Materials of International VACTRAIN/3-rd Swedish-Ukrainian Conference on Cancer Diseases January 16–17 2017 Stockholm Sweden
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2017
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irk-123456789-1376102019-01-20T21:12:46Z Materials of International VACTRAIN/3-rd Swedish-Ukrainian Conference on Cancer Diseases January 16–17 2017 Stockholm Sweden Conference reports 2017 Article Materials of International VACTRAIN/3-rd Swedish-Ukrainian Conference on Cancer Diseases January 16–17 2017 Stockholm Sweden // Experimental Oncology. — 2017 — Т. 39, № 1. — С. 88-93. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/137610 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Materials of International VACTRAIN/3-rd Swedish-Ukrainian Conference on Cancer Diseases January 16–17 2017 Stockholm Sweden |
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Materials of International VACTRAIN/3-rd Swedish-Ukrainian Conference on Cancer Diseases January 16–17 2017 Stockholm Sweden |
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materials of international vactrain/3-rd swedish-ukrainian conference on cancer diseases january 16–17 2017 stockholm sweden |
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Materials of International VACTRAIN/3-rd Swedish-Ukrainian Conference on Cancer Diseases January 16–17 2017 Stockholm Sweden // Experimental Oncology. — 2017 — Т. 39, № 1. — С. 88-93. — англ. |
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Experimental Oncology |
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88 Experimental Oncology 39, 88–93, 2017 (March)
MATERIALS OF INTERNATIONAL VACTRAIN/
3-rd SWEDISH-UKRAINIAN CONFERENCE ON CANCER DISEASES,
JANUARY 16–17, 2017, STOCKHOLM, SWEDEN
The scientific conference ‘‘International VACTRAIN/
3-rd Swedish-Ukrainian conference on cancer diseases’’
was held on January 16–17, 2017 at Karolinska Institutet,
Stockholm, Sweden. These 1.5 days were filled with
the lectures by the invited speakers and oral presenta-
tions selected on the basis of submitted abstracts from
younger researchers. The meeting was focused on what
we have learnt over the recent times, on the current situ-
ation, and on the important challenges for the future with
focus on combating cancer.
The Vice-Chancellor of Karolinska Institutet Profes-
sor Karin Dahlman-Wright gave an overview on the can-
cer research at Karolinska Institutet. Cancer is an enemy
that requires attack from all sides and with all available
resources. Collaboration is always important to make
big scientific breakthroughs and clearly cancer research
is no exception. It is vital that scientific discoveries are
translated efficiently into new treatment methods and
programs, new medicines and techniques. Karolinska
Institutet is at the forefront of cancer research in the
world. More than half of the research at Karolinska Insti-
tutet is carried out together with an international party.
As Ukraine strengthens its ties to the European Union,
collaborations like this workshop are vital.
The head of R.E. Kavetsky Institute of Experimental
Pathology, Oncology and Radiobiology (IEPOR), the
National Academy of Sciences of Ukraine academi-
cian Vasyl Chekhun gave a brief overview on research
and collaborations at IEPOR. He also mentioned that
the VACTRAIN grant allow the researchers at IEPOR,
especially younger generation to grow profession-
ally. Dr. Kateryna Tyminska, representing Embassy
of Ukraine in the Kingdom of Sweden stressed the
importance of scientific collaboration in relationship
of two countries. Plenty of the new data and ideas
were presented by researchers from Ukraine, Sweden,
and Poland.
Professors Klas Wiman, Sonia Lain and Galina
Selivanova presented the data on the new cancer
medicine, based on activation of the TP53 protein. The
main topic of the lectures by Professors Lars Holm-
gren, Pontus Aspenström and Manuel Patarroyo was
the involvement of laminins, cadherins and integrins
in the structure and migration of the cancer cells. Pro-
fessor Yihai Cao told about the antiangiogenic cancer
therapy. Professor Boris Zhivotovsky explained the
mechanism of chemoresistance of lung adenocarci-
noma. Professor Ingemar Ernberg presented his con-
cept of cancer “attractor” cells, at the edge of tumor
biology, mathematical modeling and system biology.
Professors Hans-Gustaf Ljunggren and Britta Wahren
described their work on anti-cancer immunotherapy,
including the clinical trials. The lecture by Professor
Svitlana Sidorenko was devoted to a putative target
for immunotherapy, CD150.
Many interesting reports, concerning the choice
of the targets for cancer therapy were presented by other
Swedish and Ukrainian researchers (see selected ab-
stracts). Cancer therapy and the problems of persona-
lized cancer therapy were also in the spot of the interest.
During conference, largely supported by the VACTRAIN
of Horizon 2020 program, there were also the poster
sessions, a good platform to discuss the new data criti-
cally. Over 70 researchers took part in the work of this
conference. Definitely, this meeting helped to broaden
the knowledge in medical science and to plan the new
fruitful scientific works with the aim to combat cancer.
SELECTED ABSTRACTS
ENDOPLASMIC RETICULUM STRESS
AS A KEY FACTOR OF GENOME
REPROGRAMMING IN CANCER CELLS
O.O. Ratushna*, D.O. Minchenko,
O.O. Riabovol, O.Y. Luzina, O.H. Minchenko
Palladin Institute of Biochemistry, NAS of Ukraine,
Kyiv 01601, Ukraine
*E-mail: Oksana_Ratushna@hotmail.com
The endoplasmic reticulum (ER) stress represents
the unfolded protein response to cope with the accu-
mulation of unfolded or misfolded proteins. It is required
to maintain the functional integrity of the ER, which
is a dynamic intracellular organelle with exquisite sensi-
tivity to alterations in homeostasis. The unfolded protein
response is a key player in the development of differ-
ent malignant tumors. Depending on the duration and
severity of the ER stress, it leads to cell adaptation
or demise. This stress is a fundamental phenomenon,
which provides a secure protection of the cells from
different environmental challenges and is transduced
by three major ER resident stress sensors. Activation
of these ER stress sensors leads to transcriptional
reprogramming of the cells. The signaling pathways
elicited by those stress sensors have connections with
metabolic pathways and with other plasma membrane
receptor signaling networks. As such, the ER has an es-
sential position as a signal integrator in the cell and is in-
strumental in the different phases of tumor progression.
Inositol requiring enzyme 1 (IRE1) is the most con-
served transducer of the unfolded protein response
and produces either adaptive (preserve ER homeo-
Exp Oncol 2017
39, 1, 88–93
CONFERENCE REPORT
Experimental Oncology 39, 88–93, 2017 (March)39, 88–93, 2017 (March) (March) 89
stasis) or death signals through both kinase and
endoribonuclease, including unconventional splicing
of XBP1 mRNA and regulated IRE1-dependent decay
of mRNA (RIDD). Splice variant of XBP1 controls
the expression of hundreds of the unfolded protein
response-specific genes upon global translational re-
pression to preserve ER homeostasis. We have shown
that inhibition of IRE1 suppresses glioma cell prolife-
ration and tumor growth by affecting the expression
of genes encoding the tumor suppressors, TNF recep-
tors and related proteins, key transcription factors and
protein kinases, as well as numerous mitochondrial
proteins. Moreover, inhibition of IRE1 endoribonucle-
ase activity only has stronger effect on glial cell pro-
liferation in vitro and also on glioma growth (in mouse
orthotopic brain model) and gene expression profile.
It concerns especially genes, encoding key regula-
tory factors, controlling cell proliferation, distinct from
inhibition of both enzymatic activities of IRE1.
Furthermore, we have shown that hypoxia, which
is an obligate interconnected component of malignant
tumor growth, affects almost all studied genes. Thus,
inhibition of IRE1 signaling network mostly modifies the
expression of proliferation related genes, contributing
to the rate of glioma cell proliferation. We have identi-
fied several perspective genes, whose expression was
significantly changed in glioma cells with inhibited
both, enzymatic activities of IRE1 and only its endori-
bonuclease. These genes, as well as IRE1 endoribo-
nuclease and kinase can be perspective targets to the
design the novel compounds for therapeutic strategies
to manipulate levels of ER stress in diseases. A better
understanding of the biological role of IRE1 signaling
network is needed to build an integrated systematic
view on IRE1 signaling. This will be applied to develop
novel, innovative inhibitors and activators of this sig-
naling enzyme.
THE ADAPTOR PROTEIN
Ruk/CIN85 PARADOXICALLY ENHANCES
EMT OF TRIPLE NEGATIVE MOUSE BREAST
ADENOCARCINOMA 4T1 CELLS
I. Horak*, D. Shytikov, D. Geraschenko,
L. Knopfova, L. Borsig, L. Drobot
Palladin Institute of Biochemistry, NAS of Ukraine,
Kyiv 01601, Ukraine
*E-mail: iryna.horak@gmail.com
To coordinate cellular responses, cell surface re-
ceptors employ receptor-associated adaptor proteins
that are composed exclusively of domains and motives
involved in intermolecular interactions. The assembling
of adaptor proteins-mediated supramolecular com-
plexes is regulated in dynamic and selective fashion,
thereby influencing processing of information through
signaling networks. Adaptor protein Ruk/CIN85 con-
sists of three SH3 domains, four blocks of proline-rich
motives and C-terminal coiled-coil region, and acts
as a transducer platform that participates in control
of various physiological processes, such as apopto-
sis, ligand-induced endocytosis of receptor tyrosine
kinases, vesicular trafficking, cell adhesion, moti-
lity, and invasion. It has been shown that high levels
of Ruk/CIN85 contribute to the conversion of weakly
invasive human breast adenocarcinoma MCF-7 cells
into a more malignant phenotype. However, a number
of issues with respect to the role of Ruk/CIN85 in breast
carcinogenesis remain still open.
To establish syngeneic mouse model suitable for
experiments in vivo we stably overexpressed or down-
regulated Ruk/CIN85 in triple negative mouse ad-
enocarcinoma 4T1 cells (RukUp or RukDown cells,
respectively). As it turned out, RukUp cells acquired
a rounded shape, whereas RukDown cells had a more
distinct epithelial phenotype indicating that possible
EMT/MET processes occurred in these cells. To elu-
cidate this issue, RukUp and RukDown cells were
studied for their adhesive and invasive properties
in vitro and also for the expression of several marker
EMT-associated genes.
Adhesion assay of 4T1 cells showed inverse depen-
dence of adhesion to collagen type I and fibronectin
on Ruk/CIN85 expression. We also demonstrate that
overexpression of Ruk/CIN85 is associated with in-
creased migration and invasion through Matrigel, colla-
gen type I and fibronectin, as well as through endothelial
cells layer. Importantly, Ruk/CIN85 down-regulation
led to decrease in 4T1 cells motility and invasiveness
in vitro.
Using western-blot analysis of main EMT mark-
ers (vimentin and E-cadherin) we found high levels
of vimentin and low of E-cadherin in RukUp cells,
and the opposite results in RukDown cells. We used
RT2 Profiler PCR array (Qiagen) for 84 EMT-associated
genes in order to find additional molecules involved
in Ruk/CIN85-mediated EMT. Ruk/CIN85 was found
to positively regulate expression of such transcription
factors as Mitf, Zeb2, Twist, Sparc, as well as osteo-
pontin, EGFR and TGF-β. Negative effect of Ruk/
CIN85 was demonstrated for Bmp-7, Wnt, E-cadherin,
FoxC, keratins genes Krt-7, Krt-14 and Krt-19, and
MMP-9. These data suggest that adaptor protein
Ruk/CIN85 is a critical regulatory component involved
in EMT of breast cancer cells.
BIOLOGICAL CHARACTERISTICS OF TUMOR
CELLS AT THE DIFFERENT STAGES OF EMT
UPON EXPOSURE TO ANTICANCER DRUGS
AND CYTOKINES
N. Bezdieniezhnykh*, O. Lykhova, R. Kocherga,
Yu. Kudryavets
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine,
Kyiv 03022, Ukraine
*E-mail: cellbank@ukr.net
Formation of a highly malignant metastatic tumor
cell phenotype and resistance to anticancer drugs
are associated with the implementation of epithelial-
mesenchymal transition (EMT). Thus, monitoring
of EMT and the possible inhibition of this process could
be helpful to inhibit the tumor progression. It is quite
90 Experimental Oncology 39, 88–93, 2017 (March)
often that anti-tumor treatment is not effective, so new
modalities and new targets to combat cancer should
be developed. Established cell lines, primary cultures
of malignant cells obtained from biopsies or ascites
of patients with epithelial cancers (breast, colorectal
and ovarian), immunohistochemistry, and statistical
methods were used.
We found that the majority of the primary tumor cells
(of ascitic fluids) showed expression of mesenchymal
markers, namely vimentin and N-cadherin. Upon cul-
turing in vitro, on the adhesively active extracellular
matrix, expression of mesenchymal markers, for ex-
ample, the Twist and Slug transcriptional factors, was
down-regulated. At the same time, a number of cells
expressing the epithelial markers, namely E-cadherin,
pan-keratin, etc. was increased. Moreover, the cells
altered their sensitivity to anticancer drugs, despite
the various mechanisms of drug action (ta xanes, vinca
alkaloids, antimetabolites). Modification of the extra-
cellular matrix, i.e. growth on collagen or in spheroids,
also significantly influenced EMT marker expression
and sensitivity of cells to the drugs.
Next, we investigated the influence of components
of the microenvironment, especially cytokines inter-
feron, tumor necrosis factor, and interleukins on the
established cell lines (MCF-7, MDA-MB-231, Colo 205,
HT-29) that show different EMT profiles. We wanted
to find the most effective inhibitors of the EMT process,
thus, reducing the malignancy of tumor cells. The an-
ticancer drugs, such as platinum-containing, campto-
thecins, and some others were also tested.
Concluding, we have observed a lability of phe-
notypic characteristics of the primary tumor cells,
i.e. changes in morphology, expression of markers,
associated with EMT, which correlated with their sen-
sitivity to anticancer drugs. We identified a number
of biologically active agents that might shift EMT
to either side, thus inhibiting the malignant phenotype
of tumor cells.
ALTERNATIVE DIRECTION OF INHIBITION
OF MALIGNANT PROPERTIES
IN TUMOR CELLS IN VITRO AND IN VIVO
BY GENE THERAPY WITH INF-β GENE
IN RECOMBINANT BACULOVIRUS VECTOR
O. Lykhova1,*, L. Strokovska2, O. Kovaleva1,
N. Bezdieniezhnykh1, N. Semesiuk1,
I. Adamenko1, A. Vorontsova1, Yu. Kudryavets1
1R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine,
Kyiv 03022, Ukraine
2Institute of Molecular Biology and Genetics,
NAS of Ukraine, Kyiv 03680, Ukraine
*E-mail: AlexxDNA@gmail.com
The aim of the study was to investigate the influ-
ence of recombinant baculovirus containing the
interferon-β gene (rBV/IFN) on phenotypic charac-
teristics of tumor cells in vitro: morphology, growth,
cytogenetic characteristics and expression of proteins
associated with proliferative activity, cell cycle regula-
tion, epithelial-mesenchymal transition (EMT), inva-
siveness, and the migration potential. Tumorigenicity
and the metastatic potential of tumor cells after their
transduction with rBV/IFN were studied in vivo, using
the mouse models.
The mouse Lewis lung carcinoma cells (LL cell line)
and melanoma cells (MM-4 cell line) were used.
Transduction of melanoma cells and lung carcinoma
cells with rBV/IFN leads to reduction of the cell number,
the growth rate, cell migration ability, soft agar colony
formation, and also in inhibition of the ability to grow
in a serum-free condition in vitro. The LL/rBV/IFN and
MM-4/rBV/IFN cells are arrested in G0/G1 and S phases.
Transduction of these cells with the IFN-β gene was
accompanied with the accumulation of apoptotic cells,
an increased level of nuclear anomalies in MM-4 cells
and the increased frequency of mitotic pathology
in LL cells. Moreover, transduction of LL cells with rBV/
IFN causes significant genotoxic effects: the levels
of chromosome aberrations were increased, as well
as the frequency of appearance of nuclear protrusions,
polyploidy and endoreduplication.
A l s o , t h e p r o d u c t i o n o f re c o m b i n a n t
IFN-β by MM-4 and LL cells was accompanied
by the changes in the expression of proteins associ-
ated with the cell cycle regulation, EMT, invasiveness
and migration ability: expression of p19ARF and
p21WAF1 was increased, and of N-cadherin was
decreased in MM-4 cells. The number of E-cadherin-
positive LL cells was increased. In addition, trans-
duction of lung carcinoma cells with rBV/IFN leads
to significant inhibition of expression of Slug and Twist.
These changes indicate a significant inhibition of ЕМТ
process and reversion of tumor cells to the “normal-
like” epithelial phenotype.
In result, transduction of LL carcinoma and
MM-4 melanoma cells with rBV/IFN in vitro inhibits
their ability to form solid tumors and lung metastases
in the mice. Therapeutic intravenous administration
of the rBV/IFN results in significant reduction of the
number and volume of lung metastases in the mice.
Concluding, transduction of lung carcinoma and
melanoma cells with rBV/IFN inhibits their sings of ma-
lignancy in vitro and in vivo, provides genotoxic effects
in LL cells and leads to suppression of ЕМT in these
cells.
CD150/SLAMF1 AS A NEW POTENTIAL
TARGET FOR ANTI-TUMOR THERAPY
S.P. Sidorenko*, O. Romanets-Korbut,
I. Gordiienko, L. Kovalevska, L. Shlapatska
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine,
Kyiv 03022, Ukraine
*E-mail: svitasyd@yahoo.com
CD150/SLAMF1 is a prototype member of SLAM
family within the immunoglobulin superfamily of sur-
face receptors that are widely expressed on cells within
hematopoietic system. Six of nine SLAMF receptors
have a paired unique immunoreceptor tyrosine-based
Experimental Oncology 39, 88–93, 2017 (March)39, 88–93, 2017 (March) (March) 91
switch motif (ITSM) that serves as a docking site for
SH2-containing proteins. In T and B lymphocytes,
natural killer cells, macrophages and dendritic cells
CD150 is a co-receptor molecule that mediates dif-
ferent signal transduction pathways depending on the
availability of downstream signaling elements, espe-
cially, the adaptor protein SH2D1A/SAP. Due to highly
glycosylated and sialylated extracellular Ig domains,
CD150 is involved in homotypic interactions and could
be considered as a pattern-recognizing receptor.
It is a major entry receptor for several Morbilliviruses,
including measles virus, and also a bacterial sensor that
control the killing of Gram-negative bacteria. Function-
ally it serves as a bridge between innate and adaptive
immunity.
More than 90% of lymphoid leukemia and lympho-
mas in adults have B-cell origin. Within B-cell lineage
cell surface receptor CD150/SLAMF1 is broadly
expressed starting from pre-B cells with up-regu-
lation toward plasma cells. However, expression
of CD150 is rather limited on the surface of malignant
B cells with the block of differentiation at the different
stages of maturation. The high level of CD150 surface
expression is observed in hairy cell leukemia, clas-
sical Hodgkin lymphoma, subtype of diffuse large
B-cell lymphoma with activated B cell phenotype
(ABC-DLBCL), primary cutaneous follicular centre
B-cell lymphoma, and in 60% of chronic lymphocytic
leukemia cases. Expression and functions of this an-
tigen outside of the hematopoietic system were not
fully explored. We found that CD150 was expressed
in several tumors of ectodermal origin (e.g. squamous
cell carcinoma of uterine cervix, rectum and oral ca-
vity, basalioma), but not in their normal counterparts.
Recently we found CD150 expression in malignant
cells of central nervous system (CNS) tumors. Al-
though CD150 was not found in different regions
of normal brain tissues, our immunohistochemical
study revealed its expression in 77.6% of human CNS
tumors, inclu ding glioblastoma, anaplastic astrocy-
toma, diffuse astrocytoma, ependymoma, and others.
CD150 was detected in the cytoplasm, but not on the
cell surface of glioma cell lines, and it was colocalized
with the endoplasmic reticulum and Golgi complex
markers. In addition to the full length mRNA of the
conventional mCD150 splice isoform, in glioma cells
we found a highly expressed novel CD150 transcript
(nCD150), containing an 83 bp insert. The insert is de-
rived from a previously unrecognized exon designated
Cyt-new, which is located 510 bp downstream of the
transmembrane region exon, and is a specific feature
of primate SLAMF1. Since CD150 is not revealed
in normal brain tissues, but is expressed in 77.6%
of CNS tumors, CD150 could be considered as a novel
diagnostic marker for CNS tumors and a potential
target for the therapy of gliomas, especially measles
virus-based oncolytic therapy.
CD150/SLAMF1 ANTIGEN IN MOLECULAR
PATHOBIOLOGY OF CHRONIC LYMPHOCYTIC
LEUKEMIA
I. Gordiienko*, L. Shlapatska, V. Kholodniuk,
L. Sklyarenko, S. Sidorenko
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine,
Kyiv 03022, Ukraine
*E-mail: imgordiienko@gmail.com
Recent finding indicate that CD150 cell surface
expression can be used as a surrogate prognostic
marker of chronic lymphocytic leukemia (CLL) favo-
rable outcome. It was shown that CLL patients with high
CD150 expression level on malignant B cells has longer
treatment free survival and overall survival compared
to patients, which lost CD150 cell surface expression.
However, the mechanisms that underlie dependent
on CD150 expression biological properties of CLL
B cells are not fully understood. Present study was
focused on characterization of CD150 topology and
isoforms expression, as well as study of CD150 medi-
ated signaling pathways in CLL B cells.
For the first time, we found that in cell surface
CD150 negative (csCD150−) CLL cases CD150 antigen
was expressed on the protein level and was localized
in the cytoplasm. Within cytoplasm it was colocalized
with markers of endoplasmic reticulum, Golgi appa-
ratus and endosomes, but not lysosomes. Exclusive
cytoplasmic CD150 expression in the part of CLL cases
was not associated with endoplasmic reticulum stress
or ceramide metabolism. Ligation of CD180, but not
CD40 or BCR, leaded to slight CD150 up-regulation
in csCD150− CLL B cells. CD150 cell surface expression
was positively correlated with expression of cell death
receptor CD95, BCR negative regulator — CD22 and
receptor of Toll-like family, potential prognostic marker
of favorable outcome — CD180. The highest level
of CD150 colocalization on the cell surface of CLL B cells
was observed for CD180. We found that conventional
transmembrane mCD150 is a predominant CD150 iso-
form in CLL. However, in 20% of studied CLL cases
nCD150 isoform was prevalent. Moreover, elevated
mRNA expression of sCD150 isoforms was detected
in all CLL cases, compared to normal B cells subsets.
In csCD150+ CLL cases the basal level of tyrosine phos-
phorylation and phosphorylation of serine/threonine
specific motifs, which are substrates for AMPK, Akt,
PKA, PKC, CDK kinases, was higher compared to that
in csCD150− CLL B cells. Outcome of CD150 mediated
Akt and MAPK kinases phosphorylation in CLL B cells
was dependent on basal phosphorylation level of these
kinases and often demonstrated rapid p38MAPK phos-
phorylation and bimodal kinetics of Akt, ERK1/2 and
JNK1/2 activation. Examining the transcription factors
expression profile in CLL B cells revealed that high
PU.1 protein expression level positively correlated
with CD150 cell surface expression in CLL. Further-
more, CD150 signaling was involved in regulation
of PU.1 mRNA level in csCD150+ CLL cases. We also
found that signals via CD150, in contrary to BCR,
92 Experimental Oncology 39, 88–93, 2017 (March)
CD40 and CD180, significantly down-regulated mRNA
expression levels of CCL3, CCL4 and IL-10 cytokines
in CLL B cells. This may contribute to favorable clinical
outcome of csCD150+ CLL cases.
Taken together, CLL cases are heterogeneous in cell
surface CD150 expression as well as in CD150 iso-
forms expression.
CHANGES IN EXPRESSION OF miRNA-122,
-200b, AND -320a AS PROGNOSTIC
BIOMARKERS FOR BREAST CANCER
T. Borikun*, N. Lukianova, V. Chekhun
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine,
Kyiv 03022, Ukraine
*E-mail: tborikun@gmail.com
High heterogeneity and diversity of breast tumors
make the molecular characterization based on new
biological markers very important. Recent studies have
shown that not only genes and proteins are important
players in tumorigenesis, but also a new class of po-
tential epigenetic tumor markers such as microRNAs
(miRNAs). An important advantage of miRNA molecules
is their stability in both plasma and tumor tissues.
The aim of the study was to identify the features of on-
cosuppressive miRNA-122, -200b, and -320a in breast
cancer (BC) patients and to monitor the possible correla-
tion between their expression and cancer progression.
The study was conducted using 110 tumor samples
and serum of the BC patients and 14 serum samples
of healthy volunteers. The levels of miRNAs were as-
sessed using q-PCR. The expression levels of estrogen
receptor, progesterone receptor, Her2/neu, Ki-67,
E-cadherin, and N-cadherin were monitored by im-
munohistochemical analysis.
Each of miRNAs has a great network of targets
involved is cellular processes, that makes them
very promising diagnostic markers. Among variety
of cancer-related miRNAs we choose several that
can be used as molecular markers in BC progno-
sis and are responsible for migration, proliferation,
epithelial-mesenchymal transition, and drug sensibil-
ity. We have found that the majority of BC tissues are
characterized by a significant decrease in expression
of miRNA-122 and -200b (in 93.2% and 83.1% of cas-
es, respectively). Down-regulation of miRNA-122,
-200b, and -320a in BC tissue was associated with
a higher staging, enhanced prolife ration, and also with
the absence of hormone receptors (p < 0.05). Serum
levels of miRNA-122 and -200b were associated with
the pathological stage (p = 0.005 and p = 0.01, re-
spectively). Decrease of miRNA-320a levels in cancer
tissue compared to normal samples was associated
with the triple negative BC phenotype as well, and
also with the active proliferation, hence, with cancer
aggressiveness. Low levels of miRNA-320a in serum
were observed in patients with lymph node metastases
(p = 0.03).
Concluding, the established profiles of circulating
and tumor miRNAs are associated with aggressive
clinical course of BC. It makes the determination
of expression of the miRNA-122, -200b, and -320a
in the serum the base for development of noninvasive
screening tools for BC prognosis.
THE MRPS18–2 PROTEIN AS A PUTATIVE
MARKER OF CARCINOGENESIS
M. Mints1, M. Mushtaq2, N. Iurchenko3,
L. Kovalevska3, L. Buchynska3, E. Kashuba2,3,*
1Department of Women’s and Children’s Health
(KBH), Karolinska Institutet, Stockholm 17176,
Sweden
2Department of Microbiology, Tumor and Cell
Biology (MTC), Karolinska Institutet, Box 280,
Stockholm 17177, Sweden
3R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine,
Kyiv 03022, Ukraine
*E-mail: Elena.Kashuba@ki.se
Endometrial cancer (EC) is one of the most
frequent causes of cancer death among women
in developed countries. Histopathological diagnosis
and imaging techniques for EC are limited, thus new
prognostic markers are needed to offer patients the
best treatment and follow-up.
We showed that the level of mitochondrial riboso-
mal protein MRPS18–2 (S18–2) increased in EC com-
pared with the normal endometrium and hyperplasia,
based on a study of 42 patient biopsies. Importantly,
high expression of free E2F1 in EC correlates well with
high S18–2 expression. The EC cell line HEC-1-A,
which overexpresses S18–2 constitutively, showed
an increased proliferation capacity in vitro and in vivo
(in SCID mice). Moreover, pan-keratin, β-catenin
and E-cadherin signals are diminished in these cells,
compared to the parental HEC-1-A line, in contrast
to vimentin signal that is increased. This may be as-
sociated with epithelial-mesenchymal transition.
We conclude that high expression of S18–2 and
free E2F1, and low pan-keratin, β-catenin, and E-
cadherin signals might be a good set of prognostic
markers for EC.
HIGH EXPRESSION LEVELS
OF MRPS18–2 AND PRESENCE
OF THE RB PROTEIN ARE REQUIRED
FOR THE MAINTENANCE OF THE STEM CELL
PHENOTYPE
M. Mushtaq1,*, L. Kovalevska2, G. Klein1,
E. Kashuba1,2
1Department of Microbiology, Tumor and Cell
Biology (MTC), Karolinska Institutet, Box 280,
Stockholm 17177, Sweden
2R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine,
Kyiv 03022, Ukraine
*E-mail: Muhammad.Mushtaq@ki.se
We have found that mitochondrial ribosomal protein
MRPS18–2 (S18–2) is involved in regulation of the
RB-dependent pathway. It binds to both hypo- and hy-
perphosphorylated RB. The binding between RB and
Experimental Oncology 39, 88–93, 2017 (March)39, 88–93, 2017 (March) (March) 93
S18–2 is promoted when cytoplasmic S18–2 is tar-
geted to the nucleus, and this disrupts the association
of E2F1 with RB, as indicated by the increased level
of free E2F1 in the nucleus. This presumably enhances
the RB-dependent block to S-phase entry in the cell
cycle. We have also found overexpression of the hu-
man S18–2 in immortalized primary rat embryonic
fibroblasts (REFs) that showed properties of embry-
onic stem cells. Elevated expression of S18–2 in stem
cells (our fin dings and analysis of published microar-
ray data) raises the question of whether this protein
co-operates with the RB protein in differentiation and
carcinogenesis. We wanted to seek a relation between
the expression of RB and S18–2 in Rb1−/− MEFs and
stemness. We hypothesized that simultaneous expres-
sion of both proteins at the high levels might support
stemness.
We have used transfections, inoculation into SCID
mice, directed differentiation, quantitative polymerase
chain reaction, immunostaining, immunohistochemis-
try, western blotting. We showed that S18–2 protein,
together with RB, plays a crucial role in cell de-
differentiation. We have found that overexpression
of S18–2 and RB is needed for maintenance of cell
stemness. Such cells can differentiate into various cell
lineages under certain conditions.
Concluding, the presence of RB and simultaneous
expression of S18–2 at high levels are required for the
cell stemness.
IDENTIFICATION OF NOVEL MOLECULAR
AND GENETIC MARKERS FOR EARLY
DETECTION AND PROGNOSIS OF EPITHELIAL
TUMORS
O. Mankovska*, V. Kashuba
Institute of Molecular Biology and Genetics,
NAS of Ukraine, Kyiv 03680, Ukraine
*E-mail: mankovsska@gmail.com
The main topic of our research is the identifica-
tion of molecular and genetic markers for the early
detection of epithelial tumors and prognosis of the
course of disease. Early detection of carcinogenic
process can lead to more successful treatment, thus,
significantly increasing the chances for recovery. The
investigation of non-invasive diagnostic tools, which
can be used in clinical practice, is now the main focus
of oncology. This problem can be solved by using
of molecular nucleic acid markers that are present
in biological fluids (serum, urine, semen etc).
We have developed several useful and effective
approaches for investigation of molecular and genetic
markers. We are studying both, genetic and epigenetic
changes in cancers of different types, using a broad
scale screening of their presence in clinical samples.
The techniques and methods we use allow us to obtain
reliable results and propose them for implementation
in clinical practice.
GENOTOXIC SENSITIVITY
TO 4-HYDROXYESTRADIOL IN PERIPHERAL
BLOOD LYMPHOCYTES OF ENDOMETRIAL
CANCER PATIENTS
O. Brieieva*, I. Nesina, L. Buchynska
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine,
Kyiv 03022, Ukraine
*E-mail: olha.brie@gmail.com
The endometrial cancer (EC) pathogenesis to a large
extent may be determined by genotoxic effects of estro-
gen metabolites, among which the 4-hydroxyestradiol
(4OHE2) is characterized by the most prominent DNA-
damaging properties. Genomic instability and sensitivity
to genotoxic estrogen metabolites in cells of EC patients
depend on the DNA repair efficiency.
The aim of the study was to analyze the level of DNA
damage in peripheral blood lymphocytes (PBL) of EC pa-
tients after treatment with the 4OHE2 and to evaluate the
repair efficiency of induced DNA damage.
Total 33 EC patients (mean age 60.2 ± 1.5 years)
and 20 healthy women (mean age 55.7 ± 3.1 years)
were included in the study. The level of DNA damage
in PBL was measured, using comet assay and ex-
pressed by % tail DNA (% tDNA). Estradiol (E2) level
in blood serum was assessed by ELISA (pg/ml).
Large variability in basal level of DNA damage was
observed in PBL of EC patients (1.8–20.5% tDNA)
as well as healthy donors (1.1–9.2% tDNA). In aver-
age, EC patients had significantly higher basal level
of DNA damage (7.9 ± 0.9% tDNA) compared to healthy
women (3.5 ± 0.5% tDNA) (p < 0.05). After incubation
with 4OHE2 there was an increase in the % tDNA in PBL
of EC patients (8.0–57.7% tDNA) as well as of healthy
donors (7.9–46.8% tDNA). However, the average level
of induced DNA damage in EC patients was significantly
higher (38.9 ± 2.7% tDNA) compared to healthy women
(25.8 ± 2.2% tDNA) (p < 0.05). To investigate the associa-
tion between the sensiti vity of PBL to 4OHE2 and estra-
diol level in the blood, we measured the concentration
of E2 in the blood serum. We found that the blood level
of E2 was higher (54.9 ± 4.1 pg/ml) in EC patients with
high sensitivity of PBL to 4OHE2 than in patients with low
sensitivity (38.2 ± 4.3 pg/ml). The higher level of unre-
paired DNA damage in PBL was observed in EC patients
(18.6 ± 2.6% tDNA) compared to control group (13.1 ±
1.4% tDNA). Furthermore, it was found that patients with
high sensitivity of PBL to 4OHE2 had greater amount
of unrepaired DNA damage (24.1 ± 4.4% tDNA) than
women with low sensitivity (13.1 ± 1.8% tDNA).
These results indicate that PBL of EC patients
are characterized by hypersensitivity to 4OHE2 and
impaired DNA damage repair that is associated with
hormonal status of women. It can be assumed that
increased level of 4OHE2-induced genome instability
may contribute to pathogenesis of EC.
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