Tumor Growth Research Articles

HMGA1 regulates trabectedin sensitivity in advanced soft-tissue sarcoma (STS): A Spanish Group for Research on Sarcomas (GEIS) study

HMGA1 is a structural epigenetic chromatin factor that has been associated with tumor progression and drug resistance. Here, we reported the prognostic/predictive value of HMGA1 for trabectedin in advanced soft-tissue sarcoma (STS) and the effect of inhibiting HMGA1 or the mTOR downstream pathway in trabectedin activity. The prognostic/predictive value of HMGA1 expression was assessed in a cohort of 301 STS patients at mRNA (n = 133) and protein level (n = 272), by HTG EdgeSeq transcriptomics and immunohistochemistry, respectively. The effect of HMGA1 silencing on trabectedin activity and gene expression profiling was measured in leiomyosarcoma cells. The effect of combining mTOR inhibitors with trabectedin was assessed on cell viability in vitro studies, whereas in vivo studies tested the activity of this combination. HMGA1 mRNA and protein expression were significantly associated with worse progression-free survival of trabectedin and worse overall survival in STS. HMGA1 silencing sensitized leiomyosarcoma cells for trabectedin treatment, reducing the spheroid area and increasing cell death. The downregulation of HGMA1 significantly decreased the enrichment of some specific gene sets, including the PI3K/AKT/mTOR pathway. The inhibition of mTOR, sensitized leiomyosarcoma cultures for trabectedin treatment, increasing cell death. In in vivo studies, the combination of rapamycin with trabectedin downregulated HMGA1 expression and stabilized tumor growth of 3-methylcholantrene-induced sarcoma-like models. HMGA1 is an adverse prognostic factor for trabectedin treatment in advanced STS. HMGA1 silencing increases trabectedin efficacy, in part by modulating the mTOR signaling pathway. Trabectedin plus mTOR inhibitors are active in preclinical models of sarcoma, downregulating HMGA1 expression levels and stabilizing tumor growth.

Benserazide, a cystathionine beta-synthase (CBS) inhibitor, potentially enhances the anticancer effects of paclitaxel via inhibiting the S-sulfhydration of SIRT1 and the HIF1-α/VEGF pathway

Cancer targeted therapy is essential to minimize damage to normal cells and improve treatment outcomes. The elevated activity of Cystathionine beta-synthase (CBS), an enzyme responsible for producing endogenous hydrogen sulfide (H2S), plays a significant role in promoting tumor growth, invasiveness, and metastatic potential. Consequently, the selective inhibition of CBS could represent a promising therapeutic strategy for cancer. Currently, there is much interest in combining paclitaxel with other drugs for cancer treatment. This study aimed to investigate the efficacy of combining benserazide, a CBS inhibitor, with paclitaxel in treating tumors. Firstly, we demonstrated CBS is indeed involved in the progression of multiple cancers. Then it was observed that the total binding free energy between the protein and the small molecule is −98.241 kJ/mol. The release of H2S in the group treated with 100 μM benserazide was reduced by approximately 90% compared to the negative control, and the thermal denaturation curve of the complex protein shifted to the right, suggesting that benserazide binds to and blocks the CBS protein. Next, it was found that compared to paclitaxel monotherapy, the combination of benserazide with paclitaxel demonstrated stronger antitumor activity in KYSE450, A549, and HCT8 cells, accompanied by reduced cell viability, cell migration and invasion, as well as diminished angiogenic and lymphangiogenic capabilities. In vivo studies showed that the combined administration of benserazide and paclitaxel significantly reduced the volume and weight of axillary lymph nodes in comparison to the control group and single administration group. Further mechanistic studies revealed that the combination of benserazide and paclitaxel significantly suppressed the S-sulfhydration of SIRT1 protein, thereby inhibiting the expression of SIRT1 protein and activating SIRT1 downstream Notch1/Hes1 signaling pathway in KYSE450, A549, and HCT8 cells. Meanwhile, we observed that benserazide combined with paclitaxel induced a more significant downregulation of HIF-1α, VEGF-A, VEGF-C, and VEGF-D proteins expression levels in KYSE450, A549, and HCT8 cells compared to paclitaxel alone. These findings indicated that benserazide enhances the anticancer effects of paclitaxel via inhibiting the S-sulfhydration of SIRT1 and down-regulating HIF-1α/VEGF signaling pathway. This study suggests that benserazide may have potential as a chemosensitizer in cancer treatment.

Integrated analysis reveals critical cisplatin-resistance regulators E2F7 contributed to tumor progression and metastasis in lung adenocarcinoma

BackgroundDrug resistance poses a significant challenge in cancer treatment, particularly as a leading cause of therapy failure. Cisplatin, the primary drug for lung adenocarcinoma (LUAD) chemotherapy, shows effective treatment outcomes. However, the development of resistance against cisplatin is a major obstacle. Therefore, identifying genes resistant to cisplatin and adopting personalized treatment could significantly improve patient outcomes.MethodsBy examining transcriptome data of cisplatin-resistant LUAD cells from the GEO database, 181 genes associated with cisplatin resistance were identified. Using univariate regression analysis, random forest and multivariate regression analyses, two prognostic genes, E2F7 and FAM83A, were identified. This study developed a prognostic model utilizing E2F7 and FAM83A as key indicators. The Cell Counting Kit 8 assay, Transwell assay, and flow cytometry were used to detect the effects of E2F7 on the proliferation, migration, invasiveness and apoptosis of A549/PC9 cells. Western blotting was used to determine the effect of E2F7 on AKT/mTOR signaling pathway.ResultsThis study has pinpointed two crucial genes associated with cisplatin resistance, E2F7 and FAM83A, and developed a comprehensive model to assist in the diagnosis, prognosis, and evaluation of relapse risk in LUAD. Analysis revealed that patients at higher risk, according to these genetic markers, had elevated levels of immune checkpoints (PD-L1 and PD-L2). The prognostic and diagnosis values of E2F7 and FAM83A were further confirmed in clinical data. Furthermore, inhibiting E2F7 in lung cancer cells markedly reduced their proliferation, migration, invasion, and increased apoptosis. In vivo experiments corroborated these findings, showing reduced tumor growth and lung metastasis upon E2F7 suppression in lung cancer models.ConclusionOur study affirms the prognostic value of a model based on two DEGs, offering a reliable method for predicting the success of tumor immunotherapy in patients with LUAD. The diagnostic and predictive model based on these genes demonstrates excellent performance. In vitro, reducing E2F7 levels shows antitumor effects by blocking LUAD growth and progression. Further investigation into the molecular mechanisms has highlighted E2F7’s effect on the AKT/mTOR signaling pathway, underscoring its therapeutic potential. In the era of personalized medicine, this DEG-based model promises to guide clinical practice.

IDH inhibition in gliomas: from preclinical models to clinical trials.

Gliomas are the most common malignant primary brain tumours in adults and cannot usually be cured with standard cancer treatments. Gliomas show intratumoural and intertumoural heterogeneity at the histological and molecular levels, and they frequently contain mutations in the isocitrate dehydrogenase 1 (IDH1) or IDH2 gene. IDH-mutant adult-type diffuse gliomas are subdivided into grade 2, 3 or 4 IDH-mutant astrocytomas and grade 2 or 3 IDH-mutant, 1p19q-codeleted oligodendrogliomas. The product of the mutated IDH genes, D-2-hydroxyglutarate (D-2-HG), induces global DNA hypermethylation and interferes with immunity, leading to stimulation of tumour growth. Selective inhibitors of mutant IDH, such as ivosidenib and vorasidenib, have been shown to reduce D-2-HG levels and induce cellular differentiation in preclinical models and to induce MRI-detectable responses in early clinical trials. The phase III INDIGO trial has demonstrated superiority of vorasidenib, a brain-penetrant pan-mutant IDH inhibitor, over placebo in people with non-enhancing grade 2 IDH-mutant gliomas following surgery. In this Review, we describe the pathway of development of IDH inhibitors in IDH-mutant low-grade gliomas from preclinical models to clinical trials. We discuss the practice-changing implications of the INDIGO trial and consider new avenues of investigation in the field of IDH-mutant gliomas.

P37 Drug repurposing for treatment of recessive dystrophic epidermolysis bullosa cutaneous squamous cell carcinoma

Abstract Background Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited skin disorder that leads to severe wounding and blistering of the skin. Patients with RDEB develop aggressive cutaneous squamous cell carcinoma (cSCC) with a mortality rate of approximately 90% by age 55 years. The current primary treatment is wide excision of tumours, often leading to amputations. Identifying new therapeutic avenues for RDEB cSCC remains a priority. Here, we approach this in two ways. Firstly, we identified a SMAD4-dependent sphingosine pathway response to endogenous transforming growth factor (TGF)-β, essential for RDEB cSCC proliferation which could be targeted as a potential therapeutic strategy. Secondly, we utilized a US Food and Drug Administration (FDA) screen of > 3000 drugs to identify potential druggable pathways that could be used to treat RDEB cSCC. Methods RDEB cSCC cell lines were used to test Siponimod, a sphingosine-1 receptor modulator, on proliferation, clonogenicity and invasion in vitro. Siponimod was used in vivo to test tumour growth effect of targeting the sphingosine pathway. We carried out an FDA-approved drug screen on four RDEB cSCC cell lines using the Opera Phenix high-throughput microscope to identify targets. Results Treatment of RDEB cSCC cell lines with Siponimod showed compromised proliferation compared with untreated cells in addition to significant reduction in colony formation. There was a significant decrease in the invasive capability of RDEB SCC cells. Treatment of normal human keratinocytes showed no effect on proliferation. Our FDA-approved screen identified multiple drugs that target MEK, mechanistic target of rapamycin complex, histone deacetylase, signal transducer and activator of transcription, and topoisomerase I/II in the top 50 hits. The most common target was DNA damage/repair pathways, but cytoskeletal signalling, transmembrane transporters and proteases were identified as other potential avenues for treatment of RDEB cSCC. Conclusions There is an unmet need for therapeutic options in RDEB cSCC. Here, we have demonstrated the potential of repurposing previously licensed drugs to identify treatment avenues for RDEB cSCC.

Nano-Pulse Stimulation Treatment Inhibits Pan02 Murine Pancreatic Tumor Growth and Induces a Long-Term Adaptive Immune Response with Abscopal Effects When Combined with Immune-Enhancing Agents

Nano-Pulse Stimulation Treatment Inhibits Pan02 Murine Pancreatic Tumor Growth and Induces a Long-Term Adaptive Immune Response with Abscopal Effects When Combined with Immune-Enhancing Agents

Abstract PR009: Exploring the functional consequences of APOBEC3-induced non-coding hotspot mutations in bladder cancer using massively parallel reporter assays and CRISPR-mediated base editing

Abstract Cancer research has traditionally focused on protein-coding genes, but approximately 98% of the human genome is non-coding, comprising gene regulatory elements such as enhancers, silencers, and promoters. Mutations within these regions can foster tumorigenesis by modulating the expression of oncogenes. Recent whole-genome sequencing studies have revealed several recurrently mutated non-coding genomic regions, including in bladder cancer, but their role in tumorigenesis remains unknown. In this study, we delved into whole-genome sequencing data from the bladder cancer TCGA cohort and an independent set of bladder tumors, uncovering hundreds of recurrent mutations. Mutational signature analysis identified over 65% of these mutations as APOBEC3-induced. We substantiated the involvement of APOBEC3 enzymes by using conventional deamination assays and novel approach dubbed as APOBEC3-mediated mutation capture assay. Predominantly, these mutations occurred in the hairpin loop, a favored motif for APOBEC3 enzymes. To systematically assess which of the mutations are functional and prioritize them for biological characterization, we developed Massively Parallel Reporter Assays (MPRA). Two types of MPRA libraries were constructed, one for evaluating promoter activity and the other for assessing enhancer activity of the mutations. Each library construction entailed 36,000 oligonucleotides, comprehensively representing all mutation hotspots and their corresponding wild-type alleles. The application of MPRA resulted in the identification of a substantial number of functionally relevant mutations. Zooming in on the top 20 genomic regions, we employed CRISPR-based approaches to pinpoint target genes and elucidate their functional consequences. Most of these regions co-localized with enhancers, as determined by analysis of ATAC-seq, ChIP-seq, and Hi-C data from bladder cancer cell lines. Perturbation of hotspots regions resulted in dysregulation of multiple genes. The co-regulation patterns of genes were further supported by strong correlation of their mRNA expression in RNA-seq datasets (TCGA, SRA, etc.). Notable examples included twin hotspot mutations upstream of the LEPROTL1 gene, elevating the expression of LEPROTL1, DCTN6 and SARAF, resulting in aggressive tumor growth in in vitro functional experiments. These three genes emerged as novel oncogenes in cancer. Another functional hotspot influenced the expression of two known long non-coding RNAs, NEAT1 and MALAT1, previously characterized as cancer genes. Additionally, some recurrently mutated regions or their target genes were associated with worse overall survival in the TCGA cohort. In conclusion, our findings propose that non-coding mutations likely drive tumorigenesis, contributing to tumor heterogeneity. Employing a combination of functional, multi-omics, and in vivo approaches, we aim to comprehensively characterize the role of hotspot non-coding mutations in bladder cancer and assess their clinical significance. Citation Format: Rouf Banday, Bilal Lone, Kelly Butler, Dhanusha Yesudhas, Alexandra Dobbins, Arup Chakraborty. Exploring the functional consequences of APOBEC3-induced non-coding hotspot mutations in bladder cancer using massively parallel reporter assays and CRISPR-mediated base editing [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR009.

Investigating the Effect of Liver Regeneration on Tumor Growth: Development of a Mouse Model with Orthotopic Implantation of Patient-derived HCC Organoids

Abstract Background Recurrence after hepatocellular carcinoma (HCC) treatment is a significant problem. It affects more than 70% of patients undergoing liver resection. Recurrence can arise from undetected micro metastasis or de novo cancer. Clinical and experimental studies suggest that liver regeneration ensuing surgical resection may activate occult micro-metastasis leading to regeneration induced tumor recurrence. However, in vivo patient-derived organoid (PDO) HCC models that are translatable to patient tumors are sparse. Aims Here, we aim to establish a new in vivo model to understand the impact of liver regeneration on HCC tumor growth. Methods PDOs were generated from HCC tissue obtained from patients undergoing liver resection at the Clarunis University Digestive Health and Care Center and transduced with firefly luciferase. Implantation was performed via laparotomy and orthotopic injection of the organoids in the right superior liver lobe. Tumor growth is monitored by in vivo bioluminescence imaging until the endpoint of the experiment. As a primary endpoint tumor growth two weeks after liver resection was defined. Experimental groups underwent either minor (30%) or major (65%) hepatectomy of tumor-free liver, whereas the control group received a re-laparotomy with subsequent closure of the abdomen. Normal liver and tumor tissue were characterized using immunohistochemistry. Results Two HCC-PDO lines were successfully implanted. Compared to the control, after minor and major liver resection the mean weight increased significantly to 0.82% (p < 0.0001) respectively 0.99% (p < 0.0001). Preliminary data about the tumor volume have yet to be concluded. Histology and immunohistochemistry staining for HCC confirmed the origin of the PDOs from the original patient tumor. Conclusion The establishment of an orthotopic xenograft mouse model for HCC PDO was successful. Through liver resection, a regenerative environment could be achieved, to investigate the molecular behavior of HCC recurrence after surgery. This offers the basis to study potential new targets and mechanisms to improve HCC-treatment.

An Anti-VEGF-B Antibody Reduces Abnormal Tumor Vasculature and Enhances the Effects of Chemotherapy

The vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key regulators of blood vessel formation, including in tumors, where their deregulated function can promote the production of aberrant, leaky blood vessels, supporting tumor development. Here we investigated the VEGFR1 ligand VEGF-B, which we demonstrate to be expressed in tumor cells and in tumor stroma and vasculature across a range of tumor types. We examined the anti-VEGF-B-specific monoclonal antibody 2H10 in preclinical xenograft models of breast and colorectal cancer, in comparison with the anti-VEGF-A antibody bevacizumab. Similar to bevacizumab, 2H10 therapy was associated with changes in tumor blood vessels and intra-tumoral diffusion consistent with normalization of the tumor vasculature. Accordingly, treatment resulted in partial inhibition of tumor growth, and significantly improved the response to chemotherapy. Our studies indicate the importance of VEGF-B in tumor growth, and the potential of specific anti-VEGF-B treatment to inhibit tumor development, alone or in combination with established chemotherapies.

Human lung cancer-derived mesenchymal stem cells promote tumor growth and immunosuppression

BackgroundThe presence of mesenchymal stem cells has been confirmed in some solid tumors where they serve as important components of the tumor microenvironment; however, their role in cancer has not been fully elucidated. The aim of this study was to investigate the functions of mesenchymal stem cells isolated from tumor tissues of patients with non-small cell lung cancer.ResultsHuman lung cancer-derived mesenchymal stem cells displayed the typical morphology and immunophenotype of mesenchymal stem cells; they were nontumorigenic and capable of undergoing multipotent differentiation. These isolated cells remarkably enhanced tumor growth when incorporated into systems alongside tumor cells in vivo. Importantly, in the presence of mesenchymal stem cells, the ability of peripheral blood mononuclear cell-derived natural killer and activated T cells to mediate tumor cell destruction was significantly compromised.ConclusionCollectively, these data support the notion that human lung cancer-derived mesenchymal stem cells protect tumor cells from immune-mediated destruction by inhibiting the antitumor activities of natural killer and T cells.

Hemangiosarcoma Cells Promote Conserved Host-Derived Hematopoietic Expansion.

Hemangiosarcoma and angiosarcoma are soft-tissue sarcomas of blood vessel-forming cells in dogs and humans, respectively. These vasoformative sarcomas are aggressive and highly metastatic, with disorganized, irregular blood-filled vascular spaces. Our objective was to define molecular programs which support the niche that enables progression of canine hemangiosarcoma and human angiosarcoma. Dog-in-mouse hemangiosarcoma xenografts recapitulated the vasoformative and highly angiogenic morphology and molecular characteristics of primary tumors. Blood vessels in the tumors were complex and disorganized, and they were lined by both donor and host cells. In a series of xenografts, we observed that the transplanted hemangiosarcoma cells created exuberant myeloid hyperplasia and gave rise to lymphoproliferative tumors of mouse origin. Our functional analyses indicate that hemangiosarcoma cells generate a microenvironment that supports expansion and differentiation of hematopoietic progenitor populations. Furthermore, gene expression profiling data revealed hemangiosarcoma cells expressed a repertoire of hematopoietic cytokines capable of regulating the surrounding stromal cells. We conclude that canine hemangiosarcomas, and possibly human angiosarcomas, maintain molecular properties that provide hematopoietic support and facilitate stromal reactions, suggesting their potential involvement in promoting the growth of hematopoietic tumors.

Analysis of tumor microenvironment composition in vestibular schwannomas: insights into NF2-associated and sporadic variations and their clinical correlations

ObjectiveVestibular schwannomas (VS), benign tumors stemming from the eighth cranial nerve’s Schwann cells, are associated with Merlin gene mutations, inflammation, and the tumor microenvironment (TME), influencing tumor initiation, maintenance, and potential neural dysfunction. Understanding TME composition holds promise for systemic therapeutic interventions, particularly for NF2-related schwannomatosis.MethodologyA retrospective analysis of paraffin-embedded tissue from 40 patients (2013-2020), evenly divided by neurofibromatosis type 2 status, with further stratification based on magnetic resonance imaging (MRI) progression and hearing function. Immunohistochemistry assessed TME components, including T-cell markers (CD4, CD8, CD25), NK cells (CD7), and macrophages (CD14, CD68, CD163, CCR2). Fiji software facilitated image analysis.ResultsT-cell markers (CD4, CD8, CD7) exhibited low expression in VS, with no significant NF2-associated vs. sporadic distinctions. Macrophage-related markers (CD14, CD68, CD163, CCR2) showed significantly higher expression (CD14: p = 0.0187, CD68: p < 0.0001, CD163: p = 0.0006, CCR2: p < 0.0001). CCR2 and CD163 significantly differed between NF2-associated and sporadic VS. iNOS, an M1-macrophage marker, was downregulated. CD25, a regulatory T-cell marker, correlated significantly with tumor growth dynamics (p = 0.016).DiscussionImmune cells, notably monocytes and macrophages, crucially contribute to VS pathogenesis in both NF2-associated and sporadic cases. Significant differences in CCR2 and CD163 expression suggest distinct immune responses. Regulatory T-cells may serve as growth dynamic markers. These findings highlight immune cells as potential biomarkers and therapeutic targets for managing VS.

Risk assessment model based on nucleotide metabolism-related genes highlights SLC27A2 as a potential therapeutic target in breast cancer.

Breast cancer (BC) is the most prevalent malignant tumor worldwide among women, with the highest incidence rate. The mechanisms underlying nucleotide metabolism on biological functions in BC remain incompletely elucidated. MATERIALSAND METHODS: We harnessed differentially expressed nucleotide metabolism-related genes from The Cancer Genome Atlas-BRCA, constructing a prognostic risk model through univariate Cox regression and LASSO regression analyses. A validation set and the GSE7390 dataset were used to validate the risk model. Clinical relevance, survival and prognosis, immune infiltration, functional enrichment, and drug sensitivity analyses were conducted. Our findings identified four signature genes (DCTPP1, IFNG, SLC27A2, and MYH3) as nucleotide metabolism-related prognostic genes. Subsequently, patients were stratified into high- and low-risk groups, revealing the risk model's independence as a prognostic factor. Nomogram calibration underscored superior prediction accuracy. Gene Set Variation Analysis (GSVA) uncovered activated pathways in low-risk cohorts and mobilized pathways in high-risk cohorts. Distinctions in immune cells were noted between risk cohorts. Subsequent experiments validated that reducing SLC27A2 expression in BC cell lines or using the SLC27A2 inhibitor, Lipofermata, effectively inhibited tumor growth. We pinpointed four nucleotide metabolism-related prognostic genes, demonstrating promising accuracy as a risk prediction tool for patients with BC. SLC27A2 appears to be a potential therapeutic target for BC among these genes.

MiR-1976/NCAPH/P65 axis inhibits the malignant phenotypes of lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is a malignancy with an abysmal survival rate. High metastasis is the leading cause of the low survival rate of LUAD. NCAPH, an oncogene, is involved in the carcinogenesis of LUAD. However, the regulation of NCAPH in LUAD remains controversial. In this work, we identified an up-regulation of NCAPH in LUAD tissues. Patients who expressed more NCAPH had shorter overall survival (OS). Furthermore, NCAPH overexpression promoted LUAD cell migration while inhibiting apoptosis. MiR-1976 and miR-133b were predicted to target NCAPH expression by searching TargetScan and linkedomics databases. Following that, we confirmed that miR-1976 suppressed NCAPH by directly targeting a 7-bp region of NCAPH 3' untranslated regions (UTR). In addition, increased expression of miR-1976 decreased the proliferation & migration and promoted apoptosis of LUAD cells, and the re-introduction of NCAPH reversed these influences. Furthermore, the xenograft and metastasis mouse models also confirmed that miR-1976 inhibited tumor growth and metastasis in vivo by targeting NCAPH. Finally, we found that MiR-1976 targeting NCAPH blocked the activation of NF-κB. In conclusion, miR-1976 inhibits NCAPH activity in LUAD and acts as a tumor suppressor. The miR-1976/NCAPH/NF-κB axis may, in the future, represent crucial diagnostic and prognostic biomarkers and promising therapeutic options.

Zebularine potentiates anti-tumor immunity by inducing tumor immunogenicity and improving antigen processing through cGAS-STING pathway

DNA methylation is an important epigenetic mechanism involved in the anti-tumor immune response, and DNA methyltransferase inhibitors (DNMTi) have achieved impressive therapeutic outcomes in patients with certain cancer types. However, it is unclear how inhibition of DNA methylation bridges the innate and adaptive immune responses to inhibit tumor growth. Here, we report that DNMTi zebularine reconstructs tumor immunogenicity, in turn promote dendritic cell maturation, antigen-presenting cell activity, tumor cell phagocytosis by APCs, and efficient T cell priming. Further in vivo and in vitro analyses reveal that zebularine stimulates cGAS-STING-NF-κB/IFNβ signaling to enhance tumor cell immunogenicity and upregulate antigen processing and presentation machinery (AgPPM), which promotes effective CD4+ and CD8+ T cell-mediated killing of tumor cells. These findings support the use of combination regimens that include DNMTi and immunotherapy for cancer treatment.

E3 ubiquitin ligase UBR5 promotes gemcitabine resistance in pancreatic cancer by inducing O-GlcNAcylation-mediated EMT via destabilization of OGA.

Pancreatic cancer (PC) is among the deadliest malignancies, with an extremely poor diagnosis and prognosis. Gemcitabine (GEM) remains the first-line drug for treating PC; however, only a small percentage of patients benefit from current immunotherapies or targeted therapies. Resistance to GEM is prevalent and affects long-term survival. We found that ubiquitin-protein ligase E3 module N-recognition 5 (UBR5) is a therapeutic target against GEM resistance. UBR5 was markedly upregulated in clinical GEM-resistant PC samples and GEM-resistant PC cells. UBR5 knockdown markedly increased GEM sensitivity in GEM-resistant PC cell lines. UBR5-mediated GEM resistance was accompanied by activation of epithelial-mesenchymal transition (EMT) and could be mitigated by inhibiting EMT. Further analysis revealed that UBR5 promoted GEM resistance in PC cells by enhancing O-GlcNAcylation-mediated EMT. In addition, UBR5 knockdown resulted in increased O-GlcNAase (OGA) levels, an essential negatively regulated enzyme in the O-GlcNAcylation process. We identified a negative association between OGA and UBR5 levels, which further supported the hypothesis that O-GlcNAcylation-mediated GEM resistance induced by UBR5 is OGA-dependent in PC cells. Mechanistic studies revealed that UBR5 acts as an E3 ubiquitin ligase of OGA and regulates O-GlcNAcylation by binding and modulating OGA, facilitating its degradation and ubiquitination. Additionally, high-throughput compound library screening using three-dimensional protein structure analysis and drug screening identified a Food and Drug Administration drug, Y-39983 dihydrochloride, as a potent GEM sensitiser and UBR5 inhibitor. The combination of Y-39983 dihydrochloride and GEM attenuated tumour growth in a mouse xenograft tumour model. Collectively, these data demonstrated that UBR5 plays a pivotal role in the sensitisation of PC to GEM and provides a potential therapeutic strategy to overcome GEM resistance.

Disruption of NADPH homeostasis by total flavonoids from Adinandra nitida Merr. ex Li leaves triggers ROS-dependent p53 activation leading to apoptosis in non-small cell lung cancer cells

Ethnopharmacological relevanceAdinandra nitida Merr. ex Li leaves serve as a herbal tea and hold a significant role in traditional Chinese medicine, being applied to assist in tumor treatment. Flavonoids present the primary bioactive constituents in Adinandra nitida Merr. ex Li leaves. Aim of the studyTo explore the potential of total flavonoids from Adinandra nitida Merr. ex Li Leaves (TFAN) in inhibiting non-small cell lung cancer (NSCLC) and further elucidate the underlying mechanisms. Materials and methodsHuman NSCLC cell lines and normal lung cell line were employed to assess the impact of TFAN (0–160 μg/mL for 24, 28 and 72 h) on cell proliferation in vitro. Immunofluorescence (IF) staining gauged p53 expression changes in NSCLC cells under TFAN present condition (150 μg/mL for 24 h). In vivo study utilized NSCLC cell derived xenograft tumors in nude mice, administering TFAN orally (200 and 400 mg/kg) for 14 days. Immunohistochemistry assessed Cleaved Caspase 3 expression change in A549 xenograft tumors treated with TFAN (400 mg/kg for 14 days). RNA-seq and KEGG analysis identified gene expression changes and enriched processes in A549 xenograft tumors treated with TFAN. CM-H2DCFDA and metabolomics assessed ROS level and GSH/GSSG pool changes in A549 cells under TFAN present condition. Cell viability assay and IF staining assessed A549 cell proliferation and p53 expression changes under H2O2-induced oxidative stress (0–40 μM for 24 h) and TFAN present conditions. GSEA and N-Acetyl-L-cysteine (NAC) rescue (0–1 μM for 24 h) analyzed the impact of TFAN on GSH de novo synthesis. NADPH/NADP+ pool measurement and NADPH rescue (0–10 μM for 24 h) analyzed the impact of TFAN on GSH salvage synthesis. GC-FID and HPLC-MS were utilized to detect ethanol and ethyl acetate residues, and to characterize the chemical constituents in TFAN, respectively. The total flavonoid content of TFAN was determined using a 330 nm wavelength. ResultsTFAN significantly inhibited A549 cells (wild-type p53) but not NCI–H1299 cells (p53-deficient), NCI–H596 cells (p53-mutant) or BEAS-2B in vitro. IF staining validated p53 genotype for the cell lines and revealed an increase in p53 expression in A549 cells after TFAN treatment. In vivo, TFAN selectively inhibited A549 xenograft tumor growth without discernible toxicity, inducing apoptosis evidenced by Cleaved Caspase 3 upregulation. RNA-seq and KEGG analysis suggested ROS biosynthesis was involved in TFAN-induced p53 activation in A549 cells. Elevated ROS level in TFAN-treated A549 cells were observed. Moreover, TFAN sensitized A549 cells to H2O2-induced oxidative stress, with higher p53 expression. Additionally, A549 cells compensated with GSH de novo synthesis under TFAN present condition, confirmed by GSEA and NAC rescue experiment. TFAN disrupted NADPH homeostasis to impair GSH salvage biosynthesis, supported by NADPH/NADP+ change and NADPH rescue experiment. The chemical constituents of TFAN, with acceptable limits for ethanol and ethyl acetate residues and a total flavonoid content of 68.87%, included Catechin, Epicatechin, Quercitroside, Camellianin A, and Apigenin. ConclusionThe disruption of NADPH homeostasis by TFAN triggers ROS-dependent p53 activation that leads to apoptotic cell death, ultimately suppressing NSCLC growth. These findings offer potential therapeutic implications of Adinandra nitida Merr. ex Li leaves in combating NSCLC.

Two-Photon Absorption Aggregation-Induced Emission Luminogen/Paclitaxel Nanoparticles for Cancer Theranostics.

Multifaceted nanoplatforms integrating fluorescence imaging and chemotherapy have garnered acknowledgment for their potential potency in cancer diagnosis and simultaneous in situ therapy. However, some drawbacks remain for traditional organic photosensitizers, such as poor photostability, short excitation wavelength, and shallow penetration depth, which will greatly lower the chemotherapy treatment efficiency. Herein, we present lipid-encapsulated two-photon active aggregation-induced emission (AIE) luminogen and paclitaxel (PTX) nanoparticles (AIE@PTX NPs) with bright red fluorescence emission, excellent photostability, and good biocompatibility. The AIE@PTX NPs exhibit dual functionality as two-photon probes for visualizing blood vessels and tumor structures, achieving penetration depth up to 186 and 120 μm, respectively. Furthermore, the tumor growth of the HeLa-xenograft model can be effectively prohibited after the fluorescence imaging-guided and PTX-induced chemotherapy, which shows great potential in the clinical application of two-photon cell and tumor fluorescence imaging and cancer treatment.

Expression of LASS2 Can be Regulated by Dihydroartemisinin to Regulate Cisplatin Chemosensitivity in Bladder Cancer Cells.

The aim of this study was to investigate the potential of dihydroartemisinin to augment the efficacy of cisplatin chemotherapy through the modulation of LASS2 expression. TCMSP, CTR-DB, TCGA-BLC, and other databases were used to analyze the possibility of LASS2 as the target gene of dihydroartemisinin. Cell experiments revealed the synergistic effect of DDP and DHA. Animal experiments showed that DHA inhibited the growth of DDP-treated mice. In addition, WB, real-time PCR, and immunohistochemical analysis showed that DHA enhanced LASS2 (CERS2) expression in bladder cancer cells and DDP-treated mice. LASS2 is associated with cisplatin chemosensitivity.LASS2 expression levels are different between BLC tissues and normal tissues. COX analysis showed that patients with high LASS2 expression had a higher cumulative overall survival rate than those with low LASS2 expression. The Sankey plot showed that LASS2 expression is lower in BLC tissues with more advanced stage and distant metastasis. The docking score of DHA and LASS2 reached the maximum value of -5.5259, indicating that DHA had a strong binding affinity with LASS2 targets. CCK8 assay showed that the most effective concentration ratio of DHA to DDP was 2.5μg/ml + 10μg/ml. In vivo experiments showed that DHA inhibited tumor growth in cisplatin-treated mice. In addition, WB, RT-qPCR, and immunohistochemical analysis showed that DHA was able to enhance LASS2 expression in BLC cells and DDP-treated mice. The upregulation of LASS2 (CERS2) expression in bladder cancer cells by DHA has been found to enhance cisplatin chemosensitivity.

Low serum apolipoprotein A1 impairs antitumor immunity of CD8+ T cells via the HIF-1α-glycolysis pathway.

An immunosuppressive microenvironment promotes the occurrence and development of tumors. Low apolipoprotein A1 (ApoA1) is closely related to tumor development, but the underlying mechanisms are unclear. This study investigated the association between serum ApoA1 levels and the immune microenvironment in endometrial, ovarian, and lung cancers. The serum ApoA1 level was decreased significantly in patients with endometrial and ovarian cancers compared with healthy controls. In endometrial cancer tissues, the low serum ApoA1 group showed increased CD163+ macrophage infiltration and decreased CD8+ T-cell infiltration compared with the normal serum ApoA1 group. Compromised tumor-infiltrating CD8+ T-cell functions and decreased CD8+ T-cell infiltration also were found in tumor-bearing ApoA1-knockout mice. CD8+ T-cell depletion experiments confirmed that ApoA1 exerted its antitumor activity in a CD8+ T cell-dependent manner. In vitro experiments showed that the ApoA1 mimetic peptide L-4F directly potentiated the antitumor activity of CD8+ T cells via a HIF-1α-mediated glycolysis pathway. Mechanistically, ApoA1 suppressed ubiquitin-mediated degradation of HIF-1α protein by downregulating HIF-1α subunit α inhibitor. This regulatory process maintained the stability of HIF-1α protein and activated the HIF-1α signaling pathway. Tumor-bearing ApoA1 transgenic mice showed an increased response to anti-PD-1 therapy, leading to reduced tumor growth along with increased infiltration of activated CD8+ T cells and enhanced tumor necrosis. The data reported herein demonstrate critical roles for ApoA1 in enhancing CD8+ T-cell immune functions via HIF-1α-mediated glycolysis and support clinical investigation of combining ApoA1 supplementation with anti-PD-1 therapy for treating cancer.