Background
TP53 is recognized as the most frequently mutated gene in human cancers [
1,
2]. Furthermore, germline heterozygous mutations in
TP53 cause the rare cancer predisposing Li-Fraumeni syndrome [
3,
4] and 100% of mice lacking
Trp53 develop tumours, primarily lymphomas or sarcomas highlighting the pivotal role of TP53 as a tumour suppressor [
5‐
7]. TP53 operates as a transcription factor, responding to a broad range of stress signals by binding to the DNA in a sequence-specific manner and activating many effector genes, ranging from several hundred to over a thousand [
8‐
12]. These genes play crucial roles in multiple cell protective pathways, such as apoptosis, cell cycle arrest, senescence, DNA damage and repair mechanisms [
8‐
10,
13]. Therefore, identifying the molecular mechanisms underlying TP53's function in tumour suppression is vital for understanding cancer development. Although some TP53-dependent genes, such as genes coding for the cell cycle inhibitor p21 or the pro-apoptotic proteins PUMA and Noxa, have been broadly studied in TP53-mediated tumour suppression [
8,
14‐
19], numerous others still have an unknown relevance in the TP53 network. Several studies have uncovered the importance of such undervalued players of TP53-dependent tumour suppression, including ZMAT3 [
20‐
23], ABCA1 [
24], TIGAR [
16] or GLS2 [
25] among others. Recently, it has been described how the knock-down of several TP53-dependent proteins that have a role in DNA damage response, such as CAV1, MLH1, MSH2, DDIT4, POLK, ERCC5, FANCC or RNF144B, was enough to accelerate
Eμ Myc-driven lymphoma [
23,
26]. Remarkably, knockdown of
Rnf144b substantially accelerated lymphoma development at a rate similar to knockdown of p53 itself. Moreover, mutations in RNF144B are largely mutually exclusive with mutations in p53 in several cancers, consistent with a notion that RNF144B and TP53 could function in the same pathway [
23]. Importantly RNF144B role as a tumour suppressor in other cellular and oncogene driven contexts is still unknown.
RNF144B, also known as p53-inducible RING-finger protein (p53RFP), is an E3-ubiquitin ligase enzyme from the ubiquitin-ligases RBR (RING-in-between-RING) family [
27‐
29] and is therefore partially involved in the proteasomal degradation of its targets by ubiquitin transference [
28,
30]. RNF144A, homologous of RNF144B [
28,
30], is a TP53-activated ubiquitin-ligase and it has been proposed as a tumour suppressor because it promotes proteasomal degradation of cytosolic DNA-PKc proteins and consequent apoptosis following DNA damage [
31]. Previous studies have shown that RNF144B is strongly related to the TP53 family of transcription factors, including TP53 itself [
23,
32,
33], TP63[
34] and TP73 [
35]. RNF144B regulates epithelial homeostasis and differentiation through degradation of the cell cycle inhibitor p21 [
33] and modulates apoptosis [
36,
37]. Due to its potentially important role as a tumour suppressor [
23], it is important to investigate further the cellular functions of RNF144B and its role in TP53-mediated tumour progression.
Here, we investigate the role of RNF144B as a TP53-regulated tumour suppressor in different cellular and oncogenic contexts than Eμ Myc-driven lymphoma. Our studies coupled with in vivo, 3D or 2D cellular models’ analysis and clinical data, demonstrate that RNF144B suppresses cell proliferation and transformation, in particular in the context of lung cancer. Molecular analysis showed a novel function of RNF144B in maintaining genomic stability, resulting in effects on DNA repair and mitotic progression. Finally, RNF144B deficient cells gained resistance to cell cycle and chromosomal instability inducing drugs, commonly used in the clinics.
Methods
Cell culture
Human lung adenocarcinoma cell line A549 (ATCC, CRL-3588) and the colon carcinoma cell line HCT116 (ATCC, CCL-247) were obtained from Hospital del Mar Research Institute and authenticated using Short Tandem Repeat profiling (CSIC-UAM, Madrid, Spain). Mouse
KRASG12V lung cancer cell lines (mKLC) were a gift from D. Santamaria (CIC, Spain) [
38] and were grown in DMEM (L0102, Biowest) containing 10% Fetal Bovine Serum (FBS, S181BH, Biowest) and 100 µg/ml penicillin/ streptomycin (15,140,122, Gibco). HBEC3-KT (ATCC, CRL-4051) immortalized bronchial epithelial cells were a gift from Silvestre Vicent (CIMA, Spain) [
39,
40]. HBEC3-KT cells were cultured in KSFM media (17,005,042, Gibco) containing 50 µg/mL of Bovine Pituitary Extract (BPE, 13,028,014, Gibco) and 5 ng/mL of human epidermal growth factor (hEGF, E9644, Gibco). HBEC3-KT cells expressing
KRASG12D and sgRNA knockout populations were also cultured in RPMI-1640 (L0500, Biowest) media supplemented with 10% FBS and 100 µg/ml penicillin/ streptomycin. Mouse embryonic fibroblasts (MEFs) were generated from E13.5 C57BL/6J embryos. MEFs were grown in DMEM containing 10% FBS, 100 µg/ml penicillin/ streptomycin, 100 μM asparagine (A4159, Sigma) and 50 μM 2-mercaptoethanol (63,689, Sigma). Cells were grown in 5% CO
2 at 37ºC. All cell lines were regularly tested for mycoplasma. Only mycoplasma-negative cells were used.
Virus production and transduction
To generate CRISPR knockout bulk populations or clones, cell lines were transduced with a two-construct lentiviral pFUGW-derived system: a constitutive vector with an mCherry-labeled Cas9 [
41] and a sgRNA expression vector [
23] expressing CFP. sgRNAs sequences were cloned after BsmbI (R0580S, NEB) digestion. sgRNAs targeting human genes were the following: For human
Tp53: 5’-GGCAGCTACGGTTTCCGTCT-3’, and for human
Rnf144b: 5’-TGACATGGTGTGCCTAAACC-3’. A non-targeting control sgRNA was used (sgCTRL: 5’-CCAGTTGCTCTGGGGGAACA-3’).
shRNAs GFP-labeled targeting mouse RNF144B (shRNF144B: 5’-TGCTGTTGACAGTGAGCGCCAGGTTATTTACATACTTTCATAGTGAAGCCACAGATGTATGAAAGTATGTAAATAACCTGATGCCTACTGCCTCGGA-3’), TRP53 (5’-TGCTGTTGACAGTGAGCGCCCACTACAAGTACATGTGTAATAGTGAAGCCACAGATGTATTACACATGTACTTGTAGTGGATGCCTACTGCCTCGGA-3’) or the shRenilla control (5’TGCTGTTGACAGTGAGCGCAGGAATTATAATGCTTATCTATAGTGAAGCCACAGATGTATAGATAAGCATTATAATTCCTATGCCTACTGCCTCGGA-3’) were generated into LMS (LTR/MCSV/SV40-puro-IRES-GFP) retroviral vector [
23]. 3KT cells were infected with a Lenti-CMV-KRAS
G12D construct [
39]. To immortalize MEFs cell cultures, retroviral vectors expressing
E1a and
HrasG12V were used [
23]. For the in vivo competition assay, control MEFs were transduced with a lentiviral plex-Renilla-mCherry (gift from Dr A. Celià-Terrassa, Hospital del Mar Research Institute, Spain). To perform drug response analysis of live cells, a construct expressing a nuclear localization signal (NLS) coupled to GFP was used: pTRIP-SFFV-EGFP-NLS (NLS-GFP) was a gift from Nicolas Manel (Addgene plasmid # 86677). For the overexpression studies, RNF144B cDNA construct (NM_182757.4) was generated in a pcDNA3.1( +)-C-6His vector (Genscript, Netherlands).
Lentiviral supernatant was generated by transient transfection of HEK293T (ATCC, CRL-3216) cells with the following packaging constructs: pMDL (5 μg), pRSV-rev (2.5 μg) and pVSV-G (3 μg)[
23]. For retroviral particle production GAG (4.8 µg), and pENV (2.4 µg) constructs were used [
23]. 10 μg of vector DNA was transfected using calcium phosphate precipitation. Viral supernatant from HEK293T cells was collected after 48 h, filtered, transferred to cell cultures, and centrifuged at 2200 rpm at 32ºC during 2 h. After 48-72 h, cells were FACS-sorted for the corresponding fluorescence using a BD Influx cell sorter (BD Biosciences). If needed, CRISPR single cell clones were seeded in 96 well plates and expanded to generate isogenic populations. MEFs infected with
E1a and
HrasG12V were selected with Puromycin (3 μg/ml, P7255, Sigma) and Hygromycin (200 μg/ml, 400,052, Sigma) for 72 h. MEF immortalized cell lines were used at low passage (passage 6–14) to avoid phenotypes arising from prolonged passaging.
Animal experiments
All animal experiments are compliant with ethical regulations regarding animal research and were conducted under the approval of the Ethics Committee for Animal Experiments (CEEA-PRBB, Barcelona, Spain). All animals were euthanized before or at the moment of achieving maximum tumour volume. Subcutaneous tumour models were performed by injection of 1 million cells suspended in 100 μl of PBS in both flanks of 7–10-week-old female Athymic Nude-Foxn1nu mice (Envigo). Tumours were grown for approximately 3 weeks and harvested at the endpoint. For in vivo competition assay, MEF cells were infected with the plex-Renilla-mCherry lentiviral construct or with GFP-labeled shRNA targeting RNF144B or TRP53. Cells were mixed 1:1, evaluated by FACS (LSR Fortessa, BD Biosciences) and 1 million cells were injected subcutaneously into the flanks. After 3 weeks tumours were harvested, minced, and digested in a solution of DMEM, 0,3 mg/ml Collagenase I (C1-BIOC, Sigma) and 10 μg/ml DNAse I (DN25, Sigma) at 37ºC while shaking for 2 h. Digested tumours were filtered through a 45 μM mesh, cleaned of red blood cells with Red Blood Lysis Buffer (11,814,389,001, Roche) and analyzed by cytometry (Fortessa). Subcutaneous tumour growth was followed by caliper measurements and the following formula applied to measure tumour volume: volume = 1/2(length × width2). In the case that tumours did not grow in the flank, measurement was excluded from the comparative analysis.
Intercostal intrapulmonary model was performed by injecting 200.000 A549 cells suspended in 10 μl of PBS through the ribcage into the left lung with a 29G insulin needle and a depth of 4–4.5 mm. 10–12-week-old female Athymic Nude-Foxn1nu mice were used for this study. Weight was monitored biweekly, and animals were euthanized at 6 weeks post-inoculation. Only mice with localized intrapulmonar tumours were considered for tumour burden analysis.
3KT experiments were performed by injecting 1 million cells in 100ul PBS intravenously in the tail vein and after 5 months, animals were euthanized to study lung lesions.
Lungs were inflated with 4% paraformaldehyde (PFA, sc-281692, SCBT) through the trachea and fixed overnight for histological evaluation. Lung sections were performed and scanned with an Aperio ScanScope (Leica) at the Anatomy Department (Hospital del Mar). Tumour area and lung area were measured with ImageJ to calculate tumour burden. Those mice that didn’t present any tumour growth, or that had tumoural growth outside the lung and into the thoracic space were excluded from the analysis. Mice were housed in groups of 5 per cage and irradiated chow and water were provided ad libitum.
Proliferation analysis
50.000 3KT cells were seeded in 6 well plates in triplicates and after 6 days of growth, cells were counted using Trypan Blue staining and a Countess 3 Automatic cell counter (Thermofisher). The experiment was performed in five independent replicas.
3.000 3KT cells were seeded in 6 well plates and after 8 days of growth, cells were fixed using 4% PFA for 10 min and stained with 0.5% crystal violet solution (V5265, Sigma-Aldrich) for 1 h. Plates were scanned with an Amersham Typhoon™ (Cytiva). Crystal violet was dissolved with 10% acetic acid and absorbance was read at 590 nm in a Biotek Synergy HTXmachine (Agilent). The experiment was performed in four independent replicas.
Spheroid cultures
1.000 3KT cells were resuspended in 50 μl of cold Matrigel GFR (354,230, Corning) and seeded as a drop in the wells of a 24 well plate. Soon after seeding the Matrigel domes, the plate was turned upside down and placed in the incubator for 30 min. Afterwards, 1 ml of KSFM media with 20% FBS and 1% penicillin/ streptomycin was added. Spheroids were monitored for 7 days and pictures were taken using a brightfield Olympus CKX53 microscope and an Olympus EP50 camera. Pictures were taken of 4–5 random fields per well with a 10 × objective. Spheroid diameter was analyzed by ImageJ. The number of spheroids quantified was between 180 and 410 depending on the cell line. Experiment was repeated twice and performed in three technical replicates each time.
Immunoblotting
Cells were lysed in RIPA buffer containing protease inhibitors (cOmplete protease inhibitor cocktail, 11,836,170,001, Roche). Protein extracts were quantified using the Protein Assay Dye Reagent (5,000,006, BioRad) and 20 μg were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Cytiva Amersham). Membranes were blocked for 1 h in 5% milk in PBS-T (PBS with 0.1% Tween 20) and incubated overnight with the corresponding primary antibody in PBS-T 5% milk. For probing antibodies against TRP53 (NCL-L-p53-CM5p, Leica Biosystems), TP53 (sc-126, SCBT), p-γH2AX (9718 T, CST), βACTIN (sc-47778, SCBT), His-Tag (66,005–1-Ig, ThermoFisher), and secondary antibodies anti-rabbit (sc2357, SCBT), and anti-mouse (sc-516102, SCBT) were used. Membranes were developed using the ECL Prime system (RPN2232, Cytiva) and imaged using a ChemiDoc MP (BioRad).
Overexpression analysis
2,5 × 105 A549 or 5 × 105 3KT cells were seeded in 6 well plates. 24 h after seeding were transfected with 1500 ng of the empty vector (pcDNA3.1 + C-6His) or the OE-RNF144B vector (RNF144B_OHu07981C_pcDNA3.1( +)-C-6His) using Lipofectamine 2000 reagent (11,668,027, ThermoFisher) following manufacturer instructions. Cells were counted after 72 h using Trypan Blue staining and the automatic cell counter Countess 3. A pellet of cells was collected to perform western blot and confirm overexpression. Experiment was repeated thrice and performed in triplicates.
Immunohistochemistry
Tissues were collected and fixed in 4% PFA overnight and processed for paraffin-embedding. Slides were stained for Hematoxylin and Eosin (H&E) using standard protocols. Immunohistochemistry was performed with antibodies against Ki67 (12202s, CST) and pH3 (3377 T, CST). Briefly, paraffin sections were re-hydrated and antigen retrieval was performed in a pressure cooker with Sodium Citrate Buffer pH6 for 20 min. 3% H
2O
2 was used to quench the peroxidase for 15 min and blocking was done with PBS / BSA 1% (A9647, Sigma) / 0,3 Triton-X (11,332,481,001, Sigma) for 30 min. Slides were incubated overnight at 4ºC in a humid chamber with primary antibody. The next day, sections were incubated with the 2º antibody (Impress HRP Goat Anti-Rabbit, MP-7451–15, Vector Laboratories) for 1.30 h and afterwards incubated with DAB peroxidase kit (K346711-2, Agilent) and hematoxylin. Slides were mounted with DPX mounting media (06522 Sigma). A Cell Observer (Zeiss) microscope was used for imaging. Images were analyzed and quantified using Qupath [
42] (v0.3.2).
Amplicon sequencing of sgRNA target sites
A549 (isogenic clones) and 3KT cells (bulk population) carrying Cas9 and sgNT or sgRNF144B were evaluated by amplicon sequencing to detect INDELs in the sgRNA target site. Genomic DNA was extracted from the cells using the DNeasy Blood and Tissue kit (69,504, Qiagen). The sgRNA target sites were PCR amplified using primers flanking the site of interest with recommended overhangs (Fwd:5’-ACACTCTTTCCCTACACGACGCTCTT CCGATCTGTGGCTGAAATGTGTGAGCA-3’ and Rev: 5’-GACTGGAGTTCAGACGTG TGCTCTTCCGATCTCTGTATTTTCTTGCTAGACTCC-3’). PCR was performed to ensure a single band was amplified and PCR products were purified using the QIAquick PCR Purification Kit (28,104, Qiagen) and sent to Genewiz (Leipzig, Germany) using Amplicon-EZ service, able to read from 150-500 bp.
qRT-PCR
Cells were treated with 10 μM Nutlin-3a for 6 h to stimulate TP53 activation or left untreated, depending on experiment. Total RNA was isolated from cells using TRIzol reagent (15,596,018, ThermoFisher) and reverse transcribed using SuperScript III (18,080,400, ThermoFisher) or SuperScript IV (18,090,050, ThermoFisher), Reverse Transcriptase and Oligo-d(T) primers (18,418,020, ThermoFisher). qRT–PCR was performed using either SYBR green (Roche, 4,707,516,001) or Taqman Gene Expression assays (ThermoFisher). For Taqman: Human TP53 (Hs01034249_m1), mouse TRP53 (Mm01731287_m1), human CDKN1A (Hs00355782_m1), mouse CDKN1A (Mm00432448_m1), human RNF144B (Hs00403456_m1), mouse RNF144B (Mm00461356_m1), housekeeping human HMBS (Hs00609297_m1) and mouse HMBS (Mm01143545_m1). For SYBR green the primers were as follows: Human RNF144B: 5´-TTGTCCTGCCAACAGAGCAC-3´ and human GAPDH: 5´-GCACAGTCAAGGCCGAGAAT-3´. Samples were analyzed in QuantStudio 12 K equipment (Applied Biosystems). The mRNA expression levels of TP53 target genes of interest were standardized with corresponding housekeeping genes and normalized to the untreated control.
1,5 million cells were seeded in 10cm2 plates and treated the following day with 0.3 mg/ml of colcemid (10,295,892,001, Roche) for 3 h. Cells were collected and resuspended dropwise with KCL 0.056 M and incubated during 20 min at RT. Cells were then fixed in cold methanol:glacial acetic acid solution (3:1) and washed 3 more times with the fixative solution. Cells were dropped on glass slides from 1,5 m height, dried and stained with 3% Giemsa (GS500, Sigma). After washing, coverslips were mounted and pictures were captured using a brightfield Olympus CKX53 microscope and an Olympus EP50 camera, using a 40 × objective. Chromosomes were counted manually with ImageJ Software. At least 25 cells were analyzed per cell line/genotype.
Cell cycle assay
450.000 cells were seeded in 6 well plates and the next day, cells were trypsinized, washed with PBS and fixed with cold ethanol (70%) in a dropwise manner while vortexing. After 2 h of fixation, cells were pelleted, washed twice with PBS and resuspended in working solution, containing 15 μg/ml of Propidium Iodide (00–6990-50, ThermoFisher) and 300 μg/ml RNAse A (10,109,142,001, Sigma). Cells were incubated for 2 h at room temperature (RT) and cell cycle distribution was analyzed by flow cytometry using a BD LSRII-B cytometer (BD Biosciences). Data was analyzed using FlowJo software.
Edu incorporation
Edu incorporation was performed using the Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit (C10424, ThermoFisher). Between 250.000–350.000 cells were seeded in 12 well plates and pulsed with 10 μM EdU for 2 h. Next, cells were harvested, fixed, permeabilized and stained using the Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit following manufacturer’s instructions. Cells were co-stained with a solution containing Propidium Iodide (15 μg/ml) and RNAse (300 μg/ml) to measure DNA content. Samples were analyzed using BD LSRII-B cytometer and FlowJo Software.
DNA repair quantification by immunofluorescence
15.000 cells were seeded in Phenoplate (6,055,302, PerkinElmer) black well plates and the following day cells were gamma-irradiated at 5 Gy with an IBL-437C (CIS Biointernational). Control plate was left untreated. Cells were fixed with 4% PFA. Afterwards, blocking and permeabilization was performed with PBS/5% BSA/0,3% Triton-X during 1 h at RT. Staining with the primary antibody p-γH2AX (9718 T, CST) dissolved in PBS/1% BSA/0,3% Triton-X was performed overnight at 4ºC. The following day, cells were washed × 3 with PBS and secondary anti-rabbit Alexa Fluor 647 (A21244, Invitrogen) was added during 2 h at RT in the dark. Cells were washed again × 2 with PBS and incubated with 1 μg/ml DAPI (D9542, Sigma) for 10 min. After washing, cells were imaged with the Opera or Operetta High Content Screening System (Perkin Elmer), using the 40 × objective. Segmentation of the nuclei using the DAPI signal and quantification of the number of p-γH2AX foci per cell was done using Harmony® High-Content Imaging and Analysis Software.
Cell viability assays
1 × 105 MEFs, A549 and 3KT cells were seeded in a 24-well flat bottom plate in medium containing 10% FCS. 24 h after seeding, the cells were incubated with Doxorubicin (0.05 μg/ml or 0.2 μg/ml), Nutlin-3a (10 μM) or with 0% FBS media, respectively. For 0% FBS experiments, cells were washed 3 × with PBS to remove any residual FBS before addition of medium. Cells were harvested 24 h or 72 h after, stained with APC Annexin V kit (640,920, Biolegend) and 1 μg/ml DAPI and analyzed with an BD LSRII-B cytometer and FlowJo Software.
Immunofluorescence imaging of mitotic cells
Between 120.000 and 150.000 cells were grown in 24-well plates. The day after cells were treated or not with 15 µM RO-3306 for 18 h at 37 °C, 5% CO2. Cells were washed with PBS, fixed with 4% PFA for 10 min at RT and permeabilized with PBS / 0.1% Triton X-100 for 5 min at RT. Blocking (RT, 20 min) and incubations with antibodies (RT, 1 h) were performed with 10% FBS in PBS 0.1% / Triton X-100 and washes were done with PBS 0.1% / Triton X-100 at RT for 3 × 5 min. The antibodies targeted α-tubulin (T9026, Sigma) and γ-tubulin (T6557, Sigma). An Alexa 555 Goat anti-Mouse antibody (A-21424, Invitrogen) was used as a secondary antibody. Nuclei were counterstained with 1 μg/mL DAPI for 2 min at RT and cells were mounted using the ProLong Gold antifade reagent (P10144, Thermofisher). Confocal microscopy pictures were taken with a Leica STELLARIS microscope. For counting lagging chromosomes, DNA bridges, multipolar mitosis or centrosome numbers, at least 200 cells were analyzed by eye for each condition.
Micronuclei assay
150.000 cells were grown in 24-well plates. The day after, cells were washed with PBS and fixed in freshly prepared 4% PFA for 10 min at RT. Nuclei were counterstained with 1 μg/mL DAPI in PBS for 2 min at RT and cells were mounted using the ProLong Gold antifade reagent. Confocal microscopy pictures were acquired in a z-stack mode with a Leica STELLARIS microscope. Micronuclei analysis has been made with Fiji software and for each field (45 random field/sample) the number of micronuclei were divided by the number of nuclei.
Live cell imaging of mitotic cells
100.000 cells were grown on 4-well chambered coverslips (80,426, Ibidi). The day after, cells were treated with 15 µM RO-3306 for 18 h. One hour before imaging, siR-Hoechst (SC007, Spirochrome) was added to the media at 1 µM and cells were incubated at 37 °C and 5% CO2. Just before imaging, media was replaced by FluoroBrite DMEM (A1896701, ThermoFisher) supplemented with 10% FBS and siR-Hoechst. Time-lapse live-cell imaging was performed using a Leica STELLARIS confocal system with white light laser inverted microscope maintaining temperature at 37 °C and CO2 at 5%. Images were taken every 4 min with a × 64 objective. Exposure time was optimized so that no phototoxicity or photobleaching was caused to cells. Image processing was performed using FIJI software.
In vitro cell growth assay
A549 and 3KT cells expressing Cas9 and sgNT, sgRNF144B or sgTP53 were infected with the NLS-GFP construct and sorted for GFP + cells. 5 × 10
3 cells were seeded in 96 Phenoplate black well plates. 24 h after seeding were treated with Palbociclib (1–3 μM, 3 μM, Hospital del Mar), Abemaciclib (0,5–3 μM, Hospital del Mar), Paclitaxel (10–20 nM, S1150, Selleckchem), Docetaxel (5–20 nM, Hospital del Mar), Etoposide (10–20 μM, 341,205, Sigma), Doxorubicin (0,05–0,2 μg/ml, N31815, Sigma), Carboplatin (50–100 μM, Hospital del Mar), RO-3306 (5 μM, HY-12529, MedChem) and Nutlin-3a (20 μM). Imaging was performed as described previously [
43] with the Operetta High Content Screening System using the × 20 magnification. Cell number represented by the sum of the nuclear GFP intensity/well was quantified with the Harmony Software at day 0 (prior to drug treatment) and after 48 or 72 h, depending on the cell line. Cell confluency was normalized to that of day 0 of the same well.
LC–MS/MS Proteomics and analysis
MEFs were infected with the corresponding shRNAs in three independent biological replicas and sorted for GFP by flow cytometry using a BD Influx cell sorter (BD Biosciences). Afterwards, cells were washed with PBS, scrapped with 6 M Urea and 200 mM Ammonium Bicarbonate and sonicated at 4ºC. 10 μl of each sample at 1 mg/ml was submitted for analysis. The samples were digested with Trypsin and LysC and 2 μg were analyzed by LC–MS/MS using a 90 min gradient in the Orbitrap Eclipse. Raw MS files were processed in Proteome Discoverer version 2.3.0.523 (Thermo Scientific, Waltham, MA,) [
44]. Samples have been searched against SP_Mouse database (June 2020), using the search algorithm Mascot v2.6 (
http://www.matrixscience.com/). Peptides have been filtered based on FDR and only peptides showing an FDR lower than 5% have been retained. Normalized protein abundances with “Total Peptide Amount” from Proteome Discoverer were used as input for the analysis with the DEP R package [
45].
6513 quantified protein profiles were expressed on 9 samples. We only kept proteins that were based at least in two unique peptides, leading to a final protein quantification data matrix of 5389 proteins. Proteins with missing values showed a lower expression in reference to those without missing values. A full normalized matrix of protein expression values was obtained by imputing missing quantifications with a mixed methodology. Proteins with missing at random (MAR) values were imputed with k-nearest neighbors (knn) algorithm and missing not at random (MNAR) values were imputed with random draws from a Gaussian distribution centered around a minimal value (MinProb). We conducted a protein differential expression analysis based on protein-wise linear models and empirical Bayes statistics using limma [
46]. Proteins with
p-value < 0.05 and a minimum fold-change of 50% were considered as statistically significant. 5039 proteins had no significant change in expression while 350 proteins were differentially expressed between the control replicates and the RNF144B knockdown cells.
Functional enrichment analysis of the biological processes was conducted with the Gene Ontology (GO) database using the clusterProfiler package [
47]. Significant GO terms are shown with an associated
p-adjusted value (determined by circle color) and GeneRatio (Number of differentially abundant proteins associated with the GO terms / number of input differentially abundant proteins). The circle size is given by the count of proteins detected that are involved in each GO term.
RNA-Seq analysis
MEFs were infected with the corresponding shRNAs in three independent biological replicas and sorted for GFP with a BD Influx cell sorter. After, cells were trypsinized and the pellet was snap frozen. RNA from 1 million cells was extracted using the Purelink RNA kit (10,307,963, Invitrogen) and submitted for analysis. Libraries were prepared using the TruSeq stranded mRNA Library Prep (20,020,594, Illumina) according to the manufacturer's protocol. Briefly, 1000–500 ng of total RNA were used for poly(A)-mRNA selection using poly-T oligo attached magnetic beads using two rounds of purification. RNA was fragmented under elevated temperature and primed with random hexamers for cDNA synthesis. Then, cDNA was synthesized using reverse transcriptase (SuperScript II, 18,064–014, Invitrogen) and random primers. The addition of Actinomycin D to the First Strand Synthesis Act D mix (FSA) prevents spurious DNA-dependent synthesis, improving strand specificity. After that, second strand cDNA was synthesized, incorporating dUTP in place of dTTP to generate ds cDNA using DNA Polymerase I and RNase H. A corresponding single T nucleotide on the 3’ end of the adapter provided a complementary overhang for ligating the adapter to the fragments. It was followed by subsequent ligation of the multiple indexing adapter to the ends of the ds cDNA. Finally, PCR was performed with a PCR Primer Cocktail. Final libraries were analyzed using Bioanalyzer DNA 1000 or Fragment Analyzer Standard Sensitivity (DNF-473, Agilent), and were then quantified by qPCR using the KAPA Library Quantification Kit KK4835 (07960204001, Roche) prior to the amplification with Illumina’s cBot. Libraries were sequenced 1 * 50 + 8 bp on Illumina's HiSeq2500.
We performed a quality control on the 9 raw single-end reads samples using the nf-core/rnaseq (
v. 3.10.1) [
48,
49]. Raw FASTQ files were aligned to the GRCm38.p6 version of the reference mouse genome using STAR (
v. 2.7.6a) [
50] with default parameters except for –sjdbOverhang 49, producing a set of 9 BAM files. Aligned reads in BAM files were reduced to a table of 55,487 genes by 9 samples. Genes were annotated using the GENCODE vM25 GTF file. Following previously established recommendations [
51,
52], we filtered out lowly expressed genes by discarding those that did not show a minimum reliable level of expression of 20 counts per million reads of the smallest library size, in at least all the samples of the smallest group, which was 3. After the filtering, we ended up with a final table of counts of 14,668 genes by 9 samples. The DESeq2 package (
v. 1.40.0) [
52,
53] was used for the differential expression analysis. Surrogate variables were calculated with SVA [
54]. Genes with adjusted p-value < 0.05 (5% FDR) and absolute log2FC > 1 were considered as statistically significant.
Integrative transcriptomics vs proteomics analysis was conducted to show the expression relationship patterns of differentially expressed genes vs differentially abundant proteins. Results are represented with Log2 expression ratio. The cut-offs are a minimum fold-change of 50% for the proteomics expression profile and minimum fold-change of 100% for the transcriptomics expression profile.
RNF144B differential expression study
To access comprehensive data on GTEx, GDC and TCGA Pan-Cancer normalized gene expression, phenotypic information, and somatic mutations, we utilized XenaBrowser [
55] to extract publicly available data from The Cancer Genome Atlas (TCGA) (
https://www.cancer.gov/tcga). The combined cohort of TCGA, and GTEx [
56] samples were employed to investigate gene expression differences between normal and tumour samples. To study RNF144B expression in
Tp53 wild type or mutated tumors, the GDC-TCGA Pancancer dataset was utilized. Samples were stratified depending on their classification as
Tp53 wild type or mutant and
Tp53 was considered wild type in the following conditions: no mutation present, synonymous variants (silent) or located in the intronic, 5' UTR, or 3' UTR regions.
Tp53 was considered mutant in the following conditions: splice mutations, frameshift, stop codon gain, missense mutation, coding sequence variants, inframe insertions and loss of start or stop point mutations. The considerations for
Tp53 status stratification and the specific mutations present in the patient samples are shown in Supplementary Table
1. When analyzing RNF144B expression data, we focused on cancer types that contained a minimum of 20 samples, with both
Tp53 wild type and
Tp53 mutated entries present in the gene expression matrix. The cancer types that didn’t reach the minimum 20 samples per group are: Ovarian (OV), uterine (UCEC), testicular (TGCT), papillary kidney (KIRP), cervical (CESC), thymus (THYM), mesothelioma (MESO), skin melanoma (SKCM), bile duct (CHOL), clear cell kidney (KIRC), thyroid (THCA), myeloid leukemia (LAML), rectum (READ), B-cell lymphoma (DLBC), uterine (UCS), adrenocortical (ACC), pheocromocytoma (PCPG) and uveal melanoma (UVM) malignancies. Unpaired two-tailed t-test was conducted to evaluate the statistical differences in log-normalized read counts of RNF144B between tissues or cancer types. In order to facilitate visual comparison across TCGA datasets with wild type or mutant
Tp53, the expression of RNF144B was mean centered to zero prior to plotting.
GDC-TCGA Pancan datasets were analyzed with
www.xenabrowser.net for Kaplan Meyer analysis. Samples were stratified by
Tp53 status, and by the gene expression levels of RNF144B (being high expression those samples with normalized expression values equal or above the median value and low expression the lower half). Samples containing null data were excluded. Kaplan Meier plots for 10-year overall survival were plotted for remaining samples. Comparison between groups was evaluated with a log rank test.
Analysis of RNF144B as a
Tp53 target gene in different mouse and human databases was performed using the TargetGeneRegulation database [
57].
CERES effect
RNF144B dependencies in human lung cancer cell lines were analyzed using the Achilles DepMap dataset (DepMap Public 22Q4 + Score, Chronos) [
58]. Cell lines were categorized in
Tp53 mutant or
Tp53 wild type and the CERES dependency score was plotted for each of them.
ChIP-sequencing data analysis
To perform the analysis of ChIP-seq data, the FASTQ files were acquired from the Sequence Read Archive (SRA) within the Gene Expression Omnibus (GEO) public repository. The specific accession numbers GSE71175 [
59] and GSE55727 [
60] were utilized to retrieve the FASTQ files corresponding to mouse and human cells, respectively. We used the nf-core/chipseq pipeline (
v. 1.2.2) [
48,
49]. FASTQ reads were aligned to the GRCm38.p6 reference genome. MACS2 [
61] was used to call peaks in the narrowPeak mode. Peaks with a q-value < 10e-5 were considered statistically significant. The resulting data was visualized using the Gviz R package [
62].
Aneuploidy scores analysis
For the assessment of aneuploidy scores, we utilized the gene expression dataset from the TCGA Pan-Cancer (PANCAN) cohort. Aneuploidy scores were directly obtained from [
63]. RNF144B log-normalized read counts were stratified in high and low expression using the median as cut-off. Samples were also stratified by TP53 status, following the criteria used in TCGA Pan-Cancer dataset. The term "PANCAN" denotes the analysis across all cancer types together.
GSEA analysis
GSEA [
64] was carried out in R using the fgsea package v1.24.0 [
65], using as a gene set the list of 70 genes (CIN70) with the highest levels of consistent correlation with ‘total functional aneuploidy’ (tFA) from [
66]. The C2 curated collection from the Molecular Signatures Database (MSigB) portal was associated with the CIN70 signature. GSEA was used to test for enrichment of specific gene sets within a ranked list based on
p-value and log
2FC to define whether the chromosomal instability profile is enriched among the overexpressed proteins of our analysis.
Discussion
TP53, a crucial tumour suppressor, regulates diverse cellular processes, including apoptotic cell death, cell cycle arrest and senescence, giving rise to distinct mechanisms proposed for TP53-mediated tumor suppression in different contexts [
8‐
10,
13]. Through an in vivo genetic screening in hematopoietic stem/progenitor cells, RING Finger Protein RNF144B was identified as a critical factor contributing to TRP53-mediated tumor suppression, in the context of blood cancers [
23]. We focused on better understanding RNF144B, which has been a poorly characterized tumor suppressor identified as one of the most significant hits in the screen. Here we show that RNF144B deficiency enhances growth of epithelial non-transformed and tumour derived cells, in particular lung cells, and its enforced expression is capable of inhibiting lung cancer cell proliferation driven by TP53 loss. The growth-suppressive role for RNF144B in human LUAD cancers, particularly when TP53 is intact, has been further emphasized by our human cancer genome analysis, that indicate tight correlation between TP53 status, RNF144B expression, and the prognosis of the LUAD patients. This highlights the pivotal role of RNF144B in cancer cell proliferation and transformation across various contexts.
Previous studies have shown that RNF144B is an E3-ubiquitin ligase enzyme [
27‐
29] and is therefore involved in the proteasomal degradation of its targets. Here, to understand RNF144B function we employed a proteomic and RNA-sequencing analysis. Notably, we have found that RNF144B, via its putative ubiquitin ligase activity, could regulate proteins essential for cellular processes involved in preserving genomic stability, mitotic progression, and DNA damage. The targets identified include a known RNF144B target, p21, that prompts cell cycle arrest [
34,
78], TPX2 microtubule nucleation factor required for normal assembly of mitotic spindles [
79], TOP2A, a DNA topoisomerase that is required for mitotic chromosome condensation and segregation [
80] and RAD21, a cohesin complex protein that regulates sister chromatid cohesion and separation [
81]. Notably, we observed elevated TRP53 protein levels in non-transformed RNF144B deficient cells MEFs and to lesser extent in 3KT cells, while such elevation was not detected in A549 LUAD cancer cells with abrogated RNF144B. This suggests that TP53 regulation is likely independent of RNF144B or may occur in a tissue- or stage-specific manner. However, it is clear that the ubiquitin–proteasome pathway is one of the main factors in p53 regulation during tumor development [
82,
83]. The multiple layers of negative and positive regulation governing TP53 presents challenges to understand the pathways crucial for regulating TP53 stability. Consequently, further studies are needed to validate whether RNF144B modulates TP53 expression through its E3-ligase activity in lung adenocarcinomas.
Consistent with a notion that RNF144B has a role in DNA repair [
23], we have shown increased DNA double strand breaks in response to γ-IR in RNF144B-deficient cells, suggesting a potential deficiency in DNA repair. Beyond contributing to the DNA double strand break repair, RNF144B deficiency induced chromosomal instability, a clear hallmark of aggressive and refractory cancers [
66,
84,
85]. RNF144B inactivation leads to substantial abnormalities during cell division, such as the presence of lagging chromosomes, DNA bridges and micronuclei, further emphasizing its significance in maintaining genomic integrity. While the precise mechanism by which RNF144B contributes to genomic stability maintenance remains to be fully elucidated, our study shows that cells lacking RNF144B have a higher proportion of cycling tetraploid cells, suggesting that the tetraploidy checkpoint could be partially abrogated.
Tetraploidy followed by aneuploidy is a frequent occurrence in
Tp53 mutant cancers [
26,
73,
86‐
90]. Here we showed that cells with low RNF144B that present increased ploidy, DNA damage and chromosomal aberrations can survive and progress even in the presence of wild-type
Tp53. Furthermore, analysis of the aneuploidy score indicated that lower levels of RNF144B mRNA correlated with chromosomal abnormalities in LUAD patients with wild-type TP53. This supports previous findings suggesting that TP53 may not be fully essential for maintaining a correct ploidy, and whole-genome doubling can occur even in the presence of functional TP53 [
91‐
93]. In most of the cases reported, poor checkpoint regulation due to overexpression of specific cyclins or spindle assembly factors were the cause for the appearance of mitotic slippage and subsequent tetraploidization [
91,
93‐
96]. Our results show that RNF144B could be involved in regulating the levels of several cell cycle and spindle assembly proteins, including TPX2 [
79], MAP4 [
97], BCCIP [
98], CCNB2 [
99], RAD21 [
81] and KIF4 [
100].
Our results provide preclinical evidence that dysregulation of DNA repair and mitotic fidelity caused by RNF144B deficiency enables lung cancer cells to evade the cytotoxic effects of drugs that cause CIN, DNA damage (through topoisomerase II inhibition) or mitotic alterations. Numerous reports suggest that the cellular response to cytotoxic agents is influenced by TP53 [
77]. However, our findings indicate that TP53 alone may not be the sole determinant, as RNF144B deficient 3KT and A549 lung oncogenic expressing cells exhibited similar responses to cytotoxic agents, but variable levels of TP53. Thus, resistance to drugs that cause DNA damage or CIN likely arises from other factors that contribute to tolerance to DNA damage and genomic instability, such as improper cell cycle checkpoint control. However, further studies are needed to elucidate these mechanisms. This finding may have important implications for clinical practice, as the levels of RNF144B could serve as a prognostic marker and potentially identify patients who will not benefit from DNA damage or CIN-inducing therapies, specifically in
Tp53 wild-type LUAD cancers.
In summary, we show that RNF144B limits chromosomal instability and enables DNA damage response in the context of oncogene expressing cells. These multifaceted functions of RNF144B contribute significantly in maintaining genomic integrity. Importantly, RNF144B deficiency in lung adenocarcinoma cells induces their resistance to DNA damage, CIN or cell cycle based anti-cancer therapies.
Limitations
The precise molecular mechanism and target genes involved in RNF144B-mediated maintenance of genomic stability and tumor prevention are yet to be determined. Assessment of the direct causative relationships between RNF144B and TP53 in regulating genomic stability are not defined. An additional constraint of our study was the lack of specificity of available anti-RNF144B antibodies.
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