Affymetrix Microarray Gene Expression Research Articles

Elucidating gastric cancer mechanisms and therapeutic potential of Adociaquinone A targeting EGFR: A genomic analysis and Computer Aided Drug Design (CADD) approach.

Gastric cancer predominantly adenocarcinoma, accounts for over 85% of gastric cancer diagnoses. Current therapeutic options are limited, necessitating the discovery of novel drug targets and effective treatments. The Affymetrix gene expression microarray dataset (GSE64951) was retrieved from NCBI-GEO data normalization and DEGs identification was done by using R-Bioconductor package. Gene Ontology (GO) analysis of DEGs was performed using DAVID. The protein-protein interaction network was constructed by STRING database plugin in Cytoscape. Subclusters/modules of important interacting genes in main network were extracted by using MCODE. The hub genes from in the network were identified by using Cytohubba. The miRNet tool built a hub gene/mRNA-miRNA network and Kaplan-Meier-Plotter conducted survival analysis. AutoDock Vina and GROMACS MD simulations were used for docking and stability analysis of marine compounds against the 5CNN protein. Total 734 DEGs (507 up-regulated and 228 down-regulated) were identified. Differentially expressed genes (DEGs) were enriched in processes like cell-cell adhesion and ATP binding. Eight hub genes (EGFR, HSPA90AA1, MAPK1, HSPA4, PPP2CA, CDKN2A, CDC20, and ATM) were selected for further analysis. A total of 23 miRNAs associated with hub genes were identified, with 12 of them targeting PPP2CA. EGFR displayed the highest expression and hazard rate in survival analyses. The kinase domain of EGFR (PDBID: 5CNN) was chosen as the drug target. Adociaquinone A from Petrosia alfiani, docked with 5CNN, showed the lowest binding energy with stable interactions across a 50 ns MD simulation, highlighting its potential as a lead molecule against EGFR. This study has identified crucial DEGs and hub genes in gastric cancer, proposing novel therapeutic targets. Specifically, Adociaquinone A demonstrates promising potential as a bioactive drug against EGFR in gastric cancer, warranting further investigation. The predicted miRNA against the hub gene/proteins can also be used as potential therapeutic targets.

Inhibiting the glycerophosphodiesterase EDI3 in ER-HER2+ breast cancer cells resistant to HER2-targeted therapy reduces viability and tumour growth

BackgroundIntrinsic or acquired resistance to HER2-targeted therapy is often a problem when small molecule tyrosine kinase inhibitors or antibodies are used to treat patients with HER2 positive breast cancer. Therefore, the identification of new targets and therapies for this patient group is warranted. Activated choline metabolism, characterized by elevated levels of choline-containing compounds, has been previously reported in breast cancer. The glycerophosphodiesterase EDI3 (GPCPD1), which hydrolyses glycerophosphocholine to choline and glycerol-3-phosphate, directly influences choline and phospholipid metabolism, and has been linked to cancer-relevant phenotypes in vitro. While the importance of choline metabolism has been addressed in breast cancer, the role of EDI3 in this cancer type has not been explored.MethodsEDI3 mRNA and protein expression in human breast cancer tissue were investigated using publicly-available Affymetrix gene expression microarray datasets (n = 540) and with immunohistochemistry on a tissue microarray (n = 265), respectively. A panel of breast cancer cell lines of different molecular subtypes were used to investigate expression and activity of EDI3 in vitro. To determine whether EDI3 expression is regulated by HER2 signalling, the effect of pharmacological inhibition and siRNA silencing of HER2, as well as the influence of inhibiting key components of signalling cascades downstream of HER2 were studied. Finally, the influence of silencing and pharmacologically inhibiting EDI3 on viability was investigated in vitro and on tumour growth in vivo.ResultsIn the present study, we show that EDI3 expression is highest in ER-HER2 + human breast tumours, and both expression and activity were also highest in ER-HER2 + breast cancer cell lines. Silencing HER2 using siRNA, as well as inhibiting HER2 signalling with lapatinib decreased EDI3 expression. Pathways downstream of PI3K/Akt/mTOR and GSK3β, and transcription factors, including HIF1α, CREB and STAT3 were identified as relevant in regulating EDI3 expression. Silencing EDI3 preferentially decreased cell viability in the ER-HER2 + cells. Furthermore, silencing or pharmacologically inhibiting EDI3 using dipyridamole in ER-HER2 + cells resistant to HER2-targeted therapy decreased cell viability in vitro and tumour growth in vivo.ConclusionsOur results indicate that EDI3 may be a potential novel therapeutic target in patients with HER2-targeted therapy-resistant ER-HER2 + breast cancer that should be further explored.

GPM6A expression is suppressed in hepatocellular carcinoma through miRNA-96 production

AbstractGPM6A is a glycoprotein in endothelial cells, and its biological function in the development of hepatocellular carcinoma (HCC) is unknown. Through Affymetrix gene expression microarray and bioinformatic analysis, very low GPM6A expression was found in HCC tissue. The present study aims to explore the function and regulatory mechanism of GPM6A in HCC development and progression. Levels of GPM6A expression in HCC specimens from different disorders and various hepatoma cell lines were determined, and its role on cell proliferation was evaluated in hepatoma cells stably overexpressing GPM6A. Modulation of a specific microRNA (miRNA) on its expression and function was evaluated with miRNA mimetic transfection. Herein, it is reported that much lower GPM6A levels were found in HCC tissues than pericancerous liver tissues and correlated to a poor prognosis. GPM6A overexpression inhibited cell proliferation, suppressed colony formation, migration and invasion in two hepatoma cell types. Available evidence does not support that genetic and epigenetic dysregulation contributes significantly to GPM6A inactivation in HCC. Additional findings demonstrated that miR-96-5p acted directly on the 3′-UTR of the GPM6A gene and significantly decreased its mRNA and protein levels. MiR-96-5p transfection promoted proliferation, migration and invasion of SMMC-7721 and MHCC-97H hepatoma cells; whereas the function of oncogenic microRNA-96 was significantly inhibited in GPM6A-overexpressed hepatoma cells. In conclusion, GPM6A expression in HCC is commonly suppressed regardless its base disease types, and its low expression in HCC tissues is most likely attributed to upregulated miR-96-5p. GPM6A may function as a valuable biomarker for HCC progression and prognosis.

O-141 Mapping of SARS-CoV-2-associated receptors and proteases mRNA in human endometrium during natural and stimulated cycles

Study questionCovid-19 pandemic has significantly affected the assisted reproductive technology (ART) practice. Understanding whether SARS-CoV-2 could infect endometrial tissues during ART is crucial for risk mitigationSummary answerAnalyses of gene expression profiles of SARS-CoV-2 host entry candidates from microarray data suggest that endometrium should be considered as potential target for SARS-CoV-2 infection.What is known alreadyVery few studies analyzed the gene expression profiles of SARS-CoV-2-associated receptors and proteases, mainly focusing on ACE2 and TMPRSS2 expression, resulting incomplete knowledge in different specimens from female genital tract. However, no studies have analyzed the potential impact of controlled ovarian stimulation (COS) protocols during ART procedure on the endometrial gene expression profiles of SARS-CoV-2-associated receptors and proteasesStudy design, size, durationTo address this question, we retrospectively examined the gene expression profile of SARS-CoV-2-associated receptors and proteases in endometrial biopsies of a cohort of ART candidates using Affymetrix microarray dataParticipants/materials, setting, methodsHuman endometrial tissue under natural (n = 62) and COS cycles (n = 42) were analyzed. A focus was particularly made on the renin-angiotensin system relates genes with a prominent role in the virus infection, and gene expression levels of receptors and proteases closely related to SARS-CoV-2 infectionwas also studied.Main results and the role of chanceUsing our large cohort of endometrial samples, we reported a high prevalence of genes related to the ACE2 pathway, including AGT, AGTR1, ANPEP, CTSA, ENPEP, LNPEP, MME, NLN, THOP1, BSG and CTSL during both phases(early- and mid-secretory phase), and mainly during the mid-secretory phase for ACE2. The highest signal intensities were found for CTSA, LNPEP, MME, NLN, BSG and CTSL. The most representative of dual coexpression of SARS-CoV-2-associated receptor and protease in endometrium was BSG-CSTL and BSG-CTSA. It s also important to note high variation of SARS-CoV-2 receptors inter-patients under natural cycle.Globally, the impact of COS on endometrial gene expression profile of SARS-CoV-2-associated receptors and proteases of non Covid-19 patients is low, suggesting no additional potential risks of SARS-CoV-2 infection during stimulated ART procedure compared with natural cycles.Limitations, reasons for cautionAnalyses of Affymetrix microarray gene expression data were performed in non-COVID-19 patients. Whether the SARS-CoV-2 infection changes the endometrial gene expression profile of SARS-CoV-2-associated receptors and proteases is under investigationWider implications of the findingsSpecimens from female genital tract may be considered as potential targets for SARS-CoV-2.Trial registration numbernot applicable

P–242 Gene expression profiles of SARS-CoV–2-associated receptors and proteases in human early embryonic development and follicular cells

Study questionAre the oocytes, embryos, granulosa and cumulus cells, used during ART, susceptible to the SARS-CoV–2 infection?Summary answerTranscriptomic analyses of SARS-CoV–2-associated receptors and proteases strongly suggest that blastocysts are most permissive to SARS-CoV–2 compared with mature oocytes and day 3 embryos.What is known alreadyVery few studies analyzed the gene expression profiles of SARS-CoV–2-associated receptors and proteases, mainly focusing on ACE2 and TMPRSS2 expression, resulting in partial knowledge in different specimens from female genital tract. To date, the gene expression profile of SARS-CoV–2 host entry candidates in the entire preimplantation embryos is scarcely available. Moreover, reports on oocyte and granulosa cells susceptibilityto SARS-CoV–2 are very sparse.Study design, size, durationTo address this question, we retrospectively examined the gene expression profiles of SARS-CoV–2-associated receptors and proteases in human granulosa cells (GCs), cumulus cells (CCs), mature oocytes, day 3 embryos, blastocysts and trophectoderm cells obtained from our previously described Affymetrix microarray data.Participants/materials, setting, methodsHuman GCs and CCs (n = 17), mature oocytes (n = 6), and preimplantation embryos (n = 20) were analyzed. The comparison of gene expression levels of receptors and proteases closely related to SARS-CoV–2 infection. For each gene, the number of samples with the probe set ‘present’, based on the detection call, was analyzed. Each probe set was classified according to the signal intensity value median, as low (<100), medium (100–200) or high expression level (>200).Main results and the role of chanceACE2, BSG, CTSL, CTSA were detectable at high expression level in all mature oocyte samples, while only CTSL was strongly expressed in all day 3 embryos. The most representative dual co-expression of SARS-CoV–2-associated receptor and protease (60% of samples) during the embryonic genome activation stage (EGA) was ACE2-CTSL and BSG-CTSL. In blastocysts, ACE2, BSG, CTSL, CTSA and FURIN were detectable in the entire cohort at high expression level, and the prevalence of the different dual co-expression of SARS-CoV–2-associated proteases and receptors was optimal (100% of samples). Interestingly, only CTSL was detectable in all trophectoderm samples and a prevalence of 60% was found for the BSG-CTSL co-expression. ACE2, BSG, CTSL and CTSA were present at high expression level in CCs samples. In contrast, ACE2 and BSG expression was very low while CTSL and CTSA showed a high expression level in GCs.A prevalence of 100% was reported for ACE2-CTSL, ACE2-CTSA co-expression for both cell types. In addition, BSG-CTSL and BSG-CTSA co-expression were also present in all CCs against ∼70% in GCs samples. This data suggest a potential risks of SARS-CoV–2 infection either GC or early embryo development.Limitations, reasons for cautionAnalyses of Affymetrix microarray gene expression data were performed in non-COVID–19 patients. Whether the SARS-CoV–2 infection change the gene expression profile of SARS-CoV–2-associated receptors and proteases is under investigation.Wider implications of the findings: Specimens from female genital tract may be considered as potential targets for SARS-CoV–2.Trial registration numberNot applicable

Identification of key genes and pathways for differential diagnosis between rheumatoid arthritis and osteoarthritis based on systems biology approaches

Rheumatoid arthritis (RA) and osteoarthritis (OA) are two of the most prevalent forms of arthritis and may exhibit common etiology and clinical manifestations. Distinguishing between them is very important in determining effective treatment and management strategies.In the present study, the Affymetrix microarray gene expression dataset (GSE55457) was retrieved from 10 healthy controls, 13 patients with RA, and 10 OA patients and analyzed to identify hub genes so as to distinguish between RA and OA by weighted gene co-expression network (WGCNA) analysis and functional enrichment analysis. The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database was used to construct a protein-protein interaction (PPI) network.The most significant immune pathways associated with RA disease obtained from functional enrichment analysis were Th17, Th1, and Th2 cell differentiation and cytokine-cytokine receptor interaction pathways. The results of the present study demonstrated that two hub genes, IL2RB and HLA-DOB, may help differentiate patients with RA from those with OA at the time of diagnosis. This study introduces potential pathways and candidate biomarker genes to discriminate between RA and OA.

Abstract 5970: Function and regulatory mechanism of GPM6A in hepatocellular carcinoma development and progression

Abstract BACKGROUND: GPM6A is a glycoprotein found in endothelial cells and is considered to be a malignant lymphoma oncogene. However, the role of GPM6A in the development of hepatocellular carcinoma (HCC) is unknown. Through Affymetrix gene expression microarray and bioinformatics analysis, very low GPM6A expression was found in HCC tissue. The present study aims to explore the function and regulatory mechanism of GPM6A in HCC development and progression. METHODS: Levels of GPM6A expression was determined in various hepatoma cell lines and HCC specimens from different base disorders, and its role on cell proliferation was investigated in GPM6A stably over-expressing cell lines. Modulating effects of a specific miRNA were evaluated with miRNA mimetic transfection. RESULTS: Much lower GPM6A levels were found in HCC occuring in HBV-infection or nonalcoholic steatohepatitis. TCGA data confirmed that low GPM6A expression in HCC correlates with a poor overall survival and progression-free survival rate (p=0.018 and 0.004). Over-expression of GPM6A inhibited proliferation in SMMC-7721 and MHCC-97H cells by 31% and 30%, respectively (p&amp;lt;0.01), and led to decreased colony formation by 40%. Overexpression of GPM6A was found to significantly reduce wound healing and migration rates in these two cell types. miR-96-5p was expressed at 6 to 61-fold higher in HCC tissues than adjacent tissues (p&amp;lt;0.01); whereas GPM6A gene expression was at 3 to 55-fold lower (p&amp;lt;0.01) in a negative correlation between miR-96-5p levels and GPM6A levels in HCC specimens (r=-0.64, p&amp;lt;0.05). Further experiments demonstrated that miR-96-5p acted directly on the 3'UTR of the GPM6A gene, and transfection of miR-96-5p significantly decreased GPM6A mRNA expression by 50% in two hepatoma cell lines. Moreover, MiR-96-5p transfection promoted proliferation (66%, 59%, p&amp;lt;0.01), migration (10%, 9%, p&amp;lt;0.05) and invasion (557±66 vs. 226±19; 235±36 vs. 153±17, p&amp;lt;0.05-0.01) of SMMC-7721 and MHCC-97H hepatoma cells; whereas the function of oncogenic microRNA-96 was significantly inhibited in GPM6A-overexpressed hepatoma cells. CONCLUSIONS: GPM6A expression in HCC is commonly suppressed, and correlated to a poor prognosis. Its low expression in HCC tissues is most likely attributed to upregulated miR-96-5p, which may function as an oncolytic regulator through suppressed GMP6A that may block proliferation, cloning formation, migration and invasion of hepatoma cells. Citation Format: Rui Zong Li, Gang Xu, Min Ji Zhu, Jian Wu, Jian Wu. Function and regulatory mechanism of GPM6A in hepatocellular carcinoma development and progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5970.

Abstract 5443: Modulating plasticity in aggressive variant prostate cancers

Abstract Aggressive Variant Prostate Cancers (AVPC) are a clinically and molecularly distinct subset of prostate cancers with a poor prognosis characterized by androgen-indifference, platinum-sensitivity and combined defects in Tp53, RB1 and PTEN, for which novel therapeutic approaches are urgently needed. Defects observed in these tumor suppressor pathways have been linked to lineage plasticity in genetically engineered mouse models. We hypothesized that this plasticity can be identified in human tumor samples, and can be modulated to serve as the foundation for effective combination therapies for the AVPC subset. We previously defined a gene expression signature derived from mouse embryonic day 6.5 that is representative of extensive cellular plasticity (PMID:26123483). We determined the plasticity scores in 12 patient-derived xenograft (PDX) models representative of the spectrum of prostate cancer biology (using available RNA sequencing profiles), and in 26 macrodissected tumor biopsies obtained from patients who received platinum-based chemotherapy for aggressive prostate cancer (using ALMAC Affymetrix gene expression microarray). As expected, the AVPC PDX models exhibited high plasticity scores as did 11 (42.3%) of the patient samples, associated with patient progression free- and overall survival. We next treated a well characterized AVPC PDX model (144-4) with the anti-EMT drug (p38MAPK inhibitor) SB203580 at 10mg/kg, and observed a decrease in stem-cell and EMT factors associated with plasticity. These data confirm the association between plasticity and AVPC in human tumor samples, and suggest that changes in plasticity can be measured in tumor samples to determine the value of its modulation in clinical trials. Acknowledgments: ALMAC Diagnostics Services, UK, and UTMDACC ATGC Core Lab (CCSG-CA016672-ATGC) for RNA sequencing services; UTMDACC Prostate Cancer SPORE (RS, SAM, AA), UTMDACC IRG (RS), and the UTMDACC Prostate Cancer Moonshot Program (AA) for financial support. Citation Format: Rama Soundararajan, Petra den Hollander, Peter Shepherd, Nora Navone, Daniele Lorenzini, Caddie Laberiano-Fernandez, Wei Lu, Jianling Zhou, Jingjing Liu, Jianhua Zhang, Luisa Maren Solis-Soto, Sendurai A. Mani, Ana Aparicio. Modulating plasticity in aggressive variant prostate cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5443.

Abstract 4955: Impact of PD-1, PD-L1 and EBI3 on prognosis in a cohort of localized high grade undifferentiated pleomorphic sarcoma patients

Abstract Introduction: Studies of soft-tissue sarcomas (STS) and therapeutic outcomes are limited by their rarity and heterogeneity. Despite intensive treatment, including chemotherapy, surgery and radiation therapy, 30% of patients develop recurrent disease, and the outcome of the patients with recurrent or metastatic sarcomas remains poor. The SARC028 phase II, multicenter trial of pembrolizumab reported promising activity in selected histologic subtypes of advanced STS, including undifferentiated pleomorphic sarcoma (UPS). On the other hand, multicentre phase II trial (PEMBROSARC) found that PD-1 inhibition has limited activity in selected STS (UPS and LMS and GIST). The application of immune checkpoint blockade in sarcoma is in its infancy and we do not yet understand which patients will benefit from these therapies. Methods: We explored the degree of PD-L1, PD-1, and CD8 expression in tumor tissue and microenvironment and their clinical impact in a retrospective analysis of 50 UPS sarcoma samples, at diagnosis. PD-L1 status was assessed on whole sections of fixed tissue. We also studied expression by tumors cells of two heterodimeric cytokines IL-27 (EBI3 / p28) and IL-35 (EBI3 / p35), known for inducing the expression of immune checkpoints. Their expression was investigated by analyzing the expression of their shared subunit EBI3 by immunohistochemistry. Patients presented with grade 3 FNCLCC, localized, sarcoma of the extremities, with a high risk of local and distant recurrence and were treated in a relatively homogeneous way (surgery followed by adjuvant radiation therapy and chemotherapy). Results: The median overall survival was 63.4 months after a median follow-up of 42.0 months (range, 3.7- 108.3 months). The PD-L1 score on tumor cells (E1L3N, Cell Signaling) was negative in 94% of cases, with only 1 that had more than 10% PD-L1-positive tumor cells. Three semi-quantitative scores were performed on the immune cells: PD-L1 (same antibody): score 0 (62%), 1 (34%), 2 (4%); PD-1 (PD-1 NAT, Abcam): score 0 (52%), 1 (32%), 2 (16%) and CD8 + (C8/144B, Dako): score 0 (4%), 1 (36%), 2 (58%), NE (2%). PD-1 expression was associated with CD 8 infiltration (chi2 p=0.047). The EBI3 score on tumor cells was positive (&amp;gt;30% positive cells) in 6% of cases and correlated with worse overall survival (p=0.04) and progression-free survival (p&amp;lt;0.01), whereas PD1 (expressed by T cells), PD-L1 (expressed by infiltrating immune cells or tumor cells) and CD8 infiltration lacked prognostic significance. We are now integrating data from Affymetrix microarray gene expression and whole exome to investigate the molecular differences on tumors. Conclusion: This study suggests that targeting immune checkpoints downstream of IL-27/IL-35, different from PD1 may benefit to some UPS patients. Note: This abstract was not presented at the meeting. Citation Format: Pascaline Boudou-Rouquette, Anne Jouinot, Virginie Audard, Franck Letourneur, Béatrice Parfait, François Goldwasser, Benoit Terris, Pierre Laurent-Puig, Jérôme Alexandre, Karen Leroy, Eric Pasmant, Odile Devergne, Frederique Larousserie. Impact of PD-1, PD-L1 and EBI3 on prognosis in a cohort of localized high grade undifferentiated pleomorphic sarcoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4955.

Pre‐clinical identification of potential molecular diagnostic biomarkers of secondary ischemia in microvascular fasciocutaneous flaps

Compromise of surgical microvascular flaps due to acute or late post‐operative circulatory complications remains a significant clinical problem worldwide. Timely detection of vessel occlusion may salvage a flap from necrosis, but monitoring of physical flap appearance is not always feasible and the etiology of compromise is hard to identify. Here we used a translational laboratory animal model of abdominal wall repair and Affymetrix gene expression microarray platform to reveal the molecular tissue and wound fluid markers of the arterial (AO), venous (VO) or mixed arterio‐venous (AVO) thrombosis of the pedicled flap in vivo. Bilateral 7×2.5cm epigastric flaps were raised in ~12 month old male and female Sprague‐Dawley retired breeder rats. Baseline fasciocutaneous tissue sample biopsies were obtained from the upper abdominal quadrants of sixteen sex‐matched rats prior to flap elevation. All flaps were then subjected to primary ischemia (PI), which was induced for 4 h by temporary clamping of superficial inferior epigastric artery (SIEA) and vein (SIEV) vessels to mimic free tissue transfer and anastomosis. Normoxic control (NC) samples were obtained from flaps after 24 h of non‐obstructed reperfusion. AO, VO or AVO samples were retrieved from paired flaps undergoing secondary ischemia (SI), in which SIEA, SIEV or both vessels were ligated for 4 h following 20 h of reperfusion. Total RNA was isolated from individual animal samples (n=8/gender group) and then pooled at equal concentrations prior to assessment of RNA quality. We observed 5011 (in males) and 4457 (in females) differentially expressed genes (DEGs) that demonstrated more than 2‐fold change in NC versus baseline conditions. There were 1403, 1643 and 2351 DEGs in male AO, VO and AVO flaps when compared to NC. In females, AO, VO and AVO‐associated DEG numbers were smaller (1074, 828 and 1518, respectively). Independently of gender, downregulation of keratinocyte‐specific small proline‐rich protein 4 (Sprr4) and upregulation of ankyrin repeat domain 37 (Ankrd37), previously identified as a target of hypoxia inducible factor‐1 (HIF‐1) gene, was significantly associated with the arterial SI. Venous SI was characterized by overexpression of major hematopoietic cell proteins (Gypa, Slc4a1, Lgals5, Hbq1b, Hemgn, Add2, Alas2), anti‐inflammatory bioactive lipid mediator arachidonate 15‐lipoxygenase (Alox15), CDK‐activating dual specificity phosphatase Cdc25B and Neuropeptide Y (Npy) ‐ an important neurotransmitter in skin vascular conductance. Upregulation of the latter genes distinguished VO not only from AO, but also from AVO. Finally, we identified 58 “master” DEGs shared by both genders that were present in SI regardless of its type (AO, VO, and AVO). Of these, “first‐tier” proteins included leukemia inhibitory factor (LIF), Depp1 autophagy regulator, adrenomedullin (Adm), growth arrest and DNA‐damage‐inducible, gamma (Gadd45g) and DNA‐damage‐inducible transcript 4 (Ddit4). Change in selected gene expression was further confirmed at protein or mRNA level by Western blotting or RT‐PCR analyses. In summary, our data revealed several SI‐associated diagnostic biomarkers that merit further investigation and, ultimately, validation in the human clinical trial setting.Support or Funding InformationThis work has been funded and supported by the Department of Otolaryngology ‐ Head &amp; Neck Surgery, Thomas Jefferson UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 1321: Transcriptome-wide association study of prostate cancer risk

Abstract Though more than 150 single nucleotide polymorphisms (SNPs) associated with prostate cancer have been identified through GWAS, we still do not understand the functional consequences of common genetic variation in this disease. Genetic variants may influence complex diseases like prostate cancer through gene expression. However, very large studies evaluating gene expression in prostate cancer are currently infeasible due to the limited availability of prostate cancer tissue and expense of collecting expression data. Alternative approaches have been developed to identify expression-trait associations in study populations without directly measured expression data. One such approach (Gusev et al. Nat Genet. 2016) uses a set of reference individuals with both gene expression and SNP data to develop genetic prediction models of gene expression values. These models are then used to impute cis genetic components of gene expression for a much a larger set of individuals with only genotype and phenotype information. We used Affymetrix gene expression microarray data from normal prostate and prostate tumor tissue and genotype data from prostate cancer patients in the Health Professionals’ Follow-up Study and the Physicians’ Health Study (N=187) as a reference panel for creating the predicted models of the genetic components of expression. We imputed gene expression values into Oncoarray GWAS summary statistics from the PRACTICAL consortium (N ~ 75,000) to assess the association between predicted expression and prostate cancer risk. We present the results of our transcriptome-wide association study in normal prostate and prostate tumor tissue, identifying genes significantly associated with prostate cancer risk after correcting for multiple testing. These genes may provide insight into how genetic variants influence prostate cancer through their effect on gene expression. Citation Format: Maxine Chen, Alexander Gusev, The PRACTICAL Consortium, Massimo Loda, Lorelei A. Mucci, Meir J. Stampfer, Peter Kraft, Kathryn L. Penney. Transcriptome-wide association study of prostate cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1321. doi:10.1158/1538-7445.AM2017-1321

Abstract P2-05-34: Risk stratification with EndoPredict signature for luminal subtype breast cancers: Re-analyzing microarray experiments with Han Chinese origin

Abstract Introduction: Breast cancer is a heterogeneous disease in terms of molecular aberrations. Luminal breast cancers, most of which are estrogen receptor (ER) positive without human epidermal growth factor receptor 2 (HER2) over expression clinically, constitute the majority of human breast cancers with better prognosis compared with basal-like or HER2-enriched subtype. The aim of the study is to evaluate the prognostication of EndoPredict signature, for which high- and low-risk group is defined based on a multi-gene assay. EndoPredict signature is supposed to guide adjuvant therapy for ER+/HER2- luminal breast cancers with up to three positive lymph nodes, while the test has not been validated for Han Chinese population yet. Materials and methods: Our microarray experiments (partially published under GSE48391) and two publicly available microarray studies (GSE5460 and GSE20685) constituted the combined dataset of 565 breast cancers with Han Chinese origin, of which 280 were ER+/HER2- by immunohistochemical analysis. Transformation of Affymetrix microarray gene expression values to RT-qPCR-based expression values were conducted with the mathematical formula provided by the EndoPredict investigators, with gene-specific transformation factor and offset. Each enrolled patient was categorized into either high- or low-risk group based on the result of EndoPredict (EP) algorithm. Results: Direct adaptation of the EP algorithm for microarray gene expression data resulted in over inflation of EP scores, and most cases were categorized into the high-risk group with the predefined threshold of EP score of 5 and adjustments with rescaling and relocations of microarray-based EP scores were performed. The proportion was 88% for low-risk group and 74% for high-risk group during the 10-year follow up period, with disease-specific survival advantage reported for those with EP-predicted low-risk group patients. On the other hand, borderline overall survival discrepancy was observed (89% for low-risk and 80% for high-risk group). In addition, patients with high-risk EP scores were associated with larger tumor size, higher nuclear grade, and more involved lymph nodes. Risk stratificaiton of Taiwanese breast cancers by EP algorithm: disease-specific survivalRisk groupTotal numberFailed numberCensored numberPercentage of disease-specific survival censored patientsLow-risk6485687.5%High-risk1564011674.4%P=0.01, log-rank test Risk stratificaiton of Taiwanese breast cancers by EP algorithm: overall survivalRisk groupTotal numberFailed numberCensored numberPercentage of overall survival censored patientsLow-risk6475789%High-risk1563112580.1%P=0.05, log-rank test Discussion and conclusion: The study provides a solution to enhance the comparability between the FFPE/RT-qPCR based EP algorithms and fresh frozen microarray gene expression data. The statistical framework presented here provides an “in-silicon” validation for EP algorithm and further studies taking clinical parameters into consideration will augment the clinical applicability of EP scores and EPclin scores to ascertain the prognostic power of multi-gene assay for luminal breast cancers in Taiwan. Citation Format: Huang C-C, Tsai M-L, Tu S-H, Huang C-S. Risk stratification with EndoPredict signature for luminal subtype breast cancers: Re-analyzing microarray experiments with Han Chinese origin [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-05-34.

Abstract POSTER-TECH-1112: Quantitate epithelial-mesenchymal transition in ovarian cancer

Abstract Purpose: Metastases account for more than 90% of cancer deaths and hence, inhibiting metastasis is an attractive therapeutic option. Accumulating evidences show that invasion and metastasis of ovarian carcinoma could be driven by Epithelial-Mesenchymal Transition (EMT), a developmental program being employed during carcinoma progression. As EMT is a reversible process, inhibiting or reversing EMT thus emerges as a potential anti-metastasis strategy. EMT is a spectrum consisting of the binary epithelial and mesenchymal states as well as transitional intermediate states. Therefore, reversing EMT from the mesenchymal state would result in the sliding along an EMT Spectrum. Currently, there is no quantitative tool available to assess the EMT spectrum. In this study, we describe a bioinformatics method for quantitating EMT. Experimental procedure: By using immunofluorescence staining of E-cadherin and N-cadherin, we defined the most epithelial and most mesenchymal cells in a panel of ovarian carcinoma cell lines. We derived an EMT signature from Affymetrix gene expression microarrays of these cell lines. Subsequently, with a Kolmogorov-Smirnov-based method, we computed EMT score that assesses the similarity of an ovarian carcinoma cell transcriptome with the derived EMT signature. This method scores ovarian carcinoma cell lines in the range of [-1.0,+1.0] which corresponded to the extreme epithelial and mesenchymal states respectively along the EMT spectrum. We thus applied the EMT scoring to estimate EMT status in ovarian carcinoma tissues. Results: The EMT signature derived included known EMT inducers and markers such as ZEB1, ZEB2, SNAI2 and VIM. Expressions of selected markers such as ZEB1, VIM and FN1 were confirmed by immunofluorescence staining. Ovarian carcinoma cell lines displaying a mesenchymal morphology were accurately reflected as having high EMT scores. Cell lines with high EMT scores were anoikis resistant. In addition, ovarian carcinoma tissues displaying high EMT scores had poorer overall survival than those of low EMT scores. Correlation analysis of EMT scores and pathways revealed that ovarian carcinoma with high EMT scores are associated with increased TGFβ, WNT, NOTCH, and SRC signaling pathways. This suggested that inhibitors targeting these pathways could potentially reverse EMT. We thus treated mesenchymal ovarian carcinoma cell lines SKOV3, HEY, DOV13 with a SRC inhibitor, AZD0530. We observed a partial reversal of EMT evident by decreased EMT scores (-0.11 to -0.17) in these cells. Conclusions: EMT scoring could reflect both the EMT status of a sample and the transitional changes along the EMT spectrum upon intervening with inhibitors. Thus, EMT scoring presents a promising tool to assess the effectiveness of anti-EMT or EMT reversing compounds. Citation Format: Tuan Zea Tan, Ruby Yun-Ju Huang, Qing Hao Miow, Jieru Ye, Meng Kang Wong, Jieying Amelia Lau, Seiichi Mori, Jean Paul Thiery. Quantitate epithelial-mesenchymal transition in ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-TECH-1112.

Abstract 3578: Alternative RNA splicing and protein products in leukemia outcome

Abstract Tumors and leukemias have dramatically increased levels of aberrant RNA splicing, which generates a large population of transcripts that could encode variant proteins. This increased complexity of RNA products could be due to “noisy” splicing that is increased in transformed cells, but has no functional consequence. Alternatively, the variant RNAs and the proteins they encode could actively contribute to the transformed phenotype. For example, alternative RNA splicing produces variant forms of Myb proteins whose expression correlate with poor survival. Recurrent mutations in RNA splicing factors have been detected in some leukemias, and overexpression of some RNA splicing factors can transform cells. But there is still little direct evidence linking increased levels of alternative RNA splicing to leukemogenesis or transformation. We are testing the hypothesis that increased levels of alternative RNA splicing leads to the synthesis of aberrant protein products that contribute to the transformed phenotype. To test this, we are developing a whole molecule “deep” RNA sequencing approach that will capture the full-length structures of individual whole transcripts and predict the proteins they would encode. The long-term goal is to use the new assay to analyze gene expression and alternative RNA splicing of all transcripts in a well-defined cohort of high-risk childhood B-progenitor ALL patients who routinely fail the standard therapies. We will compare whether RNA levels or predicted protein levels are better predictors of outcome, as a test of whether the encoded variant proteins are contributing to the development of leukemia. As a starting point, we have conducted conventional RNA sequencing using Human Embryonic Kidney (HEK) 293T and Jurkat T-cells, using the Ion Torrent Proton sequencing platform followed by statistical analysis with the R-based EdgeR package. We identified more than 1600 differentially expressed genes. We compared the RNA-seq results to Affymetrix microarray gene expression profiles for the same samples and found relatively similar gene expression patterns. These data sets serve as the benchmark for development of the new single-molecule sequencing assay. With the improved assay we have obtained full-length sequence information for most expressed genes starting with as little as 1 ng of total RNA. We are currently developing the method further along with the bioinformatics, computational and statistical tools to analyze the single-molecule data. If increased alternative splicing is found to play a role in tumor development, these studies could identify novel and potentially “druggable” targets for the development of new therapies or interventions for these patients. Citation Format: Hideaki Suzuki. Alternative RNA splicing and protein products in leukemia outcome. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3578. doi:10.1158/1538-7445.AM2014-3578

Abstract 5138: Histone deacetylase 2 and N-Myc reduce p53 protein phosphorylation at serine 46 by repressing gene transcription of tumor protein 53-induced nuclear protein 1

Abstract Myc oncoproteins and histone deacetylases (HDACs) exert oncogenic effects by modulating gene transcription. Paradoxically, N-Myc induces p53 gene expression. Tumor protein 53-induced nuclear protein 1 (TP53INP1) phosphorylates p53 protein at serine 46, leading to enhanced p53 activity, transcriptional activation of p53 target genes and programmed cell death. We aimed to identify the mechanism through which N-Myc overexpressing p53 wild-type neuroblastoma cells acquired resistance to apoptosis.Gene and protein expression was analysed by real-time RT-PCR and immunoblot. Cell survival/death was examined by flow cytometry study of Annexin V-staining. The prognostic value of TP53INP1 expression in tumor tissues was investigated in three independent cohorts of neuroblastoma patients. TP53INP1 was one of the genes most significantly repressed by HDAC2 and N-Myc according to Affymetrix microarray gene expression datasets. HDAC2 and N-Myc reduced TP53INP1 gene expression by direct binding to the TP53INP1 gene promoter, leading to transcriptional repression of TP53INP1, p53 protein de-phosphorylation at serine 46, neuroblastoma cell proliferation and survival. Moreover, low levels of TP53INP1 expression in human neuroblastoma tissues correlated with high levels of N-Myc expression and poor patient outcome, and the BET bromodomain inhibitors JQ1 and I-BET151 reduced N-Myc expression and reactivated TP53INP1 expression in neuroblastoma cells. These findings identify TP53INP1 repression as an important co-factor for N-Myc oncogenesis, and provide further evidence for the potential application of BET bromodomain inhibitors in the therapy of N-Myc-induced neuroblastoma. Citation Format: Jeyran Shahbazi, Christopher J. Scarlett, Murray Norris, Bing Liu, Michelle Haber, Andrew E. Tee, Alice Carrier, Andrew V. Biankin, Wendy B. London, Glenn M. Marshall, Richard Lock, Tao Liu. Histone deacetylase 2 and N-Myc reduce p53 protein phosphorylation at serine 46 by repressing gene transcription of tumor protein 53-induced nuclear protein 1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5138. doi:10.1158/1538-7445.AM2014-5138

Abstract 1835: Transcriptome and metabolome reprogramming in EGFR-mutant NSCLC early adaptive drug-escape against erlotinib

Abstract Background. Advanced NSCLC with sensitizing EGFR mutation is being treated with EGFR inhibitor first-line. Despite initial tumor response, targeted therapy invariably fails due to acquired resistance. Classical studies of acquired drug resistance focusing on late clinical events in rebiopsy studies have identified some resistance mechanisms including T790M-EGFR, MET amplification and AXL upregulation. Methods. HCC827 and PC-9 NSCLC cells (sensitizing EGFR exon 19-del) were used in vitro and in vivo drug-sensitive models. MTS viability assay, TLVM cell mobility assay, immunoblot and immunofluorescence and in vivo xenograft IHC analysis were performed. Transcriptome analysis of HCC827 cells under erlotinib inhibition time-course was performed using Affymetrix microarray gene expression profiling (Human GeneChip 1.0 ST arrays) in triplicate at 0 hr, 8 hr, 9 days of erlotinib-treatment, and 9 days pretreatment with erlotinib followed by 7 days drug-washout. Data analysis was performed using PCA, heatmap, BAMarray and PathwayStudio 6.0 analysis. We also performed mass-spectrometry-based global profiling analysis of cellular metabolome in 5 replicate. Results. We analyzed drug-sensitive HCC827 and PC-9 cells under erlotinib inhibition to characterize the adaptive response that engenders drug resistance. We identified an early adaptive drug-escape that emerged after 9 days of erlotinib, characterized with MET-independent mitochondrial BCL-2/BCL-xL prosurvival priming to result in 100-fold IC50 increase towards resistance. These cells displayed a quiescence state associated with retarded cell proliferation/cytoskeletal functions. Transcriptome profiling analysis of the early adaptive resistant HCC827 cells revealed a remarkable genome-wide adaptive reprogramming of gene expression signature, involving pathways of cell adhesion, cell cycle regulation, cell division/mitosis, glycolysis and gluconeogenesis, response to DNA damage stimulus, and DNA repair. Metabolomic profiling revealed a global adaptive metabolic reprogramming also, with resultant suppression of glucose and TCA cycle metabolism, and lipid bioenergetics. Our studies also identified autocrine TGFβ2 signaling pathway in mediating the early adaptive resistance in the mutated-EGFR lung cancer cells that escaped erlotinib inhibition through a combination of quiescence-state and EMT, cellular metabolic reprogramming and STAT3/BCL-2/BCL-xL mitochondrial prosurvival priming. Conclusion. Early adaptive drug-escape emerges within a subpopulation of EGFR-mutant NSCLC under erlotinib inhibition. This constitutes an early-stage minimal residual disease leading to eventual resistant tumor relapse. These early drug-escaping tumor cells undergo global cellular reprogramming with upregulated mitochondrial prosurvival priming, representing attractive therapeutic targets to eradicate drug resistance. Citation Format: Patrick C. Ma, Praveena S. Thiagarajan, Patrick Leahy, Rakesh Bagai, Ivy Shi, Wei Zhang, Yan Feng, Martina L. Veigl, Daniel Lindner, David Danielpour, Lihong Yin. Transcriptome and metabolome reprogramming in EGFR-mutant NSCLC early adaptive drug-escape against erlotinib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1835. doi:10.1158/1538-7445.AM2014-1835

Histone deacetylase 2 and N-Myc reduce p53 protein phosphorylation at serine 46 by repressing gene transcription of tumor protein 53-induced nuclear protein 1.

Myc oncoproteins and histone deacetylases (HDACs) exert oncogenic effects by modulating gene transcription. Paradoxically, N-Myc induces p53 gene expression. Tumor protein 53-induced nuclear protein 1 (TP53INP1) phosphorylates p53 protein at serine 46, leading to enhanced p53 activity, transcriptional activation of p53 target genes and programmed cell death. Here we aimed to identify the mechanism through which N-Myc overexpressing p53 wild-type neuroblastoma cells acquired resistance to apoptosis. TP53INP1 was found to be one of the genes most significantly repressed by HDAC2 and N-Myc according to Affymetrix microarray gene expression datasets. HDAC2 and N-Myc reduced TP53INP1 gene expression by direct binding to the TP53INP1 gene promoter, leading to transcriptional repression of TP53INP1, p53 protein de-phosphorylation at serine 46, neuroblastoma cell proliferation and survival. Moreover, low levels of TP53INP1 expression in human neuroblastoma tissues correlated with high levels of N-Myc expression and poor patient outcome, and the BET bromodomain inhibitors JQ1 and I-BET151 reduced N-Myc expression and reactivated TP53INP1 expression in neuroblastoma cells. These findings identify TP53INP1 repression as an important co-factor for N-Myc oncogenesis, and provide further evidence for the potential application of BET bromodomain inhibitors in the therapy of N-Myc-induced neuroblastoma.

Comparison of gene expression profiles in the blood, hippocampus and prefrontal cortex of rats.

BackgroundThe comparability of gene expression between blood and brain tissues is a central issue in neuropsychiatric research where the analysis of molecular mechanisms in the brain is of high importance for the understanding of the diseases and the discovery of biomarkers. However, the accessibility of brain tissue is limited. Therefore, knowledge about how easily accessible peripheral tissue, e. g. blood, is comparable to and reflects gene expression of brain regions will help to advance neuropsychiatric research.DescriptionGene expression in the blood, hippocampus (HC) and prefrontal cortex (PFC) of genetically identical rats was compared using a genome-wide Affymetrix gene expression microarray covering 29,215 expressed genes. A total of 56.8% of 15,717 expressed genes were co-expressed in blood and at least one brain tissue, while 55.3% of all genes were co-expressed in all three tissues simultaneously. The overlapping genes included a set of genes of relevance to neuropsychiatric diseases, in particular bipolar disorder, schizophrenia and alcohol addiction. These genes included CLOCK, COMT, FAAH, NPY, NR3C1, NRGN, PBRM1, TCF4, and SYNE.ConclusionsThis study provides baseline data on absolute gene expression and differences between gene expression in the blood, HC and PFC brain tissue of genetically identical rats. The present data represents a valuable resource for future studies as it might be used for first information on gene expression levels of genes of interest in blood and brain under baseline conditions. Limitations of our study comprise possible contamination of brain tissue with blood and the non-detection of genes with very low expression levels. Genes that are more highly expressed in the brain than in the blood are of particular interest since changes in their expression, e.g. due to disease status, or treatment, are likely to be detected in an experiment. In contrast, genes with higher expression in the blood than in the brain are less informative since their higher baseline levels could superimpose variation in brain.

Integrated analysis of genome-wide DNA methylation and gene expression profiles in molecular subtypes of breast cancer

Aberrant DNA methylation of CpG islands, CpG island shores and first exons is known to play a key role in the altered gene expression patterns in all human cancers. To date, a systematic study on the effect of DNA methylation on gene expression using high resolution data has not been reported. In this study, we conducted an integrated analysis of MethylCap-sequencing data and Affymetrix gene expression microarray data for 30 breast cancer cell lines representing different breast tumor phenotypes. As well-developed methods for the integrated analysis do not currently exist, we created a series of four different analysis methods. On the computational side, our goal is to develop methylome data analysis protocols for the integrated analysis of DNA methylation and gene expression data on the genome scale. On the cancer biology side, we present comprehensive genome-wide methylome analysis results for differentially methylated regions and their potential effect on gene expression in 30 breast cancer cell lines representing three molecular phenotypes, luminal, basal A and basal B. Our integrated analysis demonstrates that methylation status of different genomic regions may play a key role in establishing transcriptional patterns in molecular subtypes of human breast cancer.

Murine Microenvironment Metaprofiles Associate with Human Cancer Etiology and Intrinsic Subtypes

Ionizing radiation is a well-established carcinogen in rodent models and a risk factor associated with human cancer. We developed a mouse model that captures radiation effects on host biology by transplanting unirradiated Trp53-null mammary tissue to sham or irradiated hosts. Gene expression profiles of tumors that arose in irradiated mice are distinct from those that arose in naïve hosts. We asked whether expression metaprofiles could discern radiation-preceded human cancer or be informative in sporadic breast cancers. Affymetrix microarray gene expression data from 56 Trp53-null mammary tumors were used to define gene profiles and a centroid that discriminates tumors arising in irradiated hosts. These were applied to publicly available human cancer datasets. Host irradiation induces a metaprofile consisting of gene modules representing stem cells, cell motility, macrophages, and autophagy. Human orthologs of the host irradiation metaprofile discriminated between radiation-preceded and sporadic human thyroid cancers. An irradiated host centroid was strongly associated with estrogen receptor-negative breast cancer. When applied to sporadic human breast cancers, the irradiated host metaprofile strongly associated with basal-like and claudin-low breast cancer intrinsic subtypes. Comparing host irradiation in the context of TGF-β levels showed that inflammation was robustly associated with claudin-low tumors. Detection of radiation-preceded human cancer by the irradiated host metaprofile raises possibilities of assessing human cancer etiology. Moreover, the association of the irradiated host metaprofiles with estrogen receptor-negative status and claudin-low subtype suggests that host processes similar to those induced by radiation underlie sporadic cancers.