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Programmed ribosomal frameshifting (PRF) is a key mechanism that viruses use to generate essential proteins for replication, and as a means of regulating gene expression. PRF generally involves recoding signals or frameshift stimulators to elevate the occurrence of frameshifting at shift-prone 'slippery' sequences. Given its essential role in viral replication, targeting PRF was envisioned as an attractive tool to block viral infection. However, in contrast to controlled-PRF mechanisms, recent studies have shown that ribosomes of many human cancer cell types are prone to frameshifting upon amino acid shortage; thus, these cells are deemed to be sloppy. The resulting products of a sloppy frameshift at the 'hungry' codons are aberrant proteins the degradation and display of which at the cell surface can trigger T cell activation. In this review, we address recent discoveries in ribosomal frameshifting and their functional consequences for the proteome in human cancer cells.

Accumulating evidence identifies non-genetic mechanisms substantially contributing to drug resistance in cancer patients. Preclinical and clinical data implicate the transcriptional co-activators YAP1 and its paralog TAZ in resistance to multiple targeted therapies, highlighting the strong need for therapeutic strategies overcoming YAP1/TAZ-mediated resistance across tumor entities. Here, we show particularly high YAP1/TAZ activity in MITFlow/AXLhigh melanomas characterized by resistance to MAPK pathway inhibition and broad receptor tyrosine kinase activity. To uncover genetic dependencies of melanoma cells with high YAP1/TAZ activity, we used a genome-wide CRISPR/Cas9 functional screen and identified SLC35B2, the 3′-phosphoadenosine-5′-phosphosulfate transporter of the Golgi apparatus, as an essential gene for YAP1/TAZ-driven drug resistance. SLC35B2 expression correlates with tumor progression, and its loss decreases heparan sulfate expression, reduces receptor tyrosine kinase activity, and sensitizes resistant melanoma cells to BRAF inhibition in vitro and in vivo. Thus, targeting heparan sulfation via SLC35B2 represents a novel approach for breaking receptor tyrosine kinase-mediated resistance to MAPK pathway inhibitors.

PDF file, 732K, Differentially expressed genes group A microRNAs.

PDF file, 711K, Differentially expressed genes group B microRNAs.

PDF file - 155K, Sequencing statistics

PDF file, 96K, Supplementary Figure S1 First validation of the identified 19 microRNAs that seemed to elicit a tumor-selective lethal effect. Supplementary Figure S2 Effect of the six tumor-selective microRNAs on non-HNSCC cell lines. Supplementary Figure S3 Phenotypic effect of miR-181a overexpression in time, either when introduced by retroviral transduction or transient transfection in HNSCC cell line VU-SCC-120. Supplementary Figure S4 ATM expression after transient transfection of expression constructs with ATM and a kinase-dead ATM mutant lacking the 3'UTR of ATM.

PDF file - 3.2MB, PAS_J values and p-values describing APA during colon cancer progression

PDF file, 153K, Predicted binding sites of the six tumor-selective microRNAs in ATM and CD40.

PDF file - 87K, 3'PCR of proximal and distal PASs isoforms in relation to beta-actin. *p

PDF file - 97K, Correlation between APA and mRNA abundance. Negative PAS_J values indicate 3'UTR shortening, and vice versa

PDF file - 580K, 3'PCR analysis of APA events in tissue subtypes. A=adenoma without chromosomal aberrations, Adenoma +Ab.=adenoma with chromosomal aberrations, MSS=microsatellite stable, MSI=microsatellite instable. *p

Purpose: The prognosis of head and neck squamous cell carcinomas (HNSCC) remains disappointing and the development of novel anti-cancer agents is urgently awaited. We identified by a functional genetic screen microRNAs that are selectively lethal for head and neck cancer cells but not for normal cells. We further investigated the genes targeted by these microRNAs.Experimental Design: A retroviral expression library of human microRNAs was introduced in HNSCC cell lines and normal oropharyngeal keratinocytes to identify tumor-selective lethal microRNAs. Potential downstream gene targets of these microRNAs were identified by gene expression profiling and validated by functional assays.Results: We identified six microRNAs that selectively inhibit proliferation of head and neck cancer cells. By gene expression profiling and 3′-untranslated region (UTR) luciferase reporter assays, we showed that the ataxia telangiectasia mutated (ATM) gene is a common target for at least two and likely three of these microRNAs. Specific inhibition of ATM resulted in a similar tumor-specific lethal effect, whereas the phenotype was reverted in rescue experiments.Conclusions: These six microRNAs might be developed as novel anti-cancer agents and highlight ATM as an interesting novel therapeutic target for head and neck cancer. Clin Cancer Res; 19(20); 5647–57. ©2013 AACR.

PDF file, 107K, Data of primary screen.

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PDF file - 99K, APA events during colon cancer progression. A. UTR-APA and CR-APA events. B. 3'UTR shortening versus lengthening in UTR-APA

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PDF file, 437K, Genes predicted to have potential binding sites for the six tumor-slective lethal microRNAs.

Purpose: Alternative cleavage and polyadenylation (APA) of mRNAs is a phenomenon that alters 3′-untranslated region length leading to altered posttranscriptional regulation of gene expression. Changing APA patterns have been shown to result in misregulation of genes involved in carcinogenesis; therefore, we hypothesized that altered APA contributes to progression of colorectal cancer, and that measurement of APA may lead to discovery of novel biomarkers.Experimental Design: We used next-generation sequencing to directly measure global patterns of APA changes during colorectal carcinoma progression in 15 human patient samples. Results were validated in a larger cohort of 50 patients, including 5 normal/carcinoma pairs from individuals.Results: We discovered numerous genes presenting progressive changes in APA. Genes undergoing untranslated region (3′UTR) shortening were enriched for functional groups such as cell-cycle and nucleic acid–binding and processing factors, and those undergoing 3′UTR lengthening or alternative 3′UTR usage were enriched for categories such as cell–cell adhesion and extracellular matrix. We found indications that APA changes result from differential processing of transcripts because of increased expression of cleavage and polyadenylation factors. Quantitative PCR analysis in a larger series of human patient samples, including matched pairs, confirmed APA changes in DMKN, PDXK, and PPIE genes.Conclusions: Our results suggest that genes undergoing altered APA during human cancer progression may be useful novel biomarkers and potentially targeted for disease prevention and treatment. We propose that the strategy presented here may be broadly useful in discovery of novel biomarkers for other types of cancer and human disease. Clin Cancer Res; 18(19); 5256–66. ©2012 AACR.

Supplementary Figure 1 from Regulation of the Adenomatous Polyposis Coli Gene by the miR-135 Family in Colorectal Cancer

Supplementary Figure and Table Legends from Regulation of the Adenomatous Polyposis Coli Gene by the miR-135 Family in Colorectal Cancer

Supplementary Figure 3 from Regulation of the Adenomatous Polyposis Coli Gene by the miR-135 Family in Colorectal Cancer

Supplementary Table 1 from Regulation of the Adenomatous Polyposis Coli Gene by the miR-135 Family in Colorectal Cancer

Supplementary Figure 2 from Regulation of the Adenomatous Polyposis Coli Gene by the miR-135 Family in Colorectal Cancer

mRNA translation is a highly conserved and tightly controlled mechanism for protein synthesis and is well known to be altered by oncogenes to promote cancer development. This distorted mRNA translation is accompanied by the vulnerability of cancer to inhibitors of key mRNA translation components. Novel studies also suggest that these alternations could be utilized for immunotherapy. Ribosome heterogeneity and alternative responses to nutrient shortages, which aid cancer growth and spread, are proposed to elicit aberrant protein production but may also result in previously unidentified therapeutic targets, such as the presentation of cancer-specific peptides at the surface of cancer cells (neoepitopes). This review will assess the driving forces in tRNA and ribosome function that underlie proteome diversification due to alterations in mRNA translation in cancer cells.

Immune-checkpoint blockade therapy has been successfully applied to many cancers, particularly tumors that harbor a high mutational burden and consequently express a high abundance of neoantigens. However, novel approaches are needed to improve the efficacy of immunotherapy for treating tumors that lack a high load of classic genetically derived neoantigens. Recent discoveries of broad classes of nongenetically encoded and inducible neoepitopes open up new avenues for therapeutic development to enhance sensitivity to immunotherapies. In this review, we discuss recent work on neoantigen discovery, with an emphasis on novel classes of noncanonical neoepitopes.

The use of alternative promoters, splicing, and cleavage and polyadenylation (APA) generates mRNA isoforms that expand the diversity and complexity of the transcriptome. Here, we uncovered thousands of previously undescribed 5' uncapped and polyadenylated transcripts (5' UPTs). We show that these transcripts resist exonucleases due to a highly structured RNA and N6-methyladenosine modification at their 5' termini. 5' UPTs appear downstream of APA sites within their host genes and are induced upon APA activation. Strong enrichment in polysomal RNA fractions indicates 5' UPT translational potential. Indeed, APA promotes downstream translation initiation, non-canonical protein output, and consistent changes to peptide presentation at the cell surface. Lastly, we demonstrate the biological importance of 5' UPTs using Bcl2, a prominent anti-apoptotic gene whose entire coding sequence is a 5' UPT generated from 5' UTR-embedded APA sites. Thus, APA is not only accountable for terminating transcripts, but also for generating downstream uncapped RNAs with translation potential and biological impact.

Tumor relapse as a consequence of chemotherapy resistance is a major clinical challenge in advanced stage breast tumors. To identify processes associated with poor clinical outcome, we took a mass spectrometry‐based proteomic approach and analyzed a breast cancer cohort of 113 formalin‐fixed paraffin‐embedded samples. Proteomic profiling of matched tumors before and after chemotherapy, and tumor‐adjacent normal tissue, all from the same patients, allowed us to define eight patterns of protein level changes, two of which correlate to better chemotherapy response. Supervised analysis identified two proteins of proline biosynthesis pathway, PYCR1 and ALDH18A1, that were significantly associated with resistance to treatment based on pattern dominance. Weighted gene correlation network analysis of post‐treatment samples revealed that these proteins are associated with tumor relapse and affect patient survival. Functional analysis showed that knockdown of PYCR1 reduced invasion and migration capabilities of breast cancer cell lines. PYCR1 knockout significantly reduced tumor burden and increased drug sensitivity of orthotopically injected ER‐positive tumor in vivo, thus emphasizing the role of PYCR1 in resistance to chemotherapy., Proteomic profiling of matched tumor and normal samples, associates distinct proteomic patterns with patient prognosis in breast cancer. Functional studies in vivo support the effectiveness of PYCR1 suppression in combination with chemotherapeutics in clinical settings.

Genetic variants associated with common diseases are usually located in noncoding parts of the human genome. Delineation of the full repertoire of functional noncoding elements, together with efficient methods for probing their biological roles, is therefore of crucial importance. Over the past decade, DNA accessibility and various epigenetic modifications have been associated with regulatory functions. Mapping these features across the genome has enabled researchers to begin to document the full complement of putative regulatory elements. High-throughput reporter assays to probe the functions of regulatory regions have also been developed but these methods separate putative regulatory elements from the chromosome so that any effects of chromatin context and long-range regulatory interactions are lost. Definitive assignment of function(s) to putative cis-regulatory elements requires perturbation of these elements. Genome-editing technologies are now transforming our ability to perturb regulatory elements across entire genomes. Interpretation of high-throughput genetic screens that incorporate genome editors might enable the construction of an unbiased map of functional noncoding elements in the human genome.

Summary Transcription and translation are two main pillars of gene expression. Due to the different timings, spots of action, and mechanisms of regulation, these processes are mainly regarded as distinct and generally uncoupled, despite serving a common purpose. Here, we sought for a possible connection between transcription and translation. Employing an unbiased screen of multiple human promoters, we identified a positive effect of TATA box on translation and a general coupling between mRNA expression and translational efficiency. Using a CRISPR-Cas9-mediated approach, genome-wide analyses, and in vitro experiments, we show that the rate of transcription regulates the efficiency of translation. Furthermore, we demonstrate that m6A modification of mRNAs is co-transcriptional and depends upon the dynamics of the transcribing RNAPII. Suboptimal transcription rates lead to elevated m6A content, which may result in reduced translation. This study uncovers a general and widespread link between transcription and translation that is governed by epigenetic modification of mRNAs., Graphical Abstract, Highlights • Transcription rates of mRNAs positively correlate with rates of their translation • Dynamics of RNA polymerase II impact the deposition of m6A on mRNAs • Suboptimal transcription enhances m6A modification of mRNAs • Excessive m6A modification is detrimental for the translation process, Slowly transcribed mRNAs show increased levels of N6-adenosine methylation and reduced translation efficiency, setting up a nuclear control on protein abundance.

Genome-wide chromatin studies identified the tumor suppressor p53 as both a promoter and an enhancer-binding transcription factor. As an enhancer factor, p53 can induce local production of enhancer RNAs, as well as transcriptional activation of distal neighboring genes. Beyond the regulation of protein-coding genes, p53 has the capacity to regulate long intergenic noncoding RNA molecules (lincRNAs); however, their importance to the p53 tumor suppressive function remains poorly characterized. Here, we identified and characterized a novel p53-bound intronic enhancer that controls the expression of its host, the lincRNA00475 (linc-475). We demonstrate the requirement of linc-475 for the proper induction of a p53-dependent cell cycle inhibitory response. We further confirm the functional importance of linc-475 in the maintenance of CDKN1A/p21 levels, a cell cycle inhibitor and a major p53 target gene, following p53 activation. Interestingly, loss of linc-475 reduced the binding of both p53 and RNA polymerase II (RNAPII) to the promoter of p21, attenuating its transcription rate following p53 activation. Altogether, our data suggest a direct role of p53-bound enhancer domains in the activation of lincRNAs required for an efficient p53 transcriptional response.Oncogene advance online publication, 18 January 2016; doi:10.1038/onc.2015.502.

microRNA (miRNA) dysregulation is a common feature of cancer cells, but the complex roles of miRNAs in cancer are not fully elucidated. Here, we used functional genomics to identify oncogenic miRNAs in non-small cell lung cancer and evaluate their impact on response to epidermal growth factor (EGFR)-targeting therapy. Our data demonstrate that miRNAs with an AAGUGC motif in their seed sequence increase both cancer cell proliferation and sensitivity to EGFR inhibitors. Global transcriptomics, proteomics and target prediction resulted in the identification of several tumor suppressors involved in the G1/S transition as AAGUGC-miRNA targets. The clinical implications of our findings were evaluated by analysis of AAGUGC-miRNA expression in multiple cancer types, supporting the link between this miRNA seed family, their tumor suppressor targets and cancer cell proliferation. In conclusion, we propose the AAGUGC seed motif as an oncomotif and that oncomotif-miRNAs promote cancer cell proliferation. These findings have potential therapeutic implications, especially in selecting patients for EGFR-targeting therapy.

Ovarian cancer accounts for the highest number of gynecologic-associated deaths in the developed world, and resistance to platinum-based therapy represents a major clinical and societal challenge in patients’ management. Since metabolism is intertwined with signaling pathways controlling cell death, we aimed to investigate to what extent metabolic adaptations could contribute to the development of the resistant phenotype. By performing isotope-labeled 13C-glucose tracer analysis in vitro, we found that, when they become resistant to platinum, ovarian cancer cells stop to synthetize serine and are characterized by significantly lower intracellular levels of this amino acid compared to sensitive cells. However, serine is required for cellular growth and survival, and resistant cells increase its uptake from the medium, becoming exquisitely vulnerable to serine starvation. We showed that, although resistant cells accumulate DNA damage upon platinum treatment, they have a higher capacity of repairing it because of increased poly(ADP)-ribose polymerase (PARP) activity, compared to the sensitive ones. Since PARP enzymes are major oxidized nicotinamide adenine dinucleotide (NAD+)-consuming enzymes, we collected evidence that serine synthesis downregulation, as a consequence of central carbon metabolic reshuffling, provides resistant cells with the advantage of sparing NAD+, thus sustaining PARP activation and allowing a more efficient DNA repair. We confirmed that downregulation of serine synthesis is a peculiar trait of resistant tumors also in vivo, using patient-derived xenografts (PDX) ovarian cancer models subjected to serine/glycine free diet. Moreover, analysis of The Cancer Genome Atlas Consortium (TCGA) ovarian cancer dataset revealed that tumors from platinum-resistant patients are characterized by a downregulation of serine biosynthetic enzymes, suggesting that serine auxotrophy could represent a novel and exploitable vulnerability of platinum-resistant ovarian cancers. Citation Format: Tom Van Nyen, Joao A.G. Duarte, Matteo Rossi, Mélanie Planque, Esther Zaal, Ali Talebi, Stijn Moens, Guy Eelen, Hugo Horlings, Johan Swinnen, Celia Berkers, Peter Carmeliet, Reuven Agami, Sarah-Maria Fendt, Diether Lambrechts, Daniela Annibali, Frederic Amant. Serine auxotrophy: A novel metabolic vulnerability of platinum-resistant ovarian cancer? [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A75.

Estrogen receptor alpha (ERα) activity is associated with increased cancer cell proliferation. Studies aiming to understand the impact of ERα on cancer-associated phenotypes have largely been limited to its transcriptional activity. Herein, we demonstrate that ERα coordinates its transcriptional output with selective modulation of mRNA translation. Importantly, translational perturbations caused by depletion of ERα largely manifest as "translational offsetting" of the transcriptome, whereby amounts of translated mRNAs and corresponding protein levels are maintained constant despite changes in mRNA abundance. Transcripts whose levels, but not polysome association, are reduced following ERα depletion lack features which limit translation efficiency including structured 5'UTRs and miRNA target sites. In contrast, mRNAs induced upon ERα depletion whose polysome association remains unaltered are enriched in codons requiring U34-modified tRNAs for efficient decoding. Consistently, ERα regulates levels of U34-modifying enzymes and thereby controls levels of U34-modified tRNAs. These findings unravel a hitherto unprecedented mechanism of ERα-dependent orchestration of transcriptional and translational programs that may be a pervasive mechanism of proteome maintenance in hormone-dependent cancers.

Systematic identification of noncoding regulatory elements has, to date, mainly relied on large-scale reporter assays that do not reproduce endogenous conditions. We present two distinct CRISPR-Cas9 genetic screens to identify and characterize functional enhancers in their native context. Our strategy is to target Cas9 to transcription factor binding sites in enhancer regions. We identified several functional enhancer elements and characterized the role of two of them in mediating p53 (TP53) and ERα (ESR1) gene regulation. Moreover, we show that a genomic CRISPR-Cas9 tiling screen can precisely map functional domains within enhancer elements. Our approach expands the utility of CRISPR-Cas9 to elucidate the functions of the noncoding genome.