Computers in Biology and Medicine 134 (2021) 104470

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Computers in Biology and Medicine

journal homepage: www.elsevier.com/locate/compbiomed

New drug candidates for osteosarcoma: Drug repurposing based on gene

expression signature
Raissa Coelho Andrade a, b, Mariana Boroni c, d, Marion Kielmanowicz Amazonas a,
Fernando Regla Vargas a, b, *
a
Birth Defects Epidemiology Laboratory, Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, Brazil
b
Genetics and Molecular Biology Department, Federal University of the State of Rio de Janeiro (UNIRIO), Rio de Janeiro, Brazil
c
Bioinformatics and Computational Biology Lab, Division of Experimental and Translational Research, Brazilian National Cancer Institute (INCA), Rio de Janeiro, Brazil
d
Experimental Medicine Research Cluster (EMRC), University of Campinas (UNICAMP), Campinas, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Osteosarcoma (OS) is an aggressive bone malignancy and the third most common cancer in adolescence. Since
Osteosarcoma the late 1970s, OS therapy and prognosis had only modest improvements, making it appealing to explore new
Microarray expression data tools that could help ameliorate the treatment. We present a meta-analysis of the gene expression signature of
Gene expression signature
primary OS, and propose small molecules that could reverse this signature. The meta-analysis was performed
Drug repurposing
Afatinib
using GEO microarray series. We first compared gene expression from eleven primary OS against osteoblasts to
obtain the differentially expressed genes (DEGs). We later filtered those DEGs by verifying which ones had a
concordant direction of differential expression in a validation group of 82 OS samples versus 30 bone marrow
mesenchymal stem cells (BM-MSC) samples. A final gene expression signature of 266 genes (98 up and 168 down
regulated) was obtained. The L1000CDS2 engine was used for drug repurposing. The top molecules predicted to
reverse the signature were afatinib (PubChem CID 10184653), BRD-K95196255 (PubChem CID 3242434), DG-
041 (PubChem CID 11296282) and CA-074 Me (PubChem CID 23760717). Afatinib (Gilotrif™) is currently used
for metastatic non-small-cell lung cancer with EGFR mutations, and in vitro evidence shows antineoplastic po­
tential in OS cells. The other three molecules have reports of antineoplastic effects, but are not currently FDA-
approved. Further studies are necessary to establish the potential of these drugs in OS treatment. We believe
our results can be an important contribution for the investigation of new therapeutic genetic targets and for
selecting new drugs to be tested for OS.

1. Introduction malignancy [4].

Osteosarcoma (OS) is an aggressive bone malignancy that typically
occurs in the second decade of life, during the pubertal growth spurt [1].
It is the third most common cancer in adolescence, with approximately
60% of the cases occurring before the age of 25 years old [2,3]. A sec­
ondary incidence peak is observed in patients over 65 years old,
although in these cases it is most commonly associated with a previous
bone disease, while in young individuals it is considered a primary
In adolescents and young adults, the most common sites of OS are the
metaphysis of the distal femur, the proximal tibia and the proximal
humerus, with the most common clinical presentation being chronic
pain with indolent evolution in the affected area [1]. Prognosis of OS has
significantly improved with the introduction of chemotherapy in the late
1970s, but since then, therapy has remained essentially the same [5–7],
and the current 5-year overall survival is still less than 70% in localized
disease, and less than 20% in 3 years for metastatic disease, that occurs

Abbreviations: BM-MSC, bone marrow mesenchymal stromal/stem cells; DEGs, Differentially expressed genes; ECM, extracellular matrix; FDR, false discovery
rate; GEO, Gene Expression Omnibus (database); GSE, GEO series; GSEA, Gene Set Enrichment Analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes;
L1000CDS2, LINCS L1000 characteristic direction signatures; ORA, Overrepresentation analysis; OS, Osteosarcoma; WebGestalt, WEBbased GEne SeT AnaLysis
Toolkit.
* Corresponding author. Birth Defects Epidemiology Laboratory, Oswaldo Cruz Foundation (FIOCRUZ), Av. Brasil 4365 – Pavilhão Leônidas Deane Sala 617,
Manguinhos, Rio de Janeiro, 21040-360, Brazil.
E-mail address: fernando.vargas@ioc.fiocruz.br (F.R. Vargas).

https://doi.org/10.1016/j.compbiomed.2021.104470
Received 25 February 2021; Received in revised form 1 May 2021; Accepted 2 May 2021
Available online 7 May 2021
0010-4825/© 2021 Elsevier Ltd. All rights reserved.
R.C. Andrade et al.
most commonly in the lungs [7]. Surgery is the pillar of the treatment,
associated to neoadjuvant and adjuvant chemotherapy. Four chemo­
therapy agents are included in the standard protocols as first line
treatment: methotrexate with leucovorin rescue, doxorubicin, cisplatin,
and ifosfamide [2].
The etiology of OS remains obscure. Risk factors for the young pa­
tients include taller stature, rapid bone growth, male sex, high birth
weight and some genetic conditions such as Li-Fraumeni syndrome,
hereditary retinoblastoma and syndromes related to mutations in the
RECQL family of genes (like Rothmund–Thomson syndrome) [1,8].
Previous exposure to ionizing radiation is the only recurrently reported
environmental risk factor for the young [1,8].
Histologically, osteosarcomas are marked for the production of
osteoid tissue and the most common type is the conventional OS, rep­
resenting >80% of the cases, and characterized by different amounts of
osteoblastic, chondroblastic, and fibroblastic cells [2]. Two types of cells
have been suggested as the tumor cell-of-origin: mesenchymal stem cells
and a more differentiated/committed osteoblastic cell population, both
of which could go through malignant transformation within the appro­
priate molecular signals [9,10]. The typical genomic profile of OS is
described as chaotic and complex, including a high somatic mutation
rate and kataegis, complex chromosomal rearrangements, inactivating
mutations in TP53 and RB1, and activating mutations in PI3K/Akt/m­
TOR pathway genes [11]. Commonly gene expression alterations
include the Notch and Wnt pathway upregulation, overexpression of
Runx2 and Osterix (transcription factors required for osteoblast differ­
entiation), EZR (responsible for linking the cytoskeleton to the cell
membrane), downregulation of Fas and Fas ligand (regulators of cellular
apoptosis) and receptor tyrosine kinase genes like ALX and HER-2 [1,
12].
Despite the many studies developed to decode the molecular causes
of OS, translational applicability has remained a challenge. The insuf­
ficient improvement in chemotherapy regimens and survival over the
last decades makes it appealing to explore new tools for discovering
small molecules that could help ameliorate OS treatment [7]. In this
study, we present a meta-analysis of the gene expression signature from
primary OS tumors, and propose small molecules that could reverse this
signature, based on a drug repurposing bioinformatics tool. We believe
the results presented here can serve as a groundwork for further inves­
tigation targeting OS altered pathways.
2. Methods
2.1. Data collection
We searched the GEO database (www.ncbi.nlm.nih.gov/geo/) to
retrieve different series of samples that accessed the mRNA expression
profiles in osteosarcoma and normal bone samples. Our filters included:
organism: “Homo sapiens” and study type: “Expression profiling by
array”, focused in the Affymetrix platforms. The search terms included
“osteosarcoma”, “bone marrow mesenchymal stromal/stem cells” (or
“BM-MSC”), and “osteoblasts”. For the OS samples, we only considered
studies that used primary specimens, i.e.: biopsies and surgical samples,
excluding studies that used cell lines derived from tumor tissue. OS
samples from metastases were also excluded. For all the data series,
median age of the patients and healthy donors should be < 40 years old,
and the number of relevant samples in each series should be ≥ 3. Other
tissue types investigated in each series were not included in the analysis.
Our analysis was divided into two steps: test group and validation group.
Test group: we searched for series that included both tumor and
normal samples in the same experiment, to properly remove the batch
effect while performing the meta-analysis.
Validation group: we validated the results using independent series
that included either primary tumor tissues from patients with OS or BM-
MSC samples from healthy controls. For the BM-MSC samples, we only
considered studies that used early passage MSCs from bone marrow
2
Computers in Biology and Medicine 134 (2021) 104470
samples extracted and processed in loco, excluding studies that used
purchased BM-MSCs. We also excluded studies that used BM-MSCs
extracted from patients with osteoarthritis or osteoporosis.
2.2. Data processing and differential gene expression
The analysis were performed with the statistical computing envi­
ronment R. Raw data from each of the selected GEO series (microarray.
CEL files) were converted to expression measures and normalized using
either affy or oligo packages [13,14]. For integrating datasets from
different platforms, the probe IDs were converted to the respective Gene
Symbols, retrieved from the annotation package of each Affymetrix
platform. When more than one probe for the same gene was present, the
mean expression value of the probes was calculated.
Test group: the expression matrices generated by the two datasets in
the test group were combined. Only the genes in common between the
two different Affymetrix platforms were kept for further analysis. The
ComBat function (sva package [15]) was applied to the samples. This
function removes the batch effect, allowing data from different experi­
ments to be comparable in the same analysis, which increases the
robustness of the results obtained. Then, differential gene expression
analysis between osteosarcoma and osteoblasts was conducted using the
Limma package [16]. For the identification of differentially expressed
genes in microarray data, a combination of thresholds for adjusted
p-value (FDR) and log fold change yields better results than just the
p-value or fold change alone [17]. In this study, DEGs were defined by
applying a log fold change (logFC) threshold of >2, and a false discovery
rate (FDR) threshold of <0.05, which are common thresholds for this
analysis [18–22].
Validation group: as in the test group, the expression matrices of the
eight datasets in the validation group were combined, genes that were
not present in all the different Affymetrix platforms were filtered out,
and the ComBat function was applied to the samples. Except that here,
each dataset included exclusively tumor or normal samples. Therefore,
since the ComBat function would also remove the expression differences
caused by the biological status of the samples, we applied the function in
two steps: first in the tumor samples, then in the normal samples. To
minimize the possible biases inserted by this approach, we did not use
the same statistical criteria applied to the test group to define differen­
tially expressed genes. Instead, after performing the analysis with
Limma, we explored the expression consistency of the DEGs identified in
the test group by verifying if these genes had a concordant direction of
differential expression in the validation group. The genes with a
concordant differential expression were considered validated and
referred to as the gene expression signature for OS. Heat maps were
constructed considering the OS gene signature for both the test and
validation groups using the pheatmap package version 1.0.12. Genes
(rows) in the heatmap of the test group were clustered using the 1-Pear­
son correlation coefficient as the distance metrics and “average” as the
agglomeration method; in the heatmap of the validation group, the
genes were arranged in the same order of the test group. The samples
(columns) were also clustered using the 1-Pearson correlation coeffi­
cient as the distance metrics and “average” as the agglomeration method
for both the heatmaps.
2.3. Functional analysis
The differential expression analysis of the test group was assessed for
enriched pathways using the Gene Set Enrichment Analysis (GSEA),
with the respective log fold change scores for each gene. Then, the
validated gene signature was assessed for enriched pathways through
the overrepresentation analysis (ORA) methodology. Both in­
vestigations were performed in the WEBbased GEne SeT AnaLysis
Toolkit (WebGestalt) using the Kyoto Encyclopedia of Genes and Ge­
nomes (KEGG) database [23]. The p-values were controlled for false
discovery rate (FDR) using the Bonferroni method, considered
R.C. Andrade et al. Computers in Biology and Medicine 134 (2021) 104470

significant when <0.05.
2.4. Drug repurposing
The gene signature obtained was used as an input in the LINCS L1000
characteristic direction signatures search engine (L1000CDS2) [24] to
search for drugs and small molecules that could reverse the expression
signature of OS samples. This engine is part of the Library of Integrated
Network-based Cellular Signatures (LINCS) project, which investigates
expression profiles generated by small molecules (perturbagens) in
human cells. The gene expression perturbations in the LINCS mRNA
profiling assay come from tests with multiple cell lines, mostly
cancerous ones, including the U2OS cell line, originated from osteo­
sarcoma [24,25]. When up/down regulated gene lists are submitted to
L1000CDS2, they are compared to the genes computed from the LINCS
L1000 data and the top 50 molecules which either mimic or reverse
(according to the user’s selection) the input expression profile are
returned, with a score for each drug. The score is the number of over­
lapping genes between the input and the database signature for the drug
divided by the effective input (i.e., the genes from the input that are
present in the L1000 database). The top results were manually refer­
enced in the database PubChem [26] for further analysis of the drugs of
interest.
3. Results
3.1. Selected data
Our selection criteria led us to 10 independent GEO series, charac­
terized in Table 1. The test group was composed by GSE12865 and
GSE14359, which included, together, eleven primary OS tumor samples
in duplicate and two osteoblast cell lines in duplicate. The validation
group included eight independent series: three series with a total of 82
primary tumor tissues from patients with OS (GSE14827, GSE16091,
GSE87437) and five series with a total of 30 BM-MSC samples from
healthy controls (GSE84881, GSE18698, GSE110359, GSE113736,
GSE122778).
down-regulated in OS (Fig. 1A). We then assessed the expression con­
sistency of these DEGs by verifying if they had a concordant direction of
differential expression in the validation group (i.e.: same signed logFC).
Of the 283 genes, 280 had matched gene symbols across all the different
Affymetrix array platforms used in the validation groups (106 of the up-
regulated and 174 of the down-regulated genes). These 280 genes
yielded a 95% (266/280) differential expression concordance rate be­
tween the test and validation groups: 98/106 up-regulated genes and
168/174 down-regulated genes (Fig. 1B). The heatmaps comparing the
expression patterns of these 280 genes between test and validation
groups are exhibited in Fig. 1C. The top 20 up and down-regulated genes
observed in the test group are shown in box 1. The full list with the up
and down-regulated genes is available at Supplementary Tables 1 and 2
3.3. Functional analysis
Gene set enrichment analysis for KEGG pathways in the test group is
represented in Fig. 2A and Table 2. The over-representation analysis,
performed with the subset of 98 up- and the 168 down-regulated vali­
dated genes is exhibited in Fig. 2B (top and bottom graphics, respec­
tively). Positively enriched pathways were associated to inflammatory
response, while negatively enriched pathways included p53-signaling.
The ORA analysis revealed additional down-regulated pathways such
as extracellular matrix (ECM) receptor interaction, proteoglycans in
cancer and focal adhesion. The concomitant pathways between the
GSEA and ORA analysis are shown in bold in Table 2.
3.4. Drug repositioning in OS
The LINCS L1000 Characteristic Direction Signature Search Engine
was utilized to query the best molecules which could reverse the gene
expression signature for OS (i.e., the validated differentially expressed
genes), and thus be investigated for the treatment of this neoplasm.
Fig. 2C exhibits the clustergram with the best 50 matches of small
molecules, of which 40 were unique (the same molecule can have
multiple entries in the L1000CDS2 database, one for each cell line it was
tested). Four drug candidates were predicted to reverse the expression of

Table 1
Characteristics of the microarray GSE series used in this study.
GEO series Type of sample No of samples Naive tumor samples?a Median age of patients/controls (years) Affymetrix platform

Test group
GSE12865 Primary tumor/osteoblasts 6/1b Yes NA (all pediatric) HuGene-1_0-st
GSE14359 Primary tumor/osteoblasts 5/1b NA 17 HG-U133A
Validation group
GSE14827 Primary tumor 27 Yes 15 HG-U133_Plus_2
GSE16091 Primary tumor 34 Yes 12,5 HG-U133A
GSE87437 Primary tumor 21 Yes 15 HG-U133_Plus_2
GSE84881 BM-MSC 4 – 22,5 HG-U133_Plus_2
GSE18698 BM-MSC 3 – 36 HuEx-1_0-st
GSE110359 BM-MSC 5 – 14,6c HTA-2_0
GSE113736 BM-MSC 4d – 26,5 HuEx-1_0-st
GSE122778 BM-MSC 14 – 36 HuGene-1_0-st

NA: non-available information. BM-MSC: Bone marrow mesenchymal stem cells.

a
Naive tumor samples refer to tumors collected before chemotherapy. b Number of original samples. The duplicates available in GEO were also used. c Mean age
(median age not available)d Only the first replicate of each sample of GSE113736 was used.

3.2. Differential gene expression between osteosarcoma and normal bone
samples
We searched for differentially expressed genes in osteosarcoma by
comparing gene expression levels between osteosarcoma samples and
osteoblasts in our test group. A total of 12,040 genes were analyzed.
After applying a log fold change threshold of >2 and an FDR threshold of
<0.05, 283 genes were identified: 108 were up-regulated and 175 were
at least 10% of our input genes. Of these molecules, only one is currently
FDA-approved: afatinib (S1011; PubChem CID 10184653), which was
also the best scored match in our search, and is already used as an
antineoplastic agent. The gene modulation of afatinib in OS predicted by
our analysis is exhibited in Table 3. Besides afatinib, three molecules
met our selection criteria (Table 3): BRD-K95196255 (PubChem CID
3242434), which is a hydroxyquinoline with cytotoxic activity; DG-041
(or DTSI; PubChem CID 11296282), that acts as a prostaglandin receptor

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R.C. Andrade et al. Computers in Biology and Medicine 134 (2021) 104470

Fig. 1. Identification of OS gene signature. A) Volcano plot of the gene expression profile identified in the test group. Thresholds for DEGs were log fold change
>2/< − 2 and FDR <0.05. B) Diagram evidencing the consistency of expression direction of these genes between the test and validation groups. C) Heatmaps of DEGs
in test and validation groups. Columns correspond to the samples and rows correspond to the genes. Each batch represents one GEO series. Left: Heatmap of the test
group, exhibiting the expression patterns of 280 DEGs between 11 primary OS samples and two osteoblast cell lines. Batch 1: GSE14359; batch 2: GSE12865. Right:
Heatmap of the validation group, exhibiting the expression patterns of 280 DEGs between 82 primary OS samples and 30 BM-MSC samples. Batch 1: GSE14827; batch
2: GSE16091; batch 3: GSE84881; batch 4: GSE87437; batch 5: GSE110359; batch 6: GSE113736; batch 7: GSE122778; batch 8: GSE18698. The concordance rate of
direction of expression (i.e.: same-signed logFC) between test and validation group was 95% (266 of 280 genes).

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Box 1
Top up and down-regulated genes in OS. LogFC and FDR are correspondent to the test group.

Top 20 up and down-regulated genes in OS

Up-regulated genes Down-regulated genes
Gene symbol logFC FDR Gene symbol logFC FDR
MDFI 2.556284 2.767355e-12 TPD52L1 − 3.338841 6.711174e-17
A2M 5.909319 2.304380e-11 PENK − 4.364210 8.209018e-16
RNF144A 2.140703 5.632540e-10 SYNC − 3.262922 1.739980e-15
ARHGDIB 3.775558 1.039917e-09 FGF2 − 3.480656 2.936067e-15
MCAM 2.426295 4.577510e-09 GREM2 − 2.648890 1.134880e-14
ALPL 4.137270 8.122120e-09 LRRC2 − 2.798352 1.260484e-14
LCP1 2.659893 9.478168e-09 SLC14A1 − 4.672709 1.260484e-14
CPVL 3.240216 3.238323e-08 FGF5 − 4.050403 4.140875e-14
LYZ 4.689291 3.847037e-08 ISLR − 3.886011 5.889290e-14
HLA-DRA 5.400430 5.187442e-08 PHLDA3 − 2.310864 1.324531e-13
RASGRP3 2.216738 1.275734e-07 IL26 − 2.670094 2.534270e-13
CD93 2.396420 1.678738e-07 AVPI1 − 2.987813 2.895176e-13
HLA-DRB1 3.936381 2.185956e-07 RPS6KA2 − 3.129784 4.639098e-13
PECAM1 3.413068 2.585345e-07 QSOX1 − 3.214736 8.528371e-13
LAPTM5 3.777067 3.172885e-07 MOXD1 − 2.830525 1.025251e-12
CD53 3.143652 4.841996e-07 SERPINE1 − 4.864428 1.226166e-12
ELMO1 2.538471 8.119163e-07 AOX1 − 2.199167 1.285913e-12
GPC4 2.459877 1.500226e-06 CITED2 − 3.383479 1.584868e-12
CPE 4.395745 1.535049e-06 VGLL3 − 3.279918 1.584868e-12
MEF2C 3.308009 1.584478e-06 BST1 − 2.245540 2.041588e-12

antagonist; and CA-074 methyl ester (PubChem CID 23760717), which
is a cathepsin inhibitor. The full list of molecules is available in Sup­
plementary Table 3.
4. Discussion
Osteosarcoma is an aggressive primary malignancy of the bone, with
very limited information regarding environmental risk factors associ­
ated to its etiology [27,28]. Also, due to its rarity and peak incidence in
adolescence, osteosarcoma may often be missed on an initial physical
exam, since the primary symptom of chronic pain may be wrongly
attributed to athletics or rapid bone growth [27,29]. Those factors may
difficult the development of prevention and early detection strategies.
Indeed, since the mid-1980s, only modest progress has been made
concerning the survival of patients diagnosed with osteosarcoma [5].
Currently, the 5-year event-free survival of patients is 54%, even if
complete surgical resection is possible at diagnosis [6]. A better un­
derstanding of OS molecular features may enhance the development of
therapeutic strategies and have an impact on the morbidity and out­
comes for this disease [30].
In this study, we searched for a gene expression signature for OS, by
performing a meta-analysis from different GEO microarray series. We
therefore managed to analyze a large number of samples of a rare tumor
while also applying stringent selection criteria to the data. As a first step,
we compared gene expression from eleven primary OS samples against
osteoblasts to obtain the significantly differentially expressed genes. We
later filtered those DEGs by verifying which ones had a concordant di­
rection of differential expression in a validation group of 82 primary OS
samples versus 30 BM-MSC samples from healthy controls. Our analysis
yielded a 95% differential expression concordance rate between the test
and validation groups, with a final number of 266 genes (98 up and 168
down-regulated) deemed to constitute the gene expression signature.
An important approach in this work was the use of different cell types
as normal bone controls in the test and validation groups. There has
been a debate over which is the cell of origin of osteosarcoma. The two
best candidates are mesenchymal stem cells and a more differentiated/
committed osteoblastic cell population [9,10]. The primary location of
OS within metaphysis of long bones and its peak occurrence during rapid
growth phases in adolescence imply that rapidly dividing cells are
involved in this process. Both mesenchymal stem cells and
pre-osteoblasts fit this requirement, and there are in vitro and in vivo
evidences in favor of both cell types as possible cells of origin [9,10]. The
most likely scenario is that either cell type may originate OS under the
influence of proper genetic background, microenvironmental or epige­
netic signals [9]. Indeed, the results obtained here show a concordant
direction of differential expression of OS DEGs when compared both to
osteoblasts and BM-MSCs, favoring this hypothesis.
Also importantly, our analysis only considered primary tumor sam­
ples for evaluation, excluding OS cell lines, which can eliminate false
positive results due to differences in expression pattern between tumor
and tumor cell line samples [31]. Likewise, we selected series of patients
with a median age <40 years. It is known that OS has two peak in­
cidences through life: adolescents/young adults and elderly [27]. In the
elderly, OS often arises in the context of a previous condition, such as the
malignant transformation of Paget disease of bone or post radiotherapy
[32]. Thus, our selection criteria can give a more trustworthy expression
profile of the primary OS tumors of young individuals. Another advan­
tage is that at least 88 of the 93 OS samples analyzed in this study were
naive samples, avoiding possible alterations in gene expression that
could be caused by chemotherapeutic treatment.
Among the DEGs identified in our analysis MDFI was the most up-
regulated gene. This gene encodes the MDFI protein, a MyoD family
inhibitor [33], thus inhibiting myogenesis differentiation of mesen­
chymal lineage cells. MyoD can upregulate expression of p21 and
maintain the tumor suppressor pRb in its active state. Thus, MyoD both
induces myogenic phenotype and promotes cell cycle arrest, which
emphasizes the interconnection between cell differentiation and growth
arrest [34]. The disruption of these mechanisms by MDFI up-regulation

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Fig. 2. Enriched pathways for OS and drugs predicted to reverse OS signature. A) GSEA analysis for the OS test group. Top 10 up-regulated KEGG pathways (in
blue) and top 10 down-regulated KEGG pathways (in orange). B) Over-representation (ORA) pathway analysis of the 266 validated genes. The top 10 up-regulated
pathways (top graphic) and the top 10 down-regulated pathways (bottom graphic). C) Clustergram with the top ranked L1000 molecules predicted to reverse the OS
gene expression signature. Columns include the best ranked drugs, in descending order. Each blue cell represents an up-regulated gene in OS which is downregulated
by the respective drug in the column. Each red cell represents a down-regulated gene in OS which is upregulated by the respective drug in the column.

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R.C. Andrade et al. Computers in Biology and Medicine 134 (2021) 104470

Table 2 Table 2 (continued )

Gene set enrichment analysis results for gene expression profile in the test Description NES FDR Genes
group. The top 20 results are shown, ranked by FDR. The GSEA was performed
using the KEGG database. Pathways that were also enriched in the ORA analysis BECN1, CALCOCO2,
HIF1A, ATG9A, RELA,
(Fig. 2B) are highlighted in bold letters.
MAPK9, RAB7A, ATF4
Description NES FDR Genes Galactose metabolism − 1.8345 0.0067080 PFKP, PGM1, HK1, GLB1,
GAA, UGP2, AKR1B1,
Ashtma 2.5295 0 HLA-DRA, HLA-DRB1,
G6PC3, PFKM, GALT,
HLA-DRB5, HLA-DPA1,
B4GALT2, B4GALT1, PFKL,
FCER1G, HLA-DQA1,
AKR1B10, GALE
HLA-DPB1, HLA-DMB,
p53 signaling ¡1.8398 0.0085862 SERPINE1, IGFBP3,
HLA-DMA, HLA-DQB1
pathway CDKN1A, PERP, THBS1,
Type I diabetes 2.2913 0 HLA-DRA, CPE, HLA-
CCND1, FAZ, ZMAT3,
mellitus DRB1, HLA-DRB5, HLA-
TP53, GADD45A, CCNG1,
DPA1, HLA-DQA1, HLA-
DDB2, BAX, BCL2L1
DPB1, HLA-DMB, HLA-
Nicotinate and − 1.7658 0.017530 NT5E, BST1, AOX1, NNMT,
DMA, HLA-DQB1, CD86
nicotinamide NMRK1
Autoimmune thyroid 2.2776 0 HLA-DRA, HLA-DRB1, HLA-
metabolism
disease DRB5, HLA-DPA1, HLA-
Amino sugar and − 1.7493 0.019012 UGDH, CYB5R3, GFPT2,
DQA1, HLA-DPB1, HLA-
nucleotide sugar PGM1, HK1, PMM1, GMDS,
DMB, HLA-DMA, HLA-
metabolism NAGK, GNPDA1, UGP2,
DQB1, CD86
UAP1L1, HEXB, GFPT1,
Staphylococcus 2.2761 0 HLA-DRA, HLA-DRB1,
GALT, GMPPA, TSTA3,
aureus infection C1QA, HLA-DRB5, HLA-
CYB5R2, UAP1, CYB5R1,
DPA1, FCGR2A, C1QB,
AMDHD2, HEXA, GMPPB,
HLA-DQA1, HLA-DPB1,
GALE, UXS1, PGM3, MPI
C3, FCGR2B, HLA-DMB,
Glycolysis/ − 1.7662 0.021036 BPGM, PFKP, PGM1, HK1,
C3AR1, HLA-DMA, HLA-
Gluconeogenesis ADH5, LDHA, PCK2,
DQB1, FPR3, FCGR1A,
ALDH3A2, ALDH1A3,
C5AR1, ITGB2, ITGAM
ALDH9A1, PKM, ALDH3B1,
Leishmaniasis 2.2696 0 HLA-DRA, HLA-DRB1,
G6PC3, ALDH7A1,
HLA-DRB5, HLA-DPA1,
ALDH1B1, PDHB, ALDOA,
FCGR2A, HLA-DQA1,
ADH1B, PFKM, DLD,
HLA-DPB1, CYBB, ITGA4,
ALDH2, GAPDH, PFKL,
C3, HLA-DMB, HLA-DMA,
PGAM1, ENO1, ALDOC
MARCKSL1, HLA-DQB1,
Steroid biosynthesis − 1.7130 0.031125 DHCR24, MSMO1, LSS,
TGFB3, TLR2, FCGR1A,
DHCR7, CYP51A1, FDFT1,
NCF2, FOS, ITGB2, NCF4,
SOAT1, SC5D
ITGAM, PTPN6
Ascorbate and aldarate − 1.7013 0.033868 UGDH, ALDH3A2,
Allograft rejection 2.2370 0 HLA-DRA, HLA-DRB1,
metabolism ALDH9A1, ALDH7A1,
HLA-DRB5, HLA-DPA1,
ALDH1B1, RGN, ALDH2
HLA-DQA1, HLA-DPB1,
Melanoma ¡1.6734 0.047439 FGF5, FGF7, FGF2,
HLA-DMB, HLA-DMA,
CDKN1A, CCND1, MET,
HLA-DQB1, CD86
TP53, GADD45A, MAPK3,
Systemic lupus 2.1488 0.00010467 HLA-DRA, HLA-DRB1,
DDB2, BAX, EGFR,
erythematosus C1QA, HLA-DRB5, HLA-
MAP2K2, AKT3, HRAS,
DPA1, FCGR2A, C1QB,
MAPK1, PDGFRB,
HLA-DQA1, HLA-DPB1,
PDGFRA
C3, HLA-DMB, HLA-DMA,
HLA-DQB1, FCGR1A, NES: normalized enrichment score.
CD86
Antigen processing and 2.2060 0.00011962 HLA-DRA, CD74, HLA-
presentation DRB1, HLA-DRB5, HLA- is plausible, since the pRb pathway is the most frequently inactivated
DPA1, HLA-DQA1, HLA- pathway in OS, and represents a particularly interesting therapeutic
DPB1, HLA-DMB, CTSS, target [34]. MDFI also binds to the axin complex, resulting in increased
HLA-DMA, HLA-DQB1,
levels of free β-catenin in the cell [33]. High levels of β-catenin were
CD4, LGMN, IFI30, HSPA6,
HSPA2, KLRC4, KLRC2 identified in osteosarcoma tissues compared to normal bone and were
Graft-versus-host 2.0068 0.00075361 HLA-DRA, HLA-DRB1, associated with worse prognosis and lung metastasis [35,36]. High
disease HLA-DRB5, HLA-DPA1, expression of MDFI is associated with unfavorable prognosis in renal
HLA-DQA1, HLA-DPB1, cancer, lung cancer and melanoma [37].
HLA-DMB, HLA-DMA,
HLA-DQB1, CD86
The top down-regulated gene was TPD52L1 (D53), involved in cell
Intestinal immune 2.0141 0.00083735 HLA-DRA, HLA-DRB1, proliferation and calcium signaling. This gene encodes a member of the
network for IgA HLA-DRB5, HLA-DPA1, D52-like family of adaptor proteins that contain a coiled-coil domain,
production CXCR4, HLA-DQA1, HLA- and may form hetero- or homomers. Unlike other D52-like family
DPB1, ITGA4, HLA-DMB,
members, TPD52L1 is not over-expressed in tumor samples [38,39],
HLA-DMA, HLA-DQB1,
CD86 having a more compatible role as a tumor suppressor gene [40]. It also
Steroid hormone − 1.9950 0.0010733 AKR1C3, CYP1B1, interacts with the mitogen-activated protein kinase 5 (MAP3K5/ASK1)
biosynthesis AKR1C2,AKR1C1, STS, and positively regulates MAP3K5-induced apoptosis. One of its isoforms
HSD17B12, COMT activates apoptosis signal-regulating kinase 1 (ASK1), leading to the
Mitophagy − 1.9288 0.0021465 CITED2, RRAS, TP53,
OPTN, SQSTM1, BCL2L1,
activation of the caspase-3-dependent apoptosis pathway [41]. It is
CSNK2A2, TFE3, PINK1, down-regulated in recurrent nasopharyngeal cancer [40], and when
HRAS, RRAS2, NBR1, highly expressed in renal and thyroid cancer, it is associated with
GABARAP, BNIP3L, favorable prognosis [37].
TBC1D17, FIS1, BCL2L13,
Among the commonly reported genes with altered expression in OS,
we found the overexpression of HEY1 (Supplementary Table 1), which is

7
R.C. Andrade et al.
Table 3
Drugs predicted to reverse at least 10% of OS gene signature and the respective genes with the expression modified by them.
Drug PubChem OS up/drug down
CID
Afatinib 10184653 ARHGDIB, CD24, CD93, CXCR4, HLA-DPA1, HLA-
DQA1, LAPTM5, LRRC17, RGS5, SPARCL1,
TMSB15A
BRD- 3242434 A2M, CD74, HEY1, HLA-DPA1, HLA-DPB1, HLA-
K95196255 DRA, IQGAP2, NES, PTPRZ1, SNX10, TSPAN13
DG-041 (DTSI) 11296282 A2M, CD14, CD36, CD93, CXCL14, GMFG, LRRC17,
PECAM1, RGS5, TNFSF10, TYROBP, WIF1
CA-074 methyl 23760717 CA2, CD24, CD74, CXCL14, HEY1, HLA-DPA1, HLA-
ester DPB1, HLA-DQA1, HLA-DRA, LYZ, RGS5
a downstream effector in the Notch pathway. The Notch pathway
upregulation has been described in OS, with a possible correlation with
invasiveness potential [12]. Other commonly reported genes, like
Runx2, EZR, and HER-2 [12], were not identified as DEGs in our study.
These discrepancies may be due to the enlargement of the sample size in
our analysis when compared to individual studies, as well as to intra­
tumor and intertumor heterogeneity.
The complex microenvironment of OS plays an important role in the
malignant transformation and maintenance of the tumor. It includes
different types of cells, like bone, vascular and immune cells, which
communicate through physical contact, soluble factors release or
extracellular vesicles [9,42]. In this study, many of the upregulated
pathways were associated to inflammatory response and immunomo­
dulation. There is a crucial dialogue between bone cells and cells of the
immune system. Osteosarcomas secrete osteoclastogenic cytokines, like
RANKL and macrophage colony-stimulating factor (CSF1) that induce
bone resorption, while tumor growth is maintained by factors generated
during osteolysis [8]. The CSF1 receptor (CSF1R) was upregulated in our
analysis, together with the downregulation of TNFRSF11B (Supple­
mentary Tables 1 and 2), which encodes a negative regulator of bone
resorption that is inhibited by RANKL.
One of the modifications that occur in the in microenvironment of OS
is the malignant osteoid formation in the extracellular matrix (ECM),
responsible for the tumor radiographic “sunburst” pattern [42], which is
consistent with DEGs involved in processes such as ECM-receptor
interaction, as revealed by our ORA analysis. Another important alter­
ation is the down-regulation of the p53 pathway, also revealed in our
ORA analysis, since the inactivation of TP53 is considered one of the
major driver events in OS. Germline mutations in TP53 are associated
with the autosomal dominant hereditary cancer condition known as
Li-Fraumeni syndrome (LFS) that predisposes to a high risk for the
development of many cancers. Osteosarcoma is one of the core cancers
of LFS, affecting ~16% of the individuals with TP53 germline mutations
[43]. Besides, somatic rearrangements of intron 1 of TP53 are thought to
be a specific genetic alteration for OS, occurring in ~16% of the sporadic
OS samples [44].
We assessed possible new candidate drugs for the treatment of OS
based on the gene signature obtained in our meta-analysis. For that
intent, we used the LINCS CDS2 repurposing tool, which is considered an
updated and curated tool for drug repurposing based on gene expression
signature [24,45–48]. For further discussion, we selected the top four
drug candidates obtained using the LINCS CDS2 engine, which were
predicted to reverse the expression of at least 10% of our input genes (i.
e.: with a drug score higher than 10%). There is not a threshold deter­
mined in terms of significance for this score [24]. The users of the LINCS
CDS2 engine can select, for further discussion/filtering, all the 50 drugs
in the output, the top drugs among them, or even the ones that have
more published information available, independently of the score
[49–52]. The best ranked drug in our study was afatinib (S1011; Pub­
Chem CID 10184653), a selective tyrosine kinase inhibitor that cova­
lently binds to and irreversibly blocks signaling from the ErbB family
members EGFR (ErbB1), HER2 (ErbB2) and HER4 (ErbB4) [53,54]. Oral
8
Computers in Biology and Medicine 134 (2021) 104470
OS down/drug up
ADM, AKR1C1, AKR1C2, AKR1C3, ANPEP, CCND1, CDKN1A, CRYAB, CYP1B1, G6PD,
HMOX1, IGFBP3, IGFBP6, ITGBL1, KRT19, LRP10, MFAP5, NQO1, PENK, PTX3, STC2,
TMEM47, UGDH, VAT1
ADM, CDKN1A, CHI3L1, COMP, DDAH1, FAS, FHL2, GAS6, GEM, GREM1, HMOX1,
IGFBP3, NT5E, PHLDA3, SERPINE1, STC2, SULF1, TNFRSF12A
ADAMTS1, ADM, AOX1, BPGM, CAV1, EMP1, FHL2, FST, GEM, PRSS23, SEMA3C,
SERPINE1, STC2, THBS1, THBS2, TNFRSF12A
ADM, AKR1C1, AKR1C2, AKR1C3, C1S, CDKN1A, CHI3L1, FAZ, G6PD, GCLM, HMOX1,
IGFBP6, IL6, ISLR, NPR3, NQO1, PLIN3, PTX3, SPOCK1, STC2, TM4SF1, UGDH
afatinib (Gilotrif™) was first approved by the FDA in 2013 and it is still
currently used as a first line treatment for metastatic non-small-cell lung
cancer (NSCLC) with EGFR non-resistant activating mutations (most
commonly exon 19 deletions or exon 21 point mutation L858R) [53,54].
Afatinib has recently demonstrated to decrease proliferation, migration,
and invasion of non-metastatic and metastatic OS cell lines [55], and it is
currently being investigated in a phase II clinical trial for pediatric tu­
mors (https://clinicaltrials.gov/ct2/show/NCT02372006). A different
methodology of drug repurposing, using sarcospheres from highly
metastatic human OS cell lines and an anticancer drug library, also
found afatinib as one of the main drugs with therapeutic potential [56].
Interestingly, the Erb pathway was not enriched in our analysis, and the
genes predicted to have its expression altered by afatinib in the samples
evaluated were different from the ones usually reported in the literature.
Thus, afatinib may have an inhibitory effect on osteosarcoma cells by
acting in more than one molecular pathway.
The three other top molecules assessed in our analysis are in earlier
phases of study than afatinib. None of them is currently approved by the
FDA. BRD-K95196255 is a hydroxyquinoline with in vitro effects that
include cytotoxic activity and lung tumor cell growth inhibition [26].
DG-041 is an antagonist of the prostaglandin (PG)E2 EP3 receptor and
inhibits platelet aggregation. A potential antimetastatic effect by
avoiding platelets-induced mesenchymal-like changes in cancer cells has
been reported [57]. CA-074me is a methyl ester derivative of CA-074
with better cell permeability, that has inhibitory activity over the pro­
teases Cathepsin B and L [58]. The degradation of the extracellular
matrix induced by cathepsins seems to contribute to the development
and metastasis of tumors, making them interesting therapeutic targets
[58–60]. Importantly, CA-074 methyl ester has been shown to inhibit
osteoclastogenesis in vitro and in vivo, by mechanisms that seem inde­
pendent from cathepsins, and its use could be advantageous in treating
osteolytic conditions, including bone cancers [61].
In conclusion, this study brings new insights into the biological
knowledge of osteosarcoma, an aggressive cancer with limited thera­
peutic options. We were able to identify a gene expression signature for
this tumor using data from a large number of tumor samples from young
individuals and comparing them to both candidate cells of origin: os­
teoblasts and mesenchymal stem cells. We were then able to propose
new therapeutic small molecules based on this gene expression signa­
ture. Clearly, drug repurposing based solely on bioinformatics approach
has limitations, since the efficacy of a drug depends on several variables
other than gene expression signature, and further in vitro and in vivo
studies are necessary to establish the potential of these drugs in OS
treatment. However, given the modest improvement in OS treatment
over the last decades, we believe our results can be an important
contribution for the investigation of new therapeutic genetic targets and
for selecting new drugs to be tested for this disease.
Declaration of competing interest
The authors declare no conflicts of interest.
R.C. Andrade et al.
Acknowledgments
We thank Nicole de Miranda Scherer, Daniel Andrade Moreira and
Jéssica Gonçalves Vieira da Cruz for the orientations during the
computational analyses. We also thank Inova Fiocruz for the financial
support. This work is dedicated to the memory of Nilton Barreto dos
Santos, dear friend and great scientist.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.compbiomed.2021.104470.
Authors contributions
Conception and design: RCA, MB and FRV; Data survey and selec­
tion: RCA and MKA; Analysis and interpretation: RCA and MB; Drafting
of the article: RCA; Review and editing of the article: MB, MKA and FRV.
Funding source
Inova Fiocruz/Fundação Oswaldo Cruz, Rio de Janeiro, Brazil (Ju­
nior Postdoctoral fellowship 2019-2020). The funding source was not
involved in the study design, data analysis and preparation of the article.
References
[1] D.M. Gianferante, L. Mirabello, S.A. Savage, Germline and somatic genetics of
osteosarcoma - connecting aetiology, biology and therapy, Nat. Rev. Endocrinol. 13
(2017) 480–491, https://doi.org/10.1038/nrendo.2017.16.
[2] A. Misaghi, A. Goldin, M. Awad, A.A. Kulidjian, Osteosarcoma: a comprehensive
review, SICOT-J. 4 (2018), https://doi.org/10.1051/sicotj/2017028.
[3] S. Chen, L. Yang, F. Pu, H. Lin, B. Wang, J. Liu, Z. Shao, High birth weight increases
the risk for bone tumor: a systematic review and meta-analysis, Int. J. Environ. Res.
Publ. Health 12 (2015) 11178–11195, https://doi.org/10.3390/ijerph120911178.
[4] S. Prater, B. McKeon, Cancer, Osteosarcoma, StatPearls Publishing, 2019. http
://www.ncbi.nlm.nih.gov/pubmed/31751058. (Accessed 2 July 2020).
[5] M.S. Isakoff, S.S. Bielack, P. Meltzer, R. Gorlick, Osteosarcoma: current treatment
and a collaborative pathway to success, J. Clin. Oncol. 33 (2015) 3029–3035,
https://doi.org/10.1200/JCO.2014.59.4895.
[6] S. Smeland, S.S. Bielack, J. Whelan, M. Bernstein, P. Hogendoorn, M.D. Krailo,
R. Gorlick, K.A. Janeway, F.C. Ingleby, J. Anninga, I. Antal, C. Arndt, K.L.B. Brown,
T. Butterfass-Bahloul, G. Calaminus, M. Capra, C. Dhooge, M. Eriksson, A.
M. Flanagan, G. Friedel, M.C. Gebhardt, H. Gelderblom, R. Goldsby, H.E. Grier,
R. Grimer, D.S. Hawkins, S. Hecker-Nolting, K. Sundby Hall, M.S. Isakoff, G. Jovic,
T. Kühne, L. Kager, T. von Kalle, E. Kabickova, S. Lang, C.C. Lau, P.J. Leavey, S.
L. Lessnick, L. Mascarenhas, R. Mayer-Steinacker, P.A. Meyers, R. Nagarajan, R.
L. Randall, P. Reichardt, M. Renard, C. Rechnitzer, C.L. Schwartz, S. Strauss,
L. Teot, B. Timmermann, M.R. Sydes, N. Marina, Survival and prognosis with
osteosarcoma: outcomes in more than 2000 patients in the EURAMOS-1 (European
and American Osteosarcoma Study) cohort, Eur. J. Canc. 109 (2019) 36–50,
https://doi.org/10.1016/j.ejca.2018.11.027.
[7] R.D. Roberts, M.M. Lizardo, D.R. Reed, P. Hingorani, J. Glover, W. Allen-Rhoades,
T. Fan, C. Khanna, E.A. Sweet-Cordero, T. Cash, M.W. Bishop, M. Hegde, A.
R. Sertil, C. Koelsche, L. Mirabello, D. Malkin, P.H. Sorensen, P.S. Meltzer, K.
A. Janeway, R. Gorlick, B.D. Crompton, Provocative questions in osteosarcoma
basic and translational biology: a report from the Children’s Oncology Group,
Cancer 125 (2019) 3514–3525, https://doi.org/10.1002/cncr.32351.
[8] M. Kansara, M.W. Teng, M.J. Smyth, D.M. Thomas, Translational biology of
osteosarcoma, Nat. Rev. Canc. 14 (2014) 722–735, https://doi.org/10.1038/
nrc3838.
[9] A. Abarrategi, J. Tornin, L. Martinez-Cruzado, A. Hamilton, E. Martinez-Campos, J.
P. Rodrigo, M.V. González, N. Baldini, J. Garcia-Castro, R. Rodriguez,
Osteosarcoma: cells-of-origin, cancer stem cells, and targeted therapies, Stem Cell.
Int. (2016), https://doi.org/10.1155/2016/3631764 (2016) 3631764.
[10] A.J. Mutsaers, C.R. Walkley, Cells of origin in osteosarcoma: mesenchymal stem
cells or osteoblast committed cells? Bone 62 (2014) 56–63, https://doi.org/
10.1016/j.bone.2014.02.003.
[11] A.L. Sinatro, Osteosarcoma: Molecular Etiology, Pathophysiology, Clinical
Presentation, Diagnosis, and Treatment, Boston University, 2018.
[12] E. Tirtei, M. Cereda, E. De Luna, P. Quarello, S.D. Asaftei, F. Fagioli, Omic
approaches to pediatric bone sarcomas, Pediatr. Blood Canc. 67 (2020), https://
doi.org/10.1002/pbc.28072.
[13] L. Gautier, L. Cope, B.M. Bolstad, R.A. Irizarry, Affy - analysis of Affymetrix
GeneChip data at the probe level, Bioinformatics 20 (2004) 307–315, https://doi.
org/10.1093/bioinformatics/btg405.
9
Computers in Biology and Medicine 134 (2021) 104470
[14] B.S. Carvalho, R.A. Irizarry, A framework for oligonucleotide microarray
preprocessing, Bioinformatics 26 (2010) 2363–2367, https://doi.org/10.1093/
bioinformatics/btq431.
[15] T.L. Leek Jt, W.E. Johnson, H.S. Parker, E.J. Fertig, A.E. Jaffe, Y. Zhang, J.
D. Storey, Sva: Surrogate Variable Analysis, in: R Package, 2020, version 3.36.0.
[16] M.E. Ritchie, B. Phipson, D. Wu, Y. Hu, C.W. Law, W. Shi, G.K. Smyth, Limma
powers differential expression analyses for RNA-sequencing and microarray
studies, Nucleic Acids Res. 43 (2015) e47, https://doi.org/10.1093/nar/gkv007.
[17] E. Karatzas, G. Kolios, G.M. Spyrou, An Application of Computational Drug
Repurposing Based on Transcriptomic Signatures, in: Methods Mol. Biol., Humana
Press Inc., 2019, pp. 149–177, https://doi.org/10.1007/978-1-4939-8955-3_9.
[18] W. Yang, Y. Li, F. He, H. Wu, Microarray profiling of long non-coding RNA
(lncRNA) associated with hypertrophic cardiomyopathy, BMC Cardiovasc. Disord.
15 (2015) 1–9, https://doi.org/10.1186/s12872-015-0056-7.
[19] P. Wang, Y. Wang, B. Hang, X. Zou, J.H. Mao, A novel gene expression-based
prognostic scoring system to predict survival in gastric cancer, Oncotarget 7 (2016)
55343–55351, https://doi.org/10.18632/oncotarget.10533.
[20] K. Zhang, H. Shi, H. Xi, X. Wu, J. Cui, Y. Gao, W. Liang, C. Hu, Y. Liu, J. Li,
N. Wang, B. Wei, L. Chen, Genome-wide lncRNA microarray profiling identifies
novel circulating lncrnas for detection of gastric cancer, Theranostics 7 (2017)
213–227, https://doi.org/10.7150/thno.16044.
[21] S. Zhang, X. Zeng, T. Ding, L. Guo, Y. Li, S. Ou, H. Yuan, Microarray profile of
circular RNAs identifies hsa-circ-0014130 as a new circular RNA biomarker in non-
small cell lung cancer, Sci. Rep. 8 (2018) 2878, https://doi.org/10.1038/s41598-
018-21300-5.
[22] Y.J. Zhu, B. Zheng, G.J. Luo, X.K. Ma, X.Y. Lu, X.M. Lin, S. Yang, Q. Zhao, T. Wu, Z.
X. Li, X.L. Liu, R. Wu, J.F. Liu, Y. Ge, L. Yang, H.Y. Wang, L. Chen, Circular RNAs
negatively regulate cancer stem cells by physically binding FMRP against CCAR1
complex in hepatocellular carcinoma, Theranostics 9 (2019) 3526–3540, https://
doi.org/10.7150/thno.32796.
[23] Y. Liao, J. Wang, E.J. Jaehnig, Z. Shi, B. Zhang, WebGestalt, Gene set analysis
toolkit with revamped UIs and APIs, Nucleic Acids Res. 47 (2019) W199–W205,
https://doi.org/10.1093/nar/gkz401, 2019.
[24] Q. Duan, S.P. Reid, N.R. Clark, Z. Wang, N.F. Fernandez, A.D. Rouillard,
B. Readhead, S.R. Tritsch, R. Hodos, M. Hafner, M. Niepel, P.K. Sorger, J.T. Dudley,
S. Bavari, R.G. Panchal, A. Ma’Ayan, LINCS L1000 characteristic direction
signatures search engine, L1000CDS2, Npj Syst. Biol. Appl. 2 (2016), https://doi.
org/10.1038/npjsba.2016.15.
[25] NIH, LINCS data portal | cells - L1000 mRNA profiling assay. http://lincsportal.ccs.
miami.edu/cells/#/catalog?query=assays:L1000 mRNA profiling assay (accessed
April 18, 2021).
[26] S. Kim, J. Chen, T. Cheng, A. Gindulyte, J. He, S. He, Q. Li, B.A. Shoemaker, P.
A. Thiessen, B. Yu, L. Zaslavsky, J. Zhang, E.E. Bolton, PubChem 2019 update:
improved access to chemical data, Nucleic Acids Res. 47 (2019) D1102–D1109,
https://doi.org/10.1093/nar/gky1033.
[27] E. Simpson, H.L. Brown, Understanding osteosarcomas, JAAPA 31 (2018) 15–19,
https://doi.org/10.1097/01.JAA.0000541477.24116.8d.
[28] K.M. Makielski, L.J. Mills, A.L. Sarver, M.S. Henson, L.G. Spector, S. Naik, J.
F. Modiano, Risk factors for development of canine and human osteosarcoma: a
comparative review, Vet. Sci. 6 (2019), https://doi.org/10.3390/vetsci6020048.
[29] D.C. Stefan, M. Harif, Pediatr. Cancer Africa, Osteosarcoma, Springer International
Publishing, Cham, 2017, pp. 71–80, https://doi.org/10.1007/978-3-319-17936-0_
6.
[30] B. Otoukesh, B. Boddouhi, M. Moghtadaei, P. Kaghazian, M. Kaghazian, Novel
molecular insights and new therapeutic strategies in osteosarcoma 11 Medical and
Health Sciences 1112 Oncology and Carcinogenesis, Canc. Cell Int. 18 (2018) 1–23,
https://doi.org/10.1186/s12935-018-0654-4.
[31] A. Ertel, A. Verghese, S.W. Byers, M. Ochs, A. Tozeren, Pathway-specific
differences between tumor cell lines and normal and tumor tissue cells, Mol. Canc.
5 (2006) 55, https://doi.org/10.1186/1476-4598-5-55.
[32] A. Longhi, C. Errani, D. Gonzales-Arabio, C. Ferrari, M. Mercuri, Osteosarcoma in
patients older than 65 years, J. Clin. Oncol. 26 (2008) 5368–5373, https://doi.org/
10.1200/JCO.2007.14.9104.
[33] A. Bateman, UniProt: a worldwide hub of protein knowledge, Nucleic Acids Res. 47
(2019) D506–D515, https://doi.org/10.1093/nar/gky1049.
[34] S.E. Ballatori, P.W. Hinds, Osteosarcoma: prognosis plateau warrants
retinoblastoma pathway targeted therapy, Signal Transduct. Target. Ther. 1 (2016)
16001, https://doi.org/10.1038/sigtrans.2016.1.
[35] W. Liu, Z. Zhao, Y. Wang, W. Li, Q. Su, Q. Jia, J. Zhang, X. Zhang, J. Yin, J. Shen,
Dioscin inhibits stem-cell-like properties and tumor growth of osteosarcoma
through Akt/GSK3/β-catenin signaling pathway, Cell Death Dis. 9 (2018), https://
doi.org/10.1038/s41419-018-0363-x.
[36] Y. Lu, G.F. Guan, J. Chen, B. Hu, C. Sun, Q. Ma, Y.H. Wen, X.C. Qiu, Y. Zhou,
Aberrant CXCR4 and β-catenin expression in osteosarcoma correlates with patient
survival, Oncol. Lett. 10 (2015) 2123–2129, https://doi.org/10.3892/
ol.2015.3535.
[37] M. Uhlén, L. Fagerberg, B.M. Hallström, C. Lindskog, P. Oksvold, A. Mardinoglu,
Å. Sivertsson, C. Kampf, E. Sjöstedt, A. Asplund, I.M. Olsson, K. Edlund,
E. Lundberg, S. Navani, C.A.K. Szigyarto, J. Odeberg, D. Djureinovic, J.O. Takanen,
S. Hober, T. Alm, P.H. Edqvist, H. Berling, H. Tegel, J. Mulder, J. Rockberg,
P. Nilsson, J.M. Schwenk, M. Hamsten, K. Von Feilitzen, M. Forsberg, L. Persson,
F. Johansson, M. Zwahlen, G. Von Heijne, J. Nielsen, F. Pontén, Tissue-based map
of the human proteome, Science 80– (2015) 347, https://doi.org/10.1126/
science.1260419.
[38] M. Shehata, I. Bièche, R. Boutros, J. Weidenhofer, S. Fanayan, L. Spalding, N. Zeps,
K. Byth, R.K. Bright, R. Lidereau, J.A. Byrne, Nonredundant functions for tumor
R.C. Andrade et al.
protein D52-like proteins support specific targeting of TPD52, Clin. Canc. Res. 14
(2008) 5050–5060, https://doi.org/10.1158/1078-0432.CCR-07-4994.
[39] D. Barbaric, K. Byth, L. Dalla-Pozza, J.A. Byrne, Expression of tumor protein D52-
like genes in childhood leukemia at diagnosis: clinical and sample considerations,
Leuk. Res. 30 (2006) 1355–1363, https://doi.org/10.1016/j.leukres.2006.03.009.
[40] Z. Huang, W. Li, S. Lin, X. Fang, C. Zhang, Z. Liao, Identification of novel tumor
suppressor genes down-regulated in recurrent nasopharyngeal cancer by DNA
microarray, Indian J. Otolaryngol. Head Neck Surg. 66 (2014) 120–125, https://
doi.org/10.1007/s12070-011-0359-7.
[41] S. Cho, H.M. Ko, J.M. Kim, J.A. Lee, J.E. Park, M.S. Jang, S.G. Park, D.H. Lee, S.
E. Ryu, B.C. Park, Positive regulation of apoptosis signal-regulating kinase 1 by
hD53L1, J. Biol. Chem. 279 (2004) 16050–16056, https://doi.org/10.1074/jbc.
M305758200.
[42] H.K. Brown, K. Schiavone, F. Gouin, M.F. Heymann, D. Heymann, Biology of bone
sarcomas and new therapeutic developments, Calcif. Tissue Int. 102 (2018)
174–195, https://doi.org/10.1007/s00223-017-0372-2.
[43] G. Bougeard, M. Renaux-Petel, J.-M. Flaman, C. Charbonnier, P. Fermey,
M. Belotti, M. Gauthier-Villars, D. Stoppa-Lyonnet, E. Consolino, L. Brugières,
O. Caron, P.R. Benusiglio, B. Bressac-de Paillerets, V. Bonadona, C. Bonaïti-Pellié,
J. Tinat, S. Baert-Desurmont, T. Frebourg, Revisiting Li-fraumeni syndrome from
TP53 mutation carriers, J. Clin. Oncol. 33 (2015) 2345–2352, https://doi.org/
10.1200/JCO.2014.59.5728.
[44] S. Ribi, D. Baumhoer, K. Lee, Edison, A.S.M. Teo, B. Madan, K. Zhang, W.
K. Kohlmann, F. Yao, W.H. Lee, Q. Hoi, S. Cai, X.Y. Woo, P. Tan, G. Jundt, J. Smida,
M. Nathrath, W.K. Sung, J.D. Schiffman, D.M. Virshup, A.M. Hillmer, TP53 intron 1
hotspot rearrangements are specific to sporadic osteosarcoma and can cause Li-
Fraumeni syndrome, Oncotarget 6 (2015) 7727–7740, https://doi.org/10.18632/
oncotarget.3115.
[45] A.B. Keenan, S.L. Jenkins, K.M. Jagodnik, S. Koplev, E. He, D. Torre, Z. Wang, A.
B. Dohlman, M.C. Silverstein, A. Lachmann, M.V. Kuleshov, A. Ma’ayan,
V. Stathias, R. Terryn, D. Cooper, M. Forlin, A. Koleti, D. Vidovic, C. Chung, S.
C. Schürer, J. Vasiliauskas, M. Pilarczyk, B. Shamsaei, M. Fazel, Y. Ren, W. Niu, N.
A. Clark, S. White, N. Mahi, L. Zhang, M. Kouril, J.F. Reichard, S. Sivaganesan,
M. Medvedovic, J. Meller, R.J. Koch, M.R. Birtwistle, R. Iyengar, E.A. Sobie, E.
U. Azeloglu, J. Kaye, J. Osterloh, K. Haston, J. Kalra, S. Finkbiener, J. Li, P. Milani,
M. Adam, R. Escalante-Chong, K. Sachs, A. Lenail, D. Ramamoorthy, E. Fraenkel,
G. Daigle, U. Hussain, A. Coye, J. Rothstein, D. Sareen, L. Ornelas, M. Banuelos,
B. Mandefro, R. Ho, C.N. Svendsen, R.G. Lim, J. Stocksdale, M.S. Casale, T.
G. Thompson, J. Wu, L.M. Thompson, V. Dardov, V. Venkatraman, A. Matlock, J.
E. Van Eyk, J.D. Jaffe, M. Papanastasiou, A. Subramanian, T.R. Golub, S.
D. Erickson, M. Fallahi-Sichani, M. Hafner, N.S. Gray, J.R. Lin, C.E. Mills, J.
L. Muhlich, M. Niepel, C.E. Shamu, E.H. Williams, D. Wrobel, P.K. Sorger, L.
M. Heiser, J.W. Gray, J.E. Korkola, G.B. Mills, M. LaBarge, H.S. Feiler, M.A. Dane,
E. Bucher, M. Nederlof, D. Sudar, S. Gross, D.F. Kilburn, R. Smith, K. Devlin,
R. Margolis, L. Derr, A. Lee, A. Pillai, The library of integrated network-based
cellular signatures NIH program: system-level cataloging of human cells response
to perturbations, Cell Syst 6 (2018) 13–24, https://doi.org/10.1016/j.
cels.2017.11.001.
[46] A. Musa, L.S. Ghoraie, S.-D. Zhang, G. Galzko, O. Yli-Harja, M. Dehmer, B. Haibe-
Kains, F. Emmert-Streib, A review of connectivity map and computational
approaches in pharmacogenomics, Briefings Bioinf. 19 (2017), https://doi.org/
10.1093/bib/bbw112 bbw112.
[47] K.M. Kingsmore, A.C. Grammer, P.E. Lipsky, Drug repurposing to improve
treatment of rheumatic autoimmune inflammatory diseases, Nat. Rev. Rheumatol.
16 (2020) 32–52, https://doi.org/10.1038/s41584-019-0337-0.
10
Computers in Biology and Medicine 134 (2021) 104470
[48] O.S. Kwon, W. Kim, H.J. Cha, H. Lee, Silico drug repositioning: from large-scale
transcriptome data to therapeutics, Arch Pharm. Res. (Seoul) 42 (2019) 879–889,
https://doi.org/10.1007/s12272-019-01176-3.
[49] P. Fagone, R. Caltabiano, A. Russo, G. Lupo, C.D. Anfuso, M.S. Basile, A. Longo,
F. Nicoletti, R. De Pasquale, M. Libra, M. Reibaldi, Identification of novel
chemotherapeutic strategies for metastatic uveal melanoma, Sci. Rep. 7 (2017),
https://doi.org/10.1038/srep44564.
[50] Z. Wang, A. Ma’ayan, An open RNA-Seq data analysis pipeline tutorial with an
example of reprocessing data from a recent Zika virus study [version 1; referees: 3
approved, F1000Research (5) (2016), https://doi.org/10.12688/
F1000RESEARCH.9110.1.
[51] A.A. Pezzulo, R.A. Tudas, C.G. Stewart, L.G. Vargas Buonfiglio, B.D. Lindsay, P.
J. Taft, N.D. Gansemer, J. Zabner, HSP90 inhibitor geldanamycin reverts IL-13–and
IL-17–induced airway goblet cell metaplasia, J. Clin. Invest. 129 (2019) 744–758,
https://doi.org/10.1172/JCI123524.
[52] B. Turanli, K. Karagoz, G. Bidkhori, R. Sinha, M.L. Gatza, M. Uhlen, A. Mardinoglu,
K.Y. Arga, Multi-omic data interpretation to repurpose subtype specific drug
candidates for breast cancer, Front. Genet. 10 (2019) 420, https://doi.org/
10.3389/fgene.2019.00420.
[53] R.T. Dungo, G.M. Keating, Afatinib: first global approval, Drugs 73 (2013)
1503–1515, https://doi.org/10.1007/s40265-013-0111-6.
[54] D.S. Wishart, Y.D. Feunang, A.C. Guo, E.J. Lo, A. Marcu, J.R. Grant, T. Sajed,
D. Johnson, C. Li, Z. Sayeeda, N. Assempour, I. Iynkkaran, Y. Liu, A. MacIejewski,
N. Gale, A. Wilson, L. Chin, R. Cummings, Di Le, A. Pon, C. Knox, M. Wilson,
DrugBank 5.0: a major update to the DrugBank database for 2018, Nucleic Acids
Res. 46 (2018) D1074–D1082, https://doi.org/10.1093/nar/gkx1037.
[55] M. Cruz-Ramos, Y. Zamudio-Cuevas, D. Medina-Luna, K. Martínez-Flores,
G. Martínez-Nava, J. Fernández-Torres, A. López-Reyes, F. Solca, Afatinib is active
in osteosarcoma in osteosarcoma cell lines, J. Canc. Res. Clin. Oncol. 146 (2020)
1693–1700, https://doi.org/10.1007/s00432-020-03220-y.
[56] Annual meeting of the orthopaedic research society orlando, FL march 5-8, 2016,
2016, J. Orthop. Res. 34 (2016) S1, https://doi.org/10.1002/jor.23247. S1
(abstract 0384, by Collier CD et al).
[57] P. Guillem-Llobat, M. Dovizio, A. Bruno, E. Ricciotti, V. Cufino, A. Sacco,
R. Grande, S. Alberti, V. Arena, M. Cirillo, C. Patrono, G.A. FitzGerald,
D. Steinhilber, A. Sgambato, P. Patrignani, Aspirin prevents colorectal cancer
metastasis in mice by splitting the crosstalk between platelets and tumor cells,
Oncotarget 7 (2016) 32462–32477, https://doi.org/10.18632/oncotarget.8655.
[58] H. Ruan, S. Hao, P. Young, H. Zhang, Targeting Cathepsin B for Cancer Therapies,
in: Horizons Cancer Res., Nova Science Publishers, Inc., 2015, pp. 23–40. PMID:
26623174.
[59] D.R. Sudhan, D.W. Siemann, Cathepsin L targeting in cancer treatment, Pharmacol.
Ther. 155 (2015) 105–116, https://doi.org/10.1016/j.pharmthera.2015.08.007.
[60] D.R. Sudhan, C. Pampo, L. Rice, D.W. Siemann, Cathepsin L inactivation leads to
multimodal inhibition of prostate cancer cell dissemination in a preclinical bone
metastasis model, Int. J. Canc. 138 (2016) 2665–2677, https://doi.org/10.1002/
ijc.29992.
[61] N. Patel, S. Nizami, L. Song, M. Mikami, A. Hsu, T. Hickernell,
C. Chandhanayingyong, S. Rho, J.T. Compton, J.M. Caldwell, P.B. Kaiser, H. Bai, H.
G. Lee, C.R. Fischer, F.Y. Lee, CA-074Me compound inhibits osteoclastogenesis via
suppression of the NFATc1 and c-FOS signaling pathways, J. Orthop. Res. 33
(2015) 1474–1486, https://doi.org/10.1002/jor.22795.