Integrative multi-omics analysis identifies a prognostic miRNA signature and a targetable miR-21-3p/TSC2/ mTOR axis in metastatic pheochromocytoma/ paraganglioma

Rationale: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors that present variable outcomes. To date, no effective therapies or reliable prognostic markers are available for patients who develop metastatic PPGL (mPPGL). Our aim was to discover robust prognostic markers validated through in vitro models, and define specific therapeutic options according to tumor genomic features. Methods: We analyzed three PPGL miRNome datasets (n=443), validated candidate markers and assessed them in serum samples (n=36) to find a metastatic miRNA signature. An integrative study of miRNome, transcriptome and proteome was performed to find miRNA targets, which were further characterized in vitro. Results: A signature of six miRNAs (miR-21-3p, miR-183-5p, miR-182-5p, miR-96-5p, miR-551b-3p, and miR-202-5p) was associated with metastatic risk and time to progression. A higher expression of five of these miRNAs was also detected in PPGL patients’ liquid biopsies compared with controls. The combined expression of miR-21-3p/miR-183-5p showed the best power to predict metastasis (AUC=0.804, P=4.67·10-18), and was found associated in vitro with pro-metastatic features, such as neuroendocrine-mesenchymal transition phenotype, and increased cell migration rate. A pan-cancer multi-omic integrative study correlated miR-21-3p levels with TSC2 expression, mTOR pathway activation, and a predictive signature for mTOR inhibitor-sensitivity in PPGLs and other cancers. Likewise, we demonstrated in vitro a TSC2 repression and an enhanced rapamycin sensitivity upon miR-21-3p expression. Conclusions: Our findings support the assessment of miR-21-3p/miR-183-5p, in tumors and liquid biopsies, as biomarkers for risk stratification to improve the PPGL patients’ management. We propose miR-21-3p to select mPPGL patients who may benefit from mTOR inhibitors

Cytochrome P450 3A5 is highly expressed in normal prostate cells but absent in prostate cancer.

Testosterone is essential for the growth and function of the luminal prostate cells, but it is also critical for the development of prostate cancer, which in the majority of the cases derives from luminal cells. Cytochrome P450 3A (CYP3A) enzymes hydroxylate testosterone and dehydroepiandrosterone to less active metabolites, which might be the basis for the association between CYP3A polymorphisms and prostate cancer. However, it is unknown whether the CYP3A enzymes are expressed at relevant levels in the prostate and which polymorphisms could affect this tissue-specific CYP3A activity. Thus, we measured CYP3A4, CYP3A5, CYP3A7, and CYP3A43 mRNA in 14 benign prostatic hyperplasias and ten matched non-tumoral/tumoral prostate samples. We found that CYP3A5 mRNA in non-tumoral prostate tissue was 10% of the average amount of liver samples, whereas the expression of the other CYP3A genes was much lower. Similarly to liver, CYP3A5*3 polymorphism decreased CYP3A5 mRNA content 13-fold. CYP3A5 protein was detected in non-tumoral prostate microsomes by western blot, and immunohistochemistry (IHC) localized CYP3A5 exclusively in the basolateral prostate cells. In contrast to the normal tissue, IHC and RT-PCR showed that tumoral tissue lacked CYP3A5 expression. In conclusion, prostate basolateral cells express high levels of CYP3A5 which dramatically decrease in tumoral tissue. This finding supports an endogenous function of CYP3A5 related to the metabolism of intra-prostatic androgens and cell growth, and that polymorphisms affecting CYP3A5 activity may result in altered prostate cancer risk and aggressiveness.The authors thank Lydia Sanchez and the CNIO Immunohistochemical Unit for expert technical assist-ance. Tissue samples were provided by the TissueBank Network coordinated by the Molecular Path-ology Program of the Spanish National Cancer Centre (CNIO), with the collaboration of the Tumor Bank of the Department of Pathology, Universitary Hospital Virgen de las Nieves from Granada, Spain. This study was supported by Cristina Rodrı´guez-Antona’s Marie Curie Reintegration Fellowship of the European Community contract number MERG-CG-6-2005-014881, project SAF2006-01139 and the ’Ramon y Cajal’ program both from the Spanish Ministry ofEducation and Science. Susanna Leskela¨ has a fellow-ship from the Spanish Ministry of Education and Science AP2005-4514. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.S

Fine mapping of porcine chromosome 6 QTL and LEPR effects on body composition in multiple generations of an Iberian by Landrace intercross

The leptin receptor gene (LEPR) is a candidate for traits related to growth and body composition, and is located on SSC6 in a region where fatness and meat composition quantitative trait loci (QTL) have previously been detected in several F2 experimental designs. The aims of this work were (i) to fine map these QTL on a larger sample of animals and generations (F3 and backcross) of an Iberian x Landrace intercross and (ii) to examine the effects of LEPR alleles on body composition traits. Eleven single nucleotide polymorphisms (SNPs) were detected by sequencing LEPR coding regions in Iberian and Landrace pig samples. Three missense polymorphisms were genotyped by pyrosequencing in 33 F0, 70 F1, 418 F2, 86 F3 and 128 individuals coming from the backcross of four F2 males with 24 Landrace females. Thirteen microsatellites and one SNP were also genotyped. Traits analysed were backfat thickness at different locations (BFT), intramuscular fat percentage (IMFP), eye muscle area (EMA), loin depth (LOD), weight of shoulder (SHW), weight of ribs (RIBW) and weight of belly bacon (BBW). Different statistical models were applied in order to evaluate the number and effects of QTL on chromosome 6 and the possible causality of the LEPR gene variants with respect to the QTL. The results support the presence of two QTL on SSC6. One, at position 60-100 cM, affects BFT and RIBW. The other and more significant maps in a narrow region (130-132 cM) and affects BFT, IMFP, EMA, LOD, SHW, RIBW and BBW. Results also support the association between LEPR alleles and BFT traits. The possible functional implications of the analysed polymorphisms are considered. © 2005 Cambridge University Press

Comparing k-independent and right censored samples based on the likelihood ratio

Likelihood ratio, k-Sample tests, Weighted tests, Censored data,

Integrative analysis of miRNA and mRNA expression profiles in pheochromocytoma and paraganglioma identifies genotype-specific markers and potentially regulated pathways.

Pheochromocytoma (PCC) and paraganglioma (PGL) are rare neuroendocrine neoplasias of neural crest origin that can be part of several inherited syndromes. Although their mRNA profiles are known to depend on genetic background, a number of questions related to tumor biology and clinical behavior remain unanswered. Since microRNAs are key players in the modulation of gene expression, their comprehensive analysis could resolve some of these issues. Through characterization of microRNA profiles in 69 frozen tumors with germline mutations in the genes SDHD, SDHB, VHL, RET, NF1, TMEM127, and MAX, we identified microRNA signatures specific to, as well as common among, the genetic groups of PCC/PGLs. MicroRNA expression profiles were validated in an independent series of 30 composed of VHL-, SDHB-, SDHD- and RET-related formalin-fixed paraffin-embedded PCC/PGL samples using qRT-PCR. Up-regulation of miR-210 in VHL- and SDHB-related PCC/PGL, while miR-137 and miR-382 were confirmed as generally up-regulated in PCC/PGL (except in MAX-related tumors). Also, we confirmed over-expression of miR-133b as VHL-specific, miR-488 and miR-885-5p as RET-specific, and miR-183 and miR-96 as SDHB-specific microRNAs.To determine the potential roles microRNAs play in PCC/PGL pathogenesis, we performed bioinformatic integration and pathway analysis using matched mRNA profiling data that indicated a common enrichment of pathways associated with neuronal and neuroendocrine-like differentiation. We demonstrated that miR-183 and/or miR-96 impede NGF-induced differentiation in PC12 cells. Finally, global proteomic analysis in SDHB and MAX-tumors allowed us to determine that microRNA regulation occurs primarily through mRNA degradation in PCC/PGL, which partially confirmed our miRNA-mRNA integration results