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Background & Aims This study examined whether the regulation of resistin and visfatin could reduce damage and improve regeneration in both steatotic and non-steatotic livers undergoing partial hepatectomy under ischemia-reperfusion, a procedure commonly applied in clinical practice to reduce bleeding. Methods Resistin and visfatin were pharmacologically modulated in lean and obese animals undergoing partial hepatectomy under ischemia-reperfusion. Results No evident role for these adipocytokines was observed in non-steatotic livers. However, obese animals undergoing liver surgery showed increased resistin in liver and plasma, without changes in adipose tissue, together with visfatin downregulation in liver and increment in plasma and adipose tissue. Endogenous resistin maintains low levels of visfatin in the liver by blocking its hepatic uptake from the circulation, thus regulating the visfatin detrimental effects on hepatic damage and regenerative failure. Indeed, the administration of anti-resistin antibodies increased hepatic accumulation of adipocyte-derived visfatin, exacerbating damage and regenerative failure. Interestingly, treatment with anti-visfatin antibodies protected steatotic livers, and similar results were obtained with the concomitant inhibition of resistin and visfatin. Thus, when visfatin was inhibited, the injurious effects of anti-resistin antibodies disappeared. Herein we show that upregulation of visfatin increased NAD levels in the remnant steatotic liver, whereas visfatin inhibition decreased them. These later observations suggest that visfatin may favour synthesis of NAD instead of DNA and induces alterations in amino acid metabolism-urea cycle and NO production, overall negatively affecting liver viability. Conclusions Our results indicate the clinical potential of visfatin blocking-based therapies in steatotic livers undergoing partial hepatectomy with ischemia-reperfusion.

Acid ceramidase (AC) catalyzes the hydrolysis of ceramide into sphingosine, in turn a substrate of sphingosine kinases that catalyze its conversion into the mitogenic sphingosine- 1-phosphate. AC is expressed at high levels in several tumor types and has been proposed as a cancer therapeutic target. Using a model derived from PC-3 prostate cancer cells, the highly tumorigenic, metastatic, and chemoresistant clone PC-3/Mc expressed higher levels of the AC ASAH1 than the nonmetastatic clone PC-3/S. Stable knockdown of ASAH1 in PC-3/Mc cells caused an accumulation of ceramides, inhibition of clonogenic potential, increased requirement for growth factors, and inhibition of tumorigenesis and lung metastases. We developed de novo ASAH1 inhibitors, which also caused a dose-dependent accumulation of ceramides in PC-3/Mc cells and inhibited their growth and clonogenicity. Finally, immunohistochemical analysis of primary prostate cancer samples showed that higher levels of ASAH1 were associated with more advanced stages of this neoplasia. These observations confirm ASAH1 as a therapeutic target in advanced and chemoresistant forms of prostate cancer and suggest that our new potent and specific AC inhibitors could act by counteracting critical growth properties of these highly aggressive tumor cells. Copyright © 2013 by the American Society for Biochemistry and Molecular Biology, Inc., This work was supported by Ministry of Science and Innovation Grants SAF2008-00706 and SAF2011-22444 (to G.F.); Agència de Gestió d'Ajuts Universitaris i de Recerca de la Generalitat de Catalunya Grant 2009SGR1072 (to G.F.); Ministry of Science and Innovation Grants SAF2008-04136-C02-01 and SAF2011-24686 (to T.M.T.); Ministry of Economy and Competitivity Grant SAF2012-40017-C02-01 (to T.M.T.); Agència de Gestió d'Ajuts Universitaris i de Recerca de la Generalitat de Catalunya Grant 2009SGR1482 (to T.M.T.); Xarxa de Bancs de Tumours de Catalunya-Pla Director d'Oncologia and Fondo Europeo de Desarrollo Regional (FEDER), Unión Europea “Una manera de hacer Europa” and Ministry of Economy and Competitivity Grant SAF2012-40017-C02-02 (to P.L.F.); Red Nacional de Biobancos (ReTBioH) (to P.L.F. and R.B.); Generalitat de Catalunya Grant 2009SGR1072 (to L.C.), Ministry of Science and Innovation FPU fellowship (to O.M-C.); I3P fellowship (CSIC) (to T.C-T.); and Generalitat de Catalunya FI fellowship (to F.C.)

MicroRNAs are critical regulators of gene networks in normal and abnormal biological processes. Focusing on invasive ductal breast cancer (IDC), we have found dysregulated expression in tumor samples of several microRNAs, including the miR200 family, along progression from primary tumors to distant metastases, further reflected in higher blood levels of miR-200b and miR-7 in IDC patients with regional or distant metastases relative to patients with primary node-negative tumors. Forced expression of miR-200s in MCF10CA1h mammary cells induced an enhanced epithelial program, aldehyde dehydrogenase (ALDH) activity, mammosphere growth and ability to form branched tubuloalveolar structures while promoting orthotopic tumor growth and lung colonization in vivo. MiR-200s also induced the constitutive activation of the PI3K-Akt signaling through downregulation of PTEN, and the enhanced mammosphere growth and ALDH activity induced in MCF10CA1h cells by miR-200s required the activation of this signaling pathway. Interestingly, the morphology of tumors formed in vivo by cells expressing miR-200s was reminiscent of metaplastic breast cancer (MBC). Indeed, the epithelial components of MBC samples expressed significantly higher levels of miR-200s than their mesenchymal components and displayed a marker profile compatible with luminal progenitor cells. We propose that microRNAs of the miR-200 family promote traits of highly proliferative breast luminal progenitor cells, thereby exacerbating the growth and metastatic properties of transformed mammary epithelial cells, This work was supported by grants to MICINN (FIS-PI080274), MINECO (SAF2012-40017-C02-02) and RTICC (RD12/0036/0036) to PLF; MICINN (SAF2011-24686), MINECO (SAF2012-40017-C02-01 and SAF2015-66984-C2-1-R), Catalan Agència d’Ajuts Universitaris i de Recerca (AGAUR; 2009SGR1482), and Xarxa de Referència en Biotecnologia to TMT; CIBERBBN and Fundación ECO to TMT, MM, PLF and JB; and RTICC (RD12/0037/0016) and AGAUR (2014SGR660) to MFS. The Hospital Clínic-IDIBAPS Biobank is part of the Xarxa de Bancs de Tumors de Catalunya (XBTC), financed by the Pla Director d’Oncologia de Catalunya, and the Red Nacional de Biobancos (RNBB, ReTBioH), funded by the ISCIII (RETICS RD09-0076/0038).

The main challenge for clinical management of prostate cancer is to distinguish tumors that will progress faster and will show a higher tendency to recur from the more indolent ones. We have compared expression profiles of 18 prostate cancer samples (seven with a Gleason score of 6, eight with a Gleason score of 7, and three with a Gleason score of ≥8) and five nonneoplastic prostate samples, using the Affymetrix Human Array GeneChip Exon 1.0 ST. Microarray analysis revealed 99 genes showing statistically significant differences among tumors with Gleason scores of 6, 7, and ≥8. In addition, mRNA expression of 29 selected genes was analyzed by real-time quantitative RT-PCR with microfluidic cards in an extended series of 30 prostate tumors. Of the 29 genes, 18 (62%) were independently confirmed in the extended series by quantitative RT-PCR: 14 were up-regulated and 4 were down-regulated in tumors with a higher Gleason score. Twelve of these genes were differentially expressed in tumors with a Gleason score of 6 to 7 versus ≥8. Finally, IHC validation of the protein levels of two genes from the 12-gene signature (SEC14L1 and TCEB1) showed strong protein expression levels of both genes, which were statistically associated with a high combined Gleason score, advanced stage, and prostate-specific antigen progression. This set of genes may contribute to a better understanding of the molecular basis of prostate cancer. TCEB1 and SELC14L1 are good candidate markers for predicting prognosis and progression of prostate cancer.

et al., Malignant progression in cancer requires populations of tumor-initiating cells (TICs) endowed with unlimited self renewal, survival under stress, and establishment of distant metastases. Additionally, the acquisition of invasive properties driven by epithelial-mesenchymal transition (EMT) is critical for the evolution of neoplastic cells into fully metastatic populations. Here, we characterize 2 human cellular models derived from prostate and bladder cancer cell lines to better understand the relationship between TIC and EMT programs in local invasiveness and distant metastasis. The model tumor subpopulations that expressed a strong epithelial gene program were enriched in highly metastatic TICs, while a second subpopulation with stable mesenchymal traits was impoverished in TICs. Constitutive overexpression of the transcription factor Snai1 in the epithelial/ TIC-enriched populations engaged a mesenchymal gene program and suppressed their self renewal and metastatic phenotypes. Conversely, knockdown of EMT factors in the mesenchymal-like prostate cancer cell subpopulation caused a gain in epithelial features and properties of TICs. Both tumor cell subpopulations cooperated so that the nonmetastatic mesenchymal-like prostate cancer subpopulation enhanced the in vitro invasiveness of the metastatic epithelial subpopulation and, in vivo, promoted the escape of the latter from primary implantation sites and accelerated their metastatic colonization. Our models provide new insights into how dynamic interactions among epithelial, self-renewal, and mesenchymal gene programs determine the plasticity of epithelial TICs., T. Celià-Terrassa is a recipient of a doctoral fellowship from the CSIC. Ó. Meca-Cortés, A. Arnal-Estapé, and C. Estarás are recipients of doctoral fellowships and F. Mateo of a Juan de la Cierva postdoctoral fellowship from the Ministerio de Ciencia e Innovación (MICINN). R.R. Gomis is a researcher of the Institució Catalana de Recerca i Estudis Avançats. Financial support was provided to T.M. Thomson by MICINN (SAF2008-04136-C02-01 and SAF2011-24686), the Agència de Gestió d’Ajuts Universitaris i de Recerca (2009SGR1482), the Agencia Española de Cooperación Internacional y Desarrollo (A/023859/09), and the Xarxa de Referencia en Biotecnologia; to P.L. Fernández by MICINN (FIS-PI080274), the Fondo de Investigaciones de la Seguridad Social (PI080274), the Spanish National Biobank Network, the Instituto de Salud Carlos III (ISCIII-RETIC RD06/0020), the Xarxa de Bancs de Tumours de Catalunya-Pla Director d’Oncologia, and the Fondo Europeo de Desarrollo Regional (FEDER) — Unión Europea “Una manera de hacer Europa”; and to R.R. Gomis by the BBVA Foundation and MICINN (SAF2007-62691).

Despite its high incidence as the second most common tumor in males worldwide, primary prostate cancer has been associated with few recurrent chromosomal gains and deletions that are consistent across various studies. Few studies have explored how chromosomal alterations are coupled to abnormal gene expression. Here, we review the major genomic aberrations associated with prostate cancer and describe how detailed transcriptional and computational analyses allowed us to discover a recurrent chromosomal gain in a small region on chromosome 17. Fluorescent in situ hybridization confirmed the presence of a copy number gain in 17q25.3 in tumor-associated preneoplastic lesions of the prostate, 65% of primary tumors, and metastatic samples. These results suggest the involvement of this gain at all steps of prostate cancer progression.

Francesca Mateo et al., [Background] Tumor cell subpopulations can either compete with each other for nutrients and physical space within the tumor niche, or co-operate for enhanced survival, or replicative or metastatic capacities. Recently, we have described co-operative interactions between two clonal subpopulations derived from the PC-3 prostate cancer cell line, in which the invasiveness of a cancer stem cell (CSC)-enriched subpopulation (PC-3M, or M) is enhanced by a non-CSC subpopulation (PC-3S, or S), resulting in their accelerated metastatic dissemination. Methods: M and S secretomes were compared by SILAC (Stable Isotope Labeling by Aminoacids in Cell Culture). Invasive potential in vitro of M cells was analyzed by Transwell-Matrigel assays. M cells were co-injected with S cells in the dorsal prostate of immunodeficient mice and monitored by bioluminescence for tumor growth and metastatic dissemination. SPARC levels were determined by immunohistochemistry and real-time RT-PCR in tumors and by ELISA in plasma from patients with metastatic or non-metastatic prostate cancer., [Results] Comparative secretome analysis yielded 213 proteins differentially secreted between M and S cells. Of these, the protein most abundantly secreted in S relative to M cells was SPARC. Immunodepletion of SPARC inhibited the enhanced invasiveness of M induced by S conditioned medium. Knock down of SPARC in S cells abrogated the capacity of its conditioned medium to enhance the in vitro invasiveness of M cells and compromised their potential to boost the metastatic behavior of M cells in vivo. In most primary human prostate cancer samples, SPARC was expressed in the epithelial tumoral compartment of metastatic cases., [Conclusions] The matricellular protein SPARC, secreted by a prostate cancer clonal tumor cell subpopulation displaying non-CSC properties, is a critical mediator of paracrine effects exerted on a distinct tumor cell subpopulation enriched in CSC. This paracrine interaction results in an enhanced metastatic behavior of the CSC-enriched tumor subpopulation. SPARC is expressed in the neoplastic cells of primary prostate cancer samples from metastatic cases, and could thus constitute a tumor progression biomarker and a therapeutic target in advanced prostate cancer. © 2014 Mateo et al.; licensee BioMed Central Ltd., FM is a Juan de la Cierva Investigator, OM is a FPU Fellow from the Spanish Ministries of Science and Innovation (MICINN) and Economy and Competitiveness (MINECO) and LS is the recipient of a IDIBAPS fellowship. This work was supported by grants to TMT from MICINN (SAF2011-24686) and MINECO (SAF2012-40017-C02-01), Catalan Agència d’Ajuts Universitaris i de Recerca (AGAUR; 2009SGR1482), and Xarxa de Referència en Biotecnologia; to RP from Instituto de Salud Carlos III (ISCIII; RETICS RD06-0020/0058) and MICINN (SAF2011-30496); to IA from MINECO (PI11/079 and IPT-090000-2010-001; the latter cofinanced by the European Regional Development Fund) and AGAUR (SGR157); and to PLF from MICINN (FIS-PI080274) and MINECO (SAF2012-40017-C02-02). The Hospital Clínic-IDIBAPS Biobank is part of the Xarxa de Bancs de Tumors de Catalunya (XBTC), financed by the Pla Director d’Oncologia de Catalunya, and the Red Nacional de Biobancos (RNBB, ReTBioH), financed by the ISCIII (RETICS RD09-0076/0038), We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

Molecular characterization of radical prostatectomy specimens after systemic therapy may identify a gene expression profile for resistance to therapy. This study assessed tumor cells from patients with prostate cancer participating in a phase II neoadjuvant docetaxel and androgen deprivation trial to identify mediators of resistance. Transcriptional level of 93 genes from a docetaxel-resistant prostate cancer cell lines microarray study was analyzed by TaqMan low-density arrays in tumors from patients with high-risk localized prostate cancer (36 surgically treated, 28 with neoadjuvant docetaxel + androgen deprivation). Gene expression was compared between groups and correlated with clinical outcome. VIM, AR and RELA were validated by immunohistochemistry. CD44 and ZEB1 expression was tested by immunofluorescence in cells and tumor samples. Parental and docetaxel-resistant castration-resistant prostate cancer cell lines were tested for epithelial-to-mesenchymal transition (EMT) markers before and after docetaxel exposure. Reversion of EMT phenotype was investigated as a docetaxel resistance reversion strategy. Expression of 63 (67.7%) genes differed between groups (P < 0.05), including genes related to androgen receptor, NF-κB transcription factor, and EMT. Increased expression of EMT markers correlated with radiologic relapse. Docetaxel-resistant cells had increased EMT and stem-like cell markers expression. ZEB1 siRNA transfection reverted docetaxel resistance and reduced CD44 expression in DU-145R and PC-3R. Before docetaxel exposure, a selected CD44+ subpopulation of PC-3 cells exhibited EMT phenotype and intrinsic docetaxel resistance; ZEB1/CD44+ subpopulations were found in tumor cell lines and primary tumors; this correlated with aggressive clinical behavior. This study identifies genes potentially related to chemotherapy resistance and supports evidence of the EMT role in docetaxel resistance and adverse clinical behavior in early prostate cancer. Mol Cancer Ther; 13(5); 1270–84. ©2014 AACR.

Oncogene advance online publication.-- et al., Prostate tumor overexpressed-1 (PTOV1), a modulator of the Mediator transcriptional regulatory complex, is expressed at high levels in prostate cancer and other neoplasias in association with a more aggressive disease. Here we show that PTOV1 interacts directly with receptor of activated protein C kinase 1 (RACK1), a regulator of protein kinase C and Jun signaling and also a component of the 40S ribosome. Consistent with this interaction, PTOV1 was associated with ribosomes and its overexpression promoted global protein synthesis in prostate cancer cells and COS-7 fibroblasts in a mTORC1-dependent manner. Transfection of ectopic PTOV1 enhanced the expression of c-Jun protein without affecting the levels of c-Jun or RACK1 mRNA. Conversely, knockdown of PTOV1 caused significant declines in global protein synthesis and c-Jun protein levels. High levels of PTOV1 stimulated the motility and invasiveness of prostate cancer cells, which required c-Jun, whereas knockdown of PTOV1 strongly inhibited the tumorigenic and metastatic potentials of PC-3 prostate cancer cells. In human prostate cancer samples, the expression of high levels of PTOV1 in primary and metastatic tumors was significantly associated with increased nuclear localization of active c-Jun. These results unveil new functions of PTOV1 in the regulation of protein translation and in the progression of prostate cancer to an invasive and metastatic disease., This study was supported by Ministry of Science and Innovation and MINECO SAF2008- 03936, SAF2011-30496, TV3 Telemarathon, and Instituto Carlos III RD06/0020/0058 RETICS (to RP); SAF2008-04136-C02-01 and SAF2011-24686 (to TMT); AGAUR 2009SGR1482 (to RP and TMT); Red Nacional de Biobancos, Instituto Carlos III (to RB); and Fondo de Investigaciones de la Seguridad Social PI20231 (to PLF).

Steatotic livers show increased hepatic damage and impaired regeneration after partial hepatectomy (PH) under ischemia/reperfusion (I/R), which is commonly applied in clinical practice to reduce bleeding. The known function of retinol-binding protein 4 (RBP4) is to transport retinol in the circulation. We examined whether modulating RBP4 and/or retinol could protect steatotic and nonsteatotic livers in the setting of PH under I/R. Steatotic and nonsteatotic livers from Zucker rats were subjected to PH (70%) with 60 minutes of ischemia. RBP4 and retinol levels were measured and altered pharmacologically, and their effects on hepatic damage and regeneration were studied after reperfusion. Decreased RBP4 levels were observed in both liver types, whereas retinol levels were reduced only in steatotic livers. RBP4 administration exacerbated the negative consequences of liver surgery with respect to damage and liver regeneration in both liver types. RBP4 affected the mobilization of retinol from steatotic livers, and this revealed actions of RBP4 independent of simple retinol transport. The injurious effects of RBP4 were not due to changes in retinol levels. Treatment with retinol was effective only for steatotic livers. Indeed, retinol increased hepatic injury and impaired liver regeneration in nonsteatotic livers. In steatotic livers, retinol reduced damage and improved regeneration after surgery. These benefits of retinol were associated with a reduced accumulation of hepatocellular fat. Thus, strategies based on modulating RBP4 could be ineffective and possibly even harmful in both liver types in the setting of PH under I/R. In terms of clinical applications, a retinol pretreatment might open new avenues for liver surgery that specifically benefit the steatotic liver. Liver Transpl 18:1198-1208, 2012.

Docetaxel-based chemotherapy is the standard first-line therapy in metastatic castration-resistant prostate cancer (CRPC). However, most patients eventually develop resistance to this treatment. In this study, we aimed to identify key molecular genes and networks associated with docetaxel resistance in two models of docetaxel-resistant CRPC cell lines and to test for the most differentially expressed genes in tumor samples from patients with CRPC. DU-145 and PC-3 cells were converted to docetaxel-resistant cells, DU-145R and PC-3R, respectively. Whole-genome arrays were used to compare global gene expression between these four cell lines. Results showed differential expression of 243 genes (P < 0.05, Bonferroni-adjusted P values and log ratio > 1.2) that were common to DU-145R and PC-3R cells. These genes were involved in cell processes like growth, development, death, proliferation, movement, and gene expression. Genes and networks commonly deregulated in both DU-145R and PC-3R cells were studied by Ingenuity Pathways Analysis. Exposing parental cells to TGFB1 increased their survival in the presence of docetaxel, suggesting a role of the TGF-β superfamily in conferring drug resistance. Changes in expression of 18 selected genes were validated by real-time quantitative reverse transcriptase PCR in all four cell lines and tested in a set of 11 FFPE and five optimal cutting temperature tumor samples. Analysis in patients showed a noteworthy downexpression of CDH1 and IFIH1, among others, in docetaxel-resistant tumors. This exploratory analysis provides information about potential gene and network involvement in docetaxel resistance in CRPC. Further clinical validation of these results is needed to develop targeted therapies in patients with CRPC that can circumvent such resistance to treatment. Mol Cancer Ther; 11(2); 329–39. ©2011 AACR.

The introduction of high-throughput techniques is increasingly providing abundant information on molecular alterations requiring validation at the posttranscriptional level. Protein expression is now efficiently evaluated in large series of tumors included in tissue microarrays. We propose, describe and validate a technique to elaborate paraffin-embedded cell line microarrays (PECLIMA) from fixed cell cultures, which can be processed like standard surgical pathology biopsies prior to immunophenotyping. Our results show a reliable protein immunoexpression profiling in six widely used cell lines under different fixation conditions. This technique permits the simultaneous analysis of multiple antigens in multiple cell lines under different experimental conditions. Additional features of these arrays are long-term storage, their suitability for a variety of techniques including immunocytochemistry and in situ hybridization and their low cost.

Background Transcriptional profiling of prostate cancer (PC) has unveiled new markers of neoplasia and allowed insights into mechanisms underlying this disease. Genomewide analyses have also identified new chromosomal abnormalities associated with PC. The combination of both classes of data for the same sample cohort might provide better criteria for identifying relevant factors involved in neoplasia. Here we describe transcriptional signatures identifying distinct normal and tumoral prostate tissue compartments, and the inference and demonstration of a new, highly recurrent copy number gain on chromosome 17q25.3. Methods We have applied transcriptional profiling to tumoral and non-tumoral prostate samples with relatively homogeneous epithelial representations as well as pure stromal tissue from peripheral prostate and cultured cell lines, followed by quantitative RT-PCR validations and immunohistochemical analysis. In addition, we have performed in silico colocalization analysis of co-regulated genes and validation by fluorescent in situ hybridization (FISH). Results The transcriptomic analysis has allowed us to identify signatures corresponding to non-tumoral luminal and tumoral epithelium, basal epithelial cells, and prostate stromal tissue. In addition, in silico analysis of co-regulated expression of physically linked genes has allowed us to predict the occurrence of a copy number gain at chromosomal region 17q25.3. This computational inference was validated by fluorescent in situ hybridization, which showed gains in this region in over 65% of primary and metastatic tumoral samples. Conclusion Our approach permits to directly link gene copy number variations with transcript co-regulation in association with neoplastic states. Therefore, transcriptomic studies of carefully selected samples can unveil new diagnostic markers and transcriptional signatures highly specific of PC, and lead to the discovery of novel genomic abnormalities that may provide additional insights into the causes and mechanisms of prostate cancer.