RNA-binding Protein Gene Research Articles

A Novel RNA Binding Protein-Related Prognostic Signature for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a highly malignant and aggressive cancer with high recurrence rates and mortality. Some studies have illustrated that RNA binding proteins (RBPs) were involved in the carcinogenesis and development of multiple cancers, but the roles in HCC were still unclear. We downloaded the RNA-seq and corresponding clinical information of HCC from The Cancer Genome Atlas (TCGA) database, and 330 differentially expressed RBPs were identified between normal and HCC tissues. Through series of the univariate, the least absolute shrinkage selection operator (LASSO), and the stepwise multivariate Cox regression analyses, six prognosis-related key RBPs (CNOT6, UPF3B, MRPL54, ZC3H13, IFIT5, and PPARGC1A) were screened out from DE RBPs, and a six-RBP gene risk score signature was constructed in training set. Survival analysis indicated that HCC patients with high-risk scores had significantly worse overall survival than low-risk patients, and furthermore, the signature can be used as an independent prognostic indicator. The good accuracy of this prognostic signature was confirmed by the ROC curve analysis and was further validated in the International Cancer Genome Consortium (ICGC) HCC cohort. Besides, a nomogram based on six RBP genes was established and internally validated in the TCGA cohort. Gene set enrichment analysis demonstrated some cancer-related phenotypes were significantly gathered in the high-risk group. Overall, our study first identified an RBP-related six-gene prognostic signature, which could serve as a promising prognostic biomarker and provide some potential therapeutic targets for HCC.

Predicting the Clinical Outcome of Lung Adenocarcinoma Using a Novel Gene Pair Signature Related to RNA-Binding Protein.

Adenocarcinoma is the most common type of lung cancer, and patients have varying prognoses. RNA-binding proteins (RBP) are deemed to be closely associated with tumorigenesis and development, but the exact mechanism is currently unknown. This study was aimed at constructing a new robust prognostic model based on RNA-binding protein-related gene pair scores for better clinical guidance. The model for this study was constructed based on data of lung adenocarcinoma from The Cancer Genome Atlas (TCGA) database. Prognosis-related RBP gene pair models were created based on differentially expressed genes, and the accuracy of the models was verified in a different age, staging, and other subdatasets. A total of 379 RNA-binding protein-related genes were differentially expressed in tumor tissue. From these genes, we constructed a prognostic model consisting of 33 gene pairs, which were found to be significantly associated with survival in TCGA dataset (P < 0.0001, hazard ratio (HR) = 4.380 (3.139 to 6.111)) and different subdatasets. As expected, the results were verified in the GEO validation cohort (P = 7.8 × 10−3, HR = 1.597 (1.095 to 2.325)). We found that the signature exhibited an independent prognostic factor in both the univariate and multivariate Cox regression analyses (P < 0.001). CIBERSORT was applied to estimate the fractions of infiltrated immune cells in bulk tumor tissues. CD8 T cells, activated dendritic cells, regulatory T cells (Tregs), and activated CD4 memory T cells presented a significantly lower fraction in the high-risk group (P < 0.01). Patients in the high-risk group had significantly higher tumor mutational burden (TMB) (P = 4.953e − 04) and lower levels of immune cells (P = 3.473e − 05) and stromal cells (P = 0.005) in the tumor microenvironment than those in the low-risk group. Furthermore, the Protein-protein interaction (PPI) network and various enrichment analyses have genuinely uncovered the interrelationships and potential functions of the RBP genes within the model. The results of the present study validated the importance of RNA-binding proteins in tumorigenesis and progression and support the RBP gene-related signature as a promising marker for prognosis prediction in lung adenocarcinoma.

Effects of RNA Binding Proteins on the Prognosis and Malignant Progression in Prostate Cancer.

Prostate cancer (PCa) is a common lethal malignancy in men. RNA binding proteins (RBPs) have been proven to regulate the biological processes of various tumors, but their roles in PCa remain less defined. In the present study, we used bioinformatics analysis to identify RBP genes with prognostic and diagnostic values. A total of 59 differentially expressed RBPs in PCa were obtained, comprising 28 upregulated and 31 downregulated RBP genes, which may play important roles in PCa. Functional enrichment analyses showed that these RBPs were mainly involved in mRNA processing, RNA splicing, and regulation of RNA splicing. Additionally, we identified nine RBP genes (EXO1, PABPC1L, REXO2, MBNL2, MSI1, CTU1, MAEL, YBX2, and ESRP2) and their prognostic values by a protein–protein interaction network and Cox regression analyses. The expression of these nine RBPs was validated using immunohistochemical staining between the tumor and normal samples. Further, the associations between the expression of these nine RBPs and pathological T staging, Gleason score, and lymph node metastasis were evaluated. Moreover, these nine RBP genes showed good diagnostic values and could categorize the PCa patients into two clusters with different malignant phenotypes. Finally, we constructed a prognostic model based on these nine RBP genes and validated them using three external datasets. The model showed good efficiency in predicting patient survival and was independent of other clinical factors. Therefore, our model could be used as a supplement for clinical factors to predict patient prognosis and thereby improve patient survival.

The RNA-Binding Protein CELF2 Inhibits Ovarian Cancer Progression by Stabilizing FAM198B

An increasing number of studies have clarified the functional roles of RNA-binding proteins (RBPs) in driving post-transcriptional mechanisms of cancer progression. In this study, we integrated data from the RBP database and Gene Expression Omnibus (GEO) data with RNA sequencing (RNA-seq) data from 10 ovarian cancer tissues and 8 normal ovarian tissues and identified an RBP, CUGBP- and ETR-3-like family 2 (CELF2). We found that CELF2 expression was downregulated in ovarian cancer and positively correlated with the overall survival (OS) and progression-free survival (PFS) of patients with ovarian cancer. Altered CELF2 expression led to changes in the proliferation, migration, and invasion of ovarian cancer cells in vitro and in vivo. CELF2 expression increased the stability of its target, FAM198B, by binding to AU/U-rich elements (AREs) in the 3′ untranslated region (3′ UTR). FAM198B knockdown restored the CELF2-mediated suppression of proliferation and migration. We also found that CELF2/FAM198B may repress ovarian cancer progression by inhibiting the mitogen-activated protein kinase/extracellular-regulated protein kinase (MAPK/ERK) signaling pathway. Finally, a curcumin-induced increase in CELF2 expression resulted in increased ovarian cancer cell sensitivity to cisplatin. Our study elucidated a novel mechanism by which the CELF2/FAM198B axis regulates proliferation and metastasis in ovarian cancer, providing novel, potential therapeutic targets for ovarian cancer.

Development and validation of a six-RNA binding proteins prognostic signature and candidate drugs for prostate cancer

The dysregulation of RNA binding proteins (RBPs) regulates the progression of several cancers. However, information on the overall functions of RBPs in prostate cancer (PCa) remains largely understudied. Therefore, based on the TCGA dataset, this study identified 144 differentially expressed RBPs in tumors compared to normal tissues. Subsequently, through univariate, LASSO and multivariate Cox regression analysis, 6 RBP genes among them, MSI1, MBNL2, LENG9, REXO2, RNASE1, and PABPC1L were screened as prognostic hub genes and prognostic signature was further identified. Further analysis indicated that the high-risk group was significantly associated with poor RFS, which was validated in the MSKCC cohort. Besides, patients in the high-risk group were closely associated with dysregulation of DNA damage repair pathway, copy number alteration, tumor burden mutation, and low-response to cisplatin (P < 0.001), and bicalutamide (P < 0.001). Using the Connectivity Map, we finally predicted 3 drugs including, ribavirin, carmustine, and carbenoxolone. In summary, we identified six-RBP gene signature and 3 potential drugs against PCa, which might promote the individualized treatment strategies and further improve the quality of life among PCa patients.

Development and validation of a RNA binding protein gene pair-associated prognostic signature for prediction of overall survival in hepatocellular carcinoma

BackgroundIncreasing evidence has demonstrated the correlation between hepatocellular carcinoma (HCC) prognosis and RNA binding proteins (RBPs) dysregulation. Thus, we aimed to develop and validate a reliable prognostic signature that can estimate the prognosis for HCC.MethodsGene expression profiling and clinical information of 374 HCC patients were derived from the TCGA data portal. The survival-related RBP pairs were determined using univariate cox-regression analysis and the signature was built based on LASSO analysis. All patients were divided patients into high-and low-risk groups according to the optimal cut off of the signature score determined by time-dependent receiver operating characteristic (ROC) curve analysis. The predictive value of the signature was further validated in an independent cohort.ResultsA 37-RBP pairs signature consisting of 61 unique genes was constructed which was significantly associated with the survival. The RBP-related signature accurately predicted the prognosis of HCC patients, and patients in high-risk groups showed poor survival in two cohorts. The novel signature was an independent prognostic factor of HCC in two cohorts (all P < 0.001). Furthermore, the C-index of the prognostic model was 0.799, which was higher than that of many established risk models. Pathway and process enrichment analysis showed that the 61 unique genes were mainly enriched in translation, ncRNA metabolic process, RNA splicing, RNA modification, and translational termination.ConclusionThe novel proposed RBP-related signature based on relative expression orderings could serve as a promising independent prognostic biomarker for patients with HCC, and could improve the individualized survival prediction in HCC.

S1 Domain RNA-Binding Protein CvfD Is a New Posttranscriptional Regulator That Mediates Cold Sensitivity, Phosphate Transport, and Virulence in Streptococcus pneumoniae D39.

Posttranscriptional gene regulation often involves RNA-binding proteins that modulate mRNA translation and/or stability either directly through protein-RNA interactions or indirectly by facilitating the annealing of small regulatory RNAs (sRNAs). The human pathogen Streptococcus pneumoniae D39 (pneumococcus) does not encode homologs to RNA-binding proteins known to be involved in promoting sRNA stability and function, such as Hfq or ProQ, even though it contains genes for at least 112 sRNAs. However, the pneumococcal genome contains genes for other RNA-binding proteins, including at least six S1 domain proteins: ribosomal protein S1 (rpsA), polynucleotide phosphorylase (pnpA), RNase R (rnr), and three proteins with unknown functions. Here, we characterize the function of one of these conserved, yet uncharacterized, S1 domain proteins, SPD_1366, which we have renamed CvfD (conserved virulence factor D), since loss of the protein results in attenuation of virulence in a murine pneumonia model. We report that deletion of cvfD impacts the expression of 144 transcripts, including the pst1 operon, encoding phosphate transport system 1 in S. pneumoniae We further show that CvfD posttranscriptionally regulates the PhoU2 master regulator of the pneumococcal dual-phosphate transport system by binding phoU2 mRNA and impacting PhoU2 translation. CvfD not only controls expression of phosphate transporter genes but also functions as a pleiotropic regulator that impacts cold sensitivity and the expression of sRNAs and genes involved in diverse cellular functions, including manganese uptake and zinc efflux. Together, our data show that CvfD exerts a broad impact on pneumococcal physiology and virulence, partly by posttranscriptional gene regulation.IMPORTANCE Recent advances have led to the identification of numerous sRNAs in the major human respiratory pathogen S. pneumoniae However, little is known about the functions of most sRNAs or RNA-binding proteins involved in RNA biology in pneumococcus. In this paper, we characterize the phenotypes and one target of the S1 domain RNA-binding protein CvfD, a homolog of general stress protein 13 identified, but not extensively characterized, in other Firmicutes species. Pneumococcal CvfD is a broadly pleiotropic regulator, whose absence results in misregulation of divalent cation homeostasis, reduced translation of the PhoU2 master regulator of phosphate uptake, altered metabolism and sRNA amounts, cold sensitivity, and attenuation of virulence. These findings underscore the critical roles of RNA biology in pneumococcal physiology and virulence.

Comprehensive Analysis of the Roles and Prognostic Value of RNA-Binding Proteins in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a malignancy with relatively high incidence and poor prognosis. RNA-binding proteins (RBPs) were reported to be dysregulated in multiple cancers and were closely associated with tumor initiation and progression. However, an integrated analysis of the roles of RBPs in HNSCC has not been conducted. In the present study, we obtained transcriptome data and corresponding clinical information of HNSCC patients from The Cancer Genome Atlas database and screened out differentially expressed RBPs between tumor and normal tissues. Subsequently, we utilized a series of bioinformatics analyses to elucidate the potential functions and prognostic value of these RBPs in HNSCC. As a result, a total of 88 aberrantly expressed RBPs were identified, including 63 downregulated and 25 upregulated RBPs. Functional enrichment analysis suggested that the differentially expressed RBPs mainly participated in mRNA metabolic processes, RNA processing, RNA transport, regulation of RNA stability, RNA degradation, and mRNA surveillance pathway. Three RBP genes (NOVA1, EZH2, and RBM24) were determined as prognosis-related hub genes from which EZH2 and NOVA1 were selected to construct a prognostic signature based on LASSO Cox regression algorithm. Further analysis demonstrated that the high-risk patient group stratified by the risk signature has advanced tumor grade and poorer overall survival when compared with low-risk group. Moreover, univariate analysis showed that the risk score, tumor stage, T stage, and N stage were significantly associated with patient overall survival and the multivariate analysis results indicated that the risk score and age were greatly correlated with patient prognosis. Overall, this study provided a comprehensive landscape of RBPs in HNSCC and identified an effective gene signature for predicting the clinical outcomes of HNSCC patient, which may contribute to clinical decision making and individualized cancer treatment.

Identification of splice isoforms in T helper cells using Nanopore-based cDNA sequencing

Abstract The T helper (Th) cell compartment of the adaptive immune response provides a central mechanism of balance between inflammation and immune tolerance. The differentiation of naïve Th cells into both regulatory and effector Th subsets is regulated through complex gene regulatory networks that lead to distinct transcriptomes. Alternative RNA splicing is a major component of cellular differentiation that allows protein heterogeneity and mechanistic diversity across cell types. However, our understanding of alternative splicing control is still limited. Here we show differential expression of RNA binding Proteins (RBP) genes among Th cell subsets suggesting a regulatory role of RBPs in RNA splicing and Th differentiation. In order to interrogate the regulatory gene network, we utilized innovative Nanopore-based sequencing to generate a single molecule transcriptome map of naïve CD4 T cells, and differentiated Treg and Th1 cells to characterize full-length cDNA transcripts and alternatively spliced isoforms. Gene expression was compared across four major gene categories; RBPs, Transcription Factors (TFs), Other Protein Coding (OPC) and LncRNA. Our study revealed that RBPs exhibited high splicing potential while LncRNA exhibited relatively less splicing potential in comparison to other gene categories in all cell types. Overall, we identify RBPs that are both differentially expressed in Th subsets and differentially spliced themselves. This might represent an important control mechanism of functional protein diversity in regulating immune responses.

Characteristic Features of FUS Inclusions in Spinal Motor Neurons of Sporadic Amyotrophic Lateral Sclerosis.

Alterations of RNA metabolism caused by mutations in RNA-binding protein genes, such as transactivating DNA-binding protein-43 (TDP-43) and fused in sarcoma (FUS), have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Unlike the accumulation of TDP43, which is accepted as a pathological hall mark of sporadic ALS (sALS), FUS pathology in sALS is still under debate. Although immunoreactive inclusions of FUS have been detected in sALS patients previously, the technical limitation of signal detection, including the necessity of specific antigen retrieval, restricts our understanding of FUS-associated ALS pathology. In this study, we applied a novel detection method using a conventional antigen retrieval technique with Sudan Black B treatment to identify FUS-positive inclusions in sALS patients. We classified pathological motor neurons into 5 different categories according to the different aggregation characteristics of FUS and TDP-43. Although the granular type was more dominant for inclusions with TDP-43, the skein-like type was more often observed in FUS-positive inclusions, suggesting that these 2 proteins undergo independent aggregation processes. Moreover, neurons harboring FUS-positive inclusions demonstrated substantially reduced expression levels of dynactin-1, a retrograde motor protein, indicating that perturbation of nucleocytoplasmic transport is associated with the formation of cytoplasmic inclusions of FUS in sALS.

Integrated Analysis of the Functions and Prognostic Values of RNA Binding Proteins in Lung Squamous Cell Carcinoma.

Lung cancer is the leading cause of cancer-related deaths worldwide. Dysregulation of RNA binding proteins (RBPs) has been found in a variety of cancers and is related to oncogenesis and progression. However, the functions of RBPs in lung squamous cell carcinoma (LUSC) remain unclear. In this study, we obtained gene expression data and corresponding clinical information for LUSC from The Cancer Genome Atlas (TCGA) database, identified aberrantly expressed RBPs between tumors and normal tissue, and conducted a series of bioinformatics analyses to explore the expression and prognostic value of these RBPs. A total of 300 aberrantly expressed RBPs were obtained, comprising 59 downregulated and 241 upregulated RBPs. Functional enrichment analysis indicated that the differentially expressed RBPs were mainly associated with mRNA metabolic processes, RNA processing, RNA modification, regulation of translation, the TGF-beta signaling pathway, and the Toll-like receptor signaling pathway. Nine RBP genes (A1CF, EIF2B5, LSM1, LSM7, MBNL2, RSRC1, TRMU, TTF2, and ZCCHC5) were identified as prognosis-associated hub genes by univariate, least absolute shrinkage and selection operator (LASSO), Kaplan–Meier survival, and multivariate Cox regression analyses, and were used to construct the prognostic model. Further analysis demonstrated that high risk scores for patients were significantly related to poor overall survival according to the model. The area under the time-dependent receiver operator characteristic curve of the prognostic model was 0.712 at 3 years and 0.696 at 5 years. We also developed a nomogram based on nine RBP genes, with internal validation in the TCGA cohort, which showed a favorable predictive efficacy for prognosis in LUSC. Our results provide novel insights into the pathogenesis of LUSC. The nine-RBP gene signature showed predictive value for LUSC prognosis, with potential applications in clinical decision-making and individualized treatment.

Transcriptomic Analysis Identifies RNA Binding Proteins as Putative Regulators of Myelopoiesis and Leukemia.

Acute myeloid leukemia (AML) is a common and aggressive hematological malignancy. Acquisition of heterogeneous genetic aberrations and epigenetic dysregulation lead to the transformation of hematopoietic stem cells (HSC) into leukemic stem cells (LSC), which subsequently gives rise to immature blast cells and a leukemic phenotype. LSCs are responsible for disease relapse as current chemotherapeutic regimens are not able to completely eradicate these cellular sub-populations. Therefore, it is critical to improve upon the existing knowledge of LSC specific markers, which would allow for specific targeting of these cells more effectively allowing for their sustained eradication from the cellular milieu. Although significant milestones in decoding the aberrant transcriptional network of various cancers, including leukemia, have been achieved, studies on the involvement of post-transcriptional gene regulation (PTGR) in disease progression are beginning to unfold. RNA binding proteins (RBPs) are key players in mediating PTGR and they regulate the intracellular fate of individual transcripts, from their biogenesis to RNA metabolism, via interactions with RNA binding domains (RBDs). In this study, we have used an integrative approach to systematically profile RBP expression and identify key regulatory RBPs involved in normal myeloid development and AML. We have analyzed RNA-seq datasets (GSE74246) of HSCs, common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), monocytes, LSCs, and blasts. We observed that normal and leukemic cells can be distinguished on the basis of RBP expression, which is indicative of their ability to define cellular identity, similar to transcription factors. We identified that distinctly co-expressing modules of RBPs and their subclasses were enriched in hematopoietic stem/progenitor (HSPCs) and differentiated monocytes. We detected expression of DZIP3, an E3 ubiquitin ligase, in HSPCs, knockdown of which promotes monocytic differentiation in cell line model. We identified co-expression modules of RBP genes in LSCs and among these, distinct modules of RBP genes with high and low expression. The expression of several AML-specific RBPs were also validated by quantitative polymerase chain reaction. Network analysis identified densely connected hubs of ribosomal RBP genes (rRBPs) with low expression in LSCs, suggesting the dependency of LSCs on altered ribosome dynamics. In conclusion, our systematic analysis elucidates the RBP transcriptomic landscape in normal and malignant myelopoiesis, and highlights the functional consequences that may result from perturbation of RBP gene expression in these cellular landscapes.

Abstract 3390: RNA interactome topology perturbation analysis reveals candidate driver mutations in cancer

Abstract Identification of the driver genomic variants is important for precision therapy of human cancer. Interaction between RNA-binding proteins (RBPs) and RNA plays important roles in regulating cellular function. Decoding genome-wide protein-RNA regulatory networks as well as how cancer-related mutations impair RNA regulatory activities in cancer will help prioritizing candidate driver mutations. Here, we first explored the genetic alteration patterns of RBPs and found that candidate driver mutations are likely to occur on the surface of RBPs. This observations indicate that the driver mutations may perturb the RBP regulatory network in cancer. We then constructed protein-RNA interactome networks by integration of target binding screens and expression profiles. Regulatory network analysis highlights regulatory principles among interacting RBPs. In addition, somatic mutations selectively target functionally important genes (cancer genes, core fitness genes or conserved genes) in cancer. These regulatory patterns were further validated using independent data. A computational method (MERIT) and a web-based user friendly resource were further proposed to analyze the RBP-gene regulatory network perturbation induced by mutations across cancer types. Pan-cancer analysis also suggests that cancer cells selectively target "vulnerability" genes to perturb protein-RNA interactome that is involved in cancer hallmark-related functions. Based on the expression of perturbed RBP and target genes, we identified three subtypes of liver cancer with different survival rates. Specifically, we experimentally validated four pairs of RBP-gene interactions perturbed by mutations, which play critical roles in cell proliferation. Taken together, our results provide a valuable resource for characterizing somatic mutation-perturbed protein-RNA regulatory networks in cancer, yielding valuable insights into the genotype-phenotype relationships underlying human cancer, and potential biomarkers for precision medicine. Note: This abstract was not presented at the meeting. Citation Format: Yongsheng Li, Daniel J. McGrail, Juan Xu, Junyi Li, Song Yi, Nidhi Sahni. RNA interactome topology perturbation analysis reveals candidate driver mutations in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3390.

Abstract 1646: Beyond BRCA: Discovery of novel drivers of homologous recombination deficiencies in cancer

Abstract Since the discovery of BRCA1 and BRCA2 mutations as cancer risk factors, we have gained much understanding of their role in maintaining genomic stability through homologous recombination (HR) DNA repair. However, mutations in BRCA1/2 and other classical HR proteins such as RAD51 and PALB2 only identify 10-20% of TCGA patients who display HR deficiencies, indicating that we do not understand the vast majority of HR defects. Here, we leveraged the abundance of molecular characterization from TCGA patients for network analysis to fill this knowledge void. We discovered that over half of HR deficiencies originate outside of canonical DNA damage response genes, with particular enrichment for RNA binding proteins (RBPs). Experimental techniques validated over 90% of our predictions in a panel of 50 genes tested by siRNA, as well as 30/31 additional engineered mutations identified in TCGA patients. We further cross-validated these findings in independent patient cohorts, finding that the identified RNA processing mutations again enriched for HR deficient patients to an equal or greater degree than mutations in DNA damage genes. Using a series of experimental approaches, including protein interactome screening, RNA sequencing, and quantitative imaging cytometry, we probed how loss of RBP function induced global DNA damage response rewiring, including changes in RNA splicing, protein-protein interactions, and recruitment of repair factors to DNA damage sites. Clinically, defects in HR are known to promote cancer initiation, but also sensitize cells to targeted therapies such as PARP inhibition. We found that depletion RBPs from PARP-resistant triple-negative breast cancer cells induced sensitivity to multiple PARP inhibitors, indicating that this could identify new cohorts of patients who may benefit from PARP inhibition, beyond the small number of BRCA1/2-mutant patients. Moreover, we find that the identified RBP genes are significantly enriched for genes associated with cancer risk identified through GWAS. In patients, HR deficiencies were not equally distributed across all demographics, so preferential screening of those most at risk could further heighten this benefit. Thus, these novel drivers may show clinical relevance both for treatment stratification and for identifying individuals at high risk for cancer development to improve patient outcomes. Taken together, this exhaustive study greatly expands our repertoire of known drivers of HR deficiencies and mechanisms of damage repair, which may impact research from the most basic biology studies to clinical screening and stratification. Citation Format: Nidhi Sahni, Daniel McGrail, Yongsheng Li, Gordon Mills, Song (Stephen) Yi. Beyond BRCA: Discovery of novel drivers of homologous recombination deficiencies in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1646.

Genetic variants in RNA binding protein (RBP) to predict outcome in metastatic colorectal cancer (mCRC): Data from FIRE-3, TRIBE, and MAVERICC trials.

3545 Background: RNA binding proteins (RBPs) post-transcriptionally regulate gene expression by stabilizing or destabilizing target messenger RNA. Although alteration of RBPs affects many steps of cancer development, its clinical implication in mCRC remains unclear. Methods: We analyzed data from mCRC patients (pts) enrolled in three first-line randomized trials (FIRE-3, TRIBE, and MAVERICC). Genomic DNA from blood samples of pts was genotyped through the OncoArray, a custom array manufactured by Illumina. Candidate 30 SNPs in 10 RBP genes (MSI1, MSI2, ELAVL1, RBM3, LIN28A, LIN28B, IGF2BP1, IGF2BP2, IGF2BP3, ZFP36) were tested on association with progression-free survival (PFS) and overall survival (OS). To evaluate prognostic effects and heterogeneities across treatment arms, meta-analysis approach using the METASOFT software was conducted. We also tested interaction between each SNP and treatment within each trial, i.e. FIRE-3 cohort (FOLFIRI+cetuximab (Cet) vs FOLFIRI+bevacizumab (Bev)) and MAVERICC cohort (FOLFIRI+Bev vs FOLFOX6+Bev). For multiple testing, p values were adjusted by the false discovery rate (FDR) method. Results: A total of 884 pts’ SNPs data were available (FIRE-3: n = 236, TRIBE: n = 324, and MAVERICC: n = 324). Meta-analysis combining three trials showed RBM3 rs926152 (adjusted p = 0.045) and RBM3 rs2249585 (adjusted p = 0.016) were significantly prognostic for PFS. Whereas, in terms of OS, only LIN28B rs314277 (adjusted p = 0.045) was significant, and RBM3 rs926152 (adjusted p = 0.057) and RBM3 rs2249585 (adjusted p = 0.059) had a trend. Interaction test showed several SNPs were potentially predictive (raw p &lt; 0.05), although without any significance after FDR adjustment: in FIRE-3 cohort, MSI2 rs1822381, RBM3 rs926152, LIN28B rs221635, IGF2BP1 rs2969 for OS; in MAVERICC cohort, MSI1 rs1179442 and MSI2 rs3826301 for OS, ELAVL1 rs4804244 for PFS. Conclusions: Our results indicate prognostic potential of SNPs in RBP genes, such as RBM3 and LIN28B, in mCRC. However, we find no distinct evidence that these SNPs can predict differential effect between Cet and Bev, or between oxaliplatin- and irinotecan-based chemotherapy.

MERIT: Systematic Analysis and Characterization of Mutational Effect on RNA Interactome Topology.

The interaction between RNA‐binding proteins (RBPs) and RNA plays an important role in regulating cellular function. However, decoding genome‐wide protein–RNA regulatory networks as well as how cancer‐related mutations impair RNA regulatory activities in hepatocellular carcinoma (HCC) remains mostly undetermined. We explored the genetic alteration patterns of RBPs and found that deleterious mutations are likely to occur on the surface of RBPs. We then constructed protein–RNA interactome networks by integration of target binding screens and expression profiles. Network analysis highlights regulatory principles among interacting RBPs. In addition, somatic mutations selectively target functionally important genes (cancer genes, core fitness genes, or conserved genes) and perturb the RBP–gene regulatory networks in cancer. These regulatory patterns were further validated using independent data. A computational method (Mutational Effect on RNA Interactome Topology) and a web‐based, user‐friendly resource were further proposed to analyze the RBP–gene regulatory networks across cancer types. Pan‐cancer analysis also suggests that cancer cells selectively target “vulnerability” genes to perturb protein–RNA interactome that is involved in cancer hallmark–related functions. Specifically, we experimentally validated four pairs of RBP–gene interactions perturbed by mutations in HCC, which play critical roles in cell proliferation. Based on the expression of perturbed RBP and target genes, we identified three subtypes of HCC with different survival rates. Conclusion: Our results provide a valuable resource for characterizing somatic mutation‐perturbed protein–RNA regulatory networks in HCC, yielding valuable insights into the genotype–phenotype relationships underlying human cancer, and potential biomarkers for precision medicine.

Transcriptome‐Wide Analysis Reveals the Landscape of Aberrant Alternative Splicing Events in Liver Cancer

Alternative splicing (AS) is assumed to be a pivotal determinant for the generation of diverse transcriptional variants in cancer. However, the comprehensive dysregulation of AS and the prospective biological and clinical relevance in hepatocellular carcinoma (HCC) remain obscure. Here, we identified and depicted the AS landscape in HCC by performing reference-based assembly of sequencing reads from over 600 RNA sequencing (RNA-seq) libraries. We detected various differentially spliced ASEs across patients covering not only protein-coding genes, but also considerable numbers of noncoding genes. Strikingly, alternative transcription initiation was found to frequently occur in HCC. These differential ASEs were highly related to "cancer hallmarks" and involved in metabolism-related pathways in particular. In addition, 243 differential ASEs were identified as risk predictors for HCC patient survival. The isoform switch of metabolism-related gene UGP2 (UDP-glucose pyrophosphorylase 2) might play an essential role in HCC. We further constructed regulatory networks between RNA-binding protein (RBP) genes and the corresponding ASEs. Further analysis demonstrated that the regulated networks were enriched in a variety of metabolism-related pathways. Conclusion: Differential ASEs are prevalent in HCC, where alternative transcription initiation was found to frequently occur. We found that genes having differential ASEs were significantly enriched in metabolism-related pathways. The expression variations, binding relations, and even mutations of RBP genes largely influenced differential ASEs in HCC.

Corrigendum: Large-scale analysis of genome and transcriptome alterations in multiple tumors unveils novel cancer-relevant splicing networks

Alternative splicing is regulated by multiple RNA-binding proteins and influences the expression of most eukaryotic genes. However, the role of this process in human disease, and particularly in cancer, is only starting to be unveiled. We systematically analyzed mutation, copy number, and gene expression patterns of 1348 RNA-binding protein (RBP) genes in 11 solid tumor types, together with alternative splicing changes in these tumors and the enrichment of binding motifs in the alternatively spliced sequences. Our comprehensive study reveals widespread alterations in the expression of RBP genes, as well as novel mutations and copy number variations in association with multiple alternative splicing changes in cancer drivers and oncogenic pathways. Remarkably, the altered splicing patterns in several tumor types recapitulate those of undifferentiated cells. These patterns are predicted to be mainly controlled by MBNL1 and involve multiple cancer drivers, including the mitotic gene NUMA1 We show that NUMA1 alternative splicing induces enhanced cell proliferation and centrosome amplification in nontumorigenic mammary epithelial cells. Our study uncovers novel splicing networks that potentially contribute to cancer development and progression.

MiR394 Acts as a Negative Regulator of Arabidopsis Resistance to B. cinerea Infection by Targeting LCR.

Gray mold of tomato is caused by the pathogen Botrytis cinerea. MicroRNAs play a crucial role in the biotic and abiotic stress responses of plants and regulate their targets by gene silencing. miR394 is an ancient and conserved miRNA in plants, and it participates in the regulation of plant development and stress responses. In our previous study, miR394 was found to respond to B. cinerea infection in tomato, but the roles and regulatory mechanisms of miR394 in B. cinerea-infected tomato remain unclear. miR394 was down-regulated in tomato in response to B. cinerea infection, showing an expression pattern opposite to the previous finding that miR394 was up-regulated in tomato cv. Jinpeng 1 infected by B. cinerea. We obtained transgenic Arabidopsis overexpressing miR394, which resulted in low expression levels of its target LEAF CURLING RESPONSIVENESS (LCR). Leaf lesion size and trypan blue staining showed that miR394 overexpression led to increased sensitivity of transgenic Arabidopsis to B. cinerea compared to wild type. We also detected changes in the expression levels of stress-related miRNAs, including miR159, miR156, miR168, and miR172. In the transgenic plants, it indicated potential cross talk between these miRNAs and miR394, except for miR159. miR394 also enhanced the expression of ARGONAUTE 1 (AGO1), DSRNA-BINDING PROTEIN 4 (DRB4) and the RNA-binding protein gene DAWDLE (DDL), which are involved in the pathways of miRNA biosynthesis and regulation, suggesting that miR394 overexpression has a feedback effect on these genes. Our data indicate that overexpression of miR394 in Arabidopsis increased the susceptibility of plants to B. cinerea by affecting the expression of its target gene LCR along with a number of key genes involved in plant miRNA metabolism (AGO1). Thus, miR394 is a negative regulator of Arabidopsis resistance to B. cinerea infection by targeting LCR.

Synthetic Genetic Interaction (CRISPR-SGI) Profiling in Caenorhabditis elegans.

Genetic interaction screens are a powerful methodology to establish novel roles for genes and elucidate functional connections between genes. Such studies have been performed to great effect in single-cell organisms such as yeast and E. coli (Schuldiner et al., 2005; Butland et al., 2008; Costanzo et al., 2010), but similar large-scale interaction studies using targeted reverse-genetic deletions in multi-cellular organisms have not been feasible. We developed a CRISPR/Cas9-based method for deleting genes in C. elegans and replacing them with a heterologous fluorescent reporter (Norris et al., 2015). Recently we took advantage of that system to perform a large-scale, reverse genetic screen using null alleles in animals for the first time, focusing on RNA binding protein genes (Norris et al., 2017). This type of approach should be similarly applicable to many other gene classes in C. elegans. Here we detail the protocols involved in generating a library of double mutants and performing medium-throughput competitive fitness assays to test for genetic interactions resulting in fitness changes.