The phospholipid transporter PITPNC1 links KRAS to MYC to prevent autophagy in lung and pancreatic cancer.

BACKGROUND: The discovery of functionally relevant KRAS effectors in lung and pancreatic ductal adenocarcinoma (LUAD and PDAC) may yield novel molecular targets or mechanisms amenable to inhibition strategies. Phospholipids availability has been appreciated as a mechanism to modulate KRAS oncogenic potential. Thus, phospholipid transporters may play a functional role in KRAS-driven oncogenesis. Here, we identified and systematically studied the phospholipid transporter PITPNC1 and its controlled network in LUAD and PDAC. METHODS: Genetic modulation of KRAS expression as well as pharmacological inhibition of canonical effectors was completed. PITPNC1 genetic depletion was performed in in vitro and in vivo LUAD and PDAC models. PITPNC1-deficient cells were RNA sequenced, and Gene Ontology and enrichment analyses were applied to the output data. Protein-based biochemical and subcellular localization assays were run to investigate PITPNC1-regulated pathways. A drug repurposing approach was used to predict surrogate PITPNC1 inhibitors that were tested in combination with KRASG12C inhibitors in 2D, 3D, and in vivo models. RESULTS: PITPNC1 was increased in human LUAD and PDAC, and associated with poor patients' survival. PITPNC1 was regulated by KRAS through MEK1/2 and JNK1/2. Functional experiments showed PITPNC1 requirement for cell proliferation, cell cycle progression and tumour growth. Furthermore, PITPNC1 overexpression enhanced lung colonization and liver metastasis. PITPNC1 regulated a transcriptional signature which highly overlapped with that of KRAS, and controlled mTOR localization via enhanced MYC protein stability to prevent autophagy. JAK2 inhibitors were predicted as putative PITPNC1 inhibitors with antiproliferative effect and their combination with KRASG12C inhibitors elicited a substantial anti-tumour effect in LUAD and PDAC. CONCLUSIONS: Our data highlight the functional and clinical relevance of PITPNC1 in LUAD and PDAC. Moreover, PITPNC1 constitutes a new mechanism linking KRAS to MYC, and controls a druggable transcriptional network for combinatorial treatments.

On the identifiability of the isoform deconvolution problem: application to select the proper fragment length in an RNA-seq library.

MOTIVATION: Isoform deconvolution is an NP-hard problem. The accuracy of the proposed solutions is far from perfect. At present, it is not known if gene structure and isoform concentration can be uniquely inferred given paired-end reads, and there is no objective method to select the fragment length to improve the number of identifiable genes. Different pieces of evidence suggest that the optimal fragment length is gene-dependent, stressing the need for a method that selects the fragment length according to a reasonable trade-off across all the genes in the whole genome. RESULTS: A gene is considered to be identifiable if it is possible to get both the structure and concentration of its transcripts univocally. Here, we present a method to state the identifiability of this deconvolution problem. Assuming a given transcriptome and that the coverage is sufficient to interrogate all junction reads of the transcripts, this method states whether or not a gene is identifiable given the read length and fragment length distribution. Applying this method using different read and fragment length combinations, the optimal average fragment length for the human transcriptome is around 400-600 nt for coding genes and 150-200 nt for long non-coding RNAs. The optimal read length is the largest one that fits in the fragment length. It is also discussed the potential profit of combining several libraries to reconstruct the transcriptome. Combining two libraries of very different fragment lengths results in a significant improvement in gene identifiability. AVAILABILITY AND IMPLEMENTATION: Code is available in GitHub (https://github.com/JFerrer-B/transcriptome-identifiability). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Brain ventricular enlargement in human and murine acute intermittent porphyria.

The morphological changes that occur in the central nervous system of patients with severe acute intermittent porphyria (AIP) have not yet been clearly established. The aim of this work was to analyze brain involvement in patients with severe AIP without epileptic seizures or clinical posterior reversible encephalopathy syndrome, as well as in a mouse model receiving or not liver-directed gene therapy aimed at correcting the metabolic disorder. We conducted neuroradiologic studies in 8 severely affected patients (6 women) and 16 gender- and age-matched controls. Seven patients showed significant enlargement of the cerebral ventricles and decreased brain perfusion was observed during the acute attack in two patients in whom perfusion imaging data were acquired. AIP mice exhibited reduced cerebral blood flow and developed chronic dilatation of the cerebral ventricles even in the presence of slightly increased porphyrin precursors. While repeated phenobarbital-induced attacks exacerbated ventricular dilation in AIP mice, correction of the metabolic defect using liver-directed gene therapy restored brain perfusion and afforded protection against ventricular enlargement. Histological studies revealed no signs of neuronal loss but a denser neurofilament pattern in the periventricular areas, suggesting compression probably caused by imbalance in cerebrospinal fluid dynamics. In conclusion, severely affected AIP patients exhibit cerebral ventricular enlargement. Liver-directed gene therapy protected against the morphological consequences of the disease seen in the brain of AIP mice. The observational study was registered at Clinicaltrial.gov as NCT02076763.

Combined targeting of TGF-ß1 and integrin ß3 impairs lymph node metastasis in a mouse model of non-small-cell lung cancer.

BACKGROUND: Transforming Growth Factor beta (TGF-ß) acts as a tumor suppressor early in carcinogenesis but turns into tumor promoter in later disease stages. In fact, TGF-ß is a known inducer of integrin expression by tumor cells which contributes to cancer metastatic spread and TGF-ß inhibition has been shown to attenuate metastasis in mouse models. However, carcinoma cells often become refractory to TGF-ß-mediated growth inhibition. Therefore identifying patients that may benefit from anti-TGF-ß therapy requires careful selection. METHODS: We performed in vitro analysis of the effects of exposure to TGF-ß in NSCLC cell chemotaxis and adhesion to lymphatic endothelial cells. We also studied in an orthotopic model of NSCLC the incidence of metastases to the lymph nodes after inhibition of TGF-ß signaling, ß3 integrin expression or both. RESULTS: We offer evidences of increased ß3-integrin dependent NSCLC adhesion to lymphatic endothelium after TGF-ß exposure. In vivo experiments show that targeting of TGF-ß and ß3 integrin significantly reduces the incidence of lymph node metastasis. Even more, blockade of ß3 integrin expression in tumors that did not respond to TGF-ß inhibition severely impaired the ability of the tumor to metastasize towards the lymph nodes. CONCLUSION: These findings suggest that lung cancer tumors refractory to TGF-ß monotherapy can be effectively treated using dual therapy that combines the inhibition of tumor cell adhesion to lymphatic vessels with stromal TGF-ß inhibition.

The role of the pulmonary veins on left atrial flow patterns and thrombus formation.

Atrial fibrillation (AF) is the most common human arrhythmia, forming thrombi mostly in the left atrial appendage (LAA). However, the relation between LAA morphology, blood patterns and clot formation is not yet fully understood. Furthermore, the impact of anatomical structures like the pulmonary veins (PVs) have not been thoroughly studied due to data acquisition difficulties. In-silico studies with flow simulations provide a detailed analysis of blood flow patterns under different boundary conditions, but a limited number of cases have been reported in the literature. To address these gaps, we investigated the influence of PVs on LA blood flow patterns and thrombus formation risk through computational fluid dynamics simulations conducted on a sizeable cohort of 130 patients, establishing the largest cohort of patient-specific LA fluid simulations reported to date. The investigation encompassed an in-depth analysis of several parameters, including pulmonary vein orientation (e.g., angles) and configuration (e.g., number), LAA and LA volumes as well as their ratio, flow, and mass-less particles. Our findings highlight the total number of particles within the LAA as a key parameter for distinguishing between the thrombus and non-thrombus groups. Moreover, the angles between the different PVs play an important role to determine the flow going inside the LAA and consequently the risk of thrombus formation. The alignment between the LAA and the main direction of the left superior pulmonary vein, or the position of the right pulmonary vein when it exhibits greater inclination, had an impact to distinguish the control group vs. the thrombus group. These insights shed light on the intricate relationship between PV configuration, LAA morphology, and thrombus formation, underscoring the importance of comprehensive blood flow pattern analyses.

Design and validation of a tunable inertial microfluidic system for the efficient enrichment of circulating tumor cells in blood.

The analysis of circulating tumor cells (CTCs) in blood is a powerful noninvasive alternative to conventional tumor biopsy. Inertial-based separation is a promising high-throughput, marker-free sorting strategy for the enrichment and isolation of CTCs. Here, we present and validate a double spiral microfluidic device that efficiently isolates CTCs with a fine-tunable cut-off value of 9 µm and a separation range of 2 µm. We designed the device based on computer simulations that introduce a novel, customized inertial force term, and provide practical fabrication guidelines. We validated the device using calibration beads, which allowed us to refine the simulations and redesign the device. Then we validated the redesigned device using blood samples and a murine model of metastatic breast cancer. Finally, as a proof of principle, we tested the device using peripheral blood from a patient with hepatocellular carcinoma, isolating more than 17 CTCs/ml, with purity/removal values of 96.03% and 99.99% of white blood cell and red blood cells, respectively. These results confirm highly efficient CTC isolation with a stringent cut-off value and better separation results than the state of the art.

Complement C5a induces the formation of neutrophil extracellular traps by myeloid-derived suppressor cells to promote metastasis.

Myeloid-derived suppressor cells (MDSCs) play a major role in cancer progression. In this study, we investigated the mechanisms by which complement C5a increases the capacity of polymorphonuclear MDSCs (PMN-MDSCs) to promote tumor growth and metastatic spread. Stimulation of PMN-MDSCs with C5a favored the invasion of cancer cells via a process dependent on the formation of neutrophil extracellular traps (NETs). NETosis was dependent on the production of high mobility group box 1 (HMGB1) by cancer cells. Moreover, C5a induced the surface expression of the HMGB1 receptors TLR4 and RAGE in PMN-MDSCs. In a mouse lung metastasis model, inhibition of C5a, C5a receptor-1 (C5aR1) or NETosis reduced the number of circulating-tumor cells (CTCs) and the metastatic burden. In support of the translational relevance of these findings, C5a was able to stimulate migration and NETosis in PMN-MDSCs obtained from lung cancer patients. Furthermore, myeloperoxidase (MPO)-DNA complexes, as markers of NETosis, were elevated in lung cancer patients and significantly correlated with C5a levels. In conclusion, C5a induces the formation of NETs from PMN-MDSCs in the presence of cancer cells, which may facilitate cancer cell dissemination and metastasis.

Tumor ENPP1 (CD203a)/Haptoglobin Axis Exploits Myeloid-Derived Suppressor Cells to Promote Post-Radiotherapy Local Recurrence in Breast Cancer.

ABSTRACT: Locoregional failure (LRF) in patients with breast cancer post-surgery and post-irradiation is linked to a dismal prognosis. In a refined new model, we identified ectonucleotide pyrophosphatase/phosphodiesterase 1/CD203a (ENPP1) to be closely associated with LRF. ENPP1hi circulating tumor cells (CTC) contribute to relapse by a self-seeding mechanism. This process requires the infiltration of polymorphonuclear myeloid-derived suppressor cells and neutrophil extracellular trap (NET) formation. Genetic and pharmacologic ENPP1 inhibition or NET blockade extends relapse-free survival. Furthermore, in combination with fractionated irradiation, ENPP1 abrogation obliterates LRF. Mechanistically, ENPP1-generated adenosinergic metabolites enhance haptoglobin (HP) expression. This inflammatory mediator elicits myeloid invasiveness and promotes NET formation. Accordingly, a significant increase in ENPP1 and NET formation is detected in relapsed human breast cancer tumors. Moreover, high ENPP1 or HP levels are associated with poor prognosis. These findings unveil the ENPP1/HP axis as an unanticipated mechanism exploited by tumor cells linking inflammation to immune remodeling favoring local relapse. SIGNIFICANCE: CTC exploit the ENPP1/HP axis to promote local recurrence post-surgery and post-irradiation by subduing myeloid suppressor cells in breast tumors. Blocking this axis impairs tumor engraftment, impedes immunosuppression, and obliterates NET formation, unveiling new opportunities for therapeutic intervention to eradicate local relapse and ameliorate patient survival. This article is highlighted in the In This Issue feature, p. 1171.

Modeling the Mechanobiology of Cancer Cell Migration Using 3D Biomimetic Hydrogels.

Understanding how cancer cells migrate, and how this migration is affected by the mechanical and chemical composition of the extracellular matrix (ECM) is critical to investigate and possibly interfere with the metastatic process, which is responsible for most cancer-related deaths. In this article we review the state of the art about the use of hydrogel-based three-dimensional (3D) scaffolds as artificial platforms to model the mechanobiology of cancer cell migration. We start by briefly reviewing the concept and composition of the extracellular matrix (ECM) and the materials commonly used to recreate the cancerous ECM. Then we summarize the most relevant knowledge about the mechanobiology of cancer cell migration that has been obtained using 3D hydrogel scaffolds, and relate those discoveries to what has been observed in the clinical management of solid tumors. Finally, we review some recent methodological developments, specifically the use of novel bioprinting techniques and microfluidics to create realistic hydrogel-based models of the cancer ECM, and some of their applications in the context of the study of cancer cell migration.

CRMP2 as a Candidate Target to Interfere with Lung Cancer Cell Migration.

Collapsin response mediator protein 2 (CRMP2) is an adaptor protein that adds tubulin dimers to the growing tip of a microtubule. First described in neurons, it is now considered a ubiquitous protein that intervenes in processes such as cytoskeletal remodeling, synaptic connection and trafficking of voltage channels. Mounting evidence supports that CRMP2 plays an essential role in neuropathology and, more recently, in cancer. We have previously described a positive correlation between nuclear phosphorylation of CRMP2 and poor prognosis in lung adenocarcinoma patients. In this work, we studied whether this cytoskeleton molding protein is involved in cancer cell migration. To this aim, we evaluated CRMP2 phosphorylation and localization in the extending lamella of lung adenocarcinoma migrating cells using in vitro assays and in vivo confocal microscopy. We demonstrated that constitutive phosphorylation of CRMP2 impaired lamella formation, cell adhesion and oriented migration. In search of a mechanistic explanation of this phenomenon, we discovered that CRMP2 Ser522 phospho-mimetic mutants display unstable tubulin polymers, unable to bind EB1 plus-Tip protein and the cortical actin adaptor IQGAP1. In addition, integrin recycling is defective and invasive structures are less evident in these mutants. Significantly, mouse xenograft tumors of NSCLC expressing CRMP2 phosphorylation mimetic mutants grew significantly less than wild-type tumors. Given the recent development of small molecule inhibitors of CRMP2 phosphorylation to treat neurodegenerative diseases, our results open the door for their use in cancer treatment.

Development and multimodal characterization of an elastase-induced emphysema mouse disease model for the COPD frequent bacterial exacerbator phenotype.

Chronic obstructive pulmonary disease (COPD) patients undergo infectious exacerbations whose frequency identifies a clinically meaningful phenotype. Mouse models have been mostly used to separately study both COPD and the infectious processes, but a reliable model of the COPD frequent exacerbator phenotype is still lacking. Accordingly, we first established a model of single bacterial exacerbation by nontypeable Haemophilus influenzae (NTHi) infection on mice with emphysema-like lesions. We characterized this single exacerbation model combining both noninvasive in vivo imaging and ex vivo techniques, obtaining longitudinal information about bacterial load and the extent of the developing lesions and host responses. Bacterial load disappeared 48 hours post-infection (hpi). However, lung recovery, measured using tests of pulmonary function and the disappearance of lung inflammation as revealed by micro-computed X-ray tomography, was delayed until 3 weeks post-infection (wpi). Then, to emulate the frequent exacerbator phenotype, we performed two recurrent episodes of NTHi infection on the emphysematous murine lung. Consistent with the amplified infectious insult, bacterial load reduction was now observed 96 hpi, and lung function recovery and disappearance of lesions on anatomical lung images did not happen until 12 wpi. Finally, as a proof of principle of the use of the model, we showed that azithromycin successfully cleared the recurrent infection, confirming this macrolide utility to ameliorate infectious exacerbation. In conclusion, we present a mouse model of recurrent bacterial infection of the emphysematous lung, aimed to facilitate investigating the COPD frequent exacerbator phenotype by providing complementary, dynamic information of both infectious and inflammatory processes.

Targeting aberrant DNA methylation in mesenchymal stromal cells as a treatment for myeloma bone disease.

Multiple myeloma (MM) progression and myeloma-associated bone disease (MBD) are highly dependent on bone marrow mesenchymal stromal cells (MSCs). MM-MSCs exhibit abnormal transcriptomes, suggesting the involvement of epigenetic mechanisms governing their tumor-promoting functions and prolonged osteoblast suppression. Here, we identify widespread DNA methylation alterations of bone marrow-isolated MSCs from distinct MM stages, particularly in Homeobox genes involved in osteogenic differentiation that associate with their aberrant expression. Moreover, these DNA methylation changes are recapitulated in vitro by exposing MSCs from healthy individuals to MM cells. Pharmacological targeting of DNMTs and G9a with dual inhibitor CM-272 reverts the expression of hypermethylated osteogenic regulators and promotes osteoblast differentiation of myeloma MSCs. Most importantly, CM-272 treatment prevents tumor-associated bone loss and reduces tumor burden in a murine myeloma model. Our results demonstrate that epigenetic aberrancies mediate the impairment of bone formation in MM, and its targeting by CM-272 is able to reverse MBD.

The use of mixed collagen-Matrigel matrices of increasing complexity recapitulates the biphasic role of cell adhesion in cancer cell migration: ECM sensing, remodeling and forces at the leading edge of cancer invasion.

The migration of cancer cells is highly regulated by the biomechanical properties of their local microenvironment. Using 3D scaffolds of simple composition, several aspects of cancer cell mechanosensing (signal transduction, EMC remodeling, traction forces) have been separately analyzed in the context of cell migration. However, a combined study of these factors in 3D scaffolds that more closely resemble the complex microenvironment of the cancer ECM is still missing. Here, we present a comprehensive, quantitative analysis of the role of cell-ECM interactions in cancer cell migration within a highly physiological environment consisting of mixed Matrigel-collagen hydrogel scaffolds of increasing complexity that mimic the tumor microenvironment at the leading edge of cancer invasion. We quantitatively show that the presence of Matrigel increases hydrogel stiffness, which promotes ß1 integrin expression and metalloproteinase activity in H1299 lung cancer cells. Then, we show that ECM remodeling activity causes matrix alignment and compaction that favors higher tractions exerted by the cells. However, these traction forces do not linearly translate into increased motility due to a biphasic role of cell adhesions in cell migration: at low concentration Matrigel promotes migration-effective tractions exerted through a high number of small sized focal adhesions. However, at high Matrigel concentration, traction forces are exerted through fewer, but larger focal adhesions that favor attachment yielding lower cell motility.

Modulation of Haemophilus influenzae interaction with hydrophobic molecules by the VacJ/MlaA lipoprotein impacts strongly on its interplay with the airways.

Airway infection by nontypeable Haemophilus influenzae (NTHi) associates to chronic obstructive pulmonary disease (COPD) exacerbation and asthma neutrophilic airway inflammation. Lipids are key inflammatory mediators in these disease conditions and consequently, NTHi may encounter free fatty acids during airway persistence. However, molecular information on the interplay NTHi-free fatty acids is limited, and we lack evidence on the importance of such interaction to infection. Maintenance of the outer membrane lipid asymmetry may play an essential role in NTHi barrier function and interaction with hydrophobic molecules. VacJ/MlaA-MlaBCDEF prevents phospholipid accumulation at the bacterial surface, being the only system involved in maintaining membrane asymmetry identified in NTHi. We assessed the relationship among the NTHi VacJ/MlaA outer membrane lipoprotein, bacterial and exogenous fatty acids, and respiratory infection. The vacJ/mlaA gene inactivation increased NTHi fatty acid and phospholipid global content and fatty acyl specific species, which in turn increased bacterial susceptibility to hydrophobic antimicrobials, decreased NTHi epithelial infection, and increased clearance during pulmonary infection in mice with both normal lung function and emphysema, maybe related to their shared lung fatty acid profiles. Altogether, we provide evidence for VacJ/MlaA as a key bacterial factor modulating NTHi survival at the human airway upon exposure to hydrophobic molecules.

ß3 integrin expression is required for invadopodia-mediated ECM degradation in lung carcinoma cells.

Cancer related deaths are primarily due to tumor metastasis. To facilitate their dissemination to distant sites, cancer cells develop invadopodia, actin-rich protrusions capable of degrading the surrounding extracellular matrix (ECM). We aimed to determine whether ß3 integrin participates in invadopodia formed by lung carcinoma cells, based on our previous findings of specific TGF-ß induction of ß3 integrin dependent metastasis in animal models of lung carcinoma. In this study, we demonstrate that lung carcinoma cells form invadopodia in response to TGF-ß exposure. Invadopodia formation and degradation activity is dependent on ß3 integrin expression since ß3 integrin deficient cells are not able to degrade gelatin-coated surfaces. Even more, transient over-expression of SRC did not restore invadopodia formation in ß3 integrin deficient cells. Finally, we observed that blockade of PLC-dependent signaling leads to more intense labeling for ß3 integrin in invadopodia. Our results suggest that ß3 integrin function, and location, in lung cancer cells are essential for invadopodia formation, and this integrin regulates the activation of different signal pathways necessary for the invasive structure. ß3 integrin has been associated with poor prognosis and increased metastasis in several carcinoma types, including lung cancer. Our findings provide new evidence to support the use of targeted therapies against this integrin to combat the onset of metastases.

The human lncRNA LINC-PINT inhibits tumor cell invasion through a highly conserved sequence element.

BACKGROUND: It is now obvious that the majority of cellular transcripts do not code for proteins, and a significant subset of them are long non-coding RNAs (lncRNAs). Many lncRNAs show aberrant expression in cancer, and some of them have been linked to cell transformation. However, the underlying mechanisms remain poorly understood and it is unknown how the sequences of lncRNA dictate their function. RESULTS: Here we characterize the function of the p53-regulated human lncRNA LINC-PINT in cancer. We find that LINC-PINT is downregulated in multiple types of cancer and acts as a tumor suppressor lncRNA by reducing the invasive phenotype of cancer cells. A cross-species analysis identifies a highly conserved sequence element in LINC-PINT that is essential for its function. This sequence mediates a specific interaction with PRC2, necessary for the LINC-PINT-dependent repression of a pro-invasion signature of genes regulated by the transcription factor EGR1. CONCLUSIONS: Our findings support a conserved functional co-dependence between LINC-PINT and PRC2 and lead us to propose a new mechanism where the lncRNA regulates the availability of free PRC2 at the proximity of co-regulated genomic loci.

Characterization of three-dimensional cancer cell migration in mixed collagen-Matrigel scaffolds using microfluidics and image analysis.

Microfluidic devices are becoming mainstream tools to recapitulate in vitro the behavior of cells and tissues. In this study, we use microfluidic devices filled with hydrogels of mixed collagen-Matrigel composition to study the migration of lung cancer cells under different cancer invasion microenvironments. We present the design of the microfluidic device, characterize the hydrogels morphologically and mechanically and use quantitative image analysis to measure the migration of H1299 lung adenocarcinoma cancer cells in different experimental conditions. Our results show the plasticity of lung cancer cell migration, which turns from mesenchymal in collagen only matrices, to lobopodial in collagen-Matrigel matrices that approximate the interface between a disrupted basement membrane and the underlying connective tissue. Our quantification of migration speed confirms a biphasic role of Matrigel. At low concentration, Matrigel facilitates migration, most probably by providing a supportive and growth factor retaining environment. At high concentration, Matrigel slows down migration, possibly due excessive attachment. Finally, we show that antibody-based integrin blockade promotes a change in migration phenotype from mesenchymal or lobopodial to amoeboid and analyze the effect of this change in migration dynamics, in regards to the structure of the matrix. In summary, we describe and characterize a robust microfluidic platform and a set of software tools that can be used to study lung cancer cell migration under different microenvironments and experimental conditions. This platform could be used in future studies, thus benefitting from the advantages introduced by microfluidic devices: precise control of the environment, excellent optical properties, parallelization for high throughput studies and efficient use of therapeutic drugs.

A large-scale analysis of alternative splicing reveals a key role of QKI in lung cancer.

Increasing interest has been devoted in recent years to the understanding of alternative splicing in cancer. In this study, we performed a genome-wide analysis to identify cancer-associated splice variants in non-small cell lung cancer. We discovered and validated novel differences in the splicing of genes known to be relevant to lung cancer biology, such as NFIB, ENAH or SPAG9. Gene enrichment analyses revealed an important contribution of alternative splicing to cancer-related molecular functions, especially those involved in cytoskeletal dynamics. Interestingly, a substantial fraction of the altered genes found in our analysis were targets of the protein quaking (QKI), pointing to this factor as one of the most relevant regulators of alternative splicing in non-small cell lung cancer. We also found that ESYT2, one of the QKI targets, is involved in cytoskeletal organization. ESYT2-short variant inhibition in lung cancer cells resulted in a cortical distribution of actin whereas inhibition of the long variant caused an increase of endocytosis, suggesting that the cancer-associated splicing pattern of ESYT2 has a profound impact in the biology of cancer cells. Finally, we show that low nuclear QKI expression in non-small cell lung cancer is an independent prognostic factor for disease-free survival (HR = 2.47; 95% CI = 1.11-5.46, P = 0.026). In conclusion, we identified several splicing variants with functional relevance in lung cancer largely regulated by the splicing factor QKI, a tumor suppressor associated with prognosis in lung cancer.