Xenograft Assay Research Articles

METTL3 mediates m6A methylation modification of ULBP2 and affects the progression of cervical cancer

BackgroundCervical cancer (CC) is one of the most prevalent malignancies in women, posing a significant challenge globally. However, the precise molecular mechanism regulating CC progression through methyltransferase-like protein 3 (METTL3) and UL16 Binding Protein 2 (ULBP2) remains largely unknown.MethodsBioinformatic analysis was used to identify the effect of ULBP2 expression in CC tissues. RT-qPCR and western blotting were employed to assess the mRNA and protein expression in CC cells and tissues. Methylthiazolyldiphenyl-tetrazolium bromide (MTT), 5‑Ethynyl‑2’‑deoxyuridine (EdU), wound healing, and transwell assays were utilized to estimate cell viability, proliferation, and metastasis, respectively. Cell apoptosis was detected by flow cytometry. CC cells were treated with different doses of radiotherapy. The m6A level was measured using methylated RNA immunoprecipitation (MeRIP) assay. A xenograft assay was conducted to further verify the roles of ULBP2 in CC.ResultsULBP2 was upregulated in CC. Downregulation of ULBP2 restrained the proliferation, metastasis and radiotherapy resistance of CC cells. METTL3 regulated m6A methylation modification of ULBP2. Insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) promoted m6A methylation modification of ULBP2. METTL3 influenced the expression of ULBP2 and impacted the biological function of the CC cells. Silencing ULBP2 reduced the radioresistance of CC in vivo. Radiotherapy altered the gut microbiota in CC patients.ConclusionMETTL3 modulated the m6A methylation of ULBP2, affecting the oncogenic properties and radioresistance of CC cells.

RNFT2 promotes malignancy of triple-negative breast cancer and predicts poor outcomes.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer, and has a high recurrence rate. RING finger transmembrane-domain containing protein 2 (RNFT2) is a RING-finger E3 ubiquitin ligase that exerts oncogene functions in multiple malignant tumors such as bladder cancer and gastric cancer. However, RNFT2's role in TNBC is still unclear. Here, we investigated RNFT2's function and mechanism in TNBC. RNFT2 expressions in different stages of breast cancer were evaluated using the Gene Expression Profiling Interactive Analysis database. Overall survival (OS) in TNBC patients with high RNFT2 expressions was assessed with the Kaplan-Meier plotter database. Meanwhile, RNFT2 expressions in TNBC cells and tissues were determined using Western blot and quantitative real-time PCR. The relationship between RNFT2 and TNBC patients' OS rates was examined with Kaplan-Meier curve analysis. The correlation between RNFT2 expressions and TNBC clinicopathological data was estimated by the chi-square test. Moreover, RNFT2 functions in TNBC were determined using loss-of-function assay, Cell Counting Kit-8 analysis, Transwell, and tube formation assay. Furthermore, RNFT2's mechanism in TNBC was evaluated by prediction software, dual-luciferase reporter assay, and rescue experiments. Additionally, RNFT2's roles in TNBC in vivo were identified with cell-derived xenograft, hematoxylin-eosin staining, and immunohistochemical assays. RNFT2 was elevated in breast cancer, and owned different degrees of overexpression in breast cancer at different stages. Meanwhile, OS of TNBC patients with high RNFT2 expressions was poor. Also, RNFT2 expressions were positively correlated with size, TNM stage, and lymph node metastasis of TNBC. Functionally, silencing RNFT2 repressed TNBC cell proliferation, invasion, and angiogenesis. Mechanistically, miR-211-5p targeted RNFT2, and RNFT2 was negatively regulated by miR-211-5p in TNBC cells. Rescue assays further validated that miR-211-5p overexpression restrained TNBC cell proliferation, invasion, and angiogenesis, yet these impacts were abolished after RNFT2 overexpression. Meanwhile, animal experimental data further implied that RNFT2 knockdown reduced TNBC cell proliferation in vivo. RNFT2 facilitated TNBC development and predicted its adverse outcomes.

Epigenetic agents, zebularine and valproic acid, inhibit the growth of the oral squamous cell carcinoma cell line HSC4 in vitro and in vivo

ObjectivesThis study explored the potential of Zebularine (Zeb), a DNA methyltransferase inhibitor (DNMTi), and Valproic acid (Vpa), a histone deacetylase inhibitor (HDACi), as a combined treatment strategy for OSCC.Materials and methodsOSCC cell lines, HSC4 (well-differentiated type) and SAS (poorly differentiated type), were cultured and treated with Zeb, Vpa, and their combinations. Cell viability, mRNA expression of P16, P21, NPY, and RASSF1 using quantitative reverse transcription polymerase chain reaction (qRT-PCR), DNA methylation using methylation-specific PCR (qMSP), and in situ HDAC activity were analyzed in vitro. In vivo, a xenograft tumor formation assay was conducted using male BALB/Slc-nu nude mice, in accordance with the Basel Declaration and The ARRIVE guidelines 2.0. Tumor samples were analyzed by qRT-PCR, and qMSP.ResultsIn vitro experiments using the HSC4 and SAS cell lines revealed significant cytotoxic effects and upregulation of tumor suppressor genes (P16, P21, NPY, and RASSF1) after treatment with Zeb + Vpa. In vivo xenograft assay in nude mice treated with Zeb + Vpa demonstrated reduced tumor volume in HSC4 cell-transplanted tumors without significant adverse effects on the body weights of the mice, whereas no significant reduction in tumor size was observed in SAS cell-transplanted tumors compared with controls. Molecular analysis confirmed elevated gene expression levels and reduced DNA methylation percentages in the treated tumors, with a more pronounced effect in HSC4 compared to SAS.ConclusionsThese findings suggest that the combination of Zeb and Vpa holds promise as an effective and low-toxicity therapeutic strategy for well-differentiated type of OSCC.

LMO2 confers value as a potential immunotherapy marker in pan-cancer analysis and inhibits progression of Clear Cell Renal Cell Carcinoma.

LMO2 confers value as a potential immunotherapy marker in pan-cancer analysis and inhibits progression of Clear Cell Renal Cell Carcinoma.

ARID1A deficiency promotes malignant proliferation of hepatocellular carcinoma by activating HDAC7/ENO1 signaling pathway

Background:Epigenetic dysregulation constitutes one of the mechanisms in the pathogenesis of HCC, thereby underscoring its critical scientific importance for early diagnosis and therapeutic interventions. The chromatin remodeling factor AT-rich interaction domain 1A (ARID1A) serves as an essential epigenetic regulator and is frequently subject to inactivating mutations across various cancer types. Nevertheless, the precise molecular pathways by which ARID1A deficiency facilitates the progression of HCC remain inadequately elucidated.Methods:RNA sequencing (RNA-seq) was employed to identify genes that were significantly upregulated following the knockdown of ARID1A. Immunohistochemistry was used to assess the expression levels of ARID1A and histone deacetylase 7 (HDAC7) in HCC samples. Chromatin immunoprecipitation and luciferase reporter assays were conducted to validate PU.1 as a positive transcriptional regulator of HDAC7. Protein immunoprecipitation coupled with mass spectrometry was used to identify potential substrates of HDAC7. A xenograft assay was performed to evaluate the therapeutic potential of HDAC7 inhibitors in ARID1A-deficient HCC.Results:Our investigation demonstrated that the loss of ARID1A in HCC cells led to an upregulation of HDAC7 expression. Mechanistic analyses revealed that ARID1A deficiency facilitated PU.1-mediated transcriptional regulation of HDAC7. Additionally, the overexpression of HDAC7 resulted in a reduced acetylation level of Enolase 1 (ENO1), thereby enhancing the malignant proliferation of HCC cells. Targeting HDAC7 effectively inhibited the growth of ARID1A-deficient tumors in tumor-bearing mice.Conclusions:This study provides novel insights into the regulatory interplay between chromatin remodeling and acetylation modification, 2 pivotal epigenetic processes influencing tumorigenesis. It also suggests that pharmacological inhibition of HDAC7 offers a promising therapeutic strategy for treating HCC with ARID1A mutations.

MFAP2 promotes the progress of esophageal squamous cell carcinoma by enhancing PTGS2 signaling

Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent malignancies, accounting for over 85% of all esophageal cancers worldwide and over 90% in China. Due to the absence of effective early diagnostic tools and therapeutic approaches, the 5-year survival rate remains below 30%. Thus, it is crucial to further investigate the molecular mechanisms underlying ESCC. By analyzing differentially expressed genes in esophageal adenocarcinoma (EAC) and ESCC, we identified 127 genes that were significantly upregulated in ESCC and enriched in extracellular matrix organization. Notably, MFAP2 (microfibril associated protein 2), a matrix-related molecule with unclear function, was found to be highly expressed in ESCC in both the TCGA database and our RNA sequencing data. Its elevated levels were associated with cancer progression. Western blot, immunofluorescence, and immunohistochemistry revealed that MFAP2 protein was highly expressed in ESCC and predominantly distributed in the extracellular matrix, cytoplasm, and partially in the nucleus. In vitro functional experiments demonstrated that overexpressing MFAP2 had no significant effect on cell proliferation but inhibited cell migration and invasion. In vivo xenograft assays showed that MFAP2 enhanced the growth of the KYSE-450 cancer cell line, though no statistical difference was observed in KYSE-140. Bioinformatics analysis revealed a positive correlation between MFAP2 expression and anti-tumor (M1 type) macrophages in EAC tissues, whereas in ESCC tissues, MFAP2 correlated positively with non-activated (M0 type) macrophages. RNA sequencing indicated that MFAP2 is involved in immune pathways and can promote PTGS2 expression. Collectively, this study preliminarily evaluates the function and potential molecular mechanism of MFAP2 in ESCC, offering new therapeutic targets and ideas for ESCC treatment.

Abstract PS13-07: Enhancing T-DXd Efficacy in HER2-positive Breast Cancer Resistant to HER2 ADC by Non-biased Kinase-related Target Screening

Abstract Background: Anti-HER2 antibody-drug conjugate (HER2-ADC) therapies, such as trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd), significantly prolong survival in patients with HER2-positive metastatic breast cancer (BC) compared to physician’s choice of chemotherapy with trastuzumab (The EMILIA, TH3RESA, and DESTINY-Breast clinical trials). Intrinsic or acquired resistance to HER2-ADC therapies remains a substantial clinical challenge since there is no established standard of care following progression on T-DXd. This study aimed to elucidate novel therapeutic targets that can overcome resistance to HER2-ADC therapies using unbiased approaches with synthetic lethal kinome RNA interference screening. Methods: We conducted targeted DNA sequencing (n=15) and whole transcriptome sequencing (n=11) to investigate genetic aberrations following anti-HER2 and/or HER2-ADC therapy (T-DM1 or T-DXd) in BC patient tissue samples. We generated HER2-positive human BC cell lines resistant to T-DM1 or T-DXd. To determine the effect of the T-DM1 and T-DXd on the ERBB2 gene and HER2 protein expression, we used fluorescence in situ hybridization, droplet digital PCR, and Western blotting assay. To discern resistance mechanisms of T-DXd and identify kinase targets whose inhibition might synergistically enhance the efficacy of T-DXd, we conducted whole-genome sequencing, cDNA microarray analysis, and synthetic lethal kinome RNA interference screening using T-DXd resistant HER2-positive BC cell lines. To determine the synergistic antitumor effect of T-DXd combined with targeted therapy, we conducted Bliss synergy analysis, colony formation, and xenograft assays by mammary fat pad injection of T-DM1- or T-DXd-resistant BC cells in nude mice. Results: Increased amplification of DNA repair-related genes (TOP2A, RAD21, RAD52, and CDK12) was found in patient tissue samples after progression to HER2 antibodies, T-DM1, or T-DXd. Significant enrichment of DNA damage repair-related gene sets was observed in the transcriptome of post-treatment human samples. Further, ERBB2 gene or HER2 protein expression was reduced compared to levels before such treatment. In HER2-ADC-resistant HER2-positive BC cell lines, we had similar upregulation of DNA repair-related genes (PCNA, ATM, RAD52) and reduction of ERBB2 gene and HER2 protein expression. By synthetic lethal kinome RNA interference screening, this non-bias screening identified the PI3 kinase, cell cycle, and DNA repair canonical pathways as potential targets to enhance the efficacy of T-DXd therapy in HER2-ADC–resistant HER2-positive BC. We confirmed that ectopic HER2 expression does not improve the efficacy of HER2 ADC in HER2-ADC–resistant HER2-positive BC. After further screening all potential targets, we found that the drugs targeting the DNA damage repair pathway were the most effective in enhancing the efficacy of T-DXd. Indeed, ATR inhibitor elimusertib led to significant HER2-ADC–resistant BC cell death in vitro (Bliss synergy score >5.0, P<0.01) and in four xenografts in vivo (P<0.01) compared to cell death with T-DXd alone. Importantly, we further observed the synergy of elimusertib and T-DXd in parental HER2-positive BC cell lines, ensuring the observed effects are not limited to resistant cell lines only. Conclusions: Our findings indicate that resistance to HER2-ADC therapies is associated with increased DNA repair-related genes. By non-biased screening, this study provides robust evidence that targeting DNA repair pathways can significantly enhance the efficacy of T-DXd in HER2-ADC-resistant HER2-positive BC. The potential of combining ATR inhibitors with T-DXd to overcome resistance and improve patient outcomes is a promising avenue that warrants further exploration in a clinical trial for patients with HER2-ADC-resistant- HER2-positive BC. Citation Format: Jangsoon Lee, Kumiko Kida, JiwonKoh, Huey Liu, GanirajuC. Manyam, Young JinGi, Dileep Reddy, Asha S. Multani, Jing Wang, Gitanjali Jayachandran, Dae-Won Lee, James M. Reuben, AysegulSahin, Lei Huo, DebuTripathy, Seock-Ah Im, and Naoto T. Ueno. Enhancing T-DXd Efficacy in HER2-positive Breast Cancer Resistant to HER2 ADC by Non-biased Kinase-related Target Screening [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr PS13-07.

TTI1 contributes to radioresistance by activating ATM pathway in rectal cancer

BackgroundTTI1 has been reported as a critical protein in oncogenesis, migration and invasion of various cancers. And its expression level was increased in rectal cancer (RC). However, the biological role of TTI1 in RC is unclear. In the course of exploratory study, we found that TTI1 expression was elevated in radioresistant RC. Therefore, the study was designed to investigate the role of TTI1 in the radiotherapy of RC.MethodsThe proliferative activity of RC cells treated by irradiation was performed using MTT assay and colony formation assay in vitro as well as nude mouse tumour xenograft assay in vivo. Various methods including western blot, qRT-PCR, flow cytometry, and comet assay were used to exploring the molecular mechanism of TTI1 in radiotherapy of RC. The organoid and PDX models were used to analyse the radiotherapy sensitization effect of suppressing ATM signaling pathway.ResultsTTI1 expression was significantly increased in radioresistant RC. The clinical study demonstrated that significantly elevated TTI1 expression resulted in a poor tumour response in RC patients treated with irradiation. TTI1 knockdown could enhance sensitivity of RC cells to irradiation while TTI1 overexpression had the opposite effect. TTI1 could enhance irradiation-induced DNA damage repair through activating ATM signaling pathway. Blocking ATM signaling pathway could enhance the sensitivity of RC tissue to irradiation.ConclusionsTTI1 was identified as a critical factor in regulating resistance of RC to irradiation. TTI1 could be regarded as a credible biomarker for predicting radiotherapeutic effect and prognosis. Inhibition of ATM signaling pathway may be a novel method to overcome radioresistance.

CircZFR involves propofol-triggered ferroptosis in lung cancer cells through the IGF2BP2/GPX4 axis.

This study aims to delineate the underlying mechanism by which propofol triggers ferroptosis in lung cancer cells through the inhibition of the circZFR/IGF2BP2/GPX4 axis. The expression levels of circZFR, insulin-like growth factor 2 binding protein 2 (IGF2BP2), and glutathione peroxidase 4 (GPX4) in lung cancer cells were assessed using quantitative real-time polymerase chain reaction and Western blot analysis. Cell viability was evaluated with the cell counting kit-8 assay, and ferroptosis-related indicators were measured using appropriate kits. The interactions between circZFR and IGF2BP2, as well as between GPX4 and IGF2BP2, were investigated through RNA pull-down and RNA immunoprecipitation assays, and their effects on ferroptosis were analyzed using rescue assays. In addition, xenograft assays in nude mice were conducted to evaluate the impact of propofol on tumor growth and ferroptosis in vivo. Propofol treatment induced cell ferroptosis, as evidenced by decreased cell viability and elevated levels of malondialdehyde (MDA), Fe2+, and lipid reactive oxygen species in H1299 and SPC-A-1 cells. In addition, propofol reduced the expression of circZFR and GPX4 in lung cancer cells. Notably, the overexpression of circZFR inhibited propofol-induced ferroptosis in these cells. CircZFR interacts with IGF2BP2 to regulate the stability of GPX4 mRNA and its protein expression. Furthermore, circZFR inhibited GPX4-mediated ferroptosis by enhancing IGF2BP2 expression in both H1299 and SPC-A-1 cell lines. Moreover, propofol inhibited tumor growth in nude mice, downregulated the expression of circZFR, IGF2BP2, and GPX4, and increased MDA and Fe2+ levels in tumor tissues. Propofol downregulates circZFR to inhibit the expression of GPX4 by interacting with IGF2BP2, thereby triggering ferroptosis in lung cancer cells.

Targeting CALR reduces energy metabolism of esophageal cancer cells and inhibits tumor-associated fibroblast infiltration

Calreticulin (CALR) supports the induction of dendritic cell maturation, which makes it a key target for effective esophageal squamous cell carcinoma (ESCC) immunotherapy. The mechanism of CALR in the immunotherapy of ESCC is not fully studied. The aim of the present study was to explore the contributing role of CALR in ESCC progression. The association of CALR expression with calnexin (CANX) and protein disulfide isomerase A3 (PDIA3) expression in ESCC was analyzed. The functions of CALR in ESCC cells were examined by detection of cell migration, endoplasmic reticulum (ER) stress, mitochondrial function, cytoskeletal remodeling, cell proliferation and apoptosis. The effects of CALR on tumor growth and tumor-associated fibroblast infiltration were examined by subcutaneous xenograft assay. The expression of CALR, CANX and PDIA3 in ESCC tissue significantly increased and the expression of PDIA3 was positively associated with CANX. Overexpression of CALR resulted in enhanced cell proliferation, migration, ER stress, mitochondrial function and cytoskeletal remodeling; knockdown of CALR expression had the opposite effect. In the subcutaneous xenograft assay, knockdown CALR significantly inhibited the growth of esophageal cancer tumors, suppressed the invasion of tumor-associated fibroblasts and decreased the expression of α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), fibroblast specific protein-1 (FSP1), platelet-derived growth factor and transforming growth factor beta (TGF-β) in tumor tissue. These findings suggested that CALR promotes the progression of ESCC by regulating ER stress and mitochondrial function to mediate ATP production, cytoskeletal remodeling, cell proliferation and apoptosis through CANX and PDIA3. Knockdown CALR significantly inhibited tumor-associated fibroblast infiltration and is a potential drug target for ESCC.

Association of High Tumor-Stroma Ratio with Prostate Cancer Progression: Insights from Clinical and Genomic Data.

Tumor stroma ratio (TSR) is a prognostic factor in various cancers, but its role in prostate adenocarcinoma (PRAD) remains unclear. This study investigates TSR's prognostic value in PRAD using clinicopathological data, bulk/single-cell RNA sequencing to explore tumor-stroma interactions and identify therapeutic targets. Two PRAD cohorts (The Cancer Genome Atlas cohort, TCGA; Lanzhou University Second Hospital, LUSH) were analyzed for TSR associations with clinicopathological features and biochemical recurrence (BCR). TSR was assessed via digital image analysis and expert pathologist review. Publicly available bulk/single-cell RNA sequencing data were analyzed to identify TSR-associated genes and predict drug targets, pathways, and immunotherapy responses. Quantitative real-time PCR validated mRNA expression. In vitro assays assessed cell proliferation, growth, and migration, while in vivo xenograft assays validated BGN's role in promoting tumorigenesis. TSR significantly correlated with clinicopathological features (age, Gleason score, stage, seminal vesicle invasion, BCR) in both TCGA (n = 453) and LUSH (n = 320) cohorts. High TSR independently predicted BCR in multivariable Cox regression. High TSR was associated with copy number variations, differentially expressed miRNAs/transcription factors, and metabolic pathways. Predicted anti-cancer drug targets, like Ki8751, showed potential benefit in high-TSR patients. High TSR may correlate with poor immunotherapy response. Notably, downregulation of BGN in cancer-associated fibroblasts (CAFs) significantly suppressed cell proliferation, migration, and invasion in vitro, and in vivo xenograft assays confirmed that BGN downregulation inhibited tumor growth. This study highlights TSR's prognostic significance in prostate cancer and its association with adverse clinical outcomes and complex tumor-stroma interactions, identifying BGN, a stromal cell-related gene, as a potential therapeutic target for CAFs. However, these findings are limited by the retrospective design, necessitating prospective validation.

Downregulation of MGAT3 Promotes Benzo[a]pyrene-Mediated Lung Carcinogenesis by Regulating Cell Invasion and Migration Activity.

Environmental chemical carcinogens are major factors in the induction of lung cancer, with benzo[a]pyrene (B[a]P) being one of the most widespread and highly carcinogenic among them. Although studies have reported that B[a]P exerts its carcinogenic effects by causing mutations, inducing cytotoxicity, and inhibiting DNA synthesis, the early molecular regulatory events and mechanisms involved in B[a]P-induced tumor initiation remain unclear. This study found that the MGAT3 gene was significantly downregulated in B[a]P-induced mouse lung tumorigenesis, suggesting its important tumor-suppressive function. Further investigation revealed that suppression of MGAT3 expression promoted the invasion and migration abilities of lung cancer cells, while overexpression of MGAT3 in these cells inhibited these effects. Western blot analysis also showed that MGAT3 regulated the expression of epithelial-mesenchymal transition markers, thereby affecting the motility of lung cancer cells. Xenograft assay also confirmed the inhibitory effect of MGAT3 overexpression on tumor proliferation. Analysis of lung cancer tissue expression further validated that MGAT3 is significantly downregulated in lung cancer tissues, and this decrease in expression is associated with a poor prognosis in lung cancer patients. Our research indicates that the suppression of MGAT3 expression and its downstream regulatory molecules plays a crucial role in lung cancer development induced by environmental chemical carcinogens.

Abstract 3209: IND enabling study of NRT-YHD_001, a macrophage immune checkpoint inhibitor in liver cancer

Abstract NRT-YHD_001, a modified antisense miRNA targeting let-7i-5p for liver cancer therapy, acts as a key regulator of macrophage activity within the tumor microenvironment. Through in vitro pharmacology studies, we identified anticancer effects not only on cell growth, proliferation, viability, migration, and invasion but also through the enhancement of macrophage phagocytosis. To evaluate the in vivo efficacy of NRT-YHD_001, we used a Ras-transgenic mouse liver cancer model which develops spontaneous liver cancer mass after 15 ∼ 20 weeks of birth. Based on dose-dependent and injection interval studies, the optimal regimen was determined to be 1 mg/kg administered weekly. We confirmed that weekly intravenous injections of 1mg/kg NRT-YHD_001 showed significantly greater therapeutic efficacy than the sorafenib-treated group. Additionally, xenograft assay using human liver cancer cells in athymic-nude mice showed that weekly injection of NRT-YHD_001 led to improved survival and tumor growth inhibition compared to the untreated group. We performed Absorption, Metabolism, Distribution, and Excretion studies for NRT-YHD_001, stability studies in serum and liver homogenate, and metabolite identification and profiling analysis were completed in mice and cynomolgus monkeys. In maximum tolerated dose (MTD) studies conducted in mice and cynomolgus monkeys, no toxicity of NRT-YHD_001 was observed at doses up to 500 mg/kg administered intravenously. Furthermore, comprehensive evaluations across 4-week repeated dose toxicity and toxicokinetic in mice and monkey, safety pharmacology, and genetic toxicity studies revealed no adverse findings with NRT-YHD_001. For NRT-YHD_001, Drug Substance (DS) and Drug Product (DP) were produced by a global CDMO with experience in U.S. FDA approval of oligonucleotide drugs, and the analysis method was established.These results enabled us to complete the IND-enabling study package for NRT-YHD_001, a liver cancer therapy candidate with FTO analysis completed and intellectual property (IP) fully secured. Regulatory approval submissions to the Korea MFDS and the U.S. FDA are planned for 2025. Citation Format: Suk Woo Nam, Jin Woong Ha, Sang Yean Kim, Min Jeong Na, Soyoung Jeon, Bee Yoo, In Seop Yoon, Hyunmin Lee, Chang Won Park. IND enabling study of NRT-YHD_001, a macrophage immune checkpoint inhibitor in liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3209.

Abstract 2890: Anti-tumor effect and mechanism of action of a nuclear transportable antibody drug conjugate Y-TR-1: Anti CD26 antibody conjugated with RNA polymerase II inhibitor triptolide

Abstract Introduction: CD26 is a multifunctional transmembrane glycoprotein that is overexpressed in many cancers. We established the humanized anti-CD26 monoclonal antibody YS110, which has a direct anti-tumor effect through nuclear translocation of CD26 and transcriptional suppression of the POLR2A gene, a subunit of RNA polymerase II (Pol II). Subsequently, we developed an antibody-drug conjugate Y-TR-1 with YS110 and the Pol II inhibitor triptolide and evaluated its in vitro and in vivo antitumor efficacy and toxicity. We then elucidated the mechanism of action of Y-TR-1. Experimental procedures: 5 triptolide molecules on average were crosslinked to YS110 by the heterobifunctional linker SMCC (designated Y-TR-1). The in vitro cytotoxicity of Y-TR-1 was analyzed in CD26-positive or -negative cancer cells and normal cells. The in vivo antitumor effect of Y-TR-1 was assessed using a xenograft assay. Pharmacokinetics/pharmacodynamics studies and toxicity tests were conducted using cynomolgus monkeys. Western blotting and immunofluorescence imaging were performed to confirm the nuclear translocation of Y-TR-1. The inhibitory effect of Y-TR-1 against Pol II was analyzed using quantitative polymerase chain reaction and a live cell imaging assay. The molecular mechanism of Y-TR-1 nuclear translocation was elucidated by immunofluorescence imaging. Results: A conjugation method of Y-TR-1 was established, and the average drug-to-antibody ratio (DAR) was 4.76 by LC/MS. Y-TR-1 showed in vitro cytotoxicity on CD26-positive cancer cells, whereas non-cancer cells were less susceptible to Y-TR-1. The in vivo antitumor effect of Y-TR-1 was confirmed in a xenograft study using three CD26-positive cancer cell lines. Toxicity studies of cynomolgus monkeys with cross-reactivity to Y-TR-1 revealed no severe toxicity. The animals that received 30 mg/kg body weight of Y-TR-1 showed some decline in the reticulocyte count and slight increase in the thrombocyte count, whereas no histological damage was observed in the liver, kidney, heart, or lungs. Nuclear translocated Y-TR-1 was confirmed in the nuclear fraction of CD26-positive cancer cells by western blotting or immunofluorescence imaging. Quantitative polymerase chain reaction and live cell imaging assays revealed Pol II inhibition by Y-TR-1. Y-TR-1 was internalized via caveolae-dependent endocytosis and subsequently colocalized with endosomes before translocation to the nucleus. Conclusions: Y-TR-1 is an ADC with a novel mechanism of action, i.e., nuclear translocation and subsequent Pol II inhibition. Transcriptional repression of POLR2A expression by nuclear-localized CD26 and Pol II inhibition by internalized triptolide are expected to have additive effects. Y-TR1 exhibited potent antitumor activity against CD26-positive cancer cells in vitro and in vivo. Citation Format: Mutsumi Hayashi, Hiroko Madokoro, Koji Yamada, Ryo Hatano, Chikao Morimoto, Taketo Yamada. Anti-tumor effect and mechanism of action of a nuclear transportable antibody drug conjugate Y-TR-1: Anti CD26 antibody conjugated with RNA polymerase II inhibitor triptolide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2890.

RASSF4 Suppresses Gastric Tumor Growth through Activation of Chk2-p53 Signaling Axis.

Ras association domain family 4 (RASSF4) is a putative tumor suppressor that is frequently inactivated in multiple human cancers. However, its candidacy as a suppressor in gastric tumorigenesis remains undefined. To understand the role for RASSF4 in gastric tumorigenesis, we investigated its expression status in cancer cell lines and tissues and regulatory role in tumor growth. RASSF4 expression was analyzed in 13 cancer cell lines and 20 carcinoma tissues using PCR and immunoblot assays. RASSF4 effect on cell proliferation and apoptosis was examined by flow cytometry, colony formation, and [3H]thymidine incorporation assays and its regulation of p53 was determined using cycloheximide chase, promoter reporter, and immunoprecipitation assays. Mouse xenograft assay was performed to verify RASSF4 effect on tumor growth and therapeutic response. RASSF4 expression is epigenetically inactivated in 8 of 13 (61.5%) cancer cell lines and 15 of 20 (75%) primary carcinomas. RASSF4 suppresses cell proliferation by inducing a G2/M cell-cycle arrest and enhances apoptotic response to therapeutic drugs. RASSF4 is induced in response to genotoxic agents to facilitate stress-induced apoptosis in a highly p53-dependent fashion. Mechanistically, RASSF4 stabilizes p53 through Chk2 activation and its apoptotic function is profoundly impaired by depletion of either p53 or Chk2. RASSF4 attenuates xenograft tumor growth and enhances tumor response to 5-FU. Clinically, RASSF4 expression correlates strongly with the overall survival of gastric cancer patients. RASSF4 suppresses gastric tumor growth through the activation of the Chk2-p53 axis, illuminating the mechanistic consequence of its inactivation in gastric tumorigenesis.

Oroxylin A may promote cell apoptosis and inhibit epithelial-mesenchymal transition in endometrial cancer, associated with the ERβ/PI3K/AKT pathway

Endometrial cancer (EC) is a prevalent gynecological cancer worldwide, often associated with poor prognosis after recurrence or metastasis. Oroxylin A (OA) is an active flavonoid compound with a strong anti-tumor function. However, the effects of OA on EC remain unknown. In this study, we planned to investigate the anti-EC effects of OA and explore its mechanisms. Five cell lines were used for in vitro experiments, and female BALB/c nude mice were applied for xenograft experiments. The cytotoxicity and experimental concentration of OA were detected by CCK-8. Wound healing, transwell, and colony formation assays were used to evaluate the anti-metastatic and anti-proliferative activities of OA on EC cells. TUNEL assay and flow cytometry were applied for the evaluation of apoptosis. Network pharmacology was used to explore potential targets, and molecular dynamics simulations and dockings were applied for the quantification of binding energy, and stability of OA. RT-qPCR, WB, and immunofluorescence were applied for the detection of localization and expression of correlated markers. The results showed that OA notably inhibited the proliferation, migration, and invasion of Ishikawa cells. Meanwhile, in vivo Ishikawa xenograft assays demonstrated that OA notably inhibited growth and promoted apoptosis of EC. Mechanistically, after treatment with OA, the expressions of Cleaved Caspase-3, BAX, E-cadherin, and ERβ were increased, while the expressions of Bcl-2, Vimentin, N-cadherin, MMP2, MMP9, PI3K and phospho-AKT (Ser473) were decreased. Therefore, OA may exhibit significant anti-EC effects by regulating the ERβ/PI3K/AKT pathway to promote apoptosis and inhibit epithelial-mesenchymal transition (EMT).

RNF135 promotes the stemness of breast cancer cells by ubiquitinating and degrading DDX58.

RNF135 promotes the stemness of breast cancer cells by ubiquitinating and degrading DDX58.

PTOV1 interacts with ZNF449 to promote colorectal cancer development

PTOV1 is recognized to have a significant role in various human cancers, including prostate cancer. However, it remains unclear what its clinical significance and biological role are in colorectal cancer (CRC). TCGA, NCBI/GEO, and Kaplan–Meier plot database mining provided important clues into the function and clinical importance of PTOV1 in CRC. Western blotting, immunohistochemistry, and immunofluorescence were utilized to discover PTOV1 protein levels in CRC cell lines and tissues. To explore the involvement of PTOV1 in the development of CRC and the underlying mechanisms, several in-vitro and in-vivo studies were executed, such as CCK-8 assays, colony formation, transwell assays, qRT-PCR, Co-IP, GST pull-down, immunostaining, and mouse xenograft assays. It was shown that PTOV1 expression level was upregulated in the tissues and cells of human CRC. PTOV1 high-expression level was associated with short survival. ZNF449 interacted with PTOV1 and accelerated CRC development in vitro and in vivo. Through Co-IP and GST pull-down studies, the physical interaction of PTOV1/ZNF449 was demonstrated. Furthermore, PTOV1 directly bound ZNF449, and this complex synergistically promoted the transcription of MYC. In addition, the PTOV1/ZNF449 interaction was disrupted by the TAT- PTOV1 (125–283 aa) protein leading to inhibit the CRC development in a xenografted mouse model. According to these findings, PTOV1 has an essential role in CRC progression, and PTOV1/ZNF449 interaction could be a possible therapeutic target for CRC.

Modular Synthesis of Bioactive Selenoheterocycles for Efficient Cancer Therapy via Electrochemical Selenylation/Cyclization.

A green, efficient, and environmentally friendly electrochemical strategy was developed for synthesizing a series of selenoheterocyclic compounds. The antitumor activities of these compounds were evaluated, revealing that compounds 4o, 5n, and 5o demonstrated remarkable antitumor efficacy. These compounds effectively inhibited lung cancer by inducing cell apoptosis, causing DNA damage, and suppressing the progression of epithelial-mesenchymal transition. Notably, compound 5o was identified as the first inhibitor of DEAD-box helicase 10 (DDX10). An in vivo xenograft assay further confirmed the therapeutic potential of compound 5o, demonstrating tumor growth inhibition rates of 60%, 78%, and 88% at doses of 5 mg/kg, 10 mg/kg, and 20 mg/kg, respectively. This study highlights a promising chemotherapeutic agent for the effective treatment of lung cancer.

Cinobufagin Enhances the Sensitivity of Cisplatin-Resistant Lung Cancer Cells to Chemotherapy by Inhibiting the PI3K/AKT and MAPK/ERK Pathways.

Lung cancer patients always develop serious chemotherapy resistance after long-term use of cisplatin treatment. It has been demonstrated that the combination of cisplatin (DDP) with other chemotherapy drugs may significantly reduce drug resistance. Cinobufagin (CB) showed potent anti-tumour effect against lung cancer. However, the relevance of CB and DDP resistance in lung cancer remains unclear. This article will study the effects of CB on reversing lung cell resistance. The apoptosis was rescued by flow cytometry analysis and TUNEL staining. The invasiveness was rescued by invasion assay. The mRNA and apoptosis-related proteins were estimated by qRT-PCR analysis and Western blot analysis, respectively. Invivo antitumor activities were investigated by subcutaneous xenograft assay. The present study firstly demonstrated that the sensitivity of DDP in DDP-resistant A549 (A549/DDP) cells was enhanced when treated with CB. Moreover, CB combined with DDP weakened the proliferation and increased apoptosis of A549/DDP cells. In addition, the expression level of Bcl-2 was increased, whereas Bax and caspase-3 were activated when A549/DDP cells were treated with both drugs. After treatment with IGF1 or PMA and mixed drugs (CB + DDP), the expressions of P-AKT, P-PI3K, P-MEK1/2 and P-ERK1/2 were increased. Finally, the results of invivo experiments showed that the combination of DDP and CB significantly reduced the growth of tumours derived from A549/DDP cells. The combination of CB and DDP can be considered an effective strategy to increase the sensitivity of DDP-resistant lung cancer cells to DDP by inhibiting the PI3K/AKT and MAPK/ERK pathways.