Oncotarget

Many factors including ionizing radiation and iodine deficiency are known to increase thyroid carcinogenesis risk. Our dataset analysis of The Cancer Genome Atlas (TCGA) showed that lower mRNA expression of NK2 homeobox 1 (NKX2-1) transcription factor, a master regulator of genesis, homeostasis, and function of thyroid, is linked to poor prognosis of papillary thyroid cancer patients. Here we provide the findings that thyroid-specific Nkx2-1 conditional knockout (Nkx2-1{Delta}T) mice develop thyroid adenoma and carcinoma in higher frequency with combined exposure to radiation and iodine deficiency than control Nkx2-1fl/fl mice. Iodine deficiency caused oxidative stress, which subsequently resulted in DNA damage, leading to transformation of thyroid follicular cells. RNA-seq gene set enrichment analysis indicated higher production of reactive oxygen species (ROS) in the thyroids of Nkx2-1{Delta}T as compared to Nkx2-1fl/fl mice with combined exposure to radiation and iodine deficiency. This was accompanied by a feedback induction of SOD3 (superoxide dismutase 3) and GPX2 (glutathione peroxidase 2). These antioxidants were naturally expressed at higher levels in the thyroids of Nkx2-1{Delta}T than Nkx2-1fl/fl mice without iodine deficiency or radiation. Nkx2-1{Delta}T thyroids exhibited abnormal follicle architecture and up-regulation of Acox2 (encoding acyl-CoA oxidase 2), which produces hydrogen peroxide. These results suggest that loss of NKX2-1 may contribute to excess ROS production, which elevates basal oxidative stress resulting in the promotion of ROS-induced carcinogenesis. We propose a role for NKX2-1 as a regulator of ROS production homeostasis in the thyroid. Its disturbance would dispose thyroid follicular cells more vulnerable to the ROS-producing carcinogens.

Background Zinc finger protein 36-like 1 (ZFP36L1) is an AU-rich element-binding RNA-binding protein that regulates post-transcriptional gene expression and has been implicated in tumor progression, cell-cycle regulation, and DNA damage responses. However, its functional role in triple-negative breast cancer (TNBC) remains poorly understood. This study investigated the effects of CRISPR/Cas9-mediated ZFP36L1 knockout on cell proliferation, doxorubicin (DOX) sensitivity, cell-cycle progression, and DNA damage responses in MDA-MB-231 TNBC cells. Methods Wild-type (WT) and CRISPR/Cas9-generated ZFP36L1 knockout (KO) MDA-MB-231 cells were cultured under standard conditions. Cellular proliferation was evaluated by cell counting over three weeks. Cell viability following DOX treatment was determined using the MTT assay, and half-maximal inhibitory concentration (IC50) values were calculated. Cell-cycle distribution was assessed by propidium iodide flow cytometry after 24 h of DOX exposure, while DNA damage was quantified by {gamma}-H2AX flow cytometric analysis. Statistical significance was determined using Student's t-test with P < 0.05 considered significant. Results ZFP36L1 knockout reduced the proliferative capacity of MDA-MB-231 cells compared with WT cells. Both cell lines exhibited dose-dependent decreases in viability following DOX treatment. KO cells demonstrated a higher mean IC50 than WT cells (9.64 vs. 8.40 M), indicating a trend toward reduced DOX sensitivity; however, this difference was not statistically significant (P = 0.569). Flow cytometric analysis revealed enhanced accumulation of KO cells in the S and G2/M phases following DOX treatment, suggesting altered cell-cycle checkpoint regulation. Furthermore, KO cells exhibited elevated basal {gamma}-H2AX expression and greater DOX-induced {gamma}-H2AX accumulation than WT cells, indicating increased DNA damage and impaired maintenance of genomic stability. Conclusions CRISPR/Cas9-mediated loss of ZFP36L1 suppresses proliferation, alters cell-cycle checkpoint dynamics, and enhances DNA damage accumulation in MDA-MB-231 TNBC cells. These findings indicate that ZFP36L1 plays a context-dependent role in regulating genomic stability and cellular responses to genotoxic stress, highlighting its potential as a biomarker and therapeutic target in triple-negative breast cancer.

BackgroundPartner and Localizer of BRCA2 (PALB2) is a key tumor suppressor gene involved in homologous recombination-mediated DNA repair through its interactions with BRCA1 and BRCA2. Germline alterations in PALB2 have been associated with hereditary breast cancer risk; however, its broader molecular role in breast cancer progression and prognosis requires further investigation. MethodsA comprehensive in-silico analysis of PALB2 was performed using publicly available databases and bioinformatics platforms. Differential expression of PALB2 in breast cancer were evaluated using GEPIA2. Prognostic significance was assessed through Kaplan-Meier analyses for overall survival (OS) and disease-free survival (DFS). Protein-protein interaction (PPI) networks were constructed using STRING. Functional enrichment analyses of PALB2-associated genes were conducted using g. Mutational profiling of PALB2 in breast cancer was performed using cBioPortal with data from TCGA breast cancer cohorts. ResultsPALB2 expression was elevated in breast tumor tissues compared with normal breast tissues. Survival analyses revealed no statistically significant association between PALB2 expression and either overall survival (HR = 0.88, p = 0.44) or disease-free survival (HR = 0.74, p = 0.11). Protein interaction analysis revealed strong interactions between PALB2 and major DNA repair proteins including BRCA1, BRCA2, RAD51, RAD51C, FANCD2, and BRIP1. Functional enrichment analysis showed limited significant pathway enrichment, with only marginal transcription factor motif enrichment observed. Mutational analysis demonstrated diverse genomic alterations including missense mutations, truncating mutations, copy number gains, and shallow deletions. ConclusionThe findings support the biological relevance of PALB2 in breast cancer through its elevated expression and strong connectivity within DNA repair pathways. However, PALB2 expression alone does not appear to serve as an independent prognostic indicator. Further studies integrating genomic, transcriptomic, and clinical parameters are required to clarify its role in breast cancer progression and therapeutic response.

Cisplatin resistance remains a major barrier in head and neck squamous cell carcinoma (HNSCC) treatment. ATP-binding cassette (ABC) transporters contribute to chemoresistance by limiting intracellular drug accumulation. Bitter taste receptor 10 (T2R10) has been implicated in ABC transporter regulation, but its role in HNSCC remains undefined. HNSCC cell lines were treated with T2R10-agonist caffeine (100 or 200 M), cisplatin, or a combination, and viability was assessed by crystal violet assay. T2R10 promoter activity and expression following caffeine exposure were evaluated using a promoter-driven mCherry reporter and RT-qPCR. ABC transporter expression was measured after caffeine treatment and T2R10 gene (TAS2R10) knockdown or overexpression. Associations between tumor TAS2R10 expression and survival were assessed using TCGA data through GEPIA2. Caffeine enhanced the cisplatin-associated reduction in viability in a cell line- and concentration-dependent manner, with the strongest effect seen in UM-SCC47. A significant effect was observed in FaDu at 200 M of caffeine, and minimal response in RPMI 2650. RPMI 2650 cells and FaDu cells exhibited lower baseline TAS2R10 expression and RPMI 2650 cells did not demonstrate enhanced cisplatin sensitivity following caffeine treatment. Caffeine treatment increased TAS2R10 promoter activity and expression and was associated with decreased ABCG2 expression. TAS2R10 knockdown increased ABCG2 and ABCF1 expression, whereas TAS2R10 overexpression reduced ABCG2 and ABCC1 expression. High tumor TAS2R10 expression was associated with improved disease-free survival (log-rank p=0.0071; HR=0.61) but not overall survival. Caffeine enhances cisplatin sensitivity in selected HNSCC models. Caffeine exposure is associated with increased TAS2R10 expression and reduced expression of chemoresistance-associated transporters, particularly ABCG2.

Classic Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) are hematopoietic stem cell cancers that result in aberrant trilineage myeloid cell proliferation, bone marrow fibrosis, and increased risk of acute myeloid leukemia. MPNs are driven by deregulated activity of the JAK2 kinase, induced by mutations in the JAK2, CALR, and MPL genes, but approved JAK2 inhibitors primarily offer palliative effects, not remission. Cell models that demonstrate MPN oncogene driven JAK2 activity requisite for cell proliferation are important research tools for the development of anti-JAK2 and anti-JAK2 signaling therapeutics for MPN. SET2 and UKE1 cells are two such cell lines, as they express JAK2-V617F, one of the major driving mutations of MPN, and require signaling by JAK2 for their growth and viability. These cell lines are AML cell lines that were derived from patients with a previous diagnosis of MPN before they developed AML. Our previous studies demonstrated that the SHP2 phosphatase may be a therapeutic target for MPNs, and here we report our identification and characterization of an activating point mutation of SHP2 (encoded by the PTPN11 gene), SHP2-F71L, in UKE1 cells. Given SHP2 functions downstream of JAK2 and mediates JAK2 activation of RAS, we set out to determine the effect of mutational activation of SHP2 on the sensitivity of MPN model cells to JAK2 inhibition. We used CRISPR-Cas9 to edit this mutation in UKE1 cells back to wildtype such that these cells only express wildtype SHP2. These cells exhibited enhanced sensitivity to SHP2 inhibition and, notably, enhanced sensitivity to the JAK2 inhibitor ruxolitinib. This altered sensitivity was reverted by exogenous expression of SHP2-F71L but not SHP2-WT, indicating expression of an activated SHP2 may alter sensitivity to JAK2 inhibition in MPN model cells. We further explored this by genetically editing SET2 cells to express SHP2-F71L but observed no change in SHP2 inhibitor or JAK2 inhibitor sensitivity in cells with a SHP2-F71L encoding allele of PTPN11. Using the cytokine dependent BaF3 cell line where deregulation of JAK2 signaling by expression of JAK2-V617F induces cytokine independent transformation that remains dependent on this JAK2 signaling, we observed no effect of the expression of an activated SHP2 mutant on the sensitivity of the growth and viability of these cells to ruxolitinib. Recent studies have demonstrated activation of RAS signaling can antagonize JAK2 inhibition in pre-clinical MPN models, and the presence of RAS pathway mutations associates with patients whose disease advances on ruxolitinib therapy. Such mutations include activating mutations in PTPN11, as SHP2 is an upstream activator of RAS signaling. Our results suggest that activating PTPN11 mutations have the potential to desensitize the effects of JAK2 inhibition therapy in patients undergoing therapy and may be dependent on unknown cell and molecular profile contexts.

Small cell lung cancer (SCLC) is an aggressive thoracic malignancy with a 5-year survival rate under 7%. Lack of meaningful improvement of survival rates despite advances in treatment highlights the need for novel therapeutic approaches to improve patient outcomes. Currently, carboplatin + etoposide chemotherapy is the backbone of treatment for most patients. Lurbinectedin is a cytotoxic drug with unique activity against small cell lung cancers in patients with extensive disease and acquired resistance to carboplatin + etoposide. Our preliminary experiments in human SCLC cell lines treated with lurbinectedin demonstrated a dose-dependent increase in Chk1 and Chk2 protein phosphorylation. A consequence of the frequent TP53 inactivation in SCLC is tumor cell reliance on G2/M cell cycle checkpoints involving Chk1/Chk2 to maintain genomic integrity and allow cell survival following DNA damage. We hypothesised that inhibition of Chk1/Chk2-dependent responses with dual-inhibitor prexasertib (ACR-368), would potentiate tumor cell killing by lurbinectedin potentially in a synergistic manner. SCLC cells underwent cell death following single agent prexasertib exposure and this further increased with prexasertib + lurbinectedin combination. Highest Single Agent (HSA) synergy score calculations based on cell viability measurements suggested synergistic action between prexasertib and lurbinectedin at select dose combinations. Western blot analysis of intracellular proteins from SCLC cells treated with both drugs demonstrate dynamic, dose-dependent effects on Chk2, Chk1 and downstream effector Wee1, with lurbinectedin increasing intracellular levels of pChk1 and pChk2, while co-treatment with prexasertib deregulates this process across multiple human-derived cell lines. Synergistic killing was associated with elevated {gamma}-H2AX levels indicative of DNA double strand breaks and PARP-cleavage due to apoptotic caspase activation. Despite some heterogeneity among treated SCLC cells, the increased phosphorylation of Chk1 was noted at several kinase-activating sites including Serine 296, 317, and 345 while Chk2 Tyrosine 68 phosphorylation was consistently upregulated by lurbinectedin. The results provide a preclinical mechanistic rationale for overcoming a pro-survival, drug resistance- promoting checkpoint pathway to enhance the unique efficacy of single-agent lurbinectedin in patients with SCLC.

Emerin (EMD), an inner nuclear membrane protein essential for nuclear architecture integrity, gene expression, cellular signaling, and chromatin stability, interacts with the LINC complex and participates in cytoskeleton-nucleoskeleton communication by binding to nuclear actin filaments. EMD is implicated in migration, invasion, and metastasis in some tumors, but its role in glioblastoma (GBM) remains unclear. This study evaluated the effects of EMD knockdown and overexpression in GBM cell lines following genotoxic treatment with cisplatin. In both wild-type p53 (U87-MG) and mutant p53 (U138-MG) GBM cells, EMD expression is high, and cisplatin treatment did not affect these protein levels. EMD knockdown in U87-MG cells significantly increased cisplatin IC50, viability, and proliferation. Conversely, stable overexpression of EMD in U87-MG cells led to reduced cisplatin IC50, viability, proliferation, and migration. EMD knockdown or overexpression did not affect any U138-MG phenotypes, with or without cisplatin treatment. Modulation of EMD levels causes morphological changes in stress fiber cytoskeleton, whereas overexpression of EMD in U87-MG cells promotes an increase and a decrease in nuclear and cytoplasmic actin levels, respectively. These biological responses of U87-MG cells overexpressing EMD were coincidentally associated with alterations in the levels of pH2AX(Ser139), p-p53(Ser15), p53, and p21Kip1 proteins after cisplatin exposure. In sum, modulation of EMD levels affects the viability, migration, and proliferation of wild-type p53 GBM cells treated with cisplatin, suggesting unknown roles in the DNA damage response and repair. This work highlights EMD as a potential regulator of GBM chemoresistance and a target for therapeutic intervention.

In cancer cells, septins assemble into enigmatic higher-order structures of 300-700 nanometers, including long needle-like filaments, thick perinuclear rings, and cytoplasmic bundles or aggregates. The absence of genetic or pharmacological tools to recapitulate these architectures in-vitro has impeded mechanistic studies of their formation, function, and therapeutic targeting. Here, first, determining the overexpression of septin-2 in epithelial ovarian cancer (EOC) and its association with increased mortalities and dependencies, we select SKOV-3 ovarian cancer cells as a tractable model in which septin supramolecular assemblies can be recreated in-vitro and interrogated. This system shows that the forchlorfenuron (FCF) analog UR214-9 remodels septin architecture, converting co-expressed human septin octamers (SEPT2-SEPT6-SEPT7-SEPT9-SEPT9-SEPT7-SEPT6-SEPT2) into large cytoplasmic aggregates. In parallel, transiently expressed SEPT2 is reorganized into septin-rich noodle-like filaments, perinuclear rings, and web-like networks encircling the nucleus upon UR214-9 treatment. Mechanistically, UR214-9 disrupts the incorporation of SEPT2, SEPT7, and SEPT9 into canonical septin hetero-octamers, resulting in assembly-defective or imperfect oligomers that preferentially reorganize into these aberrant higher-order structures. This aggregation likely prevents septin-2 migration during interphase-to-cleavage furrow transition in NRK-49F-SEPT2-EGFP homozygous cells and impacts SKOV-3 cytokinesis, cell proliferation, adhesion and invasion and migration while sparing ceramide transport to the Golgi, preserving ER and cis-Golgi structure. These effects manifested in reduced growth of ovarian, endometrial and breast cancer xenografts without attracting significant off-target engagements per the global transcriptomic analysis of JIMT1 breast cancer and PANC-1 pancreatic cells. UR214-9 treated animals showed observable safety in animals. Thus, a tool to recreate aberrant septin structures and identification of septins as a druggable cytoskeletal target for ovarian, endometrial, breast and pancreatic cancer by perturbing their hetero-octamerization assembly is presented. SignificanceWe provide a method to reconstruct the higher-order septin architecture observed in cancer cells, to study their assembly and functions. Intriguingly, cancer cells tolerate hetero-oligomeric septins lacking specific subunits, suggesting that compositionally deficient oligomers are not efficiently targeted for degradation, unlike unincorporated septin monomers in normal cells. This tolerance may enable accumulation of structurally aberrant septin complexes acquiring long-needles, rings or thick-aggregates in disease cells. We further show that septin oligomerization can be pharmacologically perturbed. By integrating structural, cellular, and energetic readouts using in-silico techniques, we establish a quantitative framework for septin-targeted modulation, generating UR214-9 as a new chemotype that disrupts septin oligomeric assembly via preventing incorporation of SEPT2/7/9, into canonical hetero-octamers, causes defects in cytokinesis, altered cell migration, viability, and remodels septin-actin architectures, ultimately impairing tumor cell growth. Thus, pharmacological targeting of septin assembly represents a tractable strategy to perturb septin-dependent cellular processes in cancer and neurodegenerative diseases with reported septin dysregulation.

Abstract Background: The stem cell factor receptor or c-Kit is a type III receptor tyrosine kinase, activated by its ligand Stem cell factor (SCF). Up on activation, c-kit induces signaling pathways that regulates blood cell proliferation, survival, differentiation, and migration. Several studies reported that c-Kit/SCF signaling, contributes to the development and progression of acute myeloid leukemia (AML) in patients. However, the downstream proteins regulated by c-kit activation and their clinical significance in AML remain poorly explored. Methods: Human Acute megakaryoblastic leukemia (Mo7e) cells, were-stimulated with SCF and global protein expression were profiled using two-dimensional gel electrophoresis coupled with MALDI-TOF and LC-MS/MS. Differentially expressed proteins were functionally characterized and validated using patient data from the TCGA-LAML and matched normal data from GTEx, GEO datasets, and quantitative RT-PCR. Their diagnostic and prognostic significance was assessed using ROC, Cox regression, LASSO, Kaplan Meier survival analyses, and a prognostic nomogram model. Results: Proteomic profiling identified 14 differentially expressed proteins in SCF-stimulated Mo7e cells, which are predicted to involved in cytoskeletal organization, protein folding, metabolism, vesicular trafficking, and translational regulation. Transcriptomic analysis of the TCGA-LAML cohort revealed significant dysregulation of CFL1, CCT8, HSP90B1, MDH2, EIF5A, GSN, and TPI1. Integrated ROC, Cox regression, and LASSO analyses identified CFL1, CCT8, and GSN as the most robust prognostic biomarkers associated with poor overall survival in LAML patients. Their expression patterns were validated in independent GEO datasets and by qRT-PCR in SCF stimulated Mo7e cells. Finally, a three-gene nomogram model was developed and validated to predict the overall survival probability of AML patients at 1-, 3-, and 5-year time points. Conclusions: This study identifies CFL1, CCT8, and GSN as key downstream effectors of c-Kit signaling as prognostic biomarkers for AML. These findings provide mechanistic insights into c-Kit-driven leukemogenesis and establish a clinically relevant three-gene signature for AML risk stratification and potential therapeutic targeting.

Tumour hypoxia is associated with increased invasiveness, metastasis, and drug resistance; however, its impact on drug-metabolising enzymes remains poorly understood. This study investigated the effect of hypoxia on the expression of selected aldehyde dehydrogenase (ALDH) isoforms (ALDH1A1, 1A2, 1A3, 1B1, 2, 3A1, and 7A1) in colorectal cancer (CRC) cells. CRC cell lines (HT29, DLD-1, SW480, and HCT116) were cultured under normoxic and hypoxic (0.1% O2) conditions, while HT29 and DLD-1 cells were additionally grown as multicellular spheroids (MCS). Expression of ALDH isoforms was assessed at the mRNA and protein levels. Functional studies included siRNA-mediated knockdown of ALDH1A1, ALDH3A1, and ALDH7A1, measurement of reactive oxygen species (ROS), and stable overexpression of ALDH7A1 in H1299 cells. ALDH7A1 was consistently upregulated at both transcript and protein levels in HT29 and DLD-1 cells exposed to hypoxia. Elevated ALDH7A1 expression was also observed in hypoxic regions of MCS and CRC xenografts (HT29, DLD-1, HCT116, SW620, and COLO205). Knockdown of ALDH7A1 in DLD-1 cells reduced proliferation, increased ALDH3A1 expression, and significantly elevated ROS levels, indicating a role in redox homeostasis and suggesting functional crosstalk between these isoforms. Conversely, stable overexpression of ALDH7A1 in H1299 cells markedly reduced ROS levels. Taken together, these findings identify ALDH7A1 as a hypoxia-responsive enzyme that promotes adaptation to oxidative stress and may contribute to CRC cell survival within the hypoxic tumour microenvironment.

A fraction of patients treated with radiotherapy are known to be more sensitive to ionizing radiations. Skin fibroblasts from such radiosensitive individuals exhibit a higher cellular toxicity after irradiation and a delay in DNA repair. Deciphering the molecular mechanisms underlying these cellular defects is thus of major importance. We previously observed that the transcription factor NFATc2 is expressed at a reduced level in fibroblasts from radiosensitive patients. The present work aimed to elucidate the role of NFATc2 in the regulation of DNA repair, particularly the repair of radiation-induced double-strand breaks. We demonstrate an interaction of NFATc2 with the NHEJ repair protein Ku80 and observe that the NFATc2 RHD domain is necessary and sufficient for this interaction. Moreover, we show that NFATc2-Ku80 complexes are not colocalized to DNA double-strand breaks sites suggesting an involvement upstream of the DNA repair pathway. The silencing of NFATc2 impairs the NHEJ repair activities by delaying Ku70-Ku80 interaction in the early steps of this pathway. Finally, stable over-expression of NFATc2 in patients fibroblasts partially rescues their defective DNA repair phenotype, especially in the most radiosensitive cells. Altogether, our data reveal that NFATc2 is a regulator of DNA repair in skin fibroblasts and therefore a potential modulator of cellular radiosensitivity.

Bioactive vitamin D (1,25-dihydoxyvitamin D or 1,25(OH)2D) is synthesized from its inert circulating form 25-hydroxyvitamin D (25(OH)D) by the enzyme 1--hydroxylase in the kidneys and in other tissues including breast. Because breast cancer is associated with changes in intra-tumoral lipid composition and vitamin D is known to affect lipid metabolism, we investigated the potential role of tumor-produced 1,25(OH)2D on lipid profile expression during breast tumor progression. For that purpose, we used the MMTV-PyMT mouse model which mimics the four phases of tumor progression seen in human breast cancer (hyperplasia, adenoma/mammary intraepithelial neoplasia (MIN), early carcinoma and late carcinoma). In previous studies we showed that conditional ablation of the gene encoding 1--hydroxylase (Cyp27b1), specifically in the mammary epithelium of this MMTV-PyMT mouse model, resulted in enhanced spontaneous tumor initiation and progression. In the present study, we used mass spectrometry imaging to compare lipid composition in the mammary glands of Cyp27b1 ablated and non-ablated MMTV-PyMT mice. In non-ablated control animals, we observed changes to specific lipid signals linked to stages of tumor progression. In particular, several discriminatory lipid signals were significantly up regulated throughout tumor progression. In ablated mice, absence of Cyp27b1 in the mammary epithelium was accompanied by different lipid signals in hyperplastic lesions. Several lipid signals were exclusively detected in non-ablated tumors but absent in hyperplasia. Our findings suggest that the tumor-produced 1,25(OH)2D known to play a key role in mammary tumor progression is mechanistically related to early changes in lipid composition seen prior to the development of hyperplasia.

BackgroundMeningiomas are the most common primary nervous system neoplasm in adults. There are few good cellular models, especially of high grade/malignant meningiomas, to use to identify new therapeutic agents and treatment regimens. The widely available, partially characterized, NF2-wildtype (NF2wt) Grade 3 malignant meningioma cell line IOMM-Lee can help meet this need. MethodsWe generated new data to better characterize IOMM-Lee genomic and mtDNA variants, proliferation rate and colony-forming efficiency and sensitivity to ionizing radiation as a function of ATM kinase activity. A screen of 349 anti-cancer drugs identified multiple, mechanistically distinct clinical use drugs with nanomolar IC50 values and high drug sensitivity prediction scores. ResultsExome sequencing confirmed that IOMM-Lee is NF2wt, and contains a pathogenic TERT-promoter (c.-124C>T) variant. Population doubling times (PDT) were short (19-21 hrs), and colony forming efficiency (CFE) high, of up to 87%. IOMM-Lee is comparatively radiosensitive with a D10 of [~]3.9 Gy, and could be radiosensitized by AZD-1390-mediated ATM kinase inhibition. Thirty-four anti-cancer compounds spanning several mechanistic classes were identified that potently suppressed cell proliferation at sub-micromolar IC50 values with high Breeze 2.0 Drug Sensitivity Scores. Importance of the StudyWe provide new data to better characterize IOMM-Lee, the most widely used cell line model of human Grade 3 malignant meningioma. These data identify and characterize IOMM-Lee genomic alterations and mtDNA variants; quantify growth kinetics and ionizing radiation sensitivity; and identify multiple mechanistically distinct, clinical use drugs with nanomolar IC50 values, high drug sensitivity prediction scores and potential as meningioma systemic therapies. Our data more clearly locate IOMM-Lee in the landscape of genomically-defined meningiomas, and will aid better use of this experimentally tractable cell line model to understand meningioma biology and identify more effective malignant meningioma therapies and treatment regimens. Key pointsO_LIIOMM-Lee lacks NF2 mutations, though is clearly related to but distinct from many other meningiomas and meningioma cell lines. C_LIO_LIIOMM-Lee grows rapidly, is comparatively radio-sensitive, and can be suppressed by several mechanistic classes of anti-cancer agents at clinically achievable, sub-micromolar IC50 values with high Drug Sensitivity Scores. C_LIO_LIThe experimental tractability, simplicity and versatility of IOMM-Lee can facilitate analyses of many aspects of meningioma biology and therapeutic development across a wide range of in vitro, high throughput and in vivo xenograft/organoid protocols. C_LI

Introduction: Lung cancer in never-smokers is a growing, biologically distinct entity lacking non-invasive markers. Established urinary markers - creatine riboside (CR) and N-acetylneuraminic acid (NANA) - report tumor-intrinsic metabolism, not carcinogen processing. We investigated 27-nor-5{beta}-cholestane-3,7,12,24R,25S-pentol glucuronide (CPG), a bile-acid glucuronide linked to aryl-hydrocarbon-receptor (AhR)/CYP xenobiotic metabolism. Methods: Urinary CPG was quantified by UPLC-tandem mass spectrometry in an exploratory (NCI-Maryland; n=846) and validation (Colorado; n=505) cohort of non-small-cell lung cancer cases and frequency-matched controls. Associations with case status, smoking stratum, survival, and discrimination were assessed, using tumor RNA sequencing (n=83) and gene-set enrichment analysis (GSEA). Results: Urinary CPG was higher in cases than controls in both cohorts (P<0.0001). In never-smokers, cases exceeded smoking-matched controls (P<0.001 and P<0.0001), indicating elevation independent of tobacco exposure. After mutual adjustment for CR and NANA, CPG remained independently associated with case status (exploratory OR 1.58, 95% CI 1.15-2.16; validation OR 3.92, 95% CI 2.47-6.29), with a modest gain in discrimination. High CPG identified never-smokers with worse survival in both cohorts (P<0.001 and P=0.04), remaining significant after multivariable adjustment only in the exploratory cohort. GSEA showed AhR/CYP xenobiotic and Nrf2 oxidative-stress enrichment in high-CPG tumors; the CPG aglycone carried disease-specific 24R,25S stereochemistry. Conclusions: Urinary CPG was associated with NSCLC in two retrospective case-control cohorts, including in a smoking-matched never-smoker comparison. High CPG also identified never-smokers with worse survival, remaining independently prognostic after adjustment in the exploratory cohort. Tumor expression does not establish tissue of origin. Prospective validation against CR and NANA is required.

BackgroundReactive Oxygen Species (ROS) are the small, unstable and highly reactive species, having DNA oxidizing ability. Oxidation of the DNAs purine and pyrimidine bases can lead to single or double strands in this macromolecule. In this situation, the ATM molecule, a serine-threonine kinase, targets several proteins for phosphorylation, which causes the cell cycle to stop and the DNA damage repair begins. It has previously been proven that natural polyphenols have the cancer inhibiting properties due to their high efficacy and low side effects. Silibinin is the main herbal and medical ingredient in Milk Thistle (Silybum marianum) is a polyphenol flavonolignan, which has been widely considered as an antioxidant and anticancer agent. The purpose of the present study was to investigate the ATM gene expression and measurement of reactive oxygen species (ROS) in SKBR3 cell line, treated with Silibinin. Materials and MethodsAt first, the SKBR3 cell line was cultured in RPMI1640 culture medium and MTT assay was carried out to evaluate the Silibinin cytotoxicity. Flow Cytometry was carried out for cell cycle analysis, apoptotic induction, and ROS detection. While, Real Time PCR was used to evaluate the ATM gene expression in the Silibinin-treated and un-treated SKBR3 cells. ResultsPresent results have shown that 150 {micro}M Silibinin had the most significant cytotoxicity and apoptotic induction influence after the treatment period of 48 h. Flow cytometry data have shown that Silibinin induced considerable amount of apoptosis and caused cell cycle arrest at G1/S phase and induced production of ROS. Real-time PCR results have revealed that Silibinin increased the ATM expression in SKBR3 cell line. ConclusionSilibinin causes increased ATM gene expression by inducing ROS production, which initiates cell cycle arrest and apoptotic induction in SKBR3 cells line.

Background/ObjectivesGlioblastoma is an aggressive primary brain tumor that develops within a chronically low-oxygen microenvironment, yet most preclinical studies are performed under atmospheric oxygen conditions that poorly reflect in vivo physiology. This study investigated how sustained culture under physiological oxygen tension (physioxia; 5% O{square}) influences glioblastoma cell behavior, signaling, and therapeutic response. MethodsMultiple patient-derived glioblastoma models were cultured under normoxia (21% O{square}) or sustained physioxia (5% O{square}) for at least seven days before experimentation. Cell migration, proliferation, cell cycle distribution, expression of the epithelial-to-mesenchymal transition-associated transcription factor Slug (SNAI2), PDGFR{beta}-associated signaling, and sensitivity to 5-fluorouracil were evaluated using transwell migration assays, cell counting, flow cytometry, RT-qPCR, immunoblotting, and BrdU incorporation assays. Additional patient-derived cultures established and maintained continuously under physioxia were used to examine the effects of oxygen history. ResultsSustained physioxia consistently increased migration across all glioblastoma models while reducing proliferation in normoxia-adapted cell lines through increased G0/G1 cell cycle arrest. Physioxia significantly increased Slug expression in all models and enhanced PDGFR{beta}, AKT, and ERK phosphorylation in a cell line-dependent manner. Therapeutic sensitivity to 5-fluorouracil was also altered, with physioxia conferring increased resistance in selected glioblastoma models but not universally. Patient-derived cultures maintained continuously under physioxia retained enhanced migratory capacity and exhibited increased proliferation compared with normoxia, indicating that prior oxygen exposure influences proliferative responses while the pro-migratory phenotype remains conserved. ConclusionsPhysiological oxygen tension is a major regulator of glioblastoma cell behavior, influencing migration, proliferation, signaling, and therapeutic response. These findings demonstrate that conventional normoxic culture conditions can obscure biologically relevant phenotypes and support incorporating physioxia into experimental design to improve the physiological and translational relevance of preclinical glioblastoma research.

Whole-genome sequencing identifies focal DNA amplifications with base-pair resolution but cannot determine whether amplified sequences reside on extrachromosomal DNA (ecDNA, also known as double minutes) or within chromosomally integrated homogeneously staining regions (HSRs). DNA fluorescence in situ hybridization (DNA-FISH) metaphase spreads remain the gold standard for distinguishing these amplification states at single-cell resolution. Here, we present a detailed protocol for DNA-FISH metaphase spreads using human cancer cell lines, encompassing cell culture, metaphase arrest, hypotonic treatment, fixation, chromosome spreading, fluorescent probe hybridization, and fluorescence imaging. The protocol incorporates intermediate quality-control steps to verify successful chromosome dispersion and optimize metaphase spread quality, making the workflow accessible to laboratories without specialized cytogenetics expertise. Results demonstrate clear visualization of ecDNA and HSR amplification states using locus-specific probes and illustrate common technical artifacts that can affect interpretation. This protocol provides a robust and reproducible approach for studying the structural organization of oncogene amplification in cancer cells.

Background and PurposePARP inhibitors have been evaluated in clinical trials for several cancers and Olaparib is FDA approved for treating BRCA deficient ovarian cancer. Numerous reports have suggested Poly(ADP-ribose) polymerase1(PARP1) overexpression in a variety of cancers including breast carcinomas and proposed the role of PARP1 in metastasis. However, the mechanism of PARP1 in regulating metastatic process in BRCA proficient and deficient TNBC is not studied thoroughly. In this study, we propose that PARP1 mediated breast carcinoma progression is gene transcription mediated, where it regulates several steps of pro-metastasis. Experimental ApproachPARP inhibitors effect on metastasis was monitored by migration and invasion assay, modulation in protein expression was assessed by proteomic analysis and further confirmed by immunoblotting. Chromatin immunoprecipitation was performed to study the transcriptional role of PARP1. Ectopic expression and siRhoGDI, and immunofluorescence assessed the cytoskeleton changes. PARP inhibitor was administered in xenograft mice to study metastasis. Immunohistochemical analysis was done on patient and mice tissues. Key resultsBreast cancer cells exhibited reduced migration and invasion due to PARP1 inhibition. PARP1 regulates expression of vimentin and RhoGDI and hence cytoskeletal rearrangement causing a change in migrating potential of a cell. Metastasis in mice was reduced upon PARP inhibition. PARP1 was identified to be a novel transcriptional regulator of RhoGDI. Furthermore, RhoGDI ectopic expression substantiated the PARP inhibitor effects, suggesting the PARP inhibitor downstream signaling to be mediated through RhoGDI. Conclusions and ImplicationsWe identified a novel aspect of PARP1 as promoter of metastasis via transcriptional regulation of RhoGDI. Assessing RhoGDI levels in TNBC patients might be useful to predict sensitivity to PARP inhibitors.

Animal cell-cultured ingredients are entering the EU and UK pet food markets under frameworks that do not require pre-market, ingredient-level safety assessments, creating an ethical need for transparent safety disclosure. We present the first public safety dossier for this sector, describing the proprietary mouse embryonic stem cell line PE25 and its derived, non-viable cellular and conditioned media ingredient produced in food and feed-grade media. PE25 characterization confirmed Mus musculus identity, sterility, absence of mycoplasma and replication-competent retroviruses, and stable growth. Doxorubicin-induced p53 stress testing, CD44/BMI1 profiling, and soft agar assays showed no cancer-like traits and a non-tumorigenic profile; the final ingredient contains no viable cells. Independent OECD TG 471 and 487 assays confirmed non-genotoxicity. Heavy metals, biogenic amines, solvents, and chemical residues were below regulatory limits. Given process variability, we recommend case-by-case safety evaluation and propose this dossier as a model for responsible commercialization.

Coherent Raman spectroscopy enables label-free biochemical fingerprinting of live cells with subcellular resolution. We previously developed a machine learning framework capable of classifying glioma FFPE tissues using Raman spectral signatures. To accelerate live cell acquisition, we previously developed RADAR (Raman Spectral Analysis Using Deep Learning for Artifact Removal), a method that increases imaging speed by an order of magnitude while preserving spectral integrity. By integrating high-speed Raman imaging with supervised machine learning, we aimed to define unique biochemical fingerprints specific to cell type. We hypothesized that intrinsic biochemical composition alone is sufficient to distinguish cellular identity and tumor subtype. To test this, we generated metabolic maps of diverse brain-derived cell types--including astrocytoma, oligodendroglioma, and glioblastoma cells--using coherent Raman spectroscopy at single-cell resolution. Patient-derived brain tumor cell lines representing genetically heterogeneous backgrounds were analyzed. Samples were stratified by IDH1 mutation status (IDH1-mutant and IDH1-wild-type) and histologically classified as oligodendroglioma or astrocytoma. Raman spectral data were acquired from 286 live single cells across the two principal molecular classes, with further subdivision into two histologic subtypes within the IDH1-mutant group. Classification was performed using an XGBoost model with shallow tree depth (1-3), a 20% held-out test set, and grouped, stratified 5-fold cross-validation to control for sample-level bias. The machine learning framework distinguished IDH1-mutant from IDH1-wild-type cells with a ROC-AUC of 0.78 and further discriminated IDH1-mutant astrocytoma from oligodendroglioma cells with a ROC-AUC of 0.81. Feature importance analysis demonstrated that separation between IDH1-mutant and IDH1-wild-type cells was driven primarily by Raman peaks associated with protein amide bands, total NADH, unsaturated fatty acids, and heme-related vibrational modes. Within the IDH1-mutant class, discrimination between oligodendroglioma and astrocytoma was driven by lipid-rich vesicle signatures, protein/polyamide amide bands, and lipid-associated spectral features. Together, these findings support the feasibility of label-free, machine learning-assisted Raman profiling to resolve clinically relevant glioma subtypes at single-cell resolution. This scalable analytical framework provides a translational platform for investigating metabolic heterogeneity, therapeutic response, co-culture systems, and patient-derived organoid models.