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RESEARCH ARTICLE
Novel tumour suppressor roles for GZMA and RASGRP1
in Theileria annulata-transformed macrophages and human
B lymphoma cells
Zineb Rchiad
1,2,3,4
| Malak Haidar
1,2,3
| Hifzur Rahman Ansari
1,5
|
Shahin Tajeri
2,3
| Sara Mfarrej
1
| Fathia Ben Rached
1
| Abhinav Kaushik
1
|
Gordon Langsley
2,3
| Arnab Pain
1,6
1
Pathogen Genomics Laboratory, BESE Division, King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia
2
Laboratoire de Biologie Comparative des Apicomplexes, Faculté de Médecine, Université Paris Descartes –Sorbonne Paris Cité, Paris, France
3
INSERM U1016, CNRS UMR8104, Cochin Institute, Paris, France
4
Centre de Coalition, Innovation, et de prévention des Epidémies au Maroc (CIPEM), Mohammed VI Polytechnic University (UM6P), Ben Guerir, Morocco
5
King Abdullah International Medical Research Center (KAIMRC), King Abdulaziz Medical City, Ministry of National Guard Health Affairs, Jeddah, Saudi Arabia
6
Global Station for Zoonosis Control, Global Institution for Collaborative Research and Education (GI-CoRE), Hokkaido University, Sapporo, Japan
Correspondence
Gordon Langsley, INSERM U1016, CNRS
UMR8104, Cochin Institute, 27 rue du
Faubourg-Saint-Jacques, 75014 Paris, France.
Email: gordon.langsley@inserm.fr
Arnab Pain, Pathogen Genomics Laboratory,
BESE Division, 4700 King Abdullah University
of Science and Technology (KAUST),
Thuwal 23955-6900, Saudi Arabia.
Email: arnab.pain@kaust.edu.sa
Present address
Zineb Rchiad, African Genome Center (AGC),
Mohammed VI Polytechnic University (UM6P),
Lot 660, Hay Moulay Rachid, Ben Guerir
43150, Morocco
Funding information
Competitive Research Grant, King Abdullah
University of Science and Technology, Grant/
Award Number: OSR-2015-CRG4-2610;
ParaFrap, Grant/Award Number: ANR-
11-LABX-0024
Abstract
Theileria annulata is a tick-transmitted apicomplexan parasite that infects and transforms
bovine leukocytes into disseminating tumours that cause a disease called tropical
theileriosis. Using comparative transcriptomics we identified genes transcriptionally
perturbed during Theileria-induced leukocyte transformation. Dataset comparisons
highlighted a small set of genes associated with Theileria-transformed leukocyte dissemi-
nation. The roles of Granzyme A (GZMA) and RAS guanyl-releasing protein 1 (RASGRP1)
were verified by CRISPR/Cas9-mediated knockdown. Knocking down expression of
GZMA and RASGRP1 in attenuated macrophages led to a regain in their dissemination in
Rag2/γC mice confirming their role as dissemination suppressors in vivo. We further
evaluated the roles of GZMA and RASGRP1 in human B lymphomas by comparing the
transcriptome of 934 human cancer cell lines to that of Theileria-transformed bovine
host cells. We confirmed dampened dissemination potential of human B lymphomas
that overexpress GZMA and RASGRP1. Our results provide evidence that GZMA and
RASGRP1 have a novel tumour suppressor function in both T. annulata-infected bovine
host leukocytes and in human B lymphomas.
KEYWORDS
GZMA, human cancer cell atlas, RASGRP1,Theileria annulata, transcriptome, tumour suppressor
Zineb Rchiad and Malak Haidar are co-first authors.
Hifzur Rahman Ansari and Shahin Tajeri are co-second authors.
Received: 9 June 2020 Revised: 3 August 2020 Accepted: 7 August 2020
DOI: 10.1111/cmi.13255
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited and is not used for commercial purposes.
© 2020 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.
Cellular Microbiology. 2020;22:e13255. wileyonlinelibrary.com/journal/cmi 1of14
https://doi.org/10.1111/cmi.13255
1|INTRODUCTION
Theileria annulata is a tick-transmitted apicomplexan parasite that
infects and transforms bovine leukocytes into disseminating tumours
that cause a widespread disease called tropical theileriosis. In coun-
tries endemic for tropical theileriosis live attenuated vaccines are pro-
duced by multiple in vitro passages of virulent, transformed
macrophages and vaccination with attenuated macrophages protects
animals from severe disease (Nene & Morrison, 2016). The transfor-
mation is reversed and leukocytes revert to a quiescence state and die
upon drug-induced parasite death making Theileria-infected leuko-
cytes a powerful cellular model to identify genes regulating cellular
transformation and dissemination (Tretina, Gotia, Mann, & Silva,
2015). This parasite-based reversible model of leukocyte transforma-
tion has allowed the identification of several cell signalling pathways
associated with the virulence of Theileria-transformed leukocytes
such as c-Jun NH2-terminal kinase/c-Jun/PI3 kinase signalling
(Lizundia et al., 2006), protein kinase A (PKA) (Haidar, Echebli, Ding,
Kamau, & Langsley, 2015), transforming growth factor beta 2 (TGF-
β2) (Chaussepied et al., 2010) (Haidar, Whitworth, et al., 2015) and
SMYD3/MMP9 (Cock-Rada et al., 2012). MMP-9 and c-Jun are asso-
ciated with proliferation, angiogenesis and dissemination/metastasis
in Theileria-induced host cell transformation and human cancer
(Adamson, Logan, Kinnaird, Langsley, & Hall, 2000; Hofmann,
Westphal, Van Muijen, & Ruiter, 2000; Lizundia et al., 2006; Vleugel,
Greijer, Bos, van der Wall, & van Diest, 2006). Epigenetic changes also
contribute to Theileria-induced leukocyte transformation (Robert
McMaster, Morrison, & Kobor, 2016). OncomiR addiction has been
described as being generated by a miR-155 feedback loop in
T. annulata-transformed B cells (Marsolier et al., 2013). Similarly, miR-
126-5p contributes to infected macrophage dissemination through
JNK-Interacting Protein-2 (JIP2)/JNK1/AP1-mediated MMP9 tran-
scription (Haidar et al., 2018).
It is well established that T. annulata infection hijacks key leuko-
cyte signalling cascades to modulate host cell gene expression. For
example, RNA extracted from T. annulata-transformed B cells was
used to screen bovine microarrays demonstrating that infection had
reconfigured host cell gene expression (Kinnaird et al., 2013). None-
theless, a systematic and genome scale transcriptional comparison
of B cells and macrophages transformed by T. annulata has been lac-
king. In this study, we used RNA-seq to define the transcriptional
landscapes of two T. annulata-transformed B cell lines and a virulent
T. annulata-transformed macrophage line (Ode) and the attenuated
live vaccine directly derived from it. High stringency bioinformatic
comparisons of the transcriptional landscapes identified four candi-
date genes (MMP9,GZMA,RASGRP1 and SEPP1), as potential
players in the dissemination of virulent T. annulata-transformed
macrophages.
Theileria annulata infection confers on its host leukocyte proper-
ties largely similar to human cancer, most notably immortalization,
independence of exogenous growth factors, uncontrolled proliferation
and dissemination. The similarity between Theileria-transformed leu-
kocytes and human leukaemia suggests that Theileria-induced
transformation could be a powerful model to elucidate common
mechanisms underpinning tumour virulence. In order to generalise our
Theileria-based observations we compared the transcriptome maps of
934 human cancer cell lines to the transcriptomes of T. annulata-
transformed B lymphocytes and provide functional evidence for
shared roles for GZMA and RASGRP1 in controlling dissemination of
both human B lymphomas and Theileria-transformed leukocytes.
2|RESULTS
2.1 |Differentially expressed bovine genes
in T. annulata-transformed leukocytes
The infection and full transformation of the BL20 cell line with
T. annulata causes profound transcriptional changes (Kinnaird et al.,
2013). We have compared the transcriptome of the T. annulata-
transformed TBL3 and TBL20 B lymphocytes to their non-infected
counterparts, BL3 and BL20, respectively. The quality of the sequenc-
ing results is shown in Figure S1. Infection of BL3 and BL20 lympho-
cytes with T. annulata provoked significant changes in host cell gene
expression (1,179 and 1,517 differentially expressed genes respec-
tively, with fold change >2 and padj < .05). Transcriptional changes
between virulent compared to attenuated Ode macrophages are less
profound (76 genes, with fold change >2 and padj < .05), likely
because the infected and transformed macrophages only appear to
differ in dissemination potential (Figure S2A).
To identify bovine genes whose transcription is perturbed by
transformation and attenuation of dissemination of T. annulata-
transformed leukocytes we concentrated on the most differentially
expressed genes (DEGs) (Figure 1 and Table S1). Many of these
genes are annotated as being implicated in cell proliferation and
metastasis. Among the top five up-regulated transcripts in TBL20 is
MMP9 (matrix metallopeptidase 9), a gene highly expressed in dif-
ferent cancer types and linked to metastasis and angiogenesis (Yu &
Stamenkovic, 2000). WC1-8 is the third most up-regulated gene in
TBL20 lymphocytes and has been described as being also up-
regulated in ovarian carcinoma cells (Mangala et al., 2009). The most
down-regulated transcripts in TBL20 cells include LAIR1 (leukocyte-
associated immunoglobulin-like receptor 1) and VPREB (pre-B lym-
phocyte 1). LAIR1 is a strong inhibitor of natural killer cell-mediated
cytotoxicity and an inhibitory receptor, which down-regulates B
lymphocyte immunoglobulin and cytokine production (Merlo et al.,
2005). Relative down-regulation of LAIR-1 was not unexpected, as
itslossofexpressionisobservedduringBcellproliferation(vander
Vuurst de Vries et al., 1999). This is because TBL20 lymphocytes
possess a higher proliferative capacity compared to their uninfected
BL20 counterpart. ZBTB32 (zinc finger and BTB domain containing
32), IL21R (interleukin 21 receptor), and MMP9 areamongthetop
five up-regulated transcripts in infected TBL3 B lymphocytes.
MMP9 and ANXA5 are among the top 10 up-regulated genes in both
TBL3andTBL20,reflectingacommonroleofthesegenesinthe
promotion of the Theileria-induced transformation phenotype.
2of14 RCHIAD ET AL.
ANXA5 was previously reported to promote proliferation, migration
andinvasioninrenalcellcancer.Thefivemostdown-regulated
transcripts in TBL3 are KRT6C (keratin 6C), MATK (megakaryocyte-
associated tyrosine kinase),IGSF9B(immunoglobulin superfamily
member 9B),A2M(alpha-2-macroglobulin) and H2AFY2 (H2A his-
tone family, member Y2). The biological functions of these genes
include inhibition of cell growth and proliferation (Kim et al., 2004),
repression of DNA transcription (Perche et al., 2000) and inhibition
of cell adhesion and migration (Kurz et al., 2017), functions that are
often dampened to allow continuous proliferation and survival of
transformed cells. Out of the top 10 up- and down-regulated genes
in TBL3, 8 are up and 3 are down-regulated in TBL20. Similarly, out
of the top 10 up- and down-regulated genes in TBL20 cells, 5 are
up- and 4 are down-regulated in TBL3. This shows that the tran-
scriptional changes induced by T. annulata are not identical between
the two lymphocyte cell lines. We confirmed by qRT-PCR biologi-
cally reproducible differential expression patterns of 21 randomly
selected genes from the BL3/TBL3 and BL20/TBL20 RNA-seq
datasets (Figure S2B).
2.2 |Identification of key genes potentially
involved in Theileria-mediated macrophage
dissemination
The most down-regulated transcripts in attenuated Ode macrophages
are SKAP2 (src kinase associated phosphoprotein 2), a gene known to
promote tumour dissemination through the regulation of podosome
formation in macrophages (Tanaka et al., 2016) and NRP2 that
regulates tumour progression by promoting TGF-β-signalling
(Grandclement et al., 2011). Down-regulation of these genes corre-
lates with decreased dissemination of attenuated macrophages, as
previously we have described loss of TGF-β-signalling as being associ-
ated with decreased dissemination (Chaussepied et al., 2010). By con-
trast, the most highly up-regulated transcripts include SEPP1
(selenoprotein P) and PTPRT (protein tyrosine phosphatase, receptor
type T). Both SEPP1 and PTPRT have been previously described as
tumour suppressor genes (Scott & Wang, 2011; Short et al., 2016).
Taken together, the identity of the most strongly up- and down-
regulated genes argues that our differential transcription screen could
FIGURE 1 Top 20 DEGs in Theileria-transformed bovine host cells. Circos plot showing the top 10 up- and down-regulated DEGs in
BL3/TBL3, BL20/TBL20 and attenuated versus virulent (Att/Vir) Ode macrophages. The circular heatmap represents the FC of the top DE genes
in BL20/TBL20, BL3/TBL3 and Att/Vir Ode in the outer, middle and inner rings, respectively, where green reflects the level of up-regulation and
orange down-regulation. The genes with biological functions related to tumorigenesis and immune regulation are tagged with coloured
rectangles. Genes with no tag are hypothetical genes or have no known function in tumorigenesis and immune regulation. DE, differentially
expressed; DEG, differentially expressed gene; FC, fold change
RCHIAD ET AL.3of14
identify novel genes regulating Theileria-transformed macrophage
dissemination.
To define the genes likely playing important roles in transforma-
tion and dissemination, we compared genes differentially expressed
(DE) in TBL3, TBL20 and attenuated Ode macrophages. We
assumed that the genes most likely to play a key role are up-
regulated after infection and down-regulated upon attenuation, and
vice versa, that is, the genes down-regulated after infection and up-
regulated after attenuation. To identify these genes we have inter-
sected the obtained DEG lists of BL3/TBL3, BL20/TBL20 and
Vir/Att Ode. This approach identified four genes with potential to
play key roles in the dissemination of Theileria-transformed leuko-
cytes (Figure 2a).
The genes are MMP9, SEPP1, GZMA and RASGRP1. MMP9 is the
only gene up-regulated after T. annulata-mediated transformation and
down-regulated after attenuation. Inversely, SEPP1,GZMA and RASGRP1
are down-regulated after transformation and up-regulated after attenua-
tion. The biological functions of these genes have been implicated in
metastasis and cell invasion (Yu & Stamenkovic, 2000), selenium trans-
port (Burk et al., 1995), peptide cleavage by immune cells (Chowdhury &
Lieberman, 2008) and regulation of B cell-development and homeostasis
and differentiation (Priatel et al., 2007), respectively (Table 1). Differential
expression of these genes was confirmed by qRT-PCR (Figure 2b). We
focused on GZMA,RASGRP1 and SEPP1,astheroleofMMP9 is associ-
ated with the virulence of a number of T. annulata-infected cell lines
(Somerville, Adamson, Brown, & Hall, 1998). CRISPR/Cas9-mediated loss
FIGURE 2 Inversely DEGs in TBL20, TBL3 and Att Ode leukocytes. (a) Venn diagrams illustrating the genes inversely DE in TBL3, TBL20 and
attenuated Ode macrophages. (b) qRT-PCR confirmation of DEGs potentially playing key roles in leukocyte transformation and dissemination. Seq
and qRT-PCT refer to sequencing and real-time quantitative reverse transcription PCR, respectively. The reactions were set in three biological
replicates and the fold change calculated with the 2
ΔΔct
method. The error bars represent SEM. DE, differentially expressed; DEG, differentially
expressed gene
4of14 RCHIAD ET AL.
of SEPP1 in attenuated macrophages resulted in a lethal phenotype. It is
possible that SEPP1 plays an essential role in transformed macrophage
survival, and so the role of SEPP1 in Theileria-induced transformation
was not further evaluated (Figure S3).
2.3 |Ablation of GZMA and RASGRP1 by CRISPR/
Cas9 knockdown
To investigate the biological functions and molecular mechanisms of
GZMA and RASGRP1, we knocked down their expression by CRISPR/
Cas9. Although the macrophage transfection efficiency is generally
not higher than 30%, analysis of the mixed population by qRT-PCR
revealed a 3.4- and 1.4-fold reduction in gene expression for
RASGRP1 and GZMA, respectively (Figure 3a,b). All subsequent phe-
notypical analyses were performed on this mixed population. Also, as
controls we transfected a CRISPR/Cas9 plasmid encoding an irrele-
vant guide RNA and demonstrated specificity of knockdown by show-
ing that CRISPR/Cas9 targeting of RASGRP1 does not knockdown
GZMA (Figure S4A).
Next, we tested whether GZMA and RASGRP1 can regulate the
capacity of attenuated macrophages to traverse Matrigel. The inhibi-
tion of their expression led to a regain in dissemination potential, as
estimated in matrigel traversal assays (Figure 3c). No effect was
observed in traversal capacity when infected macrophages were
transfected with the control CRISPR/Cas9 Plasmid (Figure S4B).
Although only a 1.4-fold decrease in GZMA expression was observed
following CRISPR/Cas9, this lead to a 3.2-fold increase in matrigel tra-
versal. A reduction of RASGRP1 expression by 3.4-fold lead to a
1.9-fold increase in matrigel traversal. Both GZMA and RASGRP1
have therefore, the potential to function as suppressors of tumour
dissemination and consistently, knockdown of GZMA also led to a
regain in the ability of attenuated macrophages to form colonies in
soft agar (Figure 3d). Taken together it strongly suggests that GZMA
and RASGRP1 function as tumour suppressors.
2.4 |GZMA and RASGRP1 dampen in vivo
dissemination of Ode macrophages
Similar to metastatic tumour cells T. annulata-transformed leukocytes
also disseminate in immunodeficient mice to distant organs and form
proliferative foci (Fell, Preston, & Ansell, 1990). Dissemination of
Theileria-transformed leukocytes has been previously attributed to
increased production of matrix metaloproteinases (MMPs) (Somerville
et al., 1998). As GZMA and RASGRP1 knockdown led to a regain in
matrigel traversal we used Rag2γC immunodeficient mice to test for a
regain in dissemination in vivo. The CRISPR/Cas9-induced ablation of
expression of GZMA gave rise to an increase in the number of
Theileria-containing tumours in kidney, while knockdown of RASGRP1
increased the number of tumours in the lung and mesentary
(Figure 4). Thus, loss of RASGRP1 and GZMA expression led to a regain
in the organ/tissue invasive capacity of T. annulata-transformed mac-
rophages into these organs.
2.5 |RASGRP1 knockdown reduces GZMA
expression
RASGRP1-activated Ras family proteins possess both pro- and anti-
oncogenic properties, depending on the downstream effector path-
way and cellular context; reviewed in (Cox & Der, 2003). Our tran-
scription profiling showed that most members of the RASGRP gene
family (RASGRP1, 2 and 4) are significantly down-regulated in TBL3
and TBL20 (Figure 5a). In order to explain the higher dissemination of
RASGRP1-knocked down macrophages in vivo, we investigated
whether loss of RASGRP1 could perhaps also provoke a drop in GZMA
expression rendering attenuated macrophages more deficient in dis-
semination. As hypothesised, we found that expression of GZMA
decreased after RASGRP1 knockdown (Figure 5b). Moreover, GZMA
and RASGRP1 expression is repressed by TGF-β(Takami, Cunha,
Motohashi, Nakayama, & Iwashima, 2018; Thomas & Massague, 2005)
TABLE 1 Biological functions of DEGs potentially playing key roles in T. annulata-mediated leukocyte transformation and dissemination
Gene
symbol
Log2 FC
(TBL20)
Adj.
p-value
Log2
FC
(TBL3)
Adj.
p-value
Log2
FC
(ATT)
Adj.
p-value Biological functions References
MMP9 8.87 0 8.29 0 −2.13 5.14 E-94 Metastasis formation, cancer
cells invasion
PMID: 10652271
SEPP1 −3.95 8.74 E-174 −1.54 3.75 E-248 2.63 1.01 E-140 Transports selenoprotein,
tumour suppressor
PMID: 26053663
PMID: 27314080
PMID: 8884283
GZMA −2.87 1.45 E-05 −2.25 1.97 E-41 1.02 9.14 E-21 Plays a role in killing
pathogen-infected cells
and cancer cells
PMID: 18304003
PMID: 15780992
RASGRP1 −3.55 0 −1.78 1.97 E-41 1.2 2.15 E-15 Required for correct
functioning of
lymphocytes in chronic
infections
PMID: 17675473
Abbreviations: DEG, differentially expressed gene; FC, fold change.
RCHIAD ET AL.5of14
and the role of TGF-βin regulating dissemination of Theileria-
transformed macrophages is well established (Chaussepied et al.,
2010; Haidar, Echebli, et al., 2015; Haidar, Whitworth, et al., 2015).
Indeed, expression of GZMA and RASGRP1 was significantly
decreased in attenuated Ode macrophages treated with TGF-β
(Table S2). Taken together, it suggests that one way TGF-βpromotes
dissemination of Theileria-transformed leukocytes could be via repres-
sion of both GZMA and RASGRP1 transcription and their impact on
dissemination that we confirmed in vivo in mice.
2.6 |GZMA loss of expression dampens H
2
O
2
levels
GZMA is a serine protease that contributes to killing of both tumours
and pathogen-infected cells via a caspase-independent pathway
(Chowdhury & Lieberman, 2008). GZMA expression induces reactive
oxygen species (Martinvalet, Zhu, & Lieberman, 2005) and attenuated
macrophages are known to be more oxidatively stressed than virulent
macrophages (Metheni et al., 2014). Indeed, H
2
O
2
output was
reduced in attenuated macrophages following CRISPR/Cas9-mediated
GZMA knockdown (Figure 5c).
2.7 |Induced expression of GZMA and RASGRP1
reduces human B lymphoma cell dissemination
In order to extend the roles of GZMA and RASGRP1 to human cancer
we sought human tumour cells displaying transcriptional signatures
similar to T. annulata-transformed leukocytes. To this end, the tran-
scriptional profiles of 934 human cancer cells were obtained from the
EBI cancer cell line expression atlas (Papatheodorou et al., 2018) and
their profiles compared to those of Theileria-transformed TBL3 and
TBL20 B lymphocytes (Figure 6a). A total of 46 out of 53 and 76 out
of 79 of the lymphoma and leukaemia cell lines respectively present in
the dataset, clustered in the subset of T. annulata-infected B cell lines
and their uninfected counterparts, representing 86 and 96% of the
total number of lymphoma/ leukaemia cell lines in the entire dataset,
FIGURE 3 Colony formation and invasiveness of Ode macrophages after RASGRP1 and GZMA knockdown. (a,b) qRT-PCR confirmation of
GZMA and RASGRP1 knockdown, respectively. (c) Matrigel chamber assay showing a regain in matrigel traversal after RASGRP1 and GZMA
knockdown. (d) Increased colony formation in soft agar following RASGRP1 knockdown. Non-transfected virulent disseminating Ode
macrophages are indicated by V, and non-transfected poorly disseminating attenuated Ode macrophages by A. Error bars represent SEM of three
biological replicates. ****p< .0005, ***p< .001, **p< .005 and *p< .05
6of14 RCHIAD ET AL.
respectively. The closely clustered cell line OCI-LY19, along with the
B cell non-Hodgkin lymphoma cell line RI-1, were used to test if
GZMA and RASGRP1 can act as suppressors of dissemination in cer-
tain types of human cancer. CRISPR-mediated transcriptional activa-
tion of GZMA and RASGRP1 resulted in decreased matrigel traversal
of the OCI-LY19 B lymphoma. By contrast, only up-regulation of
GZMA showed a statistically significant decrease in traversal of the
RI-1 B lymphoma (Figure 6c). Moreover, elevated expression of GZMA
and RASGRP1 decreased the ability of both RI-1 and OCI-LY19 B lym-
phoma cells to form colonies on soft agar (Figure 6d), providing fur-
ther functional evidence that GZMA and RASGRP1 have the potential
to act as suppressors in two independent human B lymphomas.
3|DISCUSSION
In this study, we provide a holistic view of the transcriptional land-
scape of two T. annulata-transformed B cell lines, TBL3 and TBL20,
and in addition the landscape of virulent versus attenuated Ode mac-
rophages. In order to find genes with commonly perturbed transcrip-
tion the different datasets were compared using three independent
pipelines to identify just four genes, as potential regulators of tumour
dissemination. In addition to MMP9 three other genes (SEPP1,GZMA
and RASGRP1) were identified as potentially having a role in dissemi-
nation. SEPP1 is a major selenoprotein involved in selenium transport
and cellular defence against oxidative stress (Hill, Dasouki, Phillips
3rd, & Burk, 1996). Attenuated macrophages did not survive CRISPR/
Cas9-knockdown of SEPP1 implying death might be due to a failure to
control excessive oxidative stress, since attenuated macrophages dis-
play high levels of H
2
O
2
(Metheni et al., 2014).
GZMA is known to cleave APEX1 (apurinic/apyrimidinic end-
odeoxyribonuclease 1) after Lys31 and cleavage destroys its oxidative
repair functions. APEX1 is involved in NK-cell-mediated killing via
GZMA (Fan et al., 2003; Martinvalet et al., 2005) and can suppress the
activation of PARP1 during repair of oxidative DNA damage (Peddi,
Chattopadhyay, Naidu, & Izumi, 2006), and prevent oxidative stress by
FIGURE 4 Effect of GZMA and RASGRP1 knockdown on transformed macrophage dissemination in vivo. Panels represent the copy number
of the single copy T. annulata gene (ama-1, TA02980) in the lung, mesentery and left kidney. Transformed macrophages were injected into five
Rag2γC immunodeficient mice and plotted values represent the mean of obtained T. annulata-specific ama1 gene copy number. There was no
obvious difference in proliferation rate between transfected cells and control in vitro, and no obvious difference in number of nuclei per schizont
between knockdown and control cells. Error bars represent SD of five biological replicates. ***p< .001, **p< .005 and *p< .05 compared to
virulent and attenuated Ode macrophages
RCHIAD ET AL.7of14
negatively regulating Rac1/GTPase activity (Ozaki, Suzuki, & Irani,
2002). Additionally, APEX1 directly reduces the redox-sensitive cysteine
residues of target transcription factors, enhancing their DNA binding
and transcriptional activity. Analysis of our deep RNA-seq data revealed
that APEX1 is down-regulated in attenuated macrophages and its
expression increases after TGF-βtreatment, along with an important
down-regulation of GZMA and RASGRP1 (Table S2). APEX1 is over-
expressed in many cancers (Kakolyris et al., 1998; Moore, Michael, Tritt,
Parsons, & Kelley, 2000; Yang, Irani, Heffron, Jurnak, & Meyskens,
2005) and has been implicated in growth, migration and invasion of
colon cancer both in vitro and in vivo (Kim, Kim, et al., 2013). Interest-
ingly, APEX-1 protects melanoma cells from H
2
O
2
induced apoptosis
(Yang et al., 2005). The established function of APEX1 in human cancer
sustains our novel observations on the tumour suppressor roles of
GZMA and RASGRP1. Published data and our results suggest that
reduced TGF-β2 signalling leads to an increase in the transcription of
GZMA which likely leads to APEX1 cleavage and, hence, an increase in
oxidative stress because of H
2
O
2
accumulation. This is consistent with
our data shown in Figure 5c, where CRISPR-Cas9-mediated knockdown
of GZMA caused a decrease in H
2
O
2
levels.
In order to investigate the expression levels of MMP9,GZMA,
RASGRP1 and SEPP1 in other human cancer types, we interrogated
the meta-analysis of lung cancer across 19 independent studies (Cai
et al., 2019). Interestingly, the findings suggest a striking similarity in
the differential expression profiles of three out of the four key genes.
MMP9 shows an up-regulation in lung cancer tissue [Effect size (ES):
15.22; p= 2.7E
−052
], while GZMA (ES: −4.99; p= 5.9E
−07
) and SEPP1
(ES: −11.4; p= 4.40E
−30
) show a marked down-regulation in expres-
sion profiles in comparison to healthy tissues (Cai et al., 2019). The
strong correlation in the differential expression profiles of these genes
reflects their relevance in at least two unrelated cancer types.
This study has revealed new players in dissemination and oxida-
tive stress regulation of Theileria-transformed leukocytes and has pro-
vided evidence for similar roles for GZMA and RASGRP1 in
transcriptionally matched human B lymphoma cell lines. The similarity
between Theileria-induced B cell transformation and human B
FIGURE 5 RASGRP1 and GZMA knockdown reduces GZMA expression and dampening of H
2
O
2
levels, respectively. (a) Log2FC values of
RASGRP1-4 in TBL3 and TBL20 RNAseq Log2FC values from DESeq2 of TBL3 and TBL20 compared to BL3 and BL20, respectively. (b) Effect of
RASGRP1 knockdown on GZMA expression. qRT-PCR of GZMA in virulent (V), attenuated (A) and attenuated Ode macrophages after CRISPR/
Cas9-mediated RASGRP1 knockdown. Error bars represent SD of three biological replicates *** and ### represent p< .001 compared to virulent
and attenuated Ode macrophages, respectively. (c) H
2
O
2
output by virulent (V), attenuated (A), and attenuated Ode macrophages after CRISPR/
Cas9-mediated GZMA knockdown. Error bars represent SD of three biological replicates. ** and ## represent p< .01 compared to virulent and
attenuated Ode macrophages, respectively. FC, fold change
8of14 RCHIAD ET AL.
lymphomas opens novel targets for intervention against tumour dis-
semination. This is a potent illustration of the benefits of trans-
disciplinary research in general, and of the use of infectious agents as
providers of untapped perspectives on fundamental cellular processes -
in this case, processes associated with cancer progression.
4|EXPERIMENTAL PROCEDURES
4.1 |Cell lines
The BL3 (Theilen et al., 1968), TBL3, BL20 (Morzaria, Roeder, Roberts,
Chasey, & Drew, 1984), TBL20 B lymphocytes, and Ode macrophages
(Singh et al., 2001) were cultured in RPMI 1640 medium sup-
plemented with 2 mM of L-glutamine (Lonza, catalogue number
12-702F) and 10 mM Hepes (Lonza, catalogue number 17-737E),
10% heat-inactivated FBS (Gibco, catalogue number 10082147),
100 units/mL of Penicillin and 100 μg/mL of streptomycin (Lonza, cat-
alogue number 17-602E) and 10 mM b-mercaptoethanol (Sigma-
Aldrich, catalogue number M6250) for BL3/TBL3 and BL20/TBL20.
The virulent (Vir) hyper-disseminating Ode cell line was used at low
passage (53–71), while its attenuated (Att) poorly disseminating vac-
cine counterpart corresponded to passages 309–317. The OCI-LY19
cell line (DSMZ, ACC 528) was cultured in Minimum Essential Medium
Eagle - Alpha Modification (Gibco, catalogue number 12000063) sup-
plemented with 2.2 g/L of sodium bicarbonate (Thermofisher
FIGURE 6 Effect of GZMA and RASGRP1 activation on human B lymphomas. (a) (Left panel) PCA based hierarchical clustering of 934 human
cancer cell lines and T. annulata-transformed bovine B cells and their non-infected counterparts. The cluster containing bovine cells mainly
contains leukaemic human cancer cell lines. The samples are coloured by cluster ID. (Middle cluster) The samples from the BL3/TBL3 and BL20/
TBL20 sub-cluster were reclustered by comparing the similarity of their gene expression profiles. The sample labels are coloured by their
similarity to each other. (Right panel) The sub-cluster containing bovine cell lines and human cancer cell lines with similar gene expression profile.
(b) qRT-PCR determination of GZMA and RASGRP1 expression after CRISPR-mediated gene activation in RI-1 (top panel) and OCI-LY19 (bottom
panel). The error bars represent SEM of three biological replicates. (c) Matrigel chamber assay illustrating the dissemination potential of RI-1 and
OCI-LY19 following activation of RASGRP1 and GZMA transcription. (d) Decrease in colony formation in soft agar of OCI-LY19 and RI-1 following
GZMA and RASGRP1 activation. Errors bars represent SEM values of three biological replicates *, ** and *** represent student ttest p< .05,
p< .005 and p< .0005, respectively
RCHIAD ET AL.9of14
Scientific, catalogue number 25080094), 20% FBS, 10 mM Hepes and
100 units/mL of Penicillin and 100 ug/ml of streptomycin. The RI-1
cell line (DSMZ, ACC 585) was cultured in RPMI 1640 and sup-
plemented with 10% FBS, 100 units/mL of Penicillin and 100 ug/ml
of streptomycin and 10 mM Hepes. All cell lines were incubated at
37C with 5% CO
2
. All cell lines were regularly tested for mycoplasma
contamination.
4.2 |RNA extraction
Cells were seeded in three biological replicates at a density of
2.5 ×10
5
cell/ml. RNA extraction was performed using the PureLink
RNA Mini Kit (Life technologies, catalogue number 12183018A) fol-
lowing the manufacturer's instructions. Briefly, cells were pelleted,
lysed and homogenised using a 21-gauge needle, then 70% ethanol
was added to the cell lysates and the samples were loaded on spin
cartridges to bind RNA. After three washes, RNA was eluted in
RNase-free water. The quality of extracted RNA was verified using a
Bioanalyzer 2100 and quantification carried using Qubit (Invitrogen,
catalogue number Q10210).
4.3 |Illumina library preparation and sequencing
Strand-specific RNA-sequencing (ssRNA-seq) libraries were prepared
using the illumina Truseq Stranded mRNA Sample Preparation Kit
(Illumina, catalogue number RS-122-2101) following the manufac-
turer's instructions. Briefly, 1ug of total RNA was used to purify
mRNA using poly-T oligo-attached magnetic beads. mRNA was then
fragmented and cDNA was synthesised using SuperScript III reverse
transcriptase (Thermofisher, catalogue number 18080044), followed
by adenylation on the 30end, barcoding and adapter ligation. The
adapter ligated cDNA fragments were then enriched and cleaned with
Agencourt Ampure XP beads (Agencourt, catalogue number A63880).
Libraries validation was conducted using the 1000 DNA kit on 2100
Bioanalyzer (Agilent Technologies, catalogue number 5067-1504) and
quantified using qubit (Thermofisher, catalogue number Q32850).
ssRNA libraries were sequenced on Illumina Hiseq2000 and
Hiseq4000. The sequenced reads were mapped to the Bos taurus
genome Btau 4.6.1. The quality of the sequenced libraries is shown in
Figure S1.
4.4 |Sequencing data analysis
The quality of sequence reads and other parameters were checked
using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/
fastqc/). The raw RNA-seq reads were processed for adaptor trimming
by Trimmomatic (Bolger, Lohse, & Usadel, 2014). The strand-specific
reads were mapped on to Bovine genome (bosTau7; Btau_4.6.1;
GCF_000003205.5) using Tophat2 (-g 1 --library-type fr-firststrand).
The samples with respective replicates were analysed further for
differential gene expression by three different tools, baySeq
(Hardcastle & Kelly, 2010), DESeq2 (Kim, Pertea, et al., 2013)
(fitType = ‘local’) and CuffDiff2 (Trapnell et al., 2013) with default
parameters unless mentioned specifically. The count values for
DESeq2 and baySeq were calculated from BAM files using HTSeq-
count tool (Anders, Pyl, & Huber, 2015). The transcriptome quality
plots were generated by cummeRbund package (v2.14.0) in R (http://
bioconductor.org/packages/release/bioc/html/cummeRbund.html).
The sequencing data is available in the NCBI Gene Expression Omni-
bus, GEO ID: GSE135377.
4.5 |Identification of differentially expressed
genes after infection and attenuation by comparative
transcriptome analysis
The transcriptome data was analysed with baySeq, DESeq2 and
CuffDiff2. A gene was considered as a DEG if it has a padj < .05 and a
fold change (FC) > 2. The final list of DEGs contained genes com-
monly differentially expressed in CuffDiff2, DESeq2 and baySeq. This
approach minimalizes the total number of DEGs for further analysis
and allows stringent selection of the most significant and
reproducible DEGs.
4.6 |qRT-PCR
Total RNA was reverse transcribed using the High Capacity cDNA
Reverse Transcription Kit (Applied Biosystems, catalogue number
4368814) as follows: 2 μg of total RNA, 2 μL of RT buffer, 0.8 μLof
100 mM dNTP mix, 2.0 μLof10×random primers, 1 μL of MultiScribe
reverse transcriptase and Nuclease-free water to a final volume of
20 μL. The reaction was incubated 10 min at 25C, 2 hr at 37C then
the enzyme inactivated at 85C for 5 min. Real-time PCR was per-
formed in a 10 μL reaction containing 20–30 ng cDNA template, 5 μL
2×Fast SYBR Green Master Mix and 500 nM of forward and reverse
primers. The reaction was run on the 7500 HT Fast Real-Time PCR
System (Applied Biosystems). GAPDH was used as a housekeeping
gene and the results were analysed by the 2
−ΔΔCT
method. The error
bars represent the SEM of three biological replicates. Primers were
designed and assessed for secondary structures using the Primer
Express Software v3.0. The primers of all genes are listed in Table S3.
4.7 |Transfection
Macrophages were transfected by electroporation using the
Nucleofector system (Amaxa Biosystems). A total of 5 ×10
5
cells
were suspended in 100 μL of Nucleofector V solution mix (Lonza,
VCA-1003) with 2 μgofGZMA (sc-437323), RASGRP1 (sc-437322)
CRISPR/Cas9 KO plasmids, or a negative control plasmid harbouring
non-specific guide RNA (SCBT, sc-418922) was used in nucleofection
using the cell line-specific program T-O17. CRISPR/Cas9 KO plasmids
10 of 14 RCHIAD ET AL.
consist of a pool of three plasmids, each encoding the Cas9 nuclease
and a different target-specific 20 nt guide RNA (gRNA) designed for
maximum efficiency. The human B lymphoma cells were transfected
with 2 μgofGZMA (SCBT, sc-403958-ACT) or RASGRP1 (SCBT, sc-
402120-ACT) synergistic activation mediator (SAM) transcription acti-
vation system consisting of three plasmids encoding the deactivated
Cas9 (dCas9) nuclease, the MS2-p65-HSF1 fusion protein and a
target-specific 20 nt guide RNA. A non-specific control plasmid with a
non-specific guide RNA was used as a control (SCBT, sc-437275). The
transfection was done using the T-O17 program. After transfection,
cells were suspended in fresh complete medium and incubated at
37C with 5% CO
2
.
4.8 |Matrigel chamber assay
The invasive capacity of Ode macrophages was assessed in vitro using
matrigel migration chambers, as described in (Lizundia et al., 2006).
The CultureCoat Medium basement membrane extract (BME)
96-wells cell invasion assay was performed according to Culturex
instructions (Trevigen, catalogue number 3482-096-K). After 24 hr of
incubation at 37C, each well of the top chamber was washed once in
buffer. The top chamber was placed back onto the receiver plate. One
hundred microliters of cell dissociation solution-Calcein AM was
added to the bottom chamber of each well, and the mixtures were
incubated at 37C for 1 hr with fluorescently labelled cells to dissoci-
ate the cells from the membrane before reading at 485-nm excitation
and 520-nm emission wavelengths.
4.9 |Soft agar colony forming assay
A two-layer soft agar culture system was used. Cell counts were per-
formed by ImageJ software. A total of 2,500 cells were plated in a vol-
ume of 1.5 mL (0.7% bacto Agar + 2×RPMI 20% Foetal bovine
Serum) over 1.5 mL base layer (1% bacto agar + 2×RPMI 20% Foetal
bovine Serum) in six-well plates. Cultures were incubated in humidi-
fied 37C incubators with an atmosphere of 5% CO
2
in air, and con-
trol plates were monitored for growth using a microscope. At the time
of maximum colony formation final colony numbers were counted in
image J after fixation with 0.005% Cristal Violet.
4.10 |Intracellular levels of hydrogen
peroxide (H
2
O
2
)
Cells were seeded at 1 ×10
5
cell/well in a 96 well plate and incu-
bated in complete medium for 18 hr prior to the assay. Cells were
then washed with PBS and incubated with 100 μLof5M
H2-DCFDA in PBS (Molecular Probes, catalogue number D399).
H
2
O
2
levels were assayed on a fusion spectrofluorimeter
(PackardBell) at 485 and 530 nm excitation and emission wave-
lengths respectively.
4.11 |In vivo mouse studies and quantification of
T. annulata-transformed macrophages load in mouse
tissues
Theileria annulata-infected macrophage cell lines (Virulent Ode pas-
sage 53, attenuated Ode passage 309, attenuated Ode transfected
with RASGRP1 CRISPR/Cas9 knockout plasmid and attenuated Ode
transfected with GZMA CRISPR/Cas9 knockout plasmid) were
injected into four groups of five Rag2γC immunodeficient mice that
were equally distributed on the basis of age and sex in each group.
The injection site was disinfected with ethanol and one million cells
(in 200 μL PBS) were injected under the skin after gentle shaking of
the insulin syringe. The mice were kept for 3 weeks and then they
were humanely sacrificed and dissected. Six internal organs including
heart, lung, spleen, mesentery, left kidney and liver were taken and
stored in 500 μL PBS in Eppendorf tubes at −20C. The tissues were
subjected to genomic DNA extraction using the QIAmp DNA mini kit
(Qiagen, catalogue number 51304). DNA concentrations were mea-
sured by Nanodrop 1000 spectrophotometer (Thermo Fischer scien-
tific) and before each quantitative PCR reaction samples were diluted
to give a DNA concentration of 0.5 ng/μL. Absolute copy numbers of
a single copy T. annulata gene (ama-1, TA02980) that is representative
of T. annulata-infected macrophage load in each tissue were esti-
mated by the method described in Gotia et al. (2016), with some mod-
ifications. Ama-1 was cloned into pJET 1.2/blunt cloning vector using
CloneJET PCR Cloning Kit (Thermo scientific, catalogue number
K1232). The cloned plasmid was amplified in DH5-Alpha cells and
purified with QIAfilter Plasmid Maxi Kit (Qiagen, catalogue number
12243). Plasmid concentration was measured using Qubit
(Thermofisher, catalogue number Q32850). The primers for cloning
were: forward 50-GGAGCTAACTCTGACCCTTCG-30and reverse 50-
CCAAAGTAGGCCAATACGGC-30. Quantitative PCR primers were:
forward 50-GACCGATTTCATGGCAAAGT-30and reverse 50-TTGGGG
TCATGATGGGTTAT-30.
4.12 |Transcriptome-based clustering of Theileria-
transformed bovine host cells and human cancer cell
lines
The processed and quality trimmed reads from TBL20/BL20 and
BL3/TBL3 samples were mapped to Bos taurus UMD3.1 genome
using HISAT2 software (Kim, Langmead, & Salzberg, 2015) with
default settings. The mapped reads were used for gene-level TPM
quantification using StringTie (Version 1.3.3b) (Pertea et al., 2015;
Pertea, Kim, Pertea, Leek, & Salzberg, 2016). The quantified genes
were converted into their human ortholog Ensemble gene ID by find-
ing one-to-one orthologs between human and B. taurus genomes
using OMA browser (Altenhoff et al., 2018). Subsequently, TPM
values of transcripts expressed across 934 human cancer cell lines
were obtained from EBI cancer cell line Expression Atlas
(Papatheodorou et al., 2018). The redundant transcripts in the cancer
cell line expression set were collapsed using collapseRow function
RCHIAD ET AL.11 of 14
from the WGCNA R package (Langfelder & Horvath, 2008). Using the
common human ensemble gene ID, gene expression matrices of
B. taurus and human cell lines were merged together, which was then
subjected to hierarchical clustering of samples using HCPC (Lê,
Josse, & Husson, 2008) with three Principal Components (nPCs),
which resulted in four broad clusters. The sub-cluster containing the
human cancer cell lines along with TBL20/BL20 and BL3/TBL3 were
shortlisted for further analysis. Whereby, samples were scanned for
similar gene expression profiles by computing the adjacency of each
shortlisted sample with the rest of the samples, using adjacency func-
tion (method = ‘Distance’) from WGCNA R package. The resultant
adjacency matrix was then subjected to flashClust (Lê et al., 2008)
program for computing the dendrogram for manual inspection of
TBL20/BL20 and BL3/TBL3 containing sub-cluster. A schematic of
the used pipeline is presented in Figure S5. The complete dendrogram
of the 934 human cancer cell lines and Theileria-transformed lympho-
cytes can be viewed in Figure S6.
4.13 |Ethics statement
The protocol (Burk et al., 1995; Grandclement et al., 2011; Haidar
et al., 2018; Kim et al., 2004; Kinnaird et al., 2013; Kurz et al., 2017;
Mangala et al., 2009; Marsolier et al., 2013; Merlo et al., 2005; Perche
et al., 2000; Scott & Wang, 2011; Short, Whitten-Barrett, & Williams,
2016; Tanaka et al., 2016; van der Vuurst de Vries, Clevers, Log-
tenberg, & Meyaard, 1999; Yu & Stamenkovic, 2000) was approved
by the ethics committee for animal experimentation of the University
of Paris-Descartes (CEEA34.GL.03312). The university ethics commit-
tee is registered with the French National Ethics Committee for Ani-
mal Experimentation that itself is registered with the European Ethics
Committee for Animal Experimentation. The right to perform the mice
experiments was obtained from the French National Service for the
Protection of Animal Health and satisfied the animal welfare condi-
tions defined by laws (R214-87 to R214-122 and R215-10) and GL
was responsible for all animal experimentation, as he holds the French
National Animal Experimentation permit with the authorisation num-
ber (B-75-1249). This project is also covered by the KAUST IBEC
number 19IBEC12.
ACKNOWLEDGEMENTS
This study was supported by a Competitive Research Grant
from the Office for Sponsored Research (OSR-2015-CRG4-2610) at
King Abdullah University of Science and Technology (KAUST)
awarded to A. P. and G. L. Z. R. acknowledges KAUST for awarding
her PhD studentship. S. T. was supported by a post-doctoral fellow-
ship from ParaFrap (ANR-11-LABX-0024) and in addition to ANR-
11-LABX-0024, G. L. also acknowledges core support from INSERM
and the CNRS. We thank members of the Bioscience Core Labora-
tory (BCL) at KAUST for producing the raw sequencing datasets.
We also thank Franck Letourneur of the genomics platform at the
Cochin Institute (GENOM'IC) for quantifying the pJET-ama-1
plasmid.
CONFLICT OF INTEREST
The authors declare no conflicts of interest.
AUTHOR CONTRIBUTIONS
Arnab Pain and Gordon Langsley conceived and designed the study.
Zineb Rchiad prepared the ssRNAseq libraries. Sara Mfarrej, Malak
Haidar and Zineb Rchiad ran qRT-PCR reactions. Malak Haidar per-
formed the soft agar colony formation assay and Matrigel chamber for
Theileria-infected macrophages and Zineb Rchiad and Sara Mfarrej for
human cancer cell lines. Malak Haidar performed the intracellular
H
2
O
2
levels and Matrigel chamber assays. Shahin Tajeri performed
the mouse dissemination assays. Hifzur Rahman Ansari, Abhinav
Kaushik and Zineb Rchiad performed data analysis. Zineb Rchiad and
Malak Haidar prepared the figures. Zineb Rchiad prepared the first
draft of the manuscript with input from Fathia Ben Rached that was
then edited by Malak Haidar, Gordon Langsley and Arnab Pain.
ORCID
Gordon Langsley https://orcid.org/0000-0001-6600-6286
Arnab Pain https://orcid.org/0000-0002-1755-2819
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of this article.
How to cite this article: Rchiad Z, Haidar M, Ansari HR, et al.
Novel tumour suppressor roles for GZMA and RASGRP1 in
Theileria annulata-transformed macrophages and human B
lymphoma cells. Cellular Microbiology. 2020;22:e13255.
https://doi.org/10.1111/cmi.13255
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