Treatment of Polarized Cystic Fibrosis Airway Cells With HGF Prevents VX-661-Rescued F508del-CFTR Destabilization Caused by Prolonged Co-exposure to VX-770

Cystic fibrosis (CF), the most common inherited disease in Caucasians, is caused by mutations in CFTR, the most frequent of which is F508del. F508del causes ER retention and degradation of the mutant CFTR protein, but also defective channel gating and decreased half-life at the plasma membrane. Despite the recent successes with small molecule CFTR modulator drugs, the folding-corrector/gating-potentiator drug combinations approved for CF individuals carrying F508del-CFTR have sometimes produced severe side effects. Previously, we showed that a prolonged, 15-days treatment of polarized bronchial epithelial monolayers with the VX-809+VX-770 combination resulted in epithelial dedifferentiation effects that we found were caused specifically by VX-809. Moreover, prolonged VX-770 exposure also led to the destabilization of VX-809-rescued F508del-CFTR. Notably, co-treatment with the physiological factor HGF prevented VX-809-mediated epithelial differentiation and reverted the destabilizing effect of VX-770 on VX-809-rescued CFTR. Here, we show that prolonged treatment with VX-661, a second-generation corrector developed based on VX-809 structure, does not perturb epithelial integrity of polarized bronchial epithelial monolayers. Yet, its efficacy is still affected by co-exposure to VX-770, the potentiator present in all VX-661-containing combination therapies approved in the United States and Europe for treatment of F508del-CFTR carriers. Importantly, we found that co-treatment with HGF still ameliorated the impact of VX-770 in F508del-CFTR functional rescue by VX-661, without increasing cell proliferation (Ki-67) or altering the overall expression of epithelial markers (ZO-1, E-cadherin, CK8, CK18). Our findings highlight the importance of evaluating the cellular effects of prolonged exposure to CFTR modulators and suggest that the benefits of adding HGF to current combination therapies should be further investigated.This work was supported by the Grant PTDC/BIA-CEL/28408/2017 (to PJ and PM) and Center Grant UID/MULTI/04046/2019 to BioISI, both from the Portuguese Fundação para a Ciência e a Tecnologia.info:eu-repo/semantics/publishedVersio

YES1 Kinase Mediates the Membrane Removal of Rescued F508del-CFTR in Airway Cells by Promoting MAPK Pathway Activation via SHC1

(This article belongs to the Section Molecular Biology)Recent developments in CFTR modulator drugs have had a significant transformational effect on the treatment of individuals with Cystic Fibrosis (CF) who carry the most frequent F508del- CFTR mutation in at least one allele. However, the clinical effects of these revolutionary drugs remain limited by their inability to fully restore the plasma membrane (PM) stability of the rescued mutant channels. Here, we shed new light on the molecular mechanisms behind the reduced half-life of rescued F508del-CFTR at the PM of airway cells. We describe that YES1 protein kinase is enriched in F508del-CFTR protein PM complexes, and that its interaction with rescued channels is mediated and dependent on the adaptor protein YAP1. Moreover, we show that interference with this complex, either by depletion of one of these components or inhibiting YES1 activity, is sufficient to significantly improve the abundance and stability of modulator-rescued F508del-CFTR at the surface of airway cells. In addition, we found that this effect was mediated by a decreased phosphorylation of the scaffold protein SHC1, a key regulator of MAPK pathway activity. In fact, we showed that depletion of SHC1 or inhibition of MAPK pathway signaling was sufficient to improve rescued F508del-CFTR surface levels, whereas an ectopic increase in pathway activation downstream of SHC1, through the use of a constitutively active H-RAS protein, abrogated the stabilizing effect of YES1 inhibition on rescued F508del-CFTR. Taken together, our findings not only provide new mechanistic insights into the regulation of modulator-rescued F508del-CFTR membrane stability, but also open exciting new avenues to be further explored in CF research and treatment.Funding: This work was supported by the Grant PTDC/BIA-CEL/28408/2017 (to PJ and PM) and Center Grant UID/MULTI/04046/2019 to BioISI from the Portuguese Fundação para a Ciência e a Tecnologia.info:eu-repo/semantics/publishedVersio

A new twist to ibuprofen: alternative action in alternative splicing

Ibuprofen is a nonsteroidal anti-inflammatory drug (NSAID) and is a widely used medication. One indication of NSAID use is long-term chemoprevention to decrease the risk of developing various types of cancer, in particular colorectal cancer. The molecular mechanism behind the antitumour properties of NSAID has been largely attributed to inhibition of the enzyme cyclooxygenase. In this review article, the authors highlight that additional mechanisms of NSAID, especially ibuprofen, action exist that are related to cell signalling and the modulation of gene expression, including alternative splicing. For example, the authors describe how ibuprofen inhibits expression of the tumour-related splicing variant RAC1b, which is overexpressed in a specific subset of colorectal tumours. The mechanism involves changes in the phosphorylation of splicing factors that regulate this alternative splicing event. According to recent studies, ibuprofen interferes with signal transmission via protein kinases, a process which is frequently altered in cancer cells.The work undertaken in the authors’ laboratory was supported by Fundação para a Ciência e Tecnologia (FCT) through a grant (UID/MULTI/04046/2019) made to the research unit BioISI and contract ‘FCT Investigator’ to Dr Matos.info:eu-repo/semantics/publishedVersio

A novel Rac1/PAK1/BCL-6/STAT5 pathway modulates the expression of cell-cycle-associated genes

Gene expression depends on binding of transcriptional regulators to gene promoters, a process controlled by signalling pathways. The transcriptional repressor BCL-6 downregulates genes involved in cell cycle progression and becomes inactivated following phosphorylation by the Rac1 GTPase activated protein kinase PAK1. Interestingly, the DNA motifs recognized by BCL-6 and STAT5 are similar. Because STAT5 stimulation in epithelial cells can also be triggered by Rac1 signalling, we asked whether both factors have opposing roles in transcriptional regulation and whether Rac1 signalling may coordinate a transcription factor switch. We used chromatin immunoprecipitation to show that active Rac1 promotes release of the repressor BCL-6 while increasing binding of STAT5A to a BCL-6-regulated reporter gene. We further show in colorectal cell lines that the endogenous activation status of the Rac1/PAK1 pathway correlated with the phosphorylation status of BCL-6 and STAT5A. Three cellular genes (cyclin D2, p15INK4B, SUMO1) were identified to be inversely regulated by BCL-6 and STAT5A and responded to Rac1 signalling with increased expression and corresponding changes in promoter occupancy. Together, our data show that Rac1 signalling controls a group of target genes that are repressed by BCL-6 and activated by STAT5A, providing novel insights into the modulation of gene transcription by GTPase signalling

A Signaling View into the Inflammatory Tumor Microenvironment

The development of tumors requires an initiator event, usually exposure to DNA damaging agents that cause genetic alterations such as gene mutations or chromosomal abnormalities, leading to deregulated cell proliferation. Although the mere stochastic accumulation of further mutations may cause tumor progression, it is now clear that an inflammatory microenvironment has a major tumor-promoting influence on initiated cells, in particular when a chronic inflammatory reaction already existed before the initiated tumor cell was formed. Moreover, inflammatory cells become mobilized in response to signals emanating from tumor cells. In both cases, the microenvironment provides signals that initiated tumor cells perceive by membrane receptors and transduce via downstream kinase cascades to modulate multiple cellular processes and respond with changes in cell gene expression, metabolism, and morphology. Cytokines, chemokines, and growth factors are examples of major signals secreted by immune cells, fibroblast, and endothelial cells and mediate an intricate cell-cell crosstalk in an inflammatory microenvironment, which contributes to increased cancer cell survival, phenotypic plasticity and adaptation to surrounding tissue conditions. Eventually, consequent changes in extracellular matrix stiffness and architecture, coupled with additional genetic alterations, further fortify the malignant progression of tumor cells, priming them for invasion and metastasis. Here, we provide an overview of the current knowledge on the composition of the inflammatory tumor microenvironment, with an emphasis on the major signals and signal-transducing events mediating different aspects of stromal cell-tumor cell communication that ultimately lead to malignant progression.The work in the authors’ laboratory was supported by Fundação para a Ciência e a Tecnologia (FCT), Portugal, through grant UID/MULTI/04046/2019 to Research Unit BioISI and fellowship SFRH/BD/109162/2015 to JFSP.info:eu-repo/semantics/publishedVersio

Tyrosine phosphorylation modulates cell surface expression of chloride cotransporters NKCC2 and KCC3

Introduction: Cellular chloride transport has a fundamental role in cell volume regulation and membrane potential, both in normal and tumour cells (1,2). Cellular chloride entry or exit are mediated at the plasma membrane by cotransporter proteins of the solute carrier 12 family. For example, NKCC2 resorbs chloride with sodium and potassium ions at the apical membrane of epithelial cells in the kidney, whereas KCC3 releases chloride with potassium ions at the basolateral membrane. Their ion transport activity is regulated by protein phosphorylation in response to signaling pathways. An additional regulatory mechanism concerns the amount of cotransporter molecules inserted into the plasma membrane. Experimental: Co-transporter constructs were transfected into HEK293 cells and the activity of SYK kinase modulated by incubation with SYK inhibitors or by co-transfection with siRNAs, kinase-dead, or constitutively active SYK mutants. Co-transporter abundance in the plasma membrane was analyzed by biotinylation of cell surface proteins. Results: Here we describe that tyrosine phosphorylation of NKCC2 and KCC3 regulates their plasma membrane expression levels. We identified that spleen tyrosine kinase (SYK) phosphorylates a specific N-terminal tyrosine residue in each cotransporter. Experimental depletion of endogenous SYK or pharmacological inhibition of its kinase activity increased the abundance of NKCC2 at the plasma membrane of human embryonic kidney cells. In contrast, overexpression of a constitutively active SYK mutant decreased NKCC2 membrane abundance. Intriguingly, the same experimental approaches revealed the opposite effect on KCC3 abundance at the plasma membrane, compatible with the known antagonistic roles of NKCC and KCC cotransporters in cell volume regulation. Conclusions: We identified a novel pathway modulating the cell surface expression of NKCC2 and KCC3 and show that this same pathway has opposite functional outcomes for these two cotransporters. The findings have several biomedical implications considering the role of these cotransporters in regulating blood pressure and cell volume.Funding: UID/MULTI/04046/2019 (BioISI); PTDC/BIA-CEL/28408/2017; Fellowship SFRH/BD/52488/2014 to CAL.info:eu-repo/semantics/publishedVersio

Signal transduction pathways regulating alternative splicing of tumour-related RAC1b

The expression of most genes in the human genome can yield >1 transcript through the process of alternative splicing of pre-messenger RNA. Alternatively spliced transcripts significantly increase the complexity of human gene products because they either influence transcript degradation levels or encode functional protein variants that differ in specific domains. Even with cutting-edge transcriptomic approaches, it has been extremely challenging to understand or predict the complex splicing patterns observed in tissues or in diseases such as cancer; therefore, a better understanding of how cells are able to regulate alternative splicing is required.This work was supported by the Fundação para a Ciência e Tecnologia through a centre grant (UID/MULTI/04046/2013) to BioISI, and by the Portuguese association Maratona da Saúde - Cancro 2014.info:eu-repo/semantics/publishedVersio

A pro-inflammatory microenvironment triggers overexpression of tumor-related RAC1B in polarized colorectal cancer cells

Understand how tumor cells respond to a pro-inflammatory microenvironment with changes in the alternative splicing of RAC1B.FCTN/

Signal transduction pathways regulating alternative splicing of tumour-related RAC1b

The expression of most genes in the human genome can yield >1 transcript through the process of alternative splicing of pre-messenger RNA. Alternatively spliced transcripts significantly increase the complexity of human gene products because they either influence transcript degradation levels or encode functional protein variants that differ in specific domains. Even with cutting-edge transcriptomic approaches, it has been extremely challenging to understand or predict the complex splicing patterns observed in tissues or in diseases such as cancer; therefore, a better understanding of how cells are able to regulate alternative splicing is required.This work was supported by the Fundação para a Ciência e Tecnologia through a centre grant (UID/MULTI/04046/2013) to BioISI, and by the Portuguese association Maratona da Saúde - Cancro 2014.info:eu-repo/semantics/publishedVersio

Rac1 signalling modulates a STAT5/BCL-6 transcriptional switch on cell cycle-associated target gene promoters

Gene expression depends on binding of transcriptional regulators to gene promoters, a process controlled by signalling pathways. The transcriptional repressor B-cell lymphoma (BCL)-6 downregulates genes involved in cell-cycle progression and becomes inactivated following phosphorylation by the Rac1 GTPase-activated protein kinase PAK1. Interestingly, the DNA motifs recognized by BCL-6 and signal transducers and activators of transcription 5 (STAT5) are similar. Because STAT5 stimulation in epithelial cells can also be triggered by Rac1 signalling, we asked whether both factors have opposing roles in transcriptional regulation and whether Rac1 signalling may coordinate a transcription factor switch. We used chromatin immunoprecipitation to show that active Rac1 promotes release of the repressor BCL-6 while increasing binding of STAT5A to a BCL-6-regulated reporter gene. We further show in colorectal cell lines that the endogenous activation status of the Rac1/PAK1 pathway correlated with the phosphorylation status of BCL-6 and STAT5A. Three cellular genes (cyclin D2, p15INK4B, small ubiquitin-like modifier 1) were identified to be inversely regulated by BCL-6 and STAT5A and responded to Rac1 signalling with increased expression and corresponding changes in promoter occupancy. Together, our data show that Rac1 signalling controls a group of target genes that are repressed by BCL-6 and activated by STAT5A, providing novel insights into the modulation of gene transcription by GTPase signalling.Fundação para a Ciência e Tecnologia, Portugal [PPCDT/SAU-OBS/57660/2004] (to P.J.), [PTDC/SAU-GMG/119586/2010] (to P.M.), [PEst-OE/BIA/UI4046/2011] (to the BioFig research unit