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Supplementary Figure Legends 1-3 from Microarray-Based Identification of Tenascin C and Tenascin XB, Genes Possibly Involved in Tumorigenesis Associated with Neurofibromatosis Type 1

Supplementary Tables 1-4 from Microarray-Based Identification of Tenascin C and Tenascin XB, Genes Possibly Involved in Tumorigenesis Associated with Neurofibromatosis Type 1

Purpose: Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder with a complex variety of clinical manifestations. The hallmark of NF1 is the onset of heterogeneous (dermal or plexiform) benign neurofibromas. Plexiform neurofibromas can give rise to malignant peripheral nerve sheath tumors, which are resistant to conventional therapies.Experimental Design: To identify new signaling pathways involved in the malignant transformation of plexiform neurofibromas, we applied a 22,000-oligonucleotide microarray approach to a series of plexiform neurofibromas and malignant peripheral nerve sheath tumors. Changes in the expression of selected genes were then confirmed by real-time quantitative reverse transcription-PCR.Results: We identified two tenascin gene family members that were significantly deregulated in both human NF1-associated tumors and NF1-deficient primary cells: Tenascin C (TNC) was up-regulated whereas tenascin XB (TNXB) was down-regulated during tumor progression. TNC activation is mainly due to the up-regulation of large TNC splice variants. Immunohistochemical studies showed that TNC transcripts are translated into TNC protein in TNC-overexpressing tumors. Aberrant transcriptional activation of TNC seems to be principally mediated by activator protein transcription factor complexes.Conclusion:TNXB and TNC may be involved in the malignant transformation of plexiform neurofibromas. Anti-TNC antibodies, already used successfully in clinical trials to treat malignant human gliomas, may be an appropriate new therapeutic strategy for NF1.

Supplementary Figures 1-3 from Microarray-Based Identification of Tenascin C and Tenascin XB, Genes Possibly Involved in Tumorigenesis Associated with Neurofibromatosis Type 1

Supplementary Tables 1-4 from miR-181a and miR-630 Regulate Cisplatin-Induced Cancer Cell Death

Supplementary Movie 1 from miR-181a and miR-630 Regulate Cisplatin-Induced Cancer Cell Death

Non–small cell lung cancer (NSCLC) with activating mutations in the epidermal growth factor receptor (EGFR) responds to EGFR tyrosine kinase inhibitors such as erlotinib. However, secondary somatic EGFR mutations (e.g., T790M) confer resistance to erlotinib. BMS-690514, a novel panHER/vascular endothelial growth factor receptor (VEGFR) inhibitor described here, exerted antiproliferative and proapoptotic effects on NSCLC cell lines, with prominent efficacy on H1975 cells expressing the T790M mutation. In this model, BMS-690514 induced a G1 cell cycle arrest, as well as ultrastructural hallmarks of apoptosis, mitochondrial release of cytochrome c, and activation of caspases involved in the intrinsic (e.g., caspase-2, caspase-3, caspase-7, and caspase-9), but not in the extrinsic (e.g., caspase-8), pathway. Caspase inhibition conferred partial protection against BMS-690514 cytotoxicity, pointing to the involvement of both caspase-dependent and caspase-independent effector mechanisms. Transcriptome analyses revealed the up-regulation of proapoptotic (e.g., Bim, Puma) and cell cycle inhibitory (e.g., p27Kip1, p57Kip2) factors, as well as the down-regulation of antiapoptotic (e.g., Mcl1), heat shock (e.g., HSP40, HSP70, HSP90), and cell cycle promoting [e.g., cyclins B1, D1, and D3; cyclin-dependent kinase 1 (CDK1); MCM family proteins; proliferating cell nuclear antigen (PCNA)] proteins. BMS-690514–induced death of H1975 cells was modified in a unique fashion by a panel of small interfering RNAs targeting apoptosis modulators. Down-regulation of components of the nuclear factor-κB survival pathway (e.g., p65, Nemo/IKKγ, TAB2) sensitized cells to BMS-690514, whereas knockdown of proapoptotic factors (e.g., Puma, Bax, Bak, caspase-2, etc.) and DNA damage–related proteins (e.g., ERCC1, hTERT) exerted cytoprotective effects. BMS-690514 is a new pan-HER/VEGFR inhibitor that may become an alternative to erlotinib for the treatment of NSCLC. [Cancer Res 2007;67(13):6253–62]

Supplementary Figures 1-6 from miR-181a and miR-630 Regulate Cisplatin-Induced Cancer Cell Death

Supplementary Table 1 from A Novel Epidermal Growth Factor Receptor Inhibitor Promotes Apoptosis in Non–Small Cell Lung Cancer Cells Resistant to Erlotinib

Supplementary Figure, Table and Movie Legends from miR-181a and miR-630 Regulate Cisplatin-Induced Cancer Cell Death

Supplementary Methods and Materials from A Novel Epidermal Growth Factor Receptor Inhibitor Promotes Apoptosis in Non–Small Cell Lung Cancer Cells Resistant to Erlotinib

The adaptive B cell response is driven by the expansion, somatic hypermutation, and selection of B cell clones. A high number of clones in a B cell population indicates a highly diverse repertoire, while clonal size distribution and sequence diversity within clones can be related to antigen’s selective pressure. Identifying clones is fundamental to many repertoire studies, including repertoire comparisons, clonal tracking and statistical analysis. Several methods have been developed to group sequences from high-throughput B cell repertoire data. Current methods use clustering algorithms to group clonally-related sequences based on their similarities or distances. Such approaches create groups by optimizing a single objective that typically minimizes intra-clonal distances. However, optimizing several objective functions can be advantageous and boost the algorithm convergence rate. Here we propose a new method based on multi-objective clustering. Our approach requires V(D)J annotations to obtain the initial clones and iteratively applies two objective functions that optimize cohesion and separation within clones simultaneously. We show that under simulations with varied mutation rates, our method greatly improves clonal grouping as compared to other tools. When applied to experimental repertoires generated from high-throughput sequencing, its clustering results are comparable to the most performing tools. The method based on multi-objective clustering can accurately identify clone members, has fewer parameter settings and presents the lowest running time among existing tools. All these features constitute an attractive option for repertoire analysis, particularly in the clinical context to unravel the mechanisms involved in the development and evolution of B cell malignancies.

International audience; The database JET2 Viewer, openly accessible at http://www.jet2viewer.upmc.fr/, reports putative protein binding sites for all three-dimensional (3D) structures available in the Protein Data Bank (PDB). This knowledge base was generated by applying the computational method JET 2 at large-scale on more than 20 000 chains. JET 2 strategy yields very precise predictions of interacting surfaces and unravels their evolutionary process and complexity. JET2 Viewer provides an online intelligent display, including interactive 3D visualization of the binding sites mapped onto PDB structures and suitable files recording JET 2 analyses. Predictions were evaluated on more than 15 000 experimentally characterized protein interfaces. This is, to our knowledge, the largest evaluation of a protein binding site prediction method. The overall performance of JET 2 on all interfaces are: Sen = 52.52, PPV = 51.24, Spe = 80.05, Acc = 75.89. The data can be used to foster new strategies for protein– protein interactions modulation and interaction surface redesign.

RET/PTC1 rearrangement is the most common genetic alteration identified to date in papillary thyroid carcinomas (PTC) and represents an interesting target for small interfering RNA (siRNA) strategies because it is present only in the tumor cells and not in the normal cells. Our aims were (i) to target the RET/PTC1 oncogene by siRNAs, (ii) to assess the knockdown effects on cell growth and cell cycle regulation, and (iii) to identify genes affected by the RET/PTC1 silencing.Three efficient siRNAs previously designed in our laboratory in a model of murine PTC (RP-1 cells) were used to knockdown RET/PTC1 in the TPC-1 cells. By reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative RT-PCR (Q-RT-PCR) they were found unable to silence RET/PTC1. After sequencing, we redesigned an siRNA against RET/PTC1 (siRNARET/PTC1) and compared it for its efficiency and specificity with an siRNA against RET (siRNARET) in the TPC-1 cells, in human cell lines that expressed RET (MCF-7 and BT-474 cells), and in the murine RP-1 cells. The effects on cell cycle growth (MTT tests), cell cycle (flow cytometry), and apoptosis (TUNEL method) were studied. Genes affected by the RET/PTC1 knockdown were identified by microarray analysis followed by Q-RT-PCR validation.A mutation was found by sequencing within the H4 part of the RET/PTC1 junction leading to a ²⁹⁷T→G substitution. The redesigned siRNARET/PTC1 inhibits about 85% of the oncogene expression in the human TCP-1 cells. The specificity of the siRNARET/PTC1 was confirmed by the absence of a silencing effect on the human breast MCF-7 and BT-474 cells without RET/PTC1 and the murine RP-1 with ²⁹⁷G→T mutation. The downregulation of RET/PTC1 modified the cell cycle and induced an apoptotic response. Microarray analysis revealed an inhibition of E2F2 transcription factor known to be involved in the cell cycle regulation.This study shows the impact of a point mutation within a junction oncogene on the siRNA design. In the case of a therapeutic approach by siRNA, the junction oncogene must be systematically sequenced. The E2F2 gene regulation would have a biological significance and seems to be directly mediated by RET/PTC1.

The tumor suppressor protein p53 plays a major role in preserving genomic stability. p53 suppresses a pathway leading from normal diploidy to neoplastic aneuploidy (via an intermediate metastable stage of tetraploidy) at two levels: first by preventing the generation/survival of tetraploid cells, and second by repressing their aberrant multipolar division. Here, we report the characterization of p53-/- tetraploid cells, which - at difference with both their p53-/- diploid and their p53+/+ tetraploid counterparts - manifest a marked hyperphosporylation of the mitogen-activated protein kinase MAPK14 (best known as p38α) that is particularly strong during mitosis. In p53-/- tetraploid cells, phosphorylated p38α accumulated at centrosomes during the metaphase and at midbodies during the telophase. Selective knockdown or pharmacological inhibition of p38α had a dramatic effect on p53-/- (but not p53+/+) tetraploids, causing the activation of the spindle assembly checkpoint, an arrest during the metaphase, a ma...

MicroRNAs (miRNA) are noncoding RNAs that regulate multiple cellular processes, including proliferation and apoptosis. We used microarray technology to identify miRNAs that were upregulated by non–small cell lung cancer (NSCLC) A549 cells in response to cisplatin (CDDP). The corresponding synthetic miRNA precursors (pre-miRNAs) per se were not lethal when transfected into A549 cells yet affected cell death induction by CDDP, C2-ceramide, cadmium, etoposide, and mitoxantrone in an inducer-specific fashion. Whereas synthetic miRNA inhibitors (anti-miRNAs) targeting miR-181a and miR-630 failed to modulate the response of A549 to CDDP, pre-miR-181a and pre-miR-630 enhanced and reduced CDDP-triggered cell death, respectively. Pre-miR-181a and pre-miR-630 consistently modulated mitochondrial/postmitochondrial steps of the intrinsic pathway of apoptosis, including Bax oligomerization, mitochondrial transmembrane potential dissipation, and the proteolytic maturation of caspase-9 and caspase-3. In addition, pre-miR-630 blocked early manifestations of the DNA damage response, including the phosphorylation of the ataxia-telangiectasia mutated (ATM) kinase and of two ATM substrates, histone H2AX and p53. Pharmacologic and genetic inhibition of p53 corroborated the hypothesis that pre-miR-630 (but not pre-miR-181a) blocks the upstream signaling pathways that are ignited by DNA damage and converge on p53 activation. Pre-miR-630 arrested A549 cells in the G0-G1 phase of the cell cycle, correlating with increased levels of the cell cycle inhibitor p27Kip1 as well as with reduced proliferation rates and resulting in greatly diminished sensitivity of A549 cells to the late S-G2-M cell cycle arrest mediated by CDDP. Altogether, these results identify miR-181a and miR-630 as novel modulators of the CDDP response in NSCLC. Cancer Res; 70(5); 1793–803

The oncogenic process leading to nasopharyngeal carcinoma (NPC) requires the combination of genetic and epigenetic alterations, latent infection by the Epstein-Barr virus and local inflammation. A transcriptome analysis of NPC xenografts identified the gene encoding the cellular inhibitor of apoptosis protein 2 (c-IAP2) among the top five most intensely expressed. Consistently, the very high levels of the c-IAP2 protein were detected in 11 of 13 NPC biopsies. RMT 5265, a structural analog of second mitochondria-derived activator of caspase (SMAC), induced the rapid degradation of c-IAP2 in nasopharyngeal epithelial cells, whether malignant or not, but blocked clonal cell growth in NPC cells only. In short-term experiments, RMT 5265 induced apoptosis in a fraction of NPC cells, and this apoptosis was dramatically enhanced when RMT 5265 was combined with Toll-like receptor 3 (TLR3) stimulation. By contrast, the cooperative effect with tumor necrosis factor α was only marginal. The apoptosis induced by the combination of RMT 5265 and TLR3 stimulation was mediated by caspase-8 and associated with a decrease in the cellular content of the long isoform of FLICE-like inhibitory protein. Similar caspase-8 activation was obtained when siRNA knockdown of c-IAP2 was combined with TLR3 stimulation. In conclusion, c-IAP2 has a specific protective function in NPC cells challenged by TLR3 agonists. This protective function is probably important to make NPC cells tolerant to their own production of small viral RNAs, which are potential agonists of TLR3. Our data will help to design a rational use of IAP inhibitors in NPC patients.

Non–small cell lung cancer (NSCLC) with activating mutations in the epidermal growth factor receptor (EGFR) responds to EGFR tyrosine kinase inhibitors such as erlotinib. However, secondary somatic EGFR mutations (e.g., T790M) confer resistance to erlotinib. BMS-690514, a novel panHER/vascular endothelial growth factor receptor (VEGFR) inhibitor described here, exerted antiproliferative and proapoptotic effects on NSCLC cell lines, with prominent efficacy on H1975 cells expressing the T790M mutation. In this model, BMS-690514 induced a G1 cell cycle arrest, as well as ultrastructural hallmarks of apoptosis, mitochondrial release of cytochrome c, and activation of caspases involved in the intrinsic (e.g., caspase-2, caspase-3, caspase-7, and caspase-9), but not in the extrinsic (e.g., caspase-8), pathway. Caspase inhibition conferred partial protection against BMS-690514 cytotoxicity, pointing to the involvement of both caspase-dependent and caspase-independent effector mechanisms. Transcriptome analyses revealed the up-regulation of proapoptotic (e.g., Bim, Puma) and cell cycle inhibitory (e.g., p27Kip1, p57Kip2) factors, as well as the down-regulation of antiapoptotic (e.g., Mcl1), heat shock (e.g., HSP40, HSP70, HSP90), and cell cycle promoting [e.g., cyclins B1, D1, and D3; cyclin-dependent kinase 1 (CDK1); MCM family proteins; proliferating cell nuclear antigen (PCNA)] proteins. BMS-690514–induced death of H1975 cells was modified in a unique fashion by a panel of small interfering RNAs targeting apoptosis modulators. Down-regulation of components of the nuclear factor-κB survival pathway (e.g., p65, Nemo/IKKγ, TAB2) sensitized cells to BMS-690514, whereas knockdown of proapoptotic factors (e.g., Puma, Bax, Bak, caspase-2, etc.) and DNA damage–related proteins (e.g., ERCC1, hTERT) exerted cytoprotective effects. BMS-690514 is a new pan-HER/VEGFR inhibitor that may become an alternative to erlotinib for the treatment of NSCLC. [Cancer Res 2007;67(13):6253–62]

Background: To identify new molecular markers of bone marrow dissemination in human neuroblastoma (NB), we studied the transcriptome profiles of malignant neuroblasts established from the human MYCN-amplified IGR-N-91 model. Methods: This experimental model includes human neuroblastoma cells derived from a subcutaneous stage 4 disease, myocardium (Myoc) and bone marrow (BM) metastatic cells. Results: Gene expression profiles obtained with Agilent oligo microarrays revealed a set of 107 differentially expressed genes in the metastatic neuroblasts. This set included up-regulated genes involved in chemoresistance, cell motility, neuronal structure/signaling, and the recently characterized GALNT13 gene encoding a glycosyltransferase that initiates mucin-type O-glycosylation. Because the glycosylation process is involved in the progression of primary tumor to metastatic disease, we investigated whether the most strongly up-regulated gene, GALNT13, might be a marker of bone marrow involvement in stage 4 NB patients. Importantly, in the BM of healthy adults no GALNT13 transcript was detected with analysis by quantitative (n = 3) and nested reverse transcription-PCR (n = 4) assays. In contrast, GALNT13 transcripts were detected in 23/23 cytologically involved BM samples obtained at diagnosis of stage 4 NB patients and in 5/27 cytologically noninvolved BM samples obtained from patients with stage 1–4 and 4S and treated stage 4 NB. The quantitative measurements of tyrosine hydroxylase (TH), ganglioside D2 synthase, dopa decarboxylase, and GALNT13 transcript values were compared in the same NB patients, and the results showed that GALNT13 expression was most highly correlated to poor clinical outcome at diagnosis. Conclusion: We propose ppGalNAc-T13 as a new informative marker for the molecular diagnosis of BM involvement and the follow-up of minimal residual disease in NB patients.

Follicular thyroid carcinomas (FTC) arise through oncogenic pathways distinct from those involved in the papillary histotype. Recently, a t(2;3)(q13;p25) rearrangement, which juxtaposes the thyroid transcription factor PAX8 to the peroxisome proliferator-activated receptor (PPAR) gamma1, was described in FTCs. In this report, we describe gene expression in 11 normal tissues, 4 adenomas, and 8 FTCs, with or without the PAX8-PPARgamma1 translocation, using custom 60-mer oligonucleotide microarrays. Results were confirmed by quantitative real-time polymerase chain reaction of 65 thyroid tissues and by immunohistochemistry. Statistical analysis revealed a pattern of 93 genes discriminating FTCs, with or without the translocation, that were morphologically undistinguishable. Although the expression of thyroid-specific genes was detectable, none appeared to be differentially regulated between tumors with or without the translocation. Differentially expressed genes included genes related to lipid/glucose/amino acid metabolism, tumorigenesis, and angiogenesis. Surprisingly, several PPARgamma target genes were up-regulated in PAX8-PPARgamma-positive FTCs such as angiopoietin-like 4 and aquaporin 7. Moreover many genes involved in PAX8-PPARgamma expression profile presented a putative PPARgamma-promoter site, compatible with a direct activity of the fusion product. These data identify several differentially expressed genes, such as FGD3, that may serve as potential targets of PPARgamma and as members of novel molecular pathways involved in the development of thyroid carcinomas.

Background Non-small cell lung cancer (NSCLC), a leading cause of cancer deaths, represents a heterogeneous group of neoplasms, mostly comprising squamous cell carcinoma (SCC), adenocarcinoma (AC) and large-cell carcinoma (LCC). The objectives of this study were to utilize integrated genomic data including copy-number alteration, mRNA, microRNA expression and candidate-gene full sequencing data to characterize the molecular distinctions between AC and SCC. Methods Comparative genomic hybridization followed by mutational analysis, gene expression and miRNA microarray profiling were performed on 123 paired tumor and non-tumor tissue samples from patients with NSCLC. Results At DNA, mRNA and miRNA levels we could identify molecular markers that discriminated significantly between the various histopathological entities of NSCLC. We identified 34 genomic clusters using aCGH data; several genes exhibited a different profile of aberrations between AC and SCC, including PIK3CA, SOX2, THPO, TP63, PDGFB genes. Gene expression profiling analysis identified SPP1, CTHRC1and GREM1 as potential biomarkers for early diagnosis of the cancer, and SPINK1 and BMP7 to distinguish between AC and SCC in small biopsies or in blood samples. Using integrated genomics approach we found in recurrently altered regions a list of three potential driver genes, MRPS22, NDRG1 and RNF7, which were consistently over-expressed in amplified regions, had wide-spread correlation with an average of ~800 genes throughout the genome and highly associated with histological types. Using a network enrichment analysis, the targets of these potential drivers were seen to be involved in DNA replication, cell cycle, mismatch repair, p53 signalling pathway and other lung cancer related signalling pathways, and many immunological pathways. Furthermore, we also identified one potential driver miRNA hsa-miR-944. Conclusions Integrated molecular characterization of AC and SCC helped identify clinically relevant markers and potential drivers, which are recurrent and stable changes at DNA level that have functional implications at RNA level and have strong association with histological subtypes.

Neither the molecular mechanisms whereby cancer cells intrinsically are or become resistant to the DNA-damaging agent cisplatin nor the signaling pathways that account for cisplatin cytotoxicity have thus far been characterized in detail. In an attempt to gain further insights into the molecular cascades elicited by cisplatin (leading to resistance or underpinning its antineoplastic properties), we comparatively investigated the ability of cisplatin, C2-ceramide and cadmium dichloride, alone or in the presence of an array of mitochondrion-protective agents, to trigger the permeabilization of purified mitochondria. In addition, we compared the transcriptional response triggered by cisplatin, C2-ceramide and cadmium dichloride in non-small cell lung carcinoma A549 cells. Finally, we assessed the capacity of cisplatin, C2-ceramide and cadmium dichloride to reduce the clonogenic potential of a battery of yeast strains lacking proteins involved in the regulation of cell death, DNA damage signaling and stress management. This multipronged experimental approach revealed that cisplatin elicits signaling pathways that are for the most part "private," i.e., that manifest limited overlap with the molecular cascades ignited by other inducers of mitochondrial apoptosis, and triggers apoptosis mainly in a transcription-independent fashion. Indeed, bona fide cisplatin-response modifiers that we have recently identified by a functional genome-wide siRNA screen are either not transcriptionally regulated during cisplatin-induced cell death or their transcriptional modulation reflects the activation of an adaptive response promoting cisplatin resistance.

Background Aneuploidy and chromosomal instability (CIN) are common abnormalities in human cancer. Alterations of the mitotic spindle checkpoint are likely to contribute to these phenotypes, but little is known about somatic alterations of mitotic spindle checkpoint genes in breast cancer. Methods To obtain further insight into the molecular mechanisms underlying aneuploidy in breast cancer, we used real-time quantitative RT-PCR to quantify the mRNA expression of 76 selected mitotic spindle checkpoint genes in a large panel of breast tumor samples. Results The expression of 49 (64.5%) of the 76 genes was significantly dysregulated in breast tumors compared to normal breast tissues: 40 genes were upregulated and 9 were downregulated. Most of these changes in gene expression during malignant transformation were observed in epithelial cells. Alterations of nine of these genes, and particularly NDC80, were also detected in benign breast tumors, indicating that they may be involved in pre-neoplastic processes. We also identified a two-gene expression signature (PLK1 + AURKA) which discriminated between DNA aneuploid and DNA diploid breast tumor samples. Interestingly, some DNA tetraploid tumor samples failed to cluster with DNA aneuploid breast tumors. Conclusion This study confirms the importance of previously characterized genes and identifies novel candidate genes that could be activated for aneuploidy to occur. Further functional analyses are required to clearly confirm the role of these new identified genes in the molecular mechanisms involved in breast cancer aneuploidy. The novel genes identified here, and/or the two-gene expression signature, might serve as diagnostic or prognostic markers and form the basis for novel therapeutic strategies.

The tumor suppressor protein p53 plays a major role in preserving genomic stability. p53 suppresses a pathway leading from normal diploidy to neoplastic aneuploidy (via an intermediate metastable stage of tetraploidy) at two levels: first by preventing the generation/survival of tetraploid cells, and second by repressing their aberrant multipolar division. Here, we report the characterization of p53(-/-) tetraploid cells, which-at difference with both their p53(-/-) diploid and their p53(+/+) tetraploid counterparts-manifest a marked hyperphosporylation of the mitogen-activated protein kinase MAPK14 (best known as p38alpha) that is particularly strong during mitosis. In p53(-/-) tetraploid cells, phosphorylated p38alpha accumulated at centrosomes during the metaphase and at midbodies during the telophase. Selective knockdown or pharmacological inhibition of p38alpha had a dramatic effect on p53(-/-) (but not p53(+/+)) tetraploids, causing the activation of the spindle assembly checkpoint, an arrest during the metaphase, a major increase in abnormal bipolar and monopolar mitoses, as well as an increment in the generation of multinuclear cells. We conclude that the mitotic progression of p53(-/-) (but not p53(+/+)) tetraploids heavily relies on p38alpha, revealing a novel function for this protein in the context of aneuploidizing cell divisions.

Gorlin's or nevoid basal cell carcinoma syndrome (NBCCS) causes predisposition to basal cell carcinoma (BCC), the commonest cancer in adult human. Mutations in the tumor suppressor gene PTCH1 are responsible for this autosomal dominant syndrome. In NBCCS patients, as in the general population, ultraviolet exposure is a major risk factor for BCC development. However these patients also develop BCCs in sun-protected areas of the skin, suggesting the existence of other mechanisms for BCC predisposition in NBCCS patients. As increasing evidence supports the idea that the stroma influences carcinoma development, we hypothesized that NBCCS fibroblasts could facilitate BCC occurence of the patients. WT (n = 3) and NBCCS fibroblasts bearing either nonsense (n = 3) or missense (n = 3) PTCH1 mutations were cultured in dermal equivalents made of a collagen matrix and their transcriptomes were compared by whole genome microarray analyses. Strikingly, NBCCS fibroblasts over-expressed mRNAs encoding pro-tumoral factors such as Matrix Metalloproteinases 1 and 3 and tenascin C. They also over-expressed mRNA of pro-proliferative diffusible factors such as fibroblast growth factor 7 and the stromal cell-derived factor 1 alpha, known for its expression in carcinoma associated fibroblasts. These data indicate that the PTCH1(+/-) genotype of healthy NBCCS fibroblasts results in phenotypic traits highly reminiscent of those of BCC associated fibroblasts, a clue to the yet mysterious proneness to non photo-exposed BCCs in NBCCS patients.

Stage 4 neuroblastoma (NB) are heterogeneous regarding their clinical presentations and behavior. Indeed infants (stage 4S and non‐stage 4S of age 365days). Our study aimed at: (i) identifying age‐based genomic and gene expression profiles of stage 4 NB supporting this clinical stratification; and (ii) finding a stage 4S NB signature. Differential genome and transcriptome analyses of a learning set of MYCN‐non amplified stage 4 NB tumors at diagnosis (n=29 tumors including 12 stage 4S) were performed using 1Mb BAC microarrays and Agilent 22K probes oligo‐microarrays. mRNA chips data following filtering yielded informative genes before supervised hierarchical clustering to identify relationship among tumor samples. After confirmation by quantitative RT‐PCR, a stage 4S NB's gene cluster was obtained and submitted to a validation set (n=22 tumors). Genomic abnormalities of infant's tumors (whole chromosomes gains or loss) differ radically from that of children (intra‐chromosomal rearrangements) but could not discriminate infants with 4S from those without this presentation. In contrast, differential gene expression by looking at both individual genes and whole biological pathways leads to a molecular stage 4S NB portrait which provides new biological clues about this fascinating entity.

Purpose: Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder with a complex variety of clinical manifestations. The hallmark of NF1 is the onset of heterogeneous (dermal or plexiform) benign neurofibromas. Plexiform neurofibromas can give rise to malignant peripheral nerve sheath tumors, which are resistant to conventional therapies. Experimental Design: To identify new signaling pathways involved in the malignant transformation of plexiform neurofibromas, we applied a 22,000-oligonucleotide microarray approach to a series of plexiform neurofibromas and malignant peripheral nerve sheath tumors. Changes in the expression of selected genes were then confirmed by real-time quantitative reverse transcription-PCR. Results: We identified two tenascin gene family members that were significantly deregulated in both human NF1-associated tumors and NF1-deficient primary cells: Tenascin C (TNC) was up-regulated whereas tenascin XB (TNXB) was down-regulated during tumor progression. TNC activation is mainly due to the up-regulation of large TNC splice variants. Immunohistochemical studies showed that TNC transcripts are translated into TNC protein in TNC-overexpressing tumors. Aberrant transcriptional activation of TNC seems to be principally mediated by activator protein transcription factor complexes. Conclusion: TNXB and TNC may be involved in the malignant transformation of plexiform neurofibromas. Anti-TNC antibodies, already used successfully in clinical trials to treat malignant human gliomas, may be an appropriate new therapeutic strategy for NF1.

Polyploidization is a part of the normal developmental process leading to platelet production during megakaryocyte (MK) differentiation. Ploidization is mainly involved in cell enlargement, but it is not clear whether gene expression is modified during MK ploidization. In this study, human MKs were grown from CD34+ cells in the presence of thrombopoietin and sorted according to their ploidy level. A pangenomic microarray technique was applied to compare gene expression in 2N-, 4N-, 8N-, and 16N-sorted MKs. Using hierarchical clustering, we demonstrated that 2N and 4N MKs or 8N and 16N MKs are 2 different close populations with 105 discriminating genes. In the second approach, we determined the profile of genes that were continuously down- and up-regulated during polyploidization. Among the 100 down-regulated genes, 24 corresponded to genes involved in DNA replication and repair. The great majority of up-regulated genes corresponded to genes directly involved in platelet functions, such as genes encoding specific platelet glycoproteins and α-granule proteins, actin and microtubule cytoskeleton, factors involved in signaling, and transport proteins. Together, these results suggest that MK polyploidization per se does not regulate gene expression but is intrinsically included in the differentiation process.

To define genetic determinants of tumor cell resistance to the cytotoxic action of tumor necrosis factor α (TNF), we have applied cDNA microarrays to a human breast carcinoma TNF-sensitive MCF7 cell line and its established TNF-resistant clone. Of a total of 5760 samples of cDNA examined, 3.6% were found to be differentially expressed in TNF-resistant 1001 cells as compared with TNF-sensitive MCF7 cells. On the basis of available literature data, the striking finding is the association of some differentially expressed genes involved in the phosphatidylinositol-3-kinase/Akt signaling pathway. More notably, we found that the PRNP gene coding for the cellular prion protein (PrPc), was 17-fold overexpressed in the 1001 cell line as compared with the MCF7 cell line. This differential expression was confirmed at the cell surface by immunostaining that indicated that PrPc is overexpressed at both mRNA and protein levels in the TNF-resistant derivative. Using recombinant adenoviruses expressing the human PrPc, our data demonstrate that PrPc overexpression converted TNF-sensitive MCF7 cells into TNF-resistant cells, at least in part, by a mechanism involving alteration of cytochrome c release from mitochondria and nuclear condensation.

The increasing use of DNA microarrays in biomedical research, toxicogenomics, pharmaceutical development, and diagnostics has focused attention on the reproducibility and reliability of microarray measurements. While the reproducibility of microarray gene expression measurements has been the subject of several recent reports, there is still a need for systematic investigation into what factors most contribute to variability of measured expression levels observed among different laboratories and different experimenters., SCOPUS: ar.j, info:eu-repo/semantics/published

Myeloid Metaplasia with Myelofibrosis (MMM) is a myeloproliferative disorder (MPD) associating ineffective and extramedullary hematopoiesis with progressive splenomegaly, bone marrow fibrosis and neoangiogenesis. The myeloproliferation is characterized by an increased number of circulating CD34+ cells with the prominent amplification of dystrophic megakaryocytes (Mk). We compared the transcriptome of CD34+ and Mk cell from the peripheral blood (PB) of MMM patients and from the PB and bone-marrow (BM) of unmobilized healthy donors. Application of multivariate analyses (principal component analysis and classification by partitioning around medoids algorithm) on Gene Ontology annotation of differential genes allowed a global functionally approach of the main cellular dysregulated pathways in MMM. Each sample cRNA probe was individually and differentially hybridized to the cRNA reference probe on a Human Oligo-microarray 22K (Agilent) and data were normalized by application of the local LOWESS algorithm. Comparison of the CD34+ cell transcriptome between MMM and healthy donors revealed a global down regulation of 2/3 of the expressed genes in contrast to 1/3 of genes that are up-regulated after data filtration by significance analysis microarray (SAM) algorithm (threshold p