Jean-Philippe Brosseau

Identification of Alternative Splicing Markers for Breast Cancer

Breast cancer is the most common cause of cancer death among women under age 50 years, so it is imperative to identify molecular markers to improve diagnosis and prognosis of this disease. Here, we present a new approach for the identification of breast cancer markers that does not measure gene expression but instead uses the ratio of alternatively spliced mRNAs as its indicator. Using a high-throughput reverse transcription-PCR-based system for splicing annotation, we monitored the alternative splicing profiles of 600 cancer-associated genes in a panel of 21 normal and 26 cancerous breast tissues. We validated 41 alternative splicing events that significantly differed in breast tumors relative to normal breast tissues. Most cancer-specific changes in splicing that disrupt known protein domains support an increase in cell proliferation or survival consistent with a functional role for alternative splicing in cancer. In a blind screen, a classifier based on the 12 best cancer-associated splicing events correctly identified cancer tissues with 96% accuracy. Moreover, a subset of these alternative splicing events could order tissues according to histopathologic grade, and 5 markers were validated in a further blind set of 19 grade 1 and 19 grade 3 tumor samples. These results provide a simple alternative for the classification of normal and cancerous breast tumor tissues and underscore the putative role of alternative splicing in the biology of cancer.

RBFOX2 is an important regulator of mesenchymal tissue-specific splicing in both normal and cancer tissues.

ABSTRACT Alternative splicing provides a critical and flexible layer of regulation intervening in many biological processes to regulate the diversity of proteins and impact cell phenotype. To identify alternative splicing differences that distinguish epithelial from mesenchymal tissues, we have investigated hundreds of cassette exons using a high-throughput reverse transcription-PCR (RT-PCR) platform. Extensive changes in splicing were noted between epithelial and mesenchymal tissues in both human colon and ovarian tissues, with many changes from mostly one splice variant to predominantly the other. Remarkably, many of the splicing differences that distinguish normal mesenchymal from normal epithelial tissues matched those that differentiate normal ovarian tissues from ovarian cancer. Furthermore, because splicing profiling could classify cancer cell lines according to their epithelial/mesenchymal characteristics, we used these cancer cell lines to identify regulators for these specific splicing signatures. By knocking down 78 potential splicing factors in five cell lines, we provide an extensive view of the complex regulatory landscape associated with the epithelial and mesenchymal states, thus revealing that RBFOX2 is an important driver of mesenchymal tissue-specific splicing.

Alternative splicing of SYK regulates mitosis and cell survival

Most human genes produce multiple mRNA isoforms through alternative splicing. However, the biological relevance of most splice variants remains unclear. In this study, we evaluated the functional impact of alternative splicing in cancer cells. We modulated the splicing pattern of 41 cancer-associated splicing events and scored the effects on cell growth, viability and apoptosis, identifying three isoforms essential for cell survival. Specifically, changing the splicing pattern of the spleen tyrosine kinase gene (SYK) impaired cell-cycle progression and anchorage-independent growth. Notably, exposure of cancer cells to epithelial growth factor modulated the SYK splicing pattern to promote the pro-survival isoform that is associated with cancer tissues in vivo. The data suggest that splicing of selected genes is specifically modified during tumor development to allow the expression of isoforms that promote cancer cell survival.

High-throughput quantification of splicing isoforms.

Most human messenger RNAs (mRNAs) are alternatively spliced and many exhibit disease-specific splicing patterns. However, the contribution of most splicing events to the development and maintenance of human diseases remains unclear. As the contribution of alternative splicing events to diagnosis and prognosis is becoming increasingly recognized, it becomes important to develop precise methods to quantify the abundance of these isoforms in clinical samples. Here we present a pipeline for real-time PCR annotation of splicing events (RASE) that allows accurate identification of a large number of splicing isoforms in human tissues. The RASE automatically designed specific primer pairs for 81% of all alternative splicing events in the NCBI build 36 database. Experimentally, the majority of the RASE designed primers resulted in isoform-specific amplification suitable for quantification in human cell lines or in formalin-fixed, paraffin-embedded (FFPE) RNA extract. Using this pipeline it is now possible to rapidly identify splicing isoform signatures in different types of human tissues or to validate complete sets of data generated by microarray expression profiling and deep sequencing techniques.

Tumor microenvironment–associated modifications of alternative splicing

Pre-mRNA alternative splicing is modified in cancer, but the origin and specificity of these changes remain unclear. Here, we probed ovarian tumors to identify cancer-associated splicing isoforms and define the mechanism by which splicing is modified in cancer cells. Using high-throughput quantitative PCR, we monitored the expression of splice variants in laser-dissected tissues from ovarian tumors. Surprisingly, changes in alternative splicing were not limited to the tumor tissues but were also found in the tumor microenvironment. Changes in the tumor-associated splicing events were found to be regulated by splicing factors that are differentially expressed in cancer tissues. Overall, ∼20% of the alternative splicing events affected by the down-regulation of the splicing factors QKI and RBFOX2 were altered in the microenvironment of ovarian tumors. Together, our results indicate that the tumor microenvironment undergoes specific changes in alternative splicing orchestrated by a limited number of splicing factors.

Redirecting splicing with bifunctional oligonucleotides

Ectopic modulators of alternative splicing are important tools to study the function of splice variants and for correcting mis-splicing events that cause human diseases. Such modulators can be bifunctional oligonucleotides made of an antisense portion that determines target specificity, and a non-hybridizing tail that recruits proteins or RNA/protein complexes that affect splice site selection (TOSS and TOES, respectively, for targeted oligonucleotide silencer of splicing and targeted oligonucleotide enhancer of splicing). The use of TOSS and TOES has been restricted to a handful of targets. To generalize the applicability and demonstrate the robustness of TOSS, we have tested this approach on more than 50 alternative splicing events. Moreover, we have developed an algorithm that can design active TOSS with a success rate of 80%. To produce bifunctional oligonucleotides capable of stimulating splicing, we built on the observation that binding sites for TDP-43 can stimulate splicing and improve U1 snRNP binding when inserted downstream from 5′ splice sites. A TOES designed to recruit TDP-43 improved exon 7 inclusion in SMN2. Overall, our study shows that bifunctional oligonucleotides can redirect splicing on a variety of genes, justifying their inclusion in the molecular arsenal that aims to alter the production of splice variants.

8‐BuS‐ATP derivatives as specific NTPDase1 inhibitors

Ectonucleotidases control extracellular nucleotide levels and consequently, their (patho)physiological responses. Among these enzymes, nucleoside triphosphate diphosphohydrolase‐1 (NTPDase1), −2, −3 and −8 are the major ectonucleotidases responsible for nucleotide hydrolysis at the cell surface under physiological conditions, and NTPDase1 is predominantly located at the surface of vascular endothelial cells and leukocytes. Efficacious inhibitors of NTPDase1 are required to modulate responses induced by nucleotides in a number of pathological situations such as thrombosis, inflammation and cancer.

Development of an efficient strategy for the synthesis of the ETB receptor antagonist BQ-788 and some related analogues

BQ-788 [N-cis-2,6-dimethylpiperidine-1-carbonyl-L-gamma-methylleucyl-D-1-methoxycarbonyltryptophanyl-D-norleucine sodium salt] is a very potent and selective ETB receptor antagonist. The formation of the highly hindered trisubstituted urea functionality in the peptide chain and the carbamination on the indole nitrogen of the tryptophan side chain are major challenges in the synthesis of this particular antagonist. Furthermore, the high cost of the unnatural amino acids in the sequence of BQ-788 and its reported synthesis render this pseudopeptide very expensive to produce. In order to improve the yield and to reduce the number of steps compared to previous reported syntheses, we developed an efficient strategy involving a novel one-pot procedure for the synthesis of a highly hindered trisubstituted urea. Under very mild conditions, the urea was obtained by using triphosgene and sodium iodide. This strategy allowed us to synthesize BQ-788 in seven steps with an overall yield of 53%. We also generalized the use of this powerful methodology by creating some new structural analogues of the cis-2,6-dimethylpiperidine moiety by replacing it with other bulky secondary amines. We evaluated the antagonist properties of those three new analogues of BQ-788 in two bioassays in vitro. These new antagonists were less potent than BQ-788 in an ETB rich preparation and inactive in an ETA rich preparation.

The Merit of Alternative Messenger RNA Splicing as a New Mine for the Next Generation Ovarian Cancer Biomarkers

Ovarian cancer is a low incidence cancer with high mortality rate [1]. The asymptomatic na‐ ture of this cancer and the late stage diagnosis of most tumors are the reasons for ineffective surgery and chemotherapy [2]. In this sense, intensive research aim at increasing overall pa‐ tient survival and quality of life by providing biomarkers for 1) early detection and 2) pre‐ diction of chemotherapy response and/or suggestion of alternative strategies. CA-125 is a glycoprotein that is usually expressed in a variety of epithelial cells and its serum level rise up in advance ovarian cancer [3]. However, its use as an early detection marker or as a tool to screen the general population has not been approved so far [4,5]. CA-125 level is helpful in treatment-decision making but do not retain the capacity to improve overall survival and quality of life [6,7]. Clearly, there is still great need for biomarkers or combination of bio‐ markers that could positively identify early ovarian cancer lesions with great certainty or in‐ crease patients’ survival.