Description of statistically genes significantly (p<0.005) regulated by FoxE1.

<p>Description of statistically genes significantly (p<0.005) regulated by FoxE1.</p

Experimental validation of microarray results by qRT-PCR and western blotting.

<p>Relative expression assessed by means of qRT-PCR of 6 thyroid-specific genes (panel A) and 12 additional genes differentially down- and upregulated in FoxE1-silenced PCCl3 cells (panel B). As FoxE1-dependent positive controls, we evaluated Tpo and Tg mRNA expression levels. Relative gene expression in siFoxE1 samples was calculated using the corresponding siScrambl samples as a reference ( = 1). Results are mean ± SEM of four independent experiments. Total protein extracts were prepared and submitted to western blot analysis to assess the protein levels of FoxE1 and Nis (panel C), and of Cdh1 and Duox2 (panel D). Actin was used as loading control. Representative western blot assays of four independent experiments are shown.</p

Experimental design and FoxE1 protein levels after 48 hours of silencing.

<p><i>Panel A:</i> Experimental design of expression array experiments. Two comparisons were done: FoxE1-silenced PCCl3 cells (siFoxE1 PCCl3) <i>vs.</i> scrambled siRNA-treated PCCl3 cells (siScramble PCCl3), and FoxE1-silenced PCCl3 cells <i>vs.</i> wild type PCCL3 cells (wt PCCl3). Each comparison was performed in quadruplicate and using dye swaps. <i>Panel B:</i> Western Blot of extracts from control, siScramble and siFoxE1-treated cells from quadruplicate samples used for microarray analysis. Hybridizations were done with anti-FoxE1 antibodies; anti-tubulin antibodies were used as loading controls.</p

FoxE1 and NF1/CTF binding to and transcriptional activation of the NUE.

<p>A ReChIP assay was used to analyse simultaneous binding of FoxE1 and NF1/CTF proteins to the NUE (panel A). qPCR was done to analyse chromatin immunoprecipitates of PCCl3 cells using FoxE1 and NF1 antibodies. The enrichment of target sequences was calculated as the IP ratio (arbitrary units) relative to the negative control Afm, and normalized to their relative amplification in the input sample. A sequence from the Tg promoter was used as positive control. HeLa cells were transfected with 1 µg of a FoxE1 or NF1/CTF expression vector or the empty vector, and 1.5 µg and 0.1 µg of pNIS 2.8-Luc and CMV-<i>Renilla</i> constructs respectively (panel B). Forty-eight hours after transfection, cells were collected for the measurement of luciferase and <i>renilla</i> levels. Results are shown as the mean±SD of the luciferase levels relative to the non-regulated <i>renilla</i> levels of six independent experiments. (*): p<0.05; (**): p<0.01; (***): p<0.001; two tailed t-test.</p

Summary of FoxE1 microarray results in PCCl3 cells.

<p>Number of statistically significant probes and genes (p<0.005) after Foxe1 silencing; results are shown for each type of comparison performed, as well as for the combined analysis.</p

Putative FoxE1-NF1/CTF binding sites in the <i>Duox2</i> gene and in the NUE.

<p>Chromosomal location of putative FoxE1 and NF1/CTF binding motifs in the Nis upstream enhancer (panel A) and the <i>Duox2</i> gene (panel B). Oligos used and exons are represented in italics.</p

ChIP experiments for FoxE1 binding to selected genes.

<p>qPCR analysis of chromatin immunoprecipitation performed on PCCl3 cells with FoxE1 antibody. The enrichment of target sequences was calculated as the IP ratio (arbitrary units) relative to the negative control Afm, and normalized to their relative amplification in the input sample. Sequences from the Tg and Tpo promoters were used as positive controls. Two regulatory regions were analysed in the <i>Duox2</i> promoter (called Duox2-1 and Duox2-2). Results are mean ± SEM of two independent experiments, each performed in triplicate.</p

Mutation validation.

<p>Capillary sequencing chromatograms confirming the seven putative variants. <i>KRAS</i>, <i>SMARCA2</i>, <i>PRKD3</i>, <i>STAT6</i>, <i>LIFR</i> and <i>ILB1</i> showed one point mutation each, depicted as two peaks in the tumoral DNA chromatogram. For <i>NFKBIE</i>, the 4-bp deletion was also confirmed. NT = non-tumoral, TM = tumoral. R = A or G; Y = C or T; S = C or G; K = T or G; /…./ = 4-bp deletion.</p

Analysis of Paired Primary-Metastatic Hormone-Receptor Positive Breast Tumors (HRPBC) Uncovers Potential Novel Drivers of Hormonal Resistance

<div><p>We sought to identify genetic variants associated with disease relapse and failure to hormonal treatment in hormone-receptor positive breast cancer (HRPBC). We analyzed a series of HRPBC with distant relapse, by sequencing pairs (n = 11) of tumors (primary and metastases) at >800X. Comparative genomic hybridization was performed as well. Top hits, based on the frequency of alteration and severity of the changes, were tested in the TCGA series. Genes determining the most parsimonious prognostic signature were studied for their functional role <i>in vitro</i>, by performing cell growth assays in hormonal-deprivation conditions, a setting that mimics treatment with aromatase inhibitors. Severe alterations were recurrently found in 18 genes in the pairs. However, only <i>MYC</i>, <i>DNAH5</i>, <i>CSFR1</i>, <i>EPHA7</i>, <i>ARID1B</i>, and <i>KMT2C</i> preserved an independent prognosis impact and/or showed a significantly different incidence of alterations between relapsed and non-relapsed cases in the TCGA series. The signature composed of <i>MYC</i>, <i>KMT2C</i>, and <i>EPHA7</i> best discriminated the clinical course, (overall survival 90,7 vs. 144,5 months; p = 0.0001). Having an alteration in any of the genes of the signature implied a hazard ratio of death of 3.25 (p<0.0001), and early relapse during the adjuvant hormonal treatment. The presence of the D348N mutation in <i>KMT2C</i> and/or the T666I mutation in the kinase domain of <i>EPHA7</i> conferred hormonal resistance <i>in vitro</i>. Novel inactivating mutations in <i>KMT2C</i> and <i>EPHA7</i>, which confer hormonal resistance, are linked to adverse clinical course in HRPBC.</p></div

Clinical features, evolution and biological characteristics of the ten CLL patients studied.

<p>Summary of the prognosis factors considered in the clinic and genetic lesions that the ten patients harbor. ND = not done, del = deletion.</p