Bmi-1 Gene Research Articles

Neuron stress-related genes serve as new biomarkers in hypothalamic tissue following high fat diet

ObjectiveEnergy homeostasis is modulated by the hypothalamic is essential for obesity progression, however, the gene expression profiling remains to be fully understood.MethodsGEO datasets were downloaded from the GEO website and analyzed by the R packages to obtain the DEGs. And, the WGCNA analysis and PPI networks of co-expressed DEGs were designed using STRING to get key genes. In addition, the single-cell sequencing datasets and GTEx database were utilized to receive the neuron-stress genes from the key genes. Further, high-fat diet (HFD)-induced hypothalamic tissue of mice was used as an animal model to validate the mRNA up-regulation of neuron-stress genes. In addition, the Bmi1 gene was identified as a hub gene through the LASSO model and nomogram analysis. Western blot confirmed the high expression of Bmi1 in hypothalamic tissue of HFD mice and PA-stimulated microglia. Immunofluorescence staining showed that HFD induced the activation of microglia and the expression of Bmi1 in hypothalamic tissue.ResultsWe found that six genes (Sacm1l, Junb, Bmi1, Erbb4, Dkc1, and Suv39h1) are neuron stress-related genes and increased in the HFD-induced mice obesity model, Bmi1gene was identified as a key genes that can reflect the pathophysiology of obesity.ConclusionsOur research depicted a comprehensive activation map of cell abnormality in the obese hypothalamus and Bim1 may be a diagnostic marker in the clinic, which provides a new perspective and basis for investigating the pathogenesis of obesity.

A new digital droplet PCR method for looking at epigenetics in diffuse large B-cell lymphomas: The role of BMI1, EZH2, and USP22 genes.

Epigenetics has been shown to be relevant in oncology: BMI1 overexpression has been reported in leukemias, EZH2 mutations have been found in follicular lymphoma, and USP22 seems to stabilize BMI1 protein. In this study, we measured the expression of BMI1, EZH2, and USP22 in lymph nodes from 56 diffuse large B-cell lymphoma (DLBCL) patients. A new multiplex digital droplet PCR (ddPCR) has been set up to measure the expression of 4 genes (BMI1, EZH2, USP22, and GAPDH) in the same reaction on RNA extracted from paraffin-embedded tissues. The specificity of ddPCR was confirmed by a 100% alignment on the BLAST platform and its repeatability demonstrated by duplicates. A strict correlation between expression of BMI1 and EZH2 and BMI1 and USP22 has been found, and high expression of these genes was correlated with extra-nodal lymphomas. Progression-free survival (PFS) and overall survival (OS) were conditioned by IPI, bone marrow infiltration, and the complete response achievement. High levels of BMI1 and USP22 did not condition the response to therapy, but impaired the PFS, especially for patients defined at "high risk" based on the cell of origin (no germinal center [GCB]), high BCL2 expression, and IPI 3-5. In this subgroup, the probability of relapse/progression was twice higher than that of patients carrying low BMI1 and USP22 levels. High expression of BMI1 and of USP22 might be a poor prognostic factor in DLBCL, and might represent the target for novel inhibitors.

Curcumin suppresses cell viability in breast cancer cell line by affecting the expression of miR-15a-5p

Abstract Objectives Curcumin plays a leading role as an epigenetic regulator in cancer. miR-15a-5p is a crucial non-coding RNA for breast cancer (BRCA) and various cancers due to its tumor suppressor role. In our study, we aimed to examine the curcumin/miR-15a-5p/target gene interaction in BRCA cells. Methods The effects of curcumin and miR-15a-5p on cell viability in the MCF7 cells were examined using the WST8 technique. The cell migration was determined using scratch wound assay. miR-15a-5p level was detected in curcumin-treated cells and miR-15a-5p transfected cells compared to control groups by RT-qPCR. Overexpressed genes in BRCA were found by bioinformatics tools (GSE41970 and TCGA). miR15a-5p potential target genes in the miRNet tool were selected in overlapped genes between GSE41970 and TCGA. Survival analysis of the selected genes was examined using the GEPIA2 tool. Relative expression levels of four selected genes were examined via qPCR. Results Cell viability and scratch-wound closure rate were reduced in curcumin-treated and miR-15a-5p mimic transfected MCF7 cells. miR15a-5p overexpressed in curcumin-treated and miR-15a-5p transfected cells. Eighty-three dysregulated upregulated genes were detected (in GSE41970 and TCGA). Among the possible target genes of miR-15a-5p in the miRNet tool, 10 upregulated genes were detected overlapping with GSE41970 and TCGA. CCNE1 and CHEK1 genes were found to be important for survival in BRCA. CCNE1 and BMI1 were decreased in curcumin-treated and miR-15a-5p transfected cells. Conclusions Curcumin treatment increased miR-15a-5p and downregulated selected target genes. Curcumin/miR-15a-5p interaction may be a much stronger negative regulator of the CCNE1 and BMI1 genes in BRCA.

Lactiplantibacillus plantarum JM113 alleviates deoxynivalenol induced intestinal damage by microbial modulation in broiler chickens

Deoxynivalenol (DON) contamination causes the grievous injury in public and animal health, poultry suffer from the greater toxin challenge. Probiotic have been considered as a potential way to mitigate the deleterious effects of DON. In this study, a total of 144 1-day-old Arbor Acres chickens were randomly assigned into 3 groups: control group, DON group (5 mg/kg DON diet), DJ group (1×109 cfu Lactiplantibacillus plantarum JM113/kg DON diet). The results showed that Lactiplantibacillus plantarum JM113 (L. plantarum JM113) increased the growth performance of 21-day-old broilers that challenged by the DON (P < 0.05), and the DON-induced disorder of jejunal morphology was recovered in DJ group (P < 0.05). Compared with the DON group, the mRNA and protein levels of Nrf2 and NQO-1 were upregulated in jejunum of DJ group broilers (P < 0.05). Meanwhile, administration of L. plantarum JM113 effectively increased the expression level of barrier-related genes, and the protein abundance of occludin and claudin1 (P < 0.05). L. plantarum JM113 restored the mRNA and protein abundance of PCNA, and proliferation-linked gene (Lgr5 and Bmi1) expression levels in jejunum of DON-insulted broilers (P < 0.05). Furthermore, administration of L. plantarum JM113 significantly enhanced the relative abundance of s_Limosilactobacillus_reuteri in jejuna of DON-challenged broilers (P < 0.05). Spearman correlation analysis showed that s_Limosilactobacillus_reuteri was positively associated with the jejunal barrier related genes (P < 0.05). In conclusion, L. plantarum JM113 alleviated the toxic effects of DON by regulating the jejunal function through microbial adjustment. Our findings proposed a viable approach to mitigating the adverse effects of deoxynivalenol exposure in broilers.

Abstract A112: Mechanisms underlying platinum-induced ovarian cancer stem cell plasticity

Abstract Most women diagnosed with late-stage high grade serous ovarian cancer (HGSOC) develop recurrent, platinum-resistant tumors. Ovarian cancer stem cells (OCSCs) are hypothesized to contribute to the emergence of these resistant tumors. CSCs have been postulated to reside in a plastic state, which can allow for the conversion of non-CSC to CSC. This process of dedifferentiation continues during tumor development and chemotherapeutic agents like platinum can exaggerate CSC plasticity. We have previously demonstrated that acute platinum treatment enriched for OCSCs. However, whether platinum transforms non-OCSCs into OCSCs to contribute to this subpopulation of cells remains unclear, and the underlying mechanism remains incompletely understood. To examine OCSC plasticity, aldehyde dehydrogenase (ALDH; functional marker) and fluorescence activated cell sorting were used to isolate OCSCs (ALDH+) and non-OCSCs (ALDH-) from HGSOC cell lines, OVCAR5 and OVCAR3. To determine the stability of the non-OCSC phenotype, ALDH- cells were cultured for 3 and 5 days and ALDH activity was measured using flow cytometry. At both timepoints examined, ALDH- cells remained ALDH-, with approximately less than 1% being ALDH+ (p&amp;lt;0.05). To determine if platinum can induce conversion of non-OCSC to OCSC, ALDH- cells were treated with cisplatin (12µM or 15 µM for 16h), and the percent of ALDH+ cells was measured using flow cytometry. Treatment of ALDH- cells with cisplatin resulted in conversion of approximately 4% and 10% of ALDH- cells into ALDH+ cells (p&amp;lt;0.05) in OVCAR5 and OVCAR3 cells, respectively. Furthermore, both cell lines displayed increased expression (p&amp;lt;0.05) of stemness genes BMI1, NANOG, OCT4, and SOX2 and spheroid formation was observed in converted OCSCs compared to parental ALDH+ and whole cell populations treated with cisplatin, suggesting that platinum induced the observed differences in the stemness phenotype. With the goal of targeting key genes and pathways to inhibit platinum-induced OCSC conversion, RNA-sequencing was performed using converted OCSCs. Activated pathways in converted OCSCs were identified, including protein kinase signaling, inhibition of matrix metalloproteases, SPINK1 pathway, and VDR/RXR activation. We are further investigating if these pathways contribute to the mechanism involved in OCSC plasticity and whether targeting these pathways can reduce the OCSC population, which is critical to blocking the persistence of OCSCs and ultimately reduce mortality in patients. Citation Format: Tara X. Metcalfe, Shu Zhang, Christiane Hassel, Heather M. O'Hagan, Kenneth P. Nephew. Mechanisms underlying platinum-induced ovarian cancer stem cell plasticity [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A112.

An Optimized Human Erythroblast Differentiation System Reveals Cholesterol-Dependency of Robust Production of Cultured Red Blood Cells Ex Vivo

The generation of cultured red blood cells (cRBCs) ex vivo represents a potentially unlimited source for RBC transfusion and other cell therapies. However, efficient differentiation and terminal maturation (symbolized by enucleation) into cRBCs ex vivo highly depend on replenishing human plasma. In addition to cost, this requirement results in variable potency across donors or batches and complicates consistent cRBC production required for clinical translations. Thus, exploration of key factor(s) in plasma and development of a compositionally defined medium would be in need. To investigate the role of human plasma in erythroblast terminal maturation, we first optimized a widely-used differentiation system that we and other have used in the past decade. We found that culture of erythroblasts in a newly developed medium called human plasma like medium (HPLM) greatly improves the yield and consistence of erythrocyte maturation. However, human serum (1-2%) was indispensable for efficient terminal maturation in the absence of plasma. The serum-derived factor or factors (likely a protein or complex) is essential for erythroblast growth assessed at day 3 after induction of terminal differentiation. We used size exclusion chromatography (SEC) and other protein biochemistry approaches to fractionate human serum and assessed the biological effect of each fraction. We found that the activity is fractions containing proteins or complexes of &amp;gt;100K Da. Proteomics revealed that the fractions with biological activities are enriched for low density lipoproteins (LDL)-containing proteins such as apolipoprotein B-100. In addition, we performed RNA-sequencing (RNA-seq) analysis on cultured erythroblasts with or without serum. Transcriptomic data revealed that the genes associated with de novo biosynthesis of steroids and cholesterol are upregulated in the absence of serum, although production of enzymes encoded by these genes alone is insufficient to provide cholesterol required for erythroblast differentiation and maturation. Hence, we hypothesize that cholesterol-carrying LDL that are naturally present in blood is sufficient to replace human serum for erythroblast survival and terminal differentiation ex vivo. We next examined directly whether LDL purified from human blood is sufficient for erythroid commitment and enucleation. Through LDL binding and uptake assays, we confirmed that LDL binds to erythroblasts that expressed the LDL receptor and is transported intracellularly. Furthermore, LDL purified from human plasma can essentially replace 2% unfractionated serum for efficient generation of erythrocytes in culture. In addition to using human erythroblasts derived from peripheral blood mononuclear cells (PBMCs), we also extended this optimized protocol to human erythroblasts from various sources, including cord blood-derived mononuclear cells, CD34+ HSPCs, and extensively expanded erythroblasts after BMI1 gene transduction. After induction of terminal differentiation for 8 days, erythroblasts from all these sources effectively enucleated (&amp;gt;50%) and stably produced reticulocytes with no significant difference compared to the serum-containing system. We are also in progress to substitute LDL by chemical synthetic cholesterol. In sum, we developed a compositionally defined culture medium (LDL-containing Optimized Maturation Medium, LOAM) enabling robust generation of cRBCs from multiple sources of erythroblasts with high enucleation efficiency and higher reticulocyte yield compared to culture in traditional media supplemented with human plasma or serum. Using this advanced human erythroblast differentiation model, we further revealed that cholesterol carried by LDL is critical to erythroblast terminal maturation.

Senescence ofbone marrow mesenchymal stem cells in Wistar malerats receiving normal chow/high-calorie diets with/without vitamin D.

Bone marrow mesenchymal stem cells (BM-MSCs) have a momentous function in the composition of the bone marrow microenvironment because of their many valuable properties and abilities, such as immunomodulation and hematopoiesis. The features and actions of MSCs are influenced by senescence, which may be affected by various factors such as nutritional/micronutrients status, e.g., vitamin D. This study aimed to examine the effects of a high-calorie diet (HCD) with/without vitamin D on BM-MSCs senescence. In the first phase, 48 middle-aged rats were fed a normal chow diet (NCD, n = 24) and an HCD (n = 24) for 26 weeks. Afterward, the rats in each group were randomly divided into three equal subgroups. Immediately, eight-rat from each diet group were sacrificed to assess the HCD effects on the first phase measurements. In the second phase, the remaining 4 groups of rats were fed either NCD or HCD with (6 IU/g) or without vitamin D (standard intake: 1 IU/g); in other words, in this phase, the animals were fed (a) NCD, (b) NCD plus vitamin D, (c) HCD, and (d) HCD plus vitamin D for 4 months. BM-MSCs were isolated and evaluated for P16INK4a, P38 MAPK, and Bmi-1 gene expression, reactive oxygen species (ROS) levels, SA-β-gal activity, and cell cycle profile at the end of both phases. After 26 weeks (first phase), the ROS level, SA-β-gal-positive cells, and cells in the G1 phase were significantly higher in HCD-fed rats than in NCD-fed ones (P < 0.05). HCD prescription did not significantly affect cells in the S and G2 phases (p > 0.05). Compared with the NCD-fed animals, P16INK4a and P38 MAPK gene expression were up-regulated in the HCD-fed animals; also, Bmi-1 gene expression was down-regulated (P < 0.05).BM-MSCs from vitamin D-treated rats (second phase) exhibited reduced mRNA levels of P16INK4a and P38 MAPK genes and increased Bmi-1 mRNA levels (all P<0.05). Vitamin D prescription also declined the percentage of SA-β-gal-positive cells, ROS levels, and the cells in the G1 phase and increased the cells in the S phase in both NCD and HCD-fed animals (P <0.05). The reduction of the cells in the G2 phase in rats fed with an NCD plus vitamin D was statistically non-significant (P=0.128) and significant in HCD plus vitamin D rats (P=0.002).HCD accelerates BM-MSCs senescence, and vitamin D reduces BM-MSCs senescence biomarkers.

Sanguisorba officinalis L. enhances the 5-fluorouracil sensitivity and overcomes chemoresistance in 5-fluorouracil-resistant colorectal cancer cells via Ras/MEK/ERK and PI3K/Akt pathways

Sanguisorba officinalis L., a traditional Chinese medicine (TCM) called DiYu (DY) in China, has a strong tradition of utilization as a scorching, blood-cooling, and hemostatic medication, and was used for cancer prevention and treatment due to its potential immune-enhancing and hematological toxicity-reducing effects. Previous studies have reported significant effects of DY on cancers including colorectal cancer (CRC), which is one of the most common malignancies worldwide. The first-line cure 5-fluorouracil (5-FU) plays decisive commerce in the sedative of CRC as a clinically available chemotherapeutic agent. One of the primary causes of cancer treatment failure is the acquisition of chemotherapy drug resistance. In order to successfully combat the emergence of chemoresistance, it is essential to identify herbs or traditional Chinese medicine that have adjuvant therapeutic effects on CRC. Therefore, this study aimed to determine whether DY could improve the sensitivity, conquer the chemoresistance of 5-FU-resistant CRC cells, and investigate its intrinsic mechanism. Materials and methodsMTT, Hoechst 33258 staining, and flow cytometry assays were used to determine the anticancer activity of DY alone or in combination with 5-FU against 5-FU-resistant CRC cells (RKO-R and HCT15-R) and wound healing assays were conducted to detect cell migration. Transcriptomic techniques were carried out to explore the effect and mechanism of DY on drug-resistant CRC cells. Western Blot and RT q-PCR assays were performed to validate the mechanism by which DY overcomes drug-resistant CRC cells. ResultsThese results indicated that DY alone or in combination with 5-FU significantly inhibited the proliferation and the migration of resistant CRC cells, and potentiated the susceptibility of 5-FU to drug-resistant CRC cells. GO and KEGG enrichment analysis showed that the mechanisms of drug resistance in CRC cells and DY against drug-resistant CRC cells highly overlapped, involved in the modulation of biological processes such as cell migration, positive regulation of protein binding and cytoskeleton, and MAPK (Ras-ERK-MEK), PI3K/Akt, and other signaling pathways. Moreover, DY can mediate the expression of p-R-Ras, p-ERK1/2, p-MEK1/2, p-PI3K, p-AKT, HIF-1A and VEGFA proteins. In addition, DY significantly suppressed the expression of AKT3, NEDD9, BMI-1, and CXCL1 genes in resistant CRC cells. ConclusionIn conclusion, DY could inhibit the proliferation and migration of 5-FU-resistant cells and strengthen the sensitivity of 5-FU to CRC-resistant cells. Furthermore, DY may prevail over chemoresistance through the Ras/MEK/ERK and PI3K/Akt pathways. These findings imply that DY may be a potential drug for clinical treatment or adjuvant treatment of drug-resistant CRC.

Molecular biomarkers NOTCH1, CD44, BMI1, and TP53 in oral squamous cell carcinoma

It is of interest to evaluate NOTCH1, CD44, BMI1, and TP53 genes in the epiglottis, tongue, and hard palate of oral malignancies (OM) with healthy controls. This was a prospective and cross-sectional study of 60 individuals with oral malignancies (OM) (20 each of tongue, epiglottis, and hard palate) studied at Malla Reddy Medical College and tertiary care hospitals in Hyderabad. Adults aged ≥ 18 years and diagnosed with oral cancer were included in the study. Those who had cancer in more than one area were excluded from the study. Blood samples of individuals with tongue or epiglottis or hard palate were taken for testing the expression of NOTCH1, CD44, TP53, and BMI1 genes. They were analysed by the genomic sequencing method. One-way ANOVA with Bonferroni's t-test was used for statistical analysis. Expression of NOTCH1, CD44, BMI1, and TP53 genes were significantly higher in epiglottis, tongue, and hard palate compared to healthy control samples (p &lt; 0.001). All four genes were expressed in all three areas of OM. However, they were not significant between them. Further analysis revealed that NOTCH1, CD44, TP53, and BMI1 genes did not show any difference in HPV-positive and HPV-negative samples. Comparing the T stages of cancer Notch1, gene expression was significantly higher in stages 1 and 2 compared to 3 and 4. The CD44, TP53, and BMI1 did not show any differences in the T stage. However, the difference in HPV in all T stages was very minimal. Data showed that irrespective of the areas of cancer (epiglottis, tongue, and hard palate) NOTCH1, CD44, TP53, and BMI1 genes were expressed equally. The expression was not very much dependent on HPV positive (+ve) or negative (-ve). However the T-stage was showing higher expression compared to control group. Since the expression of these genes was very high in all the three malignancies, they may be used as early biomarkers to detect cancer of epiglottis, tongue, and hard palate.

A transverse colon lipoma complicated with an adenoma is successfully and safely resected en bloc by endoscopic mucosal resection during water immersion

The B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is associated with the progression of gastric cancer (GC). However, its role in drug resistance of gastric cancer stem cell (GCSC) remains unclear. This study aimed to explore the biological function of BMI-1 in GC cells and its role in drug resistance of GCSCs.We assessed BMI-1 expression in the GEPIA database and in our collected samples from patients with GC. We silenced BMI-1 using siRNA to study the cell proliferation and migration of GC cells. We also used Hoechst 33342 staining to verify the effect of adriamycin (ADR) on side population (SP) cells, and measured the effects of BMI-1 on the expression of N-cadherin, E-cadherin, and drug-resistance-related proteins (multidrug resistance mutation 1 and lung resistance-related protein). Finally, we analyzed BMI-1-related proteins uing the STRING and GEPIA databases.BMI-1 mRNA was upregulated in GC tissues and cell lines, especially in MKN-45 and HGC-27 cells. Silencing BMI-1 reduced the proliferation and migration of GC cells. Knocking down BMI-1 significantly decreased epithelial-mesenchymal transition progression, expression levels of drug-resistant proteins, and the number of SP cells in ADR-treated GC cells. Bioinformatics analysis showed that EZH2, CBX8, CBX4, and SUZ12 were positively correlated with BMI-1 in GC tissues.Our study demonstrates that BMI-1 affects the cellular activity, proliferation, migration, and invasion of GC cells. Silencing the BMI-1 gene significantly reduces the number of SP cells and the expression of drug-resistant proteins in ADR-treated GC cells. We speculate that inhibition of BMI-1 increases the drug resistance of GC cells by affecting GCSCs, and that EZH2, CBX8, CBX4, and SUZ12 may participate in BMI-1-induced enhancement of GCSC-like phenotype and viability.

Abstract 2478: The interplay between Apurinic/apyrimidinic Endonuclease 1 (APE1) and DNA G-quadruplex (G4) in regulating triple-negative breast cancer (TNBC) metastasis

Abstract Triple-negative breast cancer (TNBC) is the most aggressive breast cancer (BC) among all the BC subtypes due to its exceptionally high metastasis rate. TNBC patients with metastasis have a short overall survival as the endocrine therapy and chemotherapy are inefficient in TNBC treatment. Hence, efforts are still needed to figure out the molecular mechanism driving TNBC metastasis and find promising therapeutic targets for TNBC treatment. A newly published study revealed that a secondary DNA structure G-quadruplex (G4) is highly enriched in TNBC signature genes’ promoters, and our lab was the first one to uncover that DNA repair protein Apurinic/apyrimidinic endonuclease 1 (APE1) binds to G4 and regulates its stability in cells, so we are aimed to investigate the interaction between APE1 and G4 in TNBC and their roles in regulating TNBC metastasis. We found that knocking out or knocking down APE1 in TNBC cell lines, MDA-MB-231 and BT-549, significantly impaired the cell migration ability. RNA-seq analysis and Gene Ontology analysis revealed that APE1 knockout downregulated expression of genes such as CXCL1, VEGFA, PIK3R3, BMI1, etc. which are mostly involved in promoting cell migration. Further, APE1 ChIP-seq analysis showed that APE1 was highly enriched in CXCL1, VEGFA, PIK3R3 and BMI1 gene promoters, and these regions contained potential G4 folding sequences, suggesting a potential interaction between APE1 and G4 in these gene promoters. Consisted with this, our study with the enzyme linked immunosorbent assays (ELISA) showed that recombinant APE1 bound with high affinity to CXCL1 and VEGFA G4 secondary structures as compared to double-strand DNA oligos in vitro, suggesting that APE1’s enrichment to the metastasis-related gene promoters attributes to the G4 structures in those promoter regions. Promoter-directed ChIP assay further revealed that downregulating APE1 inhibited transcription factor (TF) c-JUN’s occupancy to VEGFA promoters. Interestingly, we discovered that the G4-targeted small molecule TMPyP4 blocked APE1-G4 binding in vitro and reduced APE1’s occupancy at gene promoters in cells. Treatment of cells with TMPyP4 significantly downregulated CXCL1, VEGFA, PIK3R3, and BMI1 gene expression as well as inhibiting cell migration. In orthotopic TNBC metastasis mouse model, mice treating with TMPyP4 significantly reduced the frequency and volume of metastatic tumors in lung tissues. Overall, our study revealed that G4 in gene promoters recruited APE1 which further promoted transcription factors’ binding to promoters and enhanced metastasis-related gene expression. Blocking APE1-G4 interaction by TMPyP4 is a potential therapeutic approach to inhibit TNBC metastasis. Citation Format: Yingling Chen, Suravi Pramanik, Mason Tarpley, Achyuth Kalluchi, Bhopal Mohapatra, Kyle Hewitt, Michael J. Rowley, Vimla Band, Kishor Bhakat. The interplay between Apurinic/apyrimidinic Endonuclease 1 (APE1) and DNA G-quadruplex (G4) in regulating triple-negative breast cancer (TNBC) metastasis [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 2478.

B-cell-specific Moloney murine leukemia virus integration site 1 knockdown impairs adriamycin resistance of gastric cancer cells

Background and study aimsThe B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is associated with the progression of gastric cancer (GC). However, its role in drug resistance of gastric cancer stem cell (GCSC) remains unclear. This study aimed to explore the biological function of BMI-1 in GC cells and its role in drug resistance of GCSCs. Patients and methodsWe assessed BMI-1 expression in the GEPIA database and in our collected samples from patients with GC. We silenced BMI-1 using siRNA to study the cell proliferation and migration of GC cells. We also used Hoechst 33342 staining to verify the effect of adriamycin (ADR) on side population (SP) cells, and measured the effects of BMI-1 on the expression of N-cadherin, E-cadherin, and drug-resistance-related proteins (multidrug resistance mutation 1 and lung resistance-related protein). Finally, we analyzed BMI-1-related proteins uing the STRING and GEPIA databases. ResultsBMI-1 mRNA was upregulated in GC tissues and cell lines, especially in MKN-45 and HGC-27 cells. Silencing BMI-1 reduced the proliferation and migration of GC cells. Knocking down BMI-1 significantly decreased epithelial-mesenchymal transition progression, expression levels of drug-resistant proteins, and the number of SP cells in ADR-treated GC cells. Bioinformatics analysis showed that EZH2, CBX8, CBX4, and SUZ12 were positively correlated with BMI-1 in GC tissues. ConclusionOur study demonstrates that BMI-1 affects the cellular activity, proliferation, migration, and invasion of GC cells. Silencing the BMI-1 gene significantly reduces the number of SP cells and the expression of drug-resistant proteins in ADR-treated GC cells. We speculate that inhibition of BMI-1 increases the drug resistance of GC cells by affecting GCSCs, and that EZH2, CBX8, CBX4, and SUZ12 may participate in BMI-1-induced enhancement of GCSC-like phenotype and viability.

Application of mPEG-CS-cRGD/Bmi-1RNAi-PTX nanoparticles in suppression of laryngeal cancer by targeting cancer stem cells

Although surgery-based comprehensive therapy is becoming the main approach to treat laryngeal cancer, recurrence, metastasis, radiotherapy resistance and chemotherapy tolerance are still the main causes of death in patients. Targeted inhibition of laryngeal cancer stem cells has been considered as the consensus to cure laryngeal cancer. Our previous study has confirmed proto-oncogene Bmi-1 as a key regulator for self-renewal of laryngeal cancer stem cells. Targeted knockdown of Bmi-1 gene effectively inhibited the self-renewal and differentiation of laryngeal cancer stem cells, leading to the promoted sensitivity to chemotherapy including paclitaxel. However, due to off-target effects and quick degradation of the naked Bmi-1-RNAi small RNA oligo by nuclease in body fluids, it is urgently needed to develop a tumor-targeted delivery system with a protective shell. In this study, we designed and synthesized cRGD peptide-modified chitosan-polyethylene glycol slow-release nanoparticles (mPEG-CS-cRGD/Bmi-1RNAi-PTX) containing Bmi-1RNAi siRNA oligo and paclitaxel, which showed spherical in shape, 200 nm diameter in size, low cytotoxicity, strong DNA wrapping, resistance to nuclease degradation and high transfection efficiency to cells. Functional analysis indicated significant suppression of cell proliferation and migration and induction of apoptosis by the nanocomplex in laryngeal cancer cells in vitro. By application to the mouse model with laryngeal cancer, the nanocomplex inhibited tumor growth significantly in vivo. In addition, cRGD peptide, paclitaxel and Bmi-1 siRNA in the nanoparticles showed synergistic effects to suppress laryngeal cancer stem cells. In conclusion, this study not only developed a laryngeal tumor-targeted chemotherapeutic system, but also demonstrated a Bmi-1 RNAi-based chemotherapeutic strategy to inhibit cancer stem cells, having strong potential to treat laryngeal cancer patients suffering therapy resistance and/or tumor recurrence.

Integrated analysis of genomic and transcriptomic profiles to examine CD73/NT5E as a driver of drug-resistance and immunosuppression in biliary cancer (BTC).

606 Background: Despite the improved outcome achieved by the addition of the anti-PD-L1 durvalumab to standard chemotherapy, the vast majority of BTC do not benefit from chemo-immunotherapy. Immunosuppressive microenvironment is a dominant feature of BTC, involved in tumour progression and drug resistance. Here, we investigated the biological role and treatment implications of the adenosine-producing enzyme CD73 in a clinically-annotated cohort of BTC. CD73, an ecto-5’-nucleotidase (NT5E), creates an immunosuppressive tumour-promoting microenvironment by converting ATP to adenosine. Methods: Immunohistochemistry for CD73, CD4/CD8 and FOXP3 as well as whole-exome and transcriptomic sequencing were performed on resected specimens of 80 BTC (Illumina Platform). Spatial Transcriptomics was performed by using Visium Spatial Gene Expression-10x Genomics). Silencing of CD73 was obtained by transient transfection od CD73-siRNA, while genetic Knock-out by using CRISPR-Cas system technology in two BTC cell lines. Tumor growth was assessed in 2D and in 3D cell culture by using MTS assay and spheroid growth analysis before and after treatment with selected drugs. Statistical and Survival, correlation analyses were performed. Results: High CD73 expression (CD73high) was associated with older age (&gt;70 years, p=0.01), gallbladder subsite (p=0.03), and nodal involvement (p=0.04). CD73high tumours were significantly enriched in infiltrating FOXP3+ T lymphocytes (p&lt;0.001). CD73high status was independent predictor of poorer prognosis at the multivariate analysis (p=0,03) together with ECOG PS ≥2 (p=0,001) and the pathological stage (p=0,025) and was associated with a remarkably shorter RFS in patients treated with adjuvant chemotherapy (p=0,011). At the transcriptomic level, CD73high tumours were significantly enriched in upregulated EMT, TNF-alfa/NFKB, hypoxia and G2/M checkpoint signaling pathways and p53, BMI1, MEL18, EGFR and K-RAS genes. In in vitro models, siRNA-mediated depletion and CRISPR-CAS9 gene KO of CD73 sensitized both BTC 2D and 3D culture to cisplatin/gemcitabine treatment. The pharmacological inhibition of CD73 by AMCP improved the sensitivity of BTC cell lines to cisplatin/gemcitabine treatment. Finally, Spatially resolved transcriptomics of CD73high revealed a critical role of CD73 in tumor immunity and therapeutic response. Conclusions: We showed that CD73high BTC display aggressive biological features, poorer prognosis and resistance to standard chemotherapy. The therapeutic targeting of this adenosinergic ectonucleotidase by clinically-available compounds has the potential to enhance the efficacy of conventional treatment in BTC.

Molecular biomarkers NOTCH1, CD44, BMI1, and TP53 in oral squamous cell carcinoma.

It is of interest to evaluate NOTCH1, CD44, BMI1, and TP53 genes in the epiglottis, tongue, and hard palate of oral malignancies (OM) with healthy controls. This was a prospective and cross-sectional study of 60 individuals with oral malignancies (OM) (20 each of tongue, epiglottis, and hard palate) studied at Malla Reddy Medical College and tertiary care hospitals in Hyderabad. Adults aged ≥ 18 years and diagnosed with oral cancer were included in the study. Those who had cancer in more than one area were excluded from the study. Blood samples of individuals with tongue or epiglottis or hard palate were taken for testing the expression of NOTCH1, CD44, TP53, and BMI1 genes. They were analysed by the genomic sequencing method. One-way ANOVA with Bonferroni's t-test was used for statistical analysis. Expression of NOTCH1, CD44, BMI1, and TP53 genes were significantly higher in epiglottis, tongue, and hard palate compared to healthy control samples (p < 0.001). All four genes were expressed in all three areas of OM. However, they were not significant between them. Further analysis revealed that NOTCH1, CD44, TP53, and BMI1 genes did not show any difference in HPV-positive and HPV-negative samples. Comparing the T stages of cancer Notch1, gene expression was significantly higher in stages 1 and 2 compared to 3 and 4. The CD44, TP53, and BMI1 did not show any differences in the T stage. However, the difference in HPV in all T stages was very minimal. Data showed that irrespective of the areas of cancer (epiglottis, tongue, and hard palate) NOTCH1, CD44, TP53, and BMI1 genes were expressed equally. The expression was not very much dependent on HPV positive (+ve) or negative (-ve). However the T-stage was showing higher expression compared to control group. Since the expression of these genes was very high in all the three malignancies, they may be used as early biomarkers to detect cancer of epiglottis, tongue, and hard palate.

Retracted: Effects of BMI1 Gene on Regulating Apoptosis, Invasion, and Migration of HEC-1B Cells Induced by Ionizing Radiation.

[This retracts the article DOI: 10.1155/2022/7052066.].

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

Retinoic acid signaling is critical for generation of pancreatic progenitors from human embryonic stem cells

Retinoic acid (RA) is essential for gut endoderm development and has been extensively used for in vitro pancreatic differentiation from human pluripotent stem cells. However, the gene regulatory network triggered by RA signaling remains poorly addressed. Also, whether RA signals control histone modifiers such as the Polycomb group proteins during pancreatic specification remains to be explored. Here, we assess the role of RA on pancreas-specific genes during the differentiation of human embryonic stem cells (hESCs). We demonstrate that RA helps cells exit the definitive endoderm stage and proceed toward a pancreatic fate. Inhibition of the RA pathway using the pharmacological inhibitor LE135 impairs the induction of pancreatic endoderm (PE) markers FOXA2, HNF4α, HNF1β, HHEX, and PDX1. We further determine that RA signals alter the expression of epigenetic-associated genes BMI1 and RING1B in the hESC-derived pancreatic progenitors. These findings broaden our understanding of the mechanisms that drive early PE specification.

Involvement of the p53-p16/RB pathway control mechanism in early-stage carcinogenesis in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma develops through a heterogeneous process involving human papillomavirus infection, smoking, and alcohol consumption. A comprehensive genomic analysis of head and neck squamous cell carcinomas to date has identified a few single driver gene mutations, the most frequent of which involve TP53 and CDKN2A/p16. To investigate the involvement of the tumorigenesis mechanism in early-stage carcinogenesis, HPV-derived genomes E6 and E7, which are carcinogens, and stem/progenitor-associated, polycomb (PcG) genes Bmi1 and TERT were induced into human stromal cells and immortalized as the head and neck squamous cell carcinoma model. We found that Bmi1 suppressed both the p16INK4a and p16/Rb-p53 pathway cross-talks. The E7 group showed that endogenous p53 is highly expressed and eludes chromosome number aberration, even on long-term observation. Bmi1 was predominantly expressed in early head and neck squamous cell carcinoma, and PcG was essential in early cancer development. Additionally, TP53 whole exon analysis revealed categories useful for estimating malignant potential, such as poor prognosis and high recurrence at the transection site. Therefore, understanding the p53-p16/RB pathway in head and neck squamous cell carcinoma is an essential factor to elucidate the early carcinogenesis of head and neck squamous cell carcinoma.

Cellular and Molecular Biological Alterations after Photon, Proton, and Carbon Ions Irradiation in Human Chondrosarcoma Cells Linked with High-Quality Physics Data.

Chondrosarcomas are particularly difficult to treat due to their resistance to chemotherapy and radiotherapy. However, particle therapy can enhance local control and patient survival rates. To improve our understanding of the basic cellular radiation response, as a function of dose and linear energy transfer (LET), we developed a novel water phantom-based setup for cell culture experiments and characterized it dosimetrically. In a direct comparison, human chondrosarcoma cell lines were analyzed with regard to their viability, cell proliferation, cell cycle, and DNA repair behavior after irradiation with X-ray, proton, and carbon ions. Our results clearly showed that cell viability and proliferation were inhibited according to the increasing ionization density, i.e., LET, of the irradiation modes. Furthermore, a prominent G2/M arrest was shown. Gene expression profiling proved the upregulation of the senescence genes CDKN1A (p21), CDKN2A (p16NK4a), BMI1, and FOXO4 after particle irradiation. Both proton or C-ion irradiation caused a positive regulation of the repair genes ATM, NBN, ATXR, and XPC, and a highly significant increase in XRCC1/2/3, ERCC1, XPC, and PCNA expression, with C-ions appearing to activate DNA repair mechanisms more effectively. The link between the physical data and the cellular responses is an important contribution to the improvement of the treatment system.