Phase Transition Of Cell Cycle Research Articles

Placental macrophage responses to viral and bacterial ligands and the influence of fetal sex.

Bacterial and viral infections of the placenta are associated with inflammation and adverse pregnancy outcomes. Hofbauer cells (HBCs) are fetal-origin macrophages in the placenta, proposed to protect the fetus from vertical pathogen transmission. We performed quantitative proteomics on term HBCs under resting conditions and following exposure to bacterial and viral pathogen-associated molecular patterns (PAMPs), and investigated the contribution of fetal sex. Resting HBCs expressed proteins pertinent to macrophage function, including chemokines, cytokines, Toll-like receptors, and major histocompatibility complex class I and II molecules. HBCs mounted divergent responses to bacterial versus viral PAMPs but exhibited protein expression changes suggestive of a more pro-inflammatoryphenotype. A comparison between male and female HBCs showed that the latter mounted a stronger and wider response. Here, we provide a comprehensive understanding of the sex-dependent responses of placental macrophages to infectious triggers, which were primarily associated with lipid metabolism in males and cytoskeleton organization in females.

An ATP-binding cassette transporter G2 (CgABCG2) regulates the haemocyte proliferation by modulating the G1/S phase transition of cell cycle in oyster Crassostrea gigas

ATP-binding cassette transporter G2 (ABCG2) is a half-transporter of the G subfamily in ATP-binding cassette transporters (ABC transporter), which is involved in the regulation of multidrug-resistant, cell cycle, and cell proliferation. In the present study, a homologue of ABCG2 (named as CgABCG2) with the conserved AAA domain and ABC2 membrane domain was identified from the Pacific oyster Crassostrea gigas. The open reading frame (ORF) of CgABCG2 was of 1956 bp encoding a predicted polypeptide of 652 amino acids, which shared 56.7%–65.7% sequence similarities with previously identified ABCG2s from other animals. The mRNA transcripts of CgABCG2 were detected in all the tested tissues with higher expression levels in gonad and haemocytes (19.31-fold and 11.23-fold of that in adductor muscle respectively, p < 0.05). CgABCG2 was mainly distributed on the cell membrane of the haemocytes with a partial distribution in the cytoplasm and nucleus. After Vibrio splendidus stimulation, the mRNA expression level of CgABCG2 in haemocytes was significantly up-regulated at 3 h and 6 h, which was 5.22-fold and 8.60-fold (p < 0.05) of that in control, respectively. After the expression of CgABCG2 was interfered by RNAi, the number of cells with EdU positive signals was reduced in both haemocytes and the potential hematopoietic sites. And the mRNA expression level of CgPCNA, CgGATA3, CgRunx, CgSCL and CgC-kit decreased significantly (p < 0.05), which were about 0.66-, 0.37-, 0.32-, 0.50-, and 0.50-fold of that in the negative control group, respectively. While the mRNA expression level of CgCDK2 increased significantly (1.84-fold to that in control, p < 0.05) and that of stem cell-related factor CgSOX2 did not change significantly in the si-CgABCG2 oysters. Moreover, the cell cycle of haemocytes was detected by flow cytometry, which was arrested at G0/G1 phase in the si-CgABCG2 oysters. All the results collectively suggested that CgABCG2 might involve the proliferation of haemocytes by regulating the expression of haematopoiesis related transcription factors and the G1/S phase transition of the cell cycle in oyster C. gigas.

Characterizing and Targeting Genes Regulated by Transcription Factor MYBL2 in Lung Adenocarcinoma Cells.

Simple SummaryThe clinical behavior and progression of lung cancer vary case by case. Therefore, understanding the factors that contribute to aggressive lung cancer cases is crucially needed. Previously, we showed that transcription factor MYBL2 expression is associated with the survival of lung adenocarcinoma patients. In this study, we characterized the functions of MYBL2 in lung adenocarcinoma cells by generating global binding profiles of MYBL2 and transcriptome profiles upon knockdown experiments. We discovered that MYBL2 regulates cell cycle genes by binding to the promoters of highly expressed genes in lung adenocarcinoma cells working with FOXM1. Moreover, we found that a previously reported FOXM1 inhibitor, FDI-6 (forkhead domain inhibitor 6), suppresses lung adenocarcinoma cell proliferation by inhibiting the activities of MYBL2 and FOXM1 and controlling cell death and cell cycle genes. Our findings provide valuable insights into the molecular mechanisms of aggressive lung cancer and suggest potential targets and treatments for the disease.Overexpression of MYBL2 is associated with poor survival of lung adenocarcinoma patients, but the molecular mechanism by which it regulates transcription and carcinogenesis has not yet been elucidated. In this study, we performed ChIP-seq using an MYBL2-targeted antibody and discovered that MYBL2 primarily binds to the promoters of highly expressed genes in lung adenocarcinoma cells. Using a knockdown experiment of MYBL2 and global transcriptome profiling, we identified that over a thousand genes are dysregulated by MYBL2, and MYBL2 acts as a transcriptional activator in lung adenocarcinoma cells. Moreover, we revealed that the binding sites of FOXM1 are largely shared with MYBL2 binding sites, and genes involved in cell cycle phase transitions are regulated by these transcription factors. We furthermore investigated the effect of a previously reported FOXM1 inhibitor, FDI-6, in lung adenocarcinoma cells. We demonstrated that FDI-6 decreases the proliferation of lung adenocarcinoma cells and inhibits the activities of FOXM1 as well as MYBL2. Moreover, we found that genes involved in cell death and cell cycle are inhibited by FDI-6. Overall, our findings suggest that MYBL2 and FOXM1 activate cell cycle genes together, acting as oncogenic transcription factors in lung adenocarcinoma cells, and they are potential treatment targets for the disease.

Integrated bioinformatic analysis of potential biomarkers of poor prognosis in triple-negative breast cancer.

BackgroundTriple-negative breast cancer (TNBC) is a heterogeneous disease associated with late-stage diagnosis and high metastatic rates. However, a gene signature for reliable TNBC biomarkers is not available yet. We aimed to identify potential key genes and their association with poor prognosis in TNBC through integrated bioinformatics.MethodsMicroarray datasets were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) in TNBC vs. non-TNBC and TNBC vs. normal tissues were analyzed. Overlapping upregulated and downregulated DEGs were selected as inputs for Gene Ontology and pathway enrichment analyses using Metascape. Then, UALCAN and Kaplan-Meier plotter were employed to analyze the prognostic values of all overlapping DEGs.ResultsWe identified 21 upregulated and 24 downregulated overlapping DEGs in TNBC vs. non-TNBC and TNBC vs. normal breast tissue. The upregulated overlapping DEGs were mainly enriched in various pathways including chromosome segregation, cell cycle phase transition, and cell division, whereas overlapping DEGs were significantly downregulated in pathways, such as multicellular organismal homeostasis, tissue homeostasis, and negative regulation of cell population proliferation. Key genes were identified by association with poor overall survival (OS). Our results showed that high expression of CENPW and HORMAD1 was associated with poor OS of TNBC patients. Conversely, the low expression of PIP, APOD, and ZNF703 indicated worse OS.ConclusionsWe identified key genes (CENPW, HORMAD1, APOD, PIP, and ZNF703) associated with poor OS. Thus, these genes might serve as candidate prognostic markers for TNBC.

Comprehensive Analysis of the Transcriptome-wide m6A Methylome in Lung Adenocarcinoma by MeRIP Sequencing.

N6-methyladenosine (m6A) is the most abundant internal modification on eukaryotic mRNAs. There is increasing evidence that m6A plays a key role in tumor progression, so it is important to analyze m6A modifications within the transcriptome-wide in lung adenocarcinoma (LUAD). Three pairs of LUAD samples and tumor-adjacent normal tissues were obtained from the South University of Science and Technology Hospital. And then methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were used to identify differential m6A modifications between tumor and tumor-adjacent normal tissues. We identified 4041 aberrant m6A peaks, of which 1192 m6A peaks were upregulated and 2849 m6A peaks downregulated. It was found that genes with the dysregulated m6A peaks were enriched in the pathways in cancer, Rap1 signaling pathway, and insulin resistance. Additionally, 612 genes with abnormal regulation of m6A peaks and RNA expression were identified by combining MeRIP-seq and RNA-seq data. Through KEGG analysis, the 612 genes were enriched in cancer-related signaling pathways, such as the cGMP-PKG signaling pathway, and the Rap1 signaling pathway. What’s more, GSEA enrichment analysis showed these genes were enriched in cell cycle phase transition, cell division, cellular response to DNA damage stimulus, and chromosome organization. To further explore the relationship between differential m6A modified genes and clinical parameters of LUAD patients, we searched The Cancer Genome Atlas (TCGA) and identified 2 genes (FCRL5 and GPRIN1) that were associated with the prognosis and diagnosis of LUAD patients. Furthermore, we found a positive correlation between GPRIN1 and m6A reader YTHDF1 in the GEPIA2 database. It was verified that YTHDF1 binds to GPRIN1 mRNA and regulates its expression. Our study results suggest that m6A modification plays important role in the progression and prognosis of LUAD and maybe a potential new therapeutic target for LUAD patients in the future.

Identification of Hub Genes for Early Diagnosis and Predicting Prognosis in Colon Adenocarcinoma.

Colon adenocarcinoma (COAD) is among the most common digestive system malignancies worldwide, and its pathogenesis and gene signatures remain unclear. This study explored the genetic characteristics and molecular mechanisms underlying colon cancer development. Three gene expression data sets were obtained from the Gene Expression Omnibus (GEO) database. GEO2R was used to determine differentially expressed genes (DEGs) between COAD and normal tissues. Then, the intersection of the data sets was obtained. Metascape was used to perform the functional enrichment analyses. Next, STRING was used to build protein-protein interaction (PPI) networks. Hub genes were identified and analysed using Cytoscape. Next, survival analysis and expression analysis of the hub genes were performed. ROC curve analysis was performed for further test of the diagnostic efficacy. Finally, alterations in the hub genes were predicted and analysed by cBioPortal. Altogether, 436 DEGs were detected. The DEGs were mainly enriched in cell cycle phase transition, nuclear division, meiotic nuclear division, and cytokinesis. Based on PPI networks, 20 hub genes were selected. Among them, 6 hub genes (CCNB1, CCNA2, AURKA, NCAPG, DLGAP5, and CENPE) showed significant prognostic value in colon cancer (P < 0.05), while 5 hub genes (CDK1, CCNB1, CCNA2, MAD2L1, and DLGAP5) were associated with early colon cancer diagnosis and ROC curve analysis showed good diagnostic accuracy. In conclusion, integrated bioinformatics analysis was used to identify hub genes that reveal the potential mechanism of carcinogenesis and progression of colon cancer. The hub genes might be novel biomarkers for early diagnosis, treatment, and prognosis of colon cancer.

DDK: The Outsourced Kinase of Chromosome Maintenance.

Simple SummaryTo ensure the maintenance of genetic stability prior to cell division a cell’s complement of chromosomes must be duplicated. This requires not only the replication of the chromosomal DNA but also the re-establishment the chromatin environment following duplication. To ensure the equal segregation of the genetic material to progeny cells, the duplicated chromatid pairs must remain physically coupled until cell division. The regulation of chromosome duplication is under the overall control of the cyclin-dependent kinases (CDK). In addition to maintaining global control of chromosome duplication, CDK directs the activation of a second kinase, the Dbf4-dependent kinase (DDK), which functions locally to facilitate the activation DNA replication and to coordinate this with the re-establishment of chromatin and the physical coupling of the chromatids following duplication. In this review, we discuss this ‘outsourcing’ by CDK to DDK of the activities that must be coordinated to ensure chromosome maintenance during cell division.The maintenance of genomic stability during the mitotic cell-cycle not only demands that the DNA is duplicated and repaired with high fidelity, but that following DNA replication the chromatin composition is perpetuated and that the duplicated chromatids remain tethered until their anaphase segregation. The coordination of these processes during S phase is achieved by both cyclin-dependent kinase, CDK, and Dbf4-dependent kinase, DDK. CDK orchestrates the activation of DDK at the G1-to-S transition, acting as the ‘global’ regulator of S phase and cell-cycle progression, whilst ‘local’ control of the initiation of DNA replication and repair and their coordination with the re-formation of local chromatin environments and the establishment of chromatid cohesion are delegated to DDK. Here, we discuss the regulation and the multiple roles of DDK in ensuring chromosome maintenance. Regulation of replication initiation by DDK has long been known to involve phosphorylation of MCM2-7 subunits, but more recent results have indicated that Treslin:MTBP might also be important substrates. Molecular mechanisms by which DDK regulates replisome stability and replicated chromatid cohesion are less well understood, though important new insights have been reported recently. We discuss how the ‘outsourcing’ of activities required for chromosome maintenance to DDK allows CDK to maintain outright control of S phase progression and the cell-cycle phase transitions whilst permitting ongoing chromatin replication and cohesion establishment to be completed and achieved faithfully.

The correlation of EZH2 expression with the progression and prognosis of hepatocellular carcinoma

BackgroundEnhancer of Zeste homologue 2 (EZH2) is a polycomb group gene and an epigenetic regulator that inhibits transcription, a modification associated with gene silencing. EZH2 plays an essential role in humoral and cell-mediated adaptive immunity. The purpose of the current study is to investigate the prognostic potential of EZH2 and to comprehensively analyse the correlation between EZH2 and immune infiltration in multiple cancer cases, especially liver hepatocellular carcinoma.MethodsEZH2 expression across cancers was explored through Oncomine, HPA, and GEPIA2. Additionally, the prognostic value of EZH2 analysis across cancers was based on the GEPIA2, TCGA portal, Kaplan–Meier Plotter, and LOGpc databases. Based on GO and KEGG analyses, GSEA helped demonstrate the biological processes through which EZH2 might lead to HCC development. GEPIA and TIMER were adopted to detect the possible relationship of EZH2 expression with tumour-infiltrating immune cells (TIICs).ResultsEZH2 overexpression levels were associated with poor prognosis of cancer, especially hepatocellular carcinoma. A high EZH2 expression level is related to a poor prognosis of HCC, especially in disease histology and stage III. The EZH2 expression level was positively correlated with critical gene markers of TAMs, M2 macrophages, M1 macrophages, and monocytes. Further analysis revealed that EZH2 genes were mainly related to DNA recombination, mitotic cell cycle phase transition, and chromosome segregation.ConclusionEZH2 plays an essential role in the immune microenvironment and is a potential prognostic marker and immunotherapy target for hepatocellular carcinoma.

BPA disrupts meiosis I in oogonia by acting on pathways including cell cycle regulation, meiosis initiation and spindle assembly

The negative in utero effects of bisphenol A (BPA) on female reproduction are of concern since the ovarian reserve of primordial follicles is constituted during the fetal period. This time-window is difficult to access, particularly in humans. Animal models and explant culture systems are, therefore, vital tools for investigating EDC impacts on primordial germ cells (PGCs). Here, we investigated the effects of BPA on prophase I meiosis in the fetal sheep ovary. We established an in vitro model of early gametogenesis through retinoic acid (RA)-induced differentiation of sheep PGCs that progressed through meiosis. Using this system, we demonstrated that BPA (3 ×10-7 M & 3 ×10-5 M) exposure for 20 days disrupted meiotic initiation and completion in sheep oogonia and induced transcriptomic modifications of exposed explants. After exposure to the lowest concentrations of BPA (3 ×10-7 M), only 2 probes were significantly up-regulated corresponding to NR2F1 and TMEM167A transcripts. In contrast, after exposure to 3 × 10-5 M BPA, 446 probes were deregulated, 225 were down- and 221 were up-regulated following microarray analysis. Gene Ontology (GO) annotations of differentially expressed genes revealed that pathways mainly affected were involved in cell-cycle phase transition, meiosis and spindle assembly. Differences in key gene expression within each pathway were validated by qRT-PCR. This study provides a novel model for direct examination of the molecular pathways of environmental toxicants on early female gametogenesis and novel insights into the mechanisms by which BPA affects meiosis I. BPA exposure could thereby disrupt ovarian reserve formation by inhibiting meiotic progression of oocytes I and consequently by increasing atresia of primordial follicles containing defective oocytes.

FOXM1 Promotes Malignant Proliferation of Esophageal Squamous Cell Carcinoma Through Transcriptional Activating CDC6.

Forkhead box M1 (FOXM1) is a proliferative transcription factor and plays a vital role in many cancers. However, the function and molecular mechanism of FOXM1 in esophageal squamous cell carcinoma (ESCC) remain poorly understood. Hence, we aim to clarify the molecular basis of FOXM1-mediated ESCC progression. In this study, bioinformatics analysis showed that FOXM1 was mainly involved in key signal pathways, including cell proliferation, cell cycle, and homologous recombination in ESCC, and predicted that CDC6 might be a potential regulatory target gene of FOXM1. The results revealed that FOXM1 and CDC6 were significantly overexpressed in ESCC tissue and cell line, and their expression was positively correlated. Further studies showed that FOXM1 directly transcriptionally activated CDC6 by binding to its promoter region in ESCC cells. Moreover, FOXM1 mediated ESCC cell proliferation by regulating CDC6 expression, which may be related to promoting G1-S phase transition of cell cycle. Taken together, FOXM1-CDC6 axis mediates ESCC malignant proliferation and may serve as a potential biological target for ESCC treatment.

Molecular clustering based on gene set expression and its relationship with prognosis in patients with lung adenocarcinoma.

BackgroundLung adenocarcinoma (LUAD) is a subtype of lung cancer with high morbidity and mortality. While genotyping is an important determinant for the prognosis of LUAD patients, there is a paucity of studies on gene set-based expression (GSE) typing for LUAD. This current study used GSE methodology to perform gene typing of LUAD patients.MethodsClinical and genomic information of the LUAD patients were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Patients with LUAD were clustered into different molecular subtypes depending on the clinical and gene set expression characteristics. The survival rate and silhouette widths were compared between each molecular subtype. Differences in survival rate between gene sets were analyzed using Kaplan-Meier survival curves. Cox regression and Lasso regression were used to establish the prognostic gene set model based on the TCGA database, and the results were validated using the GEO dataset.ResultsA total of 10 hub genes were finally identified and clustered into 3 subtypes with a mean contour width of 0.96. There were significant differences in survival rates among the 3 subtypes (P<0.05). Gene Ontology (GO) analysis indicated that the related biological processes (BP) were mainly involved in regulation of cell cycle, mitotic cell cycle phase transition, and proteasome-mediated ubiquitin-dependent protein catabolic process. The cellular components (CC) were related to the spindle, chromosomal region, and midbody. Molecular function (MF) mainly focused on ubiquitin-like protein ligase binding, translation regulator activity, and oxidation activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the main pathways included the Epstein Barr virus infection pathway of neurogeneration, the p53 signaling pathway, and the proteome pathways. In addition, the protein-protein interaction network was analyzed using the STRING and Cytospace software, and the top 9 hub genes identified were KIF2C, DLGAP5, KIF20A, PSMC1, PSMD1, PSMB7, SNAI2, FGF13, and BMP2.ConclusionsPatients with LUAD can be clustered into three subtypes based on the expression of gene sets. These findings contribute to understanding the pathogenesis and molecular mechanisms in LUAD, and may lead to potential individualized pharmacogenetic therapy for patients with LUAD.

A hierarchical transcriptional network activates specific CDK inhibitors that regulate G2 to control cell size and number in Arabidopsis

How cell size and number are determined during organ development remains a fundamental question in cell biology. Here, we identified a GRAS family transcription factor, called SCARECROW-LIKE28 (SCL28), with a critical role in determining cell size in Arabidopsis. SCL28 is part of a transcriptional regulatory network downstream of the central MYB3Rs that regulate G2 to M phase cell cycle transition. We show that SCL28 forms a dimer with the AP2-type transcription factor, AtSMOS1, which defines the specificity for promoter binding and directly activates transcription of a specific set of SIAMESE-RELATED (SMR) family genes, encoding plant-specific inhibitors of cyclin-dependent kinases and thus inhibiting cell cycle progression at G2 and promoting the onset of endoreplication. Through this dose-dependent regulation of SMR transcription, SCL28 quantitatively sets the balance between cell size and number without dramatically changing final organ size. We propose that this hierarchical transcriptional network constitutes a cell cycle regulatory mechanism that allows to adjust cell size and number to attain robust organ growth.

A Transcriptome Sequencing Study on Genome-Wide Gene Expression Differences of Lung Cancer Cells Modulated by Fucoidan.

Fucoidan has received increasing attention in anti-(lung) tumors. However, the effect of fucoidan on the gene changes of lung cancer cells (LCCs) has not been examined systematically. Herein, we investigate the effect of fucoidan on the phenotypes of LCCs and their gene expression by transcriptome sequencing analysis. The phenotypes of LCCs are significantly inhibited by fucoidan. Importantly, compared to LCCs, 1 mg/ml fucoidan has no effect on the phenotypes of normal cells. Further, 6,930 differentially expressed genes (DEGs) in the transcriptome of LCCs (3,501 up-regulated and 3,429 down-regulated genes) are detected via RNA-sequencing between the fucoidan and control groups. Gene Ontology analysis confirms that DEGs are reflected in DNA replication, cell-substrate junction, regulation of cell cycle phase transition, apoptosis, focal adhesion, cadherin binding, and cell adhesion molecule binding. Thus, our findings on the transcriptomic level highlight the therapeutic potential of fucoidan for lung cancer treatment.

Integration of transcriptomics and metabolomics provides metabolic and functional insights into reduced insulin secretion in MIN6 β-cells exposed to deficient and excessive arginine.

Previous work demonstrated that arginine is one of the strongest insulin secretagogues. However, knowledge of the mechanisms linking chronic arginine metabolism with β-cell function and insulin secretion is relatively limited. After preliminary selection of concentration according to the cell proliferation, the MIN6 pancreatic β-cells were randomly assigned to culture in 0.04mM (low-arginine, LA), 0.4mM (standard-arginine, SA), or 8mM arginine (high-arginine, HA) for 24h. Following the treatment, a combination of transcriptomics and metabolomics, together with a series of molecular biological tests were performed to investigate the responses of β-cells to varied arginine availability. Our results showed that HA treatment reduced the chronic insulin releases, and LA and HA treatments decreased the glucose-stimulated insulin secretions (GSIS) of β-cells relative to the SA group (p<.05). Transcriptomics analysis indicated that LA administration significantly inhibited oxidative phosphorylation and ATP metabolic process but promoted DNA repair and mRNA processing in β-cells, while HA administration affected ammonium ion metabolic process and mRNA export (p<.05). Both LA and HA regulated the expressions of genes involved in DNA replication, cell-cycle phase transition, and response to oxidative stress (p<.05). Protein-protein interaction and transcription factor analyses suggested that Trp53 and Nr4a2genes may play key roles during arginine stimulation. On the contrary, metabolomics analysis demonstrated that the differentially expressed metabolites (DEM) of MIN6 β-cells induced by LA were mainly enriched in glycerophospholipid metabolism, linoleic acid metabolism, and purine metabolism, while most DEMs between LA vs. SA comparison belonged to amino acid metabolism. When combined the three groups, co-expression analysis suggested that insulin secretions had strong associations with L-pyroglutamic acid, L-glutamate, and creatine concentrations, while intracellular insulin contents were mainly correlated to L-arginine, argininosuccinic acid, and phosphorylcholine. At last, integrated analysis of transcriptomics and metabolomics showed that glycerophospholipid metabolism, biosynthesis of unsaturated fatty acids, and amino acid metabolism were the most relevant pathways in β-cells exposed to abnormal arginine supply. This descriptive bioinformatics analysis suggested that the disturbed carbohydrate, lipid, and amino acid metabolisms, as well as the increased apoptosis and elevated oxidative stress, contributed to the reduced insulin secretion and lower GSIS in β-cells induced by LA or HA treatments, while some underlying mechanisms need to be further explored.

Discovery of novel ataxia telangiectasia mutated (ATM) kinase modulators: Computational simulation, biological evaluation and cancer combinational chemotherapy study

Ataxia-telangiectasia mutated (ATM) kinase is a serine/threonine protein kinase and plays a key role in DNA double-strand breaks repair. Thus, ATM is considered a promising target for radiotherapy and chemotherapy sensitizing. Herein, we report the discovery of ATM agonist A22 and inhibitor A41 by computational methods and further biological evaluation. Among them, A22 exhibited low cytotoxicity in vitro and might serve as a useful tool for ATM research. Moreover, we firstly proved that ATM inhibitors could sensitize Irinotecan and Etoposide in a time-dependent manner on MCF-7 and SW480 cells, antagonism in a short period treatment while synergy at a long-term treatment and ATM agonist worked in an opposite way of ATM inhibitors. Further mechanism study demonstrated that the antagonism effect of ATM inhibitors with chemotherapeutic agents in a short period was resulting from inhibiting the p53/p21 axis to accelerate G1/S phase cell-cycle transition and promote cell survival. Additionally, A41 displayed antitumor effects combined with a chemotherapeutic drug in the SW480 xenograft model, indicating that A41 is a promising ATM inhibitor, which could increase the antitumor effect of chemotherapeutic drugs in vivo. All in all, these findings will guide the combination of ATM inhibitors with chemotherapeutic agents in further preclinical and clinical studies.

Abstract P2-07-02: Genomic predictors of rapid progression to first line endocrine and CDK4/6 inhibitor combination therapy in patients with estrogen receptor positive (ER+) HER-2 negative (HER2-) advanced breast cancer (ABC)

Abstract Background: Endocrine therapy with CDK 4/6 inhibitors (ET/CDK4/6i) represents the 1st line therapy for ER+/HER2- ABC. While majority of patients derive clinical benefit with combination therapy, a subset have refractory disease with progression within 6 months. However, predictive biomarkers for rapid progression are lacking. In this study, we evaluated genomic profiles associated with rapid disease progression on ET/CDK4/6i. Methods: We identified 77 patients who received 1st line ET/CDK4/6i combination therapy (AI or SERD with one of the 3 approved CDK4/6is) and had ctDNA analysis performed via plasma based genotyping utilizing the commercially available Guardant360 assay at three sites: Washington University in St. Louis, MO, Northwestern University (Chicago, IL), and Massachusetts General Hospital (Boston, MA). We aimed to look at the differences in patient characteristics and genomic profiles of the tumors assessed from baseline ctDNA specimens between the patients with rapid progression (time to progression TTP&amp;lt;=6 months) vs others. In particular, we focused on growth factor receptors (FGFR, EGFR) given that previous studies have shown that activation of FGFR1 and EGFR signaling may be implicated in resistance to endocrine based therapy in breast cancer. Time to progression was estimated by using Cox regression. Variable associations were estimated via logistic regression. Results: In the combined cohort, FGFR1 amplification (FGFR1amp) was detected in 15/77 patients (19.5%). FGFR1amp was seen in 5/10 (50%) of patients with rapid progression, consistent with existing knowledge that FGFR1amp contributes to resistance to CDK4/6i and/or ET. Presence of FGFR1amp was independently associated with shorter TTP (11.2 vs. 34.7 months, HR=3.14, p=0.02). EGFR mutations (EGFRmut) were detected in 8/77 (10.4%) patients, 3 of which were found among patients with rapid progression and another 5 among those with TTP&amp;lt;=15 months. Presence of EGFRmut was also associated with shorter TTP (8.5 vs. 31.7 months, HR=6.50, p&amp;lt;0.001) in multivariable analysis. Of the 4 patients with shortest TTP (&amp;lt;3 months) 3 harbored both FGFR1amp and EGFRmut. In another 3 patients we observed FGFR1amp and co-activation of genes implicated in G1/S phase cell cycle transition, suggesting that FGFR1 amplified cells may require a co-activating downstream event that ultimately, via multiple pathway cross-talk, renders them resistant to ET/CDK4/6 inhibition. Patients with FGFR1 amplified tumors were younger compared to those without FGFR1amp (54.3 vs. 62.7 years, p=0.04). Presence of FGFR1amp was associated with presence of liver (p=0.01) but not bone or lung metastases which could be one of the explanations why patients with higher liver tumor burden are more resistant to ET/CDK4/6i inhibition. PIK3CA and TP53 gene mutations in our cohort were frequent (found in 41% and 30% of the patients, respectively) but were independently not associated with TTP (PIK3CAmut+ HR=1.31, p=0.55, TP53mut+ HR=0.67, p=0.36). ESR1 mutations were rarely encountered (9%) as the cohort had only been exposed to adjuvant endocrine therapy. Conclusions: These findings highlight how ctDNA can be used for patient stratification prior to initiation of first line of therapy in ER+/HER2- ABC since it is evident that not all patients derive the same benefit from ET/CDK4/6i. Certain genomic alterations, particularly in FGFR1, EGFR, and G1/S phase cell cycle transition are associated with rapid progression to 1st line ET/CDK4/6i therapy, and highlight the need for clinical trials investigating combination/novel therapies for this subgroup of patients with HR+/HER2- ABC. Our findings are hypothesis-generating and require further exploration in larger datasets. Citation Format: Marko Velimirovic, Lorenzo Gerratana, Andrew A Davis, Whitney L Hensing, Katherine Clifton, Ami N Shah, Paolo D'Amico, Charles S Dai, Elyssa N Denault, Cynthia X Ma, Seth A Wander, Dejan Juric, Massimo Cristofanilli, Bruce A Chabner, Aditya Bardia. Genomic predictors of rapid progression to first line endocrine and CDK4/6 inhibitor combination therapy in patients with estrogen receptor positive (ER+) HER-2 negative (HER2-) advanced breast cancer (ABC) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-07-02.

Identification and characterization of the inhibitory effect of ART on colorectal cancer cells using Raman spectroscopy

AbstractRaman spectroscopy (RS) has been used in clinical diagnostics, tissue engineering, and toxicology experiments owing to its label‐free, low‐destructive, high sensitivity, and strong specificity. Nonetheless, their application has been limited because of the integrated analysis of biological experimental methods and RS. In this study, it was challenged that applied RS in combination with biological experiments to distinguish colorectal cancer (CC) cells from which treated with artesunate (ART), which has obvious antitumor effects. Heavy water (D2O) was used to detect changes in colorectal cancer cell metabolism. The Raman spectral intensity of the C–D band at 2100–2300 cm−1 was significantly weakened in CC (SW480, SW620, and HCT116) treated with ART. According to the CCK‐8, wound healing, and Transwell assays, the activity of SW480 and SW620 cells was significantly inhibited by ART. Through literature tracking and transcriptome sequencing, multiple signal pathways of SW480 and SW620 cells have changed significantly after treatment with ART, such as “apoptotic signaling pathway,” “cell cycle phase transition,” “mitochondrial part,” and “mRNA metabolic process.” The Raman peak area significantly decreased at 490 cm−1, 746 cm−1, 1126 cm−1, 1311 cm−1, 1337 cm−1, and 1582 cm−1 in SW480 and SW620 cells treated with ART. It showed that these Raman bands are associated with ART‐induced apoptosis‐related signaling pathways. In addition, we found that application of the RS in cell type identification will be prosperous. By integrating RS with biological mechanisms, its application in the biomedical field is increasing, and its significance in biological sample detection has become more prominent.

Overexpressed GNAZ predicts poor outcome and promotes G0/G1 cell cycle progression in hepatocellular carcinoma.

We aimed to investigate the role of G protein subunit alpha Z(GNAZ) in the progression and prognosis of patients with hepatocellular carcinoma (HCC). Oncomine, GEO, TCGA, GEPIA2, Kaplan-Meier Plotter, TIMER2, Metascape, CCLE, LinkedOmics, and UALCAN databases were used to analyze the differential expression of GNAZ in HCC and normal liver tissues, relationship between GNAZ expression and prognosis of patients with HCC, and expression of GNAZ in common human HCC cell lines. Western blotting was performed to analyze GNAZ expression, while the Cell Counting Kit 8 assay was used to determine cell proliferation, and flow cytometry was used to evaluate the cell cycle and apoptosis. Wound healing and transwell invasion assays were used to investigate cell metastasis and invasion. Using Oncomine, Gene Expression Omnibus (GEO), and GEPIA2 databases, GNAZ was found to be overexpressed in HCC tissues compared with that in adjacent normal liver tissues, and western blotting analysis showed GNAZ overexpression in seven patients with HCC who underwent surgical resection of HCC and para-cancerous tissues (p<0.01). Survival analysis revealed that high GNAZ expression was negatively associated with overall survival (OS), recurrence-free survival, progression-free survival, and disease-specific survival in patients with HCC (p<0.05). GNAZ overexpression was associated with worse 4- month, 6- month, 12- month, 24- month, 36- month, 48- month, and 60-month OS, as well as with different clinicopathological characteristics of patients with HCC, including hepatitis virus infection state; alcohol consumption state; male; female; Asian; microvascular invasion, Stage I-II, Stage II-III, and Stage III-IV; and grade II (Cox regression, p<0.05). KEGG/GO biological process enrichment indicated that the genes similar to GNAZ in HCC were mainly enriched in the cell cycle, cell cycle phase transition, DNA replication checkpoint, and regulation of G0 to G1 transition. siRNA-GNAZ significantly reduced the viability of JHH-2 and SNU-761 cells from 12 to 96h; increased the percentage of cells in the G0/G1 phase and decreased that of cells in the S and G2/M phases (p<0.05); and markedly downregulated the expression of cyclin D, cyclin E, and CDK2 protein. siRNA-GNAZ also significantly increased the percentage of JHH-2 and SNU-761 cell apoptosis at late stages, while the number of surviving cells decreased (p<0.05), and upregulated the expression of apoptosis-related proteins Bax and caspase 3 protein. Furthermore, siRNA-GNAZ remarkably reduced the healing of scratch wounds in JHH-2 and SNU-761 cells and the number of invasive cells compared with that in the control group (p<0.001). Our study demonstrated that GNAZ plays a pivotal role as a potential oncogene and predicts poor prognosis in patients with HCC. It promotes tumor proliferation via cell cycle arrest, apoptosis, migration, and invasion. Thus, GNAZ may be a potential candidate biomarker providing useful insight into hepatocarcinogenesis and aggressiveness.

G-protein Coupled Receptor 34 Promotes Gliomagenesis by Inducing Proliferation and Malignant Phenotype via TGF-Beta/Smad Signaling Pathway.

Background: G-protein coupled receptor 34 (GPR34) is involved in cell motility, differentiation, and mitosis. GPR34 was reported to be highly expressed and play an oncogenic role in several solid tumors. Here, we investigated the mechanisms underlying how GPR34 promotes glioma progression. Methods: Bioinformatic analysis was performed on RNA-seq and clinical data from the gene expression omnibus (GEO), cancer genome atlas (TCGA), and Genotype-Tissue Expression (GTEx) databases. TIMER database and single-sample GSEA (ssGAEA) method were used to investigate the association between the GPR34 expression and immune infiltration level in glioma. Cox regression analysis was employed to ascertain whether the risk signature was an independent prognostic indicator for glioma. The viability and migratory/invasive potential of glioma cells were assessed using Cell Counting Kit-8, colony formation, wound healing, and Transwell assays. Results: We found that GPR34 expression was positively correlated with immune infiltration level and that high GPR34 level may be associated with poor prognosis in glioma. We further found that GPR34 may serve as an independent prognostic marker and prediction factor for the clinicopathological features of glioma. We showed that knocking down GPR34 attenuated the viability and migratory/invasive capacity of glioma cells (U251 and LN229), while GPR34 overexpression exerted the opposite effects. Additionally, core enrichment in the GSEA analysis indicated that GPR34-mediated gliomagenesis was associated with the cell cycle arrest, epithelial–mesenchymal transition (EMT), and activation of the TGF-β/Smad pathway; furthermore, inhibiting TGF-β/Smad signaling using LY2157299, a TGF-β inhibitor, reversed the oncogenic effects and malignant phenotype associated with GPR34 overexpression. Conclusion: GPR34 enhances the malignancy and carcinogenesis of glioma by promoting an EMT-like process, G1/S phase cell cycle transition, and TGF-β/Smad signaling. Accordingly, GPR34 likely functions as an oncogene in glioma and may represent a potential therapeutic target for this cancer.

ATRX loss in glioma results in dysregulation of cell-cycle phase transition and ATM inhibitor radio-sensitization.

ATRX, a chromatin remodeler protein, is recurrently mutated in H3F3A-mutant pediatric glioblastoma (GBM) and isocitrate dehydrogenase (IDH)-mutant grade 2/3 adult glioma. Previous work has shown that ATRX-deficient GBM cells show enhanced sensitivity to irradiation, but the etiology remains unclear. We find that ATRX binds the regulatory elements of cell-cycle phase transition genes in GBM cells, and there is a marked reduction in Checkpoint Kinase 1 (CHEK1) expression with ATRX loss, leading to the early release of G2/M entry after irradiation. ATRX-deficient cells exhibit enhanced activation of master cell-cycle regulator ATM with irradiation. Addition of the ATM inhibitor AZD0156 doubles median survival in mice intracranially implanted with ATRX-deficient GBM cells, which is not seen in ATRX-wild-type controls. This study demonstrates that ATRX-deficient high-grade gliomas (HGGs) display Chk1-mediated dysregulation of cell-cycle phase transitions, which opens a window for therapies targeting this phenotype.