Xenograft Mouse Research Articles

MULTI-OMIC INTEGRATION REVEAL INTRA-TUMOUR HETEROGENEITY AND IDENTIFY DISULfiRAM AND COPPER AS A SYNERGISTIC THERAPY IN PAEDIATRIC EPENDYMOMA

Abstract AIMS Ependymoma (EPN) is the second most malignant paediatric brain tumour. The PF-A subgroup, associated with a hypoxic microenvironment, has a dismal survival rate of 50% with clinical trials failing to demonstrate a significant survival advantage from chemotherapy to date. As the lack of genomic alterations makes it difficult to uncover novel therapeutic targets, we present a multi-omic integration investigating whether spatially-distinct tumour microenvironments represent targetable metabolic niches in PF-A EPN. METHOD Surgical sampling of spatially-distinct regions was performed on eight PF-A patients. Metabolites and RNA were simultaneously extracted from the same cellular population and analysed using liquid chromatography-mass spectrometry (LC-MS) and RNAseq. Multi-omic integration was performed using Metscape3 and functional assays evaluating proliferation, invasion and siRNA-mediated knockdown were performed in 2D and 3D models of intra-tumour region patient-derived cell lines. Selected single and drug combination therapy was performed in normoxia and hypoxia, and synergy assessed using the Chou-Talalay method. RESULTS Multi-omic integration revealed 124 dysregulated metabolic pathways, encompassing 156 genes and 49 metabolites with the largest number of interactions occurring in the gluconeogenesis pathway. Each anatomical region also presented at least one unique gene-metabolite interaction demonstrating heterogeneity within and across PF-A EPN tumours. Four selected drug targets (Disulfiram, Vandetanib, Mildronate and FBP1 inhibitor) based on metabolically relevant genes (ALDH3A1, FMO3, BBOX1, FBP1) showed impairment of metabolic viability in vitro, with limited chemosensitivity observed in control human cerebellar astrocytes. The addition of Cu2+ significantly sensitized cells to Disulfiram, particularly under hypoxic conditions. This synergistic combination significantly impaired invasion in both normoxia and hypoxia. CONCLUSION This is the first instance where multi-omic data integration and intra-tumour heterogeneity has been investigated for paediatric EPN revealing novel therapeutic targets in the context of gene-metabolite correlations. Drug tolerability will be further assessed in vivo using the previously characterised PF-A EPN orthotopic mouse xenograft MAF-928.

Zinc Finger Protein 536 is a Potential Prognostic Biomarker that Promotes Neuroblastoma Progression via VEGFR2-PI3K-AKT Pathway.

Neuroblastoma (NB) is a well-known pediatric malignancy intertwined with neurodevelopment. Previously implicated in neuronal differentiation, Zinc Finger Protein 536 (ZNF536) has emerged as a promising prognostic and immune-related biomarker in our pan-cancer analysis. Single-cell RNA transcriptome sequencing, bulk transcriptome analysis, and immuno-histochemistry were used to assess ZNF536 expression and its association with prognosis. Cell proliferation, migration, invasion, and differentiation in ZNF536-knockdown NB cell lines were detected to evaluate the effect of ZNF536 on tumor cells. Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), a potential target of ZNF536, and its downstream PI3K/AKT signaling cascade were investigated using transcriptome sequencing, CUT&Tag, quantitative real-time PCR (qRT-PCR), and Western blotting. The role of ZNF536 in tumorigenesis and the potential regulation axis was evaluated in vivo using a BALB/c nude mouse xenograft tumor model. ZNF536 mRNA and protein expression were significantly higher in NB patients with poor prognosis. In vitro, ZNF536 knockdown curtailed proliferation, migration, and invasion of NB cells while fostering differentiation. ZNF536 regulated VEGFR2 expression, thus activating the PI3K-AKT pathway. In vivo, ZNF536 knockdown reduced tumor growth and proliferation via the VEGFR2-PI3K-AKT pathway. ZNF536 resulted as a novel prognostic biomarker in NB, promoting oncogenesis through VEGFR2-PI3K-AKT signaling axis modulation, suggesting its therapeutic potential in managing NB progression.

Targeting Oxidative Phosphorylation with a Novel Thiophene Carboxamide Increases the Efficacy of Imatinib against Leukemic Stem Cells in Chronic Myeloid Leukemia

Patients with chronic myeloid leukemia (CML) respond to tyrosine kinase inhibitors (TKIs); however, CML leukemic stem cells (LSCs) exhibit BCR::ABL kinase-independent growth and are insensitive to TKIs, leading to disease relapse. To prevent this, new therapies targeting CML-LSCs are needed. Rates of mitochondria-mediated oxidative phosphorylation (OXPHOS) in CD34+CML cells within the primitive CML cell population are higher than those in normal undifferentiated hematopoietic cells; therefore, the inhibition of OXPHOS in CML-LSCs may be a potential cure for CML. NK-128 (C33H61NO5S) is a structurally simplified analog of JCI-20679, the design of which was based on annonaceous acetogenins. NK-128 exhibits antitumor activity against glioblastoma and human colon cancer cells by inhibiting OXPHOS and activating AMP-activated protein kinase (AMPK). Here, we demonstrate that NK-128 effectively suppresses the growth of CML cell lines and that the combination of imatinib and NK-128 is more potent than either alone in a CML xenograft mouse model. We also found that NK-128 inhibits colony formation by CD34+ CML cells isolated from the bone marrow of untreated CML patients. Taken together, these findings suggest that targeting OXPHOS is a beneficial approach to eliminating CML-LSCs, and may improve the treatment of CML.

Making the effect visible - OX40 targeting nanobodies for in vivo imaging of activated T cells.

Human OX40 (hOX40/CD134), a member of the TNF receptor superfamily, is mainly expressed on activated T lymphocytes. Triggered by its ligand OX40L (CD252), it provides costimulatory signals that support the differentiation, proliferation and long-term survival of T cells. Besides being a relevant therapeutic target, hOX40 is also an important biomarker for monitoring the presence or infiltration of activated T cells within the tumor microenvironment (TME), the inflammatory microenvironment (IME) in immune-mediated diseases (IMIDs) and the lymphatic organs. Here, we developed novel single domain antibodies (nanobodies, Nbs) targeting hOX40 to monitor the activation status of T cells by in vivo molecular imaging. Nbs against hOX40 (hOX40-Nbs) were selected from an immunized Nb-library by phage display. The identified hOX40-Nbs were characterized in vitro, including determination of their specificity, affinity, stability, epitope recognition and their impact on OX40 signaling and T cell function. A lead candidate was site-specifically conjugated with a fluorophore via sortagging and applied for noninvasive in vivo optical imaging (OI) of hOX40-expressing cells in a xenograft mouse model. Our selection campaign revealed four unique Nbs that exhibit strong binding affinities and high stabilities under physiological conditions. Epitope binning and domain mapping indicated the targeting of at least two different epitopes on hOX40. When analyzing their impact on OX40 signaling, an agonistic effect was excluded for all validated Nbs. Incubation of activated T cells with hOX40-Nbs did not affect cell viability or proliferation patterns, whereas differences in cytokine release were observed. In vivo OI with a fluorophore-conjugated lead candidate in experimental mice with hOX40-expressing xenografts demonstrated its specificity and functionality as an imaging probe. Considering the need for advanced probes for noninvasive in vivo monitoring of T cell activation dynamics, we propose, that our hOX40-Nbs have a great potential as imaging probes for noninvasive and longitudinal in vivo diagnostics. Quantification of OX40+ T cells in TME or IME will provide crucial insights into the activation state of infiltrating T cells, offering a valuable biomarker for assessing immune responses, predicting treatment efficacy, and guiding personalized immunotherapy strategies in patients with cancer or IMIDs.

Tumor-Secreted Extracellular Vesicles Counteract Therapy Response by Triggering Inflammatory Mesenchymal Stem Cell Development.

Therapy resistance is a major clinical hurdle in bone cancer treatment and seems to be largely driven by poorly understood microenvironmental factors. Recent evidence suggests a critical role for a unique subpopulation of mesenchymal stem cells with inflammatory features (iMSC), though their origin and function remained unexplored. We demonstrate that cancer-secreted extracellular vesicles (EV) trigger the development of iMSCs, which hinder therapy response in vivo, and set out to identify strategies to counteract their function. The role of iMSCs in therapy resistance was evaluated in an orthotopic xenograft mouse model of osteosarcoma. EV-induced alterations of the MSC transcriptome were analyzed and compared with single-cell RNA sequencing data of biopsies from patients with osteosarcoma and multiple myeloma. Functional assays identified EV components driving iMSC development. We assessed the efficacy of clinical drugs in blocking iMSC-induced resistance in vivo. We found that iMSCs are induced by interaction with cancer EVs and completely abrogate the antimetastatic effect of TGFβ signaling inhibition. Importantly, EV-induced iMSCs faithfully recapitulate the inflammatory single-cell RNA signature of stromal cells enriched in biopsies from patients with multiple myeloma and osteosarcoma. Mechanistically, cancer EVs act through two distinct mechanisms. EV-associated TGFβ induces IL6 production, whereas the EV-RNA cargo enhances TLR3-mediated chemokine production. We reveal that simultaneous blockade of downstream EV-activated pathways with ladarixin and tocilizumab disrupts metastasis formation and overcomes iMSC-induced resistance. Our observations establish iMSCs as major contributors to drug resistance, reveal EVs as triggers of iMSC development, and highlight a promising combination strategy to improve therapy response in patients with bone cancer.

Unraveling the metastasis-preventing effect of miR-200c in vitro and in vivo.

Advanced breast cancer, as well as ineffective treatments leading to surviving cancer cells, can result in the dissemination of these malignant cells from the primary tumor to distant organs. Recent research has shown that microRNA 200c (miR-200c) can hamper certain steps of the invasion-metastasis cascade. However, it is still unclear whether miR-200c expression alone is sufficient to prevent breast cancer cells from metastasis formation. Hence, we performed a xenograft mouse experiment with inducible miR-200c expression in MDA-MB 231 cells. The ex vivo analysis of metastatic sites in a multitude of organs, including lung, liver, brain, and spleen, revealed a dramatically reduced metastatic burden in mice with miR-200c-expressing tumors. A fundamental prerequisite for metastasis formation is the motility of cancer cells and, therefore, their migration. Consequently, we analyzed the effect of miR-200c on collective- and single-cell migration in vitro, utilizing MDA-MB 231 and MCF7 cell systems with genetically modified miR-200c expression. Analysis of collective-cell migration revealed confluence-dependent motility of cells with altered miR-200c expression. Additionally, scratch assays showed an enhanced predisposition of miR-200c-negative cells to leave cell clusters. The in-between stage of collective- and single-cell migration was validated using transwell assays, which showed reduced migration of miR-200c-positive cells. Finally, to measure migration at the single-cell level, a novel assay on dumbbell-shaped micropatterns was performed, which revealed that miR-200c critically determines confined cell motility. All of these results demonstrate that sole expression of miR-200c impedes metastasis formation in vivo and migration in vitro and highlights miR-200c as a metastasis suppressor in breast cancer.

1-Dehydro-6-Gingerdione Exerts Anticancer Effects on MDA-MB-231 Cells and in the Xenograft Mouse Model by Promoting the Ferroptosis Pathway.

Breast cancer (BC) is the most prevalent malignancy among women, with millions of newly diagnosed cases emerging annually. Therefore, identifying novel pharmaceuticals for therapeutic purposes is imperative. Several natural compounds and their products have demonstrated potential in the treatment of cancer. This study examined the effects of the ginger derivative 1-dehydro-6-gingerdione (1-D-6-G) on BC and its mechanisms of action. MTT and colony formation assays were used to check the anticancer effect of 1-D-6-G. Then the anticancer mechanism of 1-D-6-G was predicted using proteomics analysis. The molecular pathway was verified by qRT-PCR and immunobloting analysis. Additionally, the anticancer properties of 1-D-6-G were investigated in vivo using xenograft mice model. Finally, an in silico study was conducted to examine the interaction of 1-D-6-G and pathway-related proteins. MTT and colony formation assay results indicated that 1-D-6-G has potent cytotoxic properties against BC cells. Proteomic analysis revealed that the anticancer mechanism of 1-D-6-G on MDA-MB-231 cells is associated with the ferroptosis signaling pathway. In addition, qRT-PCR and immunoblotting analyses revealed that the cytotoxic effects of 1-D-6-G on MDA-MB-231 cells were associated with ferroptosis signaling induction. Our in vivo results further confirmed the in vitro findings. The administration of 1-D-6-G for 14 days exhibited anticancer properties in xenograft mice by stimulating the ferroptosis pathway without causing damage to essential organs such as the liver and kidneys. Additionally, in silico results confirmed the structural stability of the molecular interaction between 1-D-6-G and ferroptosis target proteins. Our findings indicate that 1-D-6-G has the potential to serve as a novel therapeutic agent for inhibiting BC progression by targeting the ferroptosis pathway.

Circ_0008035 Promotes Gastric Cancer Development via the miR-429/SMAD2 Cascade.

The vital roles of circular RNAs (circRNAs) in human tumorigenesis have attracted more attention. Circ_0008035 is one of the most up-regulated circRNAs in gastric cancer (GC). Herein, we explored the associated mechanism of circ_0008035 in GC. EdU incorporation experiments were performed to monitor cell proliferation ability. Cell cycle progression, apoptosis, angiogenesis, migration, and invasion were analyzed using flow cytometry, Tube formation, and Transwell assays respectively. Protein expression was detected by Western blot. Dual-luciferase reporter experiments were applied to demonstrate the relationship between circ_0008035 or SMAD family member 2 (SMAD2) and microRNA-429 (miR-429). Mouse xenograft assays were conducted for evaluation of the role of circ_0008035 in vivo. Circ_0008035 content was elevated in GC tissues (P < .0001) and cell lines (P < .001), and its deficiency hindered GC cell proliferation (P < .01), HUVEC angiogenesis (P < .05), and GC cell metastasis (P < .01) and triggered apoptosis (P < .01). Circ_0008035 could sponge miR-429 to up-regulate SMAD2 expression (P < .0001). Circ_0008035 absence restrained tumor growth in vivo (P < .01). MiR429 was a mediator of circ_0008035 function, and miR-429 hindered GC cell malignant phenotypes by SMAD2. Circ_0008035 aggravates GC cell malignant progression partially by targeting the miR-429/SMAD2 axis. Considering the inhibitory effect of circ_0008035 deficiency on GC progression, targeting circ_0008035 may be a potential approach to prevent or treat GC.

M6A-modified circXPO1 accelerates colorectal cancer progression via interaction with FMRP to promote WWC2 mRNA decay

BackgroundRecent evidence has demonstrated the vital roles of circular RNAs (circRNAs) in the progression of colorectal cancer (CRC); however, their functions and mechanisms in CRC need to be further explored. This study aimed to uncover the biological function of circXPO1 in CRC progression.MethodsCircXPO1 was identified by Sanger sequencing, RNase R, and actinomycin D treatment assays. Colony formation, scratch, transwell assays, and mouse xenograft models were adopted to evaluate CRC cell growth and metastasis in vitro and in vivo. Subcellular expression of circXPO1 was detected by FISH and nuclear-cytoplasmic separation assays. Molecular mechanisms were investigated by MeRIP, RIP, and RNA pull-down assays. Target molecular expression was detected by RT-qPCR, Western blotting and immunohistochemical staining.ResultscircXPO1 was up-regulated in CRC tissues and cells, which indicated a poor prognosis of CRC patients. circXPO1 deficiency delayed the growth, EMT, and metastasis of CRC cells. Mechanistical experiments indicated that down-regulation of ALKBH5 enhanced IGF2BP2-mediated m6A modification of circXPO1 to increase circXPO1 expression. Furthermore, circXPO1 interacted with FMRP to reduce the mRNA stability of WWC2, which consequently resulted in Hippo-YAP pathway activation. Rescue experiments suggested that WWC2 overexpression abrogated circXPO1-mediated malignant capacities of CRC cells. The in vivo growth and liver metastasis of CRC cells were restrained by circXPO1 depletion or WWC2 overexpression.Conclusionsm6A-modified circXPO1 by ALKBH5/IGF2BP2 axis destabilized WWC2 via interaction with FMRP to activate Hippo-YAP pathway, thereby facilitating CRC growth and metastasis. Targeting circXPO1 might be a potential therapeutic strategy for CRC.

Long-Term Human Immune Reconstitution, T-Cell Development, and Immune Reactivity in Mice Lacking the Murine Major Histocompatibility Complex: Validation with Cellular and Gene Expression Profiles.

Humanized mice transplanted with CD34+ hematopoietic cells (HPCs) are broadly used to study human immune responses and infections in vivo and for testing therapies pre-clinically. However, until now, it was not clear whether interactions between the mouse major histocompatibility complexes (MHCs) and/or the human leukocyte antigens (HLAs) were necessary for human T-cell development and immune reactivity. We evaluated the long-term (20-week) human hematopoiesis and human T-cell development in NOD Scid Gamma (NSG) mice lacking the expression of MHC class I and II (NSG-DKO). Triplicate experiments were performed with HPCs obtained from three donors, and humanization was confirmed in the reference strain NOD Rag Gamma (NRG). Further, we tested whether humanized NSG-DKO mice would respond to a lentiviral vector (LV) systemic delivery of HLA-A*02:01, HLA-DRB1*04:01, human GM-CSF/IFN-α, and the human cytomegalovirus gB antigen. Human immune reconstitution was detectable in peripheral blood from 8 to 20 weeks after the transplantation of NSG-DKO. Human single positive CD4+ and CD8+ T-cells were detectable in lymphatic tissues (thymus, bone marrow, and spleen). LV delivery harnessed the detection of lymphocyte subsets in bone marrow (αβ and γδ T-cells and NK cells) and the expression of HLA-DR. Furthermore, RNA sequencing showed that LV delivery increased the expression of different human reactome pathways, such as defense responses to other organisms and viruses. Human T-cell development and reactivity are independent of the expression of murine MHCs in humanized mice. Therefore, humanized NSG-DKO is a promising new model for studying human immune responses, as it abrogates the xenograft mouse MHC interference.

Tumor-associated macrophages/C-X-C motif chemokine ligand 1 promotes breast cancer autophagy-mediated chemoresistance via IGF1R/STAT3/HMGB1 signaling

Autophagy-mediated chemoresistance is the core mechanism for therapeutic failure and poor prognosis in breast cancer. Breast cancer chemotherapy resistance is believed to be influenced by tumor-associated macrophages (TAMs), by which C-X-C motif chemokine ligand 1 (CXCL1) is the most abundant cytokine secreted. Yet, its role in mediating autophagy-related chemoresistance is still unknown. This study aimed to explore the molecular mechanisms by which TAMs/CXCL1 induced autophagy-mediated chemoresistance in breast cancer. It was found that TAMs/CXCL1 promoted chemoresistance of breast cancer cells through autophagy activation in vitro, and CXCL1 silence could enhance the chemosensitivity of paclitaxel-resistant breast cancer cells via autophagy inhibition. A high-throughput quantitative PCR chip and subsequent target validation showed that CXCL1 induced autophagy-mediated chemoresistance by inhibiting VHL-mediated IGF1R ubiquitination. The elevated IGF1R then promoted STAT3/HMGB1 signaling to facilitate autophagy. Additionally, TAMs/CXCL1 silence improved paclitaxel chemosensitivity by suppressing autophagy in breast cancer mice xenografts, and clinical studies further linked CXCL1 to IGF1R/HMGB1 signaling, as well as shorter free survival of recurrence. Taken together, these results not only uncover the crucial role of TAMs/CXCL1 signaling in mediating breast cancer chemoresistance through enhancing autophagy, but also shed novel light on the molecular mechanism of IGF1R/STAT3/HMGB1 pathway in regulating autophagy and its impact on cancer prognosis.

Functional screen for mediators of onco-mRNA translation specificity.

Oncogenic protein dosage is tightly regulated to enable cancer cells to adapt and survive. Whether this is regulated at the level of translational control and the key factors in cis and trans remain unknown. The Myc oncogene is a central paradigm of an exquisitely regulated oncogene and a major driver of pancreatic ductal adenocarcinoma (PDAC). Using a functional genome-wide CRISPRi screen in PDAC cells, we identified activators of selective MYC translation through its 5' untranslated region (5'UTR) and validated four RNA binding proteins (RBPs), including epitranscriptome modifiers. Among these RBPs, our top hit was RBM42, which is highly expressed in PDAC and predicts poor survival. Combining polysome sequencing and CLIP-seq analyses, we find that RBM42 binds and selectively regulates the translation of MYC and a precise, yet vital suite of pro-oncogenic transcripts, including JUN and EGFR . Mechanistically, employing IP-mass spectrometry analysis, we find that RMB42 is a novel ribosome-associated protein (RAP). Using DMS-Seq and mutagenesis analysis, we show that RBM42 directly binds and remodels the MYC 5'UTR RNA structure, facilitating the formation of the translation pre-initiation complex. Importantly, RBM42 is necessary for human PDAC cell growth and fitness and PDAC tumorigenesis in xenograft mouse models in a Myc-dependent manner in vivo . In PDAC patient samples, RBM42 expression is correlated with Myc protein levels and transcriptional activity. This work transforms our understanding of the translational code in cancer and offers a new therapeutic opening to target the expression of oncogenes.

Identification of targetable epigenetic vulnerabilities in uveal melanoma.

Uveal melanoma (UM) is the most prevalent primary intraocular malignancy in adults, which preferentially metastasizes to the liver in approximately half of all cases. Metastatic UM is notoriously resistant to therapy and is almost uniformly fatal. UM metastasis is most strongly associated with mutational inactivation of the BAP1 tumor suppressor gene. Given the role of BAP1 in epigenetic regulation as the ubiquitin hydrolase subunit of the polycomb repressive deubiquitinase (PR-DUB) complex, we conducted high-throughput drug screening using a well-characterized epigenetic compound library to identify new therapeutic vulnerabilities. We identified several promising new lead compounds, in particular the extra-terminal domain protein (BET) inhibitor mivebresib (ABBV-075). Mivebresib significantly improved survival rates in a metastatic uveal melanoma xenograft mouse model and entirely prevented detectable metastases to the bones, spinal cord, and brain. RNA sequencing revealed a notable overlap between the genes and pathways affected by HDAC and BET inhibition, including the reversal of gene signatures linked to high metastatic risk and upregulation of genes associated with a neuronal phenotype. Together, we found that UM cells are particularly vulnerable to class I HDAC and BET inhibition, and highlight the BET inhibitor mivebresib as a promising candidate for further clinical evaluation.

LncRNA MEG3 suppresses hepatocellular carcinoma by stimulating macrophage M1 polarization and modulating immune system via inhibiting CSF-1 in vivo/vitro studies

Hepatocellular carcinoma (HCC) is characterized by a complex tumor microenvironment (TME), and long non-coding RNAs (lncRNAs) MEG3 emerged as regulators of macrophage polarization with a negative relationship with colony-stimulating factor 1 (CSF-1). Few studies are on the interplay among MEG3, CSF-1, T helper cells (Th), and the programmed cell death protein 1 and its ligands (PD-1/PD-Ls) in TME of HCC.MEG3 expression in THP-1 macrophages, monitored polarization, and PD-1/PD-Ls expression were through flow cytometry, WB, and RT-qPCR. In co-cultures, the interaction of MEG3, macrophage, and HCC was assayed by ELISA. The invasive and migratory of HCC were assessed through experiments such as CCK-8, clonogenic assay, wound healing, and Transwell. A xenograft mouse model of HCC was established, administered with MEG3 overexpression (OE) or knockdown (KD) constructs, and monitored tumor growth. In vitro, MEG3 OE induced a robust M1 macrophage phenotype, evidenced by elevated expression of M1 markers and a significant increase in Th1 cytokines, with a concomitant decrease in Th2 cytokines. This was paralleled by reduced CSF-1 and PD-1/PD-Ls expression. In contrast, MEG3 KD promoted an M2 phenotype with increased CSF-1 and PD-1/PD-Ls expression, and an upregulation of Th2 cytokines. MEG3 OE inhibited the growth, invasion, and migration of HCC, while the opposite was observed when MEG3 was downregulated. In vivo, MEG3 OE resulted in significantly reduced tumor growth, with decreased PD-1/PD-Ls expression on macrophages and enhanced Th1 response. Conversely, MEG3 KD promoted tumor growth with increased PD-1/PD-Ls and a Th2-skewed immune response. MEG3 modulates the TME by affecting TAMs through CSF-1, thereby influencing the balance of Th1/Th2 cells and altering the expression of PD-1/PD-L1s. This study demonstrates that targeting MEG3 is an effective therapeutic strategy for HCC.

Chidamide triggers pyroptosis in T-cell lymphoblastic lymphoma/leukemia via the FOXO1/GSDME axis.

T-cell lymphoblastic lymphoma/leukemia (T-LBL/ALL) is an aggressive form of hematological malignancy associated with poor prognosis in adult patients. Histone deacetylases (HDACs) are aberrantly expressed in T-LBL/ALL and are considered potential therapeutic targets. Here, we investigated the antitumor effect of a novel HDAC inhibitor, chidamide, on T-LBL/ALL. HDAC1, HDAC2 and HDAC3 levels in T-LBL/ALL cell lines and patient samples were compared with those in normal controls. Flow cytometry, transmission electron microscopy and lactate dehydrogenase release assays were conducted in Jurkat and MOLT-4 cells to assess apoptosis and pyroptosis. A specific forkhead box O1 (FOXO1) inhibitor was used to rescue pyroptosis and upregulated gasdermin E (GSDME) expression caused by chidamide treatment. The role of the FOXO1 transcription factor was evaluated by dual-luciferase reporter and chromatin immunoprecipitation assays. The efficacy of chidamide in vivo was evaluated in a xenograft mouse. The expression of HDAC1, HDAC2 and HDAC3 was significantly upregulated in T-LBL/ALL. Cell viability was obviously inhibited after chidamide treatment. Pyroptosis, characterized by cell swelling, pore formation on the plasma membrane and lactate dehydrogenase leakage, was identified as a new mechanism of chidamide treatment. Chidamide triggered pyroptosis through caspase 3 activation and GSDME transcriptional upregulation. Chromatin immunoprecipitation assays confirmed that chidamide led to the increased transcription of GSDME through a more relaxed chromatin structure at the promoter and the upregulation of FOXO1 expression. Moreover, we identified the therapeutic effect of chidamide in vivo. Our study suggested that chidamide exerts an antitumor effect on T-LBL/ALL and promotes a more inflammatory form of cell death via the FOXO1/GSDME axis, which provides a novel choice of targeted therapy for patients with T-LBL/ALL.

KLF14 directly downregulates the expression of GPX4 to exert antitumor effects by promoting ferroptosis in cervical cancer

BackgroundCervical cancer is the fourth leading cause of cancer-related death among women worldwide, and effective therapeutic strategies for its treatment are limited. Recent studies have indicated that ferroptosis, a form of regulated cell death, is a promising therapeutic strategy. KLF14 has been shown to regulate both cell proliferation and apoptosis in cervical cancer. However, its role in modulating lipid peroxidation and ferroptosis remains largely unexplored and enigmatic.MethodsSiHa and HeLa cells were transduced with lentiviral vectors to overexpress KLF14. Protein levels were analyzed via western blotting and immunohistochemistry (IHC). LDH assays, calcein-AM/propidium iodide (PI) staining, and generation of cell growth curves using a real-time cell analysis (RTCA) system were used to detect cell damage and proliferation. Cellular ROS, lipid ROS, transmission electron microscopy (TEM), and Fe2+ assays and a xenograft mouse model were used to measure the level of ferroptosis. Proteomics combined with bioinformatics methods was used to screen target genes regulated by KLF14, and CUT&Tag and dual-luciferase assays confirmed the repression of GPX4 by KLF14 via direct binding to its promoter.ResultsKLF14 is abnormally expressed in various tumors and downregulated in cervical cancer. Overexpression of KLF14 induced ferroptosis and inhibited cell proliferation in vitro as well as xenograft tumorigenicity in vivo. Mechanistic studies revealed that KLF14 binds to the promoter of GPX4, suppressing its transcriptional activity and thereby decreasing its expression, which contributes to the induction of ferroptosis. Truncation and point mutation analyses of the GPX4 promoter revealed multiple binding sites for KLF14 within the − 1000 bp to + 35 bp region, which are responsible for its inhibitory effect on GPX4 transcription. Additionally, deletion of the zinc finger motif in KLF14 abolished its inhibitory effect on GPX4 promoter activity and cell proliferation.ConclusionOur data revealed a previously unidentified function of KLF14 in promoting ferroptosis, which results in the suppression of cell proliferation. Mechanistically, we revealed a novel regulatory mechanism by which KLF14 targets GPX4. These findings suggest a novel strategy to induce ferroptosis through the targeting of KLF14 in human cervical cancer cells.

Gingerenone A induces ferroptosis in colorectal cancer via targeting suppression of SLC7A11 signaling pathway

BackgroundColorectal cancer (CRC) is one of the most common and fatal diseases, yet effective therapeutic drugs are lacking in clinical settings. Gingerenone A (GA) is an active compound derived from ginger, has demonstrated anti-tumor properties. However, the efficacy of GA against CRC and its primary mechanism of action remain unclear. Materials and methodsMTT assay and colony formation assay were employed to evaluate cell viability. Transwell assays were utilized to assess the migratory and invasive capabilities of the cells. The effects of GA on ferroptosis related proteins were analyzed using Western blot. Levels of glutathione (GSH), malondialdehyde (MDA), Fe2+, and 4-hydroxynonenal (4-HNE) levels were measured with a biochemical index determination kit. Cellular reactive oxygen species (ROS) were quantified using flow cytometry. CETSA, pull-down, and co-immunoprecipitation (Co-IP) assays confirmed the interactions between GA and SLC7A11, as well as the ubiquitination promoted by SLC7A11. A xenograft mouse model was employed to validate the anticancer effect of GA in vivo. ResultsWe observed that GA significantly suppressed proliferation in human CRC cells. Additionally, GA treatment inhibited the migration, invasion, and colony formation of CRC cells. Subsequently, through the use of specific inhibitors, we discovered that the suppression of CRC cells by GA was dependent on ferroptosis rather than autophagy or apoptosis. Previous research has demonstrated that GA treatment significantly triggers ferroptosis. Mechanistically, GA treatment promotes the degradation of the SLC7A11 protein, which plays a crucial role in ferroptosis. Notably, the knockdown of SLC7A11 abolished the detrimental effects of GA on the proliferation of CRC cells and reversed GA-induced ferroptosis in CRC cells both in vivo and in vitro. Further research has shown that GA can directly bind to the SLC7A11 protein and promote its ubiquitination. ConclusionOur research provides compelling evidence that GA may serve as a potential agent for suppressing the progression of CRC by inducing ferroptosis and promoting the ubiquitination and degradation of SLC7A11.

Sodium selenite inhibits cervical cancer progression via ROS-mediated suppression of glucose metabolic reprogramming

AimsThis study aims to explore the inhibitory effect of selenium on cervical cancer through suppression of glucose metabolic reprogramming and its underlying mechanisms. MethodsSodium selenite (SS) treated HeLa and SiHa cells were assessed for proliferation using the CCK-8 assay and immunofluorescence. DNA synthesis was measured with the EdU assay. A nude mouse xenograft model evaluated SS's anti-cervical cancer effects. Reactive oxygen species (ROS) and mitochondrial membrane potential were measured using flow cytometry, DCFH-DA, and JC-1 probes, respectively. Apoptosis was detected via Annexin V/PI staining and Western blot. Glucose uptake, lactate production, and ATP generation were determined using 2-NBDG probes and assay kits. The mRNA and protein levels of glycolysis-related genes HK2, GLUT1, and PDK1 were measured using RT-qPCR and Western blot. Key findingsSS inhibited HeLa and SiHa cells viability in a dose- and time-dependent manner. Intraperitoneal injection of SS in nude mice significantly inhibited HeLa cell xenograft growth without evident hepatotoxicity or nephrotoxicity. SS inhibited glucose metabolic reprogramming in cancer cells primarily via ROS-mediated AKT/mTOR/HIF-1α pathway inhibition. Pretreatment with N-acetylcysteine (NAC) or MHY1485 (an mTOR activator) partially reversed the inhibitory effects of SS on glucose metabolic reprogramming, cell proliferation, and migration, as well as its pro-apoptotic effects. SignificanceSS exhibited anti-cervical cancer effects, likely through the induction of ROS generation and inhibition of glucose metabolic reprogramming in cervical cancer cells, thereby inhibiting cell proliferation and promoting apoptosis. These findings provide new insights into understanding the molecular mechanisms underlying SS for potential new drug development for cervical cancer.

Brain targeted biomimetic siRNA nanoparticles for drug resistance glioblastoma treatment

Glioblastoma multiforme (GBM), the most aggressive intracranial neoplasm, remains incurable at present, primarily due to drug resistance, which significantly contributes to elevated recurrence rates and dismal prognosis. Signal transducer and activator of transcription 3 (STAT3) is a critical gene closely associated with GBM drug resistance and the progression of GBM stem cells (GSCs), making it a promising therapeutic target. In this study, we developed cancer cell membrane-cloaked biomimetic nanoparticles to deliver STAT3 siRNA to reverse drug resistance in homologous GBM. These biomimetic nanoparticles leverage homotypic targeting, rapid endosome escape, and fast siRNA release, leading to efficient in vitro STAT3 knockdown in both temozolomide-resistant U251-TR cells and X01 GSCs. Moreover, benefited from the membrane functionalization, significant prolonged blood circulation, improved blood brain barrier (BBB) penetration and GBM tumor accumulation are achieved by these siRNA biomimetic nanoparticles. Importantly, these nanoparticles effectively inhibit tumor proliferation, significantly extending median survival time in orthotopic U251-TR (43.5 d versus 20 d for PBS control) and X01 GSC-bearing mouse xenografts (52 d versus 19.5 d for PBS control). Altogether, this biomimetic siRNA platform offers a promising strategy for gene therapy targeting drug-resistant GBM.

Development of an orthotopic medulloblastoma zebrafish model for rapid drug testing.

Medulloblastoma (MB) is one of the most common malignant brain tumors in children. Current preclinical in vivo model systems for MB have increased our understanding of molecular mechanisms regulating MB development. However, they may not be suitable for large-scale studies. The aim of this study was to investigate if a zebrafish-based xenograft model can recapitulate MB growth and enable rapid drug testing. Nine different MB cell lines or patient-derived cells were transplanted into blastula-stage zebrafish embryos. Tumor development and migration were then monitored using live imaging. RNA sequencing was performed to investigate transcriptome changes after conditioning cells in neural stem cell-like medium. Furthermore, drug treatments were tested in a 96-well format. We demonstrate here that transplantation of MB cells into the blastula stage of zebrafish embryos leads to orthotopic tumor growth that can be observed within 24 hours after transplantation. Importantly, the homing of transplanted cells to the hindbrain region and the aggressiveness of tumor growth are enhanced by pre-culturing cells in a neural stem cell-like medium. The change in culture conditions rewires the transcriptome towards a more migratory and neuronal phenotype, including the expression of guidance molecules SEMA3A and EFNB1, both of which correlate with lower overall survival in MB patients. Furthermore, we highlight that the orthotopic zebrafish MB model has the potential to be used for rapid drug testing. Blastula-stage zebrafish MB xenografts present an alternative to current MB mouse xenograft models, enabling quick evaluation of tumor cell growth, neurotropism, and drug efficacy.