Neftel et al., 2019) were analyzed to map Netrin-1 pathway gene expression across GBM cellular states. Differential gene expression and pathway enrichment analyses were performed on NEO1-defined subpopulations. Bulk RNA-seq survival analysis was conducted across three independent GBM cohorts TCGA (n=106), CGGA mRNAseq_325 (n=137), and CGGA mRNAseq_693 (n=237), totaling 480 patients. Primary analysis used continuous Cox regression (per-SD hazard ratios); meta-analysis employed fixed-effects inverse-variance weighting. ResultsIn GBM single-cell data, Netrin-1 pathway genes showed state-specific enrichment --NEO1, DCC, NTN1, and RGMB were predominantly expressed in oligodendrocyte-precursor (OPC) and neural-progenitor (NPC) states. Cells positive for NEO1 were enriched for neural differentiation programs (nervous system development, p=9.6x10-; Axon Guidance, p=2.8x10-), whereas NEO1-negative cells were dominated by ribosomal/translational and immune activation programs. In the 3-cohort survival meta-analysis, NTN1 (Netrin-1 ligand) emerged as the sole gene reaching meta-analytic significance as a risk factor (Meta HR=1.163 per SD, 95% CI 1.056-1.281, p=0.0021, I{superscript 2}=0%, 3/3 cohorts concordant), while DCC and RGMB showed directionally consistent protective trends (DCC: Meta HR=0.938, 95% CI 0.858-1.025, p=0.156; RGMB: Meta HR=0.979, 95% CI 0.881-1.087, p=0.686; both 3/3 cohorts concordant). NEO1 itself did not independently predict survival (Meta HR=1.008, 95% CI 0.885-1.147, p=0.910). After Bonferroni correction for 10 genes tested (threshold p<0.005), only NTN1 met strict significance. In exploratory sex-stratified analysis of a single cohort (CGGA 693, n=237), NEO1 and NTN1 exhibited female-specific risk enhancement (NEO1: HR=1.417, p=0.014; NTN1: HR=1.249, p=0.019), with minimal effects in males. UNC5B showed context-dependent risk in MGMT-unmethylated tumors (HR=1.331, p=0.037). These sex-dimorphic findings require independent validation. ConclusionsThe Netrin-1 pathway exhibits divergent prognostic trends in GBM, with NTN1 as a risk factor and DCC trending toward protection--consistent with the dependence receptor model. These findings, which should be interpreted as hypothesis-generating, nominate NTN1 as a candidate therapeutic target and highlight the potential importance of sex-stratified evaluation in future Netrin-1-directed trials. Independent replication in larger cohorts is warranted.

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas with poor prognosis and a strong tendency for metastasis and relapse. Surgical removal remains the mainstay of treatment but is frequently ineffective or impractical. Currently, no effective targeted therapy exists for this type of malignancy. PRMT5 has recently emerged as a promising therapeutic target in various human cancers with MTAP loss, which results in cancer cell dependency on PRMT5 activity. The frequent loss of MTAP in MPNSTs suggests that PRMT5 inhibition is a promising therapeutic option and enables the stratification of cancer patients with few treatment options. We first examined human nerve sheath tumor samples and found that increased PRMT5 expression and activity correlated with MTAP loss in 86.8% (33/38) of MPNSTs and in atypical neurofibromatous neoplasm with uncertain biologic potential (ANNUBP) (5/5). When PRMT5 activity was inhibited genetically and chemically, the cell growth of MTAP-deficient MPNST cell lines was suppressed, but not that of MTAP-proficient MPNST cell lines. Moreover, in the PRMT5-inhibited MTAP-deficient MPNST cell lines, spontaneous DNA damage accumulation was observed following G2/M cell cycle arrest. The DNA replication stress marker RPA32 decreased, and CHK1 was activated early after PRMT5 knockdown, likely contributing to the accumulation of DNA damage. In addition, we combined PRMT5 inhibition with the DNA-damaging agents doxorubicin and gemcitabine, resulting in synergistic effects and increased cancer cell death in MTAP-deficient MPNST cell lines. Together, these findings identify PRMT5 as a compelling therapeutic target in MTAP-deficient MPNSTs. This PRMT5 inhibition strategy has strong translational potential for MPNSTs. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/710638v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@15abc35org.highwire.dtl.DTLVardef@1fa4ebborg.highwire.dtl.DTLVardef@470c51org.highwire.dtl.DTLVardef@79cc3f_HPS_FORMAT_FIGEXP M_FIG C_FIG