While porcine Neonatal Pancreatic Cell Clusters (NPCCs) were potential alternative source of islets, their immaturity and heterogeneity make them inefficiently ameliorate hyperglycemia in rodents. Our previous work showed that NPCCs regress to a transitional-like state immediately after isolation and undergo dynamic transcriptional changes during short-term in vitro culture. The present study aimed to further delineate the molecular cues underlying these changes during porcine pancreatic tissue isolation and cultivation. Transcriptomic and proteomic analysis were performed to compare Neonatal Porcine Pancreata (NPP), freshly isolated NPCCs (NPCCs-0D) and 3-day in vitro cultured NPCCs (NPCCs-3D). Using an in-house analysis pipeline, molecular differences between groups were identified. NPCCs‑0D showed lower molecular abundance compared with NPP and NPCCs‑3D groups under the tested conditions. Gene Sets Enrichment Analysis (GSEA) revealed that pathways related to receptor signaling and cell–microenvironment interactions were enriched in NPP and NPCCs‑3D, highlighting the importance of external regulation in maintaining pancreatic tissue architecture. In contrast. organelle- and catalysis-associated gene sets were enriched in NPCCs‑0D, suggesting a transcriptionally active and adaptive state. Collectively, these findings indicate that molecular differences among NPP, NPCCs-0D, and NPCCs-3D reflect changes in tissue organization, cellular composition, and adaptation to isolation and culture conditions, rather than a linear differentiation process.

In 2023, a previous study identified a coumarin compound meranzin in the ethanol extract of the edible red macroalga Bangia fuscopurpurea, but only a few studies have investigated the novel bioactivities of this compound and its hydrate. In this study, we examined the potential effects of meranzin and its hydrate on glioblastoma (GBM) using artificial intelligence-based in silico tools and in vitro models. We used DIGEP-Pred 2.0 to perform enrichment analysis of proteins affected by the two test compounds in GBM. ADMETlab 3.0 was applied to evaluate their medicinal chemistry properties and to predict absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiles. We then selected the compound with higher pharmacological potential for in vitro studies in the human GBM cell line. In addition, DIGEP-Pred 2.0 was used to predict its effects on cancer stemness markers. Enrichment analysis suggested that both meranzin and its hydrate may have therapeutic potential for GBM. Based on the in silico comparisons from ADMETlab 3.0, meranzin hydrate was considered to have higher pharmacological potential. Meranzin hydrate exhibited cytotoxicity in U87 cells, but not in GBM8401 cells. In the U87 model, meranzin hydrate demonstrated anti-invasion activity (IC50 = 33.12 μM), although no inhibitory effect on colony formation was observed. Furthermore, meranzin hydrate suppressed the cancer stem cell property of sphere formation (IC50 = 21.43 μM). Moreover, in silico results showed that meranzin hydrate may downregulate the protein expression of two cancer stemness markers, ALDH1A1 and NANOG, which was further validated by in vitro Western blot analysis. This study provides in silico assessments of the pharmacological potential, together with in vitro evidence of the anti-malignancy activity of the hydrate of meranzin in a human GBM cell line.

Hepatocellular carcinoma (HCC) is one of the leading cancers worldwide, and its development is strongly associated with the tumour microenvironment, particularly fibrosis and chronic inflammation. This study aims to investigate the role of the Hedgehog (Hh) pathway, a key signalling pathway in HCC progression, in the interaction between HCC cells and monocytes, which are central players in inflammation. Using a transwell migration assay, GLI1, the downstream transcriptional effector of the Hh pathway in HCC cells, was found to promote the migration of THP-1 monocyte cells. Among the cytokines regulated by the Hh pathway in HCC cells, CCL20 was identified as a crucial factor that interacts with CCR6 in THP-1 cells to facilitate migration. Next, using a luciferase reporter assay and chromatin immunoprecipitation, GLI1 binding sites within the CCL20 promoter region were confirmed. In a xenograft tumour mouse model, tumour growth and monocyte infiltration were inhibited in GLI1 or CCL20 knockout PLC5 cells. Moreover, mRNA expressions of GLI1 and CCL20 were positively correlated in clinical samples, with patients exhibiting high CCL20 expression showing poorer overall survival. Overall, our findings highlight that the Hh pathway in HCC contributes to monocyte infiltration via the CCL20-CCR6 axis, providing potential insights for future therapeutic strategies.

The increased risk of nephrotoxicity may impact the life quality and survival outcome in cancer patients receiving sorafenib therapy. Therefore, the development of novel strategy against sorafenib nephrotoxicity is an urgent work. Sodium-glucose co-transporter-2 (SGLT2) inhibitors such as empagliflozin have been approved for renal failure treatment. So far, the potential of empagliflozin against sorafenib nephrotoxicity has not yet been reported. The SGLT2 and apoptotic marker expressions in the sorafenib-treated renal proximal tubular cells (HK-2 cells) was investigated using immunoblot analysis. The cell viability was evaluated in HK-2 cells after sorafenib ± empagliflozin treatment using Alamar blue assay. The immunoblot analysis was applied to study the effect of sorafenib ± empagliflozin treatment on ferroptotic and proinflammatory stresses in HK-2 cells. The cell death, ferroptosis, lipid peroxidation, cytokine storm, and immune cells recruitments of kidneys was investigated in mice receiving a 28-day sorafenib ± empagliflozin administration using histopathological analyses. Sorafenib exposure dose-dependently upregulated SGLT2 in HK-2 cells, and empagliflozin significantly attenuated the sorafenib-induced cell death in HK-2 cells and mouse kidneys. Moreover, the sorafenib-stimulated iron deposition, oxidative DNA damage, lipid peroxidation, and glutathione peroxidase 4 (GPX4)/ SLC7A11 (xCT)-dependent ferroptosis were significantly alleviated by empagliflozin in mouse kidneys. The sorafenib-promoted cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) signaling as a ferroptosis driver was significantly blocked by empagliflozin in HK-2 cells and mouse kidneys. Empagliflozin also attenuated the sorafenib-stimulated-HMGB1/IL-1β proinflammatory signaling in vitro and in vivo. Furthermore, the sorafenib-promoted macrophage and neutrophil infiltrations were significantly reduced by empagliflozin in mouse kidneys. Collectively, empagliflozin may serve as a potent anti-ferroptotic and anti-inflammatory agent against sorafenib nephrotoxicity by targeting COX-2/PGE2 axis.

Objectives: Mesothelin (MSLN) is overexpressed in pancreatic ductal adenocarcinoma (PDAC), promoting cell proliferation, migration, and inhibiting apoptosis. While its oncogenic properties have been documented, the role of MSLN in regulating cellular senescence—a tumor-suppressive mechanism—has remained unexplored. This study is the first to identify and characterize a novel mesothelin-associated anti-senescence (MAAS) effect in PDAC. Methods: A proteogenomic analysis of PDAC tissue samples from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) was performed to evaluate MSLN-associated senescence pathways using WebGestalt. Human and murine PDAC cell lines with modified MSLN expression were analyzed for senescence phenotypes via SA-β-gal staining, Western blotting of key regulators (P53, P21waf1, and P16ink4a), γH2AX immunoblotting, and IL-8 quantification using ELISA. Results: The CPTAC analysis revealed an inverse correlation between MSLN expression and DNA damage/repair pathways. MSLN-deficient cells exhibited classic senescence features—growth arrest, an enlarged morphology, and elevated SA-β-gal activity. The expression of P53, P21waf1, and P16ink4a was upregulated, alongside increased γH2AX levels, indicating the activation of the DNA damage response. IL-8 secretion was significantly higher in the MSLN knockdown cells and reduced in the MSLN-overexpressing cells, consistent with the modulation of the SASP. Notably, MSLN deficiency impaired cell viability without inducing overt cytotoxicity, supporting a shift toward senescence. Conclusions: Our findings uncover a previously unrecognized mechanism through which MSLN promotes tumor progression by suppressing senescence via P53-associated pathways. Targeting the MAAS pathway may offer a novel therapeutic strategy to restore tumor-suppressive senescence and enhance treatment efficacy in PDAC.

Abstract Background: Hypercoagulability is a common clinical manifestation in patients with cancers, especially in pancreatic ductal adenocarcinoma (PDAC). Over 20% of PDAC patients with advanced-stage and worse prognosis undergo a venous thromboembolism (VTE) incidence. Fibrinogen (FG) may contribute to VTE due to its high expression in advanced PDAC tumors. Here, we set to determine the biological roles of tumor-derived FG in PDAC. In addition, our previous study showed that Von Willebrand Factor (VWF) A2 domain can effectively bind to FG and reduce platelet clot formation. Therefore, the functional roles of A2 on FG in PDAC were also studied. Methods: The correlation between survival rate and FG expression in PDAC was analyzed using RNAseq profiles of 149 PDAC patient samples from the TCGA database. PDAC subtyping of Baylor College of Medicine (BCM) PDAC patient-derived xenograft (PDX) cohort was performed using RNAseq data according to Moffitt’s criteria. The FG (three chains FGA, FGB, FGG) overexpression (OE) and knock-out (KO) PDAC cell lines were established. The gene expression and biological functions of FG were performed in PDX and cell lines using IHC and immunoblot assays. The FG effect on PDAC progression in vivo was studied in mouse models. Results: TCGA analysis showed that elevated tumor-derived FG gene expression is significantly correlated with worse overall survival and disease-free survival rates in PDAC patients. PDX lines RNAseq analysis clearly separated BCM PDAC PDXs into two subtypes: basal-like and classical PDACs. Basal-like PDXs showed significantly stronger FG staining than that in classical ones. Compared to the classical subtype, the basal-like subtype showed significantly upregulated genes that are associated with the epithelial-mesenchymal transition (EMT), IL6/JAK/STAT3, and TNFα signaling pathways. Accordingly, basal-like subtype PDAC exposed to IL6 or TNFα significantly increased FG and FG receptor expression with activated STAT3 and AKT pathways and elevated EMT markers, whereas classical subtype cells have minimal changes. In addition, we found that the FG-OE cells exhibit enhanced cell proliferation and migration properties whereas FG-KO cells reduced the effects. Consistent with in vitro data, FG-OE PDAC showed significantly higher tumor burdens than that of control PDAC in both orthotopic and subcutaneous PDAC mouse models. Furthermore, A2 protein treatment can effectively block the FG function and dramatically inactivate the FG-induced EMT process and AKT signaling pathway. Conclusions: Tumor-derived fibrinogen is upregulated in a basal-like subtype of PDAC cells and may contribute to tumor growth and metastasis by activating aberrant signaling pathways and promoting EMT. Our findings imply that elevated fibrinogen in PDAC could contribute to tumor progression and the A2 protein which target to fibrinogen may be a promising therapy for basal-like subtype PDAC. Citation Format: Dongliang Liu, Lisa Brubaker, Yichi Niu, Emily LaPlante, George Van Buren, Aleksandar Milosavljevic, Changyi Chen, Chenghang Zong, Miguel Cruz, Qizhi Yao. Tumor-derived fibrinogen promotes pancreatic ductal adenocarcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2079.

Environmental antineoplastics such as sorafenib may pose a risk to humans through water recycling, and the increased risk of cardiotoxicity is a clinical issue in sorafenib users. Thus, developing strategies to prevent sorafenib cardiotoxicity is an urgent work. Empagliflozin, as a sodium-glucose co-transporter-2 (SGLT2) inhibitor for type 2 diabetes control, has been approved for heart failure therapy. Still, its cardioprotective effect in the experimental model of sorafenib cardiotoxicity has not yet been reported. Real-time quantitative RT-PCR (qRT-PCR), immunoblot, and immunohistochemical analyses were applied to study the effect of sorafenib exposure on cardiac SGLT2 expression. The impact of empagliflozin on cell viability was investigated in the sorafenib-treated cardiomyocytes using Alamar blue assay. Immunoblot analysis was employed to delineate the effect of sorafenib and empagliflozin on ferroptosis/proinflammatory signaling in cardiomyocytes. Ferroptosis/DNA damage/fibrosis/inflammation of myocardial tissues was studied in mice with a 28-day sorafenib ± empagliflozin treatment using histological analyses. Sorafenib exposure significantly promoted SGLT2 upregulation in cardiomyocytes and mouse hearts. Empagliflozin treatment significantly attenuated the sorafenib-induced cytotoxicity/DNA damage/fibrosis in cardiomyocytes and mouse hearts. Moreover, GPX4/xCT-dependent ferroptosis as an inducer for releasing high mobility group box 1 (HMGB1) was also blocked by empagliflozin administration in the sorafenib-treated cardiomyocytes and myocardial tissues. Furthermore, empagliflozin treatment significantly inhibited the sorafenib-promoted NFκB/HMGB1 axis in cardiomyocytes and myocardial tissues, and sorafenib-stimulated proinflammatory signaling (TNF-α/IL-1β/IL-6) was repressed by empagliflozin administration. Finally, empagliflozin treatment significantly attenuated the sorafenib-promoted macrophage recruitments in mouse hearts. In conclusion, empagliflozin may act as a cardioprotective agent for humans under sorafenib exposure by modulating ferroptosis/DNA damage/fibrosis/inflammation. However, further clinical evidence is required to support this preclinical finding.

ABSTRACTBackgroundThe occurrence and progression of breast cancer are closely linked to copper ion homeostasis. Both copper deficiency and excess can inhibit breast cancer growth, while copper transport systems may contribute to its progression by regulating copper ion transport and the activity of associated proteins. However, a comprehensive review of the roles and applications of copper transport systems in breast cancer remains limited. In this study, we summarize the workflow of copper transport systems and the dual role of copper in cancer, highlighting the contributions of specific members of the copper transport system to breast cancer.MethodsA comprehensive search of the PubMed database was conducted to identify articles published over the past 30 years that focus on the relationship between copper transport system members and breast cancer. The findings were synthesized to elucidate the roles and mechanisms of these transporters in the onset and progression of breast cancer.ResultsWe identified 13 members of the copper transport system associated with the occurrence, progression, and mortality of breast cancer, including SLC31A1, DMT1, ATP7A, ATP7B, MTs, GSH, ATOX1, CCS, COX17, SCO1, SCO2, and COX11. Our findings revealed that, apart from STEAP, the remaining 12 members were overexpressed in breast cancer. These members influence the onset, progression, and cell death of breast cancer by modulating biological pathways such as intracellular copper ion levels and ROS. Notably, we observed for the first time that depletion of the copper storage protein GSH leads to increased copper ion accumulation, resulting in cuproptosis in breast cancer cells.ConclusionBy integrating the members of the copper transport system in breast cancer, we offer novel insights for the treatment of breast cancer and copper‐related therapies.

Supplementary Figure 7 - PDF file 166K, Real-time RT-PCR shows the mRNA levels of all the factors in the proposed network and Linear regression analyses depict the correlations between each factor and its counterparts

Supplementary Results and Methods - PDF file 255K, Additional information on results and methods

Hepatoma-derived growth factor (HDGF) overexpression and uncontrolled reactive oxygen species (ROS) accumulation are involved in malignant transformation and poor prognosis in various types of cancer. However, the interplay between HDGF and ROS generation has not been elucidated in hepatocellular carcinoma. Here, we first analyzed the profile of HDGF expression and ROS production in newly generated orthotopic hepatomas by ultrasound-guided implantation. In situ superoxide detection showed that HDGF-overexpressing hepatomas had significantly elevated ROS levels compared with adjacent nontumor tissues. Consistently, liver tissues from HDGF-deficient mice exhibited lower ROS fluorescence than those from age-and sex-matched WT mice. ROS-detecting fluorescent dyes and flow cytometry revealed that recombinant HDGF (rHDGF) stimulated the production of superoxide anion, hydrogen peroxide, and mitochondrial ROS generation in cultured hepatoma cells in a dose-dependent manner. In contrast, the inactive Ser103Ala rHDGF mutant failed to pro -mote ROS generation or oncogenic behaviors. Seahorse metabolic flux assays revealed that rHDGF dose dependently upregulated bioenergetics through enhanced basal and total oxygen consumption rate, extracellular acidification rate, and oxidative phosphorylation in hepatoma cells. Moreover, anti-oxidants of N-acetyl cysteine and MitoQ treatment significantly inhibited HDGF-mediated cell proliferation and invasive capacity. Genetic silencing of superoxide dismutase 2 augmented the HDGF-induced ROS generation and oncogenic behaviors of hepatoma cells. Finally, genetic knockdown nucleolin (NCL) and antibody neutralization of surface NCL, the HDGF receptor, abolished the HDGF-induced increase in ROS and mitochondrial energetics. In conclusion, this study has demonstrated for the first time that the HDGF/NCL signaling axis induces ROS generation by elevating ROS generation in mitochondria, thereby stimulating liver carcinogenesis.

Pancreatic ductal adenocarcinoma (PDAC) remains an extremely aggressive disease characterized by rapidly acquired multi-drug resistance, including to first-line chemotherapeutic agent gemcitabine. Autophagy is a process that is often exploited by cancer and is one of several intrinsic factors associated with resistance to gemcitabine. We have previously found that miR-198 acts as a tumor suppressor in PDAC through the targeting of factors including Valosin-containing protein (VCP). VCP has been reported to play an important role in autophagic flux. In this study, we investigated whether the repression of VCP through miR-198 administration disrupts the autophagy process and sensitizes PDAC cells to gemcitabine treatment in vitro. Moreover, we used LGA-PEI (LPNP) nanoparticles to effectively administer miR-198 to tumors in vivo, inducing tumor sensitization to gemcitabine and leading to a significant reduction in tumor burden and metastases and a concomitant downregulation of VCP expression and autophagy maturation. Our results indicate a potential therapeutic strategy for targeting gemcitabine resistant PDAC and establishes the use of LPNPs for effective therapeutic delivery of nucleic acids in vitro and in vivo.

Supplementary Table 2 from Elevated Interleukin-6 and G-CSF in Human Pancreatic Cancer Cell Conditioned Medium Suppress Dendritic Cell Differentiation and Activation

Slow skeletal muscle troponin T (TNNT1) as a poor prognostic indicator is upregulated in colon and breast cancers. However, the role of TNNT1 in the disease prognosis and biological functions of hepatocellular carci-noma (HCC) is still unclear. The Cancer Genome Atlas (TCGA), real-time quantitative RT-PCR (qRT-PCR), immunoblot, and immunohistochemical analyses were applied to evaluate the TNNT1 expression of human HCC. The impact of TNNT1 levels on disease progression and survival outcome was studied using TCGA analysis. Moreover, the bioinformatics analysis and HCC cell culture were used to investigate the biological functions of TNNT1. Besides, the immunoblot analysis and enzyme-linked immunosorbent assay (ELISA) were used to detect the extracellular TNNT1 of HCC cells and circulating TNNT1 of HCC patients, respectively. The effect of TNNT1 neutralization on oncogenic behaviors and signaling was further validated in the cultured hepatoma cells. In this study, tumoral and blood TNNT1 was upregulated in HCC patients based on the analyses using bioinformatics, fresh tissues, paraffin sections, and serum. From the multiple bioinformatics tools, the TNNT1 overexpression was associated with advanced stage, high grade, metastasis, vascular invasion, recurrence, and poor survival outcome in HCC patients. By the cell culture and TCGA analyses, TNNT1 expression and release were positively correlated with epithelial-mesenchymal transition (EMT) processes in HCC tissues and cells. Moreover, TNNT1 neutralization suppressed oncogenic behaviors and EMT in hepatoma cells. In conclusion, TNNT1 may serve as a non-invasive biomarker and drug target for HCC management. This research finding may provide a new insight for HCC diagnosis and treatment.

Supplementary Figure 1 from Elevated Interleukin-6 and G-CSF in Human Pancreatic Cancer Cell Conditioned Medium Suppress Dendritic Cell Differentiation and Activation

The posterior superior temporal gyrus (pSTG) has been implicated in the integration auditory feedback and motor system for controlling vocal production. However, the question as to whether and how the pSTG is causally involved in vocal feedback control is currently unclear. To this end, the present study selectively stimulated the left or right pSTG with continuous theta burst stimulation (c-TBS) in healthy participants, then used event-related potentials to investigate neurobehavioral changes in response to altered auditory feedback during vocal pitch regulation. The results showed that, compared to control (vertex) stimulation, c-TBS over the right pSTG led to smaller vocal compensations for pitch perturbations accompanied by smaller cortical N1 and larger P2 responses. Enhanced P2 responses received contributions from the right-lateralized temporal and parietal regions as well as the insula, and were significantly correlated with suppressed vocal compensations. Surprisingly, these effects were not found when comparing c-TBS over the left pSTG with control stimulation. Our findings provide evidence that supports a causal relationship between right, but not left, pSTG and auditory-motor integration for vocal pitch production. This lends support to a right-lateralized contribution of the pSTG in not only detecting vocal feedback errors but also driving motor commands for error correction.

Supplementary Figure 1 - PDF file 147K, Additional evidence that miR-198 is regulated by MSLN in PC cells

Background A growing body of literature has implicated the left dorsolateral prefrontal cortex (DLPFC) in the online monitoring of vocal production through auditory feedback. Specifically, disruption of or damage to the left DLPFC leads to exaggerated compensatory vocal responses to altered auditory feedback. It is conceivable that enhancing the cortical excitability of the left DLPFC may produce inhibitory influences on vocal feedback control by reducing vocal compensations. Methods We used anodal transcranial direct current stimulation (a-tDCS) to modulate cortical excitability of the left DLPFC and examined its effects on auditory-motor integration for vocal pitch regulation. Seventeen healthy young adults vocalized vowel sounds while hearing their voice pseudo-randomly pitch-shifted by ±50 or ±200 cents, either during (online) or after (offline) receiving active or sham a-tDCS over the left DLPFC. Results Active a-tDCS over the left DLPFC led to significantly smaller peak magnitudes and shorter peak times of vocal compensations for pitch perturbations than sham stimulation. In addition, this effect was consistent regardless of the timing of a-tDCS (online or offline stimulation) and the size and direction of the pitch perturbation. Conclusion These findings provide the first causal evidence that a-tDCS over the left DLPFC can facilitate auditory-motor integration for compensatory adjustment to errors in vocal output. Reduced and accelerated vocal compensations caused by a-tDCS over left DLPFC support the hypothesis of a top–down neural mechanism that exerts inhibitory control over vocal motor behavior through auditory feedback.

During glycolysis, the muscle isoform of pyruvate kinase PKM2 produces ATP in exchange for dephosphorylation of phosphoenolpyruvate (PEP) into pyruvate. PKM2 has been considered as a tumor-promoting factor in most cancers, whereas the regulatory role of PKM2 during head and neck carcinogenesis remained to be delineated. PKM2 mRNA and protein expression was examined in head and neck tumorous specimens. The role of PKM2 in controlling cellular malignancy was determined in shRNA-mediated PKM2-deficient head and neck squamous cell carcinoma (HNSC) cells. In agreement with the results in other cancers, PKM2 expression is enriched in both mouse and human HNSC tissues. Nevertheless, PKM2 mRNA expression reversely correlated with tumor stage, and greater recurrence-free survival rates are evident in the PKM2high HNSC population, arguing that PKM2 may be tumor-suppressive. Multifaceted analyses showed a greater in vivo xenografic tumor growth and an enhanced cisplatin resistance in response to PKM2 loss, whereas PKM2 silencing led to reduced cell motility. At the molecular level, metabolic shifts towards mitochondrial metabolism and activation of oncogenic Protein kinase B (PKB/Akt) and extracellular signal-regulated kinase (ERK) signals were detected in PKM2-silencing HNSC cells. In sum, our findings demonstrated that PKM2 differentially modulated head and neck tumorigenicity via metabolic reprogramming.

Supplementary Figure 2 - PDF file 207K, PBX-1 and VCP are predicted targets for miR-198