Ferroptosis, an iron-dependent form of programmed cell death driven by lipid peroxidation, represents a new potential therapeutic target in cancer. However, emerging evidence indicates that hepatocellular carcinoma (HCC) frequently exhibits resistance to ferroptosis induction, while the underlying molecular mechanism is poorly understood. Here, we found that aldo-keto reductase family 1 member C3 (AKR1C3), a protein highly expressed in ferroptosis-resistant HCC cells, negatively regulates ferroptosis in an enzyme-independent manner. Mechanistically, AKR1C3 promotes ubiquitin-proteasomal degradation of the transferrin receptor (TFRC), which is indispensable for cellular iron uptake. AKR1C3 knockdown restores TFRC expression, increases the level of labile iron pool, and sensitizes HCC cells to ferroptosis. Furthermore, AKR1C3 acts as a scaffolding protein to promote the degradation of TFRC and reduce iron uptake by promoting nuclear export of Beta-transducin repeats-containing proteins (β-TrCP) and its binding to TFRC. Notably, AKR1C3 is upregulated in NRF2-driven sorafenib-resistant HCC, and its inhibition reversed ferroptosis and sorafenib resistance. Our work uncovers AKR1C3 suppresses ferroptosis in HCC by promoting β-TrCP-mediated TFRC degradation, positioning AKR1C3 as a promising therapeutic target to enhance ferroptosis-based anticancer strategies.

OBJECTIVE:To evaluate the predictive value of deep myometrial invasion (DMI) for lymph node metastasis (LNM) in low-grade endometrioid carcinoma (LGEC) and its implications for surgical decision-making. METHODS:A retrospective cohort study was conducted on 212 patients with pathologically confirmed LGEC (G1/G2) who underwent surgical treatment at Women's Hospital, School of Medicine, Zhejiang University between January 2018 and December 2023. Clinicopathological variables were analyzed using univariate and multivariate logistic regression to identify independent risk factors for LNM. The predictive performance of DMI and other significant variables was assessed by sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and receiver operating characteristic (ROC) curves. A combined predictive model incorporating all independent factors was developed and internally validated using calibration curves and the Hosmer-Lemeshow test. External validation was performed in an independent cohort of 54 patients. RESULTS:LNM occurred in 9.4% (20/212) of patients. Multivariate analysis identified DMI ≥50% (OR=3.58, 95% CI: 1.23-10.41, P=0.019), cervical stromal invasion (CSI; OR=5.24, 95% CI: 1.47-18.73, P=0.011), and elevated preoperative CA125 >35 U/ml (OR=4.14, 95% CI: 1.42-12.07, P=0.009) as independent predictors. The combined model achieved a sensitivity of 90% and an NPV of 99% for LNM prediction; external validation yielded an NPV of 97.44%, confirming its robustness for identifying low-risk patients. Patients with DMI were more likely to undergo laparotomy than those with superficial invasion (50.0% vs. 18.3%, P<0.001) and more frequently received postoperative adjuvant therapy (52.1% vs. 12.8%, P<0.001). CONCLUSION:DMI, CSI, and elevated CA125 are independent risk factors for LNM in LGEC. The high sensitivity and NPV of the combined predictive model support a selective lymphadenectomy strategy to avoid overtreatment in low-risk patients, while DMI also informs surgical approach selection and adjuvant therapy decisions.

Background Cholestatic liver diseases, such as primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), and biliary atresia (BA), are characterized by bile accumulation and frequently progress to liver fibrosis, cirrhosis, and organ failure. Objective Given its well-documented hepatoprotective and anti-inflammatory properties, this study aimed to investigate the therapeutic potential and underlying mechanisms of gastrodin (4-(hydroxymethyl) phenyl β-D-glucopyranoside, C13H18O7), a primary bioactive compound from Gastrodia elata, for the treatment of cholestatic liver fibrosis. Methods Two established mouse models of cholestatic fibrosis were used: the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet model and the bile duct ligation (BDL) model. An integrated analysis of single-nucleus RNA sequencing (snRNA-seq) data was performed, combining an in-house dataset from the DDC model with a public dataset. Results Gastrodin treatment significantly alleviated liver injury, reduced serum levels of total bilirubin (TBIL) and total bile acids (TBA), decreased collagen deposition, and prolonged survival in mouse models. snRNA-seq analysis revealed that gastrodin promoted hepatocyte repair by regulating key sulfotransferases (Sult2a1 and Sult1e1), enhanced detoxification, reduced the proportion of Kupffer cells, and suppressed their production of inflammatory mediators (e.g., TNF-α, IL-6), potentially via upregulation of immunomodulatory genes (Pilrb1, Ifi27l2a, Rtp4). Furthermore, gastrodin modulated hepatic stellate cell (HSC) heterogeneity by expanding a novel HSC subgroup characterized by Serpina expression and associated with anti-fibrotic traits. Conclusion Gastrodin alleviates cholestatic liver fibrosis through coordinated multi-cellular mechanisms involving hepatocytes, Kupffer cells, and HSCs. It represents a promising natural therapeutic candidate, with Sult2a1, Sult1e1, and Serpina1a identified as potential targets.

Background: The immunologically cold nature and immunosuppressive tumor microenvironment (TME) of intrahepatic cholangiocarcinoma (ICC) contribute to its poor prognosis. This study aims to identify novel biomarkers related to prognosis and TME in ICC. Methods: We first identified the high expression of m6A reader insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) in ICC through bioinformatics screening. Subsequently, a retrospective study was conducted on 224 ICC patients who had undergone radical resection. The expression levels of IGF2BP2 and programmed death ligand 1 (PD-L1) were detected in a tissue microarray (TMA) using immunohistochemistry (IHC). The co-localization of IGF2BP2, PD-L1, programmed cell death protein 1 (PD-1), and CD8+T cells was evaluated by multiple immunofluorescence techniques. Results: IHC confirmed a significant upregulation of IGF2BP2 in tumor tissues compared with normal bile duct epithelia (p < 0.05). IGF2BP2 expression was positively correlated with PD-L1 expression (TPS R = 0.215, p = 0.016; CPS R = 0.295, p = 0.008). High IGF2BP2 expression was associated with increased PD-L1/PD-1 positivity and reduced CD8+T cell infiltration. Kaplan-Meier analysis revealed significantly worse 3-year overall survival (OS: 20.56% vs. 29.91%, p = 0.0291) and recurrence-free survival (RFS: 9.72% vs. 18.56%, p = 0.0372) in the IGF2BP2-high group. Multivariate analysis identified IGF2BP2 as an independent risk factor for both OS (HR = 1.683, p = 0.044) and RFS (HR = 1.946, p = 0.042). Conclusions: IGF2BP2, as a potential biomarker and independent prognostic factor for ICC, is associated with increased PD-L1 expression.

INTRODUCTION:Peptidyl arginine deiminase 4 (PAD4) catalyzes the deamination of arginine residues to citrulline, a post-translational modification known as citrullination, which regulates protein structure, function, and localization. Despite growing evidence connects PAD4 to cancer progression, its role in intrahepatic cholangiocarcinoma (ICC) remains largely unexplored. OBJECTIVES:This study aimed to elucidate the role and underlying mechanisms of PAD4 in the regulation of intrahepatic cholangiocarcinoma proliferation, as well as to explore its clinical relevance and therapeutic potential. METHODS:Hydrodynamic tail vein injection (HTVI) and subcutaneous xenograft models were used to evaluate the role of PAD4 in vivo. In vitro functional assays were performed to assess the effects of PAD4 on ICC cell proliferation. Putative targets were identified by RNA-seq and Co-IP/MS analyses. Molecular interactions were examined using GST pull-down, surface plasmon resonance (SPR), and RNA immunoprecipitation-PCR. The clinical relevance of PAD4 was evaluated in ICC patient samples using immunohistochemical analysis. RESULTS:PAD4 was identified as a key promoter of ICC proliferation by enhancing post-transcriptional expression of the MCM complex. Mechanistically, PAD catalytic domain of PAD4 interacted with the KH1 domain of IGF2BP2 and catalyzed its citrullination at R597, which markedly increased the affinity of IGF2BP2 for m6A-modified MCM2-7 transcripts, thereby stabilizing and elevating MCM mRNAs expression. Clinically, high PAD4 expression correlated with poor prognosis in ICC patients, while PAD4 and IGF2BP2 co-expression predicted worse outcomes. In vivo, combined inhibition of PAD4 and IGF2BP2 synergistically suppressed ICC growth. CONCLUSION:Our findings identify a tumor-intrinsic PAD4-IGF2BP2 axis that drives ICC progression through citrullination-dependent stabilization of MCM transcripts. Dual targeting of PAD4 and IGF2BP2 represents a promising therapeutic strategy for ICC.

BACKGROUND:Acquired lymphedema is a global health concern with limited treatment options. While vascular endothelial growth factor C (VEGF-C) administration has shown promise for the treatment of this patient population, no small-molecule compounds have hitherto been identified to improve lymphedema by stimulating VEGF-C expression and lymphangiogenesis. OBJECTIVE:This study investigated the therapeutic effect of notoginsenoside R1 (R1) on a mouse model of tail acquired lymphedema and explored the underlying mechanisms. METHODS:C57BL/6J mice and lymphatic endothelial cells (LECs) specific VEGFR-3 knockout transgenic mice underwent surgical induction of tail acquired lymphedema. Tail circumference, lymphatic drainage function, VEGF-C expression, and lymphangiogenesis were measured. LECs' function was assessed using wound healing and tube formation assays. Quantitative PCR (q-PCR) and western blot were conducted to measure VEGF-C expression levels. In addition, RNA sequencing analysis and western blot were performed to elucidate the signal pathways involved. Luciferase reporter assays assessed VEGF-C promoter activity. RESULTS:R1 treatment improved lymphedema, lymphatic function, and lymphangiogenesis in the mouse model. R1 enhanced migration, tube formation, and VEGF-C expression of LECs. These effects were abolished by VEGF-C siRNA and VEGFR-3 inhibitors. VEGFR3 knockout in LECs completely blocked R1's ability to promote lymphangiogenesis and lymphatic drainage while partially but significantly reducing its improvement on lymphedema. R1 activated the cAMP/PKA signaling pathway, leading to PKA and CREB phosphorylation. The PKA inhibitor and CREB siRNA inhibited R1-induced VEGF-C expression. Additionally, R1 activated VEGF-C promoter activity in a CREB-dependent manner. CONCLUSION:R1 emerges as the first reported small natural compound to promote VEGF-C expression. It reduces acquired lymphedema and enhances lymphangiogenesis via the cAMP/PKA/CREB signaling pathway. These findings suggest R1 as a potential novel oral medication for treating acquired lymphedema patients.

Previous studies have identified B cell subpopulations with pro- and anti-tumoral activities, while the clinical relevance of B cell subpopulations-specific markers in pan-cancer remains understudied. We integrated 14 scRNA-seq datasets (102,504 cells from 424 patients, 15 cancer types) to identify B cell subpopulations via unsupervised clustering. We characterized their functional dynamics and prognostic relevance through analyzing single-cell, bulk and spatial transcriptomic data. Moreover, using B cell subpopulations-specific gene signatures, we constructed models for predicting cancer prognosis and immunotherapy response. We identified eight B cell subpopulations (b00-b07) which were classified into naive, plasma, memory, germinal center (GC), and cycling B cells. Trajectory analysis revealed b02-naive and b04-GC cells in early phases, evolving into b01- and b03-plasma/b05- and b06-memory/b07-cycling and b05-memory subpopulations. Anti-tumor responses were activated in early pseudotime, complement/immunoglobulin pathways peaked in mid-pseudotime, and energy metabolism increased in late-pseudotime. The enrichment of b07-cycling and b04-GC was negatively correlated with cancer prognosis, while b02-naive had a positive correlation. Spatial transcriptomic analysis showed clustered b00-b06 versus dispersed b07 cells, with b04-GC and b07-cycling cells distant from tertiary lymphoid structure cores. Based on the expression profiles of 1,047 B cell subpopulations-specific signatures, we identified three pan-cancer subtypes with distinct clinical and molecular characteristics. Using 13 B cell subpopulations-specific signatures, we constructed models to accurately predict cancer survival outcomes and immunotherapy response. Our study delineates eight B cell subpopulations with distinct prognostic relevance. Signature-based stratification and models underscore their clinical relevance in cancer outcomes and therapy response, advancing understanding of B cell heterogeneity in cancer.

BACKGROUND & AIMS:Liver fibrosis is an inevitable outcome of various manifestations of progressive chronic liver disease. Clinical evidence suggests that gut microbiota contributes to the progression of liver fibrosis via gut-liver axis. Our previous research demonstrated that GL-V9, one of flavonoids compounds, is effective in alleviating liver fibrosis in mice. Based on that, we decided to delve into the alterations in gut microbiota profiling of GL-V9-treated mice with liver fibrosis and identify the bacterial species exhibiting anti-fibrotic potential. METHODS:We explored gut microbial profile of GL-V9-treated mice with liver fibrosis via 16S rRNA gene sequencing. Mouse models of liver fibrosis were established and treated accordingly. Then, indexes of liver fibrosis and inflammation were measured by sections staining, biochemical detection and RT-qPCR. Regulatory T (Treg) cells infiltration was detected by flow cytometry. Furthermore, we detected short-chain fatty acids (SCFAs) content by GC-MS and explored the role of SCFAs in liver fibrosis. RESULTS:Firstly, Bacteroides acidifaciens were screened out by 16S rRNA gene sequencing based on GL-V9 treatment. Oral administration of Bacteroides acidifaciens attenuated CCl4-induced liver fibrosis effectively, reduced the expressions of pro-inflammatory factor and suppressed the infiltration of Treg cells. Additionally, SCFAs, the main metabolites of Bacteroides acidifaciens, were further demonstrated the suppression of fibrosis and inflammation. Mechanistically, Bacteroides acidifaciens regulated SCFAs metabolic homeostasis and subsequently inhibited Treg cells infiltration via regulating PI3K/AKT/mTOR pathway. CONCLUSIONS:In CCl4-induced liver fibrosis mice, Bacteroides acidifaciens and its derived SCFAs exhibit excellent anti-fibrosis effects, which provides a new strategy for liver fibrosis treatment.

Cardiac hypertrophy is an independent risk factor and prognosis indicator of heart failure. Early intervention of cardiac hypertrophy is crucial to prevent heart failure and improve patients' outcomes. Despite evidence that activation of AMPK (adenosine monophosphate-activated protein kinase) plays a protective role in cardiac hypertrophy, whether it plays a sustained role and the precise mechanism remains unexplored. We established in vivo model of cardiac hypertrophy by coarctation of rat abdominal aorta (AAC-CH model). SGLT2 inhibitor (SGLT2i) was used to activate AMPK and cardiac function was evaluated after 2, 4, 8, 12 weeks. Animals were killed, and cardiac tissue was examined for morphological changes, fibrosis, and ferroptosis. At 2 weeks, rats already had histopathological abnormalities including enlarged cardiomyocytes, cardiac fibrosis, and ferroptosis, which persisted overtime. However, these changes were remarkably prevented by the treatment of SGLT2i. Then, we established in vitro model of cardiac hypertrophy by treating H9C2 cells with isoproterenol (ISO,10 µM). Unexpectedly, mechanistic studies revealed that antagonism of AMPK aggravated oxidative stress and ferroptosis, reduced GPX4 (glutathione peroxidase 4) level, and partially abolished the anti-hypertrophic and anti-ferroptosis effects of SGLT2i in H9C2 cells. Taken together, the regulatory role between AMPK and ferroptosis was revealed for the first time in cardiac hypertrophy. SGLT2i counteracts ferroptosis by activating AMPK, providing a sustained protection against cardiac hypertrophy. This positions SGLT2i as a potential therapeutic agent for the treatment of cardiac hypertrophy. Besides, in addition to the downregulation of AMPK in hypertrophic heart tissue, its levels are also reduced in plasma, suggesting its potential to serve as a diagnostic marker for the early detection of ferroptosis and cardiac hypertrophy.

MiR-646, a small non-coding RNA, poorly expressed in a variety of tumors. This study aimed to clarify the role of miR-646 and its underlying mechanisms in glioblastoma (GBM). In our study, we found that miR-646 mRNA levels were lower in tumor tissues than in non-cancer tissues. The ability of glioma cells to proliferate, invade, and migrate is diminished by miR-646 overexpression in vitro and in vivo. Mechanistically, miR-646 targeted sequestosome 1 (p62) in the 3'UTR and affected the Keap1/Nrf2 pathway, thus attenuating the expression of the HO-1 gene. In conclusion, this study provided a novel finding that miR-646 tampered with gliomagenesis by regulating the p62/Keap1/Nrf2 axis, which provides a potential target for GBM therapy.

Aldo-keto reductase 1C3 (AKR1C3) plays a key role in tumor progression and chemotherapy resistance, particularly in sorafenib-resistant hepatocellular carcinoma (HCC). Targeting AKR1C3 represents a promising strategy to restore chemosensitivity in resistant HCC. Previous research identified the lead compound S07-2005 through a cascade virtual screening approach (AKR1C3 IC50 = 130 ± 30 nM, SI (selective index) > 77). Using cocrystal-guided drug design, 30 was optimized to adopt an "L"-shaped conformation targeting AKR1C3's subpocket 1 (SP1) and oxyanion site (OS), enhancing inhibitory potency and selectivity (AKR1C3 IC50 = 5 ± 1 nM, SI > 2000). It enhanced sorafenib-induced ROS generation, promoted apoptosis, and restored sorafenib sensitivity in HCC models. In combination with sorafenib, compound 30 restored sorafenib sensitivity in HCC both in vitro and in vivo. Additionally, compound 30 demonstrated a favorable safety profile and pharmacokinetic properties, suggesting its potential as an adjunct to overcome AKR1C3-mediated chemotherapy resistance in cancer treatment.

BACKGROUND:Glioma is the most prevalent and lethal tumor of the central nervous system. Routine treatment with temozolomide (TMZ) would unfortunately result in inevitable recurrence and therapy resistance, severely limiting therapeutic efficacy. Tumor-associated astrocytes (TAAs) are key components of the tumor microenvironment and increasing evidence has demonstrated that aberrant expression of connexin43 (Cx43) was closely associated with glioma progression and TMZ resistance. However, the specific role of Cx43 in mediating TMZ resistance through glioma and astrocyte interactions has not been fully explored. METHODS:The expression and prognostic value of Cx43 were evaluated in tumor samples and clinical databases. ShRNA-medicated knockdown and Gfap-Cre Cx43flox/flox gene mouse were used to assess the role and functional significance of Cx43 in vitro and in vivo. Moreover, we performed mass spectrometry analysis, chromatin immunoprecipitation, and other biochemical assays to define the molecular mechanisms by which Cx43 promotes TMZ resistance. RESULTS:We confirmed that the upregulation of Cx43 expression between TAAs and glioma cells contributed to TMZ resistance and tumor recurrence. Genetic knockdown or pharmacological inhibition of Cx43 enhanced TMZ-induced cytotoxicity. Mechanistically, elevated Cx43 expression induced β-catenin accumulation at the cell surface of glioma cells, suppressing T-cell factor/lymphoid enhancer-binding factor transcription. This led to impaired miR-205-5p expression and subsequent activation of the E2F1/ERCC1 axis, which eventually led to chemoresistance. CONCLUSIONS:Our study reveals a novel regulatory mechanism in which the Cx43/miR-205-5p/E2F1/ERCC1 axis contributes to TMZ resistance in glioma. These findings further highlight the potential of targeting Cx43 as a therapeutic strategy in glioma.

In our previous study, a chemical derivatization reagent named 5-(dimethylamino) naphthalene-1-sulfonyl piperazine (Dns-PP) was developed to enhance the chromatographic retention and the mass spectrometric response of free fatty acids (FFAs) in reversed-phase liquid chromatography coupled with electrospray ionization-mass spectrometry (RPLC-ESI-MS). However, Dns-PP exhibited strong preferences for long-chain FFAs, with limited improvement for short- or medium-chain FFAs. In this study, a new series of labeling reagents targeting FFAs were designed, synthesized, and evaluated. Among these reagents, Tmt-PP (N2, N2, N4, N4-tetramethyl-6-(4-(piperazin-1-ylsulfonyl) phenyl)-1,3,5-triazine-2,4-diamine) exhibited the best MS response and was selected for further evaluations. We compared Tmt-PP with Dns-PP and four commonly used carboxyl labeling reagents from existing studies, demonstrating the advantages of Tmt-PP. Further comparisons between Tmt-PP and Dns-PP in measuring FFAs from biological samples revealed that Tmt-PP labeling enhanced the MS response for about 80% (30/38) of the measured FFAs, particularly for short- and medium-chain FFAs. Moreover, Tmt-PP labeling significantly improved the chromatographic retention of short-chain FFAs. To ensure accurate quantification, we developed a stable isotope-labeled Tmt-PP (i.e., d12-Tmt-PP) to react with chemical standards and serve as one-to-one internal standards (IS). The method was validated for accuracy, precision, sensitivity, linearity, stability, extraction efficiency, as well as matrix effect. Overall, this study introduced a new chemical derivatization reagent Tmt-PP (d12-Tmt-PP), providing a sensitive and accurate option for quantifying FFAs in biological samples.

Osteosarcoma is the most common primary malignant bone tumor in adolescents. While treatments for osteosarcoma have improved, the overall survival has not changed for three decades, and thus, new targets for therapeutic development are needed. Recently, glucocorticoids have been reported to have antitumor effects. Mometasone furoate (MF), a synthetic glucocorticoid, is of great value in clinical application, but there are few reports on its antitumor effect. Here, we verified the effect of MF on osteosarcoma in vitro and in vivo. In vitro, cell proliferation, cell cycle progression, apoptosis and cell metastasis were detected using Cell Counting Kit-8 (CCK-8), colony formation, flow cytometry, wound-healing and transwell assays, respectively. In vivo, we generated a xenograft mouse model. To examine the potential role of the AMPK pathway, an AMPK-specific inhibitor (dorsomorphin) was used. The expression levels of factors related to the cell cycle, apoptosis and activation of the AMPK/mTOR pathway were assessed by immunohistochemistry and Western blotting. MF inhibited proliferation and metastasis and induced S phase arrest and apoptosis in osteosarcoma cells in a dose-dependent manner. In vivo, MF effectively inhibited osteosarcoma cell growth and pulmonary metastasis; however, it had no negative effect on the internal organs. Additionally, MF could activate the AMPK/mTOR pathway in osteosarcoma. Dorsomorphin significantly attenuated MF-induced antitumor activities. In summary, MF can inhibit osteosarcoma proliferation and metastasis and promote osteosarcoma cell apoptosis through the AMPK/mTOR signaling pathway in vitro and in vivo, which can provide a new rationale for subsequent academic and clinical research on osteosarcoma treatment.

Acquired drug resistance is one of the most common limitations for the clinical response of colon cancer to 5-Fluorouracil (5-FU)-based chemotherapy. The relevant molecular mechanisms might be diversity, but still not be elucidated clearly. In this study, we aimed to investigate the potential mechanisms of c-Fos, a subfamily of activator protein-1, in 5-FU chemoresistance. We determined that phosphorylated c-Fos promoted colon cancer cells resistance to 5-FU by facilitating the cancer stemness. Mechanically, 5-FU treatment induced autolysosome-dependent degradation of TMPO, which subsequently triggered ERK-mediated phosphorylation of c-Fos. Additionally, c-Fos was found to bind to the promoter of NANOG and phosphorylation of c-Fos at Ser 374 was required for its regulation of NANOG expression. NANOG ablation impaired c-Fos/p-c-Fos induced 5-FU resistance and stemness. Taken together, these findings revealed that TMPO-mediated phosphorylation of c-Fos conferred 5-FU resistance by regulating NANOG expression and promoting cell stemness in colon cancer cells. c-Fos could be as a therapeutic target for colon cancer.

BackgroundOsteosarcoma is the most common malignant bone tumor in children and adolescents. Conventional chemotherapy remains unsatisfactory due to drug toxicity and resistance issues. Therefore, there is an urgent need to develop more effective treatments for advanced osteosarcoma. In the current study, we focused on evaluating the anticancer efficacy of avermectin B1, a novel avermectin analog, against osteosarcoma cells.MethodsThe half-inhibitory concentration of avermectin B1 was calculated in three osteosarcoma cell lines. Then, functional experiments were conducted to evaluate the effects of avermectin B1 on cell proliferation, the cell cycle, apoptosis and autophagy. Moreover, the AMPK/ULK1 signaling pathway was detected by Western blot assay. Finally, the in vivo effect of avermectin B1 on tumor growth and metastasis was investigated using the xenograft mouse model. To examine the role of the AMPK/ULK1 pathway, an AMPK-specific inhibitor (dorsomorphin) was used in combination with avermectin B1.ResultsAvermectin B1 inhibited the proliferation of osteosarcoma cells in a dose-dependent manner based on CCK8 and colony formation assays. Then, it was found to inhibit migration and invasion by wound healing assay and cell migration and invasion assay. In addition, avermectin B1 induced osteosarcoma cell apoptosis and autophagy. In vivo, avermectin B1 effectively inhibited osteosarcoma cell growth and pulmonary metastasis. Mechanistically, avermectin B1 activated the AMPK/ULK1 pathway to exert antitumor activity in vitro and in vivo. Dorsomorphin significantly attenuated the Avermectin B1-induced antitumor activities.ConclusionOur study suggests that avermectin B1 is a potential agent to treat osteosarcoma cells through the AMPK/ULK1 signaling pathway.

PurposeBecause only a subset of cancer patients can benefit from immunotherapy, identifying predictive biomarkers of ICI therapy response is of utmost importance.MethodsWe analyzed the association between hemoglobin (HGB) levels and clinical outcomes in 1,479 ICIs-treated patients across 16 cancer types. We explored the dose-dependent associations between HGB levels and survival and immunotherapy response using the spline-based cox regression analysis. Furthermore, we investigated the associations across subgroups of patients with different clinicopathological characteristics, treatment programs and cancer types using the bootstrap resampling method.ResultsHGB levels correlated positively with clinical outcomes in cancer patients receiving immunotherapy but not in those without immunotherapy. Moreover, this association was independent of other clinicopathological characteristics (such as sex, age, tumor stage and tumor mutation burden (TMB)), treatment program and cancer type. Also, this association was independent of the established biomarkers of immunotherapy response, including TMB, PD-L1 expression and microsatellite instability. The combination of TMB and HGB level are more powerful in predicting immunotherapy response than TMB alone. Multi-omics analysis showed that HGB levels correlated positively with antitumor immune signatures and negatively with tumor properties directing antitumor immunosuppression, such as homologous recombination defect, stemness and intratumor heterogeneity.ConclusionThe HGB measure has the potential clinical value as a novel biomarker of immunotherapy response that is easily accessible from clinically routine examination. The combination of TMB and HGB measures have better predictive performance for immunotherapy response than TMB.

The human endometrium is a highly regenerative tissue capable of undergoing scarless repair during the menstruation and postpartum phases. This process is mediated by endometrial adult stem/progenitor cells. During the healing of endometrial injuries, swift reepithelization results in the rapid covering of the wound surface and facilitates subsequent endometrial restoration. The involvement of endogenous endometrial epithelial stem cells, stromal cells, and bone marrow-derived cells in the regeneration of the endometrial epithelium has been a subject of prolonged debate. Increasing evidence suggests that the regeneration of the endometrial epithelium mainly relies on epithelial stem cells rather than stromal cells and bone marrow-derived cells. Currently, no consensus has been established on the identity of epithelial stem cells in the epithelial compartment. Several markers, including stage-specific embryonic antigen-1 (SSEA-1), sex-determining region Y-box 9 (SOX9), neural-cadherin (N-cadherin), leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5), CD44, axis inhibition protein 2 (Axin2), and aldehyde dehydrogenase 1A1 (ALDH1A1), have been suggested as potential candidate markers for endometrial epithelial stem cells. The identification of endometrial epithelial stem cells contributes to our understanding of endometrial regeneration and offers new therapeutic insights into diseases characterized by regenerative defects in the endometrium, such as intrauterine adhesion. This review explores different perspectives on the origins of human and mouse endometrial epithelial cells. It summarizes the potential markers, locations, and hierarchies of epithelial stem cells in both human and mouse endometrium. It also discusses epithelial cell-based treatments for intrauterine adhesion, hoping to inspire further research and clinical application of endometrial epithelial stem cells.

Philadelphia chromosome-positive (Ph + ) leukemia is a fatal hematological malignancy. Although standard treatments with tyrosine kinase inhibitors (TKIs) have achieved remarkable success in prolonging patient survival, intolerance, relapse, and TKI resistance remain serious issues for patients with Ph + leukemia. Here, we report a new leukemogenic process in which RAPSYN and BCR-ABL co-occur in Ph + leukemia, and RAPSYN mediates the neddylation of BCR-ABL. Consequently, neddylated BCR-ABL enhances the stability by competing its c-CBL-mediated degradation. Furthermore, SRC phosphorylates RAPSYN to activate its NEDD8 E3 ligase activity, promoting BCR-ABL stabilization and disease progression. Moreover, in contrast to in vivo ineffectiveness of PROTAC-based degraders, depletion of RAPSYN expression, or its ligase activity decreased BCR-ABL stability and, in turn, inhibited tumor formation and growth. Collectively, these findings represent an alternative to tyrosine kinase activity for the oncoprotein and leukemogenic cells and generate a rationale of targeting RAPSYN-mediated BCR-ABL neddylation for the treatment of Ph + leukemia.

Antiangiogenesis therapy has become a hot field in cancer research. Given that tumor blood vessels often express specific markers related to angiogenesis, the study of these heterogeneous molecules in different tumor vessels holds promise for advancing anti-angiogenic therapy. Previously using phage display technology, we identified a targeting peptide named GX1 homing to gastric cancer vessels for the first time. However, GX1 also showed some non-specific binding with normal gastric vessels, which can lead to toxic side effects on normal endothelial cells. Therefore, we urgently need to adopt new screening strategies to avoid non-specific binding to normal vessels and obtain gastric cancer vascular targeting peptides with higher specificity. In this study, we designed a new strategy which combined “positive screening” in vivo and “negative screening” in vitro for the first time. An in vivo positive screening was conducted using tumor bearing nude mice to identify peptides that were specifically enriched within the vasculature of gastric cancer. Concurrently, an in vitro negative screening process was conducted on normal vasculature endothelial cells, including human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMVECs), to eliminate peptides binding to normal vasculature. After four rounds of iterative screening, a targeting peptide specifically targeting gastric cancer vasculature was obtained. In addition, an in vitro co-culture model by culturing HUVEC in tumor conditioned medium (Co-HUVEC) was established to investigate the affinity of these peptides. The targeting peptide was labeled with fluorescein isothiocyanate (FITC) for competitive and inhibitory assays. Blood vessel density analysis confirmed redundant capillary vessels in the xenografts, indicating that the mouse model was suitable for positive screening. Following four rounds of panning, a significant enrichment for phages specifically binding to gastric cancer vasculature was observed, with minimal binding to normal endothelial cells. The peptide CNTGSPYEC exhibited the highest reproducibility. In vitro immunofluorescence staining confirmed that the peptide CNTGSPYEC could specifically enrich in Co-HUVECs while showing no binding to normal vascular endothelial cells. In vivo immunofluorescence staining revealed that the peptide CNTGSPYEC could only bind to vascular endothelial cells specifically in gastric cancer but show no non-specific binding with normal tissue. Competitive and inhibitory assay also verified the targeting characteristics of the peptide with the fluorescence intensity of 17.13. As the concentration increases, the competitive inhibition rate can be incrementally raised to 93