Department of Molecular Biology
Doctoral thesis:
Alterations of FADD expression and phosphorylation in T-cell lymphoblastic lymphoma
José Luis Marín Rubio
Madrid, 2019
Department of Molecular Biology
Thesis dissertation submitted for the degree of Doctor of Philosophy:
Alterations of FADD expression and phosphorylation in T-cell lymphoblastic lymphoma
José Luis Marín Rubio, Bachelor of Science in Biology
Thesis supervisors:
María Villa-Morales, PhD Prof. José Fernández-Piqueras, PhD
Department of Cell Biology and Immunology ‒ Molecular Biology Research Center “Severo Ochoa”.
Genetics Unit of the Department of Biology ‒ Autonomous University of Madrid.
department at the Autonomous University of Madrid,
CERTIFY:
that D. José Luis Marín Rubio, Bachelor of Science in Biology by the Autonomous University of Madrid, has performed under our direction this Doctoral Thesis entitled “Alterations of FADD expression and phosphorylation in T-cell lymphoblastic lymphoma” to qualify for the degree of Doctor in the Department of Molecular Biology at the Autonomous University of Madrid.
Madrid, 10th December 2018
Signature, Signature,
José Fernández-Piqueras María Villa-Morales
and Immunology of the Molecular Biology Research Center “Severo Ochoa” (CBMSO- CSIC/UAM) and in the Genetics Unit of the Department of Biology at the Autonomous University of Madrid, under the supervision of María Villa Morales, PhD and Prof. José Fernández Piqueras, PhD.
Thesis research was supported by a predoctoral fellowship from Spanish Ministry of Education, Culture and Sports (FPU13/00338), as well as several moblity fellowships from the Journal of Cell Science (JCSTF-161105), the Federation of European Biochemical Societies (FEBS), the European Association for Cancer Research (EACR) and the Erasmus+
KA103.
This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (SAF2012-36566 and SAF2015-70561 MINECO/FEDER, UE; BES-2013- 065740), the Autonomous Community of Madrid, Spain (Oncocycle S2011/BMD-2470 and B2017/BMD-3778; LINFOMAS-CM), the Spanish Association Against Cancer (AECC, 2018; PROYE1805PIRI) and the Carlos III Health Instutite (ISCIII) (ACCI-CIBERER-17).
Institutional grants from the Fundación Ramón Areces and Banco de Santander to the CBMSO are also acknowledged.
BEN OKRI
ABSTRACT
T-cell lymphoblastic neoplasms are aggressive haematological malignancies which mainly develop in children but can also affect adults. The molecular basis of T-cell lymphoblastic lymphoma is not fully understood. In consequence, there is a lack of robust and reliable prognostic markers, thus hindering the achievement of better results in the clinic. The role of FADD in cancer is controversial, probably due to its dual role in apoptosis and other non-apoptotic functions such as proliferation and cell cycle control. Moreover, it seems that these non-canonical functions depend on FADD phosphorylation and subcellular location. Despite FADD having a relevant role in T cells, alterations affecting its expression and phosphorylation are not completely unveiled in haematological cancers. In the present work, we show that T-cell lymphoblastic lymphoma cells exhibit a significant reduction of FADD, both in mouse and human. The proteomic approach indicates that FADD reduction in tumour T cells impacts on mitochondrial processes as well as in the regulation of messenger RNA processing and maturation. However, apoptosis impairment due to FADD reduction is probably the main FADD-mediated oncogenic event in T-LBL tumour cells. Nevertheless, the phosphorylation status of the remaining FADD protein is capable of shaping its non-apoptotic functions.
Phosphorylated FADD is significantly reduced as well in T-cell lymphoblastic lymphoma. Even though, phosphorylated FADD is more stable and more prone to be nuclear. There, it would be prevented from participating in death receptors- mediated apoptosis and it would favour tumour cell proliferation. Interestingly, we have determined that the level of FADD phosphorylation can provide the basis for patient stratification. Higher levels of phosphorylated FADD in the tumour significantly correlate with shorter survival times in mice and with worse clinical prognosis in human patients. This supports that FADD phosphorylation status may serve as a predictor for aggressiveness and tumour outcome. In summary, we propose FADD phosphorylation as a new biomarker with prognostic value in T-cell lymphoblastic lymphoma.
Las neoplasias linfoblásticas de células T son neoplasias hematológicas agresivas que se desarrollan principalmente en niños pero también pueden afectar a adultos.
La base molecular del linfoma linfoblástico de células T no es del todo conocida.
Por ello, carecemos de marcadores de pronóstico robustos y fiables, lo cual impide alcanzar mejores resultados en la clínica. El papel de FADD en cáncer es controvertido, probablemente debido a su función dual en la apoptosis y en funciones no apoptóticas como la proliferación o el control del ciclo celular. Además, parece que estas funciones no canónicas dependen de la fosforilación y la localización subcelular de FADD.
Aunque FADD tiene un papel importante en las células T, las alteraciones que afectan a su expresión y su fosforilación no se han desvelado completamente en los tumores hematológicos. En este trabajo, mostramos que los linfomas linfoblásticos de células T presentan una reducción significativa de FADD, tanto en tumores humanos como de ratón. Nuestra aproximación proteómica indica que la reducción de FADD en células T tumorales tiene impacto sobre procesos mitocondriales, así como sobre la regulación del procesamiento y la maduración del ARN mensajero. Sin embargo, la disfunción de la apoptosis es probablemente el principal evento oncogénico mediado por FADD en la célula tumoral. Por otro lado, el estatus de fosforilación de la proteína FADD que queda en la célula tumoral es capaz de modular sus funciones no apoptóticas.
Encontramos que la forma fosforilada de FADD también está significativamente reducida en el linfoma linfoblástico de células T. Aunque, la forma fosforilada es más estable y con mayor tendencia a localizarse en el núcleo, donde quedaría apartada de su participación en la apoptosis mediada por receptores de muerte y además favorecería la proliferación de la célula tumoral. Observamos con gran interés que el nivel de fosforilación de FADD sirve de base para la estratificación de los pacientes de linfoma linfoblástico de células T. Los niveles más altos de FADD fosforilado en el tumor se relacionan con tiempos de supervivencia significativamente más cortos en ratones y con peor pronóstico clínico en el ser humano. Estos hechos apoyan que el estatus de fosforilación de FADD puede servir para predecir la agresividad y la resolución del tumor. En resumen, proponemos la fosforilación de FADD como un nuevo biomarcador con valor pronóstico en el linfoma linfoblástico de células T.
INDEX
ABBREVIATIONS ...21
INTRODUCTION...27
1. T-cell lymphoblastic lymphoma. ...29
2. The role of apoptosis in the immune system: Fas/Fas ligand system. ...32
3. FADD gene and its regulation. ...33
4. FADD protein and its regulation. ...34
5. FADD in cell death. ...35
6. Phosphorylation of FADD and its regulation. ...37
7. Subcellular localization of FADD. ...38
8. Non-apoptotic functions of FADD. ...39
8.1. FADD in haematopoiesis and thymopoiesis. ...40
8.2. FADD in proliferation. ...41
8.3. FADD in cell cycle control. ...41
8.4. FADD in metabolism. ...42
9. Prognostic value of FADD: chemoresistance and chemosensitivity. ...43
OBJECTIVES ...45
MATERIAL AND METHODS...49
1. Human samples. ...51
2. Murine samples. ...51
3. Cell lines. ...52
4. Lentiviral constructs and generation of stable cell lines. ...52
5. In vitro FADD reconstitution...53
6. Gene expression analysis. ...53
7. FADD and Fadd sequencing. ...54
8. Antibodies and reagents. ...55
9. Western blot (WB). ...55
10. Immunohistochemistry (IHC)/Immunocytochemistry (ICC)...56
11. Immunofluorescence (IF). ...57
12. Apoptosis assay. ...57
13. Cell proliferation assay. ...58
14. Cell cycle assay. ...58
15. Determination of protein stability...58
16. Stable isotope labelling using amino acids in cell culture (SILAC). ...59
17. Immunoprecipitation (IP) of endogenous FADD. ...59
18. Trypsin digestion. ...60
19. Mass spectrometry (MS) and data analysis. ...60
20. Proteomic analysis. ...62
21. In silico analysis of published microarray datasets. ...62
22. Gene set enrichment analysis (GSEA). ...62
23. Statistical analysis. ...63
RESULTS ...65
1. T-LBL samples exhibit a significant reduction of FADD and P-FADD. ...67
2. FADD phosphorylation regulators in T-LBL. ...72
3. T-LBL can be stratified depending on P-FADD levels. ...75
4. Influence of FADD phosphorylation in its subcellular location. ...77
5. Influence of FADD phosphorylation in its protein stability. ...80
6. Apoptosis is reduced in T-LBL samples. ...81
7. FADD presence and phosphorylation influence tumour T cell proliferation. .... 85
8. FADD expression and phosphorylation are involved in cell response to pharmacological cell cycle arrest. ...88
9. The clinical characteristics of T-LBL evidence important differences depending on FADD phosphorylation level. ...90
10. Proteomic analyses ...94
10.1. An increase of intrinsic apoptosis and energy metabolism are observed in FADD-deficient tumour T cells. ...94
10.3. Validation of endogenous FADD interactors. ...102
DISCUSSION ...103
1. The complex role of FADD in cancer. ...105
2. The phosphorylation of FADD is a signal that regulates its stability and location. ...106
3. Apoptosis is impaired in FADD-deficient T cells and this could trigger other forms of cell death. ...108
4. Proteomic approaches might unveil the non-apoptotic functions of FADD in T-cell lymphoblastic neoplasm. ...110
5. Phosphorylated FADD favours its non-apoptotic functions including proliferation and cell cycle control in T-LBL. ...114
6. The levels of phosphorylated FADD could be an indicator of aggressiveness and tumour prognosis in T-LBL. ...117
CONCLUSIONS ...121
REFERENCES ...125
APPENDIX I ...143
1. Supplementary figures. ...145
2. Supplementary tables. ...154
APPENDIX II ...161
1. Publications. ...163
ABBREVIATIONS
7-AAD 7-aminoactinomycin D
Ab Antibody
Ag Antigen
Ahr Aryl hydrocarbon receptor AML Acute myeloid leukaemia amu atomic mass unit
Ani Anisomycin
bp base pair
BSA Bovine serum albumin CASP3 Caspase-3
CD Cluster of differentiation Cdc Cell division-cycle protein CDK Cyclin-dependent kinase cDNA complementary DNA chr chromosome
CHX Cycloheximide
CI Confidence interval of ratio CKIα Casein Kinase I alpha CTD C-terminal domain CTRL Control
DAB 3,3’-diaminobenzidine
DAVID Database for annotation, visualization and integrated discovery
DD Death domain
DED Death effector domain
DIABLO Diablo IAP-binding mitochondrial protein DISC Death-inducing signalling complex DMEM Dulbecco’s modified eagle’s medium DMP Dimethylpimelimidate
DN Double negative
DNA Deoxyribonucleic acid
DP Double positive
DTT Dithiothreitol
DUSP26 Dual specificity phosphatase 26 ECL Enhanced chemiluminescence EDTA Ethylenediaminetetraacetic acid
e.g. exempli gratia
EP Electroporation
ERK Extracellular signal-regulated kinase
ES Enrichment score
ETP Early T-cell lineage progenitor FACS Fluorescence activated cell sorter FADD FAS-associated with death domain FBS Foetal bovine serum
FC Flow cytometry
FDR False discovery rate
G0 Gap 0 phase
G1 Gap 1 phase
G2 Gap 2 phase
G6pdx Glucose-6-phosphate dehydrogenase X GAPDH Glyceraldehyde-3-phosphate dehydrogenase GFP Green fluorescent protein
GO Gene ontology
GProX Graphical Proteomics Data Explorer GSEA Gene set enrichment analysis
H Heavy
HEK Human embryonic kidney
HEPES Hydroxyethyl-piperazine ethane-sulfonic acid HIPK3 Homeodomain Interacting Protein Kinase 3
Hprt1 Hypoxanthine-guanine phosphoribosyltransferase 1
HR Hazard ratio
hsa Homo sapiens
HSP90 Heat shock protein 90 ICC Immunocytochemistry IHC Immunohistochemistry IF Immunofluorescence IL Interleukin
IP Immunoprecipitation JNK c-Jun N-terminal kinase kb kilobase
KEGG Kyoto encyclopaedia of genes and genomes KO Knock-out
LFQ Label free quantification LTQ Linear trap quadrupole
M Mitosis phase
mms Mus musculus
MS Mass Spectrometry
mRNA messenger RNA miR-/miRNA microRNA n number
NEG Negative cell line
NES Nuclear export sequence NF-κB Nuclear factor kappa B
NLS Nuclear localization sequence NR Non-relapse
ns non significant P/p-value Probability value P-/Phospho Phosphorylation
PAGE Polyacrylamide gel electrophoresis PARP Poly(ADP-Ribose) Polymerase 1 PBS Phosphate-buffered saline PE Phycoerythrin
PCR Polymerase chain reaction PI Propidium iodide
PKCζ Protein kinase C zeta
PLAD Pre-ligand assembly domain PLK1 Polo-like kinase 1
PMSF Phenylmethylsulphonyl fluoride PPI Protein-protein interaction
Pr Progressive
PVDF Polyvinylidene fluoride
qPCR Quantitative polymerase chain reaction R2 Coefficient of determination
Re Recurrent
REVIGO Reduce + visualize gene ontology RIPA Radioimmunoprecipitation assay RNA Ribonucleic acid
RPMI Roswell Park Memorial Institute medium
RT Reverse transcription
RT-qPCR Reverse transcription quantitative polymerase chain reaction
S Synthesis phase
SDS Sodium dodecyl sulphate SEM Standard error of mean SF3B3 Splicing Factor 3b subunit 3
SILAC Stable isotope labelling using amino acids in cell culture snRNP small nuclear ribonucleoprotein
SP Single positive
SPSS Statistical package for the social science SRSF7 Serine and arginine Rich Splicing Factor 7
STRING Search tool for the retrieval of interacting genes/proteins T-ALL T-cell acute lymphoblastic leukaemia
TBS Tris-buffered saline TCR T-cell receptor
TdT Terminal deoxynucleotidyl transferase TNF Tumour necrosis factor
TNFR Tumour necrosis factor receptor T-LBL T-cell lymphoblastic lymphoma u unified atomic mass unit or Dalton μS micro-scan
UTR Untranslated region
v version
vs. versus
WB Western blot
WHO World Health Organization Wt Wild-type
INTRODUCTION
1. T-cell lymphoblastic lymphoma.
The development of T cells in the thymus is complex and occurs through a multistep process of proliferation, differentiation and apoptosis. The thymus is composed of thymocytes of haematopoietic origin and thymic epithelial cells of non-haematopoietic origin (Shortman et al. 1990). Ninety-eight percent of the thymocytes that develop in the thymus are eliminated by apoptosis (Pozzesi et al. 2014). Positive selection is a process mediated exclusively by the epithelial cells of the thymic cortex, which provide survival signals, rescuing thymocytes from apoptosis (Baumann et al. 2002). During the development of the thymus, immature thymocytes that generate non-functional or autoreactive antigenic receptors are eliminated by apoptosis through negative selection (Pozzesi et al.
2014) (Figure 1).
Capsule
Thymus Bone marrow
Cortex
Cortico- medullary junction Medulla capsularSub- zone
Lymphoid progenitor
Blood vessel ETP
DN2 DN3
DN4
DP
CD4+SP CD8+ SP
Pre-TCR
Cortical epithelial cell
Positive selection
Negative selection Dendritic
epithelial cell
CD4+SP CD8+ SP
Figure 1. Stages of normal T-cell development and T-cell lymphoblastic neoplasms.
Lymphoid progenitors enter the thymus at the cortico-medullary junction from the bone-marrow. These subsets migrate through the thymus (as early T-cell-lineage progenitors (ETPs)) and commit to the T-cell lineage, progressing through the double
T-cell lymphoblastic neoplasms are aggressive haematological malignancies consisting of small- to medium-sized blast cells, which mainly develop in children and adolescent males but can also affect adults (Sabattini et al. 2010). According to the World Health Organization (WHO) classification of lymphoid neoplasms, these neoplasms are sub-classified in T-cell acute lymphoblastic leukaemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL) (Sabattini et al. 2010; Arber et al. 2016). The first criterion to distinguish between these two manifestations is the level of blood or bone marrow affectation. Most commonly, an extensive bone marrow and blood affectation over 25% defines T-ALL. Less frequently, mass lesions in the thymus, anterior mediastinum or in lymph nodes together with less than 25% of blasts in the marrow or blood define T-LBL (de Leval et al.
2009). Thus, T-ALL and T-LBL appear as two entities of the same disease with different clinical presentations (Aifantis et al. 2008; Cortelazzo et al. 2017). Due to the similarity between clinical, morphological and immunophenotypic features, these two conditions are often treated in the same way (Aifantis et al. 2008;
Burkhardt 2010). However, molecular data indicate that T-ALL and T-LBL do not share identical oncogenic expression profiles, suggesting underlying differences in their biology (Raetz et al. 2006; Uyttebroeck et al. 2007; Basso et al. 2011;
Bonn et al. 2015).
The annual incidence rate of lymphoblastic neoplasms in Europe is of only 1.46 per 100,000 cases, which positions T-cell lymphoblastic neoplasms in the spectrum of rare diseases and rare cancers (RARECAREnet: http://www.rarecarenet.eu/).
Specifically, T-LBL comprises approximately 8% of all lymphomas, but 85-90% of all lymphoblastic lymphomas (Cortelazzo et al. 2017).
of differentiation based on the expression of CD44 and CD25 (DN1, CD44+CD25–; DN2, CD44+CD25+; DN3, CD44–CD25+; and DN4, CD44–CD25–). Upon successful recombination at the T-cell receptor β locus, pre-T cells acquire surface expression of the pre-TCR that promotes differentiation to the DN4 stage. Pre-TCR-selected cells reach the double positive (DP; CD4+CD8+) stage, at which point they are subjected to the processes of positive and negative selection. Single-positive (SP) CD4+ and CD8+ T cells that survive negative selection leave the thymus and populate peripheral lymphoid organs. Modified from Aifantis et al. 2008 and Belver and Ferrando 2016.
Lymphadenopathy and hepatosplenomegaly are common in T-cell lymphoblastic neoplasms. Skin, tonsil, liver, spleen, central nervous system and testis in males may be also involved, although presentation at these sites without nodal or mediastinal involvement is uncommon (Sabattini et al. 2010).
T-cell lymphoblastic neoplasms can be stratified into different stages according to the expression of T-cell receptor (TCR) components and of other antigens reflecting the immunophenotypic properties from lymphoid progenitor cells arrested at early stages of T-cell maturation (Coustan-Smith et al. 2009; de Leval et al. 2009; Sabattini et al. 2010; Belver and Ferrando 2016; Cortelazzo et al. 2017) (Table 1). Lymphoblasts in T-ALL/LBL usually express nuclear terminal deoxynucleotidyl transferase (TdT) and have a high mitotic activity (de Leval et al.
2009). Various genetic aberrations are observed in T-ALL/LBL. The most common are translocations involving TCR loci, fusion genes, deletions, gene duplications and mutations. Abnormal karyotype is detected in approximately 50% of the cases (de Leval et al. 2009; Cortelazzo et al. 2017).
Table 1. Categories of T-LBL/ALL according to their immunophenotype.
CD1a CD2 CD3 CD4 CD5 CD7 CD8 CD34 ETP
Early or Pro-T Pre-T Cortical-T Medullary-T
- Negative + Positive +/-Intermediate
- - - - +
- - - + + -
- +/-
+ + + + + + -
+ + +
+ + + + +
+/- +/- +/- - - +/-
The expression pattern of specific markers is show for ETP (early-T-cell precursor), early or pro-, pre-, cortical- and medullary-T-LBL/ALL. (-) negative expression, (+) positive expression and (+/-) intermediate expression.
The standard therapeutic option for patients with T-LBL was designed for T-ALL and is based on intensive multi-drug chemotherapy protocols. The regimens contain 7 to 10 drug combinations and they are often accompanied
2. The role of apoptosis in the immune system: Fas/Fas ligand system.
system metastasis (Cortelazzo et al. 2017; Lepretre et al. 2017). The prognosis in all groups has dramatically improved in the last decades, with cure rates of approximately 75-85% (Bonn et al. 2013). Still, treatment failure occurs in around 25% of patients and the outcome for this group remains dismal (de Leval et al.
2009). Thus, from a clinical point of view, these treatments exhibit two main weaknesses. First, acute and long-term toxicity associated to the treatments, which sometimes leads to discontinuation; and second, a terrible outcome at relapse, with survival rates inferior to 10% (Burkhardt 2010; Cortelazzo et al.
2017). Both reasons argue for the urgent need to identify new prognostic markers allowing the therapeutic stratification of patients. Moreover, the ultimate goal is to offer personalized treatment for each patient. The achievement of these objectives requires a better understanding of T-LBL.
The molecular basis of these neoplasms has been well established in T-ALL (Aifantis et al. 2008), but to a lesser extent in T-LBL (Burkhardt 2010; Bonn et al. 2013), mainly due to the scarcity of adequate tumour samples for research (Burkhardt 2010). No reliable prognostic markers are currently available for T-LBL, in contrast to what happens with T-ALL (Cortelazzo et al. 2017). Therefore, the main difficulty in the treatment of T-LBL patients is the lack of prognostic parameters.
Apoptosis plays a prominent role during embryogenesis, development and homeostasis of the immune system (Drew et al. 1998; Krammer 2000). Many diseases are associated with an excess or an insufficiency in apoptosis, such as cancer, acquired immune deficiency syndrome and autoimmune diseases (Krammer 2000).
The signs of death in vertebrates are transduced through two central pathways:
extrinsic and intrinsic pathways. In the extrinsic pathway, the activation of death receptors at the cell membrane stimulates intracellularly the formation of the death-inducing signalling complex (DISC), within which the self-proteolysis of
regulatory pro-caspases-8 and -10 is induced, to eventually activate the effector caspase-3. In the intrinsic or mitochondrial pathway, the translocation of pro- apoptotic Bcl-2 proteins, such as Bax, triggers the release of cytochrome c from the mitochondria, which stimulates the activation of Apaf-1 dependent on pro- caspase-9 in the apoptosome and finally converges with the extrinsic pathway in caspase-3 (Tourneur et al. 2003).
Death receptors belonging to the tumour necrosis factor (TNF) receptors superfamily have an important role in immune regulation. They transmit the apoptosis initiating signals by their specific death ligands and are characterized by an intracellular death domain (DD) that serves to recruit adapter proteins such as Fas-associated with death domain protein (FADD) or TNFR1-associated death domain protein (TRADD) (Hsu et al. 1995; Kischkel et al. 1995). Twenty- seven members of TNF receptors superfamily have been described (Locksley et al. 2001). Among these, the best characterized is Fas, which has a crucial role in the development, maturation and elimination of T and B cells, in the cytotoxicity mediated by immune cells and in the regulation of the immune response (Curtin and Cotter 2003; Zhang et al. 2004). The progression and metastasis of tumours is associated with resistance to Fas-mediated apoptosis (Curtin and Cotter 2003).
Fas/Fas ligand system is frequently deregulated in T-LBL and this may occur at different levels of the pathway (Villa-Morales et al. 2006; Villa-Morales et al. 2007;
Villa-Morales et al. 2010; Villa-Morales and Fernandez-Piqueras 2012; Villa-Morales et al. 2014). Thus, alterations in FADD may be another frequent mechanism whereby Fas signalling would be impaired in T-LBL (Villa-Morales et al. 2014).
3. FADD gene and its regulation.
The human FADD gene is located in the chromosomal region 11q13.3, whereas in mice it is located in chromosome 7. It is composed of two exons separated by an intron (Kim et al. 1996). The 5’ flanking region of FADD has 1 kb containing consensus sequences of regulatory elements such as AP-1, SP-1, Lyf-1, N-Myc,
4. FADD protein and its regulation.
start site (Kim et al. 1996). Other transcription regulators have been described as direct regulators of FADD expression, such as Pax2, Vax2 (Viringipurampeer et al.
2012) or BRCA1 (Nguyen et al. 2018).
The 11q13.3 locus, which contains 13 genes including FADD (Gibcus et al.
2007), is a region that undergo chromosomal amplification in various proliferative disorders such as breast, bladder, oesophagus, lung, head and neck cancer (Kim et al. 1996; Bhojani et al. 2005; Chen et al. 2005; Prapinjumrune et al. 2010;
Eytan et al. 2016).
Few other genetic alterations have been described in FADD (Maki et al. 2013;
Eun et al. 2014), but mutations in this gene have been suggested to play a role in tumorigenesis or autoimmunity (Chinnaiyan et al. 1995; Yeh et al. 1998).
Multiple microRNAs have been described as direct regulators of FADD expression, being miR-155 the most reported (Wang et al. 2011). Its deregulation has been correlated with lower survival in prostate cancer, breast cancer and acute myeloid leukaemia (Palma et al. 2014). Other microRNAs reported as direct or indirect regulators of FADD expression are: miR-17-5p (Wu et al. 2016), miR- 103/107 (Wang et al. 2015), miR-128 (Mi et al. 2007; Yamada et al. 2014), miR- 146a (Curtale et al. 2010; Sandhu et al. 2014) or miR-675 (Yan et al. 2017).
The FADD protein contains 208 amino acids in human and 205 amino acids in mouse; both sequences share 68% identity and 80% global similarity of amino acids (Zhang and Winoto 1996). Conservation is particularly high in two regions, the death domain (DD) in C-terminal and the death effector domain (DED) in N-terminal, both involved in homotypic protein-protein interactions (Zhang et al.
2004) (Figure 2). Seven proteins containing DED domains have been described:
FADD, caspase-8 and -10, cFLICE, DEDD, DEDD2 and PEA-15 (Valmiki and Ramos 2009).
In some tumour types, the levels of FADD mRNA are normal but the protein is low. This suggests that additional mechanisms may reduce the amount of FADD protein (Tourneur et al. 2005). The discrepancy between genetic alterations and protein expression suggests that FADD can be regulated by other mechanisms such as post-translational modifications affecting the stability of the protein (Chien et al. 2016). FADD undergoes ubiquitylation at K48 by MKRN1, which can induce its degradation (Feltham and Silke 2017). However, it cannot be ruled out that other E3 ligases could also ubiquitylate FADD (Lee et al. 2012). It has been also reported that some microRNAs can alter the amount of FADD protein, such as miR-149-5p (Tian and Yan 2016), miR-29a (Tiao et al. 2014) and miR-15a/16 (Cao et al. 2016); this would eventually deregulate the functions of FADD.
Death Effector Domain
MDPFLVLLHSVSSSLSSSELTELKFLCLGRVGKRKLERVQSGLDLFSMLLEQNDLEPGHT ...L.G...GND.M...RE..S...TV...R...
ELLRELLASLRRHDLLRRVDDFEAGAAAGAAPGEEDLCAAFNVICDNVGKDWRRLARQLK G...Q.L...T.TA.P...A..QV..DIV...R..K....E..
VSDTKIDSIEDRYPRNLTERVRESLRIWKNTEKENATVAHLVGALRSCQMNLVADLVQEV ..EA.M.G..EK...S.S...KV...A..K..S..G..K...T.RL...E.A QQARDLQNRSGAMSPMSWNSDASTSEAS
.ES---VSK.EN...VLRD.TV.S..TP
Death Domain
NES NLS
*
1 1 61 61 121121 181181
6060
120 120 180 180 208205
Human FADD Mouse FADD Human FADD Mouse FADD Human FADD Mouse FADD Human FADD Mouse FADD
Figure 2. Alignment between the primary sequences of mouse and human FADD proteins. The death effector domain (DED) and death domain (DD) domains are indicated in green and blue, respectively. Nuclear localization (NLS) and nuclear export signals (NES) are indicated in boxes. (*) Phosphorylation site at S194/191, in human and mouse, respectively. (·) Identity in the amino acid.
5. FADD in cell death.
Fas receptor oligomerization at the cell membrane occurs sequentially, first as a ligand-independent pre-association of three receptor molecules by their pre- ligand assembly domain (PLAD), then more tightly forming microaggregates upon binding of Fas ligand. This provokes a configuration change in Fas death domain that induces recruitment of FADD by homotypic interaction of DDs by electrostatic forces (Boldin et al. 1995; Chinnaiyan et al. 1995; Kischkel et al. 1995; Jeong et al.
DEDs by electrostatic forces (Boldin et al. 1995; Fernandes-Alnemri et al. 1996;
Muzio et al. 1996; Jeong et al. 1999). These interactions are required for the formation of the DISC, where procaspases are activated and lead to effector caspase 3 activation and to subsequent apoptotic events (Kischkel et al. 1995;
Muzio et al. 1996) (Figure 3).
FASL FAS
FADD
CASPASE
DISC
APOPTOSIS
Extracellular
Cytoplasm
Although the features and outcome of necrosis and apoptosis are very different, FADD can initiate both types of cell death. Necroptosis is a form of regulated necrosis that can be activated by ligands of death receptors and stimuli that induce the expression of ligands under conditions of apoptosis deficiency.
Necroptosis is suppressed by apoptosis mediated by the FADD-Caspase-8 axis Figure 3. Fas-signalling pathway. The activated Fas receptor recruits the adapter molecule FADD by homotypic interaction through their death domains (DD). FADD recruits the initiator pro-caspases 8/10 by the death effector domain (DED) to form the death-inducing signalling complex (DISC). Pro-caspases 8/10 are cleaved in the DISC to give rise to active caspase-8/10, which proceed with the activation of the caspase cascade and apoptotic events.
6. Phosphorylation of FADD and its regulation.
(Zhou and Yuan 2014) and FADD deficiency activates necroptosis in the cell (Viringipurampeer et al. 2012). Moreover, it induces proliferative defects in the cell and can contribute to inflammatory diseases (Ma et al. 2004).
The regulation of protein activity by phosphorylation is a common mechanism used in a variety of signal transduction pathways. FADD contains two serine clusters, one located at the N-terminus and the other at the C-terminal region.
However, FADD is phosphorylated in the C-terminal region (Scaffidi et al. 2000), which has been proposed as a third functional domain, the C-terminal domain (CTD), since it contains an important phosphorylation site (Scaffidi et al. 2000;
Barnhart et al. 2003) that appears to be key for a role of FADD in the regulation of cell growth and proliferation (Hua et al. 2003). FADD is phosphorylated specifically in a single serine residue, serine 194 in human and serine 191 in mouse, within a conserved methionine-serine-proline motif (Figure 2), suggesting that the site and function of this specific phosphorylation site is relevant and thus conserved between both species (Zhang and Winoto 1996; Scaffidi et al. 2000; Zhang et al.
2004; Bhojani et al. 2005).
To date, whether FADD phosphorylation is relevant for apoptosis or not remains a controversial issue. Most authors defend that it does not affect the transmission of the apoptotic signal (Hua et al. 2003; Chen et al. 2005; Sandu et al. 2006; Yao et al. 2015), but the opposite has been also indicated (Oh and Malter 2013).
FADD phosphorylation seems to depend on the phase of the cell cycle, being high in G2/M and low in G1/S (Alappat et al. 2005). This fact is in principle irrelevant for apoptosis, since both phosphorylated and unphosphorylated FADD can interact with Fas (Kischkel et al. 1995; Zhang and Winoto 1996; Scaffidi et al.
2000; Hua et al. 2003). Therefore, phosphorylation of FADD in serine 194 would be key for functions distinct from apoptosis (Chen et al. 2005).
7. Subcellular localization of FADD.
Different kinases have been proposed as putative regulators of FADD phosphorylation: HIPK3 (Rochat-Steiner et al. 2000; Curtin and Cotter 2004; Khan et al. 2010), CKIα (Scaffidi et al. 2000; Alappat et al. 2005; Osborn et al. 2007;
Khan et al. 2010; Papoff et al. 2010; Drakos et al. 2011; Bowman et al. 2015), PKCζ (de Thonel et al. 2001), PLK1 (Jang et al. 2011a; Jang et al. 2011b), kinases that regulate the cell cycle (Scaffidi et al. 2000; Zhang et al. 2001) and others (Kennedy and Budd 1998; Meng et al. 2003; Vilmont et al. 2015). However, it is unknown whether their action can be tissue- or cell type-specific.
The cellular equilibrium of phosphorylation is regulated through the action of kinases and phosphatases. The AK2/DUSP26 complex has been implicated in FADD dephosphorylation, apparently through DUSP26 phosphatase activity with independence of AK2 kinase activity (Kim et al. 2014).
In summary, the phosphatase(s) or kinase(s) responsible for FADD status of phosphorylation have not been well identified. Therefore, further studies are necessary to identify them and to characterize the functional effects of such regulation.
FADD is primarily known for its canonical function as a cytoplasmic adapter protein between death receptors and initiator caspases. Therefore, it is assumed that FADD is primarily a cytoplasmic protein. However, it has been described that FADD also presents a nuclear location.
Macromolecules that are unable to pass through nuclear pore complexes by diffusion are transported in or out of the nucleus in an active way mediated by nuclear location (NLS) and by nuclear export signals (NES) (Yoshida and Blobel 2001). FADD presents NLS and NES in its DED domain (Gomez-Angelats and Cidlowski 2003) (Figure 2), which are highly conserved among several species (Zhang et al. 2004). In addition to the NLS and NES motifs, it was also revealed that the accumulation of FADD in the nucleus and export to the cytoplasm implies
8. Non-apoptotic functions of FADD.
phosphorylation of the Ser194 site (Screaton et al. 2003; Ramos-Miguel et al.
2011).
The nuclear location of FADD seems to depend on cell type. It has been observed in several cell lines, as well as in different healthy and tumour tissues, but not in every cell type (Gomez-Angelats and Cidlowski 2003; Screaton et al.
2003; Frisch 2004; O’Reilly et al. 2004; Bhojani et al. 2005; Yoo et al. 2007; Khan et al. 2010; Prapinjumrune et al. 2010; Tourneur and Chiocchia 2010). It has been reported that FADD translocates into the nucleus during mitosis and promotes cell progression, implying that FADD has an apoptotic or non-apoptotic role depending on its subcellular location (Alappat et al. 2005; Cheng et al. 2014).
To get further insight into how FADD subcellular location is regulated will help to solve pending questions about the functions executed by FADD.
Although the best known and possibly the most important role of FADD is to be an adaptor in apoptosis, there is ample evidence about a role of FADD in multiple non-apoptotic functions (Figure 4). These non-apoptotic functions seem to depend on the location and phosphorylation status of FADD (Tourneur et al. 2005). Thus, changes in FADD expression and phosphorylation in cancer are specific to the cell type and can result in a loss of apoptosis or in a gain of non- apoptotic functions, stimulating tumour growth.
Figure 4. Functions of FADD. FADD has been implicated in cell death and many other non-apoptotic functions.
Apoptosis Autophagy
Necroptosis
Inflammation Nucleocytoplasmic
trafficking
Embryo development
Proliferation
Cell cycle progression
Genome surveillance
Survival Metabolism
Innate
immunity
FADD
8.1. FADD in haematopoiesis and thymopoiesis.
FADD plays a role in mediating the activation, proliferation and development of T cells (Walsh et al. 1998; Kabra et al. 2001; Zhang et al. 2001; Hua et al. 2003).
FADD-deficient bone marrow exhibits defects in the generation and maintenance of lymphoid, myeloid and erythroid cells (Rosenberg et al. 2011). FADD would be essential at early stages of haematopoiesis, but its role would be secondary in later stages of differentiation (Rosenberg et al. 2011; Zhang et al. 2011). Nevertheless, thymopoiesis is partially defective in FADD-mutant mice (Beisner et al. 2003).
Pre-TCR-induced signal transduction leads to proliferation of DN cells (CD4–CD8–) and their subsequent differentiation to DP cells (CD4+CD8+) in the thymus. This transition is a critical proliferative stage during early development of T cells (Kabra et al. 2001; Zhang et al. 2005). Signalling through FADD is critical for regulating apoptosis of those T-cell progenitors that do not overcome this checkpoint, but moreover, FADD also participates in the efficient proliferation of those cells that are successful at it. Thus, it has been proposed that FADD represents a tumour suppressor with positive and negative effects on cell growth (Newton et al. 2000).
FADD also presents a role as a mediator of proliferation during DN3 (CD44–CD25+) to pre-T stage transition in the thymus, although this role would be dispensable (Newton et al. 2000).
In summary, it has been suggested that FADD-expressing T cells have a survival advantage over FADD-deficient T cells (Kabra et al. 2001).
8.2. FADD in proliferation.
The absence of FADD blocks apoptosis, but it also inhibits T cell proliferation (Newton et al. 2000; Newton and Strasser 2000; Hua et al. 2003; Li et al. 2017).
It has been described that FADD phosphorylation is higher in proliferating than in non-proliferating cells, indicating that phosphorylation of FADD may be a marker of proliferation (Drakos et al. 2011). Therefore, FADD phosphorylation may be important for its function as a mediator of proliferation in T cells (Kabra et al.
2001).
8.3. FADD in cell cycle control.
FADD-deficient T cells not only show alterations in proliferation, but also in cell cycle progression (Papoff et al. 2010). The lack of FADD in the nuclei of T cells in the thymus, glands and tonsils associates to lower numbers of T cells in G2/M phase of the cell cycle (Patel et al. 2014). FADD-deficient T cells are able to enter the cell cycle by mitogenic stimulation, but they do so more slowly and die during cell cycle progression; therefore, FADD deficiency prevents the survival of dividing T cells (Beisner et al. 2003; Osborn et al. 2007; Tourneur and Chiocchia 2010).
FADD ability to promote T cell progression through the cell cycle seems to depend on its phosphorylation status (Hua et al. 2003; Screaton et al. 2003).
Human FADD phosphorylation at serine 194 and its murine equivalent at serine 191 are key for entrance and progression of cell cycle in T cells (Hueber et al.
2000; Zhang et al. 2001). FADD phosphorylation in T cells would be biphasic, with
8.4. FADD in metabolism.
second event of phosphorylation at G2/M (Osborn et al. 2007). Taken together, these data suggest that FADD phosphorylation status is important for G1/S and G2/M transitions (Osborn et al. 2007). Several kinases have been described to interact with phosphorylated FADD at the G2/M transition, such as PLK1, CKIα, AURKA and BUB1 (Bowman et al. 2015).
The exact role of FADD phosphorylation during cell cycle progression is unknown. To get further insight into this non-apoptotic function of FADD, it is necessary to identify the regulatory elements and the interactions that occur with FADD and its phosphorylated form in normal and tumour cells.
FADD and particularly its phosphorylated form also have an important function in the regulation of metabolic disorders (Yao et al. 2013; Zhang et al.
2014b; Yao et al. 2015). It has been described that FADD deficiency entails the deregulation of proteins involved in energy metabolism (glycolysis, fatty acid metabolism, β-oxidation of fatty acids, the Krebs cycle, oxidative phosphorylation and mitochondrial activity) and proteolysis (Zhuang et al. 2013b). In agreement, FADD-deficient mice specific for adipose tissue showed higher energy expenditure, higher fatty acid oxidation, reduced fat formation, insulin and obesity resistance (Zhuang et al. 2016).
It has been reported that FADD presence maintains the balance of redox potential by regulating the levels of anti-oxidants; thus, FADD overexpression alters mitochondrial integrity and pulverizes the membrane potential by altering the expression of Bcl-2 and cytochrome c (Ranjan and Pathak 2016).
9. Prognostic value of FADD: chemoresistance and chemosensitivity.
The role of FADD in cancer is controversial, since both its absence and presence can confer survival advantages to tumour cells, including resistance to cell death, resistance to anti-tumour drugs or proliferative advantages (Tourneur et al. 2003;
Tourneur et al. 2005; Tourneur and Chiocchia 2010). Thus, both the reduction and the increment of FADD have been reported in cancer in association with bad prognosis and poor clinical outcome (Schrijvers et al. 2012; Dent 2013; Ikeda et al. 2013; Pattje et al. 2013; Callegari et al. 2016).
Chemotherapeutic drugs commonly used in cancer therapy can induce cell death by activation of Fas-mediated apoptotic pathway. These drugs induce the expression and aggregation of Fas at the cell membrane and its interaction with FADD, in a ligand-independent manner (Micheau et al. 1999). It has been observed that FADD expression is low or absent in patients with lower sensitivity or resistance to cell death induced by chemotherapeutic drugs, suggesting a correlation between FADD expression and response to chemotherapy (Tourneur et al. 2004). Furthermore, it has been reported that the phosphorylation of FADD makes the tumour cell sensitive to several chemotherapeutic treatments such as paclitaxel (Shimada et al. 2004; Alappat et al. 2005; Jang et al. 2011a) and radiotherapy (Schrijvers et al. 2012).
In summary, a deeper knowledge regarding FADD functions is required in order to understand the clinical impact that FADD alterations may have in tumour cells.
OBJECTIVES
1. To identify alterations of FADD and its phosphorylation in T-LBL.
2. To analyse the apoptotic function of FADD in T-LBL.
3. To study the non-apoptotic functions of FADD in T-LBL.
4. To determine if FADD alterations have clinical implications.
5. To explore the consequences of FADD alterations at the proteomic level and to identify new interactors of FADD in tumour T cells.
1. Identificar las alteraciones de FADD y su fosforilación en T-LBL.
2. Analizar la función apoptótica de FADD en T-LBL.
3. Estudiar las funciones no apoptóticas de FADD en T-LBL.
4. Determinar si las alteraciones de FADD tienen implicaciones clínicas.
5. Explorar las consecuencias de las alteraciones de FADD a nivel proteómico e identificar nuevas proteínas que interaccionan con FADD en células T tumorales.
MATERIAL AND METHODS
1. Human samples.
2. Murine samples.
Thirty-two human T-LBL samples and twenty-four human thymuses were obtained from the Spanish Tumour Bank Network of the Spanish National Cancer Research Centre (Centro Nacional de Investigaciones Oncológicas, CNIO, Madrid, Spain) and Hospital de La Paz, respectively.
These samples derive from mass lesions in mediastinum or lymph nodes and are composed of small- to medium-sized blast cells expressing nuclear terminal deoxynucleotidyl transferase (TdT) (Supplementary Figure 1A). Institutional review board approval was obtained for these studies (references CEI 31-773 and CEI-70- 1260, UAM Ethics Committee for Research, and RNDdT 10/073) and the participants provided written informed consent in accordance with the Declaration of Helsinki.
Four-week-aged C57BL/6J female mice obtained from The Jackson Laboratory (The Jackson Laboratory, Bar Harbor, ME, USA) were either left untreated (healthy thymuses, control group, CTRL) or subjected to gamma irradiation to induced T-cell lymphoblastic lymphoma (T-LBL group) as previously described (Santos et al. 2009). TdT levels are determined by immunohistochemistry (IHC) as a measure for the T-LBL-characteristic blast presence (Supplementary Figure 1A’). A portion of each control thymus and each T-LBL sample was mechanically dispersed and strained through a nylon mesh (BD Biosciences, San Jose, CA, USA) to isolate the thymocytes. Another portion was fixed for IHC. We adhered to the ethical considerations dictated by the European Directive 2010/63/EU and Real Decreto 53/2013 on the protection of animals used in scientific procedures.
Freshly isolated thymocytes from mouse healthy thymuses and T-LBL samples were characterized by flow cytometry (FC). Four-colour mouse CD3/CD4/CD8/TdT analysis was performed on a FACS Calibur flow cytometer (BD Biosciences). Background levels were determined with isotype-matched control antibodies (Supplementary Table 1).
Data were analysed using FlowJo v10 (Flowjo, Ashland, OR, USA).
3. Cell lines.
4. Lentiviral constructs and generation of stable cell lines.
Jurkat clone E6 (ATCC® TIB-152™), Jurkat clone A3 (ATCC® CRL-2570™), FADD- deficient Jurkat clone I2.1 (ATCC® CRL-2572™), BW5147.3 (Thy-1-e) (ATCC® TIB- 234™) and HEK-293T (ATCC® CRL-11268™) were purchased from ATCC (Manassas, VA, USA). ATCC routinely performs cell lines authentication, using short tandem repeat profiling as a procedure. Jurkat cell lines were cultured in RPMI 1640, whereas BW5147.3 and HEK-293T cells were cultured in DMEM, both from Gibco (Life Technologies, Carlsbad, CA, USA); media were supplemented with 15% and 10% FBS, respectively (GE Healthcare Life Sciences, Velizy-Villacoublay, France), 2mM L-Glutamine (Merck Millipore, Billerica, MA, USA) and 1mM sodium pyruvate (Merck Millipore). Cultures were maintained at 37°C in 5%
CO2 humidified atmosphere. Cell experimentation was always performed within a period not exceeding six months after resuscitation and in mycoplasma free culture conditions.
We purchased the lentiviral vectors EX-V0108-Lv-225, carrying FADD cDNA, and its empty control EX-NEG-Lv225 (GeneCopoeia, Rockville, MD, USA). We performed site-directed mutagenesis to obtain S194A-FADD (non- phosphorylatable mutant) and S194D-FADD (phosphomimetic mutant), using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and following the manufacturer’s instructions. The oligonucleotide primers used for site-directed mutagenesis are (the mutated sequences are underlined): Ser194Ala (S194A): forward:
5’catgacatcggggccatggccccactc3’, reverse: 5’gagtggggccatggccccgatgtcatg 3’;
Ser194Asp (S194D): forward: 5’gttccatgacatcgggtccatggccccactcctg 3’, reverse:
5’caggagtggggccatggacccgatgtcatggaac 3’.
Jurkat clone I2.1 cells were transduced by lentiviral particles obtained after HEK-293T-mediated packaging. HEK-293T were transfected using Lipofectamine
5. In vitro FADD reconstitution.
6. Gene expression analysis.
MA, USA) with pMD2.G (Addgene, Cambridge, MA, USA), which provided the VSV-G envelope, the 2nd generation lentiviral packaging plasmid psPAX2 (Addgene), and the vectors of interest (EX-NEG-Lv225, EX-V0108-Lv-225 and S194A/D). Next day, 0.45μm-filtered supernatants were added to the Jurkat I2.1 cells, using polybrene (Sigma-Aldrich, St. Louis, MO, USA) at 8μg/ml to increase the efficiency of the transduction. Cells were centrifuged at 2100rpm at 32°C for 100min, and then incubated for 5h. Puromycin dihydrochloride (Santa Cruz Biotechnology, Dallas, TX, USA) was added at 2μg/ml for selection and at 3μg/
ml for maintenance. Transduction efficiency was followed by GFP expression, measured by flow cytometry on a FACS Canto II (Becton-Dickinson, Franklin Lakes, NJ, USA). Genetic modification of Jurkat I2.1 cell line by lentiviral transduction was subjected to the regulated and approval by the Spanish Ministry of Agriculture, Food, and Environment, managed by CBMSO’s Biological Safety Department.
FADD-deficient Jurkat I2.1 cells were electroporated with EX-V0108- Lv225 or EX-NEG-Lv225 constructs (GeneCopoeia) using a Gene Pulser MXcell Electroporation System (Bio-Rad Laboratories, Hercules, CA, USA). 107 cells in 500μl of complete medium were subjected to 280V, 950μF, with 5μg or 25μg of vector. Electroporated cells were harvested in 10ml of complete medium and analysed 48h post transfection, for protein expression, both indirectly testing GFP percentage and mean fluorescence intensity by flow cytometry with a FACS Canto II (Becton-Dickinson) and directly detecting FADD by Western blot.
Total RNA from murine or human samples was obtained using TriPure Reagent (Roche Applied Science, Indianapolis, IN, USA), following manufacturer’s instructions. Fadd and Ahr gene expressions were determined at the transcriptional level by real-time RT-qPCR from total RNA in two steps, using first the High-Capacity RNA-to-cDNA™ Kit (Applied Biosystems, Foster City, CA, USA), then the FastStart SYBR Green Master (Roche, Mannheim, Germany). Expression
7. FADD and Fadd sequencing.
Total RNA and DNA from samples were extracted using TriPure Reagent (Roche Applied Science, Indianapolis, IN, USA), following manufacturer’s instructions. To obtained cDNA, one microgram of RNA was reverse-transcribed using SuperScript®
VILO™ cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA). FADD sequencing analyses from human T-LBL samples was performed using cDNA or genomic DNA, depending on its availability. The primers and PCR conditions (Supplementary Table 2) were designed using as reference sequences the published sequence of Fadd cDNA from mouse strain C57BL/6J (accession number to GenBank NM_010175) and the sequences of FADD gene (Ensembl ID ENSG00000168040) and cDNA (Ensembl ID ENST00000301838.4) from Homo sapiens.
We purified the PCR products using Wizard SV Gel and PCR Clean-up System (Promega Corporation, Madison, WI, USA). Sequencing reactions were performed at the Genomics Platform of Parque Científico de Madrid, using an ABI Prism 310 Automated Sequencer (Applied Biosystems, Foster City, CA, USA).
For comparisons, the BioEdit v7.1.11 sotfware (Ibis Therapeutics, Carlsbad, CA, USA) was used.
values of G6pdx and Hprt1 in the same samples were used for normalization, using the 2-ΔΔCT method (Livak and Schmittgen 2001). Primers are indicated in Supplementary Table 2.
MiR-134-5p, miR-155-5p and miR-339-5p were reversed transcribed using the miRCURY LNATM Universal RT microRNA PCR system (Exiqon, Vedbaek, Denmark).
RT-qPCRs were performed using miR-134-5p (Exiqon, No.205989), miR-155- 5p (Exiqon, No.205930 for mouse and No.204308 for human) and miR-339-5p (Exiqon, No.206007) LNA™ primers, as well as FastStart SYBR Green Master (Roche). Expression values of miR-39-3p (Exiqon, No.203952), miR-103a-3p (Exiqon, No.204063) and UniSp6 (Exiqon, No.203301) in the same samples were used for normalization, using the 2-ΔΔCT method (Livak and Schmittgen 2001).
9. Western blot (WB).
Total proteins from murine samples were obtained using TriPure Reagent (Roche Applied Science), following manufacturer’s instructions. Total proteins from cell lines were obtained using RIPA cell lysis buffer. Nuclear and cytoplasmic fractionated protein extracts were obtained using cytoplasmic extract buffer (10mM HEPES, 60mM KCl, 1mM EDTA with 0.075% NP-40) and RIPA. Protein extracts were supplemented with 2mM PMSF, 2.5μl/ml Protease Inhibitor Cocktail and 10μl/ml Phosphatase Inhibitor Cocktail 2 (Roche Diagnostics GmbH, Mannheim, Germany). Ten micrograms-aliquots were electrophoresed in SDS- PAGE with β-mercaptoethanol or Mini-PROTEAN-TGXTM Precast Gels (Bio-Rad Laboratories, Hercules, CA, USA) and then electrotransferred to Immobilon-P PVDF transfer membranes (Merck Millipore) or mini-sized PVDF membranes by Tranfer Blot® TurboTM Tranfer System (Bio-Rad Laboratories). Antibodies and conditions are indicated in Supplementary Table 1. The peroxidase activity was developed using WesternBright ECL Detection System (Advansta, Menlo Park, CA, USA). ImageQuant LAS 4000 digital imaging system (GE Healthcare Europe GmbH, Freiburg, Germany) was used for acquisition of images, and Quantity One® v4.6.3 (Bio-Rad Laboratories) for band intensity densitometry.
8. Antibodies and reagents.
Primary and secondary antibodies used for immunodetection are summarized in Supplementary Table 1. Gö6976, SP600125 and NSC-87877 were purchased from Calbiochem (Merck Millipore); anisomycin, CKI-7, cycloheximide, etoposide, hydroxyurea, nocodazole, paclitaxel and thymidine were purchased from Sigma- Aldrich (Sigma-Aldrich); MG-132 from PeptaNova (PeptaNova, Sandhausen, Germany); and GW843682X from Tocris Bioscience (Tocris Bioscience, Bristol, United Kingdom).
Formalin-fixed and paraffin-embedded tissues were deparaffinised and rehydrated using standard protocols and subjected to heat-induced antigen retrieval in Tris-EDTA buffer (pH=9.0) or sodium citrate buffer with 0.05% Tween 20 (pH=9.0). Mouse tissues frozen in Tissue-Tek® O.C.T™ Compound (Sakura Finetek Europe B.V., Alphen aan den Rijn, The Netherlands) were obtained after 10% formalin-fixation for 2h, cryoprotection with 30% sucrose in phosphate buffered saline 1X (PBS) for 24h. For immunocytochemistry, cells were washed twice with tris-buffered saline 1X (TBS) before being frozen in Tissue-Tek® O.C.T™
Compound (Sakura Finetek Europe B.V., Alphen aan den Rijn, The Netherlands) and they were fixed with 10% formalin for 10min. Human tissues frozen in Tissue-Tek®
O.C.T™ Compound (Sakura Finetek Europe B.V.) were fixed with 10% formalin for 10min. Endogenous peroxidase activity was reduced by 3% H2O2-pre-treatment for 30min. Dako REAL™ antibody diluent (Dako, Glostrup, Denmark) was the blocking buffer. Antibodies and conditions are indicated in Supplementary Table 1. We used liquid DAB+ substrate chromogen system (Dako) for visualization. The sections were counterstained with Mayer’s hematoxylin (Sigma-Aldrich, St. Louis, MO, USA).
An Axiovert 200 inverted microscope (Carl Zeiss, Oberkochen, Germany) with a SPOT RT Digital Scanning Camera (Diagnostic Instruments, Sterling Heights, MI, USA) at 63× magnification was used for mouse image analysis. Olympus BX61 microscope (Olympus Corporation of the Americas, Center Valley, PA, USA) was used for human image analysis and photographic material acquisition at 20×, 40×
and 100× magnification. Irrespective of intensity, the percentages of positive cells versus total cell number were calculated using ten and five random representative fields per section, in mouse and human, respectively. Fiji-Image J free software (National Institutes of Health, Bethesda, MD, USA) was used for analysis, applying the Image/Colour deconvolution, Image/Adjust/Threshold and Analyse/Analyse Particles tools as published previously (Ruifrok and Johnston 2001).
10. Immunohistochemistry (IHC)/Immunocytochemistry (ICC).
11. Immunofluorescence (IF).
Formalin-fixed frozen tissue auto-fluorescence was blocked by immersing the sections in 0.1% BSA in PBS at 4°C overnight. Then, samples were permeabilized with 0.01% Triton X-100 (Sigma-Aldrich) in PBS, at room temperature for 30min.
Non-specific binding was blocked with 3% BSA in PBS, then antibodies were applied (Supplementary Table 1). Finally, To-Pro-3 (Thermo Fisher Scientific, Waltham, MA, USA) was used for nuclear staining and the slides were mounted with Mowiol (Merck KGaA, Darmstadt, Germany).
LSM 510 scan head integrated with the Axiovert 200 M inverted confocal microscope (Carl Zeiss, Oberkochen, Germany) at 63× magnification was used to observe and capture images. Digital pictures were analysed by Fiji-Image J free software (National Institutes of Health). Mander’s coefficients were calculated to check the co-location of S191-P-FADD with To-Pro-3 (nuclear staining S191- P-FADD) and CD3e (cytoplasmic staining S191-P-FADD), mean fluorescence intensity per cell of nuclear and cytoplasmic of S191-P-FADD was quantified by Fiji-Image J free software (National Institutes of Health).
12. Apoptosis assay.
106 cells/ml were treated for 24h with agonist anti-FAS mouse antibody clone CH11 (Merck Millipore, Billerica, MA, USA), or with irrelevant mouse IgMλ antibody (Beckman Coulter, Nyon, Switzerland) at a final concentration of 100ng/ml. Apoptosis was monitored by flow cytometry on a FACS Canto II (Becton-Dickinson, Franklin Lakes, NJ, USA), using the PE Annexin-V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA), which is based on Annexin V-PE/7-AAD staining, following the manufacturer’s instructions. Finally, the results were analysed using FlowJo v10 (Flowjo, Ashland, OR, USA). Apoptosis was also monitored by WB detection of cleaved PARP, cleaved caspases-3 and -8 as apoptosis markers (Supplementary Table 1).
The viability of cells transiently expressing FADD after electroporation was determined based on Trypan blue exclusion viability test. The viability of cells stably expressing FADD after transduction was assessed for 5 days using the Cell Proliferation Kit II (XTT) (Sigma-Aldrich) as per manufacturer’s instructions.
Following incubation, the plate was read with iMark™ Microplate Absorbance Reader (Bio-Rad Laboratories, Hercules, CA, USA) at a wavelength of 490nm. The reference wavelength 655nm was also read to control for nonspecific absorption.
In these cells, the number of mitotic cells was determined by flow cytometry (FC) as the percentage of cells positive for phosphorylated histone H3, using Alexa Fluor®488-conjugated phospho-Histone H3 (Ser10) Antibody (#9708 and
#3465, Cell Signaling Technology, Danvers, MA, USA) and a FACS Canto II (Becton- Dickinson, Franklin Lakes, NJ, USA).
Time for completion of mitosis in these cells was estimated by FC as the ratio 2n/4n DNA cell content at specific time points after G2/M-synchronization by nocodazole treatment and release, using PI/RNase Staining Buffer (BD Biosciences, San Jose, CA, USA). Time points were obtained by harvesting cells every 20min after release.
14. Cell cycle assay.
15. Determination of protein stability.
For protein stability analysis, cells were treated with cycloheximide or Cell cycle profiling using PI/RNase Staining Buffer (BD Biosciences) was performed according to the manufacturer’s instructions. Phase distribution of cell cycle was examined after pharmacological treatments specifically inducing G1/S or G2/M arrest. All analytic cytometry procedures were performed on a FACS Canto II flow cytometer (Becton-Dickinson). Watson pragmatic fitting algorithm was used to determine cell cycle phase statistics using FlowJo v10 (Flowjo).
13. Cell proliferation assay.
16. Stable isotope labelling using amino acids in cell culture (SILAC).
17. Immunoprecipitation (IP) of endogenous FADD.
proteasome in protein loss. Protein levels were determined at 2, 4, and 6h after treatment by WB. Dose- and time-course experiments were previously conducted (data not shown) to determine the optimal conditions, where no significant evidence of toxicity was observed.
Whole cells were lysed with immunoprecipitation lysis buffer (50mM Tris- HCl, pH 8.0, 1% Triton X-100, 300mM NaCl, 1mM EDTA, PhosStop (Roche) and cOmplete™ Mini Protease Inhibitor Cocktail (Roche)) for 15min on ice. Lysed cells were centrifuged at 15,000×g for 15min at 4°C and the supernatant was collected. Protein concentrations were determined using the Bradford assay (Sigma) following the manufacturer ́s instructions.
For FADD immunoprecipitation (IP), 5mg of protein extracts were pre-cleared by incubation with Dynabeads®-Protein G (ThermoFisher) for overnight at 4°C.
12μg of monoclonal anti-human FADD antibody (Clone A66-2, BD Biosciences) were covalently coupled to 1.8mg of Dynabeads®-Protein G (ThermoFisher). The beads were incubated with the antibody for 2h at 4°C and then washed twice with 10 volumes of 0.1M sodium borate, pH 9.3. Next, the beads were incubated For SILAC labelling, stable Jurkat cell lines were cultured in SILAC RPMI (Thermo Fisher Scientific) supplemented with either “light” isotopically normal 242.7mg/L L-Lysine and 50.1mg/L L-Arginine (R0K0) (Sigma-Aldrich, St. Louis, MO, USA), or “heavy” 253.7mg/L 13C6 15N4 L-Arginine and 50.1mg/L 13C6 15N2 L-Lysine (R10K8) (CK Isotopes, Leicestershire, UK), with 500mg/L L-Proline (Sigma-Aldrich), 1mM sodium pyruvate (Merck Millipore), 2mM L-glutamine (Merck Millipore), 3µg/ml puromycin (Santa Cruz Biotechnology) and 15% v/v dialyzed FBS (Labtech International, East Sussex, UK). Cultures are maintained at 37°C in 5% CO2 humidified atmosphere. Cells were grown in SILAC media for seven doubling times before being analysed for incorporation efficiency of the SILAC amino acids (Supplementary Figure 2A).
19. Mass spectrometry (MS) and data analysis.
Excised bands from Coomassie-stained gels were detained, reduced (10mM DTT) and alkylated (55mM iodoacetamide) in 25mM ammonium bicarbonate prior to overnight in-gel trypsin digestion (12.5ng/ul; Trypsin Gold; Promega, Madison, WI, USA). Digested samples were acidified by addition of trifluoroacetic acid (0.5% final volume). Peptides were desalted (Peptide concentration and desalting Macrotrap; Michrom Bioresources, Pleasanton, CA, USA) and dried by vacuum centrifugation. The resulting peptide mixtures were analysed by mass spectrometry.
18. Trypsin digestion.
twice with 10 volumes of borate buffer containing 20mM dimethylpimelimidate (Sigma-Aldrich) for 30min at room temperature. The beads were washed four times with 10 volumes of ice-cold 50mM glycine (pH 2.5) to remove unbound antibody, then neutralized for 2h with 0.2M Tris-HCl (pH 8.0). This complex was incubated with pre-cleared proteins overnight at 4°C and then washed five times with citrate phosphate buffer (pH 5.0) for use. Dynabeads®-Protein G-Ab-Ag complex from labelling proteins were then mixed and washed a further three additional times. Following addition of reduced sample buffer, protein samples were run on SDS-PAGE gel and Coomassie stained (Supplementary Figure 2B) and one-tenth aliquot was checked by WB with anti-FADD antibody (FADD (1F7):
05-486; Merck Millipore, Billerica, MA, USA) (Supplementary Figure 2C).
Data analysis from LTQ Orbitrap-Velos-Pro hybrid mass spectrometer (Thermo Scientific) for SILAC or label free-quantification (LFQ) method was performed by PEAKS Studio 8.5 (Bioinformatics Solutions Inc., Waterloo, Ontario, Canada).
SILAC data from Q-Exactive HF hybrid quadrupole-orbitrap mass spectrometer (Thermo Scientific) were processed using the MaxQuant software (v1.5.8.3) (Cox and Mann 2008).
Q-Exactive HF is a hybrid quadrupole-orbitrap mass spectrometer, which
