Repurposing econazole as a pharmacological autophagy inhibitor to treat pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is characterized by the highest mortality among carcinomas. The pathogenesis of PDAC requires elevated autophagy, inhibition of which using hydroxychloroquine has shown promise. However, current realization is impeded by its suboptimal use and unpredictable toxicity. Attempts to identify novel autophagy-modulating agents from already approved drugs offer a rapid and accessible approach. Here, using a patient-derived organoid model, we performed a comparative analysis of therapeutic responses among various antimalarial/fungal/parasitic/viral agents, through which econazole (ECON), an antifungal compound, emerged as the top candidate. Further testing in cell-line and xenograft models of PDAC validated this activity, which occurred as a direct consequence of dysfunctional autophagy. More specifically, ECON boosted autophagy initiation but blocked lysosome biogenesis. RNA sequencing analysis revealed that this autophagic induction was largely attributed to the altered expression of activation transcription factor 3 (ATF3). Increased nuclear import of ATF3 and its transcriptional repression of inhibitor of differentiation-1 (ID-1) led to inactivation of the AKT/mammalian target of rapamycin (mTOR) pathway, thus giving rise to autophagosome accumulation in PDAC cells. The magnitude of the increase in autophagosomes was sufficient to elicit ER stress-mediated apoptosis. Furthermore, ECON, as an autophagy inhibitor, exhibited synergistic effects with trametinib on PDAC. This study provides direct preclinical and experimental evidence for the therapeutic efficacy of ECON in PDAC treatment and reveals a mechanism whereby ECON inhibits PDAC growth.

Metformin inhibits pancreatic cancer metastasis caused by SMAD4 deficiency and consequent HNF4G upregulation

Pancreatic ductal adenocarcinoma (PDAC) has poor prognosis due to limited therapeutic options. This study examines the roles of genome-wide association study identified PDAC-associated genes as therapeutic targets. We have identified HNF4G gene whose silencing most effectively repressed PDAC cell invasiveness. HNF4G overexpression is induced by the deficiency of transcriptional factor and tumor suppressor SMAD4. Increased HNF4G are correlated with SMAD4 deficiency in PDAC tumor samples and associated with metastasis and poor survival time in xenograft animal model and in patients with PDAC (log-rank P = 0.036; HR = 1.60, 95% CI = 1.03-2.47). We have found that Metformin suppresses HNF4G activity via AMPK-mediated phosphorylation-coupled ubiquitination degradation and inhibits in vitro invasion and in vivo metastasis of PDAC cells with SMAD4 deficiency. Furthermore, Metformin treatment significantly improve clinical outcomes and survival in patients with SMAD4-deficient PDAC (log-rank P = 0.022; HR = 0.31, 95% CI = 0.14-0.68) but not in patients with SMAD4-normal PDAC. Pathway analysis shows that HNF4G may act in PDAC through the cell-cell junction pathway. These results indicate that SMAD4 deficiency-induced overexpression of HNF4G plays a critical oncogenic role in PDAC progression and metastasis but may form a druggable target for Metformin treatment.

Anotação e caracterização de novos transcritos expressos no adenocarcinoma de pâncreas: RNAS não-codificadores longos associados a fenótipos tumorais e clínicos e formas alternativas de splicing associados a fenótipos tumorais e clínicos / Determination of relevant transcripts in ductal adenocarcinoma of pancreas: the transcriptional landscape of the long non-coding RNAs and protein-encoding RNAs revealed by the total RNAseq analysis of pancreatic adenocarcinoma

O câncer de pâncreas (PC) é uma das doenças mais devastadoras com o pior resultado de sobrevivência quando comparada com outros tipos de câncer. É apontado como o décimo câncer mais comum e a quarta causamortis por câncer, projetando-se para ser a segunda até 2030 nos Estados Unidos e na Alemanha. O PC constitui um conjunto heterogêneo de tumores, o adenocarcinoma ductal (PDAC) constitui o tipo mais frequente da neoplasia (80%). A prevenção, detecção precoce e tratamento enfrentam sérios problemas e é urgente a identificação de novos marcadores para diagnóstico, prognóstico e estratégias terapêuticas da doença. O objetivo desse trabalho foi realizar uma análise com alta-resolução do transcriptoma do PDAC) para identificar novos RNAs não codificadores, variantes de splicing e alterações transcricionais associados à malignidade. A metodologia empregada constituiu-se de gerar bibliotecas de RNA total a partir de 14 amostras cirúrgicas pareadas de tecido tumoral (PDAC) e tecido não-tumoral adjacente para sequenciamento NGS. A partir dos dados de RNAseq foi realizada a montagem do transcriptoma utilizando-se a referência do catálogo GENCODE para classificar os transcritos. Seguiram-se análises subsequentes de avaliação de características dos transcritos: estruturais, marcas regulatórias, potencial codificador, detecção em banco de dados independente (miTrascriptome, miT). Em segundo lugar foram realizadas análises de expressão diferencial, análise de sobrevida (utilizando banco de dados públicos) para seleção de transcritos potencialmente relevantes no PDAC. Foram geradas redes de co-expressão gênicas com enriquecimento de funções biológicas. Uma série de validações foram realizadas por RT-PCR de transcritos novos intergênicos e novas formas de splicing reconstruídas e selecionadas. RT-qPCR foi utilizada para validação da expressão aberrante de lncRNAs em PDAC, silenciamentos por siRNA e subsequentes ensaios de proliferação, migração e invasão. Foi avaliada in vivo o envolvimento com crescimento tumoral por ensaio xenográfico, e avaliação da implicação em funções biológicas mais específicas, como envolvimento em reparo de DNA por ensaio cometa alcalino e avaliação de enriquecimento em tumoresferas. A montagem reconstruiu 90.522 transcritos, dos quais 41.341 são anotados no GENCODE e 6.710 transcritos são novos não anotados no GENCODE (classificado como splicingvariant, intergenic RNA, antisense RNA). Desses foram validados 6 novos lincRNAs com expressão aumentada em PDAC, dois deles (TCONS00085964 e TCONS00036574) tem a expressão correlacionada com alterações na sobrevida. Foram validadas também novas formas de splicing de MMP14, CAPN8, LIF e OCT3 com expressão aumentada em PDAC. 7 lncRNAs, anotados no GENCODE, com expressão aumentada em PDAC, desses foram implicados com fenótipo tumoral de migração, invasão e proliferação: LINC01559; LINC01133, CCAT1 e UCA1. Desses LINC01559 e CCAT1 demonstraram regular a expressão de enzimas de O-glicosilação (GALNT3 e B3GNT3) envolvidas na manutenção de células tronco-tumorais em PDAC. O lncRNA UCA1 está envolvido com reparo de DNA e envolvido com a progressão tumoral invivo. Conclui-se que a abordagem por amostras pareadas a partir de RNAseq de bibliotecas de RNA total gerou um catálogo de transcritos mais completo no PDCA e revelou IncRNAs funcionalmente implicadas na doença como LINC01559 e UCA1, ampliando o conhecimento sobre os mecanismos moleculares que sustentam fenótipos malignos no câncer de pâncreas e revelando novos biomarcadores para prognóstico

Tese de DoutoradoDOIhttps://doi.org/10.11606/T.46.2019.tde-09032020-085211DocumentoTese de DoutoradoAutorPaixão, Vinicius Ferreira da (Catálogo USP)Nome completoVinicius Ferreira da PaixãoE-mailE-mailUnidade da USPInstituto de QuímicaÁrea do ConhecimentoBioquímicaData de Defesa2019-12-04ImprentaSão Paulo, 2019OrientadorReis, Eduardo Moraes (Catálogo USP) Banca examinadoraReis, Eduardo Moraes (Presidente) Hajj, Glaucia Noeli Maroso Malnic, Bettina Panepucci, Rodrigo Alexandre Título em portuguêsAnotação e caracterização de novos transcritos expressos no adenocarcinoma de pâncreas: RNAS não-codificadores longos associados a fenótipos tumorais e clínicos e formas alternativas de splicingPalavras-chave em portuguêsAlternative splicing lncRNA PDAC Transcriptoma UCA1 Xenotumor Resumo em portuguêsO câncer de pâncreas (PC) é uma das doenças mais devastadoras com o pior resultado de sobrevivência quando comparada com outros tipos de câncer. É apontado como o décimo câncer mais comum e a quarta causamortis por câncer, projetando-se para ser a segunda até 2030 nos Estados Unidos e na Alemanha. O PC constitui um conjunto heterogêneo de tumores, o adenocarcinoma ductal (PDAC) constitui o tipo mais frequente da neoplasia (80%). A prevenção, detecção precoce e tratamento enfrentam sérios problemas e é urgente a identificação de novos marcadores para diagnóstico, prognóstico e estratégias terapêuticas da doença. O objetivo desse trabalho foi realizar uma análise com alta-resolução do transcriptoma do PDAC) para identificar novos RNAs não codificadores, variantes de splicing e alterações transcricionais associados à malignidade. A metodologia empregada constituiu-se de gerar bibliotecas de RNA total a partir de 14 amostras cirúrgicas pareadas de tecido tumoral (PDAC) e tecido não-tumoral adjacente para sequenciamento NGS. A partir dos dados de RNAseq foi realizada a montagem do transcriptoma utilizando-se a referência do catálogo GENCODE para classificar os transcritos. Seguiram-se análises subsequentes de avaliação de características dos transcritos: estruturais, marcas regulatórias, potencial codificador, detecção em banco de dados independente (miTrascriptome, miT). Em segundo lugar foram realizadas análises de expressão diferencial, análise de sobrevida (utilizando banco de dados públicos) para seleção de transcritos potencialmente relevantes no PDAC. Foram geradas redes de co-expressão gênicas com enriquecimento de funções biológicas. Uma série de validações foram realizadas por RT-PCR de transcritos novos intergênicos e novas formas de splicing reconstruídas e selecionadas. RT-qPCR foi utilizada para validação da expressão aberrante de lncRNAs em PDAC, silenciamentos por siRNA e subsequentes ensaios de proliferação, migração e invasão. Foi avaliada in vivo o envolvimento com crescimento tumoral por ensaio xenográfico, e avaliação da implicação em funções biológicas mais específicas, como envolvimento em reparo de DNA por ensaio cometa alcalino e avaliação de enriquecimento em tumoresferas. A montagem reconstruiu 90.522 transcritos, dos quais 41.341 são anotados no GENCODE e 6.710 transcritos são novos não anotados no GENCODE (classificado como splicingvariant, intergenic RNA, antisense RNA). Desses foram validados 6 novos lincRNAs com expressão aumentada em PDAC, dois deles (TCONS00085964 e TCONS00036574) tem a expressão correlacionada com alterações na sobrevida. Foram validadas também novas formas de splicing de MMP14, CAPN8, LIF e OCT3 com expressão aumentada em PDAC. 7 lncRNAs, anotados no GENCODE, com expressão aumentada em PDAC, desses foram implicados com fenótipo tumoral de migração, invasão e proliferação: LINC01559; LINC01133, CCAT1 e UCA1. Desses LINC01559 e CCAT1 demonstraram regular a expressão de enzimas de O-glicosilação (GALNT3 e B3GNT3) envolvidas na manutenção de células tronco-tumorais em PDAC. O lncRNA UCA1 está envolvido com reparo de DNA e envolvido com a progressão tumoral invivo. Conclui-se que a abordagem por amostras pareadas a partir de RNAseq de bibliotecas de RNA total gerou um catálogo de transcritos mais completo no PDCA e revelou IncRNAs funcionalmente implicadas na doença como LINC01559 e UCA1, ampliando o conhecimento sobre os mecanismos moleculares que sustentam fenótipos malignos no câncer de pâncreas e revelando novos biomarcadores para prognóstico.Título em inglêsDetermination of relevant transcripts in ductal adenocarcinoma of pancreas: the transcriptional landscape of the long non-coding RNAs and protein-encoding RNAs revealed by the total RNAseq analysis of pancreatic adenocarcinomaPalavras-chave em inglêsAlternative splicing lncRNA PDAC Transcriptome UCA1 Xenotumor Resumo em inglêsPancreatic cancer (PC) is the deadliest malignancy, one of the most devastating diseases with the worst survival outcome compared to any cancer. It is touted as the tenth most common cancer and the fourth leading cause of cancer in the United States and Germany. It is projected to be the second leading cause of cancer death in a decade. PC is a heterogeneous set of tumors with high aggressiveness and mortality. Of these tumors, ductal adenocarcinoma (PDAC) is the most frequent type of cancer (80%). Prevention, early detection and treatment face serious problems, and the identification of new markers for diagnosis, prognosis and therapeutic strategies of the disease is urgent. The aim of this study was to perform a high-resolution analysis of pancreatic adenocarcinoma (PDAC) transcriptome to identify new noncoding RNAs, splicing variants, and transcriptional changes associated with malignancy in pancreatic ductal adenocarcinoma. The methodology employed consisted of generating total RNA libraries from 14 paired surgical samples of tumor tissue (PDAC) and adjacent non-tumor tissue for NGS sequencing. From the RNAseq data, the transcriptome assembly was performed using the GENCODE catalog reference to classify the transcripts. Subsequent analyzes of evaluation of transcript characteristics were followed: structural, regulatory markers, potential encoder, independent database detection (miTrascriptome, miT). Secondly, differential expression analysis, survival analysis (using public database) were performed to select potentially relevant transcripts in the PDAC. Genic co-expression networks with biological function enrichment were generated. A series of validations were performed by RT-PCR of new intergenic transcripts and new forms of reconstructed and selected splicing. RT-qPCR was used for validation of aberrant expression of lncRNAs in PDAC, siRNA silencing and subsequent proliferation, migration and invasion assays. Involvement with tumor growth was evaluated by in vivo xenograph assay. Biological function tests to determine an involvement in DNA repair was evaluated by alkaline comet assay and was performed an evaluation of tumorsphere expression enrichment. The assembly reconstructed a total of 90,522 transcripts, of which 41,341 are noted in GENCODE. 6,710 transcripts are new (splicing variant, intergenic RNA, antisense RNA) not noted in the GENCODE reference. Of these 6 new PDAC-enhanced lincRNAs were validated, two of them (TCONS00085964 and TCONS00036574) correlated with changes in survival. New forms of splicing of MMP14, CAPN8, LIF and OCT3 with increased expression in PDAC were also validated. 7 lncRNAs noted with increased expression in PDAC were implicated with tumor migration, invasion and proliferation phenotype: LINC01559; LINC01133, LINC01614; CCAT1; LINC02577; LINC00920 and UCA1. Of these LINC01559 has been shown to regulate the expression of O-glycosylation enzymes (GALNT3 and B3GNT3) involved in maintaining CSCs in PDAC. It has been identified that UCA1 is involved with DNA repair and involved with tumor progression in vivo. It is concluded that the approach of sampling RNAseq from total RNA libraries generated a more accurate transcript catalog of PDAC and revealed IncRNAs functionally implicated in the disease, such as LINC01559 and UCA1, expanding the knowledge about the molecular mechanisms that underlie malignant phenotypes in pancreatic cancer and revealing novel prognostic biomarke

Expression of ECT2 gene in human pancreatic ductal adenocarcinoma and its effect on tumor biological characteristics / 中国肿瘤生物治疗杂志

@# Objective: To investigate the expression of ECT2 (epithelial Transforming sequence 2) gene in human pancreatic ductal adenocarcinoma (PDAC) and its effect on the proliferation and apoptosis of pancreatic cancer cells. Methods: Carcinoma tissues and corresponding para-carcinoma tissues from 35 PDAC patients at Changhai Hospital Affiliated to Naval Medical University from July 2018 to March 2019 were collected for this study. The differentially expressed genes in pancreatic cancer were screened out by using Gene Expression Omnibus (GEO) Database. Then, the related gene expression in PDAC and its relation with patients’survival were analyzed by The Cancer Genome Atlas (TCGA) database. QPCR and immunohistochemistry were used to verify the mRNAand protein expressions of ECT2 in human PDAC samples. To explore the effect of ECT2 on the biological behaviors of pancreatic cancer cells, si-RNA was used to silence the ECT2 gene in pancreatic cancer PANC-1 cells, and CCK-8 proliferation assay and Flow cytometry were used to detect the proliferation and apoptosis rate of PANC-1 cells after ECT2 silence. Finally, the expressions of apoptosis-related proteins were detected by WB. Results: The differentially expressed gene-ECT2, was screened out by analyzing the gene expression profiles of human pancreatic cancer in GEO database. TCGA database analysis showed that ECT2 was highly expressed in pancreatic cancer tissues (t=4.005, P<0.05) and significantly correlated with patients’survival (P< 0.01). Moreover, it is also verified that ECT2 was highly expressed in PDAC tissues at mRNA (1.01±0.06 vs 4.25±0.12; t=24.09， P<0.01) and protein level. After ECT2 silence in PANC-1 cells, the proliferation rate was decreased (P<0.01), while the Tamoxifeninduced apoptosis rate was increased (P<0.01), and the expressions of apoptosis-related proteins (BAX and Bcl-2) were also affected. Conclusion: ECT2 is highly expressed in human pancreatic ductal adenocarcinoma and is related with patients’survival. ECT2 promotes the proliferation and apoptosis resistance of pancreatic cancer cells, providing the basis for exploring ECT2 as a new target for the prognostic judgment and treatment of pancreatic cancer.

Experience with non-cremophor-based paclitaxel-gemcitabine regimen in advanced pancreatic cancer: Results from a single tertiary cancer centre

Background & objectives: Gemcitabine combined with non-cremophor-based paclitaxel is one of the standards of care in advanced inoperable pancreatic cancer. This study was undertaken to retrospectively evaluate real world non-trial outcomes with this combination. Methods: Patients with histologically proven advanced inoperable pancreatic adenocarcinoma (PDAC), treated with non-cremophor-based paclitaxel-gemcitabine combination (PG) (gemcitabine-nanoxel or gemcitabine-abraxane) between January 2012 and June 2015, were retrospectively analyzed. Response assessment was done every 8-12 wk with computed tomography scan and responses were measured as per the Response Evaluation Criteria in Solid Tumours 1.1 criteria where feasible. Toxicity was recorded as per the Common Terminology Criteria for Adverse Events (CTCAE) v4 criteria. Progression-free survival (PFS) and overall survival (OS) were calculated using the Kaplan-Meier method. Results: A total of 78 patients with PDAC were treated with the combination. Of these, 83.3 per cent of patients had metastatic disease. The median number of chemotherapy cycles administered was three. The objective response rate for the whole group was 30.8 per cent. Grade III/IV toxicities were seen in 35.9 per cent of patients. Median PFS was 5.6 months and median OS was 11.6 months. Interpretation & conclusions: Non-cremophor-based paclitaxel in combination with gemcitabine appeared efficacious for advanced pancreatic cancers in routine clinical practice. Within the confines of a single-centre retrospective analysis, gemcitabine-nanoxel and gemcitabine-abraxane appeared to have similar efficacy and toxicity in advanced pancreatic cancers.

Cáncer de páncreas: alteraciones genéticas, cambios morfológicos y sus implicaciones terapéuticas / Pancreas cancer: genetic alterations, morphological changes and its therapeutic implications

Resumen El cáncer de páncreas es una enfermedad mortal, principalmente porque se descubre muy tarde y es muy resistente a la quimioterapia y radioterapia. El tipo más común de cáncer de páncreas (más del 90%) se desarrolla a partir de las células exocrinas del páncreas y se denomina adenocarcinoma ductal pancreático (ACDP). Se han reconocido tres lesiones precursoras de cáncer de páncreas ductal: neoplasia intraepitelial pancreática (PanIN), neoplasia papilar-mucinosa intraductal (NPMI) y neoplasia quística mucinosa (NCM). Uno de los primeros eventos genéticos implicados en la patogénesis de ACDP es una mutación en el punto de activación en el oncogén KRAS, una mutación conductora oncogénica que se encuentra en más del 90% de todos los cánceres de páncreas. Además, se ha informado que hasta un 10% de los cánceres de páncreas están asociados con el historial familiar. Aunque la causa es multifactorial, el tabaquismo y la historia familiar son dominantes. Existe heterogeneidad morfológica en muchos cánceres humanos, pero parece ser un hallazgo particularmente común en ACDP. La cirugía solo es posible en 15-20% de los casos: tumores confinados al páncreas con posible afectación ganglionar local no muy extensa y que no produce afectación vascular o está limitada.

Abstract Pancreatic cancer is a deadly disease, mainly because it is generally discovered very late and it is very resistant to chemotherapy and radiation therapy. The most common type of pancreatic cancer (over 90%) develops from the exocrine cells of the pancreas and is named pancreatic ductal adenocarcinoma (PDAC). Three precursor lesions of ductal pancreatic cancer have been recognized: pancreatic intraepithelial neoplasia (PanIN), intraductal papillary-mucinous neoplasm (IPMN) and mucinous cystic neoplasm (MCN). One of the earliest genetic events involved in PDAC pathogenesis is an activating point mutation in the KRAS oncogene, an oncogenic driver mutation found in more than 90% of all pancreatic cancer. In addition, it has been reported that up to 10% of pancreatic cancer is associated with family history. Although the cause is multifactorial, cigarette smoking and family history are dominant. Morphological heterogeneity exists in many human cancers, but seems to be a particularly common finding in PDAC. Surgery is only possible in 15-20% of cases: tumors confined to the pancreas with possible local nodal involvement not very extensive and that do not produce vascular involvement or is limited.

CCAT2 is an oncogenic long non-coding RNA in pancreatic ductal adenocarcinoma

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is highly aggressive with poor prognosis. Long non-coding RNAs (lncRNAs), a group of non-coding RNAs, play important roles in the progression of PDAC. This study aimed to investigate the potential involvement of lncRNA CCAT2 in PDAC tumorigenesis. Methods Expression of CCAT2 was detected by quantitative real-time PCR (qRT-PCR) in 80 human PDAC tissues and three PDAC cell lines. The effects of CCAT2 silencing in PANC-1 cells on cell proliferation and invasion were studied using MTT assay and transwell assay, respectively. The effect of CCAT2 silencing on tumorigenesis was assessed by PANC-1 xenograft in vivo. Using si-KRAS, the role of KRAS to regulate CCAT2 was evaluated by qRT-PCR and luciferase reporter assay. The involvement of MEK/ERK and PI3K/AKT signaling in CCAT2 regulation was investigated by pathway inhibitors PD98059 and LY294002, respectively. Results CCAT2 was significantly elevated in high-grade PDAC tissues and higher CCAT2 expression was correlated with lower survival rate in PDAC patients. CCAT2 was up-regulated in PDAC cell lines, as compared with normal pancreatic cells. Silencing of CCAT2 inhibited cell proliferation and invasion in PANC-1 cells in vitro, and attenuated tumorigenesis of PANC-1 xenograft in vivo. Furthermore, CCAT2 was regulated by KRAS through MEK/ERK signaling pathway. Conclusions CCAT2 is an oncogenic lncRNA in PDAC likely regulated by the KRAS-MEK/ERK pathway. It could be a potential diagnostic biomarker and therapeutic target for PDAC.

Identificação e anotação funcional de RNAs longos não codificadores associados à subtipos moleculares em tumores pancreáticos / Identification of long non coding RNAs associated to molecular subtypes in pancreatic tumors

O Adenocarcinoma Pancreático Ductal (Pancreatic Ductal Adenocarcinoma - PDAC) é a sé- tima causa de mortes por câncer no mundo, com uma taxa de sobrevida de apenas 6%. Embora alguns genes estejam recorrentemente mutados em grande parte dos tumores e sejam críticos para a oncogênese, a heterogeneidade das alterações moleculares tanto no tumor quanto em componentes do microambiente tumoral se reflete em diferentes características fenotípicas com comportamentos clínicos distintos e que têm sido associados a diferentes subtipos moleculares através da análise computacional de dados de alterações somáticas e transcricionais no PDAC. RNAs não codificadores longos (lncRNAs) têm sido reconhecidos como importantes reguladores da expressão gênica em doenças proliferativas mas sua associação com subtipos em PDAC e sua contribuição para o estabelecimento de diferentes fenótipos moleculares e clínicos da doença não foi explorada até o momento. Neste trabalho, foi implementada uma abordagem computacional com o objetivo de identificar e anotar funcionalmente lncRNAs associados a subtipos moleculares de PDAC. Inicialmente, a classificação não supervisionada por Fatoração Matricial Não Negativa (Non-Negative Matrix Factorization - NMF) de dados de expressão gê- nica global de amostras clínicas disponíveis publicamente (The Cancer Genome Atlas - TCGA) resultou na identificação de quatro subgrupos distintos de PDAC, que recapitulam os fenótipos Exócrino/Endócrino, Imunogênico, Escamoso e Progenitor descritos na literatura. Uma análise de expressão diferencial permitiu a identificação de assinaturas de expressão gênica características que incluem lncRNAs associados a cada subgrupo. Através da construção de redes de coexpressão de mRNAs e lncRNAs e a identificação de módulos da rede significativamente enriquecidos em genes que participam em vias moleculares conhecidas foi possível inferir possíveis funções biológicas à lncRNAs associados aos diferentes subtipos moleculares, tais como funções exócrinas/neuroendócrinas, imunogênicas, reparo de DNA/progressão do ciclo celular e progenitoras/morfogênicas. Entre ele, o subgrupo 3, enriquecido para fenótipo Escamoso e associado a hiper-expressão do supressor tumoral TP63, possui dois lncRNAS hiper-expressos neste subgrupo em relação aos outros subgrupos, sendo que o lncRNA antissenso FAM83A-AS1 tem a predição de interagir com as proteínas FGFR2, AXIN1, PTEN, BRAF, SMAD4, TGFBR2, TP53 e CDKN2A, que exercem funções importantes na transdução de sinal e supressão tumoral no câncer incluindo o de pâncreas. Entre os lncRNAs hipo-regulados no subgrupo 3 em relação ao outros subgrupos, alguns, como FLJ42875, LOC338651, C20orf56 e LOC38838 tem predição de interação com alta afinidade à proteína BRCA2, que está envolvida no reparo de DNA e participa de processos de resistência à quimioterápicos. As informações trazidas por este estudo permitem gerar hipóteses sobre a contribuição de lncRNAs para a definição de subtipos moleculares de PDAC e priorizar candidatos e experimentos para estudos funcionais de modo a contribuir para um melhor entendimento sobre os mecanismos de ação de lncRNAs na tumorigênese e agressividade do câncer de pâncreas

Pancreatic Ductal Adenocarcinoma (PDAC) is the seventh cause of worldwide cancer related deaths, with an overall survival rate of only 6%. Some genes might be recurrently mutated in a large number of tumors, and be critical for oncogenesis, molecular alteration heterogeneity both in the tumor as all as in the tumor microenvironment is reflected in diverse phenotypic features with distinct clinical outcomes, and this distinction in multiple molecular subtypes has been drawn through transcriptional and somatic alteration computational analysis within PDAC. Long Non Coding RNAs (lncRNAs) have been recognized as important gene expression regulators in proliferative diseases, but its association to molecular subtypes in PDAC and its contribution in the establishment of diverse molecular and clinical phenotypes hasnt been explored at length until the present. This work focused on the implementation of a computational approach with the objective of lncRNA identification and functional annotation associated to distinct molecular subtypes in PDAC. Initially, Non-negative Matrix Factorization (NMF), an unsupervised classification method, applied to global gene expression data from publicly available clinical samples (The Cancer Genome Atlas - TCGA) resulted in the identification of four distinct PDAC molecular subgroups reminiscent of Exocrine/Endocrine, Immunogenic, Squamous and Progenitor phenotypes. Differential expression analysis allowed a characteristic gene expression signature identification, including distinct molecular subtype associated lncRNAs. mRNA and lncRNA containing gene co-expression modules significantly enriched annotated pathways containing the molecular subtype associated lncRNAs allowed to designate possible molecular functions of the distinct molecular subtype associated lncRNAs, such as exocrine/neuroendocrine, immunogenic, DNA repair/cell cycle progression and progenitor/morphogenic functions. Subgroup 3, enriched with a Squamous phenotype and associated to TP63 over-expression contains two lncRNAs over-expressed compared to other subgroups; furthermore, the antisense lncRNA FAM83A-AS1 yielded a predicted lncRNA-protein interaction to FGFR2, AXIN1, PTEN, BRAF, SMAD4, TGFBR2, TP53 and CDKN2A, proteins that play important signal transduction and tumor suppressor roles in several cancer types, including pancreas. Among under-expressed lncRNAs in subgroup 3 compared to the other subgroups, some, such as FLJ42875, LOC338651, C20orf56 and LOC38838 yilded a high protein interaction prediction score with BRCA2, a protein involved in DNA repair and processes resuling in chemotherapy resistance. The information brought by this study allowed to generate hypothesis on lncRNA contribution to define PDAC molecular subtypes, helping prioritize candidates and experiments for functional studies, thus contributing to a better understanding on lncRNA mechanisms related to tumor progression and aggressiveness in pancreatic cancer

Pseudolaric Acid B Inhibits Proliferation, Invasion and Epithelial-to-Mesenchymal Transition in Human Pancreatic Cancer Cell

PURPOSE: This study was aimed to investigate the effect of pseudolaric acid B (PAB) on proliferation, invasion and epithelial-to-mesenchymal transition (EMT) in pancreatic cancer cells and to explore the possible mechanism. MATERIALS AND METHODS: The pancreatic cancer cell line SW1990 was cultured and treated with PAB dose- and time-dependent manners. Cell proliferation and invasion ability were measured by MTT assay and Matrigel/Transwell test, respectively. Semi-quantitative real-time polymerase chain reaction and Western blotting were conducted to detect the expression of EMT markers and the key molecules. Finally, nude mice subcutaneous transplantation tumor model was used to confirm the therapy efficacy of PAB. RESULTS: PAB could inhibit SW1990 cell proliferation and invasion in time- and dose-dependent manners. Vimentin, fibronectin, N-cadherin, Snail, Slug, YAP, TEAD1, and Survivin were down-regulated (p < 0.01), while E-cadherin, caspase-9, MST1, and pYAP were up-regulated (p < 0.05). Combined PAB and gemcitabine treatment markedly restricted the tumor growth compared with gencitabin or PAB alone groups. CONCLUSION: PAB could inhibit the proliferation and invasion ability of pancreatic cancer cells through activating Hippo-YAP pathway and inhibiting the process of EMT.