Potential Role Research Articles

Identification of Novel Stemness-based Subtypes and Construction of a Prognostic Risk Model for Patients with Lung Squamous Cell Carcinoma.

Although cancer stem cells (CSCs) contribute to tumorigenesis, progression, and drug resistance, stemness-based classification and prognostic signatures of lung squamous cell carcinoma (LUSC) remain unclarified. This study attempted to identify stemness-based subtypes and develop a prognostic risk model for LUSC. Based on RNA-seq data from The Cancer Genome Atlas (TCGA), Gene-Expression Omnibus (GEO) and Progenitor Cell Biology Consortium (PCBC), mRNA expression-based stemness index (mRNAsi) was calculated by one-class logistic regression (OCLR) algorithm. A weighted gene coexpression network (WGCNA) was employed to identify stemness subtypes. Differences in mutation, clinical characteristics, immune cell infiltration, and antitumor therapy responses were determined. We constructed a prognostic risk model, followed by validations in GEO cohort, pan-cancer and immunotherapy datasets. LUSC patients with subtype C2 had a better prognosis, manifested by higher mRNAsi, higher tumor protein 53 (TP53) and Titin (TTN) mutation frequencies, lower immune scores and decreased immune checkpoints. Patients with subtype C2 were more sensitive to Imatinib, Pyrimethamine, and Paclitaxel therapy, whereas those with subtype C1 were more sensitive to Sunitinib, Saracatinib, and Dasatinib. Moreover, we constructed stemness-based signatures using seven genes (BMI1, CCDC51, CTNS, EIF1AX, FAM43A, THBD, and TRIM68) and found high-risk patients had a poorer prognosis in the TCGA cohort. Similar results were found in the GEO cohort. We verified the good performance of risk scores in prognosis prediction and therapy responses. The stemness-based subtypes shed novel insights into the potential roles of LUSC-stemness in tumor heterogeneity, and our prognostic signatures offer a promising tool for prognosis prediction and guide therapeutic decisions in LUSC.

The intersection of adverse childhood experiences and neighborhood determinants of health: An exploratory spatial analysis.

Adverse childhood experiences (ACEs) are associated with elevated risk for poor physical and psychological health outcomes. Nearly all of the literature on the association between ACEs and poor health focuses on the individual and family level; the potential role of neighborhood environment is overlooked. Understanding the relationship between ACEs and characteristics of the neighborhood environment is a necessary first step in determining if and how place-based, trauma-informed interventions might mitigate the negative effects of ACEs. The purpose of this exploratory study was to describe the neighborhood environment of adults who have experienced ≥ 4 versus ≤ 3 ACEs. An exploratory secondary analysis of cross-sectional and geospatial data was conducted during 2021. Data sources included 2011/2012 Philadelphia ACE Survey data, a telephone survey of 1,784 randomly sampled Philadelphia adults, linked with geospatial data on 21 neighborhood-level determinants of health. Neighborhood was defined as participant's home census tract at the time of survey. The sample for this secondary analysis included the 1,679 Philadelphia ACE Survey participants for whom home census tract was available. Bivariate logistic regression examined differences between groups (≤ 3 ACEs vs. ≥ 4 ACEs). Individuals with ≥ 4 ACEs lived in neighborhoods with higher neighborhood poverty rates, less socioeconomic resources, worse food access, poorer perceived physical and mental health, more substance overdose deaths, higher crime, and less green space. Findings suggest future work may benefit from considering neighborhood environments when examining and intervening upon the association between ACEs and poor physical and psychological health. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

A Bionic Thermosensitive Sustainable Delivery System for Reversing the Progression of Osteoarthritis by Remodeling the Joint Homeostasis.

Although the pathogenesis of osteoarthritis (OA) is unclear, inflammatory cytokines are related to its occurrence. However, few studies focused on the therapeutic strategies of regulating joint homeostasis by simultaneously remodeling the anti-inflammatory and immunomodulatory microenvironments. Fibroblast growth factor 18 (FGF18) is the only disease-modifying OA drug (DMOAD) with a potent ability and high efficiency in maintaining the phenotype of chondrocytes within cell culture models. However, its potential role in the immune microenvironment remains unknown. Besides, information on an optimal carrier, whose interface and chondral-biomimetic microenvironment mimic the native articular tissue, is still lacking, which substantially limits the clinical efficacy of FGF18. Herein, to simulate the cartilage matrix, chondroitin sulfate (ChS)-based nanoparticles (NPs) are integrated into poly(D, L-lactide)-poly(ethylene glycol)-poly(D, L-lactide) (PLEL) hydrogels to develop a bionic thermosensitive sustainable delivery system. Electrostatically self-assembled ChS and ε-poly-l-lysine (EPL) NPs are prepared for the bioencapsulation of FGF18. This bionic delivery system suppressed the inflammatory response in interleukin-1β (IL-1β)-mediated chondrocytes, promoted macrophage M2 polarization, and inhibited M1 polarization, thereby ameliorating cartilage degeneration and synovitis in OA. Thus, the ChS-based hydrogel system offers a potential strategy to regulate the chondrocyte-macrophage crosstalk, thus re-establishing the anti-inflammatory and immunomodulatory microenvironment for OA therapy.

Experimental investigations of diacetylene ice photochemistry in Titan’s atmospheric conditions

Context. A large fraction of the organic species produced photochemically in the atmosphere of Titan can condense to form ice particles in the stratosphere and in the troposphere. According to various studies, diacetylene (C4H2) condenses below 100 km where it can be exposed to ultraviolet radiation. Aims. We studied experimentally the photochemistry of diacetylene ice (C4H2) to evaluate its potential role in the lower altitude photochemistry of Titan’s atmospheric ices. Methods. C4H2 ice films were irradiated with near-ultraviolet (near-UV) photons (λ > 300 nm) with different UV sources to assess the impact of the wavelengths of photons on the photochemistry of C4H2. The evolution of the ice’s composition was monitored using spectroscopic techniques. Results. Our results reveal that diacetylene ice is reactive through singlet-triplet absorption, similar to the photochemistry of other organic ices of Titan (such as dicyanoacetylene C4N2 ice) that we investigated previously. Several chemical processes occurred during the photolysis: the hydrogenation of C4H2 to form other C4 hydrocarbons (vinylacetylene C4H4 to butane C4H10); the formation of larger and highly polymerizable hydrocarbons, such as triacetylene (C6H2); and the formation of an organic polymer that is stable at room temperature. Conclusions. The nondetection of diacetylene ice in Titan’s atmosphere or surface could be rationalized based on our experimental results that C4H2 is photochemically highly reactive in the solid phase when exposed to near-UV radiation that reaches Titan’s lower altitudes and surface. C4H2 may be one of the key molecules promoting the chemistry in the ices and aerosols of Titan’s haze layers, especially in the case of co-condensation with other organic volatiles, with which it could initiate more complex solid-phase chemistry.

Emotion malleability beliefs matter in emotion regulation: a comprehensive review and meta-analysis

ABSTRACT Individuals’ beliefs about the malleability of emotions have been theorised to play a role in their psychological distress by influencing emotion regulation processes, such as the use of emotion regulation strategies. We conducted a meta-analysis to test this idea across studies with a focus on the relationships between emotion malleability beliefs and five distinct emotion regulation strategies: cognitive reappraisal, suppression, avoidance, rumination, and acceptance. Further, using two-stage meta-analytic structural equation modelling (TSSEM), we examined whether the emotion regulation strategies mediate the cross-sectional relationship between emotion malleability beliefs and psychological distress across studies. Thirty-seven studies were included in the meta-analyses and 55 cross-sectional studies were included in the TSSEM. Results demonstrated that, across studies, emotion malleability beliefs were significantly associated with greater use of putatively helpful strategies (particularly with cognitive reappraisal) and less use of putatively unhelpful strategies (particularly with avoidance). The use of cognitive reappraisal and avoidance partially mediated the relationship between emotion malleability beliefs and psychological distress. These results highlight the importance of considering beliefs about the malleability of emotions in the context of emotion regulation. These findings suggest the potential role of emotion malleability beliefs in interventions for individuals with emotion regulation-related difficulties and psychological distress.

Biochemical Changes: Their Potential Role against Fungal Disease Resistance Development in Mustard

Rape-seed mustard is an imperative edible oilseed crop which is adversely affected by different biotic stresses causing import of edible oils from abroad. The experiment was carried out for screening mustard genotypes based on disease indexing and putative role of diverse biochemical parameters in development of resistant against three major fungal diseases. A modified 0-9 scale was used for rating of disease indicators to calculate disease incident (DI). In disease indexing, mustard genotypes L-4, GSC-7 and PC-6 were considered as immune against Alternaria brassicae, Maya, L-4, China, GSL-1, GSC-7, PC-5, PC-6 and RP-9 were classified as immune against Albugo candida while L-4 and PC-5 were found immune against Erysiphe cruciferarum. The study of biochemical constituents demonstrated that these immune genotypes accumulated higher osmolytes content including free proline content and total phenol under diseased condition showing their tolerance ability against these pathogens. The tolerant genotypes also had lesser lipid peroxidation rate as indicated by malondialdehyde content analysis to induce lesser effect on total chlorophyll, amino acids, proteins and soluble sugars including reducing and non-reducing sugars. These identified genotypes have wider scope for further phyto-pathological studies and utilization in development of fungal resistance breeding.

Electrified Interactions of Polyzwitterions with Charged Surfaces: Role of Dipole Orientation and Surface Potentials.

The zwitterionic groups possess strong dipole moments, leading to inter- or intrachain interactions among zwitterionic polymers. This study aims to demonstrate the interaction of polyzwitterions poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), and poly(carboxybetaine methacrylate) (PCBMA) with electrified surfaces, despite their electrically neutral nature. We studied the adsorption of polyzwitterions and their monomers on electrified surfaces by using an electrochemical quartz crystal microbalance with dissipation (EQCM-D). The interaction between zwitterionic molecules and charged surfaces is explored by adjusting the surface potentials. Interestingly, the adsorption of polyzwitterions can be influenced by external potential, primarily due to the formation of polyzwitterions restricting the mobility of zwitterionic groups, affecting the adsorption behavior of polyzwitterions based on the surface potential. The impact is determined by the arrangement of positive and negative ions within the zwitterionic groups, which are the dipole orientation. Additionally, surface potentials determine the adsorption rate, amount, and chain conformation of the adsorbed thin polyzwitterion layers. The effect of ionic strength was investigated by introducing electrolytes into the aqueous solutions to assess the range of influenced surface potentials.

Neutrophils in the tumor microenvironment: role in tumor progression and potential targeted therapeutic strategy

Neutrophils are known as the body's first line of defense against invading pathogens, which are the most abundant bone-marrow-derived innate immune cells accounting for 50%-70% of all white blood cells in human peripheral blood. With in-depth research in recent years, neutrophils have also been recognized as one of the main immune cells in the tumor microenvironment (TME), playing an important role in tumor development. Neutrophils can promote tumor metastasis through secretion of chemokines and cytokines and formation of neutrophil extracellular traps (NETs). Also, neutrophil targeting treatment has shown a promising anticancer effect. In this article, we summarized the recent research progress related to the significant role of neutrophils in tumor metastasis, as well as the current progression and limitations of targeting neutrophils in tumor therapeutics. This review can provide a comprehensive understanding of the role of neutrophils in the TME and the development of a new anticancer strategy.

CircITGA7 regulates malignant phenotypes in bladder cancer cells via targeting miR-330-3p/KLF10 axis.

Bladder cancer (BCa) is one of the common malignancies. Circular RNAs (circRNAs) play regulatory roles in cancer progression. CircITGA7 is a circRNA generated from several exons of ITGA7. The potential role of circITGA7 in BCa remains unknown and needs to be explored. Quantitative real time polymerase chain reaction (qRT-PCR) was used to assess circITGA7 and miR-330-3p expression in BCa tissues and cell lines. Kaplan-Meier analysis was used to evaluate the overall survival of these BCa patients. The biological function of circITGA7 was examined by overexpression of circITGA7 using CCK-8, EdU, wound-healing, and Transwell assays. Xenograft assay was performed to further validate the in vitro results. To explore the mechanism of circITGA7, luciferase reporter, RNA pull-down, fluorescence in situ hybridization (FISH) assays were employed to examine the binding interaction among circITGA7, miR-330-3p and kruppel-like factor 10 (KLF10). Western blot was used to study the protein levels of KLF10.CircITGA7 was downregulated in BCa tissues and cell lines and indicated longer overall survival. Moreover, circITGA7 restricted cell proliferation, migration and invasion of BCa through negatively regulating miR-330-3p. The in vivo model showed that circITGA7 influenced the tumor growth. Besides, the overexpression of miR-330-3p promoted cell progression by directly targeting KLF10. Mechanistically, circITGA7 inhibited BCa progression by activating KLF10 via targeting miR-330-3p.CircITGA7 alleviates BCa cell progression via circITGA7/hsa-miR-330-3p/KLF10 axis, which may provide novel therapeutic targets for BCa.

Abstract 3005: Low-density lipoprotein receptor-related protein 1B (LRP1B) depletion promotes tumorigenicity by modulating fibroblasts in the tumor microenvironment in esophageal squamous cell carcinoma

Abstract Background: Esophageal squamous cell carcinoma (ESCC) is widespread worldwide with a poor survival rate. Tumor cells shedding from tumor tissues traveling to other organs through the blood circulatory system are called circulating tumor cells (CTCs). The CTCs in the blood of ESCC patients were enriched and then subjected to CTC mutational profiling by target sequencing. Our results suggested that CTC mutations in low-density lipoprotein receptor-related protein 1B (LRP1B) may have a potential prognostic role for ESCC. LRP1B belongs to the low-density receptor (LDLR) family with diverse roles in cellular functions and development, but the function of LRP1B in ESCC remains unclear. Therefore, this study aims to do a functional characterization of LRP1B. Methods: The CRISPR cas9 system was used to functionally knock down the candidate gene in ESCC cell lines with the highest endogenous level of LRP1B. The in vitro cell proliferation rate and viability were measured by MTT assay, colony formation assay, and wound healing assay. The in vivo tumorigenicity assay was performed to evaluate the tumor suppressive effect by subcutaneous injection. To examine the clinical relevance of LRP1B, we also performed immunohistochemistry (IHC) in tumor tissue microarray (TMA). Results: The reduction or loss of LRP1B mRNA was demonstrated in 60% of a panel of 15 ESCC cell lines. The depletion of LRP1B did not influence cell proliferation, cell clonogenic ability, or cell migration in ECSS cell lines. However, LRP1B depletion significantly promoted the growth of tumors in mouse xenograft models. Therefore, we hypothesize that LRP1B may be involved in modulating the tumor microenvironment (TME) by impacting various stromal components leading to an immuno-suppressive TME. We utilized RT-qPCR to investigate markers of various stromal components and observed that fibroblast markers (acta2, pdgfrb, and vimentin) had significantly higher expression levels after LRP1B downregulation in ESCC cell-derived mouse xenografts. The higher level of fibroblast cells was then verified by IHC staining. Further molecular mechanistic studies are underway and are expected to provide novel therapeutic insights by targeting the LRP1B genes. The LRP1B ESCC TMA data analysis is underway and statistical analysis will be presented. Conclusions: LRP1B plays a tumor-suppressive role in ESCC by modulating the tumor-associated fibroblasts in the TME. Acknowledgements: TBRS grant T12-701/17R and HMRF grant 05160926 to MLL Citation Format: KaiYan Xu, Josephine Mun-yee KO, Valen Zhuo you Yu, Maria Li LUNG. Low-density lipoprotein receptor-related protein 1B (LRP1B) depletion promotes tumorigenicity by modulating fibroblasts in the tumor microenvironment in esophageal squamous cell carcinoma [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 3005.

Abstract 4590: Antitumor effects of histone deacetylase inhibitors on breast cancer cell viability and mammosphere formation

Abstract Histone deacetylases (HDACs) are overexpressed in various types of primary human cancer and have become attractive targets for cancer therapy. This study aimed to evaluate the cytotoxic properties of three HDAC inhibitors (suberoylanilide hydroxamic acid (SAHA, or Vorinostat), Valproic Acid (VPA) and CAY10603) and compare them to Paclitaxel (PTX), a chemotherapy agent used to treat Triple Negative Breast Cancers (TNBCs). In the present study, drug response in the MDA-MB-231, MDA-MB-468, and MCF-10A cell lines was evaluated by cell viability assays. Apoptosis and cell cycle distribution were analyzed by flow cytometry. Mammospheres generated from MDA-MB-231 and MDA-MB-468 breast cancer cells were also subjected to drug treatment; their response was assessed under both normoxic and hypoxic conditions. We found that SAHA and CAY10603 inhibited breast cancer cell viability without significantly affecting MCF-10A cells. SAHA, CAY10603, and VPA all arrested a significant percentage of MDA-MB-231 cells at G2/M phase after 24h compared to controls. TNBC cells experienced an increase in both early and late apoptosis following treatment with the most effective concentrations of SAHA, CAY10603, and VPA (1.8µM, 2µM, and 0.4mM, respectively). SAHA proved to be the most effective drug for induction of apoptosis compared to the control, followed by CAY10603 (8.26% and 6%, 3.67%). In both TNBCs and mammospheres under hypoxic conditions, SAHA and CAY10603 down regulated HIF-1α expression significantly, but PTX did not. In contrast, VPA and PTX down regulated CTNNB1 gene expression. This study indicates that epi-drugs exert an anti-tumor effect on TNBCs, suggesting a potential role for HDAC inhibitors as novel therapeutics for breast cancer patients. Citation Format: Golnaz Asaadi Tehrani, Becca Kubick, Meenal Datta. Antitumor effects of histone deacetylase inhibitors on breast cancer cell viability and mammosphere formation [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 4590.

Abstract 5417: PHGDH expression level impacts tumorigenesis and metastasis in human breast cancer

Abstract Background: Phosphoglycerate dehydrogenase (PHGDH) is a metabolic enzyme, which is associated with the first step of serine synthesis. The gene encoding this enzyme is often elevated in some types of human cancer, including breast cancer. There are some researches that High-PHGDH expression promotes cancer cell proliferation and tumorigenesis. However, latest study showed that although breast tumors with high expression levels of PHGDH had more possibility to undergo primary-tumor growth, were less likely to metastasize than tumors with low expression of PHGDH by using mouse cell derived mouse models and PDX models. Nevertheless, the applicability of these outcomes to human breast cancer cell lines remains uncertain. Methods: The survival analysis according to PHGDH mRNA expression was analyzed in METABRIC datasets of breast cancer and the whole-transcriptome sequencing data of 120 breast cancer patients. To investigate the effect of PHGDH in human TNBC cell lines, PHGDH overexpressing MDA-MB-231 cell lines and PHGDH knockdown MDA-MB-468 cell lines were established. Subsequent functional experiments were performed to explore the influence of PHGDH on breast cancer tumorigenesis and metastasis. Results: In the analysis using METABRIC datasets, overall survival and disease-free survival were reduced in patients with high PHGDH expression and basal type breast cancer showed the highest mRNA level of PHGDH. Using whole-transcriptome sequencing data of 120 breast cancer patients, we also found PHGDH expression level was higher in cancer tissue(n=120) than normal tissue(n=90). In particular, triple-negative breast cancer indicated higher PHGDH level than luminal type breast cancer. We also used this data to perform analysis of overall survival, disease-free survival, metastasis free survival of non-TNBC patient and it resulted in reducing survival rate when patient showed high-PHGDH expression. However, TNBC patients demonstrated diametrical results in the identical analysis. We examined the impact of PHGDH overexpression in MDA-MB-231 cells and PHGDH knockdown in MDA-MB-468 cells, observing increased proliferation and decreased migration in breast cancer cells with elevated PHGDH levels. Conversely, PHGDH knockdown exhibited opposing effects. Subsequently analysis of markers associated with epithelial-mesenchymal transition revealed a correlation with PHGDH expression. Conclusions: Our data suggest that a potential role for PHGDH in breast cancer as a marker of reduced survival in triple-negative breast cancer, and as a regulator of cancer cell proliferation and metastasis. Citation Format: Jihong Baik, Juhee Kim, Sangeun Lee, Ayoung Park, Kyoungseob Shin, Sunghoon Kwon, Hanbyoel Lee, Wonshik Han. PHGDH expression level impacts tumorigenesis and metastasis in human breast cancer [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 5417.

Abstract 6831: Single-cell-based epigenetic and transcriptomic analysis in the KPC pancreatic cancer mouse model revealed dynamiccharacteristic subtypes of cancer-associated fibroblasts

Abstract Pancreatic cancer features robust fibrosis, with 40-80% fibrotic tissue and a dense stroma, highlighting the crucial need to grasp cellular interactions in the cancer microenvironment. In this study, we used single-cell RNA sequencing (scRNA-seq) to analyze pancreatic tissues from 60-day-old normal mouse (n=1) (GSE125588) and KPC (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre) cancer model with tumor diameter of 1-2 cm (n=2) (GSE129455). By examining fibroblast clusters, we identified distinct subtypes of cancer-associated fibroblasts (CAFs), and revealed four clusters: normal fibroblasts cluster, inflammatory CAFs (iCAFs) cluster marked by increased expression of IL6 and other cytokines, myofibroblast CAFs (myCAFs) cluster solely in the KPC model, and antigen-presenting CAFs (apCAFs) cluster expressing Mesothelin. We found that expression pattern of iCAFs in the KPC appeared similar to normal cells than myCAFs, suggesting a potential role in inhibiting cancer. it is reported that mesothelial cells might transition to apCAFs under the influence of IL6 and TGFβ due to tissue damage and inflammatory effects. This transition was visualized in our apCAF cluster, suggesting a potential pathway for the transformation of mesothelial cells into apCAFs. Next, in order to analyze epigenetic profiles of fibroblasts, we conducted single-cell Assay for Transposase-Accessible Chromatin with high-throughput sequencing (scATAC-seq) using 90-day-old KPC mice (n=2) and a wild-type mouse (n=1).scATAC-seq showed that there are mainly two clusters of fibroblasts, one cluster corresponded to myCAF, enriched with PDPN, while the other represented iCAF cluster, enriched with COL1A2. These data suggest that in terms of the epigenetic state, myCAF contain several different CAFs and have the potential to differentiate into either myCAFs or other type of based on certain stimulation. Ultimately, CAF differentiated into three distinct subtypes as revealed by scRNA-seq. This highlights that despite similar epigenetic states, various transcription factors characterize the functionality of these fibroblast subtypes. Further exploration of the underlying mechanism that lead to different CAF subtypes might help in controlling the microenvironment of pancreatic cancer. Citation Format: Keita Sasaki, Keiko Shinjo, Xingxing Wang, Kohei Kumegawa, Reo Raruyama, Shinji Mii, Yoshiteru Murofushi, Miho Suzuki, Atsushi Enomoto, Yutaka Kondo. Single-cell-based epigenetic and transcriptomic analysis in the KPC pancreatic cancer mouse model revealed dynamiccharacteristic subtypes of cancer-associated fibroblasts [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 6831.

Abstract 6250: The RNA editing landscape of lung cancer in never smokers

Abstract Adenosine-to-Inosine (A-to-I) editing represents a crucial RNA modification mechanism, holding the potential to drive carcinogenesis. This sophisticated post-transcriptional process is catalyzed by a series of ADAR (adenosine deaminase acting on RNA) family members, which can profoundly reshape the cellular epitranscriptomic landscape. While previous explorations have illuminated the important role of A-to-I editing in lung cancer tumorigenesis and prognosis, most studies have focused on lung cancer from smokers. To investigate the contribution on RNA-editing in lung cancers in never smokers (LCINS) , we embarked on a rigorous dissection of A-to-I RNA editing events in 603 LCINS. Harnessing transcriptome and whole-genome sequencing technologies, we identified 15,880 A-to-I sites, of which 75% were positively linked to the enzymatic activity of ADAR1. 92.80% of detected editing sites were located at Alu repetitive regions, with the remainder in non_Alu repeat (4.1%) and non-repeat regions (3.1%). The global RNA editing levels, measured as Alu Editing Index (AEI), were found to be significantly associated with ADAR1 expression, Tumor Mutational Burden, and Neoantigens Burden. A subtle correlation between global RNA editing levels and tumor stage was also noted. Delving into cancer-associated editing events, we identified 5,539 differentially edited sites in tumors compared with normal tissues, which were located at 823 genes, including several key tumor suppressors and oncogenes, like MDM2, BRAF, TP53, BRCA2, ATM, and VHL. Subsequent pathway analysis identified TGF-beta, Notch Signaling, and DNA Damage Response as the key pathways for cancer progression. Using the characterized A-to-I editing profiles, we built a prognostic risk stratification model, with high risk scores significantly linked to poor survival outcomes, even after adjusting for other clinical factors. Our study provides a comprehensive A-to-I editing landscape of lung cancer in never smokers, highlighting the potential roles of RNA modifications in the pathogenesis of this lethal disease. Citation Format: Jian Sang, Wei Zhao, Tongwu Zhang, Maria Teresa Landi. The RNA editing landscape of lung cancer in never smokers [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 6250.

Abstract 393: RAS and PI3K activation promote glioblastoma progression and ischemia-associated tumor cell death

Abstract Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor in adults. Despite the association of tumor necrosis and a poor prognosis in GBM, the mechanisms underlying tumor necrosis formation remain unclear, likely due to the lack of appropriate experimental models to examine the development of tumor necrosis. In our study, we developed mouse models with tumor necrosis by intracranially injecting human GBM cells expressing KRAS or PI3K active mutants into immunodeficient mice. Surprisingly, despite all tumors displayed similar sizes, the control tumors expressing an empty vector (EV) did not exhibit tumor necrosis. This intriguing observation led us to explore the potential role of KRAS and PI3K in promoting cell death within the tumor microenvironment. Ischemia, which is characterized by insufficient nutrients and hypoxia, is often found in GBM. Under an in vitro ischemia mimetic condition, we found that the KRAS or PI3K activated GBM cells showed more cell death compared to EV cells. Mechanistically, ischemia activates the p38 MAPK-MK2 pathway to induce cell death, a process further enhanced by KRAS or PI3K. Notably, we observed elevated expression of p-MK2 in the peri-necrotic areas of GBMs harboring KRAS or PI3K mutants compared to the cellular tumor zones. Moreover, we revealed that ischemia induces the unfolded protein response (UPR) pathway. Among UPR-related molecules, KRAS and PI3K upregulate ERN1, which encodes the transmembrane protein kinase IRE1, in ischemic conditions. Inhibition of ERN1 not only prevented ischemia-induced cell death but also inhibited the p38 MAPK-MK2 pathway. Furthermore, we observed an increase in the expression of pro-inflammatory cytokine IL-8 under ischemic conditions, a response that was prevented by inhibitors of p38 MAPK or MK2. Lastly, we found that ischemia induces ATP release and calreticulin exposure, indicating the cell death possessing certain immunogenic cell death features. In summary, our findings revealed a pivotal role of KRAS and PI3K in inducing cell death in the ischemic GBM tumor microenvironment through activating the IRE1-p38 MAPK-MK2 pathway. The cell death under this circumstance displays pro-inflammatory and immunogenic properties. Therefore, the IRE1-p38 MAPK-MK2 pathway may be exploited for the treatment of GBM in which RAS or PI3K is activated. Citation Format: Soo Yeon Kim, Miaolu Tang, Stephen Chih, Jessica Thorpe, Tong Lu, Hong-Gang Wang, Wei Li. RAS and PI3K activation promote glioblastoma progression and ischemia-associated tumor cell death [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 393.

Abstract 4219: Establishing CAF-like and TAM-like transformation induced by TNBC culture supernatant in 3D in-vitro culture as a model for targeted drug testing

Abstract Several previous studies have highlighted the significance of cell-to-cell communication, or crosstalk, between tumor cells and non-tumorigenic cells in the context of cancer progression and metastasis. Within the tumor microenvironment (TME), these interactions have the potential to alter the phenotypes and behaviors of normal cells. The use of 2D in-vitro cultures has been limited due to their inability to accurately replicate the intricate in-vivo TME. Conditioned medium (CM) derived from cultured cancer cells contains secreted factors that may influence the phenotype and functionality of normal cells. In our investigation, the exposure of normal murine fibroblast NIH3T3 and macrophage RAW 264.7 cells to conditioned medium (CM) obtained from malignant mammary epithelial 4T1 cells (4T1CM) resulted in a modified phenotype with enhanced cell viability. Treatment with 4T1CM led to the upregulation of genes, including α-smooth muscle actin (αSMA), IL-10, CD206, and vascular endothelial growth factor (VEGF), in NIH3T3 and RAW 264.7 cells compared to their respective control cells. Additionally, NIH3T3 cells treated with 4T1CM exhibited an epithelial-mesenchymal transition (EMT) phenotype, as indicated by the regulation of EMT markers such as E-cadherin, β-catenin, N-cadherin, and Vimentin. Notably, RAW 264.7 cells treated with 4T1CM showed an upregulation of cyclooxygenase-2 (COX-2) and programmed death-ligand 1 (PDL-1), suggesting a propensity for an inhibitory immune response. Moreover, NIH3T3 cells conditioned with 4T1CM demonstrated an upregulation of stemness markers, including sex determining region Y-box 2 and Aldehyde dehydrogenase. In summary, our study highlights the potential role of 4T1CM in transforming normal NIH3T3 and RAW 264.7 cells into cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). Ongoing proteomics analysis aims to map differentially expressed proteins potentially regulating the 4T1CM-induced transformation. To further investigate, a series of experiments using mono- and co-cultures of these cells are underway, specifically targeting the transformed macrophages and fibroblasts with the aim of either re-educating or eliminating them. This treatment involves drug combinations that specifically target the identified signaling pathway intermediates, offering novel therapeutic intervention strategies. Citation Format: Biplov Sapkota, Naveen Chintalaramulu, Abhishek Pandit, Shilpa Thota, Rizwana Begum, Amirsalar Mansouri, Jiri Adamec, Joseph Francis. Establishing CAF-like and TAM-like transformation induced by TNBC culture supernatant in 3D in-vitro culture as a model for targeted drug testing [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 4219.

Abstract 6525: Modulating the immune response in cholangiocarcinoma: Targeting sulfatase-2 for therapeutic intervention

Abstract Cholangiocarcinoma (CCA) is an aggressive epithelial malignancy of the bile ducts. Recent reports have suggested a rise in the incidence of CCA globally. Current treatments for CCA have limited effectiveness, with only 20-30% response rates in patients with advanced cancer. Thus, there is a growing need to investigate new therapeutic targets for CCA. The tumor microenvironment (TME) is a potential target for novel therapies. The TME plays an important role in progression and metastasis of cancer through facilitating immune escape. Recent studies have demonstrated potential roles for heparan sulfate proteoglycans (HSPGs) in tumorigenesis. Sulfatase 2 (SULF2) is a heparan sulfate editing sulfatase that removes the 6-O sulfate moiety within the heparan sulfate (HS) chains and alters the affinity of HS chains for growth factors and cytokines and their receptors, thus modulating HSPG function in cell signaling pathways. HSPG specific sulfatases have been recently identified as a potential therapeutic target in CCA. We aimed to assess the effects of anti-Sulfatase-2 antibody on immune cells in the tumor microenvironment of syngeneic CCA xenografts in C57BL6 mice. Mice bearing syngeneic CCA xenografts were treated with an anti-sulf2 antibody, and the tumors compared to untreated control xenografts. After treatment, the tumors were harvested and analyzed by Cytometry by time of flight (Cytof) to identify changes in the types and numbers of immune cells in the tumor xenografts. Treatment with anti-sulf2 antibody resulted in a decrease in immunosuppressive cells such as tumor associated macrophages (TAMs) and myeloid derived suppressor cells (MDSC) compared to the control. There was also an increase in anti-tumor cells such as T-cells and NK cells, suggesting that suppressing sulf-2 in the microenvironment can shift the TME toward an anti-tumorigenic rather than a pro-tumorigenic environment. In summary, our work demonstrates the potential to target Sulfatase 2 as a therapeutic mechanism for immunomodulation of the tumor microenvironment. Future experiments to determine the relationship between specific clinical phenotypes and the number, activities and types of immune cells seen in the TME, and experiments in other hepatobiliary cancers models, such as hepatocellular carcinoma and gallbladder cancer, will be conducted. Citation Format: Tayla R. Brooks, Nellie Campbell, Melanie Keim, Lewis R. Roberts. Modulating the immune response in cholangiocarcinoma: Targeting sulfatase-2 for therapeutic intervention [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 6525.

Abstract 2847: Study of spatial and temporal epigenetic heterogeneity in neuroblastoma suggests role of H3K27me3-mediated repression

Abstract Introduction: Trimethylation of Histone 3 Lysine 27 (H3K27) is widely understood to be a mark associated with gene repression and linked to gene silencing during development. Neuroblastoma, a developmental pediatric extracranial solid malignancy, has a relatively silent mutational landscape. Therefore, epigenetic modifications, including H3K27me3 mediated repression, could explain underlying developmental origins and the wide clinical heterogeneity observed. While previous studies have focused on the role of distinct super enhancer histone marks and their core regulatory circuits, less is understood about repressive control. We aimed to therefore investigate the role of H3K27me3-mediated repression in Neuroblastoma patients. Methods: Genome-wide H3K27me3 ChIP-Seq profiles were obtained from 48 diagnostic tissue biopsies covering all clinical and molecular patient subgroups. Transcriptome sequencing, ChIP-Seq of additional histone marks and WES/WGS was also performed on the same samples and on a panel of Neuroblastoma cell line models, with multiomics integrative analysis to validate findings. HMCAN, deepTools, DiffBind and ROSE packages were used to analyze the ChIP-seq data. Results: A comparison of H3K27me3 regions across all patient ChIP-Seq profiles, identified commonly repressed genes specific to Neuroblastoma. These genes are enriched in development and differentiation pathways, with significantly lower RNA expression levels than other publicly available pediatric tumor datasets. When compared to publicly available single cell RNAseq of the healthy adrenal medulla, we can further highlight a potential role of H3K27me3-mediated repression in regulating cell lineage commitment and differentiation ability. In addition, we identified that patient samples cluster into their clinical subgroups based on H3K27me3 patterns and differentially repressed genes, specifically between MYCN-amplified and non-amplified high-risk groups. A CRISPR screen to further validate the functional role of these repressed genes is being carried out. Conclusions: To our knowledge, this study shows for the first time that understanding of H3K27me3 mediated repression in Neuroblastoma, can uncover new insights into both disease development and clinical subclassifications. This knowledge will be important when considering future potential novel treatment options. Citation Format: Charlotte Butterworth, Alexandra Saint-Charles, Yasmine Iddir, Jaydutt Bhalshankar, Angela Bellini, Charles Bobin, Elisa Wecht, Young-Gyu Park, Caroline Louis, Virginie Raynal, Dominique Plantaz, Hervé Sartelet, Valérie Combaret, Gaelle Pierron, Isabelle Janoueix-Lerosey, Kai-Oliver Henrich, Olivier Delattre, Frank Westermann, Gudrun Schleiermacher. Study of spatial and temporal epigenetic heterogeneity in neuroblastoma suggests role of H3K27me3-mediated repression [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 2847.

Abstract 3979: PD-1 signals regulate NKG7 that is required for CD8+ T cells to sustain cytotoxicity and durable antitumor memory responses

Abstract Cytotoxic CD8+ T cells play a key role in response to anti-PD-1/PD-L1 immune checkpoint inhibitors (ICI) therapy. However, the exact effector molecules that are regulated by PD-1 signals in cytotoxic CD8+ T cells have not been completely defined. Lack of this knowledge is a crucial problem in addressing how to overcome the low clinical response rate to ICI therapy. When exploring why certain patients not responding to ICI therapy, we found non-responders did not have an elevated NKG7 expression in CD8+ T cells as responders to ICI therapy. The difference in NKG7 expression in CD8+ T cells between non-responders and responders to anti-PD-1 therapy prompted us to investigate the potential role of PD-1 signaling in regulating NKG7 expression. To that end, we generated a CD8+ T cell-specific Pdcd1 conditional knockout mice (CD8 E8ICre-Pdcd1fl/fl, also known as CD8-PD-1 cKO) where Pdcd1 is deleted after thymic selection of single positive CD8+ T cells. We observed a delayed tumor growth and prolonged survival in CD8-PD-1 cKO mice compared to control mice after B16-OVA melanoma tumor challenge. On Day 12, when the CD8-PD-1 cKO mice demonstrated their ability to control tumor growth, we conducted single cell RNA sequencing on isolated tumor infiltrating CD8+ T cells, revealing a significant increase of Nkg7 expression in effector CD8+ T cells in the Pdcd1 deficient CD8+ T cells compared to the control wild type T cells. Pdcd1-deficient effector CD8+ T cells also had increased expression of genes linked to NFAT/Calcineurin signaling pathway including Nfatc1, S100a4, Tbet and Tox, suggesting Nkg7 could be regulated by Pdcd1 via the NFAT pathway. To determine the impact of increased NKG7 expression in CD8+ T cells, we used OVA protein as a surrogate tumor antigen in vaccination. We found a complete B16-OVA tumor rejection in CD8-PD-1 cKO mice compared to control mice when tumor cells were injected on day 7 after vaccination with OVA protein and poly (I:C) (as an adjuvant), but only a partial rejection on day 21. The CD8-PD-1 cKO mice that were tumor-free for 30-40 days rejected 2nd tumor challenge with B16-OVA tumors. This long-term protection is tumor antigen (OVA) specific because these mice did not reject B16F10 tumor cells that do not express OVA antigen. We also found effector memory CD8+ T cells with high cytotoxicity increased in the spleen of CD8-PD-1 cKO mice after OVA antigen immunization compared to control. In conclusion, we found NKG7 is one of the effector molecules in cytotoxic CD8+ T cells, which can be regulated by PD-1 signals through NFAT signaling. Targeting the PD-1-NFAT pathway may be a new option to overcome low responses of T cells to ICI therapy in treatment of refractory cancer. Citation Format: Zhiming Mao, Joanina Gicobi, Jacob Hirdler, Xin Liu, Michelle Hsu, Emilia Dellacecca, Wenjing Zhang, Fabrice Lucien-Matteoni, Hai dong. PD-1 signals regulate NKG7 that is required for CD8+ T cells to sustain cytotoxicity and durable antitumor memory responses [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 3979.

Expression of IGF-1R in oral submucous fibrosis and oral squamous cell carcinoma – An RT-PCR study

ObjectivesWith the rising number of cases of oral submucous fibrosis (OSMF) undergoing malignant transformation into oral squamous cell carcinoma (OSCC), it is needed to detect the changes at the earliest. Several molecular markers have been used for this purpose, and insulin-like growth factor receptor 1 (IGF-1R) is one of the markers in vogue for several human malignancies. Thus, we compared the expression of IGF-1R mRNA in normal oral mucosa (NOM), OSMF, and OSCC. Materials and methodsThirty biopsy tissue samples for each of the three groups, NOM, OSMF, and OSCC, were taken and analyzed histopathologically and then subjected to RT-PCR for evaluation of the expression of IGF-1R mRNA in each of them. ResultsThere was an upregulation of IGF-1R mRNA in OSMF tissue samples as compared to NOM, but a downregulation as compared to OSCC. There was a 3.24-fold upregulation of IGF-1R mRNA expression in OSMF as compared to NOM and a 1.31-fold downregulation as compared to OSCC. Likewise, there was a 4.2-fold upregulation of IGF-1R mRNA expression in OSCC as compared to NOM and 1.31-fold upregulation as compared to OSMF. Moreover, the expression was more in OSMF patients with interincisal opening (IIO) < 27 mm as compared to those with IIO >27 mm and also was found to increase with the grade of OSCC. ConclusionThe study reflected a potential role of IGF-1R in the pathogenesis of OSMF and OSCC, thus implying its possible exploitation as a diagnostic and prognostic marker and also as a therapeutic target in future.