Study of the tumorigenicity of MKN45 gastric carcinoma cells <i>in vivo</i>.

<p>Tumor growth curves. 1×10⁵ cells were subcutaneously injected in mice at day 0. The curves show tumor growth until day 21, the day on which all mice were sacrificed.* P<0.05, when compared to the MKN45-SC control cell line.</p

Quantification of cell proliferation by MTT assay.

<p>Quantification of metabolically active cells by MTT assay in MKN45-C1 and MKN45-C2 clones and MKN45-SC control at 96h in culture. Data from 24 hours was used to set time zero and results were normalized to the data obtained with the scrambled control.<i>*P</i><0.01.</p

Quantification of apoptotic cells by TUNEL assay.

<p>Apoptosis of MKN45-C1 and MKN45-C2 and scramble control (SC) were evaluated at 96h in culture by the TUNEL assay. Results were normalized to the data obtained with the scrambled control. <i>*P</i><0.01.</p

MUC1 downregulation by shRNA.

<p>(<b>A</b>) MUC1 detection by immunofluorescence with HMFG-1 antibody in MKN45-C1 and MKN45-C2 and MKN45-SC control; (<b>B</b>) MUC1 protein detection by western-blot with MUC1-Ab5 antibody of total protein extracts from MKN45-C1 and MKN45-C2 and MKN45-SC control; (<b>C</b>) Quantification of MUC1 RNA in MKN45-C1 and MKN45-C2 and MKN45-SC control by real-time PCR. MUC1 expression was corrected to the house-keeping gene 18S and normalized to the data obtained with the scrambled control. <i>*P</i><0.01.</p

Targeted next generation sequencing identifies functionally deleterious germline mutations in novel genes in early-onset/familial prostate cancer

<div><p>Considering that mutations in known prostate cancer (PrCa) predisposition genes, including those responsible for hereditary breast/ovarian cancer and Lynch syndromes, explain less than 5% of early-onset/familial PrCa, we have sequenced 94 genes associated with cancer predisposition using next generation sequencing (NGS) in a series of 121 PrCa patients. We found monoallelic truncating/functionally deleterious mutations in seven genes, including <i>ATM</i> and <i>CHEK2</i>, which have previously been associated with PrCa predisposition, and five new candidate PrCa associated genes involved in cancer predisposing recessive disorders, namely <i>RAD51C</i>, <i>FANCD2</i>, <i>FANCI</i>, <i>CEP57</i> and <i>RECQL4</i>. Furthermore, using <i>in silico</i> pathogenicity prediction of missense variants among 18 genes associated with breast/ovarian cancer and/or Lynch syndrome, followed by KASP genotyping in 710 healthy controls, we identified “likely pathogenic” missense variants in <i>ATM</i>, <i>BRIP1</i>, <i>CHEK2</i> and <i>TP53</i>. In conclusion, this study has identified putative PrCa predisposing germline mutations in 14.9% of early-onset/familial PrCa patients. Further data will be necessary to confirm the genetic heterogeneity of inherited PrCa predisposition hinted in this study.</p></div

“Likely/Potentially pathogenic” missense variants found in the 121 cases by targeted NGS panel.

<p>“Likely/Potentially pathogenic” missense variants found in the 121 cases by targeted NGS panel.</p

Pedigrees of patients carrying truncating/deleterious mutations in new candidate PrCa risk genes involved in Fanconi anemia.

<p>(A) Patient HPC186 harboring the <i>RAD51C</i> frameshift mutation c.890_899del. (B) Patient HPC447 harboring the <i>FANCD2</i> splicing mutation c.2494+2T>C. (C) Patient HPC150 harboring the <i>FANCI</i> frameshift mutation c.206del. Electropherograms of the Sanger sequencing validations are shown.</p

Pedigrees of patients harboring truncating/deleterious mutations in new candidate PrCa risk genes involved in other recessive disorders.

<p>(A) Patient HPC421 harboring the <i>CEP57</i> nonsense mutation c.791C>G. (B) Patient HPC455 harboring the <i>RECQL4</i> frameshift mutation c.2636del. Electropherograms of the Sanger sequencing validations are shown.</p

Pedigrees of patients carrying truncating/deleterious mutations in the known PrCa risk genes <i>ATM</i> and <i>CHEK2</i>.

<p>(A) Patient HPC177 harboring the <i>ATM</i> stop mutation c.652C>T. (B) Patient HPC395 harboring the <i>CHEK2</i> splicing mutation c.593-1G>T. Electropherograms of the Sanger sequencing validations are shown.</p

Potential Downstream Target Genes of Aberrant ETS Transcription Factors Are Differentially Affected in Ewing’s Sarcoma and Prostate Carcinoma

<div><p><em>FLI1</em> and <em>ERG</em>, the major ETS transcription factors involved in rearrangements in the Ewing’s sarcoma family of tumors (ESFT) and in prostate carcinomas (PCa), respectively, belong to the same subfamily, having 98% sequence identity in the DNA binding domain. We therefore decided to investigate whether the aberrant transcription factors in both malignancies have some common downstream targets. We crossed a publicly available list of all putative EWSR1-FLI1 target genes in ESFT with our microarray expression data on 24 PCa and 6 non-malignant prostate tissues (NPT) and choose four genes among the top-most differentially expressed between PCa with (PCa <em>ERG+</em>) and without (PCa ETS-) ETS fusion genes (<em>HIST1H4L</em>, <em>KCNN2, ECRG4</em> and <em>LDOC1</em>), as well as four well-validated direct targets of the EWSR1-FLI1 chimeric protein in ESFT (<em>NR0B1</em>, <em>CAV1</em>, <em>IGFBP3</em> and <em>TGFBR2</em>). Using quantitative expression analysis in 16 ESFT and seven alveolar rhabdomyosarcomas (ARMS), we were able to validate the four genes previously described as direct targets of the EWSR1-FLI1 oncoprotein, showing overexpression of <em>CAV1</em> and <em>NR0B1</em> and underexpression of <em>IGFBP3</em> and <em>TGFBR2</em> in ESFT as compared to ARMS. Although none of these four genes showed significant expression differences between PCa <em>ERG</em>+ and PCa ETS-, <em>CAV1, IGFBP3</em> and <em>TGFBR2</em> were less expressed in PCa in an independent series of 56 PCa and 15 NPT, as also observed for <em>ECRG4</em> and <em>LDOC1</em>, suggesting a role in prostate carcinogenesis in general. On the other hand, we demonstrate for the first time that both <em>HIST1H4L</em> and <em>KCNN2</em> are significantly overexpressed in PCa <em>ERG+</em> and that ERG binds to the promoter of these genes. Conversely, <em>KCNN2</em> was found underexpressed in ESFT relative to ARMS, suggesting that the EWSR1-ETS oncoprotein may have the opposite effect of ERG rearrangements in PCa. We conclude that aberrant ETS transcription factors modulate target genes differently in ESFT and PCa.</p> </div