Expression of ITSN2 and TKS5 in different subtypes of breast cancer tumors
Aim. Regardless a great progress in treatment, 15% of breast cancer cases remain lethal. One of the main problems in diagnosis and cure of this cancer type is its high clinical and genetic heterogeneity, and the identification of markers for personalized treatment is still a topical issue. Methods....
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| Cite this: | Expression of ITSN2 and TKS5 in different subtypes of breast cancer tumors / S.V. Kropyvko, L.O. Tsyba, O.V. Novokhatska, Y.M. Nemesh, L.A. Syvak, T.Ye. Tarasenko, A.N. Grabovoy, A.V. Rynditch // Вiopolymers and Cell. — 2019. — Т. 35, № 1. — С. 21-29. — Бібліогр.: 23 назв. — англ. |
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Kropyvko, S.V. Tsyba, L.O. Novokhatska, O.V. Nemesh, Y.M. Syvak, L.A. Tarasenko, T.Ye. Grabovoy, A.N. Rynditch, A.V. 2019-06-15T14:53:01Z 2019-06-15T14:53:01Z 2019 Expression of ITSN2 and TKS5 in different subtypes of breast cancer tumors / S.V. Kropyvko, L.O. Tsyba, O.V. Novokhatska, Y.M. Nemesh, L.A. Syvak, T.Ye. Tarasenko, A.N. Grabovoy, A.V. Rynditch // Вiopolymers and Cell. — 2019. — Т. 35, № 1. — С. 21-29. — Бібліогр.: 23 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.00098F https://nasplib.isofts.kiev.ua/handle/123456789/154379 577.22 Aim. Regardless a great progress in treatment, 15% of breast cancer cases remain lethal. One of the main problems in diagnosis and cure of this cancer type is its high clinical and genetic heterogeneity, and the identification of markers for personalized treatment is still a topical issue. Methods. Collection of clinical material, RNA isolation, and analysis of the ITSN2 and TKS5 isoforms expression using real-time quantitative PCR with fluorescence labeled probes. Results. The reliably reduced expression of ITSN2-S has been found in the HER2/neu-positive tumors with poor prognosis. There was no significant difference in the expression of ITSN2-L and TKS5-L in the analyzed samples. Conclusions. Our study has shown a potential use of the ITSN2 short isoform (ITSN2-S) as a breast cancer prognostic marker. Мета. Незважаючи на значний прогрес у лікуванні, 15% випадків захворювання на рак молочної заози залишаються смертельними. Однією з головних проблем у діагностиці та лікуванні цього типу раку є його висока клінічна та генетична гетерогенність, тому ідентифікація маркерів для персоналізованої терапії є актуальною проблемою. Методи. Збір клінічного матеріалу, ізоляція РНК та аналіз експресії ізоформ ITSN2 та TKS5, використовуючи кількісну ПЛР в реальному часі з флюоресцентно міченими зондами. Результати. Нами було виявлено, що ITSN2-S знижує свою експресію в HER2/neu-позитивних пухлинах з поганим прогнозом. Не було відмічено істотної різниці в експресії ITSN2-L і TKS5-L в проаналізованих зразках. Висновки. В даній роботі продемонстровано потенційну можливість використання короткої ізоформи ITSN2 (ITSN2-S) як прогностичного маркера раку молочної залози. Цель. Несмотря на значительный прогресс в лечении, 15% случаев рака молочной железы остаются смертельными. Несмотря на многолетние исследования, одной из главных проблем в диагностике и лечении этого типа рака является его высокая клиническая и генетическая гетерогенность, поэтому идентификация маркеров для персонализированной терапии является актуальной проблемой. Методы. Сбор клинического материала, изоляция РНК и анализ экспрессии изоформ ITSN2 и TKS5, используя количественную ПЦР в реальном времени с флюоресцентно мечеными зондами. Результаты. Нами было выявлено, что ITSN2-S снижает свою экспрессию в HER2/neu-позитивных опухолях с плохим прогнозом. Не было отмечено существенной разницы в экспрессии ITSN2-L и TKS5-L в проанализированных образцах. Выводы. В данной работе продемонстрировано потенциальную возможность использования короткой изоформы ITSN2 (ITSN2-S) как прогностического маркера рака молочной железы. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Biomedicine Expression of ITSN2 and TKS5 in different subtypes of breast cancer tumors Експресія ITSN2 та TKS5 у різних підтипах раку молочної залози Экспрессия ITSN2 и TKS5 в различных подтипах рака молочной железы Article published earlier |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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| title |
Expression of ITSN2 and TKS5 in different subtypes of breast cancer tumors |
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Expression of ITSN2 and TKS5 in different subtypes of breast cancer tumors Kropyvko, S.V. Tsyba, L.O. Novokhatska, O.V. Nemesh, Y.M. Syvak, L.A. Tarasenko, T.Ye. Grabovoy, A.N. Rynditch, A.V. Biomedicine |
| title_short |
Expression of ITSN2 and TKS5 in different subtypes of breast cancer tumors |
| title_full |
Expression of ITSN2 and TKS5 in different subtypes of breast cancer tumors |
| title_fullStr |
Expression of ITSN2 and TKS5 in different subtypes of breast cancer tumors |
| title_full_unstemmed |
Expression of ITSN2 and TKS5 in different subtypes of breast cancer tumors |
| title_sort |
expression of itsn2 and tks5 in different subtypes of breast cancer tumors |
| author |
Kropyvko, S.V. Tsyba, L.O. Novokhatska, O.V. Nemesh, Y.M. Syvak, L.A. Tarasenko, T.Ye. Grabovoy, A.N. Rynditch, A.V. |
| author_facet |
Kropyvko, S.V. Tsyba, L.O. Novokhatska, O.V. Nemesh, Y.M. Syvak, L.A. Tarasenko, T.Ye. Grabovoy, A.N. Rynditch, A.V. |
| topic |
Biomedicine |
| topic_facet |
Biomedicine |
| publishDate |
2019 |
| language |
English |
| container_title |
Вiopolymers and Cell |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Експресія ITSN2 та TKS5 у різних підтипах раку молочної залози Экспрессия ITSN2 и TKS5 в различных подтипах рака молочной железы |
| description |
Aim. Regardless a great progress in treatment, 15% of breast cancer cases remain lethal. One of the main problems in diagnosis and cure of this cancer type is its high clinical and genetic heterogeneity, and the identification of markers for personalized treatment is still a topical issue. Methods. Collection of clinical material, RNA isolation, and analysis of the ITSN2 and TKS5 isoforms expression using real-time quantitative PCR with fluorescence labeled probes. Results. The reliably reduced expression of ITSN2-S has been found in the HER2/neu-positive tumors with poor prognosis. There was no significant difference in the expression of ITSN2-L and TKS5-L in the analyzed samples. Conclusions. Our study has shown a potential use of the ITSN2 short isoform (ITSN2-S) as a breast cancer prognostic marker.
Мета. Незважаючи на значний прогрес у лікуванні, 15% випадків захворювання на рак молочної заози залишаються смертельними. Однією з головних проблем у діагностиці та лікуванні цього типу раку є його висока клінічна та генетична гетерогенність, тому ідентифікація маркерів для персоналізованої терапії є актуальною проблемою. Методи. Збір клінічного матеріалу, ізоляція РНК та аналіз експресії ізоформ ITSN2 та TKS5, використовуючи кількісну ПЛР в реальному часі з флюоресцентно міченими зондами. Результати. Нами було виявлено, що ITSN2-S знижує свою експресію в HER2/neu-позитивних пухлинах з поганим прогнозом. Не було відмічено істотної різниці в експресії ITSN2-L і TKS5-L в проаналізованих зразках. Висновки. В даній роботі продемонстровано потенційну можливість використання короткої ізоформи ITSN2 (ITSN2-S) як прогностичного маркера раку молочної залози.
Цель. Несмотря на значительный прогресс в лечении, 15% случаев рака молочной железы остаются смертельными. Несмотря на многолетние исследования, одной из главных проблем в диагностике и лечении этого типа рака является его высокая клиническая и генетическая гетерогенность, поэтому идентификация маркеров для персонализированной терапии является актуальной проблемой. Методы. Сбор клинического материала, изоляция РНК и анализ экспрессии изоформ ITSN2 и TKS5, используя количественную ПЦР в реальном времени с флюоресцентно мечеными зондами. Результаты. Нами было выявлено, что ITSN2-S снижает свою экспрессию в HER2/neu-позитивных опухолях с плохим прогнозом. Не было отмечено существенной разницы в экспрессии ITSN2-L и TKS5-L в проанализированных образцах. Выводы. В данной работе продемонстрировано потенциальную возможность использования короткой изоформы ITSN2 (ITSN2-S) как прогностического маркера рака молочной железы.
|
| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/154379 |
| citation_txt |
Expression of ITSN2 and TKS5 in different subtypes of breast cancer tumors / S.V. Kropyvko, L.O. Tsyba, O.V. Novokhatska, Y.M. Nemesh, L.A. Syvak, T.Ye. Tarasenko, A.N. Grabovoy, A.V. Rynditch // Вiopolymers and Cell. — 2019. — Т. 35, № 1. — С. 21-29. — Бібліогр.: 23 назв. — англ. |
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21
S. V. Kropyvko, L. О. Tsyba, О. V. Novokhatska
© 2019 S. V. Kropyvko et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Bio-
polymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium,
provided the original work is properly cited
Biomedicine ISSN 0233-7657
Biopolymers and Cell. 2019. Vol. 35. N 1. P 21–29
doi: http://dx.doi.org/10.7124/bc.00098F
UDC 577.22
Expression of ITSN2 and TKS5 in different subtypes of breast cancer
tumors
S. V. Kropyvko1, L. О. Tsyba1, О. V. Novokhatska1, Y. M. Nemesh1,
L. А. Syvak2, T. Ye. Tarasenko2, A. N. Grabovoy2, А. V. Rynditch1.
1 Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03143
2 National Cancer Institute
33/43, Lomonosova Str., Kyiv, Ukraine, 03022
s.v.kropyvko@imbg.org.ua
Aim. Regardless a great progress in treatment, 15 % of breast cancer cases remain lethal. One
of the main problems in diagnosis and cure of this cancer type is its high clinical and genetic
heterogeneity, and the identification of markers for personalized treatment is still a topical issue.
Methods. Collection and characterization of clinical material, RNA isolation and analysis of
the ITSN2 and TKS5 isoforms expression using real-time quantitative PCR with fluorescence
labeled probes. Results. The reduced expression of ITSN2-S has been found in the HER2/neu-
positive tumors with poor prognosis. There was no significant difference in the expression of
ITSN2-L and TKS5-L in the analyzed samples. Conclusions. Our study has shown a potential
usage of the ITSN2 short isoform (ITSN2-S) as a breast cancer prognostic marker.
K e y w o r d s: breast cancer, ITSN2, TKS5, mRNA expression analysis.
Introduction
Mammary gland malignant tumor is one of the
most widespread among female cancer pa-
tients. According to the data of the
GLOBOCAN project of the International
Center for Cancer Research, 6.23 million of
new breast cancer cases (36 % of all tumors
in women) were discovered in the world over
five years (from 2008 to 2012), and 15 % of
these cases were lethal. Despite many years of
research and extensive experience in the treat-
ment of this cancer type, one of the major
problems in diagnosis and cure is its high
clinical and genetic heterogeneity.
To date, from the clinical and therapeutic
point of view, and taking into account the
specificity of the marker genes expression, this
group of diseases is divided into 4 main types,
according to the expression of estrogen recep-
tors (ER), progesterone (PR), and forms of the
epidermal growth factor receptor (HER2/neu):
http://www.ncbi.nlm.nih.gov/pubmed/?term=Tsyba L%5BAuthor%5D&cauthor=true&cauthor_uid=23936226
http://www.ncbi.nlm.nih.gov/pubmed/?term=Tsyba L%5BAuthor%5D&cauthor=true&cauthor_uid=23936226
22
S. V. Kropyvko, L. О. Tsyba, О. V. Novokhatska et al.
Table 1. Breast cancer classification: histology, molecular markers and survival prognosis [2–9]
Types Subtypes Additional gene markers Prognosis
Luminal A
ER+PR+HER2/neu–
≈40–60 %*
Low Ki-67
CK8/18+, FOXA1+,
ESR1, GATA3,
KRT8, KRT18, XBP1,
FOXA1, TFF3,
CCND1, LIV1, CK5/6–,
EGFR–
Favorable
Luminal B
ER+PR+HER2/
neu+/–
≈6–20 %*
Luminal B HER2–
ER+PR+HER2/neu–
≈15–20 %*
High Ki-67 ESR1, GATA3,
KRT8, KRT18, XBP1,
FOXA1, TFF3, SQLE,
LAPTM4B, CK5/6–,
EGFR–, TP53–
Intermediate
Luminal B HER2+
ER+PR+HER2/neu+
≈6 %*
Poor/
Intermediate
HER2 enriched
ER–PR–HER2/neu+
≈10–20 %*
CK5/6+, GRB7+,
ERBB2, EGFR+/–TP53– Poor
Triple negative
ER–PR–HER2/neu–
≈5–25 %*
Adenoid cystic
carcinoma
≈1 %*
MYB-NFIB gene
fusion
EGFR+, CK5/6+,
CK14+, CK17+ HER1+,
Cyclin E+, CDKN2A+,
KRT5, CDH3, ID4,
FABP7, KRT17,
TRIM29, LAMC2,
ITGB4
Favorable
Medullar carcinoma
≈2 %* Favorable
BRCA1-associated ID4+ Poor
Basal
≈10–25 %*
BRCA1–, TP53–,
CDKN2A+, RB1,
FGFR2, stem cell
markers+, CK5/6+,
EGFR+
Poor
With low claudin
≈7–14 %*
GATA3 regulators–, cell
adhesion genes,
CDH1–, claudin–,
CK5/6+/–, EGFR+/–
СD44+, SNAI3+
Poor
Metaplastic
carcinoma
≈1 %*
GATA3 regulators–,
cell ashesion genes,
PIK3CA–, AKT– or
KRAS–, EMT+, stem
cell markers+
Intermediate
Apocrine carcinoma Intermediate
Interferon enriched
≈10 %*
STAT1+, SP110+,
interferon-regulating
genes+
Intermediate
* — percentage according to [2–9].
23
Expression of ITSN2 and TKS5 in different subtypes of breast cancer tumors
luminal A, luminal B, HER2-enriched and
triple negative (ER–PR–HER2/neu–) [1].
Table 1 summarizes the classification of
breast cancer types and subtypes, depending
on the molecular markers expression discov-
ered in recent years [2–9].
The definition of tumor aggressiveness and
the choice of strategy for the further patients
treatment are based on this classification. As
can be seen from Table 1, even within the same
type there is a certain tumor heterogeneity with
its own prognosis and sensitivity to a particular
treatment. Thus, for example, the triple nega-
tive type has at least eight subtypes according
to the latest research; among them three with
poor prognosis, three — with intermediate, and
two — with favorable [4, 7]. Similarly, the
tumors of luminal B type are divided into two
subtypes: luminal B HER2/neu+ and luminal B
HER2/neu–, which have their own treatment
specificity. Each of the subtypes, besides three
to four main markers, has a set of markers that
more specifically characterize individual tu-
mors and are useful for the personalized choice
of treatment strategy, but it is still a generaliza-
tion of data from a large number of patients. In
practice, almost every single tumor has a spe-
cific expression pattern of the markers listed in
Table 1. Thus, the idea of personalized treat-
ment of each case, depending on the individu-
al markers becomes more and more actual. As
stated above, about 15 % of breast cancer
cases are lethal, inclu ding those of incorrectly
chosen treatment stra tegy. Significant progress
has been made over the past few years thanks
to the development of gene expression profiling
technologies. Tens of new marker genes have
been identified to characterize breast tumors.
Thus, in experiments on gene expression anal-
ysis using microchips, it has been found that
the ITSN2, CXCL9 and GNAI2 genes are among
the few, the expression levels of which are
significantly different in breast cancer tumor
samples from patients with recurrences and in
patients remained healthy after tumor removal
and CMF (cyclophosphamide, methotrexate
and fluorouracil) chemotherapy. The high
ITSN2, CXCL9 and GNAI2 expression levels
correlated with the absence of distant metasta-
ses for a long period of time. Besides, it has
been found that the expression ratio of only
two genes, CXCL9/ITSN2 may be an indepen-
dent prognostic factor to predict the absence of
recurrences [10].
ITSN2 is an adaptor/scaffold protein [11],
which is involved in many cellular functions,
including clathrin-mediated endocytosis [12],
actin cytoskeleton reorganization [13], cell
signaling, and others [14]. Two main ITSN2
isoforms are expressed in tissues: short
(ITSN2-S) and long (ITSN2-L), the latter has
three additional domains. Recently, it has been
shown that ITSN2 interacts with WIP, the
marker protein of the invadopodia [15, 16]
invasive cancer cells structures responsible for
their mobility and metastasis [17].
The TKS family is another recently discov-
ered family of scaffold proteins, which in-
cludes TKS4 and TKS5 that play a key role in
the invadopodia formation [18, 19]. Likewise
ITSN2, TKS4 and TKS5 are characterized by
multiple alternative splicing. The TKS5 gene
has three additional promoters which give rise
to three short isoforms without PX domain that
is responsible for membrane binding. The ele-
va ted mRNA level of the long TKS5 isoform
(TKS5-L) containing all the domains, together
with the reduced mRNA level of short isoform,
24
S. V. Kropyvko, L. О. Tsyba, О. V. Novokhatska et al.
correlates with the progression of metastasis
and poor prognosis for lung adenocarcinoma
patients [20].
These data indicate a high potential of the
ITSN2 and TKS5 genes and their isoforms as
cancer prognostic markers, particularly for
breast cancer. Therefore, we decided to inves-
tigate the mRNA expression levels of the
ITSN2 short and long isoforms and the TKS5
long isoform in the breast tumor samples using
quantitative real-time PCR to evaluate the pos-
sibility of their use as prognostic markers for
the breast cancer.
Materials and Methods
Total RNA isolation from breast tumor sam-
ples. Breast tumor samples were obtained from
the National Cancer Institute, frozen in liquid
nitrogen immediately after surgery and stored
at –80°C. Total RNA was isolated from 0.2–
0.9 g tissue by guanidinium isothiocyanate
method using the innuSOLV reagent (Analityk
Jena) in accordance with the manufacturer’s
recommendations. Clinical information on the
obtained samples is presented in Table 2.
cDNA synthesis. 5 μg of total RNA were
pre-treated with DNase I (Fermentas), accord-
ing to the manufacturer’s recommendations,
to remove residues of genomic DNA. After
that, cDNA synthesis was performed in 20 μl
according to the manufacturer’s recommenda-
tions. The cDNAs were stored at –20°C.
PCR with fluorescence labeled probes.
PCR was performed in 25 μl of mixture con-
taining 0.2 μM of each specific primer and
0.1 μM Taq-Man probe, 1.5 mM MgCl2,
0.2 mM dNTP, 2.5 units Taq DNA polymerase
(Fermentas) and the corresponding buffer.
Amplification was performed under the fol-
lowing conditions: denaturation — +94°С,
15 s (in the first cycle — 2 min); the time and
temperature of the primers reassociation and
synthesis were combined: +60°C 1 min, for
50 cycles. Each sample was analyzed in trip-
licate. PCR was performed on iQ5 BioRad.
The TBP gene was selected as a reference
based on the analysis of literature sources that
showed its appropriateness as a control for the
genes expression analysis in breast cancer
[21–23]. Primers and probes used for PCR:
(For.TBP 634-654 5’gtgcccgaaacgccgaatata3’,
Table 2. Clinical information of tumor samples
included in the study
Histological type of tumor Sample number
Invasive lobular carcinoma 44
Invasive tubular carcinoma 4
Invasive тubulolobular carcinoma 8
Invasive undifferentiated carcinoma 1
TNM classification (tumor, nodus and metastasis)
T1 17
T2 40
N0 40
N1 9
N2 8
M0 56
M1 1
Differentiation level (G)
G1 13
G2 35
G3 8
G4 1
Receptor status
ЕR+PR+HER2/neu– 31
ER–PR–HER2/neu– 11
ER+PR+HER2/neu+ 8
ER–PR–HER2/neu+ 7
25
Expression of ITSN2 and TKS5 in different subtypes of breast cancer tumors
Taq-Man probe TBP 655-676 5’ (FAM)
atcccaagcggt (BHQ1)ttgctgcggt3’, Rev.TBP
708-688 5’ccgtggttcgtggctctctta3’); (For.
ITSN2-S 3798-3821 5’cgttaagatgacgacagact-
caga3’, Taq-Man probe ITSN2-S 3820-3844
5’(FAM)cagcacaccact (BHQ1)gttgacttggatc3’,
Rev.ITSN2-S 3866-3845 5’attgtgtccagggtttg-
cagat3’); For.ITSN2-L 4324-4345 5’cgc-
tacccactgctcatcagaa3’, Taq-Man probe
ITSN2-L 4362-4385 5’(FAM)cccggagagccat
(BHQ1)gcagaccattc3’, Rev.ITSN2-L 4382-
4403 5’agggccagctttagggaggaat3’; TKS5 For.
TKS5-L 239-262 5’tgactccacctcccagactatcta3’,
Taq-Man probe TKS5-L 354-379 5’ (BHQ2)at
ccccttcctcccaggcaagatcct(ROХ)3’, Rev.
TKS5-L 407-431 5’gatgggcttcagtctcttcacagct3’.
The primers nucleotide positions correspond
to the TBP cDNA with the GenBank accession
number NM_006277, the human ITSN2 long
and short isoforms — NM_147152 and
NM_001172085.1 respectively, TKS5 —
NM_014631.
Real time PCR results calculation. The fol-
lowing formula was used to calculate PCR
results: Exp = Etarget
–Ct(target)/Eref –Ct(ref), where
Etarget — the PCR efficacy for the target gene,
Eref – the PCR efficacy for the reference gene,
Ct(target) — mean cycle value for the target gene,
Ct(ref) — mean cycle value for the reference
gene. The PCR efficacy was determined using
the “R” software. Fluorescence values in the
interval between 10 and 35–40 PCR cycles
were entered to the program, resulting in an
indicator of the efficacy of each individual
PCR reaction The cycle values for the target
and reference genes were determined as the
point of intersection of the fluorescence curves
with the threshold, above which the fluores-
cence value is considered significant. The
threshold level was set the same for all ex-
periments at 100 RFU. Statistical processing
of the PCR data was carried out with Statistica
v.8.0 software using the ANOVA method with
Fisher’s criterion.
Results and Discussion
ITSN2 mRNA expression analysis in breast
cancer samples by quantitative RT-PCR. Specht
and co-workers [10] have demonstra ted with
quantitative RT-PCR that high ITSN2 mRNA
levels in the surgically removed breast tumor
samples correlated with the absence of distant
metastases in patients for a long period of time,
but they did not verify possible variations in
the mRNA expression of ITSN2 isoforms.
We have conducted such analysis on the
tumor samples of various stages obtained from
the National Cancer Institute. Table 2 shows
the tumor data of patients whose samples were
analyzed in this study. These samples were
divided into three groups. The first group in-
cluded [the] tumors with favorable/intermedi-
ate prognosis that had the receptor status
ЕR+PR+HER2/neu–, or luminal A type. The
second group included the samples with triple
negative status (ER–PR–HER2/neu–). This
group was the most heterogeneous, however,
the majority of tumors with this diagnosis have
a poor survival prognosis (Table 1) [4]. The
third group united the tumors of luminal B type
and HER2/neu-enriched type, the receptor
status of which was respectively ЕR+/–PR+/–
HER2/neu+. Patients with these tumors had
a poor survival prognosis.
The total ITSN2-L and ITSN2-S mRNA
content in tumor samples with receptor status
ЕR+/–PR+/–HER2/neu+was significantly lower
(p = 0.043) than in tumors with triple negative
26
S. V. Kropyvko, L. О. Tsyba, О. V. Novokhatska et al.
receptor status, and counted 1.17–5.11 a.u.
(median — 2.16 a.u.) against 1.62–7.7 a.u.
(median — 2.89 a.u.). The expression level of
two ITSN2 isoforms in the ЕR+/–PR+/–HER2/
neu+ tumors was also lower than in tumors of
patients with a favorable prognosis
ЕR+PR+HER2/neu–: 0.97–7.39 a.u. (median —
3.69 a.u.) (Fig. 1A).
Significant difference in the expression
levels of the individual ITSN2 isoforms in dif-
ferent analyzed tumor groups were detected
only for ITSN2-S. The lowest ITSN2-S mRNA
content was detected in HER2/neu-positive
tumors (0.38–2.0 a.u., median — 0.69 a.u.);
the highest — in tumors with triple negative
receptor status (0.57–5.17 a.u., median —
2.21 a.u.) (Fig. 1B). There were no diffe ren ces
in the ITSN2-L amount in the studied tumor
groups (Fig. 1C). The ITSN2-S/ITSN2-L ratio
reflects the overall picture of the study. The
lowest ratio was observed in HER2/neu-posi-
tive tumors compared to tumors with triple
negative receptor status (Fig. 1D).
We have also compared the levels of ITSN2
isoforms mRNA expression in the tumors of
patients younger or older than 55 years with
Fig. 2. The relative ITSN2-S (A, C) and ITSN2-L (B, D) mRNA content in breast tumor samples from patients of dif-
ferent age (A, B) with or without metastases in lymph nodes (C, D).
Fig. 1. The relative ITSN2-S and ITSN2-L mRNA content in breast tumor samples with different receptor status. 1 —
ER+PR+HER2/neu–; 2 — ER–PR–HER2/neu–; 3 — ER+/–PR+/–HER2/neu+; A — ITSN2-S and ITSN2-L together; B —
ITSN2-S; C — ITSN2-L; D — ITSN2-S/ITSN2-L ratio; * — р < 0.05.
27
Expression of ITSN2 and TKS5 in different subtypes of breast cancer tumors
the presence or absence of metastases in the
lymph nodes, which is also a criterion for tumor
aggressiveness, but there was no correlation of
the ITSN2 isoforms mRNA content with the
patient age or the presence of metastases in the
lymph nodes (Fig. 2). We have also confirmed
the ITSN2-S and ITSN2-L expression on the
protein level (Fig. 3). The Western blot results
reflect the general trend that every individual
tumor has its own expression pattern.
Summarizing all data, we presume that the
short ITSN2 isoform can be considered as one
of the potential prognostic markers for patients
with breast cancer. Our results have demon-
strated that it is the short ITSN2 isoform that
reduces its expression in tumor samples with
poor prognosis, which in turn leads to a de-
crease in total ITSN2 levels as found by Specht
and co-workers [10]. These results were ex-
pected, since it is the long ITSN2 isoform that
has the guanosine-metabolic activity and takes
part in the actin cytoskeleton reorganization,
which in turn is an important condition for
cancer metastasis [14, 18].
TKS5 long isoform mRNA expression anal-
ysis in breast cancer samples by quantitative
RT-PCR. TKS5, a member of the TKS family,
is a necessary component for the formation of
invasive cancer cell structures - invadopodia
[18]. The mRNA of the TKS5 gene undergoes
a number of alternative splicing events that
affect the structure of the final protein mole-
cule. Expression of the longest of these iso-
forms (TKS5-L), which has a membrane phos-
phatidylinositol phosphates-binding domain,
correlates with lung adenocarcinoma metasta-
sis [20]. Therefore, we have chosen to analyze
its expression in the breast tumor samples. To
compare the TKS5-L mRNA expression, we
have divided breast tumor samples into two
groups: the first contained 10 tumor samples
with ER+PR+HER2/neu– receptor status and
had a favorable prognosis; the second group
contained 10 tumor samples with ER+/–PR+/–
HER2/neu+ receptor status and had a poor
prognosis. The real-time PCR results have
shown that TKS5-L did not significantly alter
its expression in breast cancer tumors either
with favorable (ER+PR+HER2/neu–) or with
unfavorable (ER+/–PR+/–HER2/neu+) prognosis
(Fig. 4).
Conclusions
We have demonstrated a decrease in
the ITSN2 mRNA expression in breast tumor
Fig. 4. The relative TKS5-L mRNA content in breast tu-
mor samples. 1 — ER+PR+HER2/neu–; 2 — ER+/–PR+/–
HER2/neu+.
Fig. 3. Western blot analysis of ITSN2 protein expression in
breast tumor samples. Actin was used as a loading control.
28
S. V. Kropyvko, L. О. Tsyba, О. V. Novokhatska et al.
samples with poor survival prognosis (ЕR+/–
PR+/–HER2/neu+) which is due to a decrease
of the short ITSN2 isoform (ITSN2-S)
amount. Thus, the ITSN2-S mRNA expression
can be used as a one of the prognostic markers
for breast cancer. There were no significant
differences in the ITSN2-L and TKS5-L ex-
pression in the analyzed tumor samples.
Acknowledgements
We are very grateful to Dr. Olga Gubar for the
critical reading of the manuscript.
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Експресія ITSN2 та TKS5 у різних підтипах
раку молочної залози
С. В. Кропивко, Л. О. Циба, О. В. Новохацька,
Я. M. Немеш, Л. А. Сивак, Т. Є. Тарасенко,
О. М. Грабовий, А. В. Риндич
Мета. Незважаючи на значний прогрес у лікуванні,
15 % випадків захворювання на рак молочної заози
залишаються летальними. Однією з головних проблем
у діагностиці та лікуванні цього типу раку є його ви-
сока клінічна та генетична гетерогенність, тому іден-
тифікація маркерів для персоналізованої терапії є ак-
туальною проблемою. Методи. Збір та характеристи-
ка клінічного матеріалу, ізоляція РНК та аналіз екс-
пресії ізоформ ITSN2 та TKS5, використовуючи кіль-
кісну ПЛР в реальному часі з флюоресцентно
міченими зондами. Результати. Нами було виявлено,
що ITSN2-S знижує свою експресію в HER2/neu-
позитивних пухлинах з поганим прогнозом. Не було
відмічено істотної різниці в експресії ITSN2-L і TKS5-L
в проаналізованих зразках. Висновки. В даній роботі
продемонстровано потенційну можливість викорис-
тання короткої ізоформи ITSN2 (ITSN2-S) як прогнос-
тичного маркера раку молочної залози.
К л юч ов і с л ов а: рак молочної залози, ITSN2,
TKS5, аналіз експресії мРНК.
Экспрессия ITSN2 и TKS5 в различных
подтипах рака молочной железы
С. В. Кропивко, Л. А. Цыба, О. В. Новохацкая,
Я. M. Немеш, Л. А. Сивак, Т. Е. Тарасенко,
А. Н. Грабовой, А. В. Рындич
Цель. Несмотря на значительный прогресс в лечении,
15 % случаев рака молочной железы остаются леталь-
ными. Несмотря на многолетние исследования, одной
из главных проблем в диагностике и лечении этого
типа рака является его высокая клиническая и генети-
ческая гетерогенность, поэтому идентификация мар-
керов для персонализированной терапии является
актуальной проблемой. Методы. Сбор и хараткери-
стика клинического материала, изоляция РНК и анализ
экспрессии изоформ ITSN2 и TKS5, используя количе-
ственную ПЦР в реальном времени с флюоресцентно
мечеными зондами. Результаты. Нами было выявле-
но, что ITSN2-S снижает свою экспрессию в HER2/
neu-позитивных опухолях с плохим прогнозом. Не
было отмечено существенной разницы в экспрессии
ITSN2-L и TKS5-L в проанализированных образцах.
Выводы. В данной работе продемонстрировано по-
тенциальную возможность использования короткой
изоформы ITSN2 (ITSN2-S) как прогностического мар-
кера рака молочной железы.
К л юч е в ы е с л ов а: рак молочной железы, ITSN2,
TKS5, анализ экспрессии мРНК.
Received 01.09.2018
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