Analysis of the TCGA PanCancer study
Sorcin (SRI) and EGFR genetic alterations and expression have been evaluated on the TCGA PanCancer study by interrogating the cBioPortal repository (https://www.cbioportal.org/). We have selected on the cBioPortal the Pan-cancer analysis of whole genomes [57] including 2,583 patients. Accession was done on April 2022.
Specifically, genetic alterations were visualized by analyzing the Oncoprint and the Co-occurrence functions. Expression of SRI in relation to SRI/EGFR copy numbers was evaluated using the Plots function. Evaluation of Pearson's correlations at transcript level was carried out considering the RNAseq datasets. Evaluation of Pearson's correlations at protein/phosphoprotein level was carried out on the TCGA datasets of GBM, CRC, LUAD and OVCA, by selecting the Firehose Legacy TCGA study, for each pathology, and interrogating the proteomic RPPA dataset vs. SRI mRNA (RNAseq).
Cell Culture, Transfection And Treatment
Human Non-small lung cancer cell line H1299 and HeLa cell line (ATCC, Manassas, VA, USA) were cultured in DMEM medium (Gibco® Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 50 U/mL penicillin, 50 U/ml streptomycin and 10% heat-inactivated FBS at 37°C in incubator with humidified 5% CO2 atmosphere.
For Sorcin silencing, H1299 and HeLa cell line was transiently transfected with Dicer-substrate short interfering RNAs (DsiRNAs) for Sorcin (IDT, Belgium) and NC negative control (IDT, Belgium) at 500 pM for 48h, using Lipofectamine RNAiMAX (Gibco® Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.
After the transfection, H1299 and HeLa cell lines were starved for 2 hours to examine the effect of EGF independently of other growth factors present in fetal bovine serum and to eliminate other variables which could interfere with EGF treatment. Then, H1299 and Hela cell line were treated with: Epidermal Growth factor, EGF (Merck Millipore, Italy) and Fluorescein Conjugate EGF (#E3478 Thermo Fisher Scientific, Waltham, MA, USA) at final concentration of 1ng/mL and 5ng/mL for the indicated time; Erlotinib (#5083 Cell Signaling, Danvers, MA, USA), at final concentration of 5µM for 24h; Thapsigargin (#T9033 Sigma-Aldrich, St. Louis, MO, USA), non-competitive inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), at final concentration of 100nM for 6h; EGTA (#E4378 Sigma-Aldrich, St. Louis, MO, USA), a chelator agent with specificity for calcium ions, at final concentration of 2mM for 1h. For protein stability assays, cells were treated with 10 µg/ml cycloheximide (Sigma-Aldrich, St. Louis, MO, USA) for 6h.
Lysate Preparation And Immunoblotting Analysis
Cells were lysed in 2% SDS buffer (25 mM Tris-HCl pH 7.5, 100 mM NaCl, 3 mM EDTA, 7% Glycerol) and fresh protease inhibitors. Lysates were sonicated for 15 s, centrifuged at 12000 ×rpm for 10 min to remove cell debris and then quantified in protein content with Bradford colorimetric assay (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Western blotting was performed loading 30 µg of lysates and using the following primary antibodies: rabbit monoclonal EGFR (1:1000 in 5%BSA-T-TBS solution) (D38B1, #4267, Cell Signaling), rabbit polyclonal Phospho-EGF Receptor (1:1000 in 5%BSA-T-TBS solution) (Tyr1068, #3777 Cell Signaling, Danvers, MA, USA), rabbit polyclonal AKT (1:1000 in 5%BSA-T-TBS solution) (#9272 Cell Signaling, Danvers, MA, USA), rabbit monoclonal Phospho-Akt (1:1000 in 5%BSA-T-TBS solution) (Ser473, #4060 Cell Signaling, Danvers, MA, USA), rabbit monoclonal p44/42 MAPK (Erk1/2) (1:1000 in 5%BSA-T-TBS solution) (137F5, #4695 Cell Signaling, Danvers, MA, USA), rabbit monoclonal Phospho-ERK1/ERK2 (P44/P42 MAPK) (1:1000 in 5%BSA-T-TBS solution) (T202/Y204 # MAB-94112 Immunological Science, Italy), rabbit polyclonal anti-CHD2 (1:1000 in 5%BSA-T-TBS solution) (#ab182013, Abcam), monoclonal mouse SLUG (A-7) (1:200 in 5%BSA-T-TBS solution) (#sc-166476 Santa Cruz Biotechnology, Heidelberg, Germany) monoclonal SNAI 1 (G-7) (1:200 in 5%BSA-T-TBS solution) (#sc-271977 Santa Cruz Biotechnology, Heidelberg, Germany) monoclonal mouse anti-GAPDH (1:3000 in 5%BSA-T-TBS solution) (#TA802519, OriGene Technologies, Rockville, USA). As secondary antibodies were used goat anti-mouse (1:10000 in 5% milk-T-TBS solution) and anti-rabbit (1:5000 in 5% milk-T-TBS solution or 5%BSA-T-TBS solution for phosphorylated protein) conjugated to horseradish peroxidase (Bethyl Laboratories, Montgomery, TX, USA). Protein signals were developed by ECL detection using a ChemiDoc-It Imaging System (UVP, Upland, CA) instrument.
Total Rna Extraction From Cells, Cdna Reverse Transcriptase, And Rt-qpcr
Total RNA was extracted using the TRIzol RNA Isolation System (Invitrogen) according to manufacturer instructions. Reverse transcription to cDNA was performed with the High-Capacity RNA-to-cDNA Kit (Applied Biosystems) and cDNA was amplified using the SYBR™ Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA USA) on QuantStudio™ 7 Flex Real-Time PCR System, 384-well (Applied Biosystems). Quantification levels of Sorcin was determined with the comparative 2 − ΔΔCt method, using H3 as control. All reactions were performed in duplicate. The following oligo sequences were used: H3 FW: 5’-GTGAAGAAACCTCATCGTTACAGGCCTGGT-3’; H3 RW: 5’-CTGCAAAGCACCAATAGCTGCACTCTGGAA-3’; SRI FW: 5’-GGCCACTCTGCAAGAAGGCA-3’; SRI RW: 5’TCCGGGAGCCCCTCCATACT-3’.
Immunofluorescence Analysis
3.5x10^5 cells/well were seeded into 6-well dishes and transfected with siRNA for Sorcin and Negative control. After 48h, cells were starved for 2h and treated with EGF or thapsigargin or EGTA for indicated time and fixed with 4% formaldehyde in PBS for 10 min at room temperature (RT). Then, cells were permeabilized with 0.01% Triton X-100 in PBS containing 1% BSA for 10 min. Cells were incubated with primary antibody rabbit monoclonal EGFR (1:50 in 1% BSA-PBS solution ) (D38B1, #4267, Cell Signaling, Danvers, MA, USA) and monoclonal antibody GM130 (1:100 in 1% BSA-PBS solution ) (#610822 BD Biosciences USA), followed by incubation with Alexa fluor 488 (1:500 in 1% BSA-PBS solution) (rabbit) and Alexa fluor 555 (mouse) conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, MA USA). Then, nuclei were counterstained with Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA USA) and were mounted with Vectashield (DBA, Italy). Cells were visualized under fluorescence confocal microscopy (Zeiss, Wetzlar, Germany).
Pre-embedding Immunogold Labeling Of Egf Receptor And Tem Imaging
All reagents for pre-embedding immunogold labeling were provided from Aurion (Wageningen, The Netherlands) according to the manufacturer’s instructions. Briefly, control and treated H1299 cells were fixed with a solution of 4% paraformaldehyde/0.1% glutaraldehyde in PBS per 1h at RT, washed in PBS and incubated with 50 mM glycine in PBS for 20 minutes at RT to inactivate residual aldehyde groups. After several washes, cells were permeabilized or not with 0.05% Triton X-100 in PBS for 30 minutes at RT and incubated in blocking solution for 1h at RT. After washing in incubation solution (PBS containing 5% BSA, 5% NGS and CWFS gelatin), cells were incubated with rabbit anti-EGFR primary antibody (1:200 in incubation solution) overnight at 4°C in a rocking table recognized then by ultrasmall gold conjugated F(ab)2 fragment of goat anti-rabbit IgG secondary antibody (1:100 in incubation solution). The negative controls are obtained by incubation with the only secondary antibody. After washes first in incubation solution and after in PBS cells were post-fixed in 2% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) in PBS for 24h and embedded in Agar 100 resin according to standard procedures. After fixation and several washes, the samples were treated with 0.5% osmium tetroxide (Electron microscopy Sciences) in H2O for 15 minutes. Silver enhancement was applied after osmium fixation in agitation. Samples were then dehydrated through ascending series of ethanol and immersed firstly in absolute propylene oxide (Sigma-Aldrich) and then left in a propylene oxide/resin 1:1 solution, overnight. Finally, they were embedded in Agar 100 resin (Agar Scientific, Stansted, UK) for 48 hours at 60°C.
Resin blocks were sectioned using an Ultracut E ultramicrotome (Leica EMUC6, Wetzlar, Germany). For TEM analyses (Zeiss EM10), ultrathin sections (90-100nm) were cut with a diamond knife, mounted on nickel grids, contrasted with saturated Uranyless (uranyl acetate alternative) (TAAB Laboratories Equipment Ltd, Aldermaston, UK) and lead citrate (Electron Microscopy Sciences) and photographed. Observations were performed operating at 60 kV.
Egf-fitc Binding/internalization
3.5x10^5 cells/well were seeded into 6-well dishes and transfected with siRNA for Sorcin and Negative control. After 48h, cells were starved for 2h, washed twice with cold PBS and harvested with 0.02% EDTA in PBS. Suspension of cells was incubated in serum free medium with EGF-FITC for 60 minutes at 37°C in incubator with humidified 5% CO2 atmosphere. As negative control, cells were treated with unlabeled EGF. On the flow cytometer, samples were first measured for total EGF bound and then for the intracellular EGF bound, upon the addiction of 1 M HCL to obtain a pH between 3 and 4 in the test tube which causes a quenching of fluorescence on the cell surface. The relative quantification of EGF on the cell surface is obtained by difference of fluorescence between total EGF bound and intracellular EGF bound.
Internalization Of Egf Receptor Assay
3.5x10^5 cells/well were seeded into 6-well dishes and transfected with siRNA for Sorcin and Negative control. After 48h, cells were starved for 2h and treated with EGF for 1h. Cells were washed with ice-cold PBS, trypsinized and fixed for 10 minutes in ice-cold 4% paraformaldehyde. Then, cells were permeabilized with 0.01% Triton X-100 in PBS containing 1% BSA for 10 min and incubated with Purified Mouse Anti-Human EGF Receptor (#555996, BD Biosciences USA) for 30 minutes. The amount of cell surface and intracellular EGFR was measured in the fixed unpermeabilized and permeabilized cells by flow cytometer.
Cell Invasion Assay
Cell invasion assay was performed using a 24-well plate with a non- coated 8-mm pore size filter in the insert chamber (BD Falcon, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. Each permeable support was covered with a diluted Matrigel matrix coating solution and was incubated at 37°C for 2 hours. After 48h of transfection and 2h or starvation, 2.0x10^5 H1299 cells, treated or not with EGF 1ng/mL were added to each 24-well invasion chamber. Cells were allowed to invade for 24h into the bottom chamber containing 0.7 ml DMEM media with 20% of FBS in a humidified incubator at 37°C in 5% CO2. Then, cells were washed with PBS and nuclei were stained with Diff-Quik stain (Corning, Life Sciences Tewksbury, MA USA). For the quantification of cell invasion, stained cells were dissolved in an extraction buffer, and solutions were transferred to a 96-well culture plate for colorimetric reading of OD at 560 nm. The OD value represents the invasive ability.
Wound Healing Assay
To perform wound healing assay, cells 3.5x10^5 cells/well were seeded into 6-well dishes and transfected with siRNA for Sorcin and Negative control. After 48h, confluent cells were starved for 2h and a wound has been performed in the middle of the cell monolayer. Each well was photographed at 2.5 × magnification at time 0 and after 24h of EGF 1ng/mL treatment, with a Nikon DS-Fi1 camera (Nikon Corporation, Tokyo, Japan), coupled with a Zeiss Axiovert optical microscope (Zeiss, Oberkochen, Germany). Quantification of wound healing assay was calculated as percentage of wound closure: ((At0- At1)/ At0) x100) where At0 is the initial wound area and At1 is the wound area n hours after the initial scratch.
Facs Analysis
3.5x10^5 cells/well were seeded into 6-well dishes and transfected with siRNA for Sorcin and Negative control. After 48h, cells were starved for 2h and treated with EGF and Erlotinib for 24h. Cells were washed with ice-cold PBS, trypsinized and fixed for 10 minutes in ice-cold 4% paraformaldehyde. Then, cells were permeabilized with 0.01% Triton X-100 in PBS containing 1% BSA for 10 min and incubated with primary antibody rabbit monoclonal EGFR (1:200 in 1%BSA-PBS solution) (D38B1, #4267, Cell Signaling, Danvers, MA, USA) and rabbit polyclonal Phospho-EGF Receptor (1:200 in 1%BSA-PBS solution) (Tyr1068, #3777 Cell Signaling, Danvers, MA, USA), followed by incubation with Alexa fluor 488 (rabbit) conjugated secondary antibodies (1:500 in 1%BSA-PBS solution) (Thermo Fisher Scientific, Waltham, MA, USA). Then, expression of proteins was quantified by acquired fluorescence on flow cytometry.
Phalloidin Immunofluorescence Analysis
3.5x10^5 cells/well were seeded into 6-well dishes and transfected with siRNA for Sorcin and Negative control. After 48h, cells were starved for 2h and treated with EGF for 24h. Cells were fixed with 4% formaldehyde in PBS for 10 min at room temperature (RT) and permeabilized with 0.01% Triton X-100 in PBS for 5 min. Then, cells were incubated with Rhodamine phalloidin (Thermo Fisher Scientific, Waltham, MA USA) diluted in PBS for 30 min. Then, nuclei were counterstained with Hoechst 33342 (Life Technologies).
Surface Plasmon Resonance (SPR) experiments.
SPR experiments were carried out using a SensiQ Pioneer system (ICx Nomadics), essentially as in Genovese et al., 2020 [38]. The sensor chip (Ni-NTA) was activated chemically by a 35 µl injection of 0.1 M NiSO4 at a flow rate of 5 µl/min. Ligand, i.e. human EGFR C-terminal domain, was directionally immobilized on activated sensor chips via its N-terminal His-tag. The immobilization level of EGFR was 200 RU. Wt Sorcin and Sorcin W105G analytes were dissolved in buffer 20 mM Hepes pH 7.4, 150 mM NaCl + 0.005% surfactant P20 (HBS-P buffer) (± 100 µM CaCl2) to a concentration of 10 µM, automatically further diluted in HBS-P and injected on the sensor chip, at the following concentrations: 312 nM, 625 nM, 1.25 µM, 2.5 µM, 5 µM and 10 µM at a constant flow (nominal flow rate = 30 µl/min), in the presence of CaCl2 at 0 or 100 µM concentration. The increase in RU relative to baseline indicates complex formation; the plateau region represents the steady-state phase of the interaction (RUeq), whereas the decrease in RU after injection represents dissociation of Sorcin from immobilized EGFR after injection of buffer. Control experiments were performed in sensor chips treated as described above, in the absence of immobilized ligand. Regeneration procedures are based on two long (2000 s and 500 s) injections of buffer, separated by a brief (5 s) injection of 5 mM NaOH. Kinetic evaluation of the sensorgrams was obtained using the SensiQ Qdat program and full fitting with 1, 2 and 3 sites.
Calcium Measurements
Ca2+ measurements were performed by co-transfecting H1299 or HeLa cells in a six-well plate with 1 µg cytosolic (cytAEQ) aequorin along with 5 nM of either scramble siRNA or siRNA against Sorcin, using Lipofectamine 3000. Twenty-four hours post transfection, cells were re-plated into a 96-wells plate (PerkinElmer). Forty-eight hours post transfection, CytAEQ was reconstituted by incubating cells for 1.5 h with 5 µM coelenterazine wt (Santa Cruz Biotech) in modified Krebs Ringer Buffer (KRB: 125 mM NaCl, 5 mM KCl, 400 mM KH2PO4, 1 mM MgSO4, 20 mM Hepes, pH 7.4) supplemented with 0.1% glucose at 37°C. Luminescence measurements were carried out using a PerkinElmer EnVision plate reader equipped with two injector units. After reconstitution, cells were washed with KRB (either with 1 mM Ca2+ or 0.5 mM Ca2+, depending on the experiment) and luminescence from each well was measured for 1/1.5 min. According to the experiment, 100 µM histamine (Sigma) or (Carbachol 500 µM + His 100 µM + ATP 100 µM + Bradykinin 100 nM) or (100 ng/mL EGF + CPA 20 µM + EGTA 2 mM) (final concentrations in the wells) were injected to generate Ca2+ transients, and then a hypotonic, Ca2+-rich, digitonin-containing solution was added to discharge the remaining aequorin pool. Output data were analyzed and calibrated with a custom-made macro-enabled Excel workbook.