Improved prognostication of glioblastoma beyond molecular subtyping by transcriptional profiling of the tumor microenvironment.

Glioblastoma (GBM), the most aggressive form of brain cancer, is characterized by a high level of molecular heterogeneity, and infiltration by various immune and stromal cell populations. Important advances have been made in deciphering the microenvironment of GBMs, but its association with existing molecular subtypes and its potential prognostic role remain elusive. We have investigated the abundance of infiltrating immune and stromal cells in silico, from gene expression profiles. Two cohorts, including in-house normal brain and glioma samples (n = 70) and a large sample set from TCGA (n = 393), were combined into a single exploratory dataset. A third independent cohort (n = 124) was used for validation. Tumors were clustered based on their microenvironment infiltration profiles, and associations with known GBM molecular subtypes and patient outcome were tested a posteriori in a multivariable setting. We identified a subset of GBM samples with significantly higher abundances of most immune and stromal cell populations. This subset showed increased expression of both immune suppressor and immune effector genes compared to other GBMs and was enriched for the mesenchymal molecular subtype. Survival analyses suggested that tumor microenvironment infiltration pattern was an independent prognostic factor for GBM patients. Among all, patients with the mesenchymal subtype with low immune and stromal infiltration had the poorest survival. By combining molecular subtyping with gene expression measures of tumor infiltration, the present work contributes with improving prognostic models in GBM.

Monitoring B cell response to immunoselected phage-displayed peptides by microarrays.

Successful adaptation of microarray technology for high-throughput screening of proteins requires a large number of purified recombinant proteins, e.g., antibodies for use as capture molecules. Phage surface display technology has been used for the surface expression of proteins, peptides or cDNA repertoires expressed by tumor cells. It does not require protein purification, as recombinant phages can be spotted on glass slides and used in a high-throughput screening format. Biopanning of phage libraries on patient serum antibodies is expected to enrich for antibody-binding phages for the fabrication of diagnostic and/or prognostic B-cell epitope microarrays. In contrast to other immunological techniques, microarrays can measure the antibody levels against different epitopes in a single test. This chapter highlights the recent advances in phage-based microarray technology to profile humoral immune responses in cancer patients.

Transcriptional responses of Bacillus subtillis and thuringiensis to antibiotics and anti-tumour drugs.

DNA supercoiling is a major regulator of transcription in all organisms. This process is regulated by type I and type II DNA topoisomerases that are targets for microbial antibiotics and/or anti-tumour drugs. Despite extensive studies in this field, no information is available on the response of Bacillus (B) species to eukaryotic DNA topoisomerase inhibitors. Here we found that B. thuringiensis BMG1.7 and HD9 strains are sensitive to DNA gyrase inhibitors (e.g. ciprofloxacin, novobiocin) and to etoposide VP16, a specific inhibitor of eukaryotic type II DNA topoisomerase. Inhibitory or sub-inhibitory concentrations of VP16 induced a drug-tolerant response: an immediate inhibition of growth, followed by a prolonged (10-12 h) lag in growth, and then resumption of normal growth subsequent to overnight culture. Inhibition of the DNA gyrase ATPase activity in B. subtillis 168 by novobiocin activated 80 genes and repressed 89 genes at 20 min after drug addition (P<0.05). The altered genes belonged to various functional categories dominated by those whose products maintained DNA integrity, mediated transport of low molecular-weight compounds, and SOS response genes. Quantitative RT-PCR revealed comparable effects of novobiocin in B. subtillis and thuringiensis on gene expression. In contrast to novobiocin, VP16 treatment resulted in a moderate effect on gyrA, gyrB, and topA gene expression. However, dinB and lexA genes, involved in SOS response, displayed relatively high transcriptional levels compared to those seen in untreated cells. Furthermore, a small DNA plasmid isolated from novobicin-treated B. thuringiensis BMG 1.7 contained a ladder of partially relaxed topoisomers, while the electrophoretic mobility of its counterpart isolated from VP16-treated cells was unchanged. Collectively, the present study would assist in defining common and/or different pathways affected by eubacterial and eukaryotic type DNA topoisomerase inhibitors in the same living organism.

Gene expression analysis in blood cells in response to unmodified and 2'-modified siRNAs reveals TLR-dependent and independent effects.

Ribonucleic nucleic acid recognition by Toll-like receptors (TLRs) induces innate immune responses. However, no comprehensive analysis of gene expression in human blood cells in response to unmodified and 2'-modified immunostimulatory RNAs has been reported. Using oligonucleotide microarrays, we show that around 400 genes were significantly (P<0.001) altered in peripheral blood mononuclear cells (PBMC) in response to either single-stranded (ss) or double-stranded (ds) small interfering RNAs (siRNAs). Most of the upregulated genes encode proteins involved in innate and adaptive immune responses, including proinflammatory cytokines, interferons, chemokines and chemokine receptors. Genes encoding proteins involved in lymphocyte activation (e.g. CD80, CD40, and CD69) and in regulation of the immune responses (e.g. SOCS proteins) were upregulated. Also, genes encoding for antiviral proteins (Mx1, Mx2, TRIM proteins), and interferon regulatory factors (e.g. IRF7) were upregulated. Around 90% of the genes (140 out of 160) affected by R-848, a specific ligand for TLR7 and TLR8, were also affected by ss siRNAs or ds siRNAs, indicating that the signaling pathways activated by R-848 are also activated by immunostimulatory siRNAs. In addition to immunoactivation via TLRs, ss siRNAs and ds siRNAs induced TLR-independent gene alterations. Surprisingly, replacement of only uridine bases with either 2'-fluoro or 2'-O-methyl modified counterparts abrogated all the observed bystander effects. Collectively, these microarray data offer for the first time an insight into human PMBC response to immunostimulatory RNAs such as ss siRNAs and ds siRNAs. The data should help to define strategies to either enhance or avoid the non-specific effects of siRNAs in order to develop safe therapeutics.

Expression profiling of microRNAs in cancer cells: technical considerations.

MicroRNAs (miRNAs) represent a class of small, noncoding RNAs. These small RNAs are involved in diverse biological processes, and it has been predicted that about one third of human messenger RNAs (mRNAs) appear to be miRNA targets, underlying the major influence of miRNAs on almost all cellular pathways. Deviation from the normal pattern of miRNA expression has been implicated in several diseases. Among human diseases, it has been shown that changes in miRNA expression correlate with various human cancers. Thus, miRNA profiling could contribute to more precise tumor classification and better prediction of the therapeutic outcome. This chapter summarizes these recent findings and highlights the technical advances in miRNA probe preparation, miRNA expression profiling and target identification.

Mapping of oxidative stress responses of human tumor cells following photodynamic therapy using hexaminolevulinate.

BACKGROUND: Photodynamic therapy (PDT) involves systemic or topical administration of a lesion-localizing photosensitizer or its precursor, followed by irradiation of visible light to cause singlet oxygen-induced damage to the affected tissue. A number of mechanisms seem to be involved in the protective responses to PDT, including activation of transcription factors, heat shock proteins, antioxidant enzymes and apoptotic pathways. RESULTS: In this study, we address the effects of a destructive/lethal hexaminolevulinate (HAL) mediated PDT dose on the transcriptome by using transcriptional exon evidence oligo microarrays. Here, we confirm deviations in the steady state expression levels of previously identified early defence response genes and extend this to include unreported PDT inducible gene groups, most notably the metallothioneins and histones. HAL-PDT mediated stress also altered expression of genes encoded by mitochondrial DNA (mtDNA). Further, we report PDT stress induced alternative splicing. Specifically, the ATF3 alternative isoform (deltaZip2) was up-regulated, while the full-length variant was not changed by the treatment. Results were independently verified by two different technological microarray platforms. Good microarray, RT-PCR and Western immunoblotting correlation for selected genes support these findings. CONCLUSION: Here, we report new insights into how destructive/lethal PDT alters the transcriptome not only at the transcriptional level but also at post-transcriptional level via alternative splicing.

Protein arrays: a versatile toolbox for target identification and monitoring of patient immune responses.

Functional proteomics is a promising technique for the rational identification of novel therapeutic targets and biological markers. The studies of protein-protein interactions have been gained from the development of high-throughput technologies such as the yeast two-hybrid system, protein arrays, phage display, and systematic analysis of interaction maps for the prediction of protein functions. Because antibodies are used extensively as diagnostic and clinical tools, the characterization of their antigen specificity is of prime importance. Indeed, screening protein arrays with sera from patients with either cancer or autoimmune diseases would facilitate the identification of autoantibody signatures that can be used for diagnosis and/or prognosis of patients. The usefulness of multiplexed measurements lies not only in the ability to screen many individual marker candidates but also in evaluating the use of multiple markers in combination. Here, we review the advantage of protein and serum screening of peptides and cDNA repertoires displayed on phages as well as the fabrication of protein microarrays for probing immune responses in patients.

Epigenetic disruption of miR-130a promotes prostate cancer by targeting SEC23B and DEPDC1.

MicroRNAs (miRNAs) are small, non-coding RNAs that mediate post-transcriptional gene silencing, fine tuning gene expression. In an initial screen, miRNAs were found to be globally down-regulated in prostate cancer (PCa) cell lines and primary tumours. Exposure of PCa cell lines to a demethylating agent, 5-Aza-CdR resulted in an increase in the expression levels of miRNAs in general. Using stringent filtering criteria miR-130a was identified as the most promising candidate and selected for validation analyses in our patient series. Down-regulation of miR-130a was associated with promoter hypermethylation. MiR-130a methylation levels discriminated PCa from non-malignant tissues (AUC = 0.956), and urine samples revealed high specificity for non-invasive detection of patients with PCa (AUC = 0.89). Additionally, repressive histone marks were also found in the promoter of miR-130a. Over-expression of miR-130a in PCa cells reduced cell viability and invasion capability, and increased apoptosis. Putative targets of miR-130a were assessed by microarray expression profiling and DEPD1C and SEC23B were selected for validation. Silencing of both genes resembled the effect of over-expressing miR-130a in PCa cells. Our data indicate that miR-130a is an epigenetically regulated miRNA involved in regulation of key molecular and phenotypic features of prostate carcinogenesis, acting as a tumour suppressor miRNA.

Transcriptome changes in a colon adenocarcinoma cell line in response to photochemical treatment as used in photochemical internalisation (PCI).

The photochemical internalisation (PCI) technology liberates endocytosed macromolecules like transgenes from endocytic vesicles in response to photochemical treatment. Thereby PCI improves gene transfection and is suggested for use in gene therapy. It has been proposed that PCI might also stimulate transcription of internalised transgenes, especially if they are controlled by photochemically inducible promoters (transcriptional targeting). In order to identify inducible promoters, and to evaluate the treatments influence on cellular transcriptional activity, the effect of the photochemical treatment as used in PCI (with the photosensitizer disulfonated meso-tetraphenylporphin followed by illumination) on gene transcription in WiDr adenocarcinoma cells was evaluated using microarrays. The expression of 390 genes were identified significantly changed (89% were up-regulated), of which genes associated with DNA binding and transcriptional functions were the most represented. This may be important for the expression of a photochemically internalised transgene under a specific promoter control. Real-time PCR verified photochemical up-regulation of the HSP family genes, as well as down-regulation of EGR-1 at 2-10h post-treatment, suggesting that the HSP (particularly HSP70), in addition to the microarray-identified metallothioneins, but not the EGR-1 promoters, could be relevant promoter candidates for transcriptional targeting via PCI. The resulting overview of gene expression changes in WiDr cells exposed to the PCI-relevant photochemical treatment also provide a basis for the design of new PCI-based strategies with respect of transcriptional targeting.

MicroRNAs as growth regulators, their function and biomarker status in colorectal cancer.

Gene expression is in part regulated by microRNAs (miRNAs). This review summarizes the current knowledge of miRNAs in colorectal cancer (CRC); their role as growth regulators, the mechanisms that regulate the miRNAs themselves and the potential of miRNAs as biomarkers. Although thousands of tissue samples and bodily fluids from CRC patients have been investigated for biomarker potential of miRNAs (>160 papers presented in a comprehensive tables), none single miRNA nor miRNA expression signatures are in clinical use for this disease. More than 500 miRNA-target pairs have been identified in CRC and we discuss how these regulatory nodes interconnect and affect signaling pathways in CRC progression.

NEDD4 is overexpressed in colorectal cancer and promotes colonic cell growth independently of the PI3K/PTEN/AKT pathway.

Ubiquitination controls multiple cellular processes relevant to cancer pathogenesis. Using Gene Set Enrichment Analysis of an mRNA transcriptome dataset, we have identified genes encoding components of the ubiquitin system that are differentially expressed in colorectal cancers as compared to normal colonic mucosa. Among the significantly overexpressed genes was NEDD4 (neural precursor cell-expressed developmentally down-regulated 4), the prototype member of the HECT (homologous to E6AP C-terminus) E3 ubiquitin ligase family. Previous studies have shown that NEDD4 may act as an oncoprotein by inducing ubiquitination and degradation of the tumor suppressor protein PTEN (phosphatase and tensin homolog). To investigate its functional importance in colorectal cancer, HCT-15 and LoVo colon cancer cells were depleted of NEDD4 by small interfering RNA. The depletion resulted in reduced growth and altered cell morphology in both cell lines. However, NEDD4 depletion did not affect the PTEN protein level or PI3K/AKT signaling pathway activation. Moreover, ectopic expression of NEDD4 did not influence the PTEN subcellular localization or protein level. Collectively, these data demonstrate that NEDD4 is overexpressed in colorectal cancers, and suggest that NEDD4 promotes growth of colon cancer cells independently of PTEN and PI3K/AKT signaling.

MiR-9, -31, and -182 deregulation promote proliferation and tumor cell survival in colon cancer.

Several microRNAs (miRNAs) are known to be deregulated in colon cancer, but the mechanisms behind their potential involvement on proliferation and tumor cell survival are unclear. The present study aimed to identify miRNAs with functional implications for development of colon cancer. The cell proliferation and apoptosis were examined following perturbations of miRNA levels by employing a comprehensive miRNA library screen. miRNAs nominated for relevance to colon cancer were validated on expression and functional levels. By integrating the effect of miRNA up-regulation with the endogenous miRNA expression levels within the HT29, HCT116, and SW480 colon cancer cell lines, we identified miRNAs controlling cell proliferation (n = 53) and apoptosis (n = 93). From these functionally nominated miRNAs, we narrowed the list to 10 oncogene- and 20 tumor suppressor-like miRNAs that were also differentially expressed between colon cancer (n = 80) and normal colonic mucosa (n = 20). The differential expressions of miR-9, miR-31, and miR-182 were successfully validated in a series of colon carcinomas (n = 30) and polyps (n = 10) versus normal colonic mucosa (n = 10), whereas the functional effect was confirmed in an in-depth validation using different cell viability and apoptotic markers. Several transcription factors and genes regulating cell proliferation were identified as putative target genes by integrative miRNA/mRNA expression analysis obtained from the same colon cancer patient samples. This study suggests that deregulated expression of miR-9, miR-31, and miR-182 during carcinogenesis plays a significant role in the development of colon cancer by promoting proliferation and tumor cell survival.

Oncogenicity of the developmental transcription factor Sox9.

SOX9 [sex-determining region Y (SRY)-box 9 protein], a high mobility group box transcription factor, plays critical roles during embryogenesis and its activity is required for development, differentiation, and lineage commitment in various tissues including the intestinal epithelium. Here, we present functional and clinical data of a broadly important role for SOX9 in tumorigenesis. SOX9 was overexpressed in a wide range of human cancers, where its expression correlated with malignant character and progression. Gain of SOX9 copy number is detected in some primary colorectal cancers. SOX9 exhibited several pro-oncogenic properties, including the ability to promote proliferation, inhibit senescence, and collaborate with other oncogenes in neoplastic transformation. In primary mouse embryo fibroblasts and colorectal cancer cells, SOX9 expression facilitated tumor growth and progression whereas its inactivation reduced tumorigenicity. Mechanistically, we have found that Sox9 directly binds and activates the promoter of the polycomb Bmi1, whose upregulation represses the tumor suppressor Ink4a/Arf locus. In agreement with this, human colorectal cancers showed a positive correlation between expression levels of SOX9 and BMI1 and a negative correlation between SOX9 and ARF in clinical samples. Taken together, our findings provide direct mechanistic evidence of the involvement of SOX9 in neoplastic pathobiology, particularly, in colorectal cancer.

Phospholipase C isozymes are deregulated in colorectal cancer--insights gained from gene set enrichment analysis of the transcriptome.

Colorectal cancer (CRC) is one of the most common cancer types in developed countries. To identify molecular networks and biological processes that are deregulated in CRC compared to normal colonic mucosa, we applied Gene Set Enrichment Analysis to two independent transcriptome datasets, including a total of 137 CRC and ten normal colonic mucosa samples. Eighty-two gene sets as described by the Kyoto Encyclopedia of Genes and Genomes database had significantly altered gene expression in both datasets. These included networks associated with cell division, DNA maintenance, and metabolism. Among signaling pathways with known changes in key genes, the "Phosphatidylinositol signaling network", comprising part of the PI3K pathway, was found deregulated. The downregulated genes in this pathway included several members of the Phospholipase C protein family, and the reduced expression of two of these, PLCD1 and PLCE1, were successfully validated in CRC biopsies (n = 70) and cell lines (n = 19) by quantitative analyses. The repression of both genes was found associated with KRAS mutations (P = 0.005 and 0.006, respectively), and we observed that microsatellite stable carcinomas with reduced PLCD1 expression more frequently had TP53 mutations (P = 0.002). Promoter methylation analyses of PLCD1 and PLCE1 performed in cell lines and tumor biopsies revealed that methylation of PLCD1 can contribute to reduced expression in 40% of the microsatellite instable carcinomas. In conclusion, we have identified significantly deregulated pathways in CRC, and validated repression of PLCD1 and PLCE1 expression. This illustrates that the GSEA approach may guide discovery of novel biomarkers in cancer.

Profiling of miRNA expression and prediction of target genes.

MicroRNAs (miRNAs) represent a new class of small noncoding RNAs expressed in plants and animals. They are responsible for the regulation of up to 30% of human genes, thus underlying their influence on almost all cellular pathways. Recent studies have shown miRNAs to be differentially expressed in cancers. Furthermore, several miRNAs are associated with fragile sites, preferential sites of translation, deletion and amplification that are often altered in cancers. However, despite the progress in identifying miRNA genes, knowledge about their functions and specific target genes is still limited. This chapter highlights the technical advances in miRNA gene profiling and discusses the limitations of target prediction programs and the necessity of defining the roles that govern miRNA specificity and target discrimination in vivo.

Increased miR-21 expression during human monocyte differentiation into DCs.

Differentiation of monocytes into dendritic cells (DCs) is characterised by marked changes in gene expression. The role of microRNAs (miRNAs), a new class of small endogenous non-coding regulatory RNAs, in this process is still unclear. We identified miR-223, miR-16, miR-191, miR-24, let-7b, and miR-21 as differentially expressed between monocytes and monocyte derived DCs. We evaluated the expression levels of computationally predicted target genes of miR-21 in human monocytes following stimulation with GM-CSF and IL-4. Moreover, transfection of monocytes with synthetic miR-21 inhibited the expression of a set of genes that were also repressed during monocyte differentiation to DCs in response to GM-CSF and IL-4. Among these, we identified genes that are involved in cell cycle, apoptosis and differentiation such as PDE4B, PDCD4, ANXA1, ING3, STAG2, TGFBI, S100A12, LAT2 and NRIP1. Collectively, the present study highlights the involvement of miRNAs, particularly miR-21 in monocyte differentiation to DCs and identifies potential miR-21 target genes.

Suppression of immunostimulatory siRNA-driven innate immune activation by 2'-modified RNAs.

Single-stranded (ss) and double-stranded (ds) small interfering RNAs (siRNAs) containing immunostimulatory RNA motifs can activate innate immunity through Toll-like receptor 7/8 (TLR7/8), leading to the production of proinflammatory cytokines and type I interferon. More recently, we have noted that 2'-uridine modified ss or ds siRNAs not only evade immune activation, but can suppress TLR signaling triggered by their unmodified counterparts. Here we compared the inhibitory effects of several 2'-modifications. In contrast to 2'-deoxy uridine modified ss siRNAs, 2'-O-methyl uridine modified ss siRNAs inhibited at nanomolar concentrations the production of TNF-alpha induced by a variety of immunostimulatory RNA sequences. Using oligonucleotide microarrays, we highlight the strong suppressive effect of RNA-containing 2'-O-methyl uridines. Indeed, nearly all of the 270 genes induced by an immunostimulatory ss siRNA were completely inhibited or downregulated by cotreatment with its 2'-O-methyl modified version. Also, 2'-O-methyl modified RNAs inhibited E. coli total RNA or mitochondrial RNA to induce TNF-alpha production in human monocytes. Collectively, these data indicate that 2'-modified RNAs, in particular those containing 2'-O-methyl modification, are recognized with high affinity by TLR7/8, but do not induce downstream signaling. Therefore, this new generation of TLR antagonists can be used as immunosuppressive agents to interfere with TLR signaling.

Analysis of the humoral immune response to immunoselected phage-displayed peptides by a microarray-based method.

We describe a novel approach for high-throughput analysis of the immune response in cancer patients using phage-based microarray technology. The recombinant phages used for fabricating phage arrays were initially selected via the use of random peptide phage libraries and breast cancer patient serum antibodies. The peptides displayed by the phages retained their ability to be recognized by serum antibodies after immobilization. The recombinant phage microarrays were screened against either breast cancer or healthy donor serum antibodies. A model-based statistical method is proposed to estimate significant differences in serum antibody reactivity between patients and normals. A significant tumor effect was found with most of the selected phage-displayed peptides, suggesting that recombinant phage microarrays can serve as a tool in monitoring humoral responses towards phage-displayed peptides.