Gilles Dubuis | University of Lausanne (original) (raw)

Papers by Gilles Dubuis

Research paper thumbnail of The PI3K/Akt pathway is not a main driver in HDL-mediated cell protection

Cellular Signalling, Oct 1, 2019

High-density lipoproteins (HDLs) can protect cells against a variety of death-inducing stresses. ... more High-density lipoproteins (HDLs) can protect cells against a variety of death-inducing stresses. This is often accompanied by activation of the anti-apoptotic Akt kinase but whether this activation mediates the protective functions of HDLs is still unclear. In this study, we evaluated the roles of PI3K/Akt signaling in endoplasmic reticulum (ER) stress-and starvation-induced cell death using pharmacological and genetic approaches to gain a better understanding of the relationship between Akt-and HDL-mediated protection. Three cell models were used for this purpose, a primary endothelial cell line, an insulinoma cell line and a colon adenocarcinoma cell line. Our results show that HDLs indeed elicited mild Akt activation in all the tested cellular models. PI3K is one of the main upstream proteins involved in Akt stimulation. In the three cellular models, LY294002, a PI3K inhibitor, only slightly blunted HDLs protection, indicating that HDLs induce PI3K-independent cell protection. Furthermore, genetic ablation or silencing of Akt did not abolish the protective effects of HDLs. This study demonstrates that the PI3K-Akt signaling pathway is not the main mediator of the cell protective functions of HDLs. Further investigation is therefore needed to identify the intrinsic mechanism of HDL-mediated cell protection.

Research paper thumbnail of Role of the sub-cellular localization of RasGAP fragment N2 for its ability to sensitize cancer cells to genotoxin-induced apoptosis

Experimental Cell Research, Jul 1, 2009

The specific sensitization of tumor cells to the apoptotic response induced by genotoxins is a pr... more The specific sensitization of tumor cells to the apoptotic response induced by genotoxins is a promising way of increasing the efficacy of chemotherapies. The RasGAP-derived fragment N2, while not regulating apoptosis in normal cells, potently sensitizes tumor cells to cisplatin- and other genotoxin-induced cell death. Here we show that fragment N2 in living cells is mainly located in the cytoplasm and only minimally associated with specific organelles. The cytoplasmic localization of fragment N2 was required for its cisplatin-sensitization property because targeting it to the mitochondria or the ER abrogated its ability to increase the death of tumor cells in response to cisplatin. These results indicate that fragment N2 requires a spatially constrained cellular location to exert its anti-cancer activity.

Research paper thumbnail of HDLs extract lipophilic drugs from cells

Data from independent experiments are depicted with symbols of different motifs and 253 shading. ... more Data from independent experiments are depicted with symbols of different motifs and 253 shading. Comparisons between multiple groups were performed using two-way 254 ANOVA or one-way ANOVA followed by Dunnett's multiple comparisons test using 255 GraphPad Prism. Comparisons between two groups were performed using Student's 256 t-test in GraphPad Prism. 257 258. CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

Research paper thumbnail of CRISPR/Cas9 genome-wide screening identifies KEAP1 as a sorafenib, lenvatinib, and regorafenib sensitivity gene in hepatocellular carcinoma

Oncotarget, Dec 17, 2019

Sorafenib is the first-line drug used for patients with advanced hepatocellular carcinoma (HCC). ... more Sorafenib is the first-line drug used for patients with advanced hepatocellular carcinoma (HCC). However, acquired sorafenib resistance in cancer patients limits its efficacy. Here, we performed the first genome-wide CRISPR/Cas9-based screening on sorafenib-treated HCC cells to identify essential genes for non-mutational mechanisms related to acquired sorafenib resistance and/or sensitivity in HCC cells. KEAP1 was identified as the top candidate gene by Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK). KEAP1 disrupted HCC cells were less sensitive than wild-type cells in short-and long-term sorafenib treatments. Compared to wildtype cells, KEAP1-disrupted cells showed lower basal and sorafenib-induced reactive oxygen species (ROS) levels and were more resistant to oxidative stress-induced cell death. The absence of KEAP1 led to increased activity of Nrf2, a key transcription factor controlling antioxidant responses, as further evidenced by increased expression of Nrf2-controlled genes including NQO1, GPX2 and TXNRD1, which were positively associated with chemoresistance. In addition, KEAP1 disruption counteracted the reduction of cell viability and the elevation of ROS caused by lenvatinib, a drug that recently showed clinical efficacy as a first-line treatment for unresectable HCC. Finally, Keap1 disruption also increased the resistance of cells to regorafenib, a recently approved drug to treat HCC as a second line therapy. Taken together, our data indicate that deregulation of the KEAP1/Nrf2 pathway following KEAP1 inactivation contributes to sorafenib, lenvatinib, and regorafenib resistance in human HCC cells through up-regulation of Nrf2 downstream genes and decreased ROS levels.

Research paper thumbnail of Author Reply to Peer Reviews of HDLs extract lipophilic drugs from cells

Research paper thumbnail of Author Reply to Peer Reviews of Genetic, cellular and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore

Research paper thumbnail of HDLs extract lipophilic drugs from cells 1

Data from independent experiments are depicted with symbols of different motifs and 253 shading. ... more Data from independent experiments are depicted with symbols of different motifs and 253 shading. Comparisons between multiple groups were performed using two-way 254 ANOVA or one-way ANOVA followed by Dunnett's multiple comparisons test using 255 GraphPad Prism. Comparisons between two groups were performed using Student's 256 t-test in GraphPad Prism. 257 258. CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

Research paper thumbnail of Author response: Genetic, cellular, and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore

Research paper thumbnail of APOBEC3C, a nucleolar protein induced by genotoxins, is excluded from DNA damage sites

The FEBS Journal, 2021

The human genome contains 11 APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like) cy... more The human genome contains 11 APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like) cytidine deaminases classified into four families. These proteins function mainly in innate antiviral immunity and can also restrict endogenous retrotransposable element multiplication. The present study focuses on APOBEC3C (A3C), a member of the APOBEC3 sub-family. Some APOBEC3 proteins use their enzymatic activity on genomic DNA, inducing mutations and DNA damage, while other members facilitate DNA repair. Our results show that A3C is highly expressed in cells treated with DNA damaging agents. Its expression is regulated by p53. Depletion of A3C slightly decreases proliferation and does not affect DNA repair via homologous recombination or non-homologous end joining. The A3C interactomes obtained from control cells and cells exposed to the genotoxin etoposide indicated that A3C is a nucleolar protein. This was confirmed by the detection of either endogenous or ectopic A3C in nucleoli. Interestingly, we show that A3C is excluded from areas of DNA breaks in live cells. Our data also indicate that the C-terminal part of A3C is responsible for its nucleolar localization and exclusion from DNA damage sites.

Research paper thumbnail of Publisher Correction: Loss-of-function of the long non-coding RNA A830019P07Rik in mice does not affect insulin expression and secretion

Scientific Reports, 2020

An amendment to this paper has been published and can be accessed via a link at the top of the pa... more An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Research paper thumbnail of Loss-of-function of the long non-coding RNA A830019P07Rik in mice does not affect insulin expression and secretion

Scientific Reports, 2020

Long non-coding RNAs (lncRNAs) contribute to diverse cellular functions and the dysregulation of ... more Long non-coding RNAs (lncRNAs) contribute to diverse cellular functions and the dysregulation of their expression or function can contribute to diseases, including diabetes. The contributions of lncRNAs to β-cell development, function and survival has been extensively studied in vitro. However, very little is currently known on the in vivo roles of lncRNAs in the regulation of glucose and insulin homeostasis. Here we investigated the impact of loss-of-function in mice of the lncRNA A830019P07Rik, hereafter P07Rik, which was previously reported to be associated with reduced plasma insulin levels. Compared with wild-type littermates, male and female P07Rik mutant mice did not show any defect in glycaemia and plasma insulin levels in both fed and fasted state. Furthermore, P07Rik mutant mice displayed similar glucose and insulin levels in response to an intra-peritoneal glucose tolerance test. Ex vivo, islets from mutant P07Rik released similar amount of insulin in response to increase...

Research paper thumbnail of Genetic, cellular and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore

Cell-penetrating peptides (CPPs) allow intracellular delivery of cargo molecules. They provide ef... more Cell-penetrating peptides (CPPs) allow intracellular delivery of cargo molecules. They provide efficient methodology to transfer bioactive molecules in cells, in particular in conditions when transcription or translation of cargo-encoding sequences is not desirable or achievable. The mechanisms allowing CPPs to enter cells are ill-defined. Using a CRISPR/Cas9-based screening, we discovered that KCNQ5, KCNN4, and KCNK5 potassium channels positively modulate cationic CPP direct translocation into cells by decreasing the transmembrane potential (Vm). These findings provide the first unbiased genetic validation of the role of Vm in CPP translocation in cells. In silico modeling and live cell experiments indicate that CPPs, by bringing positive charges on the outer surface of the plasma membrane, decrease the Vm to very low values (−150 mV or less), a situation we have coined megapolarization that then triggers formation of water pores used by CPPs to enter cells. Megapolarization lowers...

Research paper thumbnail of CRISPR/Cas9 genome-wide screening identifies KEAP1 as a sorafenib, lenvatinib, and regorafenib sensitivity gene in hepatocellular carcinoma

Oncotarget, 2019

Sorafenib is the first-line drug used for patients with advanced hepatocellular carcinoma (HCC). ... more Sorafenib is the first-line drug used for patients with advanced hepatocellular carcinoma (HCC). However, acquired sorafenib resistance in cancer patients limits its efficacy. Here, we performed the first genome-wide CRISPR/Cas9-based screening on sorafenib-treated HCC cells to identify essential genes for non-mutational mechanisms related to acquired sorafenib resistance and/or sensitivity in HCC cells. KEAP1 was identified as the top candidate gene by Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK). KEAP1 disrupted HCC cells were less sensitive than wild-type cells in short-and long-term sorafenib treatments. Compared to wildtype cells, KEAP1-disrupted cells showed lower basal and sorafenib-induced reactive oxygen species (ROS) levels and were more resistant to oxidative stress-induced cell death. The absence of KEAP1 led to increased activity of Nrf2, a key transcription factor controlling antioxidant responses, as further evidenced by increased expression of Nrf2-controlled genes including NQO1, GPX2 and TXNRD1, which were positively associated with chemoresistance. In addition, KEAP1 disruption counteracted the reduction of cell viability and the elevation of ROS caused by lenvatinib, a drug that recently showed clinical efficacy as a first-line treatment for unresectable HCC. Finally, Keap1 disruption also increased the resistance of cells to regorafenib, a recently approved drug to treat HCC as a second line therapy. Taken together, our data indicate that deregulation of the KEAP1/Nrf2 pathway following KEAP1 inactivation contributes to sorafenib, lenvatinib, and regorafenib resistance in human HCC cells through up-regulation of Nrf2 downstream genes and decreased ROS levels.

Research paper thumbnail of The PI3K/Akt pathway is not a main driver in HDL-mediated cell protection

Cellular Signalling, 2019

High-density lipoproteins (HDLs) can protect cells against a variety of death-inducing stresses. ... more High-density lipoproteins (HDLs) can protect cells against a variety of death-inducing stresses. This is often accompanied by activation of the anti-apoptotic Akt kinase but whether this activation mediates the protective functions of HDLs is still unclear. In this study, we evaluated the roles of PI3K/Akt signaling in endoplasmic reticulum (ER) stress-and starvation-induced cell death using pharmacological and genetic approaches to gain a better understanding of the relationship between Akt-and HDL-mediated protection. Three cell models were used for this purpose, a primary endothelial cell line, an insulinoma cell line and a colon adenocarcinoma cell line. Our results show that HDLs indeed elicited mild Akt activation in all the tested cellular models. PI3K is one of the main upstream proteins involved in Akt stimulation. In the three cellular models, LY294002, a PI3K inhibitor, only slightly blunted HDLs protection, indicating that HDLs induce PI3K-independent cell protection. Furthermore, genetic ablation or silencing of Akt did not abolish the protective effects of HDLs. This study demonstrates that the PI3K-Akt signaling pathway is not the main mediator of the cell protective functions of HDLs. Further investigation is therefore needed to identify the intrinsic mechanism of HDL-mediated cell protection.

Research paper thumbnail of RasGAP-Derived Fragment N Increases the Resistance of Beta Cells towards Apoptosis in NOD Mice and Delays the Progression from Mild to Overt Diabetes

PLoS ONE, 2011

The caspase-3-generated RasGAP N-terminal fragment (fragment N) inhibits apoptosis in a Ras-PI3K-... more The caspase-3-generated RasGAP N-terminal fragment (fragment N) inhibits apoptosis in a Ras-PI3K-Akt-dependent manner. Fragment N protects various cell types, including insulin-secreting cells, against different types of stresses. Whether fragment N exerts a protective role during the development of type 1 diabetes is however not known. Non-obese diabetic (NOD) mice represent a well-known model for spontaneous development of type 1 diabetes that shares similarities with the diseases encountered in humans. To assess the role of fragment N in type 1 diabetes development, a transgene encoding fragment N under the control of the rat insulin promoter (RIP) was back-crossed into the NOD background creating the NOD-RIPN strain. Despite a mosaic expression of fragment N in the beta cell population of NOD-RIPN mice, islets isolated from these mice were more resistant to apoptosis than control NOD islets. Islet lymphocytic infiltration and occurrence of a mild increase in glycemia developed with the same kinetics in both strains. However, the period of time separating the mild increase in glycemia and overt diabetes was significantly longer in NOD-RIPN mice compared to the control NOD mice. There was also a significant decrease in the number of apoptotic beta cells in situ at 16 weeks of age in the NOD-RIPN mice. Fragment N exerts therefore a protective effect on beta cells within the pro-diabetogenic NOD background and this prevents a fast progression from mild to overt diabetes.

Research paper thumbnail of Impaired Akt Activity Down-Modulation, Caspase-3 Activation, and Apoptosis in Cells Expressing a Caspase-resistant Mutant of RasGAP at Position 157

Molecular Biology of the Cell, 2005

RasGAP bears two caspase-3 cleavage sites that are used sequentially as caspase activity increase... more RasGAP bears two caspase-3 cleavage sites that are used sequentially as caspase activity increases in cells. When caspase-3 is mildly activated, RasGAP is first cleaved at position 455. This leads to the production of an N-terminal fragment, called fragment N, that activates the Ras-PI3K-Akt pathway and that promotes cell survival. At higher caspase activity, RasGAP is further cleaved at position 157 generating two small N-terminal fragments named N1 and N2. We have now determined the contribution of this second cleavage event in the regulation of apoptosis using cells in which the wild-type RasGAP gene has been replaced by a cDNA encoding a RasGAP mutant that cannot be cleaved at position 157. Our results show that cleavage of fragment N at position 157 leads to a marked reduction in Akt activity. This is accompanied by efficient processing of caspase-3 that favors cell death in response to various apoptotic stimuli. In nontumorigenic cells, fragments N1 and N2 do not modulate apop...

Research paper thumbnail of HDLs extract lipophilic drugs from cells

High-density lipoproteins (HDLs) prevent cell death induced by a variety of cytotoxic drugs. The ... more High-density lipoproteins (HDLs) prevent cell death induced by a variety of cytotoxic drugs. The underlying mechanisms are however still poorly understood. Here we present evidence that HDLs efficiently protect cells against thapsigargin (a SERCA inhibitor) by extracting the drug from cells. Drug efflux could also be triggered to some extent by low-density lipoproteins (LDLs) and serum, which contains lipoproteins. HDLs did not reverse the non-lethal mild endoplasmic reticulum (ER) stress response induced by low thapsigargin concentrations or by SERCA knock-down but HDLs inhibited the toxic SERCA-independent effects mediated by high thapsigargin concentrations. HDLs were also found to extract other lipophilic compounds, such as the anti-diabetic drug glibenclamide. In contrast, hydrophilic substances (doxorubicin hydrochloride, rhodamine 123) were not extracted from cells by HDLs. This work shows that HDLs utilize their capacity of loading themselves with lipophilic compounds, akin ...

Research paper thumbnail of Splice variant-specific stabilization of JNKs by IB1/JIP1

Cellular signalling, 2007

Islet-Brain 1 (IB1) (also called JNK-interacting protein 1; JIP1) is a scaffold protein that teth... more Islet-Brain 1 (IB1) (also called JNK-interacting protein 1; JIP1) is a scaffold protein that tethers components of the JNK mitogen-activated protein kinase pathway inducing a modulation of the activity and the target specificity of the JNK kinases. Dysfunctions in IB1 have been associated with diseases such as early type II diabetes. To gain more insight in the functions of IB1, its ability to modulate the expression levels of the various JNK proteins was assessed. Each of the three JNK genes gives rise to several splice variants encoding short or long proteins. The expression levels of the short JNK proteins, but not of the long variants, were systematically higher in rat tissues and in transformed cell lines expressing high IB1 levels compared to tissues and cells with no or low IB1 expression. HEK293 cells bearing a tetracycline-inducible IB1 construct showed a specific increase of the short JNK endogenous splice variants in the presence of tetracycline. The augmented expression level of the short JNK splice variants induced by IB1 resulted from an increased stability towards degradation. Modulation of the stability of specific JNK splice variants represents therefore a newly identified mechanism used by IB1 to regulate the JNK MAPK pathway.

Research paper thumbnail of Caspase-3 protects stressed organs against cell death

Molecular and cellular biology, 2012

The ability to generate appropriate defense responses is crucial for the survival of an organism ... more The ability to generate appropriate defense responses is crucial for the survival of an organism exposed to pathogenesis-inducing insults. However, the mechanisms that allow tissues and organs to cope with such stresses are poorly understood. Here we show that caspase-3-knockout mice or caspase inhibitor-treated mice were defective in activating the antiapoptotic Akt kinase in response to various chemical and environmental stresses causing sunburns, cardiomyopathy, or colitis. Defective Akt activation in caspase-3-knockout mice was accompanied by increased cell death and impaired survival in some cases. Mice homozygous for a mutation in RasGAP that prevents its cleavage by caspase-3 exhibited a similar defect in Akt activation, leading to increased apoptosis in stressed organs, marked deterioration of their physiological functions, and stronger disease development. Our results provide evidence for the relevance of caspase-3 as a stress intensity sensor that controls cell fate by either initiating a RasGAP cleavage-dependent cell resistance program or a cell suicide response.

Research paper thumbnail of HDLs protect the MIN6 insulinoma cell line against tunicamycin-induced apoptosis without inhibiting ER stress and without restoring ER functionality

Molecular and cellular endocrinology, Jan 5, 2013

HDLs protect pancreatic beta cells against apoptosis induced by several endoplasmic reticulum (ER... more HDLs protect pancreatic beta cells against apoptosis induced by several endoplasmic reticulum (ER) stressors, including thapsigargin, cyclopiazonic acid, palmitate and insulin over-expression. This protection is mediated by the capacity of HDLs to maintain proper ER morphology and ER functions such as protein folding and trafficking. Here, we identified a distinct mode of protection exerted by HDLs in beta cells challenged with tunicamycin (TM), a protein glycosylation inhibitor inducing ER stress. HDLs were found to inhibit apoptosis induced by TM in the MIN6 insulinoma cell line and this correlated with the maintenance of a normal ER morphology. Surprisingly however, this protective response was neither associated with a significant ER stress reduction, nor with restoration of protein folding and trafficking in the ER. These data indicate that HDLs can use at least two mechanisms to protect beta cells against ER stressors. One that relies on the maintenance of ER function and one that operates independently of ER function modulation. The capacity of HDLs to activate several anti-apoptotic pathways in beta cells may explain their ability to efficiently protect these cells against a variety of insults.

Research paper thumbnail of The PI3K/Akt pathway is not a main driver in HDL-mediated cell protection

Cellular Signalling, Oct 1, 2019

High-density lipoproteins (HDLs) can protect cells against a variety of death-inducing stresses. ... more High-density lipoproteins (HDLs) can protect cells against a variety of death-inducing stresses. This is often accompanied by activation of the anti-apoptotic Akt kinase but whether this activation mediates the protective functions of HDLs is still unclear. In this study, we evaluated the roles of PI3K/Akt signaling in endoplasmic reticulum (ER) stress-and starvation-induced cell death using pharmacological and genetic approaches to gain a better understanding of the relationship between Akt-and HDL-mediated protection. Three cell models were used for this purpose, a primary endothelial cell line, an insulinoma cell line and a colon adenocarcinoma cell line. Our results show that HDLs indeed elicited mild Akt activation in all the tested cellular models. PI3K is one of the main upstream proteins involved in Akt stimulation. In the three cellular models, LY294002, a PI3K inhibitor, only slightly blunted HDLs protection, indicating that HDLs induce PI3K-independent cell protection. Furthermore, genetic ablation or silencing of Akt did not abolish the protective effects of HDLs. This study demonstrates that the PI3K-Akt signaling pathway is not the main mediator of the cell protective functions of HDLs. Further investigation is therefore needed to identify the intrinsic mechanism of HDL-mediated cell protection.

Research paper thumbnail of Role of the sub-cellular localization of RasGAP fragment N2 for its ability to sensitize cancer cells to genotoxin-induced apoptosis

Experimental Cell Research, Jul 1, 2009

The specific sensitization of tumor cells to the apoptotic response induced by genotoxins is a pr... more The specific sensitization of tumor cells to the apoptotic response induced by genotoxins is a promising way of increasing the efficacy of chemotherapies. The RasGAP-derived fragment N2, while not regulating apoptosis in normal cells, potently sensitizes tumor cells to cisplatin- and other genotoxin-induced cell death. Here we show that fragment N2 in living cells is mainly located in the cytoplasm and only minimally associated with specific organelles. The cytoplasmic localization of fragment N2 was required for its cisplatin-sensitization property because targeting it to the mitochondria or the ER abrogated its ability to increase the death of tumor cells in response to cisplatin. These results indicate that fragment N2 requires a spatially constrained cellular location to exert its anti-cancer activity.

Research paper thumbnail of HDLs extract lipophilic drugs from cells

Data from independent experiments are depicted with symbols of different motifs and 253 shading. ... more Data from independent experiments are depicted with symbols of different motifs and 253 shading. Comparisons between multiple groups were performed using two-way 254 ANOVA or one-way ANOVA followed by Dunnett's multiple comparisons test using 255 GraphPad Prism. Comparisons between two groups were performed using Student's 256 t-test in GraphPad Prism. 257 258. CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

Research paper thumbnail of CRISPR/Cas9 genome-wide screening identifies KEAP1 as a sorafenib, lenvatinib, and regorafenib sensitivity gene in hepatocellular carcinoma

Oncotarget, Dec 17, 2019

Sorafenib is the first-line drug used for patients with advanced hepatocellular carcinoma (HCC). ... more Sorafenib is the first-line drug used for patients with advanced hepatocellular carcinoma (HCC). However, acquired sorafenib resistance in cancer patients limits its efficacy. Here, we performed the first genome-wide CRISPR/Cas9-based screening on sorafenib-treated HCC cells to identify essential genes for non-mutational mechanisms related to acquired sorafenib resistance and/or sensitivity in HCC cells. KEAP1 was identified as the top candidate gene by Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK). KEAP1 disrupted HCC cells were less sensitive than wild-type cells in short-and long-term sorafenib treatments. Compared to wildtype cells, KEAP1-disrupted cells showed lower basal and sorafenib-induced reactive oxygen species (ROS) levels and were more resistant to oxidative stress-induced cell death. The absence of KEAP1 led to increased activity of Nrf2, a key transcription factor controlling antioxidant responses, as further evidenced by increased expression of Nrf2-controlled genes including NQO1, GPX2 and TXNRD1, which were positively associated with chemoresistance. In addition, KEAP1 disruption counteracted the reduction of cell viability and the elevation of ROS caused by lenvatinib, a drug that recently showed clinical efficacy as a first-line treatment for unresectable HCC. Finally, Keap1 disruption also increased the resistance of cells to regorafenib, a recently approved drug to treat HCC as a second line therapy. Taken together, our data indicate that deregulation of the KEAP1/Nrf2 pathway following KEAP1 inactivation contributes to sorafenib, lenvatinib, and regorafenib resistance in human HCC cells through up-regulation of Nrf2 downstream genes and decreased ROS levels.

Research paper thumbnail of Author Reply to Peer Reviews of HDLs extract lipophilic drugs from cells

Research paper thumbnail of Author Reply to Peer Reviews of Genetic, cellular and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore

Research paper thumbnail of HDLs extract lipophilic drugs from cells 1

Data from independent experiments are depicted with symbols of different motifs and 253 shading. ... more Data from independent experiments are depicted with symbols of different motifs and 253 shading. Comparisons between multiple groups were performed using two-way 254 ANOVA or one-way ANOVA followed by Dunnett's multiple comparisons test using 255 GraphPad Prism. Comparisons between two groups were performed using Student's 256 t-test in GraphPad Prism. 257 258. CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

Research paper thumbnail of Author response: Genetic, cellular, and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore

Research paper thumbnail of APOBEC3C, a nucleolar protein induced by genotoxins, is excluded from DNA damage sites

The FEBS Journal, 2021

The human genome contains 11 APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like) cy... more The human genome contains 11 APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like) cytidine deaminases classified into four families. These proteins function mainly in innate antiviral immunity and can also restrict endogenous retrotransposable element multiplication. The present study focuses on APOBEC3C (A3C), a member of the APOBEC3 sub-family. Some APOBEC3 proteins use their enzymatic activity on genomic DNA, inducing mutations and DNA damage, while other members facilitate DNA repair. Our results show that A3C is highly expressed in cells treated with DNA damaging agents. Its expression is regulated by p53. Depletion of A3C slightly decreases proliferation and does not affect DNA repair via homologous recombination or non-homologous end joining. The A3C interactomes obtained from control cells and cells exposed to the genotoxin etoposide indicated that A3C is a nucleolar protein. This was confirmed by the detection of either endogenous or ectopic A3C in nucleoli. Interestingly, we show that A3C is excluded from areas of DNA breaks in live cells. Our data also indicate that the C-terminal part of A3C is responsible for its nucleolar localization and exclusion from DNA damage sites.

Research paper thumbnail of Publisher Correction: Loss-of-function of the long non-coding RNA A830019P07Rik in mice does not affect insulin expression and secretion

Scientific Reports, 2020

An amendment to this paper has been published and can be accessed via a link at the top of the pa... more An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Research paper thumbnail of Loss-of-function of the long non-coding RNA A830019P07Rik in mice does not affect insulin expression and secretion

Scientific Reports, 2020

Long non-coding RNAs (lncRNAs) contribute to diverse cellular functions and the dysregulation of ... more Long non-coding RNAs (lncRNAs) contribute to diverse cellular functions and the dysregulation of their expression or function can contribute to diseases, including diabetes. The contributions of lncRNAs to β-cell development, function and survival has been extensively studied in vitro. However, very little is currently known on the in vivo roles of lncRNAs in the regulation of glucose and insulin homeostasis. Here we investigated the impact of loss-of-function in mice of the lncRNA A830019P07Rik, hereafter P07Rik, which was previously reported to be associated with reduced plasma insulin levels. Compared with wild-type littermates, male and female P07Rik mutant mice did not show any defect in glycaemia and plasma insulin levels in both fed and fasted state. Furthermore, P07Rik mutant mice displayed similar glucose and insulin levels in response to an intra-peritoneal glucose tolerance test. Ex vivo, islets from mutant P07Rik released similar amount of insulin in response to increase...

Research paper thumbnail of Genetic, cellular and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore

Cell-penetrating peptides (CPPs) allow intracellular delivery of cargo molecules. They provide ef... more Cell-penetrating peptides (CPPs) allow intracellular delivery of cargo molecules. They provide efficient methodology to transfer bioactive molecules in cells, in particular in conditions when transcription or translation of cargo-encoding sequences is not desirable or achievable. The mechanisms allowing CPPs to enter cells are ill-defined. Using a CRISPR/Cas9-based screening, we discovered that KCNQ5, KCNN4, and KCNK5 potassium channels positively modulate cationic CPP direct translocation into cells by decreasing the transmembrane potential (Vm). These findings provide the first unbiased genetic validation of the role of Vm in CPP translocation in cells. In silico modeling and live cell experiments indicate that CPPs, by bringing positive charges on the outer surface of the plasma membrane, decrease the Vm to very low values (−150 mV or less), a situation we have coined megapolarization that then triggers formation of water pores used by CPPs to enter cells. Megapolarization lowers...

Research paper thumbnail of CRISPR/Cas9 genome-wide screening identifies KEAP1 as a sorafenib, lenvatinib, and regorafenib sensitivity gene in hepatocellular carcinoma

Oncotarget, 2019

Sorafenib is the first-line drug used for patients with advanced hepatocellular carcinoma (HCC). ... more Sorafenib is the first-line drug used for patients with advanced hepatocellular carcinoma (HCC). However, acquired sorafenib resistance in cancer patients limits its efficacy. Here, we performed the first genome-wide CRISPR/Cas9-based screening on sorafenib-treated HCC cells to identify essential genes for non-mutational mechanisms related to acquired sorafenib resistance and/or sensitivity in HCC cells. KEAP1 was identified as the top candidate gene by Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK). KEAP1 disrupted HCC cells were less sensitive than wild-type cells in short-and long-term sorafenib treatments. Compared to wildtype cells, KEAP1-disrupted cells showed lower basal and sorafenib-induced reactive oxygen species (ROS) levels and were more resistant to oxidative stress-induced cell death. The absence of KEAP1 led to increased activity of Nrf2, a key transcription factor controlling antioxidant responses, as further evidenced by increased expression of Nrf2-controlled genes including NQO1, GPX2 and TXNRD1, which were positively associated with chemoresistance. In addition, KEAP1 disruption counteracted the reduction of cell viability and the elevation of ROS caused by lenvatinib, a drug that recently showed clinical efficacy as a first-line treatment for unresectable HCC. Finally, Keap1 disruption also increased the resistance of cells to regorafenib, a recently approved drug to treat HCC as a second line therapy. Taken together, our data indicate that deregulation of the KEAP1/Nrf2 pathway following KEAP1 inactivation contributes to sorafenib, lenvatinib, and regorafenib resistance in human HCC cells through up-regulation of Nrf2 downstream genes and decreased ROS levels.

Research paper thumbnail of The PI3K/Akt pathway is not a main driver in HDL-mediated cell protection

Cellular Signalling, 2019

High-density lipoproteins (HDLs) can protect cells against a variety of death-inducing stresses. ... more High-density lipoproteins (HDLs) can protect cells against a variety of death-inducing stresses. This is often accompanied by activation of the anti-apoptotic Akt kinase but whether this activation mediates the protective functions of HDLs is still unclear. In this study, we evaluated the roles of PI3K/Akt signaling in endoplasmic reticulum (ER) stress-and starvation-induced cell death using pharmacological and genetic approaches to gain a better understanding of the relationship between Akt-and HDL-mediated protection. Three cell models were used for this purpose, a primary endothelial cell line, an insulinoma cell line and a colon adenocarcinoma cell line. Our results show that HDLs indeed elicited mild Akt activation in all the tested cellular models. PI3K is one of the main upstream proteins involved in Akt stimulation. In the three cellular models, LY294002, a PI3K inhibitor, only slightly blunted HDLs protection, indicating that HDLs induce PI3K-independent cell protection. Furthermore, genetic ablation or silencing of Akt did not abolish the protective effects of HDLs. This study demonstrates that the PI3K-Akt signaling pathway is not the main mediator of the cell protective functions of HDLs. Further investigation is therefore needed to identify the intrinsic mechanism of HDL-mediated cell protection.

Research paper thumbnail of RasGAP-Derived Fragment N Increases the Resistance of Beta Cells towards Apoptosis in NOD Mice and Delays the Progression from Mild to Overt Diabetes

PLoS ONE, 2011

The caspase-3-generated RasGAP N-terminal fragment (fragment N) inhibits apoptosis in a Ras-PI3K-... more The caspase-3-generated RasGAP N-terminal fragment (fragment N) inhibits apoptosis in a Ras-PI3K-Akt-dependent manner. Fragment N protects various cell types, including insulin-secreting cells, against different types of stresses. Whether fragment N exerts a protective role during the development of type 1 diabetes is however not known. Non-obese diabetic (NOD) mice represent a well-known model for spontaneous development of type 1 diabetes that shares similarities with the diseases encountered in humans. To assess the role of fragment N in type 1 diabetes development, a transgene encoding fragment N under the control of the rat insulin promoter (RIP) was back-crossed into the NOD background creating the NOD-RIPN strain. Despite a mosaic expression of fragment N in the beta cell population of NOD-RIPN mice, islets isolated from these mice were more resistant to apoptosis than control NOD islets. Islet lymphocytic infiltration and occurrence of a mild increase in glycemia developed with the same kinetics in both strains. However, the period of time separating the mild increase in glycemia and overt diabetes was significantly longer in NOD-RIPN mice compared to the control NOD mice. There was also a significant decrease in the number of apoptotic beta cells in situ at 16 weeks of age in the NOD-RIPN mice. Fragment N exerts therefore a protective effect on beta cells within the pro-diabetogenic NOD background and this prevents a fast progression from mild to overt diabetes.

Research paper thumbnail of Impaired Akt Activity Down-Modulation, Caspase-3 Activation, and Apoptosis in Cells Expressing a Caspase-resistant Mutant of RasGAP at Position 157

Molecular Biology of the Cell, 2005

RasGAP bears two caspase-3 cleavage sites that are used sequentially as caspase activity increase... more RasGAP bears two caspase-3 cleavage sites that are used sequentially as caspase activity increases in cells. When caspase-3 is mildly activated, RasGAP is first cleaved at position 455. This leads to the production of an N-terminal fragment, called fragment N, that activates the Ras-PI3K-Akt pathway and that promotes cell survival. At higher caspase activity, RasGAP is further cleaved at position 157 generating two small N-terminal fragments named N1 and N2. We have now determined the contribution of this second cleavage event in the regulation of apoptosis using cells in which the wild-type RasGAP gene has been replaced by a cDNA encoding a RasGAP mutant that cannot be cleaved at position 157. Our results show that cleavage of fragment N at position 157 leads to a marked reduction in Akt activity. This is accompanied by efficient processing of caspase-3 that favors cell death in response to various apoptotic stimuli. In nontumorigenic cells, fragments N1 and N2 do not modulate apop...

Research paper thumbnail of HDLs extract lipophilic drugs from cells

High-density lipoproteins (HDLs) prevent cell death induced by a variety of cytotoxic drugs. The ... more High-density lipoproteins (HDLs) prevent cell death induced by a variety of cytotoxic drugs. The underlying mechanisms are however still poorly understood. Here we present evidence that HDLs efficiently protect cells against thapsigargin (a SERCA inhibitor) by extracting the drug from cells. Drug efflux could also be triggered to some extent by low-density lipoproteins (LDLs) and serum, which contains lipoproteins. HDLs did not reverse the non-lethal mild endoplasmic reticulum (ER) stress response induced by low thapsigargin concentrations or by SERCA knock-down but HDLs inhibited the toxic SERCA-independent effects mediated by high thapsigargin concentrations. HDLs were also found to extract other lipophilic compounds, such as the anti-diabetic drug glibenclamide. In contrast, hydrophilic substances (doxorubicin hydrochloride, rhodamine 123) were not extracted from cells by HDLs. This work shows that HDLs utilize their capacity of loading themselves with lipophilic compounds, akin ...

Research paper thumbnail of Splice variant-specific stabilization of JNKs by IB1/JIP1

Cellular signalling, 2007

Islet-Brain 1 (IB1) (also called JNK-interacting protein 1; JIP1) is a scaffold protein that teth... more Islet-Brain 1 (IB1) (also called JNK-interacting protein 1; JIP1) is a scaffold protein that tethers components of the JNK mitogen-activated protein kinase pathway inducing a modulation of the activity and the target specificity of the JNK kinases. Dysfunctions in IB1 have been associated with diseases such as early type II diabetes. To gain more insight in the functions of IB1, its ability to modulate the expression levels of the various JNK proteins was assessed. Each of the three JNK genes gives rise to several splice variants encoding short or long proteins. The expression levels of the short JNK proteins, but not of the long variants, were systematically higher in rat tissues and in transformed cell lines expressing high IB1 levels compared to tissues and cells with no or low IB1 expression. HEK293 cells bearing a tetracycline-inducible IB1 construct showed a specific increase of the short JNK endogenous splice variants in the presence of tetracycline. The augmented expression level of the short JNK splice variants induced by IB1 resulted from an increased stability towards degradation. Modulation of the stability of specific JNK splice variants represents therefore a newly identified mechanism used by IB1 to regulate the JNK MAPK pathway.

Research paper thumbnail of Caspase-3 protects stressed organs against cell death

Molecular and cellular biology, 2012

The ability to generate appropriate defense responses is crucial for the survival of an organism ... more The ability to generate appropriate defense responses is crucial for the survival of an organism exposed to pathogenesis-inducing insults. However, the mechanisms that allow tissues and organs to cope with such stresses are poorly understood. Here we show that caspase-3-knockout mice or caspase inhibitor-treated mice were defective in activating the antiapoptotic Akt kinase in response to various chemical and environmental stresses causing sunburns, cardiomyopathy, or colitis. Defective Akt activation in caspase-3-knockout mice was accompanied by increased cell death and impaired survival in some cases. Mice homozygous for a mutation in RasGAP that prevents its cleavage by caspase-3 exhibited a similar defect in Akt activation, leading to increased apoptosis in stressed organs, marked deterioration of their physiological functions, and stronger disease development. Our results provide evidence for the relevance of caspase-3 as a stress intensity sensor that controls cell fate by either initiating a RasGAP cleavage-dependent cell resistance program or a cell suicide response.

Research paper thumbnail of HDLs protect the MIN6 insulinoma cell line against tunicamycin-induced apoptosis without inhibiting ER stress and without restoring ER functionality

Molecular and cellular endocrinology, Jan 5, 2013

HDLs protect pancreatic beta cells against apoptosis induced by several endoplasmic reticulum (ER... more HDLs protect pancreatic beta cells against apoptosis induced by several endoplasmic reticulum (ER) stressors, including thapsigargin, cyclopiazonic acid, palmitate and insulin over-expression. This protection is mediated by the capacity of HDLs to maintain proper ER morphology and ER functions such as protein folding and trafficking. Here, we identified a distinct mode of protection exerted by HDLs in beta cells challenged with tunicamycin (TM), a protein glycosylation inhibitor inducing ER stress. HDLs were found to inhibit apoptosis induced by TM in the MIN6 insulinoma cell line and this correlated with the maintenance of a normal ER morphology. Surprisingly however, this protective response was neither associated with a significant ER stress reduction, nor with restoration of protein folding and trafficking in the ER. These data indicate that HDLs can use at least two mechanisms to protect beta cells against ER stressors. One that relies on the maintenance of ER function and one that operates independently of ER function modulation. The capacity of HDLs to activate several anti-apoptotic pathways in beta cells may explain their ability to efficiently protect these cells against a variety of insults.