Patent Application
20240173380
The contents of the electronic sequence listing (370114_401 USPC_SeqListing_Revised.txt; Size: 1,234 bytes; and Date of Creation: Oct. 30, 2023) is herein incorporated by reference in its entirety and is being submitted electronically.
The present invention refers to the medical field. Particularly, the present invention refers to Connexin 43 (Cx43) for use in the treatment of cancer wherein the cancer is characterized by the activation of a mitogen-activated protein kinase (MAPK) selected from the list consisting of: BRAF, RAS, MEK or ERK. The present invention also refers to a combination drug product comprising Cx43 and an inhibitor of a mitogen-activated protein kinase (MAPK) selected from the list consisting of: BRAF, RAS, MEK or ERK.
Mutations of MAPK, for instance BRAF mutations, are detected in many types of cancer. BRAF is a potent oncogene that is activated in more than 8% of all cancer types. Approximately about 30-50% of thyroid cancer, 3% of non-small cell lung cancer (NSCLC), 5% to 10% of patients with metastatic colorectal cancer, 100% of hairy cell leukaemia and more than 50% of melanoma patients have a mutation of the proto-oncogene BRAF, that lead to a constitutive activation of the serine/threonine kinases of the MAPK cascade.
Mutations in BRAF were initially described in 2002, with V600E being the most common mutation. These mutations lead to constitutive activation of BRAF and RAS-RAF-MEK-ERK signalling cascade, which promotes cancer growth by enhancing cell proliferation and survival while inhibiting cell death by apoptosis. Following the discovery of V600E mutation, targeted therapies including BRAF-specific small molecule inhibitors such as dabrafenib, and later the MEK inhibitors such as trametinib, were developed for the treatment of metastatic disease. BRAF/MEK inhibitors (BRAF/MEKi) are indicated for patients with unresectable or metastatic melanoma due their efficacy, and the combination of these inhibitors has improved the progression-free survival. In contrast to patients with BRAF mutated metastatic melanoma, only 5% of patients with BRAF mutated colorectal cancer responded to BRAF inhibitors monotherapy. Efforts to define resistance mechanisms to BRAF inhibition have led to new clinical trials using combined therapies in order to avoid primary and acquired resistance to BRAF inhibitors.
As previously mentioned, targeting MAPK pathway with specific BRAF/MEKi showed high initial responses in the majority of mutant BRAF melanoma patients. However, more than 50% of patients acquire resistance to the drugs and, consequently, cancer relapse. Also, some patients with BRAF mutant melanomas do not respond to treatment because of intrinsic mechanisms of resistance.
As a result, there is an unmet medical need of finding new pharmaceutical strategies for reducing the resistance to MAPK inhibitors and, consequently, for ameliorating patient relapse.
The present invention solves this problem by using Cx43, preferably in combination with MAPK inhibitors.
As explained above, the present invention refers to Cx43, preferably in combination with an inhibitor of a mitogen-activated protein kinase (MAPK) selected from the list consisting of: BRAF, RAS, MEK or ERK, for use in the treatment of cancer wherein the cancer is characterized by the activation of mitogen-activated protein kinase (MAPK) selected from the list consisting of: BRAF, RAS, MEK or ERK.
Kindly note that cancer types characterized by the activation of MAPK are well-established in the prior art, such as it is indicated for instance in this document: [A S Dhillon, et al., 2007. MAP kinase signalling pathways in cancer. Oncogene volume 26, pages 3279 3290(2007). Published: 14 May 2007].
Particularly, the inventors of the present invention have found that Cx43 induces cellular senescence in BRAF-mutated tumour and enhances BRAF/MEK-induced senescence (
The channel protein Cx43, in a channel-independent manner (
The restoration of Cx43, using a vector or via EVs, increases BRAF/MEKi efficiency, preventing and reverting drug resistance (
So, the first embodiment of the present invention refers to Cx43 for use in the treatment of cancer wherein the cancer is characterized by the activation of mitogen-activated protein kinase (MAPK) selected from the list consisting of: BRAF, RAS, MEK or ERK.
In a preferred embodiment, the cancer is characterized by the activation of BRAF or NRAS.
In a preferred embodiment, the cancer selected from: melanoma, colon cancer, lung cancer or breast cancer.
In a preferred embodiment, Cx43 is administered before, after or simultaneously to a treatment with an inhibitor of a mitogen-activated protein kinase (MAPK) selected from the list consisting of: BRAF, RAS, MEK or ERK.
In a preferred embodiment, Cx43 is administered before, after or simultaneously to a treatment with a BRAF and/or MEK inhibitor.
In a preferred embodiment, Cx43 is administered before, after or simultaneously to a treatment with dabrafenib, trametinib, vemurafenib, encorafenib, cobimetinib and/or binimetinib.
In a preferred embodiment, Cx43 is administered before, after or simultaneously to a treatment with a senolytic agent, preferably navitoclax.
The second embodiment refers to a combination drug product comprising Cx43 and an inhibitor of a mitogen-activated protein kinase (MAPK) selected from the list consisting of: BRAF, RAS, MEK or ERK.
In a preferred embodiment, the combination drug product comprises Cx43 and a BRAF and/or MEK inhibitor.
In a preferred embodiment, the combination drug product comprises Cx43 and dabrafenib, trametinib, vemurafenib, encorafenib, cobimetinib and/or binimetinib.
In a preferred embodiment, the combination drug product further comprises a senolytic agent, preferably navitoclax.
The third embodiment of the invention refers to a pharmaceutical composition comprising the combination drug product of the invention and, optionally, pharmaceutically acceptable excipients or carriers.
The fourth embodiment of the invention refers to the pharmaceutical composition of the invention for use in the treatment of cancer wherein the cancer is characterized by the activation of mitogen-activated protein kinase (MAPK) selected from the list consisting of: BRAF, RAS, MEK or ERK wherein Cx43 is administered by using a delivery vehicle.
In a preferred embodiment, the delivery vehicle is a nanoparticle, an extracellular vesicle or an expression vector which encodes Cx43. Particularly, any of the extracellular vesicles known in the prior art may be used for this purpose [Guillaume van Niel, et al., 2018. Shedding light on the cell biology of extracellular vesicles. Nature Reviews Molecular Cell Biology volume 19, pages 213 228(2018)] [Oscar P. B. Wiklander et al., 2019. Advances in therapeutic applications of extracellular vesicles. Science Translational Medicine. 15 May 2019. Vol. 11, Issue 492, eaav8521. DOI: 10.1126 scitranslmed.aav8521].
The last embodiment of the present invention refers to a method for treating cancer, wherein the cancer is characterized by the activation of mitogen-activated protein kinase (MAPK) selected from the list consisting of: BRAF, RAS, MEK or ERK, which comprises the administration of a therapeutically effective amount of Cx43, preferably in combination with any of the above defined mitogen-activated protein kinase (MAPK) inhibitors, and most preferably, also in combination with a senolytic agent (as defined above), or a pharmaceutical composition comprising thereof.
In the context of the present invention the following terms are defined:
Data are presented as mean±SEM. Mann-Whitney or Two-tailed Student's t-test were used to calculate the significance and represented as follows: *p<0.05, **p<0.01, ****p<0.0001.
The present invention is illustrated by means of the examples set below without the intention of limiting its scope of protection.
The BRAF mutated tumour cell lines A375 (melanoma), SK-Mel-28 (melanoma), MDA-MB-231 (human breast cancer) and HT-29 (human colorectal adenocarcinoma) were also used to test the role of Cx43 in drug resistance. SK-Mel147 (melanoma), NRAS mutated cell line was selected to compare the results with BRAF mutant cell lines. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Lonza) supplemented with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific) and 100 U/mL penicillin and 100 μg/ml streptomycin (Gibco, Thermo Fisher Scientific). HT-29 were cultured in McCoy'S 5A medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific) and 100 U/mL penicillin and 100 μg/ml streptomycin (Gibco, Thermo Fisher Scientific). The cells lines were maintained in sterile conditions under a flow laminar hood (Telstar BIO II A). Cells were maintained at 37° C. in 5% CO2 humidified incubator (SANYO CO2). The medium was changed every 2-3 days. To detach the cells, the medium was removed, and cells were washed with a saline solution (Fresenius Kabi). Trypsin (Gibco, Thermo Fisher Scientific) was added to the dish and incubated at 37° C. for several minutes (min). After adding DMEM supplemented with 10% FBS, cells were transferred to falcon tube and counted using the Neubauer chamber method in a light microscope. Cells were centrifuged at 1800 revolutions per minute (rpm) for 5 min at room temperature (RT) before seeding in new plates to use them for experimental analysis or to treat them with different drugs. The BRAF inhibitor Dabrafenib (D) (Tafinlar® 75 mg, Novartis) was dissolved in dimethyl sulfoxide (DMSO) at 1 mM final concentration and stored at −20° ° C. The MEK inhibitor Trametinib (T) (Mekinist® 2 mg, Novartis) was dissolved in DMSO (1 mM) and stored at −20° C. To obtain double resistance cells (DR) to T and D, tumour cells were treated for 1 to 6 months under the presence of both inhibitors (started with a low dose (0.5 nM T+30 nM D) and escalated as and when cell lines were confluent (2 nM T+200 nM D). Concentrations (initial and maintenance) used were depended on the sensibility of the cell lines to the treatments. Carbenoloxolone disodium salt (CBX) (Sigma-Aldrich) was dissolved in distilled water (dH2O) at 20 mM final concentration and stored at −4° C.
Cell lines were transfected by electroporation. The Amaxa® Cell Line Nucleofector® Kit V (Lonza) was used to transfect cells in a Cell Line Nucleofector™ Device (Lonza). A million cells were harvested and resuspended in 100 μL of Nucleofector® solution. 3 μg of the corresponding plasmid was added, and the mixture was transferred into an electroporation cuvette using the U-28 program. Cell lines were transfected with pIRESpuro2 plasmid construct (Clontech) containing the human Cx43 sequence, kindly donated by Arantxa Tabernero (Institute of Neuroscience of Castilla y León, University of Salamanca, Spain). The transfected cells were seeded in culture medium and 24 hours (h) post-transfection the medium was replaced with complete medium containing puromycin dihydrochloride (Tocris, Bioscience) at different concentrations depending of the cell line (0.5 μg/mL to 2 μg/mL).
Cells were harvested and washed twice with saline before protein isolation. Total cell lysates were obtained by disaggregating the cells with an insulin 30-gauge syringe (Omnican, Braun) in ice-cold lysis buffer composed by 150 mM NaCl, 50 mM Tris-HCL (pH 7.5), 5 mM EDTA (pH 8), 0.5% (v/v) Nonidet P-40, 0.1% (w/v) Sodium Dodecyl Sulfate (SDS), 0.5% (v/v) Sarkosyl (all from Merck) supplemented with 0.1 mM phenylmethysulfonyl fluoride (PMSF) and 1× Protease Inhibitors Cocktail (Sigma-Aldrich. Loading buffer (10% (v/v) β-mercaptoethanol (Merk); 10% SDS, 50% glycerol (v/v); 200 mM Tris-HCl pH 6.8, 0.1% bromophenol blue) was added to protein extracts and boiled at 99° C. for 10 min. Protein concentrations were measured using a Nanodrop® ND-1000 (Thermo Fisher Scientific) and 30 μg of protein samples were used to perform the SDS-PAGE electrophoresis in 10% or 15% Acrylamide/Bis-acrylamide gels and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (Inmobilon-P, Millipore) using Mini Trans-Blot Cell System (Bio-Rad Laboratories). Transfer was performed at 100 V during 1 or 2 h in ice cold. After transference, membranes were stained with ATX Ponceau S red staining solution (Merk) for 15 min at RT and then unstained with several washes with distilled water. Membranes were blocked with 5% skin milk (Merck) in tris-buffered saline with 0.05% Tween-20 (TBS-T) for 1 h at RT. Primary antibodies and secondary antibodies were diluted in 5% skin milk TBS-T incubated overnight (O/N) at 4° C. or 1 h at RT, respectively. HRP-conjugated secondary antibodies used were anti-mouse (NA-931, Sigma-Aldrich) and anti-rabbit (A6154, Sigma-Aldrich). Signal was developed using Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific) in either a LAS-3000 Imager (Fujifilm) or an Amersham Imager 600 (GE Healthcare). Image J software was used to quantify protein band intensities. For nuclear protein isolation the NE-PER™ Nuclear and Cytoplasmic Extraction kit (Thermo Fisher Scientific) was used following manufacture's protocol. For membrane (insoluble)/cytosol (soluble) protein, pelleted cells were lysed in 1% Triton X-100 (v/v) in phosphate-buffered saline (PBS: MP Biomedicals) supplemented with 0.1 PMSF and 1× Protease Inhibitors Cocktail. The cells were intermittently vortexed and kept on ice for 1 h. Lysates were centrifuged at 10,000 g for 15 min at 4° C. and supernatant was collected (cytosolic soluble proteins). The insoluble fraction was resuspended in lysis buffer (membrane). The primary antibodies used were: anti-Cx43 (#C6219), Sigma-Aldrich 1: 1000; anti-Tubulin (T9026), Sigma-Aldrich 1: 5000; anti-N-cadherin (#13116), Cell Signaling 1: 1000; anti-CDKN2A/p16INK4a (ab108349), Abcam 1: 1000; anti-p53 (Sc-126, Santa Cruz) 1: 500; anti-p44/42 MAPK (Erk1/2) (#9102), Cell Signaling 1: 1000; anti-Twist-1 (Sc-81417), Santa Cruz 1:100; anti-Lamin A (Sc-20680), Santa Cruz 1: 1000; anti-CD9 (Sc-9148), Santa Cruz 1: 1000; anti-CD63 (Sc-15363), Santa Cruz 1: 1000. The secondary antibodies used were: Goat anti-Rabbit (A6154), Sigma-Aldrich 1: 1000 to 1: 5000; Sheep anti-Mouse (NA-931), Sigma-Aldrich 1: 1000 to 1: 10000.
1×104 cells were cultured on coverslips (Thermo Fisher Scientific) until 80-90% confluence, washed with PBS and fixed with 2% (w/v) paraformaldehyde (PFA: Sigma-Aldrich) in PBS for 15 min at RT. The cells were incubated twice for 10 min each with 0.1 M glycine (Sigma-Aldrich), permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) in PBS for 10 min, washed twice with PBS and incubated with 1% bovine serum albumin (BSA; Sigma-Aldrich) at RT for 30 min. Fixed cells were incubated with primary antibody diluted in 1% BSA in PBS supplemented with 0.1% (v/v) Tween 10 (PBS-T) O/N at 4° C. Cells were then washed three times with PBS and incubated with Alexa-conjugated secondary antibodies (diluted in 1% BSA in PBS-T) at RT for 1 h in the dark. Cells were washed three times with PBS and counterstaining of nuclei with 1 μg/mL of 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich) for 5 min at RT in the dark and rinsed three times with PBS. Mounting the cover slips were done with a drop of glycergel aqueous mounting medium (Dako) on a glass microscope slide. Negative controls were obtained by omitting the primary antibody. The primary antibody used were: anti-Cx43 (#C6219), Sigma-Aldrich 1: 500; The secondary antibody used were: Goat anti-Rabbit (F-2765), Thermo Fisher Scientific 1: 100.
Cells were seeded on 6 or 12 wells and cultured until 80-100% of confluence and rinsed twice with warm PBS. Lucifer Yellow CH dilithium salt (1 mg/mL) (LY: Cell Projects Ltd© Kent) in PBS was added to the cells and parallel cuts (3 cuts per well) were performed with a surgical blade (Swann-Mortonscalpel®). After 3-5 min at 37° C. cells were washed with PBS and fixed 2% PFA in PBS. LY becomes incorporated by cells along the cut and move from the dye-loaded cells into adjacent ones connected by functional gap junction channels. The areas for LY loading were randomly selected in the central part of wells to obtain the images for quantification. Images were captured with 10× objective using a Nikon Eclipse Ti fluorescent microscope. The constant exposures were used for all images for a given experiment. Ten images per sample were quantified. The score was calculated as the ratio of the number of non-damage LY positive cells at the edge and the number of the LY positive cells outside the edge.
Cells were seeded on 12-wells plated and cultured until 80-100% of confluence. Glass micropipettes were backfilled with 133 mM of 5- or 6-carboxyfluorescein with a negative charge (−2) (Sigma-Aldrich) diluted in potassium acetate 0.1 M (Sigma-Aldrich). One cell was injected and hyperpolarizing pulses were applied with an intensity of 0.25-1 nA. Cells were excited with 488 nm in a confocal microscopy and the image was registered in Lasershap (MRC-1024, BioRad). In the image acquisition process, regions of interest (or ROI) were defined to detect the cell that was injected, the donor cells and the adjacent receptor cells. Before injecting the cell, the gain of the photomultipliers was reduced below 20% of the dynamic detection range (from 0-250), which was taken as the basal value. The fluorescence emitted was measured at 640/40 nm and 540/40 nm with two photodetectors, the time course of the passage of dye from the injected cell to the adjacent cells was studied.
Total RNA was isolated from cells using TRI Reagent® RT (Vitro Bio) according to manufacturer's protocol. 5×105 to 106 cells were harvested and lysed in 1 mL TRIzol reagent. 230 μL of chloroform were added to the tubes and they were vortexed during 1 min. Samples were incubated for 10 min at 4° C. and then centrifugated at 14,000 g for 15 min at 4° C. After centrifugation, the aqueous phase (upper) are collected and transferred to a new tube with 500 μL of isopropanol and samples were vortexed 15 sec. The tubes were incubated for 30 min at −20° C. followed by 14,000 g centrifugation for 15 min at 4° C. Supernatant was discarded, and the RNA pellets were washed with 1 mL of 70% ethanol and centrifugated for 5 min at 7,000 g at 4° C. The supernatants were discarded, and RNA pellets were air dry for 2 h at 4° C. RNA was treated with DNase I, RNase free (Thermo Fisher Scientific) following manufacturer's protocol. RNA samples were quantified in a Nanodrop® ND-1000 (Thermo Fisher Scientific). 1 μg of RNA was used to synthesize complementary DNA (cDNA) with the Superscript™ IV VILO™ Master Mix (Invitrogen, Thermo Fisher Scientific). Samples were denatured for 10 min at 65° C., and 2 μL of the master mix were added. Samples were incubated using a thermo cycler (Veriti, Applied Biosystems) for 10 min at 25° C., followed by 60 min at 42° C. and 5 min at 85° C. Finally, cDNA samples are quantified in a Nanodrop® ND-1000 (Thermo Fisher Scientific) and resuspended in DNase/RNase/Protease-free water (Sigma-Aldrich).
5 μL of cDNA (1 μg) were mixed with 0.5 μL of primer mix (10 μM each primer), 10 μL of Applied Biosystems™ PowerUP™ SYBR™ Green Master Mix (Applied Biosystems, Thermo Fisher Scientific) and complete with dH2O until 20 μL per well on LightCycler® 480 System (Roche). The program consisted on a first denaturing cycle of 10 min at 95° C. followed by 30-55 amplification cycles for 10 seconds (sec) at 95° C., 30 sec at 60° C. for annealing and 12 sec at 72° C. for extension. The primers used were: GJA1 (Cx43): Forward: SEQ ID NO: 1; Reverse: SEQ ID NO: 2. HPRT1: Forward: SEQ ID NO: 3; Reverse: SEQ ID NO: 4.
Dojindo's highly water-soluble tetrazolium salt (WST-8) is reduced by dehydrogenase activity in cells to give a yellow-color formazan dye, which is soluble in the tissue culture media. The amount of the formazan dye generated by dehydrogenases is directly proportional to the number of living cells. Equal number of cells (1000-2000 cells) were seeded per triplicate in 96-well plates and cultured in the different conditions. Medium was replaced by 100 μL of fresh medium and 10 μL of CCK-8 reagent was added per well. After an incubation for 1 to 4 h at 37° C., absorbance was measured at 450 nm in a NanoQuant microplate reader Infinite M200 (TECAN). Results were calculated as optical density (OD) 450 nm=OD of sample−OD of blank.
The E8 xCELLigence plates were prepared by addition of 100 μL of complete media to every well. After incubation at 37° C., plates were inserted into the xCELLigence station, and the base-line impedance was measured to ensure that all wells and connections were working. Following harvesting and counting, cells were diluted to the selected seeding density and 100 μl of cells in culture medium was added to each well. For the cell proliferation assays for each condition, 1×103-5×103 cells/well was seeded into 100 μL of media in the E-Plate L8 (Acea Biosciences). The attachment and proliferation of the cells were monitored every 30 min. Cell proliferation was monitored for 48-72 h. Cell Index at each time point is defined as (Rn−Rb)/15, where Rn is the cell-electrode impedance of the well when it contains cells and Rb is the background impedance of the well with the media alone. For the cell adhesion assays, for each condition, 1×103-5×103 cells/well was seeded into 100 μL of media in E-Plate L8 (Acea Biosciences). Cell proliferation was monitored for 1 h every 10 sec. The extend of cell adhesion and spreading was monitored every min for 1-3 h. Cell Index at each time point is defined as (Rn−Rb)/15, where Rn is the cell-electrode impedance of the well when it contains cells and Rb is the background impedance of the well with the media alone.
Cells proliferation by tracking new DNA synthesis was performed with Alexa Fluor 647™ Click-iT® Assays kit (Thermo Fisher Scientific), following manufacturer's instructions. Briefly, cells were previously cultured in a serum-free medium and incubated with 10 μM of 5-ethynyl-2′-deoxyuridine (EdU), a nucleoside analog to thymidine, for 2 h to be incorporated into DNA during S phase (DNA synthesis). Cells were harvested, fixed, and incubated with the Alexa 647™ picolyl azide. EdU incorporation was analyses by flow cytometry using a FACs Scalibur and acquiring 20,000 event per triplicate. Data was analyzed using the FCS Express 6 Flow software.
Colony formation assays were performed by seeding 5×103 to 5×104 cells per well onto 6-12 well plates and grown for 7-15 days. The culture medium was replaced every 48 h. The cells were washed with warm Saline Solution and fixed with cold 2% PFA for 15 min. Cells were washed with 1×PBS and stained with 0.1% of crystal violet (Sigma-Aldrich) for 15 min. After staining, the cells were washed with distilled water and dried at room temperature. The colonies were quantified by diluting crystal violet in 5% acetic acid and 100 μL of the solution was collected to measure the absorbance at 570 nm using a NanoQuant microplate reader Infinite M200 (TECAN).
The detection of SA-β-Gal activity was measured using the Senescence Cells Histochemical Staining Kit (Sigma-Aldrich). Cells were rinsed with warm PBS and fixed for 7 min at RT with a fixation buffer composed by 2% paraformaldehyde and 0.2% glutaraldehyde. After three washed with PBS, cells were incubated for 6 h or O/N at 37° C. without CO2 in a staining solution containing X-gal. This lactose analogue can be cleaved by β-galactosidase producing a blue insoluble product. Finally, cells were washed with PBS and images were taken using a light microscope.
SA-β-Gal activity was analyzed with the fluorogenic β-Galactosidase substrate di-β-D-galactopyranoside (FDG, Thermo Fisher Scientific), which is hydrolyzed by the endogenous β-galactosidase to fluorescein (FITC). Cells were harvested and incubated in a staining medium (PBS, 4% FBS, 10 mM HEPES, pH 7.2) at 37° C. for 10 min. The β-Galactosidase assay was started by adding warm 2 mM FDG to the cell suspension and after incubating for 3 min at 37° C. in the dark. The reaction was stopped with the addiction of ice-cold staining medium to the cells, which were kept on ice protected from light. 515 nm laser was used for fluorescein and acquiring 20,000 event per triplicate. Data was analyzed using the FCS Express 6 Flow software.
Supernatants (died cells) were aspirated and attached cells were harvested. Supernatants and cells were mixed, counted and 1 million cells were resuspended in 1 mL in flow cytometry (FC) buffer. Membrane viability analysis was performed by double staining PI/YO-PRO-1. YO-PRO-1 (Invitrogen) was added to the cells (150 nM) and incubated 10 min in the dark at 4° C. PI (Invitrogen) was added (2 μg/mL) and incubated for 5 min in the dark at 4° C. After the incubation time (PI/YO-PRO) samples were analyzed in a FACs Scalibur acquiring 20,000 event per triplicate. The red fluorescence emitted by PI was detected at 610 nm and the green florescence emitted by YO-PRO was detected at 515 nm. Data was analyzed using the FCS Express 6 Flow software.
Cells were seeded into 162 cm2 culture flasks (Corning, Sigma-Aldrich) until confluence (80%-90%). Cells were washed three times with PBS and cultured for 48 h in DMEM 0% FBS. Supernatants were collected and centrifuged at 1800 rpm for 5 min. Pellet was discarded and supernatants were filtered through a 0.22 μm filter and then, the filtered supernatants, were centrifuged at 100,000 g for 90 min at 4° C. (Optimal-90K ultracentrifuge) using 70 Ti rotor (Beckman Coulter). Supernatants were discarded and pellet-containing EVs were washed with PBS and centrifuged at 100,000 g for 90 min at 4° C. The pellet with EVs was resuspended in culture medium, PBS or lysis buffer, depending on the analysis performed. The EVs were characterized by scanning electron microscopy and by the NTA Nanosight NS300.
Purified EVs were fixed in 2% PFA, and 5 μL were deposited on formvar-carbon coated grids and let them air-dry for 1 h. Grids were then washed three times with PBS, followed by a 10 min incubation with 0.1 M glycine. Grids were incubated with 1% BSA in PBS for 10 min at RT. Grids were fixed with 1% glutaraldehyde for 5 min, followed by several washed with distilled water. Grids were stained in uranyl-oxalate pH 7 for 5 min and then contrasted in a mixture of 4% uranyl acetate and 2% methyl cellulose for 5 min on ice. Grids were dried and stored until visualize using JEOL JEM 1010 transmission electron microscope.
Isolated EVs were stained in 100 μL of PBS with 1 μM Dil for 1 h at 37° C. After them, EVs were washed in 10 mL of PBS and centrifuged at 100,000 g for 90 min at 4° C. The pellet was resuspended in culture medium for different experiments.
EVs from tumour cell lines A375-EV and A375-Cx43 were collected in lysis buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA (pH 8), 0.5% (v/v) Nonidet P-40, 0.1% (w/v) SDS, 0.5% (v/v) Sarkosyl) for protein isolation. In order to make global protein identification, an equal amount of protein (100 μg) from all conditions were loaded on a 10% SDS-PAGE gel. The run was stopped when the front had penetrated 3 mm into the resolving gel. The protein band was visualized by Sypro-Ruby fluorescent staining (Lonza), excited, and subjected to in-gel, manual tryptic digestion following the protocol defined by Shevchenko (Shevchenko) with minor modifications. The peptides were extracted three times by incubation in 40 μL of a solution containing 60% ACN and 0.5% formic acid (HCOOH) for 20 min. The resulting peptide extracts were pooled, concentrated in a SpeedVac and stored at −20° C.3. For protein identification by LC-MS/MS, 4 μl (aprox. 4 μg) of digested peptides of each sample were separated using Reverse Phase Chromatography. Gradient was developed using a micro liquid chromatography system (Eksigent Technologies nanoLC 400, SCIEX) coupled to high speed Triple TOF 6600 mass spectrometer (SCIEX) with a micro flow source. The analytical column used was a silica-based reversed phase column Chrom XP C18 150×0.30 mm, 3 mm particle size and 120 Å pore size (Eksigent, SCIEX). The trap column was a YMC-TRIART C18 (YMC Technologies) with a 3 mm particle size and 120 Å pore size, switched on-line with the analytical column. The loading pump delivered a solution of 0.1% formic acid in water at 10 L/min. The micro-pump provided a flow-rate of 5 μL/min and was operated under gradient elution conditions, using 0.1% formic acid in water as mobile phase A, and 0.1% formic acid in acetonitrile as mobile phase B. Peptides were separated using a 90 min gradient ranging from 2% to 90% mobile phase B (mobile phase A: 2% acetonitrile, 0.1% formic acid; mobile phase B: 100% acetonitrile, 0.1% formic acid). Data acquisition was carried out in a TripleTOF 6600 System (SCIEX) using a data dependent workflow (DDA). Source and interface conditions were as follows: ionspray voltage floating (ISVF) 5500 V, curtain gas (CUR) 25, collision energy (CE) 10 and ion source gas 1 (GS1) 25. Instrument was operated with Analyst TF 1.7.1 software (SCIEX). Switching criteria was set to ions greater than mass to charge ratio (m/z) 350 and smaller than 1400 (m/z) with charge state of 2-5, mass tolerance 250 ppm and an abundance threshold of more than 200 counts (cps). Former target ions were excluded for 15 s. Instrument was automatically calibrated every 4 h using as external calibrant tryptic peptides from pepcalMix. The mass spectromic analysis were performed in the Proteomics Facility of the Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS). After MS/MS analysis, data files were processed using ProteinPilot™ 5.01 software (SCIEX), which uses the algorithm Paragon™ for database search and Progroup™ for data grouping. Data were searched using a Human specific Uniprot database. False discovery rate (FDR) was performed using a non-lineal fitting method displaying only those results that reported a 1% Global false discovery rate or better (shilov IV and Tang WH).
Differential gene expression levels between the EVs derived from A375 control (EVs Cx43−) and EVs derived from A375 overexpressed Cx43 (EVs Cx43+) were estimated based on the false discovery rate (FDR). Proteomic analysis was performed considering FDR ≥0.82 for EVs Cx43− and FDR ≥0.79 for EVs Cx43+. All calculations were performed using GraphPad Prism 7.0. Proteomaps were constructed using a web tool based on the t-test difference values (Liebermeister et al., 2014). The enrichment analysis was done using GSEA software version 4.0.3 (Subramanian et al. 2005) (Gene Ontology (GO), KEGG Orthology and Reactome data base). For the present study, enrichment analysis was performed based on FDR of data set of each group of specific proteins (EVs Cx43− and EVs Cx43+). GSEA analysis heat maps are shown by Prism based on Normalized Enrichment Score (NES) and FDR. Scaffold (version Scaffold_4.11.0, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at greater than 99.9% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii, Al et al Anal. Chem. 2003; 75(17):4646-58). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters.
Tumour cells A375-EV and A375-Cx43 were cultured in 175 cm2 flasks until 80% confluence. 10 mL of cell culture media was collected and centrifuged at 1500×g for 5 min to remove cells and debris. The supernatant was transferred to a new 50 ml conical tube for exosome (EVs) isolation. ExoQuick-TC (System Biosciences) was added to the supernatant at 1:5 ratio (ExoQuick:Supernatant), mixed gently, and allowed to incubate O/N at 4° C. Next day, the admixture was centrifugated at 1500× g for 30 min to recover exosomes for total RNA isolation. The remaining exosomes from samples were processed for total RNA isolation using the SeraMir Exosome RNA Purification Column kit (System Biosciences) according to the manufacturer's instructions. For each sample, 1 μL of the final RNA eluate was used for measurement of small RNA concentration by Agilent Bioanalyzer Small RNA Assay using Bioanalyzer 2100 Expert instrument (Agilent Technologies). Small RNA libraries were constructed with the Clean Tag Small RNA Library Preparation Kit (TriLink) according to the manufacturer's protocol. The final purified library was quantified with High Sensitivity DNA Reagents (Agilent Technologies). The libraries were pooled, and the 140 bp to 300 bp region was size selected on an 8% TBE gel (Invitrogen, Life Technologies). The size selected library is quantified with High Sensitivity DNA 1000 Screen Tape (Agilent Technologies). High Sensitivity D1000 reagents (Agilent Technologies), and the TailorMix HT1 qPCR assay (SeqMatic), followed by a NextSeq High Output single-end sequencing run at SR75 using NextSeq 500/550 High Output v2 kit (Illumina) according to the manufacturer's instructions. The Exosome Small RNA-seq Analysis Kit initiates with a data quality check of the input sequence using FastQC, an open-source quality control (QC) tool for high-throughput sequence data. FastQC runs analyses of the uploaded raw sequence reads that quality of the data and inform the subsequent preprocessing steps in the analysis. Following QC, the analysis moves to preprocessing of the RNA-seq reads to improve the quality of data input for read mapping. The open-source tools used are FastqMcf, part of the EA-utils package, and PRINSEQ. Data preprocessing detects and removes N's at the ends of reads, trims sequencing adapters, and filters reads for quality and length. FastQC is then re-run to analyze the trimmed reads, allowing a before and after comparison. The summary report generated provides a quality assurance check to validate the processed set of input data used in the subsequent read mapping step. The improved set of sequence reads are mapped to the reference genome using Bowtie, an ultrafast, memory-efficient short read aligner, followed by the generation of a mapping summary report for review. Using the open-source software SAMtools and Picard expression analyses are carried out determination of ncRNA abundance and differential expression analysis across samples. Expression statistics are calculated and visualized using R software for statistical computing and graphics. The Exosome Small RNA-seq Analysis Kit identifies and maps miRNAs, tRNAs, small rRNAs, repeat elements, antisense transcripts and a variety of small ncRNAs. The Exosome Small RNA-seq analysis produces a summarization of results, including expression statistics and chromosome distribution, as well as genome browser tracks of read alignment and read coverage for analysis in a genomic context.
Data were analyzed considering p-value <0.05 and fold change <−3.8 and >3.8. Genes were considered as differentially expressed if the comparison of relative abundance between both groups is positive or negative. The list of differentially expressed genes in EVs Cx43− and EVs Cx43+, containing gene identifiers and corresponding expression values (fold changes), were uploaded into the miRNet 2.0 (Xia Lab, McGill) online tool. The analysis included biological processes, canonical pathways, biological processes, molecular functions and gene networks. Graphics were performed using GraphPad Prism 7.0.
The primary melanoma cell line A375 were derived from human melanoma tissue. Decreased expression of connexins (Cxs) have been observed in melanoma cell lines. Cx43 expression was analysed in A375 human metastatic melanoma cell line. To address the direct effect of Cx43 in melanoma, Cx43-expressing vector (Cx43) or empty vector (EV) were transfected in A375 cells. Cx43 overexpression was confirmed by western blot, flow cytometry analysis, RT-qPCR and immunofluorescence (IF) staining (
EVs including exosomes have been reported to be involved in the development and progression of cancer malignancy by promoting cancer proliferation, establishing a premetastatic niche, and regulating drug resistance. We have isolated EVs from A375 cell line and have characterized them by electron microscopy and by nanoparticle tracking analysis (NTA) confirming the sizes of these particles are between 20-100 nm (
Cx43 can interact with different proteins and probably with different molecules of RNA and DNA. For this reason, we have decided to investigate if the presence of Cx43 in the EVs could change the protein and RNA content of the EVs secreted by tumour cells that overexpress this channel protein. EVs have the ability to carry cell-specific cargos of proteins, lipids, and genetic materials, and can be selectively taken up by neighbouring or distant cells and modify their signalling and functions. In order to study if the presence of Cx43 in EVs could affect their content, we performed a proteomic (LC-MS/MS) and RNA sequence analysis. To determine the protein profile of EVs (Cx43 negative (−) and Cx43 positive (+) EVs), total exosomal proteins were separated by SDS-PAGE. A total of 1141 proteins were identified for both groups of EVs (Cx43− and Cx43+), 160 proteins were exclusive of EVs negative for (Cx43−) and 482 proteins were exclusive for EVs containing (Cx43+) (
On the other hand, enrichment analysis of small RNA content was analysed using MiRNet 2.0. Heatmaps show pathways and biological processes significantly enriched in EVs isolated from Cx43− (
The presence of Cx43 also changes the patterns of small RNAs that modify cellular function in recipient cells. RNA sequencing analysis showed (
In order to investigate the role of Cx43 in EVs, tumour cells were treated with EVs containing or not Cx43 and released by melanoma cells overexpressing Cx43 or by normal melanoma cells which hardly express Cx43 (
Targeting BRAF and MEK using specific inhibitors have become the standard of care for patients with melanoma and tumours with a mutation in BRAF. However, the therapeutic benefits are often limited due to the development of drug resistance. To examine the potential effect of Cx43 on the response of tumour cells to BRAF/MEK inhibitors (BRAF/MEKi) (Dabrafenib/Trametinib) treatment, A375 melanoma cell line was treated with Dabrafenib and Trametinib (
To test whether Cx43 is also involved in acquired resistance of melanoma cell to BRAF/MEKi, cells were selected for resistance to Dabrafenib and Trametinib by prolongated exposure to high concentrations of these drugs. After 4 months, double resistant cells (DR) hardly responded to high concentrations of both drugs (
When we treated melanoma tumour cells (A375) with BRAF/MEKi we detected an increase in Cx43 protein levels when cells are sensible to these treatments (
These results demonstrate that Cx43 within EVs results in (i) an effective therapeutic strategy to reduce cellular proliferation in BRAF-mutant tumour cells; (ii) increases the efficacy of BRAF/MEKi and (iii) the combination with navitoclax and BRAF/MEKi targets resistant tumour cells re-sensitive DR cells to BRAF/MEKi (
| Number | Date | Country | Kind |
|---|---|---|---|
| 20382883.5 | Oct 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/077487 | 10/6/2021 | WO |
