Array Gene Expression Profiling Research Articles

Similarities in Pathobiology of Alzheimer’s Disease and Type 2 Diabetes Mellitus: A Proof of Concept by a Case Study

AbstractBackgroundAlzheimer’s disease (AD) is the leading cause of dementia worldwide. Developing countries will have >3 times the prevalence of the developed countries by the year 2050. Our previous studies with a Pakistani population have suggested that type 2 diabetes mellitus (T2DM) is strongly associated with impaired cognitive functioning, and genetic pathways showed strong resemblance to known AD patterns. The present case studies investigated two important pathways, i.e., Amyloid Precursor Protein (APP) processing and Lipid Metabolism, using gene expression profiling arrays to compare and contrast AD and T2DM patients from Pakistan.MethodWe performed high‐throughput qRT‐PCR with preconfigured a TaqMan Low Density Profiling Array specific to Human Alzheimer’s Disease (ABI, CAT # 441887) to measure the altered gene expressions and their corresponding pathways (Ingenuity Pathway Analysis, IPA®). Both the T2DM (n = 2; M/F each) and AD (n = 2, M/F each) patients (cases) were recruited from outpatient departments of a regional hospital, and were compared with their respective matched, healthy controls.ResultOverall, we observed a differential gene expression profile in AD and T2DM participants compared to controls. CYP46A1, MAPT, CAPNS1, GAL, AGER, CAPN1, PSENEN, ST6GAL1, GRIN2B, GRIN2C, GRIN2A, and GAP43 was downregulated in both T2DM and AD patients. ACHE, CHRM3, GRIN2C and GAP43 significantly (p value <0.05) downregulated in all T2DM patients. CDC2, UCHL1, CTSG, CASP3, IDE, NAE1, CDK5R1, IL6, APBB2, GLS are the top 10 common genes that were upregulated in both AD and T2DM groups. The IPA analysis revealed important Canonical Pathways, that included, viz., Amyloid Processing, Neuroinflammation Signaling, ErB4 Signaling, nNOs Signaling. Molecular Mechanisms of Cancer. The top disease and biofunctions included Metabolic Disease, Neurological disease, Organismal Injury and Abnormalities, Psychological Disorders, Cardiovascular Disease, with the top upstream regulators PHLPP2, IL33, APP, CRP, TAC1.ConclusionThe differential gene expression patterns and associated disease pathways from this pilot study suggest that specific genes and pathways were common to both diseases and may suggest common underlying processes and causes. Information from such biochemical and genetic studies will be crucial to the development of therapeutic targets to treat AD.

Different Types of Gene Expression Profiles and Pathway Enrichment Analysis of Oral Dysplastic Keratinocyte Cells Caused by Heavy Smoking

Aim/Background: The aim of the study is to carry out survival analysis on the candidate hub genes to check out their effects on Keratinisation of mucous cells are related to the genes which are responsible for OSCC. Materials and Methods: Gene expression profiling array for two microarray data sets of keratinized and normal oral tissue has been taken from GEO then DEGs and lnc RNAs are identified in oral cancer using LIMMA R package 15 in bio-conductor followed by KEGG analysis. STRING tool used to construct the PPIN for the sub PPIN, MCODE algorithm used for our work. The prognostic correlation co efficient values of the genes responsible for the keratinisation process in cancers have been studied using TCGA project in The Encyclopedia of RNA Interactomes (ENCORI). Functional annotation and co-expression results done with these CD44, CD58, CD40, VIM, KRT19, MMP1, ANGPTL4, FABP4, ENTPD1, and FCER2 key protein coding genes. Results: Hyper-methylated promoters play a crucial role in the inactivation suppressor genes during carcinogenesis and dysregulation of the DEGs are intently related with progression of cancer as functions as reporter genes. The enrichment result of DEGs can reveal the role of some URGs shows various activated levels in keratinocyte cells of heavy smokers and keratinocytes of people who had never smoked. ENTPD1 is connected to the DEGs which are related to TNF receptor, lymphocyte receptor. Ag3 associated with lymphocytes respectively, also to KRT19 which together acts with KRT8 causes mutations and alterations of genes. Conclusion: From this study a hallmark gene ENTPD1 has been identified for oral squamous cell carcinoma along with HNSCC with the seed gene CD44 interconnected with CD40, CD58.

Effect of STIL on the Gene Expression Profile of Gastric Cancer Cells

Objective To explore the molecular mechanism underlying gastric carcinogenesis and progression by using gene expression profiling array together with bioinformatics. Methods Lentivirus short hairpin RNA targeting STIL(ShSTIL)and scrambled sequence RNA(ShCon)were transduced into the gastric cancer cell line SGC-7901.RNA extraction,complementary DNA synthesis,construction of biotin-labelled amplified RNA probes,and hybridization with gene expression profile were consecutively performed.We collected corresponding data and analyzed differentially expressing genes(DEGs),followed by the analysis of gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)enrichment,transcription factor regulating network,and protein-protein interacting networks. Results Compared with ShCon,a total of 417 and 87 genes were respectively down-regulated and up-regulated,respectively,in the ShSTIL group(P<0.05,fold change>1 or <-1).GO and KEGG enrichment analysis indicated that genes regulated by STIL were localized in cytoplasm,extracellular exosome,Golgi apparatus and various biomembranes,and were implicated in the ubiquitin-mediated proteolysis,P53 signaling pathway,and pathways regulating pluripotency of stem cells.Evaluation on genes enriched in KEGG pathways,regulation of transcription factors,and protein-protein interacting network demonstrated that IGF1R,STUB1,SKP2,and FOXO1 were localized at the centre of the network and played a key role in the development and progression of gastric cancer. Conclusion Through the protein-protein interactions,STIL may activate E3 ubiquitin ligase STUB1 or SKP2,promote the proteolysis of FOXO1-a transcription factor,regulate the expression of IGF1R,and thus promote gastric carcinogenesis and progression.

Membrane metallo-endopeptidase (Neprilysin) regulates inflammatory response and insulin signaling in white preadipocytes

ObjectiveAccumulation of visceral white adipose tissue (WAT) associates with insulin resistance, adipose tissue inflammation, and metabolic syndrome, whereas accumulation of subcutaneous WAT may be protective. We aimed to identify molecular mechanisms that might provide mechanistic insights underlying the phenotypic differences in these tissues. Membrane Metallo-Endopeptidase (MME/Neprislyin) is an extracellular, membrane-bound protease enriched in subcutaneous WAT that can target degradation of a variety of peptides, including insulin, IL6, and β-amyloids. We hypothesized that MME contributes to adipose depot-specific metabolic properties. MethodsWe performed RNA sequencing on human subcutaneous and visceral preadipocytes and array gene expression profiling in murine subcutaneous and visceral preadipocytes. We conducted several insulin signaling and inflammatory response experiments on different cellular states of MME expression. ResultsMME in white preadipocytes is expressed at a higher level in subcutaneous compared to visceral WAT and favors insulin signaling and a low inflammatory response. Thus, knockdown of MME in subcutaneous preadipocytes increased the inflammatory response to substance P and amyloid β aggregates. This associated with increased basal insulin signaling and decreased insulin-stimulated signaling. Moreover, MME differentially regulates the internalization and turnover of the α/β subunits of the insulin receptor. ConclusionMME is a novel regulator of the insulin receptor in adipose tissue. Given the clinical significance of both chronic inflammation and insulin sensitivity in metabolic disease, these results show a potentially new target to increase insulin sensitivity and decrease inflammatory susceptibility.

Long non-coding RNA NAP1L6 promotes tumor progression and predicts poor prognosis in prostate cancer by targeting Inhibin-β A.

Background/purposeLong non-coding RNAs (lncRNAs) have emerged as key molecules in initiation and progression of prostate cancer (PCa). In this study, we aimed to explore the role of lncRNA NAP1L6 in the development and progression of PCa.Materials and methodsWe identified that lncRNA NAP1L6 was over-expressed both in PCa tissues and cell lines by gene expression array profiling. The expression level of NAP1L6 in 75 PCa tissues and adjacent tissues was detected by RT-PCR. Next, the correlations between NAP1L6 expression and clinical features of patients with PCa were analyzed by paired t-test or chi-squared test, and its association with patient prognosis was assessed by the Kaplan–Meier method. The effects of NAP1L6 on PC-3 and 22RV1 cells were evaluated by Cell Counting Kit-8 (CCK-8), migration, invasion, and colony formation assays. Further analysis of the results of the microarray was performed to find downstream gene of NAP1L6. Cell function experiments were performed in order to explore the relationship between NAP1L6 and Inhibin-β A (INHBA) and the specific mechanism by which INHBA affects the development of PCa.ResultsUsing microarray analysis, we identified 412 lncRNAs and 1245 mRNAs to be significantly differentially expressed in three PCa samples when compared with adjacent non-tumor tissues (ANTT) (fold-change ≥2.0 or ≤0.5, P<0.05, false discovery rate (FDR) <0.05). NAP1L6 expression was upregulated in PCa tissues and cell lines (both P<0.05) compared with ANTT. Besides, high expression level of NAP1L6 promotes PCa cell proliferation, migration, and invasion (all P<0.05), and is significantly associated with larger tumor diameter, distant metastasis, and shorter survival time (all P<0.05). We found that NAP1L6 promoted the expression of INHBA (P<0.05), and knockdown of NAP1L6 led to the reduction of PCa cell migration, invasion, and proliferation by regulating the expression of INHBA (all P<0.05).ConclusionlncRNA NR6A1 might play an oncogenic role in PCa initiation and progression by regulating the expression of INHBA, and might act as a novel prognostic biomarker for PCa treatment.

Identification of an oncogenic network with prognostic and therapeutic value in prostate cancer.

Identifying critical pathways governing disease progression is essential for accurate prognosis and effective therapy. We developed a broadly applicable and novel systems‐level gene discovery strategy. This approach focused on constitutively active androgen receptor (AR) splice variant‐driven pathways as representative of an intractable mechanism of prostate cancer (PC) therapeutic resistance. We performed a meta‐analysis of human prostate samples using weighted gene co‐expression network analysis combined with experimental AR variant transcriptome analyses. An AR variant‐driven gene module that is upregulated during human PC progression was identified. We filtered this module by identifying genes that functionally interacted with AR variants using a high‐throughput synthetic genetic array screen in Schizosaccharomyces pombe. This strategy identified seven AR variant‐regulated genes that also enhance AR activity and drive cancer progression. Expression of the seven genes predicted poor disease‐free survival in large independent PC patient cohorts. Pharmacologic inhibition of interacting members of the gene set potently and synergistically decreased PC cell proliferation. This unbiased and novel gene discovery strategy identified a clinically relevant, oncogenic, interacting gene hub with strong prognostic and therapeutic potential in PC.

PO-365 Isothiocyanates as potent epigenetic regulators in human malignant melanoma chemoprevention

IntroductionGlucosinolates (GLs) are phytochemicals abundant in cruciferous vegetables, which are hydrolysed, by myrosinase, to a range of isothiocyanates (ITCs). These molecules are biologically active metabolites capable of mediating a plurality of anticancer effects including cell cycle arrest, inhibition of proliferation and apoptotic induction. Their wide range of biological properties may be reflected by their ability to interfere with the epigenetic machinery at both DNA and histone levels. The aim of the current study is to investigate how ITCs interact with the epigenetic pathway(s) response in order to induce apoptosis by modifying the expression of key apoptotic genes in an in vitro model of human malignant melanoma.Material and methodsOur in vitro human malignant melanoma model consists of (i) immortalised normal keratinocyte (HaCaT) cells; (ii) malignant melanoma (A375) cells and epidermoid carcinoma (A431) cells subjected to the following ITCs: R, S-Sulforaphane (SFN), Phenethyl Isothiocyanate (PEITC), Benzyl Isothiocyanate (BITC), Allyl Isothiocyanate (AITC) and Iberin (IBN) over different concentrations and time points of exposure. Apoptotic induction was confirmed by TaqMan qPCR gene expression profiling arrays while involvement of the epigenetic machinery was assessed by western immunoblotting for determining protein expression levels of histone deacetylases (HDACs), histone acetyltransferases (HATs) and various other histone modification tags.Results and discussionsOur results showed that all ITCs were capable of inducing apoptosis as evident by the differential expression of key target genes in a manner where PEITC and IBN were involved primarily in up-regulation compared to SFN and AITC both of which were involved in down-regulation of the majority of these apoptotic genes. Finally, differences in HDAC and HAT protein expression levels, among ITC treatments, were evident in addition to their differential compartmentalization between nucleus and cytosol.ConclusionOverall our results suggest an ITC-dependent cytotoxicity effect which is mediated via apoptotic induction and is underlined, at least partially, by epigenetic pathway(s) response mechanism(s). Our data support the notion that ITCs may be promising candidates in the context of epigenetic therapy for the treatment of human malignant melanoma.

Identification of novel methylated targets in colorectal cancer by microarray analysis and construction of co-expression network.

The present study was conducted to investigate novel methylated targets in colorectal cancer (CRC). The mRNA expression profiles of GSE32323 in 17 cancer and non-cancerous tissues from CRC patients, as well as expression profiles of 5 CRC cell lines prior and subsequent to 5-aza-2′-deoxycytidine (5-aza-dC) treatment, were obtained from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) in 5 CRC cell lines prior and subsequent to 5-aza-dC treatment were combined with the CRC-specific gene expression profiling array data. Context likelihood of relatedness algorithm was used to construct the co-expression network of CRC-specific gene expression profile. A sub-network of identified reverse-overlapped DEGs was selected and underwent Kyoto Encyclopedia of Genes and Genomes Pathway Analysis. A total of 6 reverse-overlapped DEGs were identified. This present study verified fibulin 2 (FBLN2) and protein phosphatase 1 regulatory inhibitor subunit 14A (PPP1R14A) to be downregulated in the CRC tissue sample but upregulated in CRC cell lines following 5-aza-dC treatment. The identified reverse-overlapped DEGs were enriched in tumor-associated signaling pathways, including cellular tumor antigen p53, cell cycle and NOD-like receptor (NLR) signaling pathway. A total of 2 silenced genes with abnormal methylation in CRC were identified, including FBLN2 and PPP1R14A. The reverse-overlapped DEGs were enriched in p53, cell cycle and NLR signaling pathways, indicating that reverse-overlapped DEGs, particularly FBLN2 and PPP1R14A, may be important tumor suppressors and that these reverse-overlapped DEGs are inactivated by methylation in CRC.

Identification of functional pathways associated with the conditional ablation of serum response factor in Dstncorn1 mice.

The aim of the present study was to investigate the signaling pathways associated with functional alterations in corneal tissues following the conditional ablation of serum response factor (Srf) in Dstncorn1 mice. The gene expression profiling array GSE49688, which includes 3 samples each from the wild-type (WT), Dstncorn1 mutant (corn1) and corn1 mice following the conditional ablation of Srf from the corneal epithelium [namely rescued (res)] mouse groups, was downloaded from the Gene Expression Omnibus database. The limma package was used to identify differentially expressed genes (DEGs) among the three mouse groups. DEGs were subsequently analyzed by dynamic comparison, hierarchical clustering and pathway enrichment analysis. Pathway alteration scores were also calculated in order to study the dynamic metergasis of each identified pathway. A total of 788 DEGs were identified between the corn1 and res groups, 1,365 DEGs were identified between the corn1 and WT groups, and 852 DEGs were identified between the res and WT groups. Among these DEGs, 228 genes were differentially expressed across all three groups, and were mainly enriched in signaling pathways involved in the regulation of the actin cytoskeleton, including the cofilin 1 (CFL1), the mitogen-activated protein kinase (MAPK) signaling pathway and focal adhesion. The dilated cardiomyopathy signaling pathway displayed the highest alteration score, and was enriched with integrin and integrin β-6 (ITGB6). In conclusion, the actin cytoskeleton regulatory pathway, MAPK and dilated cardiomyopathy signaling pathways, as well as CFL1 and ITGB6 genes, may be regulated by Srf to serve important roles in the progression of corneal disease.

Long non-coding RNA lnc-MX1-1 is associated with poor clinical features and promotes cellular proliferation and invasiveness in prostate cancer

Long non-coding RNAs (lncRNAs) are emerging as key molecules in human cancer genesis and progression, including prostate cancer. Large amount of lncRNAs have been found that differentially expressed between prostate cancer tissues and normal prostate tissues. Whether these lncRNAs could serve as a novel biomarker for prostate cancer diagnosis or prognosis, and their biological functions in prostate cancer need further investigation. In the present study, we identified that lncRNA lnc-MX1-1 is over-expressed in prostate cancer tissues compared with their adjacent normal prostate tissues by gene expression array profiling. The expression of lnc-MX1-1 in 60 prostate cancer cases was determined by real-time quantitative PCR and the correlations between lnc-MX1-1 expression and patients' clinical features were further analyzed. Next, we impaired lnc-MX1-1 expression using RNAi in LNCaP and 22Rv1 prostate cancer cells to explore the effects of lnc-MX1-1 on proliferation and invasiveness of the cells. Our results showed that there was a significant association between over-expression of lnc-MX1-1 and patients' clinical features such as PSA, Gleason score, metastasis, and recurrence free survival. Moreover, knockdown of lnc-MX1-1 reduced both proliferation and invasiveness of LNCaP and 22Rv1 cells. In conclusion, the results suggest that lnc-MX1-1 may serve as a potential biomarker and therapeutic target for prostate cancer.

E2F1 controls germ cell apoptosis during the first wave of spermatogenesis.

SummaryCell cycle control during spermatogenesis is a highly complex process owing to the control of the mitotic expansion of the spermatogonial cell population and following meiosis, induction of DNA breaks during meiosis and the high levels of physiological germ‐cell apoptosis. We set out to study how E2F1, a key controller of cell cycle, apoptosis, and DNA damage responses, functions in the developing and adult testis. We first analyzed the expression pattern of E2f1 during post‐natal testis development using RNA in situ hybridization, which showed a differential expression pattern of E2f1 in the adult and juvenile mouse testes. To study the function of E2f1, we took advantage of the E2F1−/− mouse line, which was back‐crossed to C57Bl/6J genetic background. E2f1 loss led to a severe progressive testicular atrophy beginning at the age of 20 days. Spermatogonial apoptosis during the first wave of spermatogenesis was decreased. However, already in the first wave of spermatogenesis an extensive apoptosis of spermatocytes was observed. In the adult E2F1−/− testes, the atrophy due to loss of spermatocytes was further exacerbated by loss of spermatogonial stem cells. Surprisingly, only subtle changes in global gene expression array profiling were observed in E2F1−/− testis at PND20. To dissect the changes in each testicular cell type, an additional comparative analysis of the array data was performed making use of previously published data on transcriptomes of the individual testicular cell types. Taken together, our data indicate that E2F1 has a differential role during first wave of spermatogenesis and in the adult testis, which emphasizes the complex nature of cell cycle control in the developing testis.

Abstract P2-05-02: Preliminary translational results from PROMIX, a phase II trial of preoperative chemotherapy with the addition of bevacizumab in large operable and locally advanced HER2-negative breast cancer

Abstract Background: Preoperative chemotherapy in breast cancer (bc) provides unique possibilities to evaluate effects of therapy by studying response and changes in the tumor during the course of treatment. A pathologic complete response (pCR) correlates positively with long term prognosis in high-proliferating bc. In triple negative bc (TNBC) the prognosis was still relatively serious in cases with pCR in one large meta-analysis (Cortazar Lancet 2014). Methods: 150 cases were included in this multicenter study, and treated with six cycles of epirubicin and docetaxel, adding bevacizumab from cycle 3, before surgery. Core needle biopsies were collected at free hand or with ultrasound (US) guidance and snap frozen at base-line and after cycle 2, tissue was also collected at surgery. Subtyping was performed using immunohistochemistry (IHC) of ER, Ki67 and HER2 according to modified St Gallen criteria; and using bead array gene expression profiling (GEX) according to PAM50. Results: Biopsies were successfully retrieved from 145/150 pts at baseline, 138 after cycle 2 and 139 at surgery. The mRNA yield was adequate for GEX from 123/145 (85%) at baseline, 82/138 (59%) at cycle 2 and 71/139 (51%) at surgery, the decrease being a result of tumor shrinkage during treatment. Initial PAM50 subtypes were as follows: luminal A (LA) 20%, luminal B (LB) 45%, HER2 5 %, basal like (BL) 22% and normal like (NL) 8%. PAM50 at baseline differed compared to IHC subtypes. Among IHC defined LA-like cases 15/33 (45%) were classified as LB by PAM50. Similarly, among IHC LB-like 22/57 (39%) were classified as non-LB (6 basal, 8 LA, 3 HER2 and 5 NL), while among IHC TNBC 7/28 (25%) were classified as non-BL subtypes (1 LA, 3 HER2 and 3 NL). Of the pts with a baseline GEX analysis, 17 (14%) achieved a pCR. The observed pCR rates among PAM50 subtypes were: LA 8%, LB 5%, HER2 17%, BL 53% and NL 20%. For non-pCR cases, 39/52 (75%) of the tumors changed PAM50 subtype between baseline and surgery. The majority changed to the NL subtype. 33% of the LB tumors changed to the LA subtype. Currently, after 2.2 years of follow-up, 16 pts are deceased due to bc. Among BL cases, 6/9 pts with a pCR at surgery remain alive; while 3/9 have died from bc. Exploratory analyses using functional gene modules (Desmedt Clin Cancer Res 2008) suggest that patients with BL tumors who have died have higher scores for PLAU/invasion and lower scores for STAT1/immune response compared with those who are still alive. Tumor size at baseline did not obviously correlate with outcome. Conclusion: We show that biological material can be retrieved from a substantial fraction of cases treated within a multicenter study of preoperative chemotherapy. The success rate may be ameliorated by routine use of US guidance. The distribution of subtypes differs between modified IHC St Gallen criteria and PAM50, especially within the luminal subtypes. The pCR rate is highest among cases with a BL tumor at baseline. Shift of the gene signature between different subtypes during the course of treatment is frequent. In this set of relatively large tumors, the prognosis among BL bc appears to be adverse in spite of a pCR. Citation Format: Niklas Loman, Ida Johansson, Judith Bjöhle, Jan Frisell, Tobias Lekberg, Lisa Rydén, Anna von Wachenfeldt, Jonas Bergh, Thomas Hatschek, Ingrid Hedenfalk. Preliminary translational results from PROMIX, a phase II trial of preoperative chemotherapy with the addition of bevacizumab in large operable and locally advanced HER2-negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-05-02.

Salt-inducible kinase 2 regulates mitotic progression and transcription in prostate cancer.

Salt-inducible kinase 2 (SIK2) is a multifunctional kinase of the AMPK family that plays a role in CREB1-mediated gene transcription and was recently reported to have therapeutic potential in ovarian cancer. The expression of this kinase was investigated in prostate cancer clinical specimens. Interestingly, auto-antibodies against SIK2 were increased in the plasma of patients with aggressive disease. Examination of SIK2 in prostate cancer cells found that it functions both as a positive regulator of cell-cycle progression and a negative regulator of CREB1 activity. Knockdown of SIK2 inhibited cell growth, delayed cell-cycle progression, induced cell death, and enhanced CREB1 activity. Expression of a kinase-dead mutant of SIK2 also inhibited cell growth, induced cell death, and enhanced CREB1 activity. Treatment with a small-molecule SIK2 inhibitor (ARN-3236), currently in preclinical development, also led to enhanced CREB1 activity in a dose- and time-dependent manner. Because CREB1 is a transcription factor and proto-oncogene, it was posited that the effects of SIK2 on cell proliferation and viability might be mediated by changes in gene expression. To test this, gene expression array profiling was performed and while SIK2 knockdown or overexpression of the kinase-dead mutant affected established CREB1 target genes; the overlap with transcripts regulated by forskolin (FSK), the adenylate cyclase/CREB1 pathway activator, was incomplete. This study demonstrates that targeting SIK2 genetically or therapeutically will have pleiotropic effects on cell-cycle progression and transcription factor activation, which should be accounted for when characterizing SIK2 inhibitors.

Myelin-forming cell-specific cadherin-19 is a marker for minimally infiltrative glioblastoma stem-like cells.

Glioblastoma stem-like cells (GSCs) exhibit stem-like properties, are highly efficient at forming tumor xenografts, and are resistant to many current therapies. Current molecular identifiers of GSCs are scarce and controversial. The authors describe differential cell-surface gene expression profiling to identify GSC-specific markers. Independent human GSC lines were isolated and maintained in standard neural stem cell (NSC) media and were validated for self-renewal, multipotent differentiation, and tumor initiation properties. Candidate upregulated GSCspecific plasma membrane markers were identified through differential Affymetrix U133 Plus 2.0 Array gene expression profiling of GSCs, human NSCs (hNSCs), normal brain tissue, and primary/recurrent glioblastoma multiforme samples. Results were validated by using comparative quantitative reverse transcription polymerase chain reaction and Western blot analysis of GSCs, hNSCs, normal human astrocytes, U87 glioma cell line, and patient-matched serum-cultured glioblastoma multiforme samples. A candidate GSC-specific signature of 19 upregulated known and novel plasma membrane-associated genes was identified. Preferential upregulation of these plasma membrane-linked genes was validated by quantitative polymerase chain reaction. Cadherin-19 (CDH19) protein expression was enhanced in minimally infiltrative GSC lines. Gene expression profiling of GSCs has shown CDH19 to be an exciting new target for drug development and study of GBM tumorigenesis.

Insulin Growth Factor 1 Receptor Expression Is Associated with NOTCH1 Mutation, Trisomy 12 and Aggressive Clinical Course in Chronic Lymphocytic Leukemia

Insulin growth factor 1 receptor (IGF1R) is emerging as an important gene in many solid and hematological cancers and its over expression has been reported to be associated with aggressive disease and pharmacologic resistance. Specifically, the IGF1R-IGF1-2 interaction was recently described to be involved in the constitutive activation of many important cell signaling such as NOTCH1 and PI3K/Akt pathways that play a key role in many solid and hematological cancers. In this study we performed a clinical and biological investigations about the role of IGF1R expression in a large and representative prospective series of chronic lymphocytic leukemia (CLL) in Binet A clinical stage enrolled in observation O-CLL1 protocol (clinicaltrial.gov identifier NCT00917540). Total RNA extraction, preparation of DNA single-stranded sense target, and hybridization to gene expression profiling arrays were carried out according to manufacturer’s protocols in 217 CLL patients enrolled in the multicentre O-CLL1 protocol. Gene expression data has been deposited in the National Centre for Biotechnology Information’s Gene Expression Omnibus database http://www.ncbi.nlm.mih.gov/geo and are accessible through series accession number GSE51529. High IGF1R expression was significantly associated with IGHV unmutated (IGHV-UM) status (p<0.0001), high ZAP-70 and CD38 expression (p<0.0001) and unfavorable cytogenetic deletion [i.e. del(11)(q23) and del(17)(p13)], particularly with del(11)(q23) (p=0.03). Cases with del(13)(q14) as single lesion were characterized by the lowest IGF1R gene levels. On the contrary, among the most common cytogenetic aberrations, trisomy 12 showed stronger IGF1R expression compared with the other patients (p<0.0001) and this association was independent from IGHV mutational status. Patients with stereotyped HCDR3 sequences showed a greater IGF1R expression compared to not stereotyped HCDR3 (p=0.001) even if this can be related to the high frequency of IGHV-UM among stereotyped HCDR patients. Interestingly, subset #4 patients, who are known to exhibit an indolent clinical course and distinct biological profile, were also characterized by lower IGF1R expression compared to other M-IGHV and UM-IGHV patients. NOTCH1 c.7541_7542delCT mutation was investigated by next generation sequencing Roche 454 technology in 199 (92%) patients (Lionetti et al, BJH 2014). Globally, median depth of coverage was 1510x, ranging from 605 to 2842. Mutant allele frequency estimated by NGS ranged from 0.02% to 75% of total reads per sample. The presence of NOTCH1 mutation was confirmed by ASO-PCR and Sanger sequencing in all patients with allele burden higher > 0.7% (31; 15.5%) and 7% (19; 9.5%) respectively. We considered as mutated only the 31 patients in whom the presence of the dinucleotide deletion was confirmed by ARMS-PCR. Patients carrying NOTCH1 mutation were characterized by a greater IGF1R expression compared with wild type cases (p=0.002). In addition high IGF1R expression was not significantly different comparing patients with low and high NOTCH1 mutation burden. In order to avoid the bias represented by the strong association between NOTCH1 and trisomy 12, we compared the IGF1R expression between NOTCH1 mutated and wild type cases excluding trisomy 12, and confirmed the previous association (p=0.004). IGF1R expression represented a strong clinical prognostic factor in our CLL cohort: by Kaplan-Maier analysis we observed a significant time to first treatment stratification in all CLLs, in all IGHV-UM and in all IGHV-M patients (p<0.0001). Furthermore, IGF1R retained its significance in multivariate analysis with most important clinical and molecular prognostic factors (CD38 expression, unfavorable FISH and IGHV mutational status). Overall, our study shows the importance of IGF1R expression in CLL and its strong association with specific clinical and biological features, confirming the interest for the study of this gene as a potential prognostic factor and its possible role as a therapeutic target in a specific group of CLL patients carrying trisomy 12 and NOTCH1 mutations. DisclosuresNo relevant conflicts of interest to declare.

Abstract 4111: Vitamin D for prevention of liver cancer in the setting of disrupted TGF-β signaling pathway

Abstract Hepatocellular carcinoma (HCC) is the third most common cause of cancer related death worldwide. Vitamin D (VD) has been implicated in the prevention of multiple cancers- some with inactivation of TGF-β signaling. Recently, inactivation of TGF-β signaling has been associated with HCC. We analyzed whole genome data for TGF-β signaling and examined the role of VD in the context of TGF-β inactivation in HCC. Methods: Databases of HCC Genomics (COSMIC) and transcriptomics (TCGA) were analyzed. The effect of calcitriol on cell proliferation was measured in HCC cell lines; Hep-G2 and MEF cells, with knocked down TGF-β/β2-spectrin (β2SP). Wild-type (WT), Sptbn1+/- and Smad3+/- mice were fed with diets containing 200 IU VD/kg or 10,000 IU VD/kg for 9 weeks. Hepatocyte proliferation was analyzed through Ki67 staining. The expression of cyclin D1 was evaluated by Western blotting. A whole genome gene expression profiling array was performed from liver samples of those mice using Illumina MouseWG-6 v2.0 gene expression arrays®. Lastly, liver samples from patients with HCC who had received VD supplementation were evaluated by immunohistochemistry. Results: Whole genome data revealed aberrant TGF-β signaling in ∼70% of HCCs. Treatment with calcitriol suppressed the growth of HCC cell lines, regardless of their TGF-β pathway status. Inhibition of proliferation was noted in WT MEFs as well as in the Sptbn1+/- and Sptbn1-/- cells. High dose VD reduced hepatic proliferation and cyclin D1 levels in Sptbn1+/- and Smad3+/- mutants, and in WT mice. The microarray data demonstrated the most significant changes in gene expression were associated with acute phase inflammatory response (p&amp;lt;0.005). Validation studies showed a significant fold change difference of P-PARA, OAZ1, TLR7 and TLR 9 after high dose VD diet between WT mice and the mutants (p&amp;lt;0.05). VD supplementation to patients with HCC was associated with higher expression of β2SP (p&amp;lt;.0001) and TβRII (p=.013) compared with that of patients who did not receive the supplement. Conclusions: Whole genome data revealed aberrant TGF-β signaling in ∼ 70% of HCCs. VD suppresses the proliferation of HCC cell lines as well as normal hepatocytes potentially through restoring of TGF-β tumor suppressor signaling. VD modulates inflammation markedly differently in the setting of TGF-β signaling inactivation. Taken together, these results suggest that VD treatment strategies could potentially be pivotal in prevention and treatment of HCC. Citation Format: Lior H. Katz, Andrea Cortes, Vivek Shukla, Keigo Machida, Hidekazu Tsukamoto, Kirty Shetty, Aiwu R. He, Lynt B. Johnson, Jian Chen, Randa El-Zein, Ju-Seog Lee, Lopa Mshra. Vitamin D for prevention of liver cancer in the setting of disrupted TGF-β signaling pathway. [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 4111. doi:10.1158/1538-7445.AM2014-4111

Abstract B73: Human omental-derived adipose stem cells: Modulator of ovarian cancer proliferation, migration and chemoresistance

Abstract Objectives: Adipose tissue contains a population of multipotent mesenchymal stromal cells (ASC) which exhibit tumor tropism, similar to bone marrow derived mesenchymal stem cells (MSC). Excess visceral adipose tissue increases the risk of ovarian cancer. The omentum is a prominent site for ovarian cancer metastasis. We hypothesize that the omentum serves as a source of ASC which promote ovarian cancer progression. Materials and Methods: ASC were isolated from the omentum (O-ASCs) of three patients with ovarian cancer. O-ASC1 was isolated from a patient with synchronous adenocarcinoma of endometrial and ovarian cancer without peritoneal metastasis; and O-ASC4 and O-ASC5 were isolated from patients with peritoneally disseminated serous ovarian cancer. Gene expression array profiling was performed with using Nimblegen arrays (Roche NimbleGen, Inc., Madison, WI). The impact of stromal cells on proliferation and chemoresistane and radio-protection of ovarian cancer cells was tested with co-culture assays using luciferase-labeled human ovarian cancer cell lines. Transwell migration assays were performed with conditioned media from O-ASC and control cell lines. To evaluate of O-ASCs tumor tropism in-vivo experiment was performed. Results: Human O-ASCs were detected engrafted within the stroma of human ovarian cancer xengrafts. O-ASC significantly promoted in-vitro proliferation and migration of ovarian cancer cell lines OVCA 429, OVCA 433, A2780. Co-culture of ovarian cancer cells with O-ASCs increased resistance to chemotherapeutic drugs and radiation. Gene expression array analysis revealed significant differences in expression profiles for group of 415 genes in sub-populations of O-ASCs. Conclusions: Adipose stem cells derived from human omentum promoted the proliferation, migration, chemoresistance and radioresistance of ovarian cancers. Clinical isolates demonstrate heterogenous effects in- vitro. Future studies will determine if tumor promoting effects of O-ASC can be predicted on the basis of clinical or disease related characteristics. Citation Format: Aleksandra Nowicka, Travis Solley, Frank C. Marini, Hadley J. Sharp, Russell R. Broaddus, Kolonin G. Mikhail, Samuel C. Mok, Wendy A. Woodward, Karen H. Lu, Ann H. Klopp. Human omental-derived adipose stem cells: Modulator of ovarian cancer proliferation, migration and chemoresistance. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B73.

PGC-1β and ChREBP partner to cooperatively regulate hepatic lipogenesis in a glucose concentration-dependent manner

Peroxisome proliferator-activated receptorγ coactivators (PGC-1α and PGC-1β) play important roles in the transcriptional regulation of intermediary metabolism. To evaluate the effects of overexpressing PGC-1α or PGC-1β at physiologic levels in liver, we generated transgenic mice with inducible overexpression of PGC-1α or PGC-1β. Gene expression array profiling revealed that whereas both PGC-1 family proteins induced mitochondrial oxidative enzymes, the expression of several genes involved in converting glucose to fatty acid was induced by PGC-1β, but not PGC-1α. The increased expression of enzymes involved in carbohydrate utilization and de novo lipogenesis by PGC-1β required carbohydrate response element binding protein (ChREBP). The interaction between PGC-1β and ChREBP, as well as PGC-1β occupancy of the liver-type pyruvate kinase promoter, was influenced by glucose concentration and liver-specific PGC-1β(-/-) hepatocytes were refractory to the lipogenic response to high glucose conditions. These data suggest that PGC-1β-mediated coactivation of ChREBP is involved in the lipogenic response to hyperglycemia.

C/EBPα and MYB regulate FLT3 expression in AML.

The interaction between the receptor FLT3 (FMS-like tyrosine kinase-3) and its ligand FL leads to crucial signalling during the early stages of the commitment of haematopoietic stem cells. Mutation or over-expression of the FLT3 gene, leading to constitutive signalling, enhances the survival and expansion of a variety of leukaemias and is associated with an unfavourable clinical outcome for acute myeloid leukaemia (AML) patients. In this study, we used a murine cellular model for AML and primary leukaemic cells from AML patients to investigate the molecular mechanisms underlying the regulation of FLT3 gene expression and identify its key cis- and trans-regulators. By assessing DNA accessibility and epigenetic markings, we defined regulatory domains in the FLT3 promoter and first intron. These elements permit in vivo binding of several AML-related transcription factors, including the proto-oncogene MYB and the CCAAT/enhancer binding protein C/EBPα, which are recruited to the FLT3 promoter and intronic module, respectively. Substantiating their relevance to the human disease, our analysis of gene expression profiling arrays from AML patients uncovered significant correlations between FLT3 expression level and that of MYB and CEBPA. The latter relationship permits discrimination between patients with CEBPA mono- and bi-allelic mutations, and thus connects two major prognostic factors for AML.

Intrahepatic recurrence of hepatocellular carcinoma 13 years after orthotopic liver transplantation for hepatitis C-related cirrhosis with occult hepatocellular carcinoma: A case report

Intrahepatic recurrence of hepatocellular carcinoma 13 years after orthotopic liver transplantation for hepatitis C-related cirrhosis with occult hepatocellular carcinoma: A case report