Microarray-based Expression Profiling Research Articles

Gene Expression Analysis Reveals the Concurrent Activation of Proapoptotic and Antioxidant-Defensive Mechanisms in Flavokawain B–Treated Cervical Cancer HeLa Cells

Flavokawain B (FKB) is known to possess promising anticancer abilities. This is demonstrated in various cancer cell lines including HeLa cells. Cervical cancer is among the most widely diagnosed cancer among women today. Though FKB has been shown to be effective in treating cancer cells, the exact molecular mechanism is still unknown. This study is aimed at understanding the effects of FKB on HeLa cells using a microarray-based mRNA expression profiling and proteome profiling of stress-related proteins. The results of this study suggest that FKB induced cell death through p21-mediated cell cycle arrest and activation of p38. However, concurrent activation of antioxidant-related pathways and iron sequestration pathway followed by activation of ER-resident stress proteins clearly indicate that FKB failed to induce apoptosis in HeLa cells via oxidative stress. This effect implies that the protection of HeLa cells by FKB from H2O2–induced cell death is via neutralization of reactive oxygen species.

MicroRNA-205‑5b inhibits HMGB1 expression in LPS-induced sepsis.

Inflammatory cytokines belonging to high mobility group box(HMGB)1 play a key role in sepsis through yet unknown mechanisms. The inflammatory response is modulated by microRNAs(miRNAs or miRs) at multiple levels and is poorly understood. In this study, the regulation of HMGB1 by miRNAs was evaluated using 3-(2,4-dimethoxybenzylidene)anabaseine (GTS-21) to activate the cholinergic anti-inflammatory pathway(CAP) and decrease HMGB1 expression in RAW264.7 cells. Microarray-based miRNA expression profiling of RAW264.7 cells was used to screen target miRNAs through genetic screening, GO analysis and hierarchical clustering. The expression of miRNA targets in the serum, colon, spleen, livers and lungs of BALB/c mice was quantified by RT-qPCR. Serum protein levels were quantified by ELISA. Western blot analysis and RT-qPCR were used for verification invitro. Using miRNA array analysis, we screened 3miRNAs(miR‑205‑5b, miR‑196a and miR‑193b). Animal experiments with miR‑205‑5b indicated its high degree of expression in the serum, colon, spleen, liver and lungs following the downregulation of HMGB1 in the tissues. RAW264.7 cells transfected with miR‑205‑5b mimics downregulated HMGB1 protein expression, suggesting translational regulation. HMGB1 expression negatively correlated with miR‑205‑5b expression in LPS-induced sepsis. By contrast, HMGB1 expression in LPS-stimulated RAW264.7 cells was increased following transfection with miR‑205‑5b inhibitor. miR‑205‑5b is a critical mediator of cholinergic anti-inflammatory activity in late sepsis. The upregulation of miR‑205‑5b as a potential therapeutic target for the treatment of inflammatory diseases is a possible novel therapeutic strategy against late sepsis. The mechanisms involved include the by post-transcriptional suppression of HMGB1 in cells and tissues.

Elucidation of molecular and functional heterogeneity through differential expression network analyses of discrete tumor subsets.

Intratumor heterogeneity presents a major hurdle in cancer therapy. Most current research studies consider tumors as single entities and overlook molecular diversity between heterogeneous state(s) of different cells assumed to be homogenous. The present approach was designed for fluorescence-activated cell sorting-based resolution of heterogeneity arising from cancer stem cell (CSC) hierarchies and genetic instability in ovarian tumors, followed by microarray-based expression profiling of sorted fractions. Through weighted gene correlation network analyses, we could assign enriched modules of co-regulated genes to each fraction. Such gene modules often correlate with biological functions; one such specific association was the enrichment of CD53 expression in CSCs, functional validation indicated CD53 to be a tumor-initiating cell- rather than quiescent CSC-marker. Another association defined a state of poise for stress-induced metastases in aneuploid cells. Our results thus emphasize the need for studying cell-specific functionalities relevant to regeneration, drug resistance and disease progression in discrete tumor cell fractions.

Pharmacogenomics of Scopoletin in Tumor Cells.

Drug resistance and the severe side effects of chemotherapy necessitate the development of novel anticancer drugs. Natural products are a valuable source for drug development. Scopoletin is a coumarin compound, which can be found in several Artemisia species and other plant genera. Microarray-based RNA expression profiling of the NCI cell line panel showed that cellular response of scopoletin did not correlate to the expression of ATP-binding cassette (ABC) transporters as classical drug resistance mechanisms (ABCB1, ABCB5, ABCC1, ABCG2). This was also true for the expression of the oncogene EGFR and the mutational status of the tumor suppressor gene, TP53. However, mutations in the RAS oncogenes and the slow proliferative activity in terms of cell doubling times significantly correlated with scopoletin resistance. COMPARE and hierarchical cluster analyses of transcriptome-wide mRNA expression resulted in a set of 40 genes, which all harbored binding motifs in their promoter sequences for the transcription factor, NF-κB, which is known to be associated with drug resistance. RAS mutations, slow proliferative activity, and NF-κB may hamper its effectiveness. By in silico molecular docking studies, we found that scopoletin bound to NF-κB and its regulator IκB. Scopoletin activated NF-κB in a SEAP-driven NF-κB reporter cell line, indicating that NF-κB might be a resistance factor for scopoletin. In conclusion, scopoletin might serve as lead compound for drug development because of its favorable activity against tumor cells with ABC-transporter expression, although NF-κB activation may be considered as resistance factor for this compound. Further investigations are warranted to explore the full therapeutic potential of this natural product.

Identification of the differential expression of serum microRNA in type 2 diabetes.

The identification of disease-specific alterations in miRNA expression and the ability to detect miRNAs in serum furnish the basis for identified potential research value. This study was aimed to characterize the expression of miRNAs in the serum samples from people with type 2 diabetes mellitus (T2DM) and healthy individuals in order to detect the differential expression of miRNAs in T2DM. In total, 582 participants were recruited. Microarray-based miRNA expression profiles were screened in pooled serum samples from two groups (T2DM and healthy control). The candidates' miRNAs were validated by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Five significantly different serum miRNAs were identified in T2DM patients (hsa-miR-320d, hsa-miR-4534, hsa-miR-3960, hsa-miR-451a, and hsa-miR-572) compared to those in the serum of healthy controls. This study provided evidence that serum miRNAs had differential expressions between healthy controls and T2DM patients. These five differential expression miRNAs might be of help for subsequent study in T2DM.

Evidence of TAF1 dysfunction in peripheral models of X-linked dystonia-parkinsonism.

The molecular dysfunction in X-linked dystonia-parkinsonism is not completely understood. Thus far, only noncoding alterations have been found in genetic analyses, located in or nearby the TATA-box binding protein-associated factor 1 (TAF1) gene. Given that this gene is ubiquitously expressed and is a critical component of the cellular transcription machinery, we sought to study differential gene expression in peripheral models by performing microarray-based expression profiling in blood and fibroblasts, and comparing gene expression in affected individuals vs. ethnically matched controls. Validation was performed via quantitative polymerase chain reaction in discovery and independent replication sets. We observed consistent downregulation of common TAF1 transcripts in samples from affected individuals in gene-level and high-throughput experiments. This signal was accompanied by a downstream effect in the microarray, reflected by the dysregulation of 307 genes in the disease group. Gene Ontology and network analyses revealed enrichment of genes involved in RNA polymerase II-dependent transcription, a pathway relevant to TAF1 function. Thus, the results converge on TAF1 dysfunction in peripheral models of X-linked dystonia-parkinsonism, and provide evidence of altered expression of a canonical gene in this disease. Furthermore, our study illustrates a link between the previously described genetic alterations and TAF1 dysfunction at the transcriptome level.

MicroRNA-138 promotes acquired alkylator resistance in glioblastoma by targeting the Bcl-2-interacting mediator BIM.

Glioblastoma is the most aggressive brain tumor in adults with a median survival below 12 months in population-based studies. The main reason for tumor recurrence and progression is constitutive or acquired resistance to the standard of care of surgical resection followed by radiotherapy with concomitant and adjuvant temozolomide (TMZ/RT→TMZ). Here, we investigated the role of microRNA (miRNA) alterations as mediators of alkylator resistance in glioblastoma cells. Using microarray-based miRNA expression profiling of parental and TMZ-resistant cultures of three human glioma cell lines, we identified a set of differentially expressed miRNA candidates. From these, we selected miR-138 for further functional analyses as this miRNA was not only upregulated in TMZ-resistant versus parental cells, but also showed increased expression in vivo in recurrent glioblastoma tissue samples after TMZ/RT→TMZ treatment. Transient transfection of miR-138 mimics in glioma cells with low basal miR-138 expression increased glioma cell proliferation. Moreover, miR-138 overexpression increased TMZ resistance in long-term glioblastoma cell lines and glioma initiating cell cultures. The apoptosis regulator BIM was identified as a direct target of miR-138, and its silencing mediated the induced TMZ resistance phenotype. Altered sensitivity to apoptosis played only a minor role in this resistance mechanism. Instead, we identified the induction of autophagy to be regulated downstream of the miR-138/BIM axis and to promote cell survival following TMZ exposure. Our data thus define miR-138 as a glioblastoma cell survival-promoting miRNA associated with resistance to TMZ therapy in vitro and with tumor progression in vivo.

Long-noncoding RNAs in basal cell carcinoma

Long noncoding RNAs (lncRNAs) are fundamental regulators of pre- and post-transcriptional gene regulation. Over 35,000 different lncRNAs have been described with some of them being involved in cancer formation. The present study was initiated to describe differentially expressed lncRNAs in basal cell carcinoma (BCC). Patients with BCC (n = 6) were included in this study. Punch biopsies were harvested from the tumor center and nonlesional epidermal skin (NLES, control, n = 6). Microarray-based lncRNA and mRNA expression profiles were identified through screening for 30,586 lncRNAs and 26,109 protein-coding transcripts (mRNAs). The microarray data were validated by RT-PCR in a second set of BCC versus control samples. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of mRNAs were performed to assess biologically relevant pathways. A total of 1851 lncRNAs were identified as being significantly up-regulated, whereas 2165 lncRNAs were identified as being significantly down-regulated compared to nonlesional skin (p < 0.05). Oncogenic and/or epidermis-specific lncRNAs, such as CASC15 or ANRIL, were among the differentially expressed sequences. GO analysis showed that the highest enriched GO targeted by up-regulated transcripts was "extracellular matrix." KEGG pathway analysis showed the highest enrichment scores in "Focal adhesion." BCC showed a significantly altered lncRNA and mRNA expression profile. Dysregulation of previously described lncRNAs may play a role in the molecular pathogenesis of BCC and should be subject of further analysis.

Circulating 'lncRNA OTTHUMT00000387022' from monocytes as a novel biomarker for coronary artery disease.

Long non-coding RNAs (lncRNAs) have been found to be involved in the pathogenesis of coronary artery disease (CAD). However, it remains to be established whether or not circulating lncRNAs can serve as biomarkers of CAD. Using a microarray-based lncRNA expression profiling, we found 86 lncRNAs that were differentially expressed in circulating peripheral blood monocytes and plasma from 15 CAD patients and 15 control subjects. After choosing a consistent criterion (average normalized intensity ≥7 with significance <0.005) and confirmed by quantitative PCR, only three lncRNAs (CoroMarker, BAT5, and IL21R-AS1) remained as candidate CAD biomarkers. Using the analysis of area under the curve (AUC) of the receiver-operating characteristic in another pilot group and another larger cohort, CoroMarker was found to be the best candidate biomarker for CAD with an AUC of 0.920 and 95% confidence interval of 0.892-0.947. CoroMarker was independent from known CAD risk factors and other cardiovascular diseases. In a prospective study, we found that the sensitivity and specificity of CoroMarker were 76 and 92.5%, respectively. Functional enrichment analysis showed CoroMarker to be clustered with genes positively associated with signal transduction, transmembrane transport, synaptic transmission, and innate immunity and negatively associated with inflammation. These findings were validated in THP-1 cells; CoroMarker siRNA treatment decreased the concentrations of proinflammatory cytokines [interleukin (IL)-1β, IL-6, and tumour necrosis factor α] in the culture medium. The present study suggests that CoroMarker is a novel and specific biomarker of CAD.

Study of microRNAs-21/221 as potential breast cancer biomarkers in Egyptian women

microRNAs (miRNAs) play an important role in cancer prognosis. They are small molecules, approximately 17–25 nucleotides in length, and their high stability in human serum supports their use as novel diagnostic biomarkers of cancer and other pathological conditions. In this study, we analyzed the expression patterns of miR-21 and miR-221 in the serum from a total of 100 Egyptian female subjects with breast cancer, fibroadenoma, and healthy control subjects. Using microarray-based expression profiling followed by real-time polymerase chain reaction validation, we compared the levels of the two circulating miRNAs in the serum of patients with breast cancer (n=50), fibroadenoma (n=25), and healthy controls (n=25). The miRNA SNORD68 was chosen as the housekeeping endogenous control. We found that the serum levels of miR-21 and miR-221 were significantly overexpressed in breast cancer patients compared to normal controls and fibroadenoma patients. Receiver Operating Characteristic (ROC) curve analysis revealed that miR-21 has greater potential in discriminating between breast cancer patients and the control group, while miR-221 has greater potential in discriminating between breast cancer and fibroadenoma patients. Classification models using k-Nearest Neighbor (kNN), Naïve Bayes (NB), and Random Forests (RF) were developed using expression levels of both miR-21 and miR-221. Best classification performance was achieved by NB Classification models, reaching 91% of correct classification. Furthermore, relative miR-221 expression was associated with histological tumor grades. Therefore, it may be concluded that both miR-21 and miR-221 can be used to differentiate between breast cancer patients and healthy controls, but that the diagnostic accuracy of serum miR-21 is superior to miR-221 for breast cancer prediction. miR-221 has more diagnostic power in discriminating between breast cancer and fibroadenoma patients. The overexpression of miR-221 has been associated with the breast cancer grade. We also demonstrated that the combined expression of miR-21 and miR-221can be successfully applied as breast cancer biomarkers.

Identification of miR-139-5p as a saliva biomarker for tongue squamous cell carcinoma: a pilot study.

Of all human oral carcinomas, 41% are localized to the tongue. Despite considerable improvements in both diagnosis and treatment, tongue squamous cell carcinoma (TSCC) has remained one of the most lethal types of cancer. Here, we aimed at identifying a salivary microRNA (miRNA) expression signature specific for TSCC patients. To identify putative diagnostic biomarkers, we compared the miRNA expression profiles of saliva samples from three TSCC patients and four healthy control individuals using an Agilent miRNA microarray platform (V19). Three of the differentially expressed miRNAs identified were selected for further validation using quantitative reverse-transcription PCR (qRT-PCR) in saliva samples from 25 TSCC patients and 25 healthy control individuals. Through microarray-based expression profiling, we found that 419 miRNAs were deregulated in the saliva samples from the TSCC patients compared to those from the healthy control individuals tested. Subsequent qRT-PCR analysis revealed that the expression level of miR-139-5p was significantly reduced in the TSCC validation samples compared to the controls. Further analysis of post-operative saliva samples derived from TSCC patients revealed that the miR-139-5p expression levels had turned back to normal again. In addition, we found that miR-139-5p exhibited enough power to discriminate pre-operative TSCC patients from both normal individuals (AUC: 0.805) and post-operative TSCC patients (AUC: 0.713), thereby underscoring its diagnostic potential. From our results we conclude that saliva can be used as a feasible source for routine TSCC diagnostics and that miR-139-5pmay serve as a potential biomarker for early TSCC detection.

Transfection of Sertoli cells with androgen receptor alters gene expression without androgen stimulation.

BackgroundAndrogens play an important role for the development of male fertility and gained interest as growth and survival factors for certain types of cancer. Androgens act via the androgen receptor (AR/Ar), which is involved in various cell biological processes such as sex differentiation. To study the functional mechanisms of androgen action, cell culture systems and AR-transfected cell lines are needed. Transfection of AR into cell lines and subsequent gene expression analysis after androgen treatment is well established to investigate the molecular biology of target cells. However, it remains unclear how the transfection with AR itself can modulate the gene expression even without androgen stimulation. Therefore, we transfected Ar-deficient rat Sertoli cells 93RS2 by electroporation using a full length human AR.ResultsTransfection success was confirmed by Western Blotting, immunofluorescence and RT-PCR. AR transfection-related gene expression alterations were detected with microarray-based genome-wide expression profiling of transfected and non-transfected 93RS2 cells without androgen stimulation. Microarray analysis revealed 672 differentially regulated genes with 200 up- and 472 down-regulated genes. These genes could be assigned to four major biological categories (development, hormone response, immune response and metabolism). Microarray results were confirmed by quantitative RT-PCR analysis for 22 candidate genes.ConclusionWe conclude from our data, that the transfection of Ar-deficient Sertoli cells with AR has a measurable effect on gene expression even without androgen stimulation and cause Sertoli cell damage. Studies using AR-transfected cells, subsequently stimulated, should consider alterations in AR-dependent gene expression as off-target effects of the AR transfection itself.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-015-0051-7) contains supplementary material, which is available to authorized users.

Interferon-β Modulates the Innate Immune Response against Glioblastoma Initiating Cells.

Immunotherapy targeting glioblastoma initiating cells (GIC) is considered a promising strategy. However, GIC are prone to evade immune response and there is a need for potent adjuvants. IFN-β might enhance the immune response and here we define its net effect on the innate immunogenicity of GIC. The transcriptomes of GIC treated with IFN-β and controls were assessed by microarray-based expression profiling for altered expression of immune regulatory genes. Several genes involved in adaptive and innate immune responses were regulated by IFN-β. We validated these results using reverse transcription (RT)-PCR and flow cytometry for corresponding protein levels. The up-regulation of the NK cell inhibitory molecules HLA-E and MHC class I was balanced by immune stimulating effects including the up-regulation of nectin-2. In 3 out of 5 GIC lines tested we found a net immune stimulating effect of IFN-β in cytotoxicity assays using NKL cells as effectors. IFN-β therefore warrants further investigation as an adjuvant for immunotherapy targeting GIC.

C3-induced release of neurotrophic factors from Schwann cells – potential mechanism behind its regeneration promoting activity

Previous studies revealed a peripheral nerve regeneration (PNR)1 promoting activity of Clostridium botulinum C32 exoenzyme or a 26mer C-terminal peptide fragment covering amino acids 156–181 (C3156−181),3 when delivered as one-time injection at the lesion site. The current study was performed to 1) investigate if prolonged availability of C3 and C3156−181 at the lesion site can further enhance PNR in vivo and to 2) elucidate effects of C3 and C3156−181 on Schwann cells (SCs)4in vitro.For in vivo studies, 10 mm adult rat sciatic nerve gaps were reconstructed with the epineurial pouch technique or autologous nerve grafts. Epineurial pouches were filled with a hydrogel containing i) vehicle, ii) 40 μM C3 or iii) 40 μM C3156−181. Sensory and motor functional recovery was monitored over 12 weeks and the outcome of PNR further analyzed by nerve morphometry. In vitro, we compared gene expression profiles (microarray analysis) and neurotrophic factor expression (western blot analysis) of untreated rat neonatal SCs with those treated with C3 or C3156−181 for 72 h. Effects on neurotrophic factor expression levels were proven in adult human SCs.Unexpectedly, prolonged delivery of C3 and C3156−181 at the lesion site did not increase the outcome of PNR. Regarding the potential mechanism underlying their previously detected PNR promoting action, however, 6 genes were found to be commonly altered in SCs upon treatment with C3 or C3156−181. We demonstrate significant down-regulation of genes involved in glutamate uptake (Eaac1,5Grin2a6) and changes in neurotrophic factor expression (increase of FGF-27 and decrease of NGF8). Our microarray-based expression profiling revealed novel C3-regulated genes in SCs possibly involved in the axonotrophic (regeneration promoting) effects of C3 and C3156−181. Detection of altered neurotrophic factor expression by C3 or C3156−181 treated primary neonatal rat SCs and primary adult human SCs supports this hypothesis.

Generating and evaluating a ranked candidate gene list for potential vertebrate heart field regulators

The vertebrate heart develops from two distinct lineages of cardiomyocytes that arise from the first and second heart fields (FHF and SHF, respectively). The FHF forms the primitive heart tube, while adding cells from the SHF allows elongation at both poles of the tube. Initially seen as an exclusive characteristic of higher vertebrates, recent work has demonstrated the presence of a distinct FHF and SHF in lower vertebrates, including zebrafish. We found that key transcription factors that regulate septation and chamber formation in higher vertebrates, including Tbx5 and Pitx2, influence relative FHF and SHF contributions to the zebrafish heart tube. To identify molecular modulators of heart field migration, we used microarray-based expression profiling following inhibition of tbx5a and pitx2ab in embryonic zebrafish (Mosimann & Panakova, et al, 2015; GSE70750). Here, we describe in more detail the procedure used to process, prioritize, and analyze the expression data for functional enrichment.

Genome wide microarray based expression profiles associated with BmNPV resistance and susceptibility in Indian silkworm races of Bombyx mori

The molecular mechanism involved in BmNPV resistance was investigated using a genome wide microarray in midgut tissue of Indian silkworm Bombyx mori. In resistant race (Sarupat), 735 genes up-regulated and 589 genes down-regulated at 12h post BmNPV infection. Similarly, in case of susceptible race (CSR-2), 2183 genes up-regulated and 2115 genes down-regulated. Among these, nine up-regulated and eight down-regulated genes were validated using real-time qPCR analysis. In Sarupat, vacuolar protein sorting associated, Xfin-like protein and carboxypeptidase E-like protein genes significantly up-regulated in infected midgut; prominently down-regulated genes were glutamate receptor ionotropic kainite 2-like, BTB/POZ domain and transferrin. Considerably up-regulated genes in the CSR-2 were peptidoglycan recognition protein S6 precursor and rapamycin while the conspicuous down-regulated genes were facilitated trehalose transporter and zinc transporter ZIP1-like gene. The up-regulation of genes in resistant race after BmNPV infection indicates their possible role in antiviral immune response.

Next-generation sequencing of human tumor xenografts is significantly improved by prior depletion of mouse cell

Human tumor xenografts represent the gold standard method for many research areas, including drug discovery, cancer stem cell biology, and metastasis prediction. When compared to in vitro cell culture models, human tumor xenografts show a higher validity for most assays1. During the growth phase in vivo, xenografted tissue is vascularized and infiltrated by cells of murine origin. The level of infiltration is highly dependent on multiple factors like tumor subtype, growth rate, and region of transplantation. However, even when these factors are kept constant, the amount and composition of infiltrating mouse cells is highly variable. Due to this, molecular downstream analyses such as microarray-based expression profiling are biased by cross-hybridization of mouse-derived molecules to human probes. In addition, a reduction of sensitivity caused by measuring undesired mouse signals during nextgeneration sequencing analysis can be expected. To overcome these limitations, we developed a fast and easy method allowing for the effective depletion of all cells of mouse origin by using automated tissue dissociation and magnetic cell sorting (MACS® Technology). We performed whole exome sequencing (WES) of bulk human tumor xenografts from lung, bladder, and kidney cancer, and compared the results to samples depleted of mouse cells.

Abstract 1455: Next generation sequencing of human tumor xenografts is significantly improved by prior depletion of mouse cells

Abstract Human tumor xenografts represent the gold standard method for research areas such as drug discovery, cancer stem cell biology, and metastasis prediction. When compared to in vitro cell culture models, human tumor xenografts show a higher validity for most assays (DeRose et al., 2011). During the growth phase in vivo, xenografted tissue is vascularized and infiltrated by cells of murine origin. The level of infiltration is highly dependent on multiple factors like tumor subtype, growth rate, and region of transplantation. However, even when these factors are kept constant, the amount and composition of infiltrating mouse cells is highly variable. Due to this, molecular downstream analyses such as microarray based expression profiling are challenged by cross hybridization of mouse derived molecules to human probes. In addition, a reduction of sensitivity caused by measuring mouse signals during next-generation sequencing analysis can be expected. To overcome these limitations, we have developed a fast and easy method allowing for the comprehensive depletion of all cells of mouse origin by using automated tissue dissociation and magnetic cell sorting (MACS). We have performed whole exome sequencing of bulk human tumor xenografts from lung, bladder, and kidney cancer, and compared the results to samples depleted of mouse cells. A significant increase (p &amp;lt; 0.05) in cluster density as well as an average increase in read counts of 33% was observed for the mouse cell depleted samples, indicating improved sample quality. Indeed, we observed a strong reduction of debris and dead cells upon the depletion procedure. We mapped the reads of all samples against human and mouse genomes and determined their putative origin. An average of 12% of reads derived from non-depleted samples was assigned to mouse cells. This amount could be reduced to 0.28% by prior depletion of mouse cells. As 15% of the mouse-derived reads mapped erroneously to human genome (1.9% of total reads) in the non-depleted samples, a strong positive influence of mouse cell depletion (0.04% of total reads) on downstream analyses can be expected. The number of predicted SNPs was 2-fold higher in the non-depleted samples and 56% of SNPs identified in the non-depleted samples were not present anymore after depletion of mouse cells. Furthermore, the increase in coverage from 39-fold (non-depleted) to 56-fold (depleted) resulted in a higher sensitivity of relevant SNP detection after mouse cell depletion. 16% of SNPs identified in the depleted samples were exclusively found in these samples. Taken together, removal of mouse cells significantly improves the analysis of human tumor xenografts by next generation sequencing. As this effect was observed although a human sequence specific selection has been carried out during exome enrichment, the influence on whole exome and whole transcriptome sequencing are expected to be even more prominent. Citation Format: David Agorku, Stefan Tomiuk, Kerstin Klingner, Stefan Wild, Silvia Rüberg, Lisa Zatrieb, Andreas Bosio, Julia Schueler, Olaf Hardt. Next generation sequencing of human tumor xenografts is significantly improved by prior depletion of mouse cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1455. doi:10.1158/1538-7445.AM2015-1455

Abstract 583: Colorectal cancer cell lines recapitulate molecular and pharmacological features of clinical samples

Abstract Colorectal Cancer (CRC) is characterized by wide genetic, biological and clinical heterogeneity. Oncogenic events, such as KRAS, NRAS and BRAF mutations, are known negative biomarker of response to EGFR targeted therapies. In addition to mutational analysis, transcriptional profiling has been recently exploited to identify distinct CRC molecular subtypes, associated with biological and clinical features such as cell of origin, microsatellite instability, prognosis and response to treatments. Detailed analysis of the relationships between the subtypes and clinical/biological features requires a large panel of preclinical models closely recapitulating the molecular heterogeneity of CRC. To this aim, we assembled a large collection of 151 CRC cell lines. For each line, we: (i) verified genetic identity by short tandem repeat (STR) analysis; (ii) assessed microsatellite instability (MSI) status; (iii) sequenced mutational hotspots in the KRAS, BRAF, NRAS and PIK3CA genes; (iv) performed microarray-based global mRNA expression profiling; (v) evaluated sensitivity to the EGFR-targeting drug cetuximab. STR analysis revealed that some cell lines previously thought to be unrelated are indeed derived from the same individual. This result was confirmed by cell line hierarchical clustering based on mRNA expression profiles. Overall, the mutational landscape of the compendium was concordant with what observed in patients (mutation rates: KRAS = 47%; BRAF = 17%; NRAS = 0.7%; PIK3CA = 18%). Similarly, sensitivity to cetuximab was confined to lines without RAS or BRAF mutations. Gene expression profiling was exploited to assign cell lines to molecular subtypes, according to five different published transcriptional classifiers. We found that all molecular subtypes previously identified in CRC patients were robustly maintained in the lines. Moreover, significant overlaps were detected between individual subtypes across distinct classifiers. Subtype-specific molecular and pharmacological associations previously defined in CRC samples were largely recapitulated in the compendium. In particular, MSI+ cells were significantly enriched in inflammatory and goblet subtypes and less prevalent in the Transit Amplifying (TA) and Stem groups. Cell lines carrying BRAF mutations clustered in the inflammatory subtype, while RAS mutations were equally distributed among all subtypes. Notably, in TA lines without RAS or BRAF mutations, the fraction of cetuximab-sensitive cells was strongly enriched (56%), confirming clinical data and indicating that addiction to the EGFR pathway is an intrinsic feature of this CRC subtype. In conclusion, our results describe a powerful preclinical resource reflecting the molecular and functional heterogeneity of CRC, which can be used to explore multidimensional information of potential clinical relevance. Citation Format: Gabriele Picco, Mariangela Russo, Carlotta Cancelliere, Michela Buscarino, Claudio Isella, Simona Lamba, Barbara Martinoglio, Federica Di Nicolantonio, Alberto Bardelli, Enzo Medico. Colorectal cancer cell lines recapitulate molecular and pharmacological features of clinical samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 583. doi:10.1158/1538-7445.AM2015-583

Cytotoxicity of 35 medicinal plants from Sudan towards sensitive and multidrug-resistant cancer cells

BackgroundCancer is a complex disease with multiple genetic and epigenetic alterations. Since decades, the hallmark of cancer therapy is chemotherapy. Cytotoxic drugs erase rapidly dividing cells without sufficient differentiation between normal and cancerous cells resulting in severe side effects in normal tissues. Recently, strategies for cancer treatment focused on targeting specific proteins involved in tumor growth and progression. The present study was designed to investigate the cytotoxicity of 65 crude extracts from 35 Sudanese medicinal plants towards various cancer cell lines expressing molecular mechanisms of resistance towards classical chemotherapeutics (two ATP-binding cassette transporters, ABCB1 (P-glycoprotein) and ABCB5, tumor suppressor p53, epidermal growth factors receptor (EGFR). And the aim was to identify plant extracts and isolated compounds thereof with activity towards otherwise drug-resistant tumor cells. MethodsCold maceration was performed to obtain crude extracts from the plants. The resazurin assay was used to determine cytotoxicity of the plant extracts. Microarray-based mRNA expression profiling, COMPARE, and hierarchical cluster analyses were applied to identify, which genes correlate with sensitivity or resistance to ambrosin, the main constituent of the most active extract Ambrosia maritima. ResultsThe results of the resazurin assay on different tumors showed that Lawsonia inermis, Trigonella foenum-graecum and Ambrosia maritma were the most active crude extracts. Ambrosin was selected as one active principle of A. maritima for microarray-based expression profiling. Genes from various functional groups (transcriptional regulators, signal transduction, membrane transporters, cytoskeleton organization, chaperones, immune system development and DNA repair) were significantly correlated with response of tumor cell lines to ambrosin. ConclusionThe results revealed cytotoxicity and pharmacogenomics studies of Sudanese medicinal plants provide an attractive strategy for the development of novel cancer therapeutics with activity towards cell lines that resistance to established anticancer agents.