Application of serex-analysis for identification of human colon cancer antigens
Background: Colorectal, lung and breast tumors are the most devastating and frequent malignances in clinical oncology. SEREX-analysis of colon cancer leads to identification of more than hundred antigens which are potential tumor markers. With idea that immunoscreening with pool of allogeneic sera i...
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| Опубліковано в: : | Experimental Oncology |
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| Дата: | 2015 |
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2015
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| Назва журналу: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Цитувати: | Application of serex-analysis for identification of human colon cancer antigens / O.M. Garifulin, V.O. Kykot, N.Y. Gridina, R.G. Kiyamova, I.T. Gout, V.V. Filonenko // Experimental Oncology. — 2015. — Т. 37, № 3. — С. 173-180. — Бібліогр.: 46 назв. — англ. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1860169351543390208 |
|---|---|
| author | Garifulin, O.M. Kykot, V.O. Gridina, N.Y. Kiyamova, R.G. Gout, I.T. Filonenko, V.V. |
| author_facet | Garifulin, O.M. Kykot, V.O. Gridina, N.Y. Kiyamova, R.G. Gout, I.T. Filonenko, V.V. |
| citation_txt | Application of serex-analysis for identification of human colon cancer antigens / O.M. Garifulin, V.O. Kykot, N.Y. Gridina, R.G. Kiyamova, I.T. Gout, V.V. Filonenko // Experimental Oncology. — 2015. — Т. 37, № 3. — С. 173-180. — Бібліогр.: 46 назв. — англ. |
| collection | DSpace DC |
| container_title | Experimental Oncology |
| description | Background: Colorectal, lung and breast tumors are the most devastating and frequent malignances in clinical oncology. SEREX-analysis of colon cancer leads to identification of more than hundred antigens which are potential tumor markers. With idea that immunoscreening with pool of allogeneic sera is more productive for antigen isolation, SEREX-analysis was applied to four cases of stages II–IV primary colon tumor and 22 new antigens were isolated. Objective: To characterize 22 primary colon cancer antigens isolated by SEREX-technique. Materials and Methods: Allogenic screening, real-time PCR analysis. Results: After allogeneic immunoscreening, for 5 of 22 (22%) isolated antigens were confirmed colon cancer restricted serological profile solely positive for 14% of tested colon cancer sera. Through these five antigens, KY-CC-17/β-actin has cytoskeleton function; KY-CC-14/ACTR1A and KY-CC-19/TSGA2 participate in chromosome segregation; KY-CC-12/FKBP4 regulates steroid receptor function and KY-CC-15/PLRG1 is a component of spliceosome complex. For the last four antigens tested were found aberrant mRNA expression in some cases of colon tumor. Conclusion: The exploration of identified antigens may define suitable targets for immunotherapy or diagnostic of colon cancer. Key Words: colon cancer, SEREX-analysis, allogeneic immunoscreening.
|
| first_indexed | 2025-12-07T17:58:13Z |
| format | Article |
| fulltext |
Experimental Oncology 37, 173–180, 2015 (September) 173
APPLICATION OF SEREX-ANALYSIS FOR IDENTIFICATION
OF HUMAN COLON CANCER ANTIGENS
O.M. Garifulin1, V.O. Kykot2 , N.Y. Gridina1, R.G. Kiyamova1,3*, I.T. Gout4, V.V. Filonenko1
1Institute of Molecular Biology and Genetics, NAS of Ukraine, Kyiv 03143, Ukraine
2National Cancer Institute, Kyiv 03022, Ukraine
3Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan 420008, Russian Federation
4Department of Biochemistry and Molecular Biology, Royal Free and University College Medical School,
Cower Street, London WC1E 6BT, United Kingdom
Background: Colorectal, lung and breast tumors are the most devastating and frequent malignances in clinical oncology. SEREX-analy-
sis of colon cancer leads to identification of more than hundred antigens which are potential tumor markers. With idea that immunoscreen-
ing with pool of allogeneic sera is more productive for antigen isolation, SEREX-analysis was applied to four cases of stages II–IV pri-
mary colon tumor and 22 new antigens were isolated. Objective: To characterize 22 primary colon cancer antigens isolated by SEREX-
technique. Materials and Methods: Allogenic screening, real-time PCR analysis. Results: After allogeneic immunoscreening, for
5 of 22 (22%) isolated antigens were confirmed colon cancer restricted serological profile solely positive for 14% of tested colon cancer
sera. Through these five antigens, KY-CC-17/β-actin has cytoskeleton function; KY-CC-14/ACTR1A and KY-CC-19/TSGA2 par-
ticipate in chromosome segregation; KY-CC-12/FKBP4 regulates steroid receptor function and KY-CC-15/PLRG1 is a component
of spliceosome complex. For the last four antigens tested were found aberrant mRNA expression in some cases of colon tumor. Conclusion:
The exploration of identified antigens may define suitable targets for immunotherapy or diagnostic of colon cancer.
Key Words: colon cancer, SEREX-analysis, allogeneic immunoscreening.
Colon cancer as frequent case of colorectal tumors
along with lung and breast tumors are a significant
cause of morbidity and mortality in Western popula-
tions associated with a high risk of metastases, recur-
rence, and early patient death. Colon cancer formation
through adenoma to adenocarcinoma and later to its
metastatic counterpart involved a multistep cell pro-
cesses those may associated with aberrant expression
of hundreds of genes [1, 2]. It was a long-standing
challenge in cancer research if tumor cells proteins
may specifically recognized as self-antigens (tumor
associated antigens — TAAs) by patient’s immune
system. Immunologic methods such as T cell epitope
cloning [3] and recently emerged as a powerful me-
thod for identification of TAAs serological expression
cloning (SEREX) [4] applied to different tumors have
led to the discovery of a subset of antigens recognized
by CTLs and/or by humoral system in tumor-specific
manner [5]. TAAs often represent genes with altered
normal expression, oncogene i.e. overexpressed
or with activity in tumor cells (activated only in neo-
plastic cells and silence in normal cells, overexpressed
in tumor cells, mutated genes), those can predict its
possible association with tumorigenesis [6]. Several
tumor-specific antigens such as NY-ESO-1, MAGE-1,
MAGE-3, SSX2, Melan-A, and tyrosinase proteins are
promising sensitive and specific target molecules for
cancer screening and diagnosis, cancer immuno-
therapy and development of antigen-specific cancer
vaccines [7–9].
In recent years a subset of TAAs reacted exclu-
sively or much higher frequently with sera from colon
cancer patients were uncovered by SEREX-analysis
of colon primary tumors as well as colon cancer cell
lines and colon tumor metastasis [10–12]. As it was
showed, a frequency of TAAs for human primary colon
tumors is more rare than it was found for its metasta-
ses. It is possible that patients with metastasis may
have a more augmented IgG response than patients
with original tumor [11]. Therefore, SEREX-analysis
of primary colon cancer may define new TAAs useful
for early tumor diagnosis and immunotherapy inter-
vention. Previously we described of immunogeni city
of newly identified colon cancer antigens in sera
of patients of different types of tumors [13]. Here
we describe identification of these TAAs by SEREX
methodology in more detail and characterize in terms
of gene identity, tumor-specific immunogenicity and
expression in normal colon and colon tumor tissues.
MATERIALS AND METHODS
Tissue samples. The colon primary tumor sam-
ples were obtained from the National Cancer Institute
(Kyiv, Ukraine) as surgical specimens, frozen in liquid
nitrogen and stored at −80 °C. The histological clas-
sification of tumors was confirmed by histopathological
examination at the Department of Pathology, National
Cancer Institute (Kyiv, Ukraine). Tumor serum samples
were obtained at the same Institute from 20 patients
(35–62 years old) including sera samples from 14 co-
lon cancer patients and 6 gastric cancer patients.
Control sera were obtained from 18 individuals with
no evidence of malignancy (27–62 years old). All
Submitted: August 05, 2015.
*Correspondence: E-mail: r.g.kiyamova@imbg.org.ua
Abbreviations used: SEREX — method of identification of human
tumor antigens by serological expression cloning; TAAs — tumor-
associated antigens.
Exp Oncol 2015
37, 3, 173–180
ORIGINAL CONTRIBUTIONS
174 Experimental Oncology 37, 173–180, 2015 (September)
sera samples were processed in the same way and
stored with 50% glycerol at −20 °C. Consent forms
were obtained from all patients. The study protocol
was approved by the Ethics Committee of the Institute
of Molecular Biology and Genetics.
mRNA preparation and construction of cDNA
libraries. Normal colon and colon primary tumor
samples were obtained as surgical specimens and
frozen in liquid nitrogen with following storage at
−80 °C. Total RNA was prepared by the guanidinium
thiocyanate method [14], and mRNA was puri-
fied by the Dynabeads Oligo(dT)25 kit (Dynal, Oslo,
Norway). 5 ug of mRNA was used for construction
of oligo(dT)-primed double-stranded cDNA ligated
into the lambda ZAP II expression vector (Stratagene,
La Jolla, CA). A total of four cDNA libraries were con-
structed with titers of 1–3•106, and the average inserts
size of 1.2–1.5 kb (Table 1).
Table 1. Colon cancer cDNA libraries analyzed by SEREX
cDNA
Library
Colon tumor
(histology; stage) Titer Serum type
Clones
scree-
ned
Posi-
tive
clones
Ge-
nes
1C Poorly differentiated,
metastases in lymph
nodes; stage IV
2×105 Autologous 2×105 5 2
2C Moderately differen-
tiated, lymph node
hyperplasia; stage III
2×106 Autologous 2.5×105 3 2
3C Moderately differen-
tiated; stage II
1×105 Autologous 1×105 1 1
4C Moderately differen-
tiated; stage III
0.2×105 Allogene-
ic (pool
of 6 sera
from stages
II–IV colon
cancer pa-
tients)
0.2×105 37 17
Total 46 22
Immunoscreening. Sera used for immuno-
screening were stored at −20 °C with 50% glycerol.
In order to remove antibodies reactive with the vector
system sera prior to immunoscreening were diluted
at 1:10 in TBS and absorbed through overnight incuba-
tion at 4 °C with Sepharose 4B (Pharmacia, Uppsala,
Sweden) coupled to lysates from E. coli XL1 and lamb-
da ZAP II-infected E. coli XL1 synthesized as described
previously [15]. Final sera dilutions (1:100) were pre-
pared in TBS with 0.2% non-fat dried milk (Marwell,
England) and 0.01% NaN3 as preservative. The cDNA
libraries were not amplified prior to immunoscreening
procedure. Immunoscreening of cDNA libraries was
performed as described [10], except recombinant
phage plated at concentration of 104/15 cm plate.
Allogeneic immunoscreening of positive clones
was developed with sera dilution at 1:100 as de-
scribed [10]. Serum reactivity was detected with the
alkaline phosphatase substrate, 4-nitro blue tetrazo-
lium chloride/5-bromo-4-chloro-3-indolylphosphate
(Sigma, St. Louis, MO, USA). Serum was considered
as reactive when positive staining was observed during
immunoscreening.
DNA Sequencing. Monoclonalized phage cDNA
clones were converted to pBluescript phagemids
by in vivo excision according to user manual (Stratagene,
La Jolla, CA, USA). The cDNA inserts were sequenced
with universal M13mp18 primer using the DYEnamic
ET Terminator Cycle Sequencing Kit ( Amersham Bio-
sciences, Buckinghamshire, England) and ABI Prism
automated DNA sequencer (Perkin Elmer, Norwalk, CT).
RT-PCR analysis. PCR primers (Invitrogen,
Paisley, Scotland) with melting temperature between
60–65 °C were designed to amplify cDNA fragments
of 400–500 bp represented adjacent exons of cor-
respondent genes in order to check possible genomic
DNA contamination in PCR product bands. The integrity
of synthesized cDNAs was determined by the amplifica-
tion of β-actin. RT-PCR was performed using HotStarTag
DNA polymerase (Qiagen, GmbH, Germany) with 35 cy-
cles of 1 min at 94 °C, 1 min at 65 °C, and 1 min at 72 °C,
followed by 7 min at 72 °C. The sequences of primers
used for amplification were as follows: KY-CC-8: forward,
5´CAGCGTTATTCTCATTGCCTG; reverse, 5´CTCCACT-
GACATCTTCCATTG; KY-CC-12: forward, 5´CAGAAGGTC-
CTGCAGCTCTAC; reverse, 5´GATGGACATATGTTCAT-
GTGC; KY-CC-14: forward, 5´GACAGGCTCCTGAGT-
GAAGTG; reverse, 5´CCTAGGATGGTCAGCAGCAAG;
KY-CC-15: forward, 5´TTATACGGCATTATCATGGAC;
reverse, 5´AAGATGCATGGTGCCATTGTC; KY-CC-19:
forward, 5´ACGGCCGAGCTCATTCACCTG; reverse,
5´TCTCGGCTCACTTCATCTTAG.
Real-time RT-PCR. For real-time RT-PCR were
used 6 normal colon cDNAs and 9 colon cancer cDNAs
with the same primer pairs combination as described
above. Thermal cycling and fluorescent monitoring
were performed using an ABI7700 Sequence Analyzer
(Applied Biosystems, Foster City, CA, USA). As endo-
genous control was used β-actin. Triplicate PCR reac-
tions were prepared for each cDNA sample with SYBR
Green I PCR mix (Qiagen, GmbH, Germany). PCR
consisted of 40 cycles of 94 °C denaturation (1 min),
65 °C annealing (1 min), and 72 °C extension (1 min)
with following denaturation at 65–94 °C for 20 min
to determine PCR products melting curve. The point
at which a PCR product is first detected above a fixed
threshold (Ct) was determined for each sample, and
the average of triplicate samples was calculated.
Respective Ct va lues were normalized (ΔCt) by sub-
tracting the Ct value obtained for β-actin control. For
6 normal colon cDNA samples normalized ΔCt was
calculated as the mean value. The relative concentra-
tions of genes-specific mRNAs in colon cancer cDNAs
compare to normal colon tissue (ΔΔCt) were calcu-
lated by subtracting the normalized mean ΔCt value
obtained for normal colon cDNAs from those obtained
with 9 colon tumor samples (ΔΔCt = ΔCt of tumor —
ΔCt of normal colon), and the relative concentration
was determined as 2-ΔΔCt.
RESULTS
SEREX defined cDNAs clones. Immunoscre ening
of the four primary colon cancer cDNA expression li-
braries with autologous or allogeneic serums produced
a total of 48 serum-positive clones. Sequence analysis
Experimental Oncology 37, 173–180, 2015 (September) 175
of cDNAs from isolated clones revealed 22 different
genes those have been deposited at LICR SEREX data-
base (http://www.licr.org/SEREX.html) under KY-CC-
1-KY-CC-22 designation. For libraries 1C-3C screened
with autologous serums were isolated 9 clones those
represent 5 different genes. For library 4C screened with
pool of 6 allogeneic sera obtained from colon cancer
(stages II–IV) patients were isolated 37 clones those
represent 17 different genes. The largest proportion
of antigens has been isolated during allogeneic im-
munoscreening of library 4C in contrast to the other
free libraries screened with autologous serums (see
Table 1). All of isolated antigens, except KY-CC-20 are
genes with known function (Table 2). Searching for
homology to previously SEREX-defined genes at LICR
SEREX database (http://www.licr.org/SEREX.html)
revealed that 6 (KY-CC-2, 5, 6, 15, 16, 21) through
22 antigens isolated at this work have been identi-
fied previously in different tumors and only KY-CC-16,
21 found for colon cancer. Despite as much as five
libraries have been analyzed, SEREX-defined clones
were unique for each immunoscreened library (see
Table 2). Analysis of antigen’s molecular functions
showed that at least 8 antigens are involved in the re-
alisation of the genetic information (KY-CC-1/RPL18,
KY-CC-2/se2–2, KY-CC-5/EEF1A1, KY-CC-8/BRCA2,
KY-CC-13/RPLP0, KY-CC-15/PLRG1, KY-CC-18/
GNB2L1, KY-CC-22/TRIP11) (see Table 2). The 7 anti-
gens participate in cell proliferation, development and
apoptosis (KY-CC-7/PDAP1, KY-CC-8/BRCA2, KY-
CC-10/TRIM2, KY-CC-11/BTN3A3, KY-CC-18/GNB2L1,
KY-CC-19/TSGA2, KY-CC-21/UACA), other 2 (KY-CC-3/
COX1, KY-CC-4/TALDO1) revealed metabolic activity,
6 antigens (KY-CC-9/CXCR4, KY-CC-11/BTN3A3, KY-
CC-12/FKBP4, KY-CC-16/BRAP, KY-CC-18/GNB2L1,
KY-CC-22/TRIP11) are involved in cell signalling and
5 antigens (KY-CC-6/COL1A1, KY-CC-12/FKBP4, KY-
CC-14/ACTR1A, KY-CC-16/BRAP, KY-CC-17/ACTB)
participate in restructuring of the cytoskeleton and cell
adhesion (see Table 2). It should be noted that some
antigens at least KY-CC-8/BRCA2, KY-CC-11/BTN3A3,
KY-CC-12/FKBP4, KY-CC-18/GNB2L1 and KY-CC-22/
Table 2. Characterization of antigens identified by immunoscreening of colon cancer cDNA expression libraries
Antigen Gene homology Molecular Function
KY-CC-1/RPL18 Ribosomal protein L18 (RPL18) RNA synthesis. Part of the 60S ribosomal subunit
KY-CC-2/se2–2 CEP290 gene, Aliase CTCL tu-
mor antigen se2–2
Part of the tectonic-like complex which is required for tissue-specific ciliogenesis and may regulate
ciliary membrane composition. Activates ATF4-mediated transcription
KY-CC-3/COX1 Cytochrome c oxidase subunit
I (COX1)
Cytochrome c oxidase is the component of the respiratory chain that catalyzes the reduction of oxy-
gen to water
KY-CC-4/TALDO1 Transaldolase 1 (TALDO1) The key enzyme of the pentose phosphate pathway and important for the balance of metabolites
in the pentose-phosphate pathway
KY-CC-5/EEF1A1 Translation elongation factor
1 alpha 1 (EEF1A1)
Promotes the GTP-dependent binding of aminoacyl-tRNA to the A-site of ribosomes during protein
biosynthesis
KY-CC-6/COL1A1 Collagen, type I, alpha 1
(COL1A1)
The collagen type I, alpha 1, play role in fibril forming, putative downregulated c-Myc target gene
KY-CC-7/PDAP1 PDGFA associated protein
1 (PDAP1)
Enhances PDGFA-stimulated cell growth in fibroblasts, but inhibits the mitogenic effect of PDGFB
KY-CC-8/BRCA2 BRCA2 region, mRNA se-
quence CG016
Important for cell cycle control and DNA repair through homologous recombination, also involved
in embryonic cellular proliferation
KY-CC-9/CXCR4 Chemokine (C-X-C motif), re-
ceptor 4 (fusin) (CXCR4)
Receptor for the CХC chemokine. Acts as a receptor for extracellular ubiquitin. Involved in hemato-
poiesis, cardiac ventricular septum formation and mediates LPS-induced inflammatory response
KY-CC-10/TRIM2 Tripartite motif-containing
2 (TRIM2)
UBE2D1-dependent E3 ubiquitin-protein ligase that mediates the ubiquitination of NEFL and
of phosphorylated BCL2L11. Plays a neuroprotective function
KY-CC-11/BTN3A3 Butyrophilin, subfamily 3, mem-
ber A3 (BTN3A3)
Plays a role in T-cell responses in the adaptive immune response. Also, the proteins of this family
play role in cell proliferation and development
KY-CC-12/FKBP4 FK506 binding protein 4 (59kD)
(FKBP4)
Immunophilin protein with PPIase and co-chaperone activities. Component of steroid receptors
hete rocomplexes through interaction with heat-shock protein 90 (HSP90). Acts also as a regulator
of microtubule dynamics
KY-CC-13/RPLP0 Ribosomal protein, large,
P0 (RPLP0)
Ribosomal protein (60S) is the functional equivalent of E. coli protein L10
KY-CC-14/ACTR1A ARP1 actin-related protein
1 homolog A, centractin alpha
(yeast) (ACTR1A)
Component of a multi-subunit complex involved in microtubule based vesicle motility. It is associ-
ated with the centrosome
KY-CC-15/PLRG1 Pleiotropic regulator
1 (PRL1 homolog) (PLRG1)
Component of the PRP19-CDC5L complex that forms an integral part of the spliceosome and is re-
quired for activating pre-mRNA splicing
KY-CC-16/BRAP BRCA1 associated protein
(BRAP)
Negatively regulates MAP kinase activation by limiting the formation of Raf/MEK complexes proba-
bly by inactivation of the KSR1 scaffold protein. May also act as a cytoplasmic retention protein with
a role in regulating nuclear transport
KY-CC-17/ACTB Actin, beta (ACTB) Actins are highly conserved proteins that are involved in various types of cell motility and are ubiqui-
tously expressed in all eukaryotic cells
KY-CC-18/GNB2L1 Guanine nucleotide binding
protein (G protein), beta poly-
peptide 2-like 1 (GNB2L1)
Involved in the recruitment, assembly and/or regulation of a variety of signaling molecules and plays
a role in many cellular processes. It is a part of the 40S ribosomal subunit
KY-CC-19/TSGA2 H. sapience testes specific
A2 homolog (mouse) (TSGA2)
May play an important role in male meiosis (By similarity). It is necessary for proper building of the
axonemal central pair and radial spokes
KY-CC-20/IM-
AGE:4893383
EST: IMAGE: 4893383 No data available for molecular function
KY-CC-21/UACA Uveal autoantigen with coiled-
coil domains and ankyrin re-
peats (UACA)
Regulates APAF1 expression and plays an important role in the regulation of stress-induced apop-
tosis. Promotes apoptosis by regulating three pathways, apoptosome up-regulation, LGALS3/galec-
tin-3 down-regulation and NF-kappa-B inactivation
KY-CC-22/TRIP11 TRIP11 H. sapiens thyroid hor-
mone receptor interactor 11
Binds the ligand binding domain of the thyroid receptor (THRB) in the presence of triiodothyronine
and enhances THRB-modulated transcription. Golgi auto-antigen
176 Experimental Oncology 37, 173–180, 2015 (September)
TRIP11exhibit multiple functions and were classified
in different categories.
Allogeneic immunoscreening of SEREX-de-
fined antigens. In order to determine colon cancer
related serological profile of identified antigens alloge-
neic immunoscreening have been performed with sera
obtained from 14 colon cancer and 6 gastric tract cancer
patients, as well as 18 healthy donors. Eight of 22 tested
antigens reacted with both normal and cancer sera
samples; two antigens were positive only for normal sera;
seven antigens showed no reaction with any sera and
five antigens solely positive only for colon cancer sera
(Table 3). For some antigens reacted with cancer and
normal sera previously showed their association with au-
toimmune, inflammatory-related or non-cancerous (viral)
diseases (see Table 3). In addition, KY-CC-1/RPL18,
KY-CC-8/CG016, KY-CC-18/GNB2L1 and KY-CC-22/
FLJ20542 may represent novel tumor-independent
occurring autoantigens firstly isolated in this work.
KY-CC-21/UACA, KY-CC-18/GNB2L1 and KY-CC-6/
COL1A1 autoantigens revealed the highest percentage
reactivity with both normal and cancer serums tested,
ranging from 29% to 100% (see Table 3). Five antigens
with colon-cancer specific serological profile, namely
KY-CC-12/FKBP4, KY-CC-14/ACTR1A, KY-CC-15/
PLRG1, KY-CC-19/TSGA2, KY-CC-17/β-actin were reac-
ting totally for 14% of tested colon cancer sera samples
(Table 4). Through these antigens only KY-CC-15/
PLRG1 was previously identified by SEREX-analysis
in hepatocellular carcinoma (data unpublished), for the
others have not been documented their reactivity with
any tumor patients sera.
Table 3. SEREX-defined antigens with not colon cancer related serological
profile
Antigen
Serum reactivity (number
of positive sera/number
of sera analysed)
Association with
autoimmune
dise ase2/refe-
rence
Colon
tumor1
Gastrict
tract tu-
mor
Healthy
donors
KY-CC-1/RPL18 1/14 0/6 1/18 No data
KY-CC-2/se2–2 0/14 2/6 1/18 No data
KY-CC-3/COX1 0/14 0/6 0/18 HT [16]
KY-CC-4/TALDO1 0/14 0/6 0/18 MS [17]
KY-CC-5/EEF1A1 2/14 0/6 3/18 No data
KY-CC-6/COL1A1 12/12 NT 11/11 PBC [18], AP [19]
KY-CC-7/PDAP1 0/14 0/6 0/18 No data
KY-CC-8/CG016 1/14 0/6 1/18 No data
KY-CC-9/CXCR4 0/14 0/6 0/18 No data
KY-CC-10/TRIM2 0/14 0/6 0/18 No data
KY-CC-11/BTN3A3 0/14 0/6 0/18 MS [20]
KY-CC-13/RPLP0 0/14 0/6 1/18 SLE [21]
KY-CC-16/BRAP 0/14 0/6 0/18 No data
KY-CC-18/GNB2L1 5/9 NT 9/11 No data
KY-CC-20/IMAGE:4893383 1/7 NT 2/11 No data
KY-CC-21/UACA 5/14 1/6 5/18 Panuveitis [22]
KY-CC-22/FLJ20542 1/14 0/6 2/18 No data
Note: 1Data of serum reactivity not include reactivity with autologous or pool
of allogeneic sera used for initial SEREX-analysis. 2Abbreviations: PBC — pri-
mary biliary cirrhosis; AP — adult periodontitis; MS — multiple sclerosis; SLE —
systemic lupus erythematosus; HT — Hashimoto’s thyroiditis.
mRNA expression profile of serologically de-
fined colon cancer specific antigens. KY-CC-12/
FKBP4, KY-CC-14/ACTR1A, KY-CC-15/PLRG1 and
KY-CC-19/TSGA2 defined by allogeneic immuno-
screening as colon cancer specific antigens were
tested RT-PCR and real-time RT-PCR for 6 normal
colon cDNAs and 9 colon cancer cDNAs.
Antigens tested by RT-PCR were positive for all co-
lon cancer cDNAs; KY-CC-15/PLRG1 and KY-CC-19/
TSGA2 were positive for all 6 normal colon cDNAs,
therefore KY-CC-12/FKBP4 and KY-CC-14/ACTR1A
were positive only for 4 normal colon cDNAs and very
weak bands observed for these genes for the other
2 normal colon cDNAs (data not shown). The rela-
tionship between tested antigens mRNA expression
levels and serological reactivity was not examined for
respective colon cancer patients due to their sera were
not available for typing.
According to the result of real-time RT-PCR, KY-
CC-14/ACTR1A mRNA showed increased expression
level at 2.5–7.7 times for 8 through 9 tested colon can-
cer cDNAs compare to normal colon (Figure, a). So, KY-
CC-14/ACTR1A may have slightly upregulated mRNA
expression level as mean at 3.5 times in colon tumors.
KY-CC-19
0.1
1.0
10.0
100.0
1 2 3 4 5 6 7 8 9
Colon cancer cDNAs
Fo
ld
c
ha
ng
e
KY-CC-15
0.1
1.0
10.0
100.0
1 2 3 4 5 6 7 8 9
Colon cancer cDNAs
Fo
ld
c
ha
ng
e
KY-CC-14
1
2
3
4
5
6
7
8
1 2 3 4 5 6 7 8 9
Colon cancer cDNAs
Fo
ld
c
ha
ng
e
KY-CC-12
0.01
0.10
1.00
10.00
1 2 3 4 5 6 7 8 9
Colon cancer cDNAs
Fo
ld
c
ha
ng
e
a
c
b
d
Figure. Real-time RT-PCR results for relative expression level
of identified colon cancer related antigens in 9 tested colon
cancer cDNA samples. Expression level of antigens in normal
colon referred as 1 in all cases
KY-CC-15/PLRG1 and KY-CC-19/TSGA2 showed
heterogeneous mRNA expression profile in colon tumor
samples with exceptionally high levels of transcripts com-
pare to normal colon associated with the colon cancer
case N7 (80 and 88 times elevation, correspondently)
(Figure, b, c). KY-CC-19/TSGA2 mRNA expression also
increased at 7 times in tumor case N3 and at 10 times
in tumor case N9, despite for colon cancer cases
NN 1, 2, 4, 5, 8 its normal expression down regulated
at 2–3.5 times (Figure, b). KY-CC-15/PLRG1 mRNA
in addition to tumor case N7 have slightly increased ex-
pression at 3 times in tumor case N3 and near the same
level of expression compare to normal colon in tumor
Experimental Oncology 37, 173–180, 2015 (September) 177
cases NN 1, 2, 4, 5, 6, 8, 9 (difference range at 0.48–1.96)
(Figure, c). KY-CC-15/PLRG1 and KY-CC-19/TSGA2 rep-
resent the genes with selective activation of normal
expression in some cases of colon cancer and may
be immunogenic as aberrantly expressed.
By real-time RT-PCR result KY-CC-12/FKBP4 mRNA
normal expression down regulated at 11–25 times in tu-
mor cases NN 2, 3, 8 and at 3.1 and 3.7 times in tumor
cases NN 4, 6, respectively (Figure, d). In the others
four cases of tested tumors, KY-CC-12/FKBP4 mRNA
expression was near at the same range (difference
at 0.7–1.2 times) as in normal colon. KY-CC-12/
FKBP4 showed a great down regulation of normal ex-
pression in approximately of ⅓ of colon cancer cases.
DISCUSSION
Utilization of SEREX-analysis led to identification
of a broad spectrum normal and tumor sera IgG reactive
proteins currently consisted of over 2000 enters at LICR
SEREX database (http://www.licr.org/SEREX.html).
Exploration of these proteins in term of tumor-specific
immunity discovered a subset of antigens as attrac-
tive targets for cancer diagnosis and immunotherapy.
It have been previously showed that primary colon
tumors analyzed by SEREX methodology may display
a small number antigens detected by autologous
sera [10, 12], despite metastasis counterpart more
productive for serum reactive clones [10]. Recently,
application of SEREX-analysis to colon cancer cell
lines and testis cDNA expression libraries immuno-
screened with colon cancer patients allogeneic sera
led to identification of about ten new antigens with
colon tumor restricted serological profile. It is also has
been found a decline in quantity of serum reactive anti-
gens for colon tumor stages II–III patients sera [11, 13].
With idea that serum samples from tumor patients may
differ by cancer-related IgG response and tumor con-
sists a mix of different cells, not one cell line, we used
for serological screening of one primary colon tumor
cDNA expression library pool of 6 allogeneic sera along
with autologous serum immunoscreening of the ot hers
three synthesized tumor cDNA libraries [13]. Here
we describe antigens isolated in more detail.
Of isolated 22 different antigens the largest pro-
portion (17 antigens) were derived from the cDNA
expression library screened with pool of allogeneic
sera. In contrast, only 5 antigens were obtained
from the cDNA expression libraries screened with
autologous sera (see Table 1). SEREX-defined an-
tigens were unique for each cDNA library analyzed,
i.e. no similar antigens have been found (see Ta-
ble 2). For 16 of 22 SEREX-defined antigens there
are no data about their immunogenecity in any
type of cancer according to SEREX database data
(http://www.licr.org/SEREX.html). So, it is possible
that complete list of serum IgG reactive antigens for
colon cancer should be far from completion and many
other antigens can be isolated using for immuno-
screening a pool of allogeneic sera those increases
a chance to recognize a more primary colon tumor
antigens. Antigens identified were characterized ac-
cording their molecular functions and it was shown
that the most of them are involved in the implementa-
tion of the genetic information including replication,
transcription, translation and reparation (see Table 2).
Analysis of frequency of the allogeneic sera antibody
response of healthy donors and colon cancer as well
as gastric tract tumors patients to the 22 identified
antigens showed that 5 (22%) antigens, namely KY-
CC-12/FKBP4, KY-CC-14/ACTR1A, KY-CC-15/PLRG1,
KY-CC-17/β-actin, KY-CC-19/TSGA2 have colon
cancer-restricted serological profile. Except KY-CC-15/
PLRG1, previously identified by SEREX in hepatocellular
carcinoma (data unpublished), the others four antigens
are firstly isolated at this work and all solely positive for
14% of tested colon cancer allogeneic sera (Table 4).
During allogeneic immunoscreening some antigens
showed no reactivity with any type of sera tested (see
Table 3). We suggest that using a more extended set
of normal and cancer serum samples as well as mo-
dernization of conditions for large-scale allogeneic
immunoscreening should be this problem solving.
Table 4. SEREX-defined antigens with colon cancer related serological profile
Antigen
Serum reactivity (number of positive
sera/number of sera analysed) Previously
isolated
by SEREX in2
Colon tumor1 Gastrict
tract tu-
mor
Healthy
donorsTotal Reactive
serum
KY-CC-12/FKBP4 1/14 9 0/6 0/18 No data
KY-CC-14/ACTR1A 1/14 9 0/6 0/18 No data
KY-CC-15/PLRG1 1/14 8 0/6 0/18 Hepatocellular
carcinoma
KY-CC-17/β-actin 1/14 8 0/6 0/18 No data
KY-CC-19/TSGA2 1/14 9 0/6 0/18 No data
Note: 1Data of serum reactivity not include reactivity with autologous or pool
of allogeneic sera used for initial SEREX-analysis. 2Data obtained at LICR
SEREX database.
We were surprised for housekeeping cytoskeletal
protein β-actin identified as colon cancer antigen.
Previously have been reported that some patients
with type 1 autoimmune hepatitis have anti-actin an-
tibody [23] and β-actin also a candidate autoantigen
in autoimmune inner ear disease [24]. Therefore,
recently was showed that the cause for the anti-
actin immune response in medullary breast cancer
(MCB) relates to the actin exposure on the surface
of apoptotic MCB cells, allow this intracellular an-
tigen to be exposed to the local humoral immune
system [25]. We suggest that apoptosis or intensive
necrosis of colon cancer cells may also provoke of hu-
moral immune response to β-actin. As it was showed
for the other housekeeping gene — ATP6S1, putative
accessory unit of the vacuolar H+-ATPase complex,
despite the broad tissue expression it may stimulate
humoral responses in a significant proportion of mela-
noma and non-small cell lung carcinoma patients
treated by vaccination with irradiated autologous
tumor cells without provoking clinical autoimmunity
[26]. So, study of β-actin immunogenecity and au-
toimmunity in different tumours patients may define
it is usefulness for detection of apoptotic positive colon
cancer variants or cancer treatment.
178 Experimental Oncology 37, 173–180, 2015 (September)
As it has been recorded previously, immunogene-
city of SEREX-defined antigens may depend on muta-
tion and/or overexpression of correspondent genes
in tumor cells those also can be contributed to tumor
formation [6, 27]. Sequenced cDNAs for colon cancer-
specific serum reactive clones have no any muta-
tions according to comparison with relative matches
in GenBank data. Real-time RT-PCR for KY-CC-12/
FKBP4, KY-CC-14/ACTR1A, KY-CC-15/PLRG1, KY-
CC-19/TSGA2 revealed aberrant distribution its mRNA
normal level in some tested colon cancer tissues that
can made them implications in tumor etiologic and
immunogenecity context.
KY-CC-12/FKBP4 (FKBP52) belongs to a distinct
group of structurally related immunophilins and par-
ticipates in association with Hsp90 to form chaperoned
mammalian steroid receptor complexes into a confor-
mation that is optimal for binding hormone [28–30].
FKBP4 is expressed at varying levels in all human tis-
sues as well as cultured cells tested and its expression
is reduced in fibroblastic CCL39 mutant-transformed
cells or cells able to grow in low serum-containing me-
dium, suggesting that FKBP4 might participate in the
negative feedback control to cell proliferation [31].
Also we found the reduction of FKBP4 mRNA expres-
sion in colon tumor comparing to normal colon tissue
(Figure, d). Due to one molecule of FKBP4 have stoi-
chiometry in steroid receptor heterocomplexes with
two molecules of hsp90 determined for glucocorticoid
receptor [30, 32], progesterone receptor [33], and
estrogen receptor [34] misbalance in this ratio due
to decreasing in FKBP4 expression possible for colon
cancer as well as increasing in heat shock proteins ex-
pression reported for gastrointestinal tumors [35] may
lead to appearance of nonfunctional steroid receptor.
In case of ER-β those can mediated protective effects
of estrogen replacement therapy and its protein ex-
pression is markedly and specifically reduced in colon
cancers compared to adjacent normal colon in vivo [36],
process described above may be alternative way to in-
activation of ER-β in ER-β positive colon cancer cells
as more early event in molecular etiologic of this tumor.
Down regulation of FKBP4 expression in colon cancer
cells can disrupt normal protein processing for which
multiple isoforms have been found [28], as well as con-
centration in cell to form complex with hsp90 with fol-
lowing generation of aberrant set of peptides presenting
by MHC. Recently the same mechanism was proposed
for down-regulated genes found by SEREX-analysis
as immunogeneic antigens [11].
KY-CC-14/ACTR1A (actin-related protein 1 ho-
molog A, centractin alpha (yeast)) is an actin-related
protein, and is approximately 60% identical at the amino
acid level to conventional actin. It comprises the most
abundant 42.6 kD subunit in the dynactin complex
involved in a diverse array of cellular functions, inclu-
ding ER-to-Golgi transport, the centripetal movement
of lysosomes and endosomes, spindle formation, chro-
mosome movement, nuclear positioning, and axono-
genesis [37]. We found for approximately half of tested
colon tumors increasing of ACTR1A mRNA expression
at 3–8 times compare to normal tissue (Figure, c), those
can predict immunogenecity of correspondent protein.
Recently have been found that dynactin complex may
participate in maintaining of multicentric chromosomes
during chromosome segregation in tumor cells [38]. So,
ACTR1A may associated as one of many components
for survival of cancer cells with multiple chromosome
aberration. The next study of dynactin complex in tu-
mor cells may define appropriated targets for therapy
oriented on disruption of control mechanism aberrant
chromosome segregation.
KY-CC-19/TSGA2 represents protein, also known
as RSPH1, is present in the sperm flagellum [39]. This
protein is a homolog for mouse testes specific A2 pro-
tein and has unknown function in the humans [40].
Mouse TsgA2, initially believed to be confined to the
testis in male mice [41, 42]. TsgA2 have been iso-
lated using testis-specific polyclonal antibodies from
an expression cDNA library prepared from the mouse
testis [41]. Immunohistochemical examination revealed
TsgA2 specific association with the metaphase chro-
mosomes and spindles suggesting that the protein
plays important roles in male meiosis [41]. We found
that KY-CC-19/TSGA2 mRNA expression in human not
restricted only to reproductive tissues because it was
found in testis, normal colon and colon tumors. In two
through 9 colon tumors tested TSGA2 mRNA expression
was elevated at about 10 times and for the other one
at near 90 times compare to normal colon (Figure, a).
Such overexpression of TSGA2 in some cases of colon
tumors may be a reason for its immunogenecity. Ac-
cording to postulated for TsgA2 important role in meta-
phase chromosomes segregation it is also possible,
as in case of ACTR1A, that multiple chromosome aber-
ration in tumor cells require some specific modification
in molecular machinery acted in cell division and human
TSGA2 one of that for tumorgenesis process.
KY-CC-15/PLRG1 represents human homolog
to pleiotropic regulator 1 found in Arabidopsis [43].
The human proteins CDC5L (hCDC5) and PLRG1 are
both highly conserved components of a multiprotein
complex that is a subunit of the spliceosome and
interaction between them is essential for pre-mRNA
splicing progression [44, 45]. It has been shown that
overexpression of CDC5L in mammalian cells shorte-
ned the G2 phase of the cell cycle and a dominant
negative mutant of the protein lacking the activation
domain slowed G2 progression and delayed entry
into mitosis [46] and also PLRG1 deficiency causes
an earlier cell cycle arrest, as cells cannot progress
through S phase [45]. We found that in one case of co-
lon tumor mRNA expression of KY-CC-15/PLRG1 was
elevated at 80 times compare to normal colon those
can be as reason for IgG antibody response in case
of tumors with shortened G2 phase, i.e. with rapidly
growth characteristic.
In conclusion, we found that using pool of al-
logeneic sera obtained from colon cancer patients
for SEREX-analysis of colon primary cancer may
Experimental Oncology 37, 173–180, 2015 (September) 179
be an optimal way to overcome a difficulty with isola-
tion of low number immunoreactive antigens for this
tumor. We suggest that best way to complete SEREX
characterization of all possible colon cancer antigens
is using different stage colon tumors for serum im-
munoscreening each with pools of sera obtained also
for different stage tumors. It was found that primary
colon tumors have new unique antigens in comparison
to previously isolated, because majority of the last have
been obtained for colon cancer cell lines. We con-
firmed for five through 22 antigens isolated at this work
its colon-cancer restricted serological profile requests
its next exploration to define suitable targets for colon
tumor therapy or diagnostic.
ACKNOWLEDGMENTS
We thank Dr. U. Sahin and Dr. O. Tureci at III. Medi-
zinische Klinik and Poliklinik, Johannes Gutenberg
Universitat Mainz, Germany, for introduction to us allo-
geneic SEREX-analysis procedure and help in RT-PCR
analysis of cDNAs. This study was supported by Rus-
sian Science Foundation (project 15–15–20032).
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Copyright © Experimental Oncology, 2015
|
| id | nasplib_isofts_kiev_ua-123456789-145484 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 1812-9269 |
| language | English |
| last_indexed | 2025-12-07T17:58:13Z |
| publishDate | 2015 |
| publisher | Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| record_format | dspace |
| spelling | Garifulin, O.M. Kykot, V.O. Gridina, N.Y. Kiyamova, R.G. Gout, I.T. Filonenko, V.V. 2019-01-22T12:47:53Z 2019-01-22T12:47:53Z 2015 Application of serex-analysis for identification of human colon cancer antigens / O.M. Garifulin, V.O. Kykot, N.Y. Gridina, R.G. Kiyamova, I.T. Gout, V.V. Filonenko // Experimental Oncology. — 2015. — Т. 37, № 3. — С. 173-180. — Бібліогр.: 46 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/145484 Background: Colorectal, lung and breast tumors are the most devastating and frequent malignances in clinical oncology. SEREX-analysis of colon cancer leads to identification of more than hundred antigens which are potential tumor markers. With idea that immunoscreening with pool of allogeneic sera is more productive for antigen isolation, SEREX-analysis was applied to four cases of stages II–IV primary colon tumor and 22 new antigens were isolated. Objective: To characterize 22 primary colon cancer antigens isolated by SEREX-technique. Materials and Methods: Allogenic screening, real-time PCR analysis. Results: After allogeneic immunoscreening, for 5 of 22 (22%) isolated antigens were confirmed colon cancer restricted serological profile solely positive for 14% of tested colon cancer sera. Through these five antigens, KY-CC-17/β-actin has cytoskeleton function; KY-CC-14/ACTR1A and KY-CC-19/TSGA2 participate in chromosome segregation; KY-CC-12/FKBP4 regulates steroid receptor function and KY-CC-15/PLRG1 is a component of spliceosome complex. For the last four antigens tested were found aberrant mRNA expression in some cases of colon tumor. Conclusion: The exploration of identified antigens may define suitable targets for immunotherapy or diagnostic of colon cancer. Key Words: colon cancer, SEREX-analysis, allogeneic immunoscreening. We thank Dr. U. Sahin and Dr. O. Tureci at III. Medizinische Klinik and Poliklinik, Johannes Gutenberg Universitat Mainz, Germany, for introduction to us allogeneic SEREX-analysis procedure and help in RT-PCR analysis of cDNAs. This study was supported by Russian Science Foundation (project 15–15–20032). en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Original contributions Application of serex-analysis for identification of human colon cancer antigens Article published earlier |
| spellingShingle | Application of serex-analysis for identification of human colon cancer antigens Garifulin, O.M. Kykot, V.O. Gridina, N.Y. Kiyamova, R.G. Gout, I.T. Filonenko, V.V. Original contributions |
| title | Application of serex-analysis for identification of human colon cancer antigens |
| title_full | Application of serex-analysis for identification of human colon cancer antigens |
| title_fullStr | Application of serex-analysis for identification of human colon cancer antigens |
| title_full_unstemmed | Application of serex-analysis for identification of human colon cancer antigens |
| title_short | Application of serex-analysis for identification of human colon cancer antigens |
| title_sort | application of serex-analysis for identification of human colon cancer antigens |
| topic | Original contributions |
| topic_facet | Original contributions |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/145484 |
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