The alternative ways of cancer

The alternative processing of genomic loci through alternative splicing (AS) to produce multiple transcripts is a prevalent mode of gene expression regulation in multicellular organisms. Although the specific function of the majority of alternative transcripts remains unknown, the differential production of transcript isoforms has been related to essential biological processes, such as the acquisition of tissue-specific functions, and it has been long recognized that disruption of splicing mechanisms can cause disease, including cancer. Cancer arises from genetic and epigenetic alterations that interfere with essential mechanisms of the normal life cycle of cells, such as replication control, DNA repair and cell death, and multiple cancer-related alterations have been described to induce AS changes in tumor transcriptomes. Cancer-related mutations that disrupt splicing regulatory motifs or create cryptic ones, as well as mutations and expression alterations in splicing factors and chromatin regulators, can lead to alterations in RNA processing and splicing of genes, which in turn impact their function and contribute to the pathological properties of tumors. The prevalence of AS in cancer genomes and its prominent role in cancer-related processes indicates that these alterations may be related to significant functional impacts and may explain some of the observed oncogenic properties.

AS_drivers

With the aim to address this question, we have recently performed an exhaustive analysis of the functional impacts produced by AS changes in tumors (Climente-Gonzalez et al. 2017). We described how cancer specific AS changes lead frequently to shorter protein products and sometimes, although less often, to a non-coding transcript. As a consequence, transcript isoforms that are more highly expressed in tumors encode for fewer functional domains, i.e. there is a potential loss of functional capacities of genes in cancer. Interestingly, the protein domains more frequently affected by AS belong to functional families classically affected by somatic mutations in tumors. Additionally, these functional domain losses are in fact strongly associated to protein-protein interactions, and often affect partners of classical cancer drivers. Moreover, since we analyzed the splicing changes occurring in each individual tumor sample, we were able to observe that protein affecting mutations and splicing changes that affect similar functional domain families tend to occur in different patients, suggesting an equivalence between the mutations and splicing changes. Splicing alterations may thus recapitulate similar functional impacts to those observed through genetic alterations, namely protein affecting mutations and copy number alterations, more commonly associated with cancer. Transcriptome data thus shows that alternative splicing has a functional impact similar to other alterations, and may also play a driving role in cancer progression.

Climente-González H, Porta-Pardo E, Godzik A, Eyras E. The Functional Impact of Alternative Splicing in Cancer. Cell Rep. 2017 Aug 29;20(9):2215-2226.

 

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

We have recently studied the variability of the cell transcriptome in several tumours to describe new relevant alterations in cancer. The genetic information of cells is encoded in the DNA. This information is read by the cellular machinery, which will generate the so-called RNA and then translate it into proteins. Sometimes, the same gene can give rise to different RNA molecules, which may produce proteins with potentially very different functions. This is due to a process known as alternative splicing, which influences the synthesis of most of the RNAs and proteins of eukaryotic cells and its regulation depends on the RNA-binding proteins (RBPs).

Despite the importance of alternative splicing in cell function being widely known by the scientific community, the role it plays in cancer is only beginning to be realized. This has been possible thanks to new sequencing technologies and the availability of RNA sequencing data of multiple tumours through projects such as The Cancer Genome Atlas (TCGA). Although cancer originates from mutations in DNA, they have an impact on the set of RNA molecules of the cell, known as the transcriptome, which can induce and maintain mechanisms linked to the development of cancer. We have now studied the alterations in RBPs that could cause changes in alternative splicing linked to the development of cancer.

For this analysis, we have used various tools developed in our lab. Among them, we used SUPPA a fast and accurate tool to study splicing from multiple samples. Thanks to SUPPA, we could calculate the splicing profiles of more than 4000 samples in less than one day. We then carried out a comprehensive analysis of the mutations, copy number alterations and expression differences in genes, as well as the alternative splicing changes associated to them, for these samples from eleven different types of cancer taken from the TCGA project. This analysis showed RBPs are often altered in human tumours and that these alterations determine the cell transcriptome and induce cell transformations related to the development of cancer. Until now, these alterations remained invisible to the methods used in major cancer genome analysis projects.

With the collaboration of Juan Valcárcel’s lab (CRG) and Miguel Ángel Pujana’s lab (ICO), we were able to show that introducing the identified alterations of the transcriptome in non-tumour cells, these acquire tumourigenic properties. In addition to expanding our knowledge on the role of RBPs in tumours, these results highlight the importance of alternative splicing as a complementary mechanism in the development of cancer, becoming a new relevant factor to be taken into account in the study of this disease. This research opens up new ways of understanding the biology of cancer and searching for new therapeutic strategies. The alterations in alternative splicing are particularly important in the context of those tumours that do not harbour known mutations and for which no therapy is known, and, therefore, they may open new opportunities to understand tumour biology and search for new therapies.

This study was made possible thanks to the funding from the Sandra Ibarra Foundation, the Consolider RNAREG project from MINECO, the funding from AGAUR, as well as from other projects of the Spanish Government and FEDER funds.

Sebestyén E, Singh B, Miñana B, Pagès A, Mateo F, Pujana MA, Valcárcel J, Eyras E. Large-scale analysis of genome and transcriptome alterations in multiple tumors unveils novel cancer-relevant splicing networks. Genome Res. 2016 Jun;26(6):732-44.

Figure_1Allegoric representation of the variability of the transcriptome through changes in alternative splicing in difference cancer types. Illustration by Babita Singh.

The switch of cancer

For many years, scientists have struggled to understand and cure cancer. The study of the genome of multiple tumors has been fundamental to detect recurrent alterations in several types of cancer, and has facilitated their classification and the development of new therapeutic strategies. In particular, high-throughput technologies have been applied in the context of multiple international projects to detect actionable alterations, i.e. genetic changes in the genome of cancer cells that can be used to develop new targeted therapies. These studies have highlighted the heterogeneity of genetic alterations in patients suffering from the same type of cancer, motivating the development of individualized treatments. However, known actionable alterations tend to occur at low frequency, and often a tumor sample has fewer mutations than those seemingly necessary to explain the tumoral process. Thus, there is a need to expand the catalogue of cancer signatures to integrate other molecular alterations for the characterization of individual tumors.

Most of the strategies used in cancer genome projects are based on searching for genetic alterations or changes in the expression of genes. On the other hand, there is more and more evidence that alterations in the splicing regulatory program play an important role in tumor transformation. Splicing is a process by which the long RNA molecule transcribed from the gene in the genome is processed to remove segments called introns, giving rise to an RNA transcript. Alternative splicing provides a mechanism to generate multiple RNA transcripts from the same gene by eliminating introns in different ways. This process is tightly regulated, and is known to give rise to proteins with cell-type specific or opposing functions, or even provide a way to activate or deactivate gene function. This dramatic change between conditions lead by splicing is generally called a splicing switch.

switch_of_cancer_v1

Splicing switches that are not originally present and regulated in cells can induce altered cellular states, leading to disease. Accordingly, the determination of alterations in alternative splicing in tumors can be fundamental for the development of tumor specific molecular targets for prognosis and therapy. However, this analysis is generally hindered by the heterogeneity of tumors of the same origin from different individuals, as well as by the normal variability between individuals. In our group we have developed a new computational method, robust to biological and technical variability, which identifies significant splicing switches across a large number of tumor samples and shows high accuracy on held-out datasets. Moreover, the method is capable of identifying complex alternative splicing changes that cannot be described using standard methodologies. Additionally, the method is independent of parameterizations, which is especially relevant for the analysis of RNA sequencing data from samples from multiple laboratories and technological platforms.

We have applied this method to data from the Cancer Genome Atlas (TCGA) project, which is the NIH-funded branch of the ICGC project. This is the first published large-scale analysis describing the splicing alterations in 9 cancer types using RNA sequencing data from more than 4000 samples. This is possibly the first systematic study of alternative splicing alterations in 9 difference cancer types using so many patient samples. In this work, we have discovered that there exist many splicing switches in patients with the same cancer type that can separate with high accuracy tumor and normal samples, and different types of cancer from each other, providing a predictive signature. In particular, these signatures provide simple rules based on the expression of a few RNA molecules that could allow determining the cancer type from an RNA sample of a new patient. Additionally, we found such set of rules for the triple-negative breast cancer subtype, which is one of the most aggressive subtypes of breast cancer. This new computational method reveals novel signatures of cancer in terms of RNA transcript isoforms specifically expressed in tumors, providing potential novel molecular targets for prognosis and therapy.

Further reading:

Endre Sebestyén, Michał Zawisza, Eduardo Eyras. Detection of recurrent alternative splicing switches in tumor samples reveals novel signatures of cancer. Nucleic Acids Research 2015; doi: 10.1093/nar/gku1392

News at the UPF web site: “Interruptors del càncer” (in Catalan)

 

The new voyage of the “Argonautes”

A new publication from our group describes a new function for a protein mostly characterized for its role in directing small non-coding RNAs to their targets.

Small RNA molecules, which include the so called micro-RNAs (miRNAs), provide a powerful mechanism to regulate gene expression in the cell cytoplasm either by triggering degradation of the messenger RNAs molecules (mRNA) or by inhibiting their translation into proteins. This mechanism, known as post-transcriptional gene silencing, takes place in the cell cytoplasm and has important implications for understanding developmental and disease processes. The miRNAs are guided to their targets by a molecular complex that is made of a group of proteins called Argonautes. These proteins are essential to direct the miRNAs to their mRNA targets, and their structure and role in the post-transcriptional gene silencing has been thoroughly described up to date.

In this work, we report that Argonaute proteins also play an important role in gene regulation in the cell nucleus. The published work describes how Argonaute proteins, besides their role in post-transcriptional regulation, can also affect gene expression during transcription, the cell process that makes mRNA from DNA. In particular, this work shows evidence that Argonaute can bind to specific locations of the genome. These locations are transcriptional enhancers, regions in the DNA that control of the expression of one or multiple genes by governing when these genes must be turned on or off. Enhancers are usually placed far from the genes they regulate, but they can also occur inside a gene. The activation of enhancers takes place through binding of specific factors, as well as by the acquisition of multiple chemical modifications by the chromatin, the structural packing of DNA in the cell that makes possible to hold the long DNA chains from chromosomes into small volumes in the cell nucleus. Each cell type has a specific subset of enhancers that are activated by generic biochemical modifications of the chromatin and by specific protein factors that control gene expression in that cell, and which ultimately determines the cell identity. The aberrant activation or silencing of enhancers can impact cell function and lead to cell transformation, like cancer.

AGO1_figure

This article describes how the Argonaute protein AGO1 can bind to enhancer regions inside genes when they are activated, and thereby directly affect how the RNA molecule transcribed from that gene locus is processed (see Image). In particular, using high-throughput sequencing techniques to describe the regions of the chromatin that have specific biochemical modifications, and which ones interact with Argonatue proteins, we have discovered that the Argonaute protein, specifically the member AGO1 from the Argonaute protein family, binds preferentially to active transcriptional enhancers and that this association is mediated by the RNA molecules that are produced from the active enhancers, also known as enhancer RNAs (eRNAs). Moreover, the interaction of AGO1 with enhancers occurs mostly inside genic regions and appears to affect the splicing of the host gene rather than its transcription. In summary, this work suggests that AGO1 has a function in the nucleus that is related to the activation of transcriptional enhancers, and from these sites is capable to modulate the processing of the RNA from the same locus. These results contribute to the understanding of the complex regulation of gene expression in eukaryotic cells. This work proposes that Argonautes can perform yet another voyage to the nucleus to control how genes are processed, proving to be important and versatile players in determining cell function.

This work has been funded by the Sandra Ibarra Foundation for cancer and by the European Network on alternative splicing EURASNET.

Alló M, Agirre E, Bessonovc S, Bertucci P, Gómez-Acuña L, Buggiano V, Bellora N, Singh B, Petrillo E, Blaustein M, Miñana B, Dujardin G, Pozzi B, Pelisch F, Bechara E, Agafonov D, Srebrow A, Lührmann R, Valcárcel J, Eyras E*, Kornblihtt AR*. Argonaute-1 binds transcriptional enhancers and controls constitutive and alternative splicing in human cells. PNAS doi:10.1073/pnas.1416858111 http://www.pnas.org/content/early/2014/10/08/1416858111

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