Utilitat de la Immunohistoquímica i els Tissue Micro Arrays en el coneixement de les alteracions moleculars del càncer d'endometri

Generalment el Càncer d’Endometri (EC) s’associa a bon pronòstic, no obstant el 20% dels EC associats a bon pronòstic recidiven. El propòsit d’aquesta tesi ha estat l’estudi de les principals diferències en l’expressió de gens implicats en el desenvolupament i progressió del CE i que estan relacionats amb la hipòxia, l’apoptosi i l’adaptació a la radioteràpia mitjançant la Immunohistoquímica i els Arrays matricials de teixit en CE primaris i recidives post radioteràpia. Les recidives presenten valors més elevats de p53, HIF-1α, FLIP, Ki67, p50, c-REL, Rel B i β-catenina. La hipòxia activa tant la via NF-κB canònica com l’alternativa i tant la cinasa IKKα com IKKβ són necessàries per l’acumulació de RelA (p65) i p100, mentre que el processament de p52 depèn de IKKα. La hipòxia també indueix l’expressió nuclear de HIF-1α, FLIP, β-catenina i l’augment de l’activitat de via NF-κB, en canvi això no s’observa quan la línia cel•lular Ishikawa es sotmet a radiació. Per la seva banda, ANXA2 juga un paper en promoure el procés de metàstasi en el EC i és un marcador predictiu de recidiva també en els EC de risc baix-intermedi. La classificació histològica del EC pot ser difícil en alguns carcinomes d’alt grau o mixtes. Per aquest motiu també hem determinat una firma de 9 biomarcadors que ajuda a predir el tipus histològic en el EC.Generalmente el Cáncer de Endometrio (EC) se asocia a buen pronóstico, sin embargo el 20% de los EC asociados a buen pronóstico recidivan. El propósito esta tesis doctoral ha sido el estudio de las principales diferencias en la expresión de genes implicados en el EC y que están relacionados con la hipoxia, la apoptosis i la adaptación a la radioterapia mediante la Inmunohistoquímica y los Arrays matriciales de tejido en EC primarios y recidivas post radioterapia. Las recidivas presentan valores más elevados de p53, HIF-1α, FLIP, ki 67, p50, c-REL, Rel B y β-catenina. La hipoxia activa tanto la vía NF-κB canónica como la alternativa y tanto la cinasa IKKα como IKKβ son necesarias para la acumulación de RelA (p65) y p100, mientras que el procesamiento de p52 depende de IKKα. Además, la hipoxia induce la expresión nuclear de HIF-1α, FLIP, β-catenina y el aumento de la actividad de NF-κB aunque dichos cambios no se observan cuando la línea celular Ishikawa se somete a radiación. Por otro lado ANXA2 juega un papel en la promoción del proceso de metástasis en el EC y es un marcador predictivo de recidiva incluso en los EC de riesgo bajo-intermedio. La clasificación histológica del EC puede ser difícil en algunos carcinomas de alto grado o mixtos. Por ese motivo también hemos querido determinar una firma de biomarcadores que ayude a predecir el tipo histológico en el EC.Endometrial Cancer (EC) is usually associated with good prognosis. However, 20% of the EC associated with good prognosis have recurrent disease. The aim of this work was the study of the expression of relevant genes involved in development, progression and recurrence of EC and those involved in resistance to apoptosis, hypoxia and adaptation to radiation, using Immunohistochemistry and Tissue Micro Arrays in primary EC and post radiation recurrences. Post radiation recurrences showed higher p53, HIF-1α, FLIP, Ki67, p50, c-REL, Rel B and β-catenin expression values. Hypoxia activates not only canonical NF-ΚB molecular pathway but also the alternative and both IKKα and IKKβ are necessary to RelA (p65) and p100 accumulation whereas p52 processing is IKKα dependent. Additionally, hypoxia induced nuclear expression of HIF-1α, FLIP, β-catenin and increased NF-κB activity. However, these changes were not observed when Ishikawa cell line was subjected to radiation. ANXA2 plays a role in promoting metastasis in EC and is a predictive biomarker of recurrent disease also among low-intermediate risk EC. Histological typing is difficult in some high grade or mixed endometrioid-non endometrioid EC. For this reason we have assessed an internally and externally 9-biomarker signature to predict histological type in EC

Biomimetic device and foreign body reaction cooperate for efficient tumour cell capture in murine advanced ovarian cancer

Metastasis is facilitated by the formation of pre-metastatic niches through the remodelling of the extracellular matrix (ECM) promoted by haematopoietic and stromal cells. The impact of these primed sites is pronounced for intraperitoneal metastases, where the cavity-exposed ECM supports the attachment of the disseminating tumour cells. Likewise, implantation of biomaterial scaffolds influences metastatic progression systemically through a foreign body reaction (FBR). In this study, we integrated the concept of creating an artificial niche to capture tumour cells actively disseminating in the peritoneal cavity with a therapeutic strategy modulating the interactions of metastatic cells with the ECM. The aim was to transform a disseminated disease into a focal disease. For this, we designed and developed a ‘biomimetic’ ECM composed of a nonresorbable three-dimensional scaffold with collagen coating and characterized the FBR to the implanted biomaterial. We also analysed the safety of the implanted devices and their ability to capture tumour cells in different murine preclinical models of advanced ovarian cancer. Implantation of the biomimetic devices resulted in an initial inflammatory reaction that transformed progressively into a fibrous connective tissue response. The adhesive capabilities of the scaffold were improved with the ancillary effect of the FBR and showed clinical utility in terms of the efficacy of capture of tumour cells, disease focalization and survival benefit. These results demonstrated the performance and safety of this ‘biomimetic’ ECM in preclinical models of advanced ovarian cancer. Translated into the clinical setting, this new therapeutic strategy represents the possibility for control of peritoneal carcinomatosis upon primary ovarian debulking surgery and to expand the percentage of patients who are candidates for second rescue surgeries at the time of relapse.This work was supported by Nasasbiotec

An inducible knockout mouse to model the cellautonomous role of PTEN in initiating endometrial, prostate and thyroid neoplasias

PTEN is one of the most frequently mutated tumor suppressor genes in human cancers. The role of PTEN in carcinogenesis has been validated by knockout mouse models. PTEN heterozygous mice develop neoplasms in multiple organs. Unfortunately, the embryonic lethality of biallelic excision of PTEN has inhibited the study of complete PTEN deletion in the development and progression of cancer. By crossing PTEN conditional knockout mice with transgenic mice expressing a tamoxifen-inducible Cre-ERT under the control of a chicken actin promoter, we have generated a tamoxifeninducible mouse model that allows temporal control of PTEN deletion. Interestingly, administration of a single dose of tamoxifen resulted in PTEN deletion mainly in epithelial cells, but not in stromal, mesenchymal or hematopoietic cells. Using the mT/mG double-fluorescent Cre reporter mice, we demonstrate that epithelial-specific PTEN excision was caused by differential Cre activity among tissues and cells types. Tamoxifen-induced deletion of PTEN resulted in extremely rapid and consistent formation of endometrial in situ adenocarcinoma, prostate intraepithelial neoplasia and thyroid hyperplasia. We also analyzed the role of PTEN ablation in other epithelial cells, such as the tubular cells of the kidney, hepatocytes, colonic epithelial cells or bronchiolar epithelium, but those tissues did not exhibit neoplastic growth. Finally, to validate this model as a tool to assay the efficacy of anti-tumor drugs in PTEN deficiency, we administered the mTOR inhibitor everolimus to mice with induced PTEN deletion. Everolimus dramatically reduced the progression of endometrial proliferations and significantly reduced thyroid hyperplasia. This model could be a valuable tool to study the cell-autonomous mechanisms involved in PTEN-loss-induced carcinogenesis and provides a good platform to study the effect of anti-neoplastic drugs on PTEN-negative tumors

Inhibition of WNT-CTNNB1 signaling upregulates SQSTM1 and sensitizes glioblastoma cells to autophagy blockers

WNT-CTNN1B signaling promotes cancer cell proliferation and stemness. Furthermore, recent evidence indicates that macroautophagy/autophagy regulates WNT signaling. Here we investigated the impact of inhibiting WNT signaling on autophagy in glioblastoma (GBM), a devastating brain tumor. Inhibiting TCF, or silencing TCF4 or CTNNB1/β-catenin upregulated SQSTM1/p62 in GBM at transcriptional and protein levels and, in turn, autophagy. DKK1/Dickkopf1, a canonical WNT receptor antagonist, also induced autophagic flux. Importantly, TCF inhibition regulated autophagy through MTOR inhibition and dephosphorylation, and nuclear translocation of TFEB, a master regulator of lysosomal biogenesis and autophagy. TCF inhibition or silencing additionally affected GBM cell proliferation and migration. Autophagy induction followed by its blockade can promote cancer cell death. In agreement with this notion, halting both TCF-CTNNB1 and autophagy pathways decreased cell viability and induced apoptosis of GBM cells through a SQSTM1-dependent mechanism involving CASP8 (caspase 8). In vivo experiments further underline the therapeutic potential of such dual targeting in GBM

Optimal protocol for PTEN immunostaining; role of analytical and preanalytical variables in PTEN staining in normal and neoplastic endometrial, breast, and prostatic tissues

In some tumors, phosphatase and tensin homolog (PTEN) inactivation may have prognostic importance and predictive value for targeted therapies. Immunohistochemistry (IHC) may be an effective method to demonstrate PTEN loss. It was claimed that PTEN IHC showed poor reproducibility, lack of standardization, and variable effects of preanalytical factors. In this study, we developed an optimal protocol for PTEN IHC, with clone 6H2.1, by checking the relevance of analytical variables in normal tissue and tumors of endometrium, breast, and prostate. Pattern and intensity of cellular staining and background nonspecific staining were quantified and subjected to statistical analysis by linear mixed models. The proposed protocol showed a statistically best performance (P .001). However, there was a trend of significance for decreased staining and fixation under high temperature. Moreover, staining was better in endometrial aspirates than in matched hysterectomy specimens, subjected to less controlled preanalytical variables (mean histoscores, 80 and 40, respectively; P = .002). A scoring system combining intensity of staining and percentage of positive cells was statistically associated with PTEN alterations (P = .01).The study was done according to the research collaboration with Dako Denmark A/S. The research team was also supported by grants FIS PI100922, Fundación Mutua Madrileña AP75732010, 2009SGR794, RD12/0036/0013, Fundación Asociación Española contra el Cancer, programa de intensificación de la investigación, Instituto Carlos III, Verelst Baarmoederkankerfonds, Leuven, and European Network for Individualized Treatment of Endometrial Carcinoma. F. A. is senior researcher for the research fund Flandersb. Tumor samples were obtained with the support of XarxaCatalana de Bancs de Tumors, the TumorBanc Platform of RTICC, and Red de Biobancos (RD09/0076/00059

Multilayer OMIC data in medullary thyroid carcinoma identifies the STAT3 pathway as a potential therapeutic target in RETM918T tumors

Purpose: Medullary thyroid carcinoma (MTC) is a rare disease with few genetic drivers, and the etiology specific to each known susceptibility mutation remains unknown. Exploiting multilayer genomic data, we focused our interest on the role of aberrant DNA methylation in MTC development.Experimental Design: We performed genome-wide DNA methylation profiling assessing more than 27,000 CpGs in the largest MTC series reported to date, comprising 48 molecularly characterized tumors. mRNA and miRNA expression data were available for 33 and 31 tumors, respectively. Two human MTC cell lines and 101 paraffin-embedded MTCs were used for validation.Results: The most distinctive methylome was observed for RETM918T-related tumors. Integration of methylation data with mRNA and miRNA expression data identified genes negatively regulated by promoter methylation. These in silico findings were confirmed in vitro for PLCB2, DKK4, MMP20, and miR-10a, -30a, and -200c. The mutation-specific aberrant methylation of PLCB2, DKK4, and MMP20 was validated in 25 independent MTCs by bisulfite pyrosequencing. The methylome and transcriptome data underscored JAK/Stat pathway involvement in RETM918T MTCs. Immunostaining [immunohistochemistry (IHC)] for the active form of signaling effector STAT3 was performed in a series of 101 MTCs. As expected, positive IHC was associated with RETM918T-bearing tumors (P < 0.02). Pharmacologic inhibition of STAT3 activity increased the sensitivity to vandetanib of the RETM918T-positive MTC cell line, MZ-CRC-1.Conclusions: Multilayer OMIC data analysis uncovered methylation hallmarks in genetically defined MTCs and revealed JAK/Stat signaling effector STAT3 as a potential therapeutic target for the treatment of RETM918T MTCs.This work was supported by the Fondo de Investigaciones Sanitarias (FIS) project PI14/00240 and the Comunidad de Madrid (Grant S2011/BMD-2328 TIRONET) to MR. LI-P is supported by the Centro de Investigacion Biomédica en Red de Enfermedades Raras (CIBERER). VM was supported by a predoctoral fellowship from the "la Caixa"/CNIO international PhD programme. CM-C is supported by a postdoctoral fellowship from the Fundación AECC