cDNA Microarray Data Research Articles

Do neutrophil extracellular traps implicate in atheromatous plaques from carotid endarterectomy? Re-analyzes of cDNA microarray data by surgeons.

Carotid artery stenosis is the cause of 15% of strokes. Neutrophil extracellular traps (NETs) and peptidyl arginine deiminase 4 (PAD4) are believed to be involved in thrombosis. This pilot study described the differential expression profile of NETs between atheromatous plaques and surrounding tissues. Microarray datasets of carotid plaques were obtained from Gene Expression Omnibus. The normalized data were processed into comma-separated value matrix files using spreadsheet software. Analyzes of microarray data were conducted using integrated differential expression and pathway analysis. The clustering results illustrated that the classifications of plaque and control had reasonable biological validity. Pathway analysis revealed the relevance of immune response, cell signaling, and other pathways. Differentially expressed genes were detected between carotid plaques and control specimens. However, enrichment analyzes did not reveal a difference in PAD4 expression between the groups and that NET implication was only found in one cDNA microarray dataset. This pilot study does not necessarily dismiss the possibility of a relationship between NETs and atherothrombotic stroke. Gene expression could differ between endothelial cells and atheromas, and further studies are needed.

Altered threonine tyrosine kinase (TTK) expression is associated with adverse clinical outcomes in breast tumors: An in silico approach

The most common and lethal malignancy in women on a global scale remains breast cancer. Threonine tyrosine kinase (TTK) has been shown to be a critical mitotic spindle assembly checkpoint (SAC) molecule, resulting in correct chromosome segregation and maintenance of genomic stability. Therefore, the present study was carried out to evaluate the expression pattern of threonine tyrosine kinase (TTK) in breast cancer and its potential prognostic and predictive value for therapeutic response using bioinformatics tools. Web platforms containing clinical information and cDNA microarray data were selected to perform in silico analyzes of the potential threonine tyrosine kinase (TTK) marker. The threonine tyrosine kinase (TTK) gene was found to be differentially expressed in tumor samples when compared to healthy breast tissue samples (p<0.0001) and the TNBC subtype exhibited the highest expression of threonine tyrosine kinase (TTK) relative to the other subtypes (p<0.0001). Furthermore, Kaplan-Meier curves revealed that high threonine tyrosine kinase (TTK) levels corresponded to an unfavorable outcome for overall survival (p<0.0001), as well as for recurrence-free survival (p<0.0001) and distant metastasis-free survival (p<0.0001). Finally, differential expression of threonine tyrosine kinase (TTK) was related to the response of breast cancer patients to different therapies. Our cumulative results demonstrate that threonine tyrosine kinase (TTK) may be a promising biomarker for predicting prognosis and therapeutic response in breast cancer patients.

Role of microRNA/lncRNA Intertwined With the Wnt/β-Catenin Axis in Regulating the Pathogenesis of Triple-Negative Breast Cancer.

Objective (s): In this mini-review, we aimed to discuss the Wnt/β-catenin signaling pathway modulation in triple-negative breast cancer, particularly the contribution of lncRNAs and miRNAs in its regulation and their possible entwining role in breast cancer pathogenesis, proliferation, migration, or malignancy. Background: Malignant tumor formation is very high for breast cancer in women and is a leading cause of death all over the globe. Among breast cancer subtypes, triple-negative breast cancer is rife in premenopausal women, most invasive, and prone to metastasis. Complex pathways are involved in this cancer’s pathogenesis, advancement, and malignancy, including the Wnt/β-catenin signaling pathway. This pathway is conserved among vertebrates and is necessary for sustaining cell homeostasis. It is regulated by several elements such as transcription factors, enhancers, non-coding RNAs (lncRNAs and miRNAs), etc. Methods: We evaluated lncRNAs and miRNAs differentially expressed in triple-negative breast cancer (TNBC) from the cDNA microarray data set literature survey. Using in silico analyses combined with a review of the current literature, we anticipated identifying lncRNAs and miRNAs that might modulate the Wnt/β-catenin signaling pathway. Result: The miRNAs and lncRNAs specific to triple-negative breast cancer have been identified based on literature and database searches. Tumorigenesis, metastasis, and EMT were all given special attention. Apart from cross-talk being essential for TNBC tumorigenesis and treatment outcomes, our results indicated eight upregulated and seven downregulated miRNAs and 19 upregulated and three downregulated lncRNAs that can be used as predictive or diagnostic markers. This consolidated information could be useful in the clinic and provide a combined literature resource for TNBC researchers working on the Wnt/β-catenin miRNA/lncRNA axis. Conclusion: In conclusion, because the Wnt pathway and miRNAs/lncRNAs can modulate TNBC, their intertwinement results in a cascade of complex reactions that affect TNBC and related processes. Their function in TNBC pathogenesis has been highlighted in molecular processes underlying the disease progression.

POS-328 SUPPRESSION OF ER-ASSOCIATED DEGRADATION BY INTRAGLOMERULAR CROSS-COMMUNICATION BETWEEN MESANGIAL CELLS AND PODOCYTES CAUSES PODOCYTE INJURY IN DIABETIC KIDNEY DISEASE

Mesangial lesions and podocyte injury are essential manifestations of the progression of diabetic kidney disease (DKD). Although cross-communication between mesangial cells (MCs) and podocytes has recently been suggested by the results of single cell analyses, the molecular mechanisms and its role in disease progression still remain elusive. Furthermore, our cDNA microarray data of diabetic mouse glomeruli suggested the involvement of endoplasmic reticulum (ER) stress in DKD pathophysiology. Thus, we hypothesized that the cross-communication between MCs and podocytes may affect the ER stress responses of these cells and thereby participate in the progression of DKD, especially in podocyte injury.

Uncovering the anti-angiogenic effect of semisynthetic triterpenoid CDDO-Im on HUVECs by an integrated network pharmacology approach

AimTo reveal the molecular mechanism of anti-angiogenic activity of semisynthetic triterpenoid CDDO-Im. Materials and methodsUsing re-analysis of cDNA microarray data of CDDO-Im-treated human vascular endothelial cells (HUVECs) (GSE71622), functional annotation of revealed differentially expressed genes (DEGs) and analysis of their co-expression, the key processes induced by CDDO-Im in HUVECs were identified. Venn diagram analysis was further performed to reveal the common DEGs, i.e. genes both susceptible to CDDO-Im and involved in the regulation of angiogenesis. A list of probable protein targets of CDDO-Im was prepared based on Connectivity Map/cheminformatics analysis and chemical proteomics data, among which the proteins that were most associated with the angiogenesis-related regulome were identified. Finally, identified targets were validated by molecular docking and text mining approaches. Key findingsThe effect of CDDO-Im in HUVECs can be divided into two main phases: the short early phase (0.5–3 h) with an acute FOXD1/CEBPA/JUNB-regulated pro-angiogenic response induced by xenobiotic stress, and the second anti-angiogenic step (6–24 h) with massive suppression of various angiogenesis-related processes, accompanied by the activation of cytoprotective mechanisms. Our analysis showed that the anti-angiogenic activity of CDDO-Im is mediated by its inhibition of the expression of PLAT, ETS1, A2M, SPAG9, RASGRP3, FBXO32, GCNT1 and HDGFRP3 and its direct interactions with EGFR, mTOR, NOS2, HSP90AA1, MDM2, SYK, IRF3, ATR and KIF14. SignificanceOur findings provide valuable insights into the understanding of the molecular mechanisms of the anti-angiogenic activity of cyano enone-bearing triterpenoids and revealed a range of novel promising therapeutic targets to control pathological neovascularization.

Discovery of new long noncoding RNAs associated with ulcerative colitis with a novel general microarray expression data

UC is a chronic, nonspecific disease and characterized by a chronic relapsing intestinal inflammation, which puts a person at a higher risk of developing bowel cancer, while the causes of UC are unknown. Recently, with the development of microarray technology, more and more studies are focusing on the potential roles of long noncoding RNAs (lncRNAs) in the pathogenesis of diseases. The purpose of this study is to devise a method, based on cDNA microarray probe genomics data, to computationally determine the potential function of evolutionary conserved lncRNAs in ulcerative colitis (UC). We analysed a total of 12,593 microarray probes present in the Ensembl, OMIM, UniGene, and Gene Ontology databases. We found that lncRNA n385775 was significantly higher (P < 0.001) in patients with active UC, while n336281 (P = 0.017), n341081 (P = 0.041), and n387236 (P = 0.006) were significantly lower. Then, we validated our findings by measuring the expression of lncRNAs in colon tissue samples from patients affected by UC. Moreover, we validated the expression pattern of the lncRNAs in two cell lines, Caco2/bbe and T84, treated with TNF-α. In Caco2/bbe cells, lncRNA n385775 was significantly upregulated after TNF-α treatment (P = 0.002). This study reports a novel method to re- annotate the transcriptome expression profile from existing cDNA microarray data as a potential approach to investigate the function of lncRNAs in UC.

Inhibition of CXCR2 plays a pivotal role in re-sensitizing ovarian cancer to cisplatin treatment

cDNA microarray data conducted by our group revealed overexpression of CXCL2 and CXCL8 in ovarian cancer (OC) microenvironment. Herein, we have proven that the chemokine receptor, CXCR2, is a pivotal molecule in re-sensitizing OC to cisplatin, and its inhibition decreases cell proliferation, viability, tumor size in cisplatin-resistant cells, as well as reversed the overexpression of mesenchymal epithelium transition markers. Altogether, our study indicates a central effect of CXCR2 in preventing tumor progression, due to acquisition of cisplatin chemoresistant phenotype by tumor cells, and patients’ high lethality rate. We found that the overexpression of CXCR2 by OC cells is persistent and anomalously confined to the cellular nuclei, thus pointing to an urge in developing highly lipophilic molecules that promptly permeate cells, bind to and inhibit nuclear CXCR2 to fight OC, instead of relying on the high-cost genetic engineered cells.

POS-356 Suppression of ER-associated degradation by intraglomerular crosstalk between mesangial cells and podocytes causes podocyte injury in diabetic kidney disease

Mesangial lesions and podocyte injury are essential manifestations of the progression of diabetic kidney disease (DKD). Although cross-communication between mesangial cells (MCs) and podocytes has recently been suggested by the results of single-nucleus RNA-sequencing analyses, the molecular mechanisms and role in disease progression remain elusive. Furthermore, our cDNA microarray data of diabetic mouse glomeruli suggested the involvement of endoplasmic reticulum (ER) stress in DKD pathophysiology.

Suppressed ER-associated degradation by intraglomerular cross talk between mesangial cells and podocytes causes podocyte injury in diabetic kidney disease.

Mesangial lesions and podocyte injury are essential manifestations of the progression of diabetic kidney disease (DKD). Although cross-communication between mesangial cells (MCs) and podocytes has recently been suggested by the results of single-nucleus RNA sequencing analyses, the molecular mechanisms and role in disease progression remain elusive. Our cDNA microarray data of diabetic mouse glomeruli suggested the involvement of endoplasmic reticulum (ER) stress in DKD pathophysiology. In vitro experiments revealed the suppression of the ER-associated degradation (ERAD) pathway and induction of apoptosis in podocytes that were stimulated with the supernatant of MCs cultured in high glucose conditions. In diabetic mice, ERAD inhibition resulted in exacerbated albuminuria, increased apoptosis in podocytes, and reduced nephrin expression associated with the downregulation of ERAD-related biomolecules. Flow cytometry analysis of podocytes isolated from MafB (a transcription factor known to be expressed in macrophages and podocytes)-GFP knock-in mice revealed that ERAD inhibition resulted in decreased nephrin phosphorylation. These findings suggest that an intraglomerular cross talk between MCs and podocytes can inhibit physiological ERAD processes and suppress the phosphorylation of nephrin in podocytes, which thereby lead to podocyte injury under diabetic conditions. Therapeutic intervention of the ERAD pathway through the cross talk between these cells is potentially a novel strategy for DKD.

In silico analyses identify lncRNAs: WDFY3-AS2, BDNF-AS and AFAP1-AS1 as potential prognostic factors for patients with triple-negative breast tumors.

BackgroundLong non-coding RNAs (lncRNAs) are characterized as having 200 nucleotides or more and not coding any protein, and several been identified as differentially expressed in several human malignancies, including breast cancer.MethodsHere, we evaluated lncRNAs differentially expressed in triple-negative breast cancer (TNBC) from a cDNA microarray data set obtained in a previous study from our group. Using in silico analyses in combination with a review of the current literature, we identify three lncRNAs as potential prognostic factors for TNBC patients.ResultsWe found that the expression of WDFY3-AS2, BDNF-AS, and AFAP1-AS1 was associated with poor survival in patients with TNBCs. WDFY3-AS2 and BDNF-AS are lncRNAs known to play an important role in tumor suppression of different types of cancer, while AFAP1-AS1 exerts oncogenic activity.ConclusionOur findings provided evidence that WDFY3-AS2, BDNF-AS, and AFAP1-AS1 may be potential prognostic factors in TNBC development.

Abstract A133: Role of CXCR2 in the acquisition of pan-resistant phenotype in high grade serous ovarian cancer cells

Abstract Epithelial ovarian cancer (EOC) remains the most lethal gynecologic malignancy. Indeed, EOC epidemiological scenario is partially due to its late diagnosis, when most of tumor cells have acquired metastatic and chemoresistant phenotype. Despite the initial satisfactory response of EOC cells to platinum/taxanes-based therapy, chemoresistance emerges in an uncontrolled pace, specifically when the patient carries a high-grade serous adenocarcinoma (HGS-AC). Therefore, EOC clinic urges for novel treatment possibilities, aiming the reversal of chemoresistance, the improvement of patients’ quality of life, and their survival rates. cDNA microarray data from our group demonstrated that chemokines secreted to the tumor microenvironment, as CXCL2, IL-6, and IL-8 likely play a crucial role in EOC cells chemoresistance. Thereafter, the present study focused on the role of CXCL2, which binds to its receptor CXCR2 in the membrane of target cells. Nonetheless its function in HGS-AC is unclear. Our study model was based in two HGS-AC lineages: chemosensitive A2780, and its pan-resistant derived ACRP (IC50 to cisplatin 7.3uM vs. 26,56uM, p&amp;lt;0.001; to doxuribicin 0.07uM vs. 1.5uM, p&amp;lt;0.0001, and to paclitaxel 0.22uM vs. 1.7uM, p&amp;lt;0.0001, respectively). CXCR2 was knockdown (KD), and its expression was decreased to 0.6-fold as compared to control cells (p= 0.0015). As compared to the control experimental condition (100%), CXCR2 KD HGS-AC cells described compromised cellular proliferation and metabolic viability (BrdU and Presto blue/SRB assays: A2780 21% vs. ACRP 23%, p&amp;lt;0.0001), and lower cell viability (clonogenic assay: A2780 70% vs. ACRP 49%, p&amp;lt;0.0001). Although angiogenesis does not seem to influence our observations, chicken embryo chorioallantoic membrane assay revealed that CXCR2 KD ACRP-induced tumors shrunk by 3.3-fold in comparison the control condition. Intriguingly, similar results were not observed when cells were treated with the pharmacological inhibitor of CXCR2. We lately discovered that the receptor was anomalously expressed in the cells nucleus, thus justifying our observations while opening novel avenues for future investigations in the field. We, then, argued whether CXCR2 KD would modulate carcinogenic classic signaling pathways. We observed a biological tendency of less P-AKT (0.5- fold, p=0.09), but not P-ERK (0.3-fold, p=0.7) when compared to control. We also investigated the involvement of EMT in our studied phenomena. Our findings demonstrated significative decline in the expression of SLUG (0.9-fold vs. control, p=0.022) and SNAIL (1.1-fold vs. control, p=0.005) in CXCR2 KD ACRP cells. Finally, in silico analysis including over 200 HGS-AC cases revealed that overexpression of CXCR2 is robustly correlated to the worse disease prognosis and patients’ overall survival rate, specially in late stage disease (p=0.035). Altogether, our results present innovative strategy to fight pan-chemoresistant HGS-AC by silencing anomalously overexpressed CXCR2. Citation Format: Taciane Henriques, Diandra dos Santos, Isabella Guimaraes, Nayara Tessarollo, Paulo Lyra, Patricia Mesquisa, Diana Padua, Ana Luisa Amaral, Luisa Pereira, Bruno Cavadas, Ian Silva, Raquel Almeida, Leticia Rangel. Role of CXCR2 in the acquisition of pan-resistant phenotype in high grade serous ovarian cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A133. doi:10.1158/1535-7163.TARG-19-A133

Loss of alanine-glyoxylate and serine-pyruvate aminotransferase expression accelerated the progression of hepatocellular carcinoma and predicted poor prognosis

BackgroundAccumulated studies reported abnormal gene expression profiles of hepatocellular carcinoma (HCC) by cDNA microarray. We tried to merge cDNA microarray data from different studies to search for stably changed genes, and to find out better diagnostic and prognostic markers for HCC.MethodsA systematic review was performed by searching publications indexed in Pubmed from March 1, 2001 to July 1, 2016. Studies that reporting cDNA microarray profiles in HCC, containing both tumor and nontumor data and published in English-language were retrieved. The differentially expressed genes from eligible studies were summarized and ranked according to the frequency. High frequency genes were subjected to survival analyses. The expression and prognostic value of alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT) was further evaluated in HCC datasets in Oncomine and an independent HCC tissue array cohort. The role of AGXT in HCC progression was evaluated by proliferation and migration assays in a human HCC cell line.ResultsA total of 43 eligible studies that containing 1917 HCC patients were included, a list of 2022 non redundant abnormally expressed genes in HCC were extracted. The frequencies of reported genes were ranked. We finally obtained a list of only five genes (AGXT; ALDOB; CYP2E1; IGFBP3; TOP2A) that were differentially expressed in tumor and nontumor tissues across studies and were significantly correlated to HCC prognosis. Only AGXT had not been reported in HCC. Reduced expression of AGXT reflected poor differentiation of HCC and predicts poor survival. Knocking down of AGXT enhanced cell proliferation and migration of HCC cell line.ConclusionsThe present study supported the feasibility and necessity of systematic review on discovering new and reliable biomarkers for HCC. We also identified a list of high frequency prognostic genes and emphasized a critical role of AGXT deletion during HCC progression.

Hexokinase-2 Regulates Hypoxia-Inducible Autophagy, Leading to Enhance Anti-Apoptotic Capability of Refractory Multiple Myeloma

(background) The drug resistance of multiple myeloma (MM) cells is thought to be induced by various factors of the bone marrow microenvironment. Of these factors, hypoxic stress may be associated with drug resistance in various hematologic malignancies, including MM. Hypoxic stress lead MM cells to induce distinct gene expressions. It has been reported that oncogenic transcription factors such as IRF4 and Myc are suppressed under hypoxia. Instead, accumulation of another transcription factor, HIF-1α upregulates anti-apoptotic proteins, increases glycolysis, and enhances neovascularization leading MM cells to represent anti-apoptotic phenotype. Autophagy is an intracellular process that encapsulates cytoplasmic components, which are directed to the lysosome for degradation. Autophagy and proteasomal degradation prevent apoptosis caused by endoplasmic reticulum (ER) stress. Although proteasome inhibitor such as bortezomib, is a key drug for MM, it may induce treatment resistance. This might be because autophagy is induced in hypoxic microenvironment. Autophagy associated molecules might be therapeutic target in MM cells adapted to hypoxia. (Aim and methods) To clarify the association of hypoxia inducible genes and autophagy, and to obtain rational basis for a new therapeutic strategy against MM, we performed following experiments in vitro using myeloma cell lines (MM.1S, KMS-12-PE, KMS-11, and H929) and primary samples (n=6) that were subjected to hypoxia (1% O2). (Results) First, we examined volcano plot analysis on our cDNA microarray data (GSE80545) of patient samples incubated in normoxia or hypoxia for 48 hours. 546 probes were significantly elevated in hypoxia (fold change &gt; 2.0, p &lt; 0.05). Gene ontology analysis revealed that "Glycolytic Process" contained 13 genes such as PFKFB4, ENO2, ALDOC, PFKFB3, HK2, PFKP, GPI, PGK1, LDHA, ALDOA, ENO1, PKM, and GAPDH. We focused on hexokinase-2 (HK2) because it has been reported that HK2 activates autophagy under stress conditions. Western blot analysis for patient samples revealed that HK2 expression was remarkably upregulated under hypoxia. Apoptosis assay showed that viable cells of HK2 knockdowned cell lines were significantly lower than that of control cells under hypoxia, but not under normoxia. Also, in hypoxia, we found that number of 3-bromopyruvate (3-BrPA, a HK2 inhibitor) subjected viable cells were significantly lower than that of normoxia. This suggested that HK2 contributes to anti-apoptotic phenotype of MM cells under hypoxia. Next, we examined the role of HK2 in autophagy under hypoxia. Because degradation of p62 and increase of LC3-II/LC3-I ratio is considered to be useful for autophagy detection, we examined these factors by Western blot analysis. We found that hypoxic stress decreased expression of p62 and increased the ratio of LC3-II/LC3-I in myeloma cell lines, indicating that hypoxia activates autophagy. However, under hypoxia, these changes were canceled by HK2 knockdown. We confirmed that the number of autophagosome were significantly decreased in HK2-knockdowned myeloma cells by electron microscopy analysis. These data suggested that HK2 is required for hypoxia-inducible autophagy in MM. Finally, we examined the effect of combined inhibition of HK2 and proteasome. In hypoxia, apoptosis by bortezomib was significantly increased in HK2-knockdowned myeloma cells when compared with control. Moreover, we found that the combination of 3-BrPA and bortezomib increased apoptotic cells in both normoxia and hypoxia. These results suggested that HK2-inhibition can induce apoptosis against MM cells with enhancement of sensitivity to proteasome inhibitors. (Conclusion) These results suggest that hypoxia induced HK2 promotes autophagy and inhibits apoptosis. Thus, the combination of proteasome inhibitors and HK2 inhibition may bring about a deep response against treatment resistant MM. Disclosures Ikeda: Nippon Shinyaku Research Grant: Research Funding. Takahashi:Bristol-Myers Squibb: Speakers Bureau; Eisai Pharmaceuticals: Research Funding; Pfizer: Research Funding, Speakers Bureau; Otsuka Pharmaceutical: Research Funding, Speakers Bureau; Kyowa Hakko Kirin: Research Funding; Chug Pharmaceuticals: Research Funding; Ono Pharmaceutical: Research Funding; Novartis Pharmaceuticals: Research Funding, Speakers Bureau; Astellas Pharma: Research Funding; Asahi Kasei Pharma: Research Funding.

APLNR is involved in ATRA-induced growth inhibition of nasopharyngeal carcinoma and may suppress EMT through PI3K-Akt-mTOR signaling.

The apelin receptor (APLNR) is a GPCR involved in many pathophysiological processes; however, the correlation between APLNR expression and nasopharyngeal carcinoma (NPC) has not been reported. In this study, we used cDNA microarray data to determine APLNR expression levels in NPC tissues. We found that APLNR expression was reduced in NPC tissues compared with noncancerous nasopharyngeal epithelial tissues. Subsequently, a large-scale sample of 1015 tissues was used to validate this discovery and explore the relationship between APLNR expression and prognosis of NPC. Expression levels of APLNR in NPC tissues were indeed down-regulated. Furthermore, positive expression of APLNR in NPC predicted a better prognosis (disease-free survival: P = 0.001; overall survival: P < 0.001). Moreover, ingenuity pathway analysis revealed that an indirect interaction existed between APLNR and retinoic acid (RA) in the cancer regulatory network. Consistently, after treatment with all-trans-RA (ATRA), we found that APLNR was significantly up-regulated in NPC cell lines (5-8F and HNE1), and proliferation of NPC cells was inhibited. Cell cycle arrest occurred in the G0/G1 phase. In contrast, knockdown of APLNR diminished ATRA-induced growth inhibition of NPC cells. In addition, we surprisingly found that APLNR also played an important role in migration and invasion of NPC. Wound-healing and Transwell assays revealed that APLNR overexpression led to reduced migratory and invasive properties in 2 NPC cell lines. Western blot results revealed that hallmarks of epithelial-mesenchymal transition (EMT) were altered as well, suggesting that APLNR was capable of inhibiting EMT in NPC cells. Our study further demonstrated that low expression of APLNR promoted EMT in NPC cells by activating the PI3K-protein kinase B-mammalian target of rapamycin signaling pathway. Taken together, our data suggest that APLNR could potentially predict prognosis for patients with NPC and inhibit proliferation, migration, invasion, and EMT in nasopharyngeal cancer cells.-Liu, Y., Liu, Q., Chen, S., Liu, Y., Huang, Y., Chen, P., Li, X., Gao, G., Xu, K., Fan, S., Zeng, Z., Xiong, W., Tan, M., Li, G., Zhang, W. APLNR is involved in ATRA-induced growth inhibition of nasopharyngeal carcinoma and may suppress EMT through PI3K-Akt-mTOR signaling.

SUN-297 INTRAGLOMERULAR CROSSTALK BETWEEN MESANGIAL CELLS AND PODOCYTES INHIBITS NORMAL ER-ASSOCIATED DEGRADATION PROCESSES AND INDUCES PODOCYTE INJURY IN DIABETIC KIDNEY DISEASE

We previously reported that endoplasmic reticulum (ER) stress induced by glucolipotoxicity and homeostatic inflammation through intraglomerular cellular crosstalk may play an important role in the progression of diabetic kidney disease. Crosstalks of mesangial cell (MC)-endothelial cell (EC) or podocyte-EC have been established, whereas cross-communication between podocytes and MCs is still scarce. Moreover, involvement with these consequences in podocyte injury still remains unclear. Cluster analyses were performed using cDNA microarray data set of isolated glomeruli from two distinct diabetic mouse models. By use of DAVID bioinformatics resources, commonly increased or decreased genes to both models were extracted, and made integrative analyses. Next, we conducted quantitative PCR array focused on ER stress associated genes. Using cultured mouse podocytes (MPC5) and mouse macrophages stimulated with mesangial cell-cultured medium (MC-sup) under high-glucose condition (HG), we evaluated podocyte-specific responses compared to those in macrophages. Further evaluations of ER stress and apoptotic responses of MPC5 stimulated with MC-sup were made by Western blotting, real-time PCR and TUNEL staining. The effects of an ER-associated degradation (ERAD) inhibitor Eeyarestatin I (EerI) in MPC5 and db/db mice were also examined. Commonly increased or decreased genes in both DM models were associated with apoptosis (enrichment score 3.03), inflammation (3.03), and energy metabolism (1.24), suggesting ER stress as a potential factor. Pathway-focused PCR array analysis revealed that genes related to ERAD pathway (xbp1, erp44, ero1-lb, derlin) were suppressed, but apoptotic pathway was enhanced in podocytes stimulated with HG MC-sup. Besides, these responses were also observed in isolated diabetic glomeruli. In vitro, IRE1α and spliced XBP1 were suppressed in podocytes by HG MC-sup, although apoptosis evaluated by TUNEL-staining, Bax (and Bax/Bcl-2 ratio), ATF6 and CHOP was markedly increased. These results were augmented by HG MC-sup compared to MC-sup under low-glucose condition. Other types of cells, such as proximal tubular cells or macrophages, did not show any similar responses. Of note, treatment with EerI recapitulated similar responses, namely suppressed IRE1α, spliced XBP1 and Derlin-2 in podocytes. In vivo, administration of EerI tended to exacerbate albuminuria in db/db mice. It is recently reported that ERAD pathway may play important roles in the maintenance of podocytes to avoid ER stress in several glomerular diseases including diabetic kidney disease. In the present study, we firstly reveal that intraglomerular crosstalk between MCs and podocytes inhibits normal ERAD processes, potentially causing podocyte injury in diabetic conditions.

The ion channels and transporters gene expression profile indicates a shift in excitability and metabolisms during malignant progression of Follicular Lymphoma

The definition of the gene expression profile of genes encoding Ion Channels and Transporters (ICT-GEP) represents a novel and attracting aspect in cancer. We determined the ICT-GEP of Follicular Lymphoma (FL), and compared it with that of the more aggressive Diffuse Large B Cell Lymphoma (DLBCL). cDNA microarray data were collected both from patients enrolled for this study, and from public datasets. In FL the ICT-GEP indicated the overexpression of both the K+ channel encoding gene KCNN4, and SLC2A1, which encodes the Glut1 glucose transporter. SLC2A1 turned out to represent the hub of a functional network, connecting channels and transporters in FL. Relapsed FL patients were characterised by 38 differentially expressed ICT genes, among which ATP9A, SLC2A1 and KCNN4 were under-expressed, indicating a down-regulation of both excitability and glycolysis. A completely different profile of K+ channel encoding genes emerged in DLBCL accompanied by the over-expression of the fatty acid transporter-encoding gene SLC27A1 as well as of the metabolism regulator NCoR1. This indicates a change in excitability and a shift towards an oxidative metabolism in DLBCL. Overall, the ICT-GEP may contribute to identifying novel lymphoma biomarkers related to excitability and metabolic pathways, with particular relevance for drug resistant, relapsed FL.

Abstract P3-08-06: A novel integrated, clinical-pathologic and genomic classification method segregates early from late relapsing invasive ductal luminal breast cancers (IDLBC) of the METABRIC study

Abstract Background and Objectives Endocrine therapy is one of the most effective anti-cancer treatments for HR-positive breast cancers. However, in a significant proportion of patients the disease will relapse irrespective of the treatment modality (SERM, SERD, AI). Despite the progress made with multigene signatures for molecular classification of luminal breast cancers, predicting sensitivity towards endocrine modalities remains challenging. With the help of invasive ductal luminal breast cancers (IDLBC) from the METABRIC study we have developed an integrated clinical-pathologic and genomic classification method, identifying three distinct groups (IDLBC1, -2 and -3) [1, 2]. The 3 groups differ by DNA damage load, predominant lesion type, characteristic lesion patterns and oncogenic driver mechanisms [1, 3]. Here we present characteristic gene expression profiles of IDLBCs and discuss their biologic and clinical implications. Methods Gene expression profiles based on cDNA microarray data of 1.104 IDLBCs on the METABRIC study were examined (EGAS00000000083) [3]. To identify differentially expressed genes across IDLBC1-3 groups (52%, 23%, 25%), we compiled a gene list (N=2034) from 34 gene signatures reported to be predictive of ER, PR, GATA3, FOXA1 and/or AR signalling. A multiclass significance analysis of microarrays with 100 iterations and a false discovery rate of 0.05 was used to extract differentially expressed genes. Results Of 2034 steroid receptor signalling associated genes 36 exhibited differential expression in the IDLBC1/2 and -3 groups. The IDLBC1/2 specific set included 2 ER-associated genes (WLS, SPARCL1), 1 FOXA1-associated gene (NTN4) and 2 PR-associated genes (ZBTB16, CRY2). The IDLBC3 overexpressed set included 31 genes, amongst them regulators of mitotic fidelity, as well as effectors of centromere and kinetochore functioning. Eight of these genes have been reported as ER-associated (POLR2H, ECE2, SCL7A5, KIF11, FAM83D, GPSM2, MASTL, POLE2). Additional IDLBC3 hallmarks included high proliferation activity, defect apoptosis, epithelial-mesenchymal-transformation (EMT), BRCA-ness, multiple-drug resistance (MDR) and early relapse. The estimated relapse-free-survival rate at 5 years in the IDLBC3 group was 77% [95%CI 72; 82], significantly lower than in IDLBC1/2 tumours (91% [95%CI 89; 93]) and comparable to triple negative IDLBCs on the METABRIC study (70% [95%CI: 65; 76]). Conclusions We have shown that our integrated classification method has the potential to segregate early relapsing luminal breast cancers (IDLBC3) from tumours with favourable prognosis (IDLBC1/2). The data presented here and in the abstract by Demanse et al [4] provide also an intuitive hypothesis why IDLBC3 tumours are sensitive towards Letrozole but not Tamoxifen. Prospective clinical studies will be required to validate this hypothesis and to demonstrate the value of the IDLBC classification approach.

Functional Blockade of Small GTPase RAN Inhibits Glioblastoma Cell Viability.

Glioblastoma, the most common malignant tumor in the brain, lacks effective treatments and is currently incurable. To identify novel drug targets for this deadly cancer, the publicly available results of RNA interference screens from the Project Achilles database were analyzed. Ten candidate genes were identified as survival genes in 15 glioblastoma cell lines. RAN, member RAS oncogene family (RAN) was expressed in glioblastoma at the highest level among all candidates based upon cDNA microarray data. However, Kaplan-Meier survival analysis did not show any correlation between RAN mRNA levels and patient survival. Because RAN is a small GTPase that regulates nuclear transport controlled by karyopherin subunit beta 1 (KPNB1), RAN was further analyzed together with KPNB1. Indeed, GBM patients with high levels of RAN also had more KPNB1 and levels of KPNB1 alone did not relate to patient prognosis. Through a Cox multivariate analysis, GBM patients with high levels of RAN and KPNB1 showed significantly shorter life expectancy when temozolomide and promoter methylation of O6-methylguanine DNA methyltransferase were used as covariates. These results indicate that RAN and KPNB1 together are associated with drug resistance and GBM poor prognosis. Furthermore, the functional blockade of RAN and KPNB1 by importazole remarkably suppressed cell viability and activated apoptosis in GBM cells expressing high levels of RAN, while having a limited effect on astrocytes and GBM cells with undetectable RAN. Together, our results demonstrate that RAN activity is important for GBM survival and the functional blockade of RAN/KPNB1 is an appealing therapeutic approach.

Discovery of Key Genes in Dermatomyositis Based on the Gene Expression Omnibus Database.

The aim of this study was to identify biomarkers of dermatomyositis (DM). The analysis was conducted by retrieving DM-related cDNA microarray data sets from public databases. Gene ontology, Kyoto encyclopedia of genes and genomes, and protein-protein interaction analyses were performed, together with quantitative PCR-based detection of biomarkers in muscle tissue after stimulation with serum from patients with DM or healthy controls. Our analysis of five microarray data sets identified 20 common differentially expressed genes that are closely associated with DM. PCR analysis showed that mRNAs of IFITM2, LY6E, DDX58, and IFI6 were expressed at significantly higher levels in the muscle tissue of patients with DM than in normal muscle tissues. These mRNAs were also upregulated in human skeletal muscle cells stimulated with the serum from patients with DM. The results of integrated analyses of the DM microarray data and the mRNA levels of genes showed significant differences between the muscle tissues of DM patients and controls, which could indicate key pathogenic genes and novel therapeutic targets for DM.

Downregulation of hepatic lipase is associated with decreased CD133 expression and clone formation in HepG2 cells.

The drug resistance of cancer remains a major obstacle to successful chemotherapy. New strategies for improving chemotherapeutic efficacy are urgently required. Recent studies have indicated that LIPC plays a role in promoting the liver metastasis of colorectal cancer. In the present study, we aimed to investigate the effects of LIPC on theproliferation and clone formation of colorectal cancer-derived cells, and chemoresistance in hepatoblastoma-derived HepG2 cells. The activity and expression of LIPC were determined in the cell lines by RT-qPCR and western blot analysis. HepG2 cells in which LIPC was knocked down by LIPC short hairpin RNA(shRNA) and control cells [shRNA control(shCON)] were established and analyzed for cell proliferation and colony formation rates. FACS analysis was used to explore the association between LIPC and the tumor-derived cell biomarker, CD133, and the percentages of CD133-positive cells were assessed by FACS. Additionally, shLIPC- and shCON-transfected cells were treated with various concentrations of doxorubicin and 5-floxuridine(5-FU), and cell viability was determined by MTT assay. mRNA levels in the shLIPC- and shCON-transfected cells were compared by cDNA microarray and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis. The results revealed that the HepG2 cells exhibited a relatively higher LIPC activity and expression levels compared to the other colon cancer cell lines. The downregulation of LIPC in the HepG2 cells was associated with the decreased expression of CD133, decreased cell proliferation and colony formation, as well as increased resistance to chemotherapy. KEGG analysis of the cDNA microarray data revealed increased levels in the cell adhesion molecule(CAM) pathway, including CLDN10 and CLDN1, indicating that CAMs may play a role in LIPC-mediated tumor progression. The present findings indicate a potential role of LIPC as a promising therapeutic target in cancer.