Significant Reduction In Proliferation Research Articles

Osteoking inhibits apoptosis of BMSCs in osteoporotic rats via PI3K/AKT signaling pathway.

This study aims to investigate whether Osteoking inhibits apoptosis of BMSCs in osteoporotic rats via the PI3K/AKT signaling pathway and to conduct a detailed exploration of this mechanism. The goal is to provide a theoretical basis for the clinical application of Osteoking in osteoporosis treatment. A rat model of osteoporosis was established through bilateral ovariectomy (OVX), followed by treatment with Osteoking. After ten weeks of therapy, BMD was evaluated. The biomechanics of the left tibia were measured, the left femur was sequenced, and the right tibia was stained using histomorphometric and Masson's staining methods. Peripheral serum was collected to measure bone-related markers, including E2, PINP, and CTX. RNA-Seq results were verified using the remaining bone samples. Comparative analysis demonstrated the efficacy of Osteoking in treating osteoporosis and provided preliminary insights into the underlying mechanisms. Primary BMSCs were cultured using bone marrow apposition. CCK8 assays were conducted to screen the intervention conditions of Osteoking and LY294002. Various concentrations of Osteoking-containing serum and LY294002 were tested separately to determine the optimal intervention concentration for drug delivery. The impact of Osteoking on lipid formation was also evaluated. Following treatment of BMSCs from OVX rats with Sham serum, OVX serum, OVX+LY294002 serum, and Osteoking+LY294002 serum, the expression of PI3K/AKT/mTOR, osteogenesis-related regulatory factors, and apoptosis-related regulatory factors was assessed. Flow cytometry was employed to evaluate apoptosis in BMSCs. Osteoking significantly improved whole-body BMD and bone biomechanical indices in OVX rats. It also significantly elevated the serum levels of E2 and PINP while reducing the level of CTX, which significantly improved bone microstructure and promoted new bone formation. RNA-seq analysis indicated that the therapeutic mechanism involved the PI3K/AKT signaling pathway. Osteoking increased the expression of RUNX2 and decreased the expression of PPAR-γ, a marker of lipogenesis, in OVX rats. Extraction of BMSCs for subsequent studies revealed a significant reduction in proliferation and osteogenic differentiation, along with an increase in lipogenic differentiation, in the OVX group. Osteoking treatment inhibited the expression of PPAR-γ and increased the expression of RUNX2 in BMSCs. Additionally, Osteoking reversed the LY294002-mediated inhibition of PI3K/AKT/mTOR signaling pathway activation, increased the expression of the apoptosis-protecting protein Bcl2, and decreased the expression of apoptosis-associated proteins Caspase3 and Bax. Osteoking markedly improved bone microstructure, biomechanics, and bone density in OVX rats. Osteoking-containing serum reversed the imbalance in lineage differentiation in OVX rats, characterized by reduced osteogenic differentiation and increased lipid differentiation of BMSCs. Furthermore, Osteoking-containing serum significantly increased BMSC proliferation and prevented apoptosis in OVX rats through the PI3K/AKT signaling pathway.

Development of a prognostic model for NSCLC based on differential genes in tumour stem cells

Non-small cell lung cancer (NSCLC) constitutes a significant portion of lung cancers and cytotoxic drugs (e.g. cisplatin) are currently the first-line treatment. However, NSCLC has developed resistance to this drug, which limits the therapeutic effect and thus affects prognosis. NSCLC sc-RNA-seq data were downloaded from the GEO database and Ku Leuven Laboratory for Functional Epigenetics, and bulk RNA-seq data were obtained from the TCGA database. The “Seurat” package was employed for scRNA-seq data processing, and the uniform manifold approximation and projection (UMAP) were applied for downscaling and cluster identification. Use the FindAllMarkers function to find differential genes (DEGs) for tumor stem cells. Then, we performed univariate regression analyses on the DEGs to identify potential prognostic genes. We created a machine learning framework based on potential prognostic genes, which combines 10 machine learning methods and their 101 combinations to get the optimal prognostic risk model. The model was evaluated in the training set and validation set. A nomogram was developed to provide physicians with a quantitative tool for prognosis prediction. Finally, we evaluated the expression and functionality of SLC2A1. We discovered 22 cell clusters containing 218379 cells by examining single-cell RNA sequencing datasets (GSE148071, KU_lom, GSE131907, GSE136246, GSE127465). Tumour cells were isolated for subpopulation analysis and 162 differential genes from SOX2_cancer were obtained. After univariate Cox analysis, we found 23 genes with prognostic potential prognostic value and utilized them to develop 101‑combination machine learning computational framework. We eventually picked the best performing ‘StepCox[both] + RSF’, which includes 8 genes. The model has a relatively high prediction accuracy in both TCGA and GEO datasets. In in vitro investigations, targeted suppression of the SLC2A1 gene resulted in significant reductions in proliferation, invasion and migration in A549 cells. In addition, a significant reduction in cisplatin resistance was seen in A549/DDP cells. The outcomes demonstrated the precision and credibility of the prognostic model for NSCLC, highlighting its potential significance in the treatment and prognosis of individuals affected by this disease. SLC2A1 may become a promising prognostic marker and a potential therapeutic target, offering valuable insights to inform clinical treatment decisions.

Abstract 6605: Understanding the role of sumoylated Etv1 in mammary oncogenesis

Abstract The Small Ubiquitin-like Modifier (SUMO) pathway is a major post-translational regulatory mechanism involved in a variety of cellular functions including the DNA damage response, transcriptional regulation, and carcinogenesis. Recent discoveries by our group demonstrated that inhibiting the SUMO pathway can repress the growth of breast cancer xenografts and Neu-driven mammary oncogenesis in a genetically engineered mouse model with conditional knockout (CKO) of the SUMOylation E2 conjugating enzyme, Ube2i. However, despite the consistency of the findings, there remains considerable debate concerning the mechanisms through which SUMO inhibition represses tumorigenesis. Our downstream analysis suggested ETS Translocation Variant 1, encoded by ETV1 gene, acts as a SUMO-sensitive transcription factor that drives mammary oncogenesis and cancer growth. To examine oncogenic properties of SUMO-conjugated ETV1 in breast cancer, we cloned HA-tagged WT Etv1, SUMO-resistant Etv15xKR, or SUMO mimetic SUMO-1-Etv15xKR into a lentiviral vector with a GFP cassette. Proliferation, colony formation in soft agar, and in vivo xenograft assays were evaluated in the Ube2iF/F cell line derived from a tumor in a MMTV-c-neu/Ube2ifl/fl animal. We did ChIP-seq in Ube2iF/F cells using SUMO-1 and Etv1 antibodies to examine the effects of SUMO conjugation on Etv1 genomic occupancy. RNA-seq using siEtv1 or AdCre was also performed to compare changes in gene expression with SUMO-conjugated Etv1 genomic occupancy. Inhibition of SUMOylation via AdCre caused a significant reduction in proliferation and colony formation in vitro and completely block the formation of flank xenografts in vivo. Interestingly, expressing a SUMO-resistant ETV15xKR rescued in vitro growth effects, whereas a SUMO mimetic SUMO-1-ETV15xKR had no effect on restoring proliferation. On the contrary, in vivo xenograft assays demonstrated that the Etv15xKR-SUMO1 efficiently induced tumor formation despite the loss of Ube2i while the sumo-resistant Etv15xKR was unable to rescue tumorigenesis. Bulk RNA-seq and ChIP seq performed demonstrated different patterns of gene regulation between sumoylated and unsumoylated Etv1. Gene set enrichment analysis (GSEA) showed genes associated with cancer stemness were significantly enriched in cells expressing Etv15xKR-SUMO1. Collectively, these observations support Etv1 acting as a SUMO-sensitive transcription factor that drives a stem cell phenotype necessary for mammary oncogenesis and progression. Citation Format: Zhijie Li, Kelsey E. Koch, Dakota T. Thompson, Dana M. Van der Heide, Jeremy Chang, Christopher M. Franke, Mohammed O. Suraju, Anna C. Beck, Allison W. Lorenzen, Jeffrey R. White, Mikhail V. Kulak, David K. Meyerholz, Colin Kenny, Ronald J. Weigel. Understanding the role of sumoylated Etv1 in mammary oncogenesis [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 6605.

High CD133 expression in proximal tubular cells in diabetic kidney disease: good or bad?

BackgroundProximal tubular cells (PTCs) play a critical role in the progression of diabetic kidney disease (DKD). As one of important progenitor markers, CD133 was reported to indicate the regeneration of dedifferentiated PTCs in acute kidney disease. However, its role in chronic DKD is unclear. Therefore, we aimed to investigate the expression patterns and elucidate its functional significance of CD133 in DKD.MethodsData mining was employed to illustrate the expression and molecular function of CD133 in PTCs in human DKD. Subsequently, rat models representing various stages of DKD progression were established. The expression of CD133 was confirmed in DKD rats, as well as in human PTCs (HK-2 cells) and rat PTCs (NRK-52E cells) exposed to high glucose. The immunofluorescence and flow cytometry techniques were utilized to determine the expression patterns of CD133, utilizing proliferative and injury indicators. After overexpression or knockdown of CD133 in HK-2 cells, the cell proliferation and apoptosis were detected by EdU assay, real-time cell analysis and flow analysis. Additionally, the evaluation of epithelial, progenitor cell, and apoptotic indices was performed through western blot and quantitative RT-PCR analyses.ResultsThe expression of CD133 was notably elevated in both human and rat PTCs in DKD, and this expression increased as DKD progressed. CD133 was found to be co-expressed with CD24, KIM-1, SOX9, and PCNA, suggesting that CD133+ cells were damaged and associated with proliferation. In terms of functionality, the knockdown of CD133 resulted in a significant reduction in proliferation and an increase in apoptosis in HK-2 cells compared to the high glucose stimulus group. Conversely, the overexpression of CD133 significantly mitigated high glucose-induced cell apoptosis, but had no impact on cellular proliferation. Furthermore, the Nephroseq database provided additional evidence to support the correlation between CD133 expression and the progression of DKD. Analysis of single-cell RNA-sequencing data revealed that CD133+ PTCs potentially play a role in the advancement of DKD through multiple mechanisms, including heat damage, cell microtubule stabilization, cell growth inhibition and tumor necrosis factor-mediated signaling pathway.ConclusionOur study demonstrates that the upregulation of CD133 is linked to cellular proliferation and protects PTC from apoptosis in DKD and high glucose induced PTC injury. We propose that heightened CD133 expression may facilitate cellular self-protective responses during the initial stages of high glucose exposure. However, its sustained increase is associated with the pathological progression of DKD. In conclusion, CD133 exhibits dual roles in the advancement of DKD, necessitating further investigation.

Investigating the potential of zinc oxide nanoparticles and zinc ions as promising approaches to lung cancer

Lung cancer is the leading cause of cancer casualties globally, with adverse side effects and drug resistance making current therapeutic options inadequate. Therefore, novel therapeutics require development. A major genetic contributor to these cancers is the mutant TP53 gene, whose mutated protein fails to transactivate p21, a cell cycle control protein in p53-p21-RB signaling, resulting in uncontrolled cell growth and cisplatin resistance. Recent studies suggest zinc oxide nanoparticles (ZnO NPs) show promise as a targeted, cytotoxic therapy; however, their mechanisms and efficacy are strongly debated as they aggregate in solution, reducing bioavailability while demonstrating poor aqueous solubility. As such, we hypothesized that a combined treatment using either ZnO NPs and cisplatin or zinc ions and cisplatin would induce cancer cell apoptosis and reduce cisplatin resistance. We treated mutant p53 lung cancer cells with ZnO NPs, zinc ion solution isolated from the NP suspension, and/or cisplatin; all significantly reduced proliferation individually and in combination. Increased reactive oxygen species (ROS) and p21 upregulation from ZnO NP treatment suggests mechanisms involving p21, perhaps through a p53-independent mechanism or via p53 reactivation. Zinc ion solution rendered a reduction in p21 in the presence of significantly lower ROS induced by ZnO NP, suggesting a mechanism needing further elucidation. We subjected a control cell line, HaCaT cells, to the same treatments and demonstrated no significant reduction in proliferation in the presence of ZnO NPs or zinc ions. Our research suggests the use of ZnO NPs and zinc ions as potential strategies for lung cancer treatment regimens.

MiR-325-3p Reduces Proliferation and Promotes Apoptosis of Gastric Cancer Cells by Inhibiting Human Antigen R.

Human antigen R (HuR), also known as ELAVL1, is a widely expressed RNA-binding protein (RBP) that has a significant impact on the development and advancement of tumors. Our previous study found that 5-fluorouracil (5-FU) may impede the proliferation and increase apoptosis in gastric cancer cells by reducing the nucleocytoplasmic shuttling of HuR. However, how posttranscriptional regulation influences HuR functions in gastric cancer remains to be elucidated. Here, we demonstrated that miR-325-3p has the potential to regulate the expression level of HuR by directly binding to its 3'UTR, which in turn led to a significant reduction in proliferation and an increase in apoptosis in gastric cancer cells. In addition, xenograft experiment showed that knockdown of HuR or overexpression of miR-325-3p group exhibited smaller tumor sizes after transplant of gastric cancer cells into zebrafish larvae. Thus, our findings offer new insights into the pathogenesis of gastric cancer and may potentially assist in identifying novel targets for drug therapy.

Proliferation-Related Features of the Human Mesenchymal Stem Cells Derived from Palatine Tonsils, Adipose Tissues, and Bone Marrow.

Mesenchymal stem cells (MSCs) are widely used in regenerative medicine and cell-based transplantations. However, an in-depth comparison of the different MSC origins is lacking. This study aimed to compare the expression of adipose-derived (AMSCs), bone marrow-derived (BMSCs), and tonsil-derived (TMSCs) and evaluate whether TMSCs are good alternatives for AMSCs or BMSCs. We analyzed the expression levels of 47,000 transcripts in AMSCs (n = 4), BMSCs (n = 4), and TMSCs (n = 4) using GeneChip. Microarray data were analyzed using the LIMMA package to compare the TMSCs, AMSCs, and BMSCs. Hub genes were analyzed using STRING and Cytoscape. To ascertain the functional roles of AURKA and AURKB, small interfering RNA (siRNA) molecules specifically targeting AURKA and AURKB mRNA were synthesized and employed to induce knockdown of AURKA and AURKB in TMSC and AMSC. We analyzed the expression level of OCT4, SOX-2, and NANOG genes in TMSC and AMSCs by cell culture and real-time PCR. We identified commonly increased 256 and decreased 160 genes in TMSCs from the differentially expressed genes (DEGs) between the TMSCs, AMSCs, and BMSCs. In the DEG-based protein-protein interaction and gene set enrichment analysis, hub genes (AURKA, AURKB, CDC20, and BUB1) highly expressed in TMSCs were enriched for development- and progression-related oocyte meiosis, the cell cycle, and ubiquitin-mediated proteolysis. In vitro analysis demonstrated that cells with downregulated expression of AURKA and AURKB exhibited a significant reduction in proliferation compared to control cells. However, silencing of the genes did not affect the differentiation capacity in TMSCs and AMSCs. Our study compared MSCs of different origins to better understand the similarities and differences among these cell types.

O011 Investigating the effect of supraphysiological levels of calcium on irreversible electroporation (IRE) of pancreatic cancer cell lines

Abstract Introduction Pancreatic cancer remains difficult to treat with a 5-year survival of 2%. Loco-regional therapies are being introduced such as Irreversible Electroporation (IRE); a non-thermal ablative therapy thought to cause cell death by increasing the cell membrane potential resulting in the formation of aqueous pores, leading to cell death. Mitochondrial changes have previously been reported to be a key driver of apoptosis and calcium has been implicated in this process. We aimed to study the influence of calcium in combination with IRE in pancreatic cancer. Methods Immortalised pancreatic cancer cell line, PANC-1, and pancreatic cancer patient-derived xenograft cells (PDX 185) were exposed to calcium (Ca2+) and IRE monotherapy and in combination. Cells were trypsinised and electroporated in 4mm cuvettes with a BTX generator (Harvard Apparatus). Cell proliferation and viability were analysed, and depolarised mitochondria were evaluated by TMRE and analysed using flow cytometry. Results IRE combined with calcium was more cytotoxic compared to monotherapy with cell proliferation assays demonstrating a statistically significant reduction in proliferation (p<0.0001) across both cell lines. Interestingly, mitochondrial membrane potential analyses demonstrated a depolarising effect only in cells exposed to both calcium and IRE suggesting a mitochondrial mediated effect in cell death in these groups. Conclusion Calcium in combination with IRE may act to potentiate cell death through depolarisation of the mitochondrial membrane. Future work will involve understanding the impact of IRE in combination with calcium in 3D models, allowing the evaluation of molecular cell death mechanisms.

Abstract 1192: Branched-chain keto acids exert an immune-suppressive and neurodegenerative microenvironment in CNS leptomeningeal lymphoma

Abstract Leptomeningeal disease is a devastating complication characterized by the rapid onset of debilitating neurological symptoms and a markedly poor prognosis. It is diagnosed in 5-15% of hematological cancers. Here, we aim to identify the metabolic and immune remodeling of the tumor leptomeningeal microenvironment in Non-Hodgkin lymphoma through comprehensive multi-omic analysis of patient specimens, in vivo models, and in vitro functional studies. Cerebrospinal fluid (CSF) from leptomeningeal lymphoma (LML) patients and tumor-free controls were collected and analyzed using single-cell RNA sequencing (scRNA-Seq), metabolomics, proteomics, and lipidomics. Patients’ CSF cellular components were highly enriched with heterogeneous B-cell populations. Few T-lymphocytes in LML patients showed expression of activation or proliferation markers. Poor prognosis was associated with significant infiltration of macrophages and a lack of dendritic cells. These findings were confirmed to be unique to LML using immune-competent animal models where tumor cells were injected intrathecally, and into lymph nodes. Moreover, LML patients’ CSF showed an accumulation of branched-chain keto-acids (BKAs), which are well-known neurotoxins, metabotoxins, and acidogens. Absolute quantification of individual BKAs was confirmed by high-performance liquid chromatography and showed a 30-fold increase in LML patients’ CSF. To investigate the effect of BKAs accumulation on T-lymphocytes, we examined their viability, proliferation, and activation with BKAs treatment in the context of physiological CSF. Cell trace violet-labeled T lymphocytes showed a significant reduction in proliferation and viability with increasing doses of BKAs. Additionally, a significant decline in the secretion of TNF-α, interferon-γ, granzyme B, and IL-2 was found using ELISA assays, indicating a reduction in T-lymphocyte activation. The proteomic and lipidomic analysis of patients’ CSF also revealed a significant downregulation in proteins and lipids vital for neuronal development, synaptic organization, and myelin sheath integrity. Similarly, scRNA-Seq of the leptomeningeal layer collected from the LML mouse model revealed compromised leptomeningeal integrity. Neurological scoring of the LML mouse model showed disease progression to be associated with rapid neurological decline. To investigate the direct effect of BKAs on the leptomeningeal and neuronal integrity, we tested different concentrations of BKAs on murine primary neuronal cells and human leptomeningeal cells (HMC). MTT assays showed a significant reduction in HMC metabolic activity after 72 hours and in neuronal metabolic activity after 7 days. In conclusion, our data unveil the immunosuppressive and neurodegenerative role of BKAs in the leptomeningeal tumor microenvironment. Citation Format: Mariam Lotfy Khaled, Gerald Wallace, Brittany Evernden, Zhihua Chen, Hasan Alhaddad, Yuan Ren, Oscar Ospina, Maclean Hall, Ann Chen, Timothy J. Robinson, John Koomen, Shari Pilon-thomas, Peter Forsyth, Inna Smalley. Branched-chain keto acids exert an immune-suppressive and neurodegenerative microenvironment in CNS leptomeningeal lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1192.

Abstract 2009: Gemcitabine-loaded EGFR aptamer for targeted therapy of lung cancer

Abstract Lung cancer is the leading cause of cancer-related death in the world and non-small cell lung cancer (NSCLC) is the most common types of the lung cancer. Epidermal growth factor receptor (EGFR) is an attractive therapeutic target due to its high expression in NSCLC. Gemcitabine (Gem) has shown antitumor activity against many kinds of solid tumors, including NSCLC. Aptamers emerged as promising drug delivery vehicles because of their high binding affinity for the target molecules. The objective of this study was to develop the aptamer-drug conjugates using the EGFR-specific aptamer. EGFR aptamers selected by the SELEX process and gemcitabine was internally incorporated into the EGFR aptamer. In vitro binding affinity of EGFR and Gem-loaded EGFR aptamer was evaluated on EGFR-positive A549, H460, H1299 cell lines, as well as EGFR-negative H522 cells using confocal microscopy. To determine whether the Gem-loaded EGFR aptamer was effectively internalized, Z-stack images were acquired after 1 hr incubation of Cy5-conjugated EGFR aptamer at 37°C. The ex vivo biodistribution of Cy7-conjugated EGFR aptamer was investigated in NSCLC xenograft nude mice. To confirm the antiproliferative effects of Gem-loaded EGFR aptamer, the MTT cell proliferation assay was carried out in NSCLC cells. In the confocal image of EGFR-positive cells, Gem-loaded EGFR aptamers were selectively bound to the cell surface, while no aptamer binding was observed in H522 cells. Z-stack imaging showed that internalized Gem-loaded EGFR aptamer accumulated in EGFR-positive cells. The ex vivo biodistribution data showed that the Gem-loaded EGFR aptamer exhibited higher binding in EGFR-positive A549 tumors than scramble EGFR aptamer. A549 cells showed significant reduction in proliferation following treatment with Gem-loaded EGFR aptamer. Our results indicated that Gem-loaded EGFR aptamer has a potential therapeutic agent in EGFR-positive NSCLC therapy. Citation Format: Jun Young Park, Ye Lim Cho, Ju Ri Chae, Won Jun Kang. Gemcitabine-loaded EGFR aptamer for targeted therapy of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2009.

Abstract P2-16-07: Overexpression of the alpha subunit of the epithelial sodium channel in MDAMB231 breast cancer cells reduces cell migration and proliferation

Abstract Breast cancer is the most common cancer diagnosed in women worldwide and leads to 600,000+ deaths annually. The vast majority of breast cancer-related deaths are attributed to metastasis from the primary site in the breast to visceral organs or the brain. Therefore, understanding how these cells achieve metastasis is vital. Ion channels, such as the epithelial sodium channel (ENaC), are emerging as new targets for cancer research due to their role in regulating cell functions such as the process that allows cancer cells to undergo phenotypic changes necessary for metastasis. Ion channel dysregulation may be crucial in cancer development due to the ion channels’ potential role in controlling cancer cell characteristics such as migration and proliferation. There is limited research investigating the role ENaC may have in breast cancer. We have shown that more migratory, mesenchymal breast cancer cell lines have reduced mRNA and protein expression of ENaC compared to more epithelial breast cancer cell lines. Importantly, higher expression levels of the pore-forming alpha-ENaC subunit is correlated with an increased breast cancer survival in patients (n=1705) included in the SCAN-B data set. Bioinformatic analysis, using the SCAN-B data set, showed an increase in the expression of alpha-ENaC mRNA correlated with lower expression levels of migration and proliferation markers, suggesting ENaC has a role in breast cancer cell proliferation and migration. Therefore, this research examines the influence of stably overexpressing the pore-forming subunit of ENaC, alpha-ENaC, in breast cancer cells on their cell migration and proliferation ability. We hypothesised that increasing the expression of ENaC will restore the cells to a more epithelial state, thus reducing cell migration and proliferation. MDAMB231 breast cancer cells were engineered via a stable transfection to overexpress alpha-ENaC or to express an empty vector control. Several clones were isolated and expanded and using RTqPCR, alpha-ENaC mRNA overexpression was confirmed to consistently have 200-fold increased mRNA expression compared to control cells (n=9, p=0.0006). An increase in alpha-ENaC protein expression was also confirmed via western blotting. Using an EdU assay the alpha-ENaC overexpressing cells were shown to have a significant reduction in proliferation compared to the control cell line (n=3, p=0.0048). The alpha-ENaC overexpressing cells showed reduced migratory ability when examined in two established migration assays. In scratch wound assays, the alpha-ENaC overexpressing cells had a significant reduction in cell migration at 24 hours post scratch (n=4, p=0.0001). In Boyden chamber assays, the number of alpha-ENaC-overexpressing cells that migrated through the membrane was significantly reduced compared to the control cell line (n=4, p=0.02). Our results suggest that increased sodium entry into the cell through ENaC has a role in preventing the development of metastatic breast cancer cells, and highlights ENaC as a potential target for future breast cancer therapies. Citation Format: Sarah McQueen, Adam Ware, Heather Cunliffe, Fiona McDonald. Overexpression of the alpha subunit of the epithelial sodium channel in MDAMB231 breast cancer cells reduces cell migration and proliferation [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-16-07.

Effects of Curcumin and Lactoferrin to Inhibit the Growth and Migration of Prostatic Cancer Cells.

Prostate cancer remains one of the main causes of death for men worldwide. Despite recent advances in cancer treatment, patients develop resistance after an initial period of optimal efficacy. Nowadays, it is accepted that natural compounds can result in health benefits with a preventive or adjuvant effect. The purpose of this study was to evaluate the effects of curcumin (CU), a bioactive compound in the spice turmeric, and lactoferrin (LF), a natural glycoprotein with immunomodulatory properties, on DU145 and PC3. Prostate cancer cells were cultured with and without LF (175 μM) and CU (2.5 μg/mL and 5 μg/mL), alone and in combination. Cell viability, migration ability, death receptors (DRs), and integrins (α3, β1) gene expression were evaluated, as well as human annexin V quantification and Akt phosphorylation. Differences among cells group, defined according to the treatment used, were assessed with ANOVA. The results showed that the effects of CU and LF are different between the two prostatic cell lines analyzed. In DU145, a reduction in cell proliferation and migration is reported both in the presence of single and combined treatments. In PC3 cells, there is a significant reduction in proliferation in the presence of CU alone, while the inhibition of migration is mainly related to the LF treatment and its combination with CU, compared to untreated cells. Moreover, the reduction in gene expression of integrins and Akt pathway activation were observed mostly in the presence of the CU and LF combination, including the upregulation of DR and annexin V levels, with greater significance for the DU145 cells. In conclusion, our results suggest that CU and LF may have a potentially beneficial effect, mainly when administered in combination, leading to a reduction in cancer cells' aggressiveness.

Extracellular matrix and cyclic stretch alter fetal cardiomyocyte proliferation and maturation in a rodent model of heart hypoplasia.

Birth defects, particularly those that affect development of the heart, are a leading cause of morbidity and mortality in infants and young children. Babies born with heart hypoplasia (heart hypoplasia) disorders often have a poor prognosis. It remains unclear whether cardiomyocytes from hypoplastic hearts retain the potential to recover growth, although this knowledge would be beneficial for developing therapies for heart hypoplasia disorders. The objective of this study was to determine the proliferation and maturation potential of cardiomyocytes from hypoplastic hearts and whether these behaviors are influenced by biochemical signaling from the extracellular matrix (ECM) and cyclic mechanical stretch. Congenital diaphragmatic hernia (CDH)-associated heart hypoplasia was induced in rat fetuses by maternal exposure to nitrofen. Hearts were isolated from embryonic day 21 nitrofen-treated fetuses positive for CDH (CDH+) and from fetuses without nitrofen administration during gestation. CDH+ hearts were smaller and had decreased myocardial proliferation, along with evidence of decreased maturity compared to healthy hearts. In culture, CDH+ cardiomyocytes remained immature and demonstrated increased proliferative capacity compared to their healthy counterparts. Culture on ECM derived from CDH+ hearts led to a significant reduction in proliferation for both CDH+ and healthy cardiomyocytes. Healthy cardiomyocytes were dosed with exogenous nitrofen to examine whether nitrofen may have an aberrant effect on the proliferative ability of cardiomyocyte, yet no significant change in proliferation was observed. When subjected to stretch, CDH+ cardiomyocytes underwent lengthening of sarcomeres while healthy cardiomyocyte sarcomeres were unaffected. Taken together, our results suggest that alterations to environmental cues such as ECM and stretch may be important factors in the pathological progression of heart hypoplasia.

The low-density lipoprotein receptor-mTORC1 axis coordinates CD8+ T cell activation.

Activation of T cells relies on the availability of intracellular cholesterol for an effective response after stimulation. We investigated the contribution of cholesterol derived from extracellular uptake by the low-density lipoprotein (LDL) receptor in the immunometabolic response of T cells. By combining proteomics, gene expression profiling, and immunophenotyping, we described a unique role for cholesterol provided by the LDLR pathway in CD8+ T cell activation. mRNA and protein expression of LDLR was significantly increased in activated CD8+ compared to CD4+ WT T cells, and this resulted in a significant reduction of proliferation and cytokine production (IFNγ, Granzyme B, and Perforin) of CD8+ but not CD4+ T cells from Ldlr -/- mice after in vitro and in vivo stimulation. This effect was the consequence of altered cholesterol routing to the lysosome resulting in a lower mTORC1 activation. Similarly, CD8+ T cells from humans affected by familial hypercholesterolemia (FH) carrying a mutation on the LDLR gene showed reduced activation after an immune challenge.

Abstract B014: Metabolic reprogramming in high-grade sarcomas: repurposing anti-cholesterol agents as a novel therapeutic strategy

Abstract Introduction and Objective: Undifferentiated pleomorphic sarcoma (UPS) and leiomyosarcoma (LMS) are treated with surgery, radiation, and/or chemotherapy. Current drug therapies, such as doxorubicin, have limited response rates and severe toxicities. We performed a drug screen with over 3000 compounds on four patient-derived UPS cell lines and uncovered sensitivity to simvastatin, which inhibits the rate limiting enzyme in the mevalonate pathway. Statins are well tolerated drugs used to lower cholesterol in patients with hypercholesterolemia. The mevalonate pathway can be exploited by cancer through metabolic reprogramming and phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) signaling to enhance proliferation. Thus, the goals of our study are to define the mechanism(s) responsible for simvastatin sensitivity and determine the efficacy when combined with doxorubicin in vivo. Hypothesis: Dysregulation of the mevalonate pathway and/or PI3K/mTOR pathway will render certain high-grade sarcomas sensitive to simvastatin. Methods: We confirmed simvastatin sensitivity in UPS, as well as in LMS cell lines, with dose-response curves. We investigated the mechanisms whereby simvastatin inhibits UPS and LMS viability with immunoblotting and flow cytometry. Proliferation was quantified using a cell proliferation dye. To assess in vivo efficacy and toxicity of simvastatin we performed a murine study with an established xenograft UPS model from our lab. We are currently assessing the efficacy of this novel combination in an LMS xenograft. Results: We found decreased phosphorylated AKT (pAKT ser473) and increased rates of active caspase-3 in most sensitive UPS cell lines with simvastatin (1uM) treatment. This indicates downregulation of PI3K/mTOR signaling and an increase in apoptosis, respectively. Importantly, these effects were rescued with the addition of mevalonate (200uM), an effector immediately downstream of the rate limiting enzyme that statins inhibit, indicating an on-target effect. In four sensitive LMS cell lines treated with simvastatin (1.5uM or 2uM) a decrease in pAKT ser473 is not consistently seen, revealing that downregulation of PI3K/mTOR signalling may not be a universal mechanism. Furthermore, in some UPS and LMS cell lines there was a significant reduction in proliferation with simvastatin + doxorubicin (0.5uM) treatment (p<0.05). Our in vivo study demonstrated that simvastatin (50mg/kg) in combination with doxorubicin (1.2mg/kg) reduced the volume of UPS tumors in mice. Furthermore, mice treated with combination therapy had higher concentrations of simvastatin and doxorubicin within the tumor than those treated with either single agent. However, this study was not designed to look at pharmacokinetics (PK), so to confirm these results a PK study is underway in an LMS xenograft. Conclusion: UPS and LMS are sensitive to simvastatin, suggesting these sarcomas rely on metabolic reprogramming to sustain viability. Thus, simvastatin may be a novel therapy for certain sarcoma patients. Citation Format: Jen Dorsey, Yael Babichev, Rosemarie E. Venier, Richard Marcellus, Rima Al-awar, Linda Z. Penn, Albiruni A. Razak, Brendan C. Dickson, Eric Chen, Irene L. Andrulis, Jay Wunder, Rebecca A. Gladdy. Metabolic reprogramming in high-grade sarcomas: repurposing anti-cholesterol agents as a novel therapeutic strategy [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B014.

Abstract 1315: Tumor necrosis factor related apoptosis inducing ligand fosters myeloid derived suppressor cell mediated tumor immune evasion in cholangiocarcinoma

Abstract Background and Aims: Tumor necrosis factor related apoptosis inducing ligand (TRAIL) is predominantly expressed on immune cells. Although TRAIL biology has garnered considerable attention as a potential anti-cancer strategy, TRAIL agonists have had very limited anti-cancer activity in humans. TRAIL signaling can attenuate the T lymphocyte response. However, this potential immunosuppressive function of TRAIL has not been examined in cancer biology. Cholangiocarcinoma (CCA), a highly lethal biliary tract cancer, has a dense immunosuppressive microenvironment. Accordingly, CCA provides a model to examine the potential immune regulatory function of TRAIL in cancer biology. Methods: Using a syngeneic, orthotopic murine model of CCA (PMID: 29464042), murine CCA cells (SB cells) that express both Trail and Trail receptor (Tr) were implanted into livers of WT and Tr-/- mice. Hence, in this model the host immune cells express Trail but not the receptor; therefore, they would be capable of inducing TRAIL-mediated apoptosis in CCA cells but would be resistant to TRAIL-mediated immunosuppression. After 4 weeks of tumor growth, mice were sacrificed, and tumors were characterized using flow cytometry. Results: We observed that Tr-/- mice had a significant reduction in tumor burden compared to WT mice. Moreover, tumor bearing Tr-/- mice had a significant increase in cytotoxic T lymphocytes (CTLs) and enhanced CTL effector function. Myeloid-derived suppressor cells (MDSCs) were significantly decreased in Tr-/- tumors compared to WT tumors. Furthermore, implantation of SB cells devoid of Trail (SB-Trail-/-) into WT mice resulted in a significant reduction in tumor burden and MDSC infiltration. In vitro functional studies employing SB cells deficient in Tr (SB-Tr-/-) and MDSCs were carried out. There was a significant reduction in proliferation and immunosuppressive function of MDSCs when cocultured with SB-Tr-/- compared to SB cells. These results indicate that TRAIL-TR fosters MDSC growth and immunosuppressive function. In conclusion, we have demonstrated that Tr-/- mice have a significant reduction in CCA tumor burden and MDSC infiltration. Consequently, Tr-/- tumors have enhanced CTL infiltration and function. These data suggest that the TRAIL-TR system mediates tumors immune evasion via MDSCs, and direct targeting of TRAIL on CCA cells is a potential anti-tumor strategy. Citation Format: Emilien Loeuillard, Jingchun Yang, Haidong Dong, Gregory J. Gores, Sumera I. Ilyas. Tumor necrosis factor related apoptosis inducing ligand fosters myeloid derived suppressor cell mediated tumor immune evasion in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1315.

Tet1 Suppresses p21 to Ensure Proper Cell Cycle Progression in Embryonic Stem Cells.

Ten eleven translocation 1 (Tet1) is a DNA dioxygenase that promotes DNA demethylation by oxidizing 5-methylcytosine. It can also partner with chromatin-activating and repressive complexes to regulate gene expressions independent of its enzymatic activity. Tet1 is highly expressed in embryonic stem cells (ESCs) and regulates pluripotency and differentiation. However, its roles in ESC cell cycle progression and proliferation have not been investigated. Using a series of Tet1 catalytic mutant (Tet1m/m), knockout (Tet1−/−) and wild type (Tet1+/+) mouse ESCs (mESCs), we identified a non-catalytic role of Tet1 in the proper cell cycle progression and proliferation of mESCs. Tet1−/−, but not Tet1m/m, mESCs exhibited a significant reduction in proliferation and delayed progression through G1. We found that the cyclin-dependent kinase inhibitor p21/Cdkn1a was uniquely upregulated in Tet1−/− mESCs and its knockdown corrected the slow proliferation and delayed G1 progression. Mechanistically, we found that p21 was a direct target of Tet1. Tet1 occupancy at the p21 promoter overlapped with the repressive histone mark H3K27me3 as well as with the H3K27 trimethyl transferase PRC2 component Ezh2. A loss of Tet1, but not loss of its catalytic activity, significantly reduced the enrichment of Ezh2 and H3K27 trimethylation at the p21 promoter without affecting the DNA methylation levels. We also found that the proliferation defects of Tet1−/− mESCs were independent of their differentiation defects. Together, these findings established a non-catalytic role for Tet1 in suppressing p21 in mESCs to ensure a rapid G1-to-S progression, which is a key hallmark of ESC proliferation. It also established Tet1 as an epigenetic regulator of ESC proliferation in addition to its previously defined roles in ESC pluripotency and differentiation.

Therapeutic benefits of niraparib tosylate as radio sensitizer in esophageal squamous cell carcinoma: an in vivo and in vitro preclinical study

PurposeEsophageal squamous cell carcinoma is associated with high morbidity and mortality rate for which radiotherapy is the main treatment modality. Niraparib, a Poly (ADP-ribose) polymerase 1 inhibitors (PARPi) was previously reported to confer radiosensitivity in different malignancies including non-small cell lung cancer. In this study, we assessed the in vivo ability of niraparib in conferring radiosensitivity to esophageal squamous cell carcinoma cells.Materials and methodsIn this study, KYSE-30 and KYSE-150 cell lines were selected as in vivo esophageal squamous cell carcinoma models. The experimental groups were: niraparib tosylate alone, radiotherapy alone, control (no intervention), and combination therapy (radiotherapy + niraparib tosylate). Cell cytotoxicity assay, colony formation assay, flow cytometry, immunofluorescence, Western blotting, immunohistochemistry, lentivirus transfection analysis, and xenograft models were used for confirming radiosensitizing ability of niraparib and to investigate the possible cellular mechanism involved in radiosensitization.ResultsThe colony formation efficiency of the combination group was significantly much lower than that of the single radiation group (P < 0.01). Cell cytotoxicity assay demonstrated a significant reduction in proliferation of irradiated cells after treatment with niraparib tosylate compared to niraparib tosylate alone (P < 0.01). Cell apoptosis significantly increased in the combination group compared to either niraparib tosylate or radiotherapy alone (P < 0.01). Rate of tumor suppression rate was significantly high in the combined treatment group (P < 0.01) but, significantly decreased in nude mice. Western blot and lentivirus infection model suggested overexpression of FANCG genes to confer radiosensitivity.ConclusionThese results suggest that the synergistic effect of niraparib tosylate and radiation may be related to the down-regulation of FANCG.

Regulation of Epithelial-Mesenchymal Transition of A549 Cells by Prostaglandin D2.

Despite significant advances in diagnostic and operative techniques, lung cancer remains one of the most lethal malignancies worldwide. Since prostaglandins such as prostaglandin D2 (PGD2) is involved in various pathophysiological process, including inflammation and tumorigenesis, this study aims to investigate the role of PGD2 during the process of epithelial-mesenchymal transition (EMT) in A549 cells. A549 cells were stimulated with PGD2 and expression of EMT markers was analyzed by immunoblotting and immunofluorescence. EMT-related gene, Slug expression was evaluated using quantitative real-time polymerase chain reaction (qPCR). Migration and invasion abilities of A549 cells were determined in chemotaxis and Matrigel invasion assays, respectively. We also inhibited the TGF/Smad signaling pathway using a receptor inhibitor or silencing of TGF-β1 and TGFβ type I receptor (TGFβRI), and protein expressionwas assessed by immunoblotting and immunofluorescence. Here, we found that stimulation of A549 cells with PGD2 resulted in morphological changes into a mesenchymal-like phenotype under low serum conditions. Stimulation of A549 cells with PGD2 resulted in a significant reduction in proliferation, whereas invasion and migration were enhanced. The expression of E-cadherin was markedly downregulated, while Vimentin expression was upregulated after treatment of A549 cells with PGD2. Slug expression was markedly upregulated by stimulating A549 cells with PGD2, and stimulation of A549 cells with PGD2 significantly enhanced TGF-β1 expression, and silencing of TGF-β1 significantly blocked PGD2-induced EMT and Smad2 phosphorylation. In addition, PGD2-induced Smad2 phosphorylation and EMT were significantly abrogated by either pharmacological inhibition or silencing of TGFβRI. PGD2-induced expression of Slug and EMT were significantly augmented in low nutrient and low serum conditions. Finally, the subsequent culture of mesenchymal type of A549 cells under normal culture conditions reverted the cell's phenotype to an epithelial type. Given these results, we suggest that tumor microenvironmental factors such as PGD2, nutrition, and growth factors could be possible therapeutic targets for treating metastatic cancers.

Abstract WP250: Exosomes Derived From Different Regional Cerebral Endothelial Cells Have Distinct Effects On Adult Rodent Neurogenesis

Introduction: Exosomes mediate intercellular communication. We tested the hypothesis that exosomes derived from different area cerebral endothelial cells (CECs) have distinct effects on adult neurogenesis. Methods and Results: Exosomes were isolated from CECs of cortical (Cor-CEC-exo) and subventricular zone (SVZ-CEC-exo) vessels of non-stroke and stroke rats (n=4/group). Neural stem cells (NSCs) from the non-stroke SVZ were treated with CEC-exo. Proliferation and differentiation of NSCs were measured by BrdU + cells and Tuj1 + neuroblasts. There were no significant differences of size distribution and marker proteins among studied exosomes. Treatment of NSCs with non-stroke Cor-CEC-exo did not significantly change NSC proliferation (35%± 2% vs 31%±2% in control) and neuronal differentiation (5%±2% vs 5%± 0.1% in control). However, treatment of NSCs with non-stoke SVZ-CEC-exo significantly (p&lt;0.05) increased BrdU + (42%± 2%) and Tuj1 + cells (10%± 2%). These data suggest that non-stroke Cor-CEC-exo, but not SVZ-CEC-exo, do not affect neurogenesis. RT-PCR analysis showed that Cor-CEC-exo were enriched in selective miRNAs including miR-203 that is known to negatively regulate NSC proliferation. To study whether miRNAs are involved in the effect of CEC-exo on neurogenesis, we isolated NSCs in which miRNA biogenesis gene, Dicer, was ablated. Compared to wild-type (WT) NSCs, ablation of Dicer led to significant reduction of proliferation (16%±1%) and neuronal differentiation (1%±0.1%). Treatment of Dicer knockout NSCs with Cor- or SVZ-CEC-exo significantly increased BrdU+ (25%±2% or 34%±2%) and TuJ1+ cells (3%±0.1% or 5%±0.1%), suggesting that both Cor- and SVZ-CEC-exo can rescue Dicer knockout impaired neurogenesis. Moreover, both stroke Cor- and SVZ-CEC-exo significantly augmented non-stroke WT NSC proliferation (41%±3% and 51%±4%) and differentiation (9%±0.2% and 16%±0.3). Conclusion: Our data demonstrate that under non-stroke condition, Cor-CEC-exo do not promote neurogenesis, while SVZ-CEC-exo enhance neurogenesis. After stroke, both Cor-and SVZ-CEC-exo augment neurogenesis. The effect of CEC-exo on neurogenesis is mediated by miRNAs. This study provides new insight into CEC-exo in mediating neurogenesis.