METHODS AND PHARMACEUTICAL COMPOSITIONS FOR MODULATING STEM CELLS PROLIFERATION OR DIFFERENTIATION

Abstract

The present invention relates to a method for modulating stem cells proliferation or differentiation comprising a step of contacting said stem cells with an effective amount of an activator or inhibitor of a proprotein convertase subtilisin kexin 9 (PCSK9). Inventors performed a global transcriptomic analyses in hiPSCs and showed that PCSK9 inhibition by shRNA and the intracellular PCSK9-R104C/V114A mutation negatively regulate the NODAL signaling pathway and its targets. This regulation was manifested in drastic reduction P-SMAD2/total SMAD2 protein level. This was accompanied by reduced proliferation rate where hiPSC-shPCSK9 and hiPSC-R104C/V114A demanded >1.3-fold more time to double compared to their control counterparts. They showed that PCSK9 was regulating this signaling pathway through direct physical interaction with DACT2, an intracellular attenuator of NODAL receptor and favoring its protein degradation. Thus, these findings allow to understand the differentiation and proliferation of cells.

Claims

1. A method for modulating stem cells proliferation or differentiation comprising a step of contacting said stem cells with an effective amount of an activator or inhibitor of a proprotein convertase subtilisin kexin 9 (PCSK9).

2. The method according to claim 1 is suitable to replace damaged cells and treat diseases.

3. A method for modulating cancer stem cells proliferation or differentiation comprising a step of contacting said cancer stem cells with an effective amount of an inhibitor of a proprotein convertase subtilisin kexin 9 (PCSK9).

4. A method of treating a cancer in a subject in need thereof comprising a step of administering to said subject a therapeutically effective amount of a proprotein convertase subtilisin kexin 9 (PCSK9) inhibitor.

Description

FIGURES

[0067] FIG. 1: Cell proliferation assay of hiPSC-shCt and hiPSC-shPCSK9 (upper panel; 3 different passages of each cell line performed independently), hiPSC Control and hiPSC-FL-PCSK9-V5 (overexpression) (middle panel) and UhiPSC-Control and UhiPSC PCSK9-R104CN114A (lower panel, 3 different passages of each cell line performed independently), absorbance values at 48 h, 72 h and 96 h were normalized to the absorbance of cells at 24 h after seeding. (*p<0.05, **p<0.01)

[0068] FIG. 2: Cell proliferation assay of Caco2-shCt and Caco2-shPCSK9 (upper panel; 3 different passages of each cell line performed independently), absorbance values at 48 h, 72 h and 96 h were normalized to the absorbance of cells at 24 h after seeding. Doubling time estimated based on the growth rate of Caco2 cell lines (lower panel; n=3 of each). (*p<0.05).

[0069] FIG. 3: Cell proliferation assay of HepG2-shCt and HepG2-shPCSK9 (upper panel; 3 different passages of each cell line performed independently), absorbance values at 48 h, 72 h and 96 h were normalized to the absorbance of cells at 24 h after seeding. Doubling time estimated based on the growth rate of HepG2 cell lines (lower panel; n=3 of each). (*p<0.05, **p<0.01).

[0070] FIG. 4: The impact of R104CN114A dominant negative PCSK9 mutations on the DACT2 expression. DACT2 gene expression by RT-qPCR (3 different passages). DACT2 and -tubulin protein levels detected by Western blot (3 different passages) with the corresponding quantification. (*p<0.05).

EXAMPLE

[0071] Material & Methods

[0072] 1. Cell Culture

[0073] The human induced pluripotent stem cells (hiPSC) were reprogrammed from patient-derived urine cells and characterized as previously described (Si-Tayeb et al. 2016). hiPSC were cultured on mouse embryonic fibroblasts (MEFs) in hiPSC medium composed of DMEMF12 (Life Technologies) supplemented with 20% Knockout Serum Replacer (Life Technologies), 0.5% L-Glutamine (Life Technologies) with 0.14% -mercaptoethanol (Sigma), 1% NEAA and 5 ng/ml fibroblast growth factor 2 (FGF2, Miltenyi) in hypoxia (4% O2, 5% CO2) and manually passed once a week. For feeder free culture conditions, hiPSC colonies were manually picked from MEFs and plated onto plates coated with Matrigel (Corning; 0.05 mg/ml) in StemMACS iPS-Brew medium (Miltenyi). Passages were performed using the Gentle Cell Dissociation Buffer (Stem Cell Technologies).

[0074] HepG2 cells and CACO2 cells were culture in DMEM supllumented with non essential amino acids, glutamate and 10% FCS.

[0075] 3. PCSK9 Silencing

[0076] PCSK9 gene expression has been silenced upon lentiviral transduction of specific shRNA (Sigma). The clone TRCN0000075236 cloned into the pLKO.1-Puro vector has been used to target PCSK9 while an unspecific shRNA sequence has been used as control. Upon transduction, hiPS cells K3 (Si-Tayeb, Noto, Sepac, et al. 2010), HEepG2 cells and CACO2 cells were subjected to Puromycin (TOCRIS Bioscience 4089/50) selection using a concentration up to 8 g/ml.

[0077] 4. PCSK9 Overexpression

[0078] Full length PCSK9 cDNA sequence fused with a V5 tag at the C terminus extremity was integrated in the genomic area of AAVS1 integration sites with specific TALENS and homologous sequence. Upon transfection, hiPSC (Si-Tayeb et al Disease Models and Mechanisms 2016) were subjected to Puromycin (TOCRIS Bioscience 4089/50) selection using a concentration up to 8 g/ml.

[0079] 5. Gene Expression Analysis

[0080] RNA samples were isolated using the RNeasy Mini Kit (Qiagen). Reverse transcription of 1 g of RNA into cDNA was conducted using the high-capacity cDNA reverse-transcription kit (Applied Biosystems). Conditions were as follows: 10 min at 25 C., and then 2 hours at 37 C. Quantitative Polymerase Chain Reaction (qPCR) studies were conducted in triplicate using the brilliant III Ultra-Fast Master Mix with high ROX (Agilent). Primers are listed in (Table2). Each qPCR included 2 s at 50 C., 10 s at 95 C. followed by 40 cycles of 15 s at 95 C., and 60 s at 60 C. Cycle threshold was calculated by using default settings for the real time sequence detection software (Applied Biosystems).

[0081] 6. Protein Expression Analysis

[0082] hiPSC were initially lysed in a buffer composed of 150 mM NaCl, 25 mM Tris Base, 1 mM EDTA (ethylene diaminetetraacetic acid), and 1% NP-40 (Nonidet P-40) at pH 7.4 and containing a cocktail of protease inhibitors (Sigma Aldrich) and phosphatase inhibitors (Sigma Aldrich). Total cell lysates were then passed 10 times through a fine gauge needle followed by sonication (5 pulses for 5 sec each). A protein assay was then carried out against a range of standard bovine serum albumin (BSA) using Pierce BCA Protein Assay Kit. The lysates were denatured for 10 min at 70 C. in a mixture of NuPAGE Sample Reducing Agent (10X) that contains dithiothreitol (DTT) (500 mM) and NuPAGE LDS Sample Buffer (4X) containing 2% LDS (lithium dodecyl sulfate), 10% glycerol, 141 mM Tris Base, 106 mM Tris HCl, 0.51 mM EDTA, 0.51 mM EDTA, 0.175 mM Phenol Red and pH 8.5. 25 micrograms of each sample were loaded onto a 10% polyacrylamide gel and the proteins were separated by electrophoresis in presence of SDS. After migration, the proteins are transferred to nitrocellulose membrane (Bio-Rad) using Trans-Blot Turbo Transfer System (Bio-Rad). Revelation and quantification was done by Image Lab software (Bio-Rad). The membrane was saturated for one hour in TBS-T buffer (10 mM Tris, NaCl 0.5 mM and 0.1% Tween-20) containing 5% skimmed milk lyophilized. The membrane was then incubated with primary antibody overnight at 4 C. in TBS-T milk. Horseradish peroxidase (HRP)-conjugated secondary antibody staining was performed for 1 h at room temperature (RT) in TBS-T milk. Protein bands were detected using ECL detection system (Bio-Rad).

[0083] 7. Proliferation Assay

[0084] MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium assay was used to assess the proliferation of hiPSC. Briefly, 1104 shRNA-expressing cells or 5103 PCSK9 LOF and control hiPSCs were plated onto 96-well-plates (previously coated with Matrigel 0.05 mg/ml in quintuplicates. The day after the passage, media was then changed, supplemented with puromycin for the shRNA-expressing cells (TOCRIS Bioscience, 8 g/ml). Cells were then incubated with 20 l/well of MTT (Sigma Aldrich M5655) solution (5 mg/ml in sterile PBS) for 3 h at 37 C. The resulting purple-colored formazan crystals were then solubilized using MTT solvent containing 4 mM HCl, 0.1% NP-40 in isopropanol. Finally the absorbance was read at 590 nm using Perkin Elmer VICTOR X3 Multilabel Plate Reader. The proliferation rate was monitored over 24 h, 48 h, 72 h and 96 h.

[0085] 8. Statistical Analysis

[0086] Data are expressed as means.e.m. Significant differences between mean values were determined with the Mann-Whitney U-test for comparison of two groups or paired Student's t-test if appropriate.

[0087] Results

[0088] We have showed that PCSK9 inhibition or loss of activity induced a decreased proliferation of hiPSC (FIGS. 1A and 1C) while PCSK9 overexpression increased hiPSC proliferation (FIG. 1B). PCSK9 inhibition in HepG2 cells (FIG. 2) and Caco2 cells (FIG. 3) significantly decreased their proliferation and increased their doubling time rate. Using hiPS cells carrying the dominant negative LOF mutations R104CN114A, we showed that the loss of PCSK9 activity enhanced DACT2 expression and thus decreased SMAD2 signaling (FIG. 4). These data strongly suggest that PCSK9 interacts with the scaffold protein DACT2 (Disheveled antagonist of B-catenin), a tumor supressor, and induces its degradation. Therefore PCSK9 intracellular inhibition led to a decrease in cell proliferation.

REFERENCES

[0089] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.