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Robert, C.

Publications and source records attributed to Robert, C..

At least 19 recordsLinked to original sources

RNA promotes MAPK signaling through direct interaction with BRAF

RNAs are increasingly recognized as direct regulators of protein function, yet whether RNA can control the activation of signaling kinases remains poorly understood. Here we identify RNA as a direct regulator of BRAF, a central kinase in the RAS-RAF-MEK-ERK (MAPK) pathway. Using complementary crosslinking and RNA-protein interaction approaches, we show that BRAF directly binds RNA in cells and that this interaction is enhanced upon MAPK pathway activation. Depletion of intracellular RNA reduces BRAF:CRAF complex assembly and impairs MEK1/2 phosphorylation, indicating that RNA contributes to RAF complex formation and MAPK signal transduction. We identify a positively charged surface within the BRAF kinase domain that mediates RNA binding. A separation-of-function RNA-binding mutant selectively attenuates BRAF-dependent MEK1/2 phosphorylation, demonstrating that RNA binding is required for efficient MAPK signaling. Moreover, artificial recruitment of the RNA-binding-deficient BRAF mutant to an RNA scaffold restores its signaling activity, supporting a direct role for RNA-mediated BRAF complex assembly. These data identify RNA as an active component of RAF kinase regulation and establish RNA-protein interactions as an additional determinant of MAPK pathway activation.

molecular biology↗

PCIF1 loss licenses antitumour immunity via cholesterol biosynthesis

Cholesterol biosynthesis is classically viewed as a tumour supportive metabolic programme, yet cholesterol-associated metabolic states can paradoxically coincide with improved responses to immune checkpoint blockade (ICB)1, highlighting an unresolved context-dependent relationship between cholesterol homeostasis and antitumour immunity. Here we identify PCIF1, a cap-specific RNA methyltransferase2, as a tumour-intrinsic brake on the inflammatory potential of cholesterol biosynthesis. PCIF1 depletion had limited effects on tumour cell proliferation in vitro, but suppressed tumour growth in immunocompetent, not immunodeficient, hosts and sensitized immune-refractory syngeneic tumours to ICB. Immune profiling by single-cell RNA sequencing, flow cytometry and multiplex immunofluorescence revealed increased CD8+ T cell infiltration in PCIF1-deficient tumours, and CD8+ T cell depletion restored tumour growth. Mechanistically, PCIF1 loss selectively enhanced translational engagement of SCAP, activated SREBP2-dependent cholesterol biosynthesis and promoted intracellular cholesterol accumulation. Unexpectedly, activation of this cholesterol programme induced a tumour cell inflammatory cytokine state enriched for T cell-recruiting chemokines, including CXCL10. Pharmacological perturbation of the SCAP-cholesterol axis attenuated this inflammatory programme, placing cholesterol sensing upstream of PCIF1 loss-induced tumour cell inflammatory activation. In ICB-treated patient cohorts, high PCIF1 expression in pre- and on-treatment samples was associated with poor clinical response, and across tumour types, PCIF1 expression inversely correlated with cytolytic immune signatures. These findings reveal PCIF1-mediated SCAP translational control as a tumour cell checkpoint that constrains the antitumour inflammatory activity of a canonical growth-associated cholesterol programme and shapes checkpoint therapy responsiveness.

cancer biology↗

Arginine synthesis pathway and ASS1 play a critical role in mRNA translation reprogramming and ICI resistance in cutaneous melanoma

Immune checkpoint inhibitors (ICI) have revolutionized cancer treatment, but their efficacy has now reached a plateau. ICIs are the first class of treatment targeting the crosstalk between immune and tumor cells, making it crucial to understand the complex interactions within the tumor microenvironment (TME) to enhance therapeutic responses. The elevated consumption of resources by cancer cells, coupled with limited vascularization, often results in a TME that is deficient in nutrients, leading to competition for resources between cancer and stromal cells. Consequently, targeting tumor metabolism has emerged as a promising strategy to improve the efficacy of ICIs. Through metabolomic analysis, we have identified metabolic alterations in melanoma cells that are resistant to ICIs, specifically an increase in arginine synthesis and upregulation of ASS1, the rate-limiting enzyme in this pathway. By using gain and loss of function models, as well as a pharmacological inhibitor specific for ASS1, we demonstrated that modulations in the expression or activity of ASS1 is associated with translational reprogramming, characterized by an inhibition of the cap-dependent mRNA translation mediated through mTORC1/4EBP1 axis. We also demonstrated that targeting ASS1 in vivo, resensitize tumors initially resistant to ICI. Taken together, our results highlight the interaction between modulations of arginine synthesis pathway, mRNA translation reprogramming, antitumor immunity, and restauration of sensitivity to anti-PD-1. Our work also demonstrates the therapeutic potential of targeting arginine synthesis pathway, and especially ASS1, to offer new treatments to patients suffering from cutaneous melanoma resistant to ICIs.

cancer biology↗

MELK controls tumor metabolism to promote resistance to melanoma therapy

Melanoma is the most aggressive form of skin cancer, and despite major advances in targeted and immune therapies, durable responses remain limited due to the emergence of resistance mechanisms. Metabolic reprogramming has emerged as a key driver of therapy resistance, allowing tumor cells to adapt to environmental and therapeutic pressures. Here, we identify the maternal embryonic leucine zipper kinase (MELK) as a critical mediator of resistance in melanoma. We demonstrate that MELK stimulates intracellular accumulation of amino acids, leading to activation of mTORC1 signaling and enhanced mitochondrial metabolism and biogenesis. This metabolic shift promotes resistance to immune checkpoint blockade. Importantly, preclinical MELK inhibition sensitizes resistant melanoma cells to immunotherapy, revealing a potential combinatorial strategy to overcome resistance. Our findings establish MELK as a central regulator of metabolic adaptation and therapeutic resistance in melanoma, highlighting its potential as a promising therapeutic target.

cancer biology↗

The Paraphyletic Origins of Genetic Resistance to Cabbage Stem Flea Beetle in Brassica oleracea

The cabbage stem flea beetle (CSFB) poses a growing threat to winter Brassica crops in Europe, yet the genetic basis of resistance remains poorly understood. To clarify the genetic architecture and evolutionary origins of resistance to CSFB adult feeding, we conducted a genome-wide association study (GWAS) by combining high-throughput pool-sequencing and a large-scale non-choice feeding assay on 113 Brassica oleracea accessions from wild (or feral) populations and major domesticated morphotypes. We demonstrate that resistance displays moderate heritability with a predominantly polygenic basis, revealing strong phenotypic divergence among morphotypes: B. oleracea var. capitata was generally susceptible, whereas var. botrytis and wild populations showed markedly higher resistance. Despite this polygenic background, we identified a major-effect candidate QTL on chromosome C01 with strong enrichment of resistance alleles in wild populations and susceptible alleles in var. capitata. Genome-wide F-statistics and heterozygosity scans revealed a recent selective sweep at this locus in wild lineages. Considering current evidence for the feral origin of contemporary "wild" populations, our results suggest that resistance evolved after domestication and subsequent feralization, independently of resistance in var. botrytis. This paraphyletic distribution underlines the critical importance of integrating demographic history into quantitative genetic analyses of domesticated plant systems.

genetics↗

Selective mRNA translation determines adaptative mutability of melanoma cells to anti-BRAF/MEK combination therapy

During their inevitable evolution towards acquired resistance to anti-cancer targeted therapies, cancer cells adopt distinct gene expression profiles that allow them to transiently adapt to and tolerate the treatment. Similar to bacterial cells that transiently tolerate antibiotics, cancer cells surviving therapy can increase their mutation rate, enhancing the likelihood of acquiring resistance-conferring mutations and evolving into resistant cells. This adaptive mutability has been linked to transcriptional reprogramming of DNA damage repair mechanisms and effective therapeutic strategies to target such mechanisms are currently lacking. Here we show that translational control mediates the adaptive mutability of melanoma drug-tolerant cells by regulating the translation of the error-prone non-homologous end joining (NHEJ) component 53BP1. The specific inhibition of 5UTR-driven 53BP1 mRNA translation was sufficient to impair NHEJ and mutability. We found that the eIF4A RNA helicase, a key component of the eIF4F translation complex, regulates 53BP1 mRNA translation. Consequently, targeting the eIF4A with two small molecule inhibitors significantly delays the acquisition of resistance to combination of BRAF and MEK inhibitors (BRAFi/MEKi) in BRAFV600-mutant melanoma xenograft models and cell lines by reducing the mutability of drug-tolerant cells. Our results demonstrate that a standard-of-care therapy for melanoma, by engaging non-genetic adaptation driven at the translational level, contributes to the evolution of drug-tolerant melanoma cells toward acquired resistance.

cancer biology↗

Kinetics of elastic recoil in the wings of the cicada, Dundubia rufivena

In this paper, we quantitatively characterise the kinetics of elastic recoil in the wings attached to the cicada Dundubia rufivena. We use high speed videography to deduce the velocities of recoil motion in both bending (B) and torsion (T), in downstroke and upstroke modes of flapping. Elastic recoil is faster in downstroke (DS) than it is in upstroke (US), reaching average velocities, [Formula], 244 {degrees}.s-1 in DS-B vs 6434 {degrees}.s-1 in US-B, and 5887 {degrees}.s-1 in DS-T vs 4545 {degrees}.s-1 in US-T. Our results also therefore evidence that bending velocities during elastic recoil, are higher to the velocities in torsion, and conclude that this is a result of the necessary geometrical distances that need to be covered over a stroke. The wings do not act alone as biological springs, but rather, we find that the stiffnesses of the wings (1.7-3.6 GPa) are higher when attached to the cicada body, than they are when detached. This evidences thoracical involvement as part of the biological spring enabling elastic recoil, indicating that elastic recoil of flapping wings should be approached from a systems perspective, rather than solely through a localised understanding of wing mechanics.

zoology↗

Synthetic communities of maize root bacteria interact and redirect benzoxazinoid metabolization

Plant roots are colonised by diverse microbial communities. These communities are shaped by root exudates including plant specialized metabolites. Benzoxazinoids are such secreted compounds of maize. Individual microbes differ in their ability to tolerate and metabolize antimicrobial benzoxazinoids. To investigate how these traits combine in a community, we designed two synthetic communities of maize root bacteria that share six common strains and differ in their ability to metabolize benzoxazinoids based on the seventh strain. We exposed both communities to the benzoxazinoid MBOA (6-methoxybenzoxazolin-2(3H)-one) and found that the metabolizing community did not degrade MBOA to its aminophenoxazinone, as observed for individual strains, but as a community they formed the corresponding acetamide. MBOA shaped differential compositions of both communities and increased the fraction of MBOA-tolerant strains. The benzoxazinoid metabolizing community showed a higher tolerance to MBOA and was able to utilize MBOA as their sole carbon source for growth. Hence, bacterial interaction results in alternative benzoxazinoid metabolization and increases community performance in presence of these antimicrobial compounds. Future work is needed to uncover the genetics of this metabolic interaction and ecological consequences for the bacterial community and the host plant. ImportanceWe investigated how maize root bacteria - alone or in community - tolerate and metabolize antimicrobial compounds of their host plant. We found the capacity to metabolize such a compound to impact bacterial community size and structure and most importantly, to beneeit community eitness. We also found that interacting bacteria redirected the metabolisation of the antimicrobial compound to an alternative degradation product. Our work highlights the need to study the team work of microbes to uncover their community traits to ultimately understand the ecological consequences for the bacterial community and eventually the host plant.

microbiology↗

Plasma Cell-Free DNA in Acute and Chronic Aortic Syndromes.

Acute aortic syndromes and other acute aortic events are commonly misdiagnosed and associated with high mortality and morbidity. There is need for a blood-based biomarker to indicate the acutely unstable aorta. Plasma cell-free DNA (cfDNA) is an established diagnostic marker in prenatal testing and cancer. Its use as a potential diagnostic marker in aortic disease remains to be demonstrated. We found that plasma short-fragment (50-700bp) cfDNA concentration is significantly increased in patients with both acute and chronic aortic disease compared with several control groups, and is significantly increased in acute versus chronic aortic disease. Plasma sf-cfDNA may differentiate acute aortic syndromes from other acute presentations of chest pain with good diagnostic accuracy and could prove a significant advance in the management of aortic disease.

genomics↗

Retinal organoids mirror CRISPR/Cas9 gene editing efficiency observed in vivo

Human retinal organoids are in vitro 3D structures that recapitulate key molecular and structural characteristics of the in vivo retina. They include the presence of all essential retinal cell types including photoreceptors, making them relevant models for preclinical development of gene therapies. A critical knowledge gap exists in understanding their utility for gene editing optimization, particularly for specific genetic disorders. We assessed the potential of retinal organoids for optimizing CRISPR/Cas9-mediated gene editing, focusing on the therapeutically relevant RHO gene implicated in autosomal dominant Retinitis Pigmentosa (adRP). Using retinal organoids, in vitro HEK293T cells, and two humanized mouse models carrying different RHO mutations, we compared editing efficiencies. We observed that retinal organoids have lower transfection efficiency compared to HEK293T cells. Notably, they exhibited editing efficiencies more closely aligned with those found in vivo. We also observed similar delivery patterns of CRISPR/Cas9 tools in both retinal organoids and mouse retinas. These delivery patterns and editing efficiencies remained consistent across dual AAV systems and transiently delivered ribonucleoprotein complexes. Our findings demonstrate that retinal organoids achieve editing outcomes comparable to those observed in vivo underscoring their utility as part of a preclinical testing platform for genome editing, with implications for advancing gene therapy research in inherited retinal diseases. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=156 HEIGHT=200 SRC="FIGDIR/small/630388v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@94478borg.highwire.dtl.DTLVardef@e50970org.highwire.dtl.DTLVardef@ce998corg.highwire.dtl.DTLVardef@1c2bbdf_HPS_FORMAT_FIGEXP M_FIG C_FIG eTOCRetinal organoids can be used to mirror in vivo mouse retina to develop CRISPR therapeutics. Here, Pulman and colleagues show similar ranges of gene editing and delivery dynamics between the organoids and in vivo mouse retina, highlighting the organoids underexplored potential for evaluating gene editing therapies in retinal diseases.

bioengineering↗

The Role of Thermal Stability in AAV Titration of Engineered Variants

Determining the concentration of recombinant adeno-associated virus (AAV) productions, also known as titering, is crucial not only for quality control purposes but also for comparative studies of preclinical and clinical gene therapy trials. Recently, several AAVs were engineered by inserting seven amino acids at the outermost tip of the capsids protruding VR-VIII loop. These variants have demonstrated increased transduction capabilities over naturally occurring AAV serotypes in several studies. However, they have also been shown to produce lower yields when titered using standard techniques, raising questions about their adequacy for clinical development and use. Here, we investigated why peptide insertion onto AAV capsids reduces their titer by examining viral stocks using electron microscopy and PCR-based titering. We reveal that the DNAse digestion step, performed to eliminate free-floating DNA prior to qPCR or ddPCR, adversely impacts engineered capsid stability due to exposure to heat, artificially lowering viral titers of engineered serotypes. Titering without heating yields significantly higher titers for these variants which have melting temperatures (Tm) close to the DNAse inactivation temperature, while titers for parental serotypes with higher Tm remain unchanged. Our findings provide an important new perspective for titering engineered variants with lower thermostability, especially when comparing their effectiveness to their parental serotypes.

molecular biology↗

An extra-glycolytic function for hexokinase 2 as an RNA-binding protein regulating SOX10 mRNA translation in melanoma

Several studies have reported the importance of aerobic glycolysis in melanoma development. Although metabolic benefits of glycolysis have been extensively described in tumor cells, the extra-metabolic functions linked to this energetic pathway in melanoma growth and proliferation have not been clearly established yet. Recently, some key glycolytic enzymes, such as GAPDH and PKM2, were reported to regulate mRNA translation. Translational control of gene expression is considered as a critical effector in cancer biology, representing a highly promising area of research. Here, we report that Hexokinase 2 (HK2), a glucose kinase that catalyzes the first step of glycolysis, is an RNA binding protein (RBP) that regulates mRNA translation in melanoma. We show that siRNA-mediated HK2 depletion changes the translational landscape of melanoma cells. Polysome profiling experiments and RNA-Seq indicate that the translational regulation exerted by HK2 is partly independent of the metabolic status or the glycolytic pathway. We found that HK2 specifically regulates the translation of the mRNA encoding SOX10, a transcription factor implicated in the regulation of tumor initiation, maintenance and progression in melanoma. RNA-protein interaction assays, including crosslinking immunoprecipitation (CLIP), indicate that HK2 is an RBP whose interaction with RNA is independent of its hexokinase activity or subcellular localization. We also show that HK2 specifically associates with the 5 untranslated region (5UTR) of the SOX10 mRNA, and that several deletions in this region decreases both HK2-SOX10 mRNA association and SOX10 5 UTR-mediated translation. We further show that HK2-dependent SOX10 translational regulation is involved in melanoma cell proliferation and colony formation. Collectively, our data highlight a non-metabolic function of HK2, indicating that melanoma cells may enhance glycolysis for purposes beyond simple anabolism.

cancer biology↗

Rapid loss of genetic variation and increased inbreeding in small and isolated populations of Norwegian wild reindeer

Wildlife responses to habitat loss and fragmentation are a central concern in the management and conservation of biodiversity. Small and isolated populations are vulnerable, both due to demographic and genetic mechanisms, which are often linked. Thus, understanding how (changes in) genetic diversity, effective population sizes, and levels of inbreeding relate to population size and degree of isolation is key for developing effective conservation strategies. High-density Single Nucleotide Polymorphism (SNP) arrays represent an increasingly cost-efficient tool to achieve the data needed for such analysis. Here, we present the development of a novel 625k SNP array for reindeer Rangifer tarandus and apply this array to assess conservation genetic issues across thirteen Norwegian wild reindeer populations of varying size, isolation, and genetic origin (i.e., semi-domesticated reindeer origin or a mix of wild reindeer and semi-domesticated reindeer origins). Many of these populations are currently completely isolated, with no gene flow from other populations. We genotyped n = 510 individuals sampled by hunters and found that variation in population size across the populations largely predicted their (recent loss of) genetic variation (observed heterozygosity, Ho), as well as effective population size (Ne) and (change in) level of recent inbreeding. For the smallest and most isolated populations, with total population sizes of <50-100 individuals and a high and increasing level of recent inbreeding, estimated loss of genetic variation was as high as 3-10% over the time span of a generation or less, and estimated Ne was as low as six individuals. With the current level of isolation and associated lack of gene flow, and considering their already low genetic diversity, these populations are hardly viable - neither demographically nor genetically - in the long term. These results have direct relevance for the management of Norwegian wild reindeer, recently red-listed as Near Threatened. Yet, these genetic challenges, characterizing many of the small wild reindeer populations in Norway, have been largely ignored by management thus far. Mitigation efforts such as reducing barriers would introduce substantial conservation dilemma due to the aim of avoiding further spread of chronic wasting disease (CWD), as well as potential further domestic introgression into populations with genetically wild reindeer (or mixed) origin. Nevertheless, our cost-efficient and high-density SNP array especially designed for reindeer and caribou offers a powerful genetic tool to include in future monitoring, providing important contributions to management and conservation decisions.

genetics↗

Altered NRG1/ErbB4 signaling and cholesterol metabolism dysregulation are key pathomechanisms in VRK1-related motor neuropathies and motor neuron diseases

Hereditary Motor and Sensory Neuropathy (HMSN), or Charcot-Marie-Tooth disease (CMT), are the most common group of Inherited peripheral neuropathies (IPN), characterized by a strong clinical and genetic heterogeneity. Among them, distal Hereditary Motor Neuropathy (dHMN), also known as neuronopathy, is a subgroup, where only motor nerves are affected. This subgroup is also genetically heterogeneous, with 25 genes described to date, of which VRK1, that we have recently described as responsible for dHMN, associated to upper motor neuron signs. There are now more than thirty mutations in VRK1, which cause a range of neurological diseases affecting motor neurons (mainly lower, but also upper) or their axons in the peripheral nervous system, that we design as VRK1-related motor neuron diseases. In two previous studies, we have demonstrated that dHMN due to VRK1 mutations lead to reduced levels of VRK1 in the nucleus, and that this depletion alters the dynamics of coilin, a phosphorylation target of VRK1. hiPSC-derived Motor Neurons (hiPSC-MN) from these patients, display Cajal Bodies (CBs) disassembly and defects in neurite outgrowth and branching, altered Action Potential (AP) waveform and decreased Axonal Initial Segment (AIS) length. In this study, we have further studied the link between the loss of VRK1 function and the defects observed in hiPSC-MNs, by realizing bulk mRNA-Seq sequencing in this in vitro model of the disease. Our results evidenced altered NRG1/ERBB4 signaling, leading to cholesterol metabolism dysregulation and deregulation of genes encoding the glutamate receptors AMPAR and NMDAR, which role in the Axonal Initial Segment and abnormal AP initiation in hiPSC-MNs remains to be investigated.

genetics↗

Continent-wide genomic analysis of the African buffalo (Syncerus caffer).

The African buffalo (Syncerus caffer) is a wild bovid with a historical distribution across much of sub-Saharan Africa. Genomic analysis can provide insights into the evolutionary history of the species, and the key selective pressures shaping populations, including assessment of population level differentiation, population fragmentation, and population genetic structure. In this study we generated the highest quality de novo genome assembly (2.65 Gb, scaffold N50 69.17 Mb) of African buffalo to date, and sequenced a further 195 genomes from across the species distribution. Principal component and admixture analyses provided surprisingly little support for the currently described four subspecies, but indicated three main lineages, in Western/Central, Eastern and Southern Africa, respectively. Estimating Effective Migration Surfaces analysis suggested that geographical barriers have played a significant role in shaping gene flow and the population structure. Estimated effective population sizes indicated a substantial drop occurring in all populations 5-10,000 years ago, coinciding with the increase in human populations. Finally, signatures of selection were enriched for key genes associated with the immune response, suggesting infectious disease exert a substantial selective pressure upon the African buffalo. These findings have important implications for understanding bovid evolution, buffalo conservation and population management.

genomics↗

Direct delivery of Cas9 or base editor protein and guide RNA complex enables genome editing in the retina

Genome editing by CRISPR-Cas holds promise for the treatment of retinal dystrophies. For therapeutic gene editing, transient delivery of CRISPR- Cas9 is preferable to viral delivery which leads to long-term expression with potential adverse consequences. Successful delivery of Cas9 protein and its guide RNA as ribonucleoprotein (RNP) complexes has been reported in the retinal pigment epithelium in vivo but not into photoreceptors, the main target of retinal dystrophies. Here, we investigate the feasibility of direct RNP delivery to photoreceptors and RPE cells. We show that RNPs composed of Cas9 or adenine- base editor and guide RNA, without addition of any carrier compounds, induce gene editing in retinal cells at variable rates depending on the guide RNA efficiency and on the locus. But Cas9 RNP delivery at high concentrations leads to outer retinal toxicity indicating a need to improve delivery efficiency for future therapeutic use.

bioengineering↗

The lactonase BxdA mediates metabolic adaptation of maize root bacteria to benzoxazinoids

Root exudates contain secondary metabolites that affect the plants root microbiome. How microbes cope with these bioactive compounds, and how this ability shapes root microbiomes remain largely unknown. We investigated how maize root bacteria metabolise benzoxazinoids, the main specialised metabolites of maize. Diverse and abundant bacteria metabolised the major compound (6-methoxy-benzoxazolin-2-one, MBOA) in the maize rhizosphere to 2-amino-7-methoxyphenoxazin-3-one (AMPO). By contrast, bacteria isolated from Arabidopsis, which does not produce benzoxazinoids, were unable to metabolise MBOA. Among Microbacteria strains, this differential metabolisation allowed to identify a conserved gene cluster containing the lactonase bxdA. BxdA converts MBOA to AMPO in vitro and we show that this capacity provided bacteria a growth benefit under carbon-limiting conditions. Together these results reveal that maize root bacteria - through BxdA - are metabolically adapted to the benzoxazinoids of their host. We propose that metabolic adaptation to plant-specialised compounds shapes root bacterial communities across the plant kingdom.

microbiology↗

MET functions in tumour progression and therapy resistance are repressed by intronic polyadenylation

Intronic polyadenylation (IPA) leads to the production of transcript isoforms with alternative last exons in thousands of mammalian genes. Widespread regulation of IPA isoforms was observed during oncogenic transformation and in tumours versus healthy tissues, and several IPA isoforms were involved in oncogenesis. However, little is known about the potential involvement of IPA in tumour progression, such as cancer cell invasiveness and metastasis, and in resistance to anticancer therapies. Here, we show that an IPA isoform of MET (short MET) whose production is inhibited by U1 snRNP (U1), an essential ribonucleoprotein complex that recognizes the 5 exon-intron junction of pre-mRNA, is associated with better prognosis in breast cancer. Induction of the short MET isoform, using a steric-blocking antisense oligonucleotide targeting the U1 binding site in the vicinity of the short MET alternative polyadenylation site, antagonizes cell invasiveness. U1 blockade with an antisense oligonucleotide targeting the U1 snRNA also decreases breast cancer cell invasiveness, in both human and mouse cancer cell models, and this effect involves IPA induction in MET and several genes belonging to the RAS/RAF/MAPK signalling pathway. Finally, short MET relieves melanoma cell resistance to MAPK cascade-targeted therapy in vitro and in vivo. IPA isoform levels of MET and a few other genes (mTOR, EGFR and CTNNA1) help predict such resistance in patients. Altogether, our findings provide evidence for a role of IPA in both cancer cell invasiveness and resistance to therapy. This suggests that IPA isoforms can be exploited as biomarkers and therapeutic targets to combat tumour progression.

molecular biology↗