Cell lines and organoids

HCT-116 and HT-29 cells were purchased from ATCC and cultured in DMEM (11360-070, Gibco) with 10% FBS (0270-106, Gibco) and 100 IU/mL penicillin–streptomycin (15140-122, Gibco).

CRC-stem cells (CRC-SCs) were isolated from CRC patients and propagated as tumorspheres. Briefly, human colon tissue fragments were obtained in accordance with the ethical standards of the Institutional Committee on Human Experimentation after informed consent. Tumor samples were subjected to mechanical and enzymatic dissociation using the human Tumor Dissociation Kit (130-095-929, Miltenyi Biotec) according to the manufacturer’s instructions. For magnetic separation, cells were labeled with CD44 microbeads (130-095-194, Miltenyi Biotec) 24–48 h after dissociation using the Miltenyi Biotec cell isolation kit (130-108-339, Miltenyi Biotec). CRC-SCs were maintained in DMEM/F12 Advanced (12491015, Gibco) supplemented with 6 mg/mL glucose (G8769, Sigma-Aldrich), 2 mM l-glutamine (25030081, Gibco), 10 ng/mL bFGF (F0291, Sigma-Aldrich), 20 ng/mL EGF (E9644, Sigma-Aldrich), B27 supplement (12587010, Gibco), and N-2 supplement (17502-048, Gibco). Patient-derived CRC organoids were generated using IntestiCult™-SF Organoid Growth Medium (Human) (100-0340, STEMCELL Technologies) according to the manufacturer’s instructions.

All cell lines were tested to be mycoplasma-free (117048, Minerva Biolabs). All cell cultures were performed in a 37 °C and 5% CO2 incubator.

RNA interference

HCT-116 cells were transfected with 50 nM validated siRNAs (Ambion) directed against (p38α) MAPK14 using HiPerfect reagent (301704, QIAGEN) according to the manufacturer’s instructions.

In vivo studies

For in vivo studies, normal and adenocarcinoma colon mucosa tissues were obtained from C57BL/6 mice (n = 12) and from APCMin/+ mice (n = 24) treated with azoxymethane (AOM) (A5486, Sigma-Aldrich), respectively. Four-month-old APCMin/+ male mice were administered with AOM (14 mg/kg body weight) once a week for 5 weeks; one month later, they were subjected to daily intraperitoneal injections of the p38α inhibitor SB202190 (S7067, Sigma-Aldrich) (0.05 μmol/kg body weight) or DMSO for 14 days and then sacrificed. Body weight was recorded daily. Procedures involving animals and their care were conducted in conformity with the institutional guidelines that comply with national and international laws and policies.

Histology and immunohistochemistry

Tissue specimens were fixed in 4% buffered formalin, embedded in paraffin, and sectioned at 4 mm thickness. Sections were dewaxed and rehydrated in dH2O. Endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide for 10 min. Antigen retrieval was conducted in 10 mM sodium citrate buffer (pH 6.0) for 30 min. Sections were incubated overnight with anti-p38α (1:200, 9218, Cell Signaling Technologies) or anti-β-catenin (1:400, 9562, Cell Signaling Technologies) primary antibodies. Then, they were incubated with secondary biotinylated antibodies and subsequently with streptavidin–biotin-peroxidase (Envision + System HRP anti-rabbit and anti-mouse, K8002, Agilent). Samples were developed with DAB and mounted with permanent mounting media. Negative controls were used in each experiment. p38α and β-catenin immunoreactivity was evaluated through a semiquantitative approach by two independent pathologists. Ten fields with an equal area were selected for analysis at 40 × magnification. Protein expression was assessed with ImageJ software and reported as a positivity percentage.

Chemicals

TWS-119 (S1590) and ralimetinib (S1494) were purchased from SelleckChem. Wnt3a (H17001) and SB202190 (S7067) were purchased from Sigma-Aldrich.

Immunoblotting

Nuclear and cytoplasmic fractions were obtained by using the Nuclear Extraction Kit (ab113474, Abcam) according to the manufacturer’s instructions. Immunoblots were carried out as previously described [13]. Briefly, cells were lysed in nuclear and cytoplasmatic lysis buffer and samples were electrophoresed in a 10% SDS–polyacrylamide gel and transferred to nitrocellulose membranes (1704156, Bio-Rad Laboratories). Blots were blocked for 1 h at room temperature in blocking buffer (1X TBS, 0.1% Tween-20 with 5% w/v non-fat dry milk) and washed with TBS-T. Membranes were incubated with the indicated antibodies (Cell Signaling Technologies; 1:1000) in 10 mL of primary antibody dilution buffer (1X TBS, 0.1% Tween-20 with 5% BSA) overnight at 4 °C. Then, blots were extensively washed and incubated with horseradish peroxidase-conjugated anti-rabbit/mouse secondary antibodies in blocking buffer for 45 min, washed, and developed with the ECL-Plus Chemiluminescence Reagent (RPN2232, GE Healthcare) according to the manufacturer’s instructions.

Primary antibodies: anti-p38α MAPK (9228), anti-Lamin B1 (12586S), anti-PDI (2446S), anti-β-catenin (9562S), anti-c-Myc (9402), anti-p-β-catenin (9561), anti-p-GSK3β (35480), anti-GSK3β (9315), and anti-Vinculin (13901) all from Cell Signaling Technologies. Secondary antibodies: Rabbit IgG HRP and Mouse IgG HRP (NA934V and NA931V, respectively, GE Healthcare). Densitometric evaluation was performed using ImageJ software.

Immunofluorescence

Cells were seeded on glass coverslips, fixed in 4% paraformaldehyde, and permeabilized using 0.01–0.1% Triton X-100. Coverslips were incubated with the indicated primary antibodies and then with Alexa Fluor 488 (A-11094, Thermo Fisher Scientific) and 647 (A-32728, Thermo Fisher Scientific) secondary antibodies; nuclei were counterstained using DAPI (D9542, Sigma-Aldrich). Slides were sealed using Vectashield Mounting Medium (H1000, Vector Laboratories). Images were acquired using a Zeiss fluorescence microscope. Primary antibodies: p38α (9228, Cell Signaling Technologies), β-catenin (8480, Cell Signaling Technologies), c-Myc (9402, Cell Signaling Technologies), and FLAG (F1804, Sigma-Aldrich).

Quantitative real-time PCR

Total RNA was isolated using TRIzol reagent (93289, Sigma-Aldrich) according to the manufacturer’s instructions. To avoid possible DNA contamination, RNA was treated with DNase-1 (AM2224, Ambion). RNA purity was confirmed by spectrophotometry and RNA integrity by agarose gel electrophoresis. cDNA was synthesized by retro-transcribing 1 μg of total RNA using the iScript cDNA Synthesis Kit (1708890, Bio-Rad Laboratories) according to the manufacturer’s instructions. Real-time PCR primers were designed using Primer Express software. PCR assays were performed in 96-well optical reaction plates using a QuantStudio 3 machine (Thermo Fisher Scientific). Each assay was carried out in triplicate wells. Baseline values of amplification plots were set automatically and threshold values were kept constant to obtain normalized cycle times and linear regression data. The following reaction mixture per well was used: 5 μL of SYBR Green PCR Master Mix (1725240, Bio-Rad Laboratories), 1.2 μL of primers at a final concentration of 100 nM, 2.3 μL of RNAse-free water, 1.5 μL of cDNA. For all experiments, the following PCR conditions were used: denaturation at 95 °C for 30 s, followed by 40 cycles of 15 s at 95 °C and 30 s at 60 °C. Quantitative normalization of cDNA in each sample was performed using β-actin as an internal control. Relative quantification was done using the ddCT method. Primer sequences are available upon request. Results are representative of at least three independent experiments.

Cloning and mutagenesis

The plasmids described in this manuscript were generated with specific primers, as previously described [26]. Site-directed mutagenesis was performed using the Q5® Site-Directed Mutagenesis Kit (E05545, New England Biolabs) according to the manufacturer’s instructions. The human beta-catenin pcDNA3 plasmid (16828) was purchased from Addgene. The human beta-catenin pcDNA3-S111A and human beta-catenin pcDNA3-T112A constructs were generated by site-directed mutagenesis, using the following primers:

SDM_ beta-catenin _S111A_FW 5'-P-GCAGATCCCAGCTACACAGTT,

SDM_ beta-catenin _S111A_RV 5'-P-ATGCCCTCATCTAATGTCTCA,

SDM_ beta-catenin _T112A_FW 5'-P-GATCCCATCTGCACAGTTTGATG.

SDM_ beta-catenin _T112A_ RV 5'-P- TGCATGCCCTCATCTAATG.

The pGEX-4T-3 GST-beta-catenin Wild-Type, pGEX-4T-3 GST-beta-catenin S111A, and pGEX-4T-3 GST-beta-catenin T112A constructs were generated by cloning the relevant β-catenin fragments from human beta-catenin pcDNA3, human beta-catenin pcDNA3-S111A, and human beta-catenin pcDNA3-T112A, respectively, into the pGEX-4T-3 GST Expression Vector (28-9545-52, GE Healthcare), using the BamHI and NotI enzymes.

Recombinant protein expression/purification

BL21(DE3) Competent Cells (C2527H, Biolabs) transformed with different constructs were grown in Luria Broth medium with ampicillin (A9518, Sigma) and induced with 200 nM IPTG when they reached the optical density of 0.6 (A600) at 37 °C, overnight. Cells were then collected by centrifugation, and pellets were lysed with B-PER lysis buffer (78248, Thermo Fisher Scientific). The lysate was centrifuged at 20,000×g for 20 min at 4 °C. Recombinant protein expression was confirmed by SDS-PAGE. GST-Fusion proteins were purified using Pierce Glutathione Magnetic Agarose Beads (78601, Thermo Fisher Scientific) according to the manufacturer’s instructions. GST-fused proteins were evaluated and quantified by SDS-PAGE.

Co-immunoprecipitation

Cells were collected and homogenized in lysis buffer (50 mM Tris–HCl pH 7.4, 5 mM EDTA, 250 mM NaCl, and 1% Triton X-100) supplemented with protease and phosphatase inhibitors. Coupling was performed between Dynabeads Protein A or G (10002D or 10003D, Thermo Fisher Scientific) and antibodies in 100 μL of 0.01% Tween20-1X PBS for 45 min at room temperature on a rocking platform. Cell lysates were immunoprecipitated with antibody-bead complexes. Immunocomplexes were washed extensively and boiled in Laemli sample buffer and subjected to SDS-PAGE and immunoblot analysis. Input corresponds to 10% of the lysate. IgGs were used as negative controls. Primary antibodies: p38α (9218, Cell Signaling Technologies), β-catenin (9562, Cell Signaling Technologies), GSK3β (9315, 9832 Cell Signaling Technologies), APC (2504, Cell Signaling Technologies), Axin1 (2087, Cell Signaling Technologies), FLAG-M2 (F1804, Abcam). Rabbit IgG HRP (NA934V, GE Healthcare) was used as a secondary antibody and revealed using the ECL-plus chemiluminescence reagent (RPN2232, GE Healthcare). Results are representative of at least three independent experiments.

Mass spectrometry analysis

Mass spectrometry analysis was performed by the Cogentech SRL service. Gel bands were subjected to reduction with dithiothreitol (DTT) (A39255, Thermo Fisher Scientific), alkylation with iodoacetamide (IAA) (A39271, Thermo Fisher Scientific), and digestion with trypsin (90059, Thermo Fisher Scientific). Peptides were loaded onto a TiO2 resin for phospho-enrichment. Then, the flow-through was treated with C18 Spin Tips & Columns (84850, Thermo Fisher Scientific) for desalting. The samples enriched for phospho-peptides and the desalted flow-through were further purified with SP3 and then analyzed by nLC-ESI–MS/MS on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) with a 32 min gradient. Samples were run in technical duplicate, in a positive mode with electrospray ionization. Data acquisition and processing were performed with Analyst TF (version 1.7.1, AB SCIEX). Data were analyzed using the Proteome Discoverer, Mascot, and Scaffold setting software. The parameter settings of data processing were as follows: DataBase = Uniprot_CP_Human_2020_GST_CTNB1 (Database di Uniprot_cp_Human + GST CTNB1 Human sequence, Accession Number P010101); Enzyme = Trypsin (cuts at C-term of K and also on R); Modifications = Acetyl (Protein N-term), Carbamidomethyl (C), Oxidation (M), Phosphorylation (STY); Peptide Thresholds: 95.0% minimum; Protein Thresholds: 99.0% minimum; 2 peptides/protein minimum.

Chromatin immunoprecipitation

Chromatin isolated from HT-29 and HCT-116 cells was subjected to chromatin immunoprecipitation using the MAGnify Chromatin Immunoprecipitation System (492024, Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, cells were cross-linked in 1% formaldehyde (252549, Sigma-Aldrich) for 10 min. Cross-linking was blocked with 0.125 M glycine (23390.4, Serva) for 5 min; then, cells were washed with 1 × PBS and lysed in lysis buffer. Chromatin was sonicated to a fragment length of about 200–500 bp and immunoprecipitated with 1 µg of anti-p38α (8690, Cell Signaling Technologies), anti-β-catenin (9562S, Cell Signaling Technologies), rabbit IgGs, FLAG-M2 (F1804, Abcam), or mouse IgGs. Quantitative real-time PCR was performed using SYBR Green IQ reagent (1708880, Bio-Rad Laboratories) with the CFX Connect detection system (Bio-Rad Laboratories). Primer sequences are available upon request. Results are representative of at least three independent experiments.

In vitro kinase activity

The analysis of p38α kinase activity was performed using the ADP-Glo Kinase Assay (V9101, Promega) according to the manufacturer’s instructions. 10 ng of p38α active protein (V2701, Promega) were assayed in a kinase reaction buffer with 500 ng of human recombinant β-catenin protein (β-catenin-WT, β-catenin-S111A, β-catenin-T112A), 150 μM ATP, and varying concentrations (0.01, 0.1, and 1 μM) of ralimetinib (S1494, SelleckChem). A total of 500 ng of p38 peptide substrate was used as a control. The generated luminescence was measured using a SPECTROstar Omega microplate reader (BMG Labtech).

Droplet digital PCR (ddPCR) assay

ddPCR was performed using 1 ng of RNA extracted from the HCT-116 CRC cell line or 20 ng of RNA extracted from patient-derived CRC-SC tumorspheres using the Purelink RNA Micro Kit (12183016, Thermo Fisher Scientific) and retro-transcribed to cDNA using the SuperScript VILO cDNA Synthesis Kit (11755050, Thermo Fisher Scientific) according to the manufacturer’s instructions. Reactions were prepared using the ddPCR Supermix for Probes Kit (1863024, Bio-Rad Laboratories) according to the manufacturer’s instructions. 20 µL of each reaction mix were converted into droplets with the QX200 Droplet Generator (Bio-Rad Laboratories). The droplets were transferred to a 96-well plate, sealed, and cycled in a C100 Thermocycler (Bio-Rad Laboratories) under the following cycling protocol: 25 °C for 3 min, 95 °C for 10 min followed by 40 cycles of 94 °C for 30 s and 60 °C for 1 min, a post-cycling step of 98 °C for 10 min, and infinite hold at 4 °C. The plate was then transferred into a QX200 Reader (Bio-Rad Laboratories).

For patient-derived CRC organoids, 10 ng of RNA were extracted using the Purelink RNA Micro Kit (12183016, Thermo Fisher Scientific) according to the manufacturer’s instructions. The ddPCR assay was prepared using the One-Step RT-ddPCR Advanced Kit for Probes (1864021, Bio-Rad Laboratories) according to the manufacturer’s instructions. 20 µL of each reaction mix were converted into droplets with the QX200 Droplet Generator (Bio-Rad Laboratories). The droplets were transferred to a 96-well plate, sealed, and cycled in a C100 Thermocycler (Bio-Rad Laboratories) under the following cycling protocol: 25 °C for 3 min, reverse transcription at 50 °C for 60 min, 95 °C for 10 min followed by 40 cycles of 95 °C for 30 s and 55 °C for 1 min, a post-cycling step of 98 °C for 10 min, and infinite hold at 4 °C. The plate was then transferred into a QX200 Reader (Bio-Rad Laboratories).

The data were analyzed using Bio-Rad QX Manager 1.2 Standard Edition. Probes were as follows:

c-Myc: ddPCR Gene Expression Assay, MYC, Human, Homo sapiens (dHsaCPE5051056, Bio-Rad Laboratories).

β-catenin: ddPCR Gene Expression Assay, β-catenin, Human, Homo sapiens (dHsaCPE5040214, Bio-Rad Laboratories).

TOPFlash/FOPFlash assay

To assess the transcriptional activity of β-catenin in HT-29 cells, we took advantage of the TOP/FOP reporter system using a dual-luciferase kit (Dual-GloTM Luciferase Assay System, E2920, Promega). On the day of the seeding, 6 × 105 HT-29 cells were co-transfected with 1 µg of p38 and 1 µg of β-catenin plasmids, using the Neon transfection system (NEON, ThermoFisher Scientific) according to the manufacturer’s instructions, and plated onto 24-wells. After 24 h, cells were transiently transfected with 2 ng of Renilla luciferase vector (E2231, Promega) and 100 ng of TOPFlash β-catenin-responsive firefly luciferase reporter plasmid (17285, Millipore) or the FOPFlash negative control (17285, Millipore) using Lipofectamine 3000 (L3000001, ThermoFisher Scientific) and serum-starved for 24 h. Then, cells were stimulated with Wnt3a (50 ng/mL) and TWS-119 (10 μM) for 4 h and treated or not with ralimetinib (10 μM). After 24 h in culture, cells were harvested and both firefly and Renilla luciferase activity was measured in triplicate according to the manufacturer’s instructions. Firefly luciferase activity was normalized against Renilla luciferase activity and fold increase in TOPFlash-compared to FOPFlash-transfected cells was determined.