Simple Summary Pancreatic cancers are lethal types of cancer. A majority of patients progress to an advanced and metastatic disease, which remains a major clinical problem. Therefore, it is crucial to identify critical regulators to help predict the disease progression and to develop more efficacious therapeutic approaches. In this work we found that an increased expression of the developmental factor SOX9 is associated with metastasis, a poor prognosis and resistance to therapy in pancreatic ductal adenocarcinoma patients and in cell cultures. We also found that this effect is at least in part due to the ability of SOX9 to regulate the activity of stem cell factors, such as BMI1, in addition to those involved in EMT and metastasis. Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers mainly due to spatial obstacles to complete resection, early metastasis and therapy resistance. The molecular events accompanying PDAC progression remain poorly understood. SOX9 is required for maintaining the pancreatic ductal identity and it is involved in the initiation of pancreatic cancer. In addition, SOX9 is a transcription factor linked to stem cell activity and is commonly overexpressed in solid cancers. It cooperates with Snail/Slug to induce epithelial-mesenchymal transition (EMT) during neural development and in diseases such as organ fibrosis or different types of cancer. Methods: We investigated the roles of SOX9 in pancreatic tumor cell plasticity, metastatic dissemination and chemoresistance using pancreatic cancer cell lines as well as mouse embryo fibroblasts. In addition, we characterized the clinical relevance of SOX9 in pancreatic cancer using human biopsies. Results: Gain- and loss-of-function of SOX9 in PDAC cells revealed that high levels of SOX9 increased migration and invasion, and promoted EMT and metastatic dissemination, whilst SOX9 silencing resulted in metastasis inhibition, along with a phenotypic reversion to epithelial features and loss of stemness potential. In both contexts, EMT factors were not altered. Moreover, high levels of SOX9 promoted resistance to gemcitabine. In contrast, overexpression of SOX9 was sufficient to promote metastatic potential in K-Ras transformed MEFs, triggering EMT associated with Snail/Slug activity. In clinical samples, SOX9 expression was analyzed in 198 PDAC cases by immunohistochemistry and in 53 patient derived xenografts (PDXs). SOX9 was overexpressed in primary adenocarcinomas and particularly in metastases. Notably, SOX9 expression correlated with high vimentin and low E-cadherin expression. Conclusions: Our results indicate that SOX9 facilitates PDAC progression and metastasis by triggering stemness and EMT.

The development of predictive biomarkers of response to targeted therapies is an unmet clinical need for many antitumoral agents. Recent genome-wide loss-of-function screens, such as RNA interference (RNAi) and CRISPR-Cas9 libraries, are an unprecedented resource to identify novel drug targets, reposition drugs and associate predictive biomarkers in the context of precision oncology. In this work, we have developed and validated a large-scale bioinformatics tool named DrugSniper, which exploits loss-of-function experiments to model the sensitivity of 6237 inhibitors and predict their corresponding biomarkers of sensitivity in 30 tumor types. Applying DrugSniper to small cell lung cancer (SCLC), we identified genes extensively explored in SCLC, such as Aurora kinases or epigenetic agents. Interestingly, the analysis suggested a remarkable vulnerability to polo-like kinase 1 (PLK1) inhibition inCREBBP-mutant SCLC cells. We validated this association in vitro using four mutated and four wild-type SCLC cell lines and twoPLK1inhibitors (Volasertib and BI2536), confirming that the effect ofPLK1inhibitors depended on the mutational status ofCREBBP. Besides, DrugSniper was validated in-silico with several known clinically-used treatments, including the sensitivity of Tyrosine Kinase Inhibitors (TKIs) and Vemurafenib toFLT3andBRAFmutant cells, respectively. These findings show the potential of genome-wide loss-of-function screens to identify new personalized therapeutic hypotheses in SCLC and potentially in other tumors, which is a valuable starting point for further drug development and drug repositioning projects.

The advent of RNA-seq technologies has switched the paradigm of genetic analysis from a genome to a transcriptome-based perspective. Alternative splicing generates functional diversity in genes, but the precise functions of many individual isoforms are yet to be elucidated. Gene Ontology was developed to annotate gene products according to their biological processes, molecular functions and cellular components. Despite a single gene may have several gene products, most annotations are not isoform-specific and do not distinguish the functions of the different proteins originated from a single gene. Several approaches have tried to automatically annotate ontologies at the isoform level, but this has shown to be a daunting task. We have developed ISOGO (ISOform + GO function imputation), a novel algorithm to predict the function of coding isoforms based on their protein domains and their correlation of expression along 11,373 cancer patients. Combining these two sources of information outperforms previous approaches: it provides an area under precision-recall curve (AUPRC) five times larger than previous attempts and the median AUROC of assigned functions to genes is 0.82. We tested ISOGO predictions on some genes with isoform-specific functions (BRCA1, MADD,VAMP7 and ITSN1) and they were coherent with the literature. Besides, we examined whether the main isoform of each gene -as predicted by APPRIS- was the most likely to have the annotated gene functions and it occurs in 99.4% of the genes. We also evaluated the predictions for isoform-specific functions provided by the CAFA3 challenge and results were also convincing. To make these results available to the scientific community, we have deployed a web application to consult ISOGO predictions (https://biotecnun.unav.es/app/isogo). Initial data, website link, isoform-specific GO function predictions and R code is available at https://gitlab.com/icassol/isogo.

BackgroundSplicing is a genetic process that has important implications in several diseases including cancer. Deciphering the complex rules of splicing regulation is crucial to understand and treat splicing-related diseases. Splicing factors and other RNA-binding proteins (RBPs) play a key role in the regulation of splicing. The specific binding sites of an RBP can be measured using CLIP experiments. However, to unveil which RBPs regulate a condition, it is necessary to have a priori hypotheses, as a single CLIP experiment targets a single protein.ResultsIn this work, we present a novel methodology to predict context-specific splicing factors from transcriptomic data. For this, we systematically collect, integrate and analyze more than 900 CLIP experiments stored in four CLIP databases: POSTAR2, CLIPdb, DoRiNA and StarBase. The analysis of these experiments shows the strong coherence between the binding sites of RBPs of similar families. Augmenting this information with expression changes, we are able to correctly predict the splicing factors that regulate splicing in two gold-standard experiments in which specific splicing factors are knocked-down.ConclusionsThe methodology presented in this study allows the prediction of active splicing factors in either cancer or any other condition by only using the information of transcript expression. This approach opens a wide range of possible studies to understand the splicing regulation of different conditions. A tutorial with the source code and databases is available at https://gitlab.com/fcarazo.m/sfprediction.

BACKGROUND: Aberrant alternative splicing plays a key role in cancer development. In recent years, alternative splicing has been used as a prognosis biomarker, a therapy response biomarker, and even as a therapeutic target. Next-generation RNA sequencing has an unprecedented potential to measure the transcriptome. However, due to the complexity of dealing with isoforms, the scientific community has not sufficiently exploited this valuable resource in precision medicine. FINDINGS: We present TranscriptAchilles, the first large-scale tool to predict transcript biomarkers associated with gene essentiality in cancer. This application integrates 412 loss-of-function RNA interference screens of >17,000 genes, together with their corresponding whole-transcriptome expression profiling. Using this tool, we have studied which are the cancer subtypes for which alternative splicing plays a significant role to state gene essentiality. In addition, we include a case study of renal cell carcinoma that shows the biological soundness of the results. The databases, the source code, and a guide to build the platform within a Docker container are available at GitLab. The application is also available online. CONCLUSIONS: TranscriptAchilles provides a user-friendly web interface to identify transcript or gene biomarkers of gene essentiality, which could be used as a starting point for a drug development project. This approach opens a wide range of translational applications in cancer.

Alternative splicing (AS) has shown to play a pivotal role in the development of diseases, including cancer. Specifically, all the hallmarks of cancer (angiogenesis, cell immortality, avoiding immune system response, etc.) are found to have a counterpart in aberrant splicing of key genes. Identifying the context-specific regulators of splicing provides valuable information to find new biomarkers, as well as to define alternative therapeutic strategies. The computational models to identify these regulators are not trivial and require three conceptual steps: the detection of AS events, the identification of splicing factors that potentially regulate these events and the contextualization of these pieces of information for a specific experiment. In this work, we review the different algorithmic methodologies developed for each of these tasks. Main weaknesses and strengths of the different steps of the pipeline are discussed. Finally, a case study is detailed to help the reader be aware of the potential and limitations of this computational approach.

Impedance spectroscopy analysis (IS) has awakened a great interest for many industrial applications and sectors for the implementation of novel monitoring capabilities. More specifically, microelectrode-based sensors are increasingly being used to analyze electrical or electrochemical changes in liquid samples, as well as other effects such as biofouling, particle adhesion, etc. However, real environmental conditions are usually subjected physiochemical changes that affect the impedance measurement. In this context, it is difficult to isolate the effect of only one parameter (Le., conductivity of the medium) from the other ones. This work is focused specifically on the analysis of the influence of temperature and pH on the impedance measurements. Different experiments were carried out using interdigitated microelectrodes (IDE) sensors for a geometry range in wine samples to adjust a proposed mathematical model of the impedance behavior. In the case of fermentation processes of alcoholic beverages, this methodology will help to isolate the chemical changes measured by impedance from temperature or pH variation. This model also provides the significance of the effect of each parameter on the impedance values. After the experimental validation, the model was used to correct the impedance values accordingly to the variation of each parameter showing its applicability to the real field. Finally, the proposed methodology can be easily applied and extended to other environments and sensors types. (C) 2018 Elsevier B.V. All rights reserved.

Background: RNA-seq is a reference technology for determining alternative splicing at genome-wide level. Exon arrays remain widely used for the analysis of gene expression, but show poor validation rate with regard to splicing events. Commercial arrays that include probes within exon junctions have been developed in order to overcome this problem. We compare the performance of RNA-seq (Illumina HiSeq) and junction arrays (Affymetrix Human Transcriptome array) for the analysis of transcript splicing events. Three different breast cancer cell lines were treated with CX-4945, a drug that severely affects splicing. To enable a direct comparison of the two platforms, we adapted EventPointer, an algorithm that detects and labels alternative splicing events using junction arrays, to work also on RNA-seq data. Common results and discrepancies between the technologies were validated and/or resolved by over 200 PCR experiments. Results: As might be expected, RNA-seq appears superior in cases where the technologies disagree and is able to discover novel splicing events beyond the limitations of physical probe-sets. We observe a high degree of coherence between the two technologies, however, with correlation of EventPointer results over 0.90. Through decimation, the detection power of the junction arrays is equivalent to RNA-seq with up to 60 million reads. Conclusions: Our results suggest, therefore, that exon-junction arrays are a viable alternative to RNA-seq for detection of alternative splicing events when focusing on well-described transcriptional regions.