Relationship between NF-κB, ER, PR, Her2/neu, Ki67, p53 expression in human breast cancer
Aim: The aim of the present study was to investigate expression patterns of transcription factor NF-κB (p50 and p65), ER, PR, Her2/neu, Ki-67 and p53 in tumor tissue of patients with breast cancer (BC) and analyze correlation between these markers. Patients and Methods: 62 BC patients previously not...
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| Cite this: | Relationship between NF-κB, ER, PR, Her2/neu, Ki67, p53 expression in human breast cancer / D.O. Shapochka, S.P. Zaletok, M.I. Gnidyuk // Experimental Oncology. — 2012. — Т. 34, № 4. — С. 358-363. — Бібліогр.: 38 назв. — англ. |
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Shapochka, D.O. Zaletok, S.P. Gnidyuk, M.I. 2018-06-21T13:00:16Z 2018-06-21T13:00:16Z 2012 Relationship between NF-κB, ER, PR, Her2/neu, Ki67, p53 expression in human breast cancer / D.O. Shapochka, S.P. Zaletok, M.I. Gnidyuk // Experimental Oncology. — 2012. — Т. 34, № 4. — С. 358-363. — Бібліогр.: 38 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/139879 Aim: The aim of the present study was to investigate expression patterns of transcription factor NF-κB (p50 and p65), ER, PR, Her2/neu, Ki-67 and p53 in tumor tissue of patients with breast cancer (BC) and analyze correlation between these markers. Patients and Methods: 62 BC patients previously nottreated with chemo- or radiotherapy were included in the study. All tumors belong to invasive ductal carcinoma of different grade. Expression of molecular markers was determined by immunohistochemical analysis on paraffin-embedded tissue sections. Results: The correlation between tumor grade and expression of ER, PR, Ki-67 and p53 was defined. NF-κB expression was found to be changed dependent on expression of ER, PR and p53 and also on molecular subtype (luminal, Her2-positive, hybrid, basal-like). The highest levels of NF-κB, Ki-67 and p53 were found in Her2/neu+ and basal-like tumor subtypes. Conclusion: The increase of nuclear expression of NF-κB correlates with a decrease of expression of steroid hormone receptors (ER and PR), increase of p53 accumulation, and is associated with Her2-positive and basal-like tumor types. en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Original contributions Relationship between NF-κB, ER, PR, Her2/neu, Ki67, p53 expression in human breast cancer Article published earlier |
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Relationship between NF-κB, ER, PR, Her2/neu, Ki67, p53 expression in human breast cancer |
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Relationship between NF-κB, ER, PR, Her2/neu, Ki67, p53 expression in human breast cancer Shapochka, D.O. Zaletok, S.P. Gnidyuk, M.I. Original contributions |
| title_short |
Relationship between NF-κB, ER, PR, Her2/neu, Ki67, p53 expression in human breast cancer |
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Relationship between NF-κB, ER, PR, Her2/neu, Ki67, p53 expression in human breast cancer |
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Relationship between NF-κB, ER, PR, Her2/neu, Ki67, p53 expression in human breast cancer |
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Relationship between NF-κB, ER, PR, Her2/neu, Ki67, p53 expression in human breast cancer |
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relationship between nf-κb, er, pr, her2/neu, ki67, p53 expression in human breast cancer |
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Shapochka, D.O. Zaletok, S.P. Gnidyuk, M.I. |
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Shapochka, D.O. Zaletok, S.P. Gnidyuk, M.I. |
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Original contributions |
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Original contributions |
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2012 |
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English |
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Experimental Oncology |
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Article |
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Aim: The aim of the present study was to investigate expression patterns of transcription factor NF-κB (p50 and p65), ER, PR, Her2/neu, Ki-67 and p53 in tumor tissue of patients with breast cancer (BC) and analyze correlation between these markers. Patients and Methods: 62 BC patients previously nottreated with chemo- or radiotherapy were included in the study. All tumors belong to invasive ductal carcinoma of different grade. Expression of molecular markers was determined by immunohistochemical analysis on paraffin-embedded tissue sections. Results: The correlation between tumor grade and expression of ER, PR, Ki-67 and p53 was defined. NF-κB expression was found to be changed dependent on expression of ER, PR and p53 and also on molecular subtype (luminal, Her2-positive, hybrid, basal-like). The highest levels of NF-κB, Ki-67 and p53 were found in Her2/neu+ and basal-like tumor subtypes. Conclusion: The increase of nuclear expression of NF-κB correlates with a decrease of expression of steroid hormone receptors (ER and PR), increase of p53 accumulation, and is associated with Her2-positive and basal-like tumor types.
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1812-9269 |
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https://nasplib.isofts.kiev.ua/handle/123456789/139879 |
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Relationship between NF-κB, ER, PR, Her2/neu, Ki67, p53 expression in human breast cancer / D.O. Shapochka, S.P. Zaletok, M.I. Gnidyuk // Experimental Oncology. — 2012. — Т. 34, № 4. — С. 358-363. — Бібліогр.: 38 назв. — англ. |
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AT shapochkado relationshipbetweennfκberprher2neuki67p53expressioninhumanbreastcancer AT zaletoksp relationshipbetweennfκberprher2neuki67p53expressioninhumanbreastcancer AT gnidyukmi relationshipbetweennfκberprher2neuki67p53expressioninhumanbreastcancer |
| first_indexed |
2025-11-24T20:29:35Z |
| last_indexed |
2025-11-24T20:29:35Z |
| _version_ |
1850495350547152896 |
| fulltext |
358 Experimental Oncology 34, 358–363, 2012 (December)
RELATIONSHIP BETWEEN NF-ΚB, ER, PR, HER2/NEU, KI67,
P53 EXPRESSION IN HUMAN BREAST CANCER
D.O. Shapochka*1, S.P. Zaletok1, M.I. Gnidyuk2
1R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of NAS of Ukraine, Kyiv, Ukraine
2Ivano-Frankivsk Regional Oncology Dispensary, Ivano-Frankivsk, Ukraine
Aim: The aim of the present study was to investigate expression patterns of transcription factor NF-κB (p50 and p65), ER,
PR, Her2/neu, Ki-67 and p53 in tumor tissue of patients with breast cancer (BC) and analyze correlation between these
markers. Patients and Methods: 62 BC patients previously not treated with chemo- or radiotherapy were included in the
study. All tumors belong to invasive ductal carcinoma of different grade. Expression of molecular markers was determined
by immunohistochemical analysis on paraffin-embedded tissue sections. Results: The correlation between tumor grade and
expression of ER, PR, Ki-67 and p53 was defined. NF-κB expression was found to be changed dependent on expression of ER,
PR and p53 and also on molecular subtype (luminal, Her2-positive, hybrid, basal-like). The highest levels of NF-κB, Ki-
67 and p53 were found in Her2/neu+ and basal-like tumor subtypes. Conclusion: The increase of nuclear expression
of NF-κB correlates with a decrease of expression of steroid hormone receptors (ER and PR), increase of p53 accumulation,
and is associated with Her2-positive and basal-like tumor types.
Key Words: breast cancer, receptors of steroid hormones, Her2/neu, Ki-67, p53, NF-κB, molecular subtype.
Breast cancer (BC) is by far the most frequent
cancer in women, and the main cause of death in 35–
55 years old women. Despite the impro�ement of diag�Despite the impro�ement of diag�
nostic methods and chemotherapeutic regimens o�erall
5�year sur�i�al of patients significantly depends on the
stage of disease and for the period 2000–2005 was
56.2% in Ukraine and 88.0% in the USA [1].
Such clinical characteristics as age, menstrual sta�
tus, tumor size, lymph node status and morphological
characteristics of the tumor (histological type, grade,
lymphatic/�ascular in�asion) traditionally are the most
important prognostic factors. Howe�er, in the last de�
cades understanding of tumor nature has been greatly
impro�ed by molecular biology researches that allowed
application of tumor’s molecular features for prognosis
of the disease course. Most molecular markers that are
studied today, determine the ability of cells to malig�
nant growth [2]. Estrogen and progesterone receptors
(ER, PR) were the first predicti�e molecular markers
for BC. Patients with positi�e status of these steroid
hormone receptors generally ha�e high sensiti�ity
to hormone therapy. Next marker that was included
in clinical practice is HER2/neu, which positi�e status
in patients with BC correlates with high sensiti�ity
to targeted therapy with trastuzumab. The use of these
markers in clinical practice contributed to indi�idual�
ization of treatment and choice of adequate chemo�
therapeutic schemes for patients. Howe�er, 25–50%
of ER and PR positi�e tumors are resistant to hormone
therapy [3], and tumors with Her2/neu o�erexpression
not always respond to trastuzumab therapy. There are
also about 20% of patients with BC negati�e by all three
markers mentioned abo�e, and these tumors are more
resistant to traditional therapy schemes.
Transcription factors may be considered as prom�
ising therapeutical targets because they affect the
transcription of oncogenes that could play an important
role in the formation of chemo�and radioresistance. One
of the transcription factors dysfunction of which often
occurs in malignant tumors, is a nuclear transcription
factor NF�κB. NF�κB family is composed of polypep�
tides c�rel, p50, p52, p65 (RelA), p68 (RelB), p100 and
p105, that belong to the rel�family proteins homologous
to oncogene ��rel. The best studied of them are pro�
teins p50 and p65, which form the classic NF�kappaB
heterodimer (p50/p65). In most cells, NF�κB is pres�
ent as a latent, inacti�e, IkB�bound complex in the
cytoplasm. Under external stimuli, I�κB proteins are
degraded �ia the ubiquitin�proteasome pathway, lead�
ing to release of the acti�e form of NF�κB that translo�
cates to the nucleus where it regulates the expression
of target genes. NF�κB was disco�ered as a protein that
specifically binds to a sequence of positi�e regulator
of immunoglobulin’s κ light chain gene [4–6].
Further studies ha�e established the crucial role
of NF�κB in the formation of immune response as well
in processes of tumor de�elopment and growth. In many
tumors (including BC, colon cancer, prostate cancer,
lymphoid tumors, and probably many others) NF�
kB is constituti�ely acti�e and located in cancer cell
nucleus [7]. NF�κB acti�ation is affected by a �ariety
of inducers such as TNFα [8, 9], LPS [4, 6], PMA [4],
interleukin�1 [10] and 2 [11], many �iruses [12, 13], hy�
drogen peroxide [14], ionizing radiation [15, 16] and oth�
ers. The time of NF�κB acti�ation �aries from 10 min for
TNFα [4], up to 6 h for PMA [17] and is highly dependent
on cell type. For re�inacti�ation NF�κB requires synthe�
sis of I�κB de novo that takes some time, so the action
of acti�ated NF�κB can last up to 8 h [4]. One of the most
important effects of NF�κB acti�ation is the blockade
Received: July 10, 2012.
*Correspondence: E-mail: shapochka.dm@gmail.com
Abbreviations used: BC — breast cancer; ER — estrogen receptor;
HIF-1 — hypoxia inducible factor-1; NF-κB — nuclear factor kappa B;
PR — progesterone receptor; TNFα — tumor necrosis factor alpha.
Exp Oncol 2012
34, 4, 358–363
Experimental Oncology 34, 358–363, 2012 (December)34, 358–363, 2012 (December) (December) 359
of apoptosis. Classic inducer of NF�κB acti�ation is a tu�
mor necrosis factor (TNFα). TNFα may cause two op�
posite effects: on the one hand, it acti�ates cytotoxicity
mechanisms that lead to cell death, on the other hand,
indirectly acti�ates the transcription of genes which
products can block apoptosis. NF�κB acti�ation plays
a key role in pre�ention of TNF�induced apoptosis. TNFα
is not the only agent capable to induce apoptosis and
acti�ation of NF�κB. Similar processes can be induced
by a majority of NF�κB acti�ators. It should be noted that
the effect of NF�κB on apoptosis is highly dependent
on many factors such as the type of inducer, cell type
and functional status of the cell at a gi�en time, the pre�
sence of external influences on the cell and many others
[18]. In many malignancies constituti�e le�el of NF�κB
[19–21] is increased, at the same time, dysregulation
of NF�κB in initially normal cell may lead to its malignant
transformation [22, 23]. These and other data suggest
an important role of NF�κB in cell proliferation and main�
taining the tumor cell �iability. This role has at least two
important aspects. The first is that some oncogenes
(c�myc), oncoproteins (p53) and onco�iruses are under
the influence of NF�κB, or acti�ate it during malignant
transformation (eg, ras). Another important point is the
influence of NF�κB on tumor sensiti�ity to chemotherapy
and immune reactions [24]. Action of many anticancer
drugs, as well as most defense reactions is to induce
apoptosis in cancer cells [25, 26]. At the same time,
the acti�ation of NF�κB by these apoptosis inducers
(TNFα, ionizing radiation, etc.) can protect cells from
death. Montagut et al. [27] ha�e shown that acti�ation
of NF�κB was significantly correlated with resistance
to chemotherapy in BC patients. Furthermore, in some
cases NF�κB acti�ation increased after chemotherapy
exposure, which could cause the formation of drug
resistance [27].
Also, correlation of NF�κB expression with other
markers has been studied. The need for such studies
is that the effect exerted by NF�κB on cell depends
on the expression and functional status of many pro�
teins, for example, �ery complex and �aried interactions
between NF�κB and p53, which can lead to opposite
effects in different systems. One of the most important
functions of p53 is induction of apoptosis in response
to some signals, such as �iral infection or DNA dam�
age. Due to these properties p53 is considered a tumor
suppressor what is shown in different models [28,
29]. Many studies also ha�e shown that both p53 and
NF�kB inhibit each other’s ability to stimulate gene
expression and that this process is controlled by the
relati�e le�els of each transcription factor. Expression
of either wild�type p53 or NF�kB suppresses stimulation
of transcription by the other factor. Howe�er, mutations
in the p53 gene lead to loss of its ability to regulate
the transcription and acti�ity of the targets (including
NF�kB), which causes the loss of apoptotic proper�
ties. So, it is important to compare NF�κB expression
with expression of other markers that may ha�e a sig�
nificant effect on the functional acti�ity of NF�κB. There
are also many contradictions in the results of studies
of NF�κB expression due to the absence of a standard
method of determining the NF�κB acti�ation, and the
differences in interpretation of results obtained by the
same method. Howe�er, in BC patients most results
suggest that NF�κB is acti�ated mainly in ER�negati�e
and Her2/neu�positi�e tumors [27, 30–32]. These
tumors usually possess high proliferati�e index, are
of high grade and drug resistance. These data allow
to propose that the acti�ity of NF�κB may affect the drug
sensiti�ity of tumors, and that the possibility of regula�
tion the NF�κB acti�ity open a new features in therapy
of tumors [33].
MATERIALS AND METHODS
62 patients with BC pre�iously not treated with
chemo� or radiotherapy and cured in I�ano�Franki�sk
Regional Oncology Dispensary (I�ano�Franki�sk,
Ukraine) were included in the study. All tumors belong
to in�asi�e ductal carcinoma of different grade. Clinical
data of patients and tumor characteristics are shown
in Table 1.
Table 1. Clinical data of patients and morphological characteristics of tumors
Parameter Number of cases (%)
Stage
I 4 (6.4)
II 40 (64.5)
III 7 (11.3)
IV 2 (3.2)
«X» 9 (14.5)
Axillary nodal status
+ 33 (53.2
- 29 (46.8
Tumor grade
1 8 (12.9)
2 32 (51.6)
3 16 (25.8)
“X” 6 (9.7)
Expression of molecular markers was determined
by immunohistochemical approach on formalin�fixed
paraffin�embedded 4 µm tissue sections. Antigen re�
trie�al was performed at temperature 98 °C for 30 min,
endogenous peroxidase was blocked with 0.03% hy�
drogen peroxide for 5 min. Slides were then washed
with Tris�buffered saline solution at pH 7.6 and incu�
bated for 30 min with the following primary antibodies:
Estrogen Receptor α (Monoclonal Rabbit Anti�Human,
RTU, clone SP1, Dako, Denmark), Progesterone
Receptor (Monoclonal Mouse Anti�Human, RTU,
clone PgR 636, Dako, Denmark), c�erbB2 (Poly� PgR 636, Dako, Denmark), c�erbB2 (Poly�PgR 636, Dako, Denmark), c�erbB2 (Poly� 636, Dako, Denmark), c�erbB2 (Poly�Dako, Denmark), c�erbB2 (Poly�), c�erbB2 (Poly�c�erbB2 (Poly��erbB2 (Poly�erbB2 (Poly�2 (Poly�Poly�
clonal Rabbit Anti�Human, 1:1000, Dako, Denmark),
NF�кB p50 (NLS) (Polyclonal Rabbit Anti�Human,
1:200, sc114, Santa Cruz Biotechnology, USA),
p�NF�кB p65 (Ser 536) (Polyclonal Rabbit Anti�
Human, sc�33020, Santa Cruz Biotechnology, USA),
Ki�67 (Monoclonal Mouse Anti�Human, RTU, clone
MIB�1, Dako, Denmark), p53 (Polyclonal Rabbit Anti�
Human, 1:100, RP 106, Diagnostic BioSystems, USA),
Cytokeratin 5/6 (Monoclonal Mouse Anti�Human,
RTU, clone D5/16 B4, Dako, Denmark). After rinsing,
slides were incubated with HRP from FLEX detection
system (Dako, Denmark) for 20 min, treated with DAB
for 3 min and counterstained with haematoxylin. Slides
were washed in tap water, dehydrated, and mounted
with glass co�erslips.
360 Experimental Oncology 34, 358–363, 2012 (December)
For interpretation the reaction with antibodies
against ER, PR, p50 and p65 the H�score method was
used. The score is obtained by the formula:
H= 3 x % of strongly stained nuclei + 2 x % of mode�
rately stained nuclei + % of weakly stained nuclei.
Expression le�el of marker with H>100 was classi�
fied as high, H=50�99 — moderate and H<50 — low.
Her2/neu expression le�el was determined
by reaction with antibodies against HER2/neu. For
interpretation the reaction such criteria were applied:
0 (negati�e): no staining is obser�ed or membranous
staining is obser�ed in less than 10% of the tumor
cells. 1+ (negati�e): A faint/barely perceptible staining
is detected in more than 10% of the tumor cells. The
cells are stained in part of their membrane. 2+ (equi�o�
cal): A weak to moderate complete membrane stain�
ing is obser�ed in more than 10% of the tumor cells.
3+ (positi�e): A strong complete membrane staining
is obser�ed in more than 30% of the tumor cells. Tu�
mors with expression le�el 0 and 1+ considered HER2/
neu�negati�e, and with 2+ and 3+ — positi�e.
Proliferation le�el was determined by reaction with
antibodies against Ki�67: 0 — no nuclear staining is ob�
ser�ed in tumor cells; 1 — nuclear staining is detected
in 1–10% of the tumor cells; 2 — 11–20%; 3 — 21–50%;
4 — >50%. In tumors with le�el of Ki�67 expression “0”,
“1”, “2” proliferation le�el was considered as low; and
with “3” and “4” — high.
P53�status of tumors was determined by the le�el
of accumulation in the nuclei of tumor cells. Negati�e
(0) — nuclear staining is detected in less than 10%
of the tumor cells; positi�e: 1 — 11–20; 2 — 21–50%;
3 — 51–75%; 4 — > 75%.
To check basal cell origin the expression
of CK 5/6 in tumors with three�negati�e phenotype
(ER�/PR�/Her2�) was determined. The tumors with
cytoplasmic staining of more than 10% of tumor cells
were considered as three�negati�e basal; otherwise —
three�negati�e non�basal.
For statistical analysis of the data the SPSS pro�
gram was used. To identify the correlation, the Pearson
coefficient of correlation was defined, and to �erify its
�alidity χ2 test was used.
RESULTS
In the 62 tumors of BC patients, expression le�els
of the following molecular markers were in�estigated: ER,
PR, Her2/neu, Ki�67, р53 and NF�κB subunits (p50 and
p65 proteins). 47 tumors (76%) were ER�positi�e (H > 10)
and 15 (24%) — ER�negati�e (H≤10). PR�status was
positi�e (H>10) in 43 cases (68%), and negati�e —
in 20 (32%). The Her2/neu o�erexpression was identi�
fied in 25 tumors (40%), other 37 patients had negati�e
Her2/neu status. The study of NF�кB (р50 та р65)
expression ha�e shown high le�els of p50 (H > 100) and
p65 (H > 100) in tumors, respecti�ely, in 39 (63%) and
7 (11%) patients. It should be noted that all tumors with
high expression of p65 had high le�els of p50.
Proliferati�e acti�ity of tumors was high (Ki�
67 > 20%) in 29 (47%) and low (Ki�67<20%) in 33 (53%)
patients. p53 status in tumors was positi�e in 22 (35%)
and negati�e in 40 (65%) patients.
According to molecular profile, tumors of BC pa�
tients were di�ided according to the classification of Da� Da�Da�
�id J. Dabbs [34] (Table 2). Tumors with high expression
of ER (H > 100) and negati�e Her2/neu status (0, 1+),
regardless of PR expression were attributed to luminal
A subtype. Tumors with negati�e HER2/neu status and
low�intermediate expression of ER (H = 11–99) or nega�
ti�e ER status (H ≤ 10) with positi�e PR expression
(H > 10) were attributed to luminal B subtype. Basal�like
subtype tumors had negati�e ER� ( H≤ 10), PR� (H ≤ 10)
and Her2/neu�status (0, 1+). To �erify the basal origin,
samples of triple�negati�e tumors were stained for cy�
tokeratin 5/6. CK5/6+ tumors were attributed to three
negati�e basal and CK5/6� — to triple�negati�e non�
basal. Her2/neu+ subtype is characterized by nega�2/neu+ subtype is characterized by nega�neu+ subtype is characterized by nega� is characterized by nega�is characterized by nega�characterized by nega�nega�
ti�e ER/PR status and Her2/neu hyperexpression. The
remaining tumors with positi�e status of ER, PR and
Her2/neu were referred to the hybrid luminal A/Her2/
neu or luminal B/Her2/neu subtype according to the
le�el of ER/PR expression. Due to limited number
of patients (n = 62), for statistical analysis such subtypes
of tumors were included: luminal A and B — to luminal;
triple�negati�e basal and non�basal — to basal�like;
hybrid luminal A and B/Her2/neu+ — to hybrid luminal/
Her2/neu+ subtype [35]. So, after integration, the dis�
tribution of tumors by subtypes was as follows: tumors
of 25 patients (40%) belonged to luminal subtype,
12 (20%) — to basal�like, 7 (11%) — to Her2/neu+,
18 (29%) — to hybrid (luminal/Her2/neu+) subtypes
(Table 3).
Table 2. Expression of molecular markers in BC
Experession of marker Number of cases (%)
ER
- 15 (24)
+ 47 (76)
PR
- 20 (32)
+ 42 (68)
Her2/neu
- 37 (60)
+ 25 (40)
р65 expression
Low 55 (89)
High 7 (11)
р50 expression
Low 23 (37)
High 39 (63)
Ki-67 expression
Low (< 20%) 33 (53)
High (> 20%) 29 (47)
р53
- 40 (65)
+ 22 (35)
The next relationships between clinical and mor�
phological characteristics of tumors and molecular
markers expression were found: 1) the direct cor�
relation between tumor grade and proliferation index
(Ki�67: r=1.0; p < 0.01); also, significantly increased
accumulation of p53 was found in tumors with grade
2 and 3 compared to grade 1 tumors (Fig. 1); 2) the
in�erse correlation was between tumor grade and ER
(significant correlation: r = �0.999; p < 0.05), PR (un�
Experimental Oncology 34, 358–363, 2012 (December)34, 358–363, 2012 (December) (December) 361
reliable correlation: r = �0.964; p = 0.086) (Fig. 1).
0
0,5
1
1,5
2
2,5
3
3,5
1 2 3
Grade
Ex
pr
es
si
on
o
f К
і-6
7
an
d
р5
3
Ki-67
p53
0
20
40
60
80
100
120
140
160
180
200
ER PR
Ex
pr
es
si
on
o
f E
R
an
d
PR
(H
) Grade 1
Grade 2
Grade 3
a
b
Fig. 1. Correlation between tumor grade and expression of Ki�
67 and p53 (a), ER and PR (b)
No relationship between expression of studied
markers and clinical characteristics (age, stage, axil�
lary lymph node status) was found.
The in�erse correlation between expression of ste�
roid hormone receptors and: p50 expression (ER:
p=�0.991, r<0.05; PR: p=�0.998, r<0.05); p53 ac�=�0.991, r<0.05; PR: p=�0.998, r<0.05); p53 ac�r<0.05; PR: p=�0.998, r<0.05); p53 ac�<0.05; PR: p=�0.998, r<0.05); p53 ac�PR: p=�0.998, r<0.05); p53 ac�: p=�0.998, r<0.05); p53 ac�p=�0.998, r<0.05); p53 ac�=�0.998, r<0.05); p53 ac�r<0.05); p53 ac�<0.05); p53 ac�; p53 ac�
cumulation (ER: p=�0.986, r=0.053; PR: p=�0.998,
r<0.05) was found. The correlation between p65 and
these markers was not significant.
The �ast majority of tumors with high expression
of p50 and p65 were found to be ER (5 of 7) and PR
(6 of 7) negati�e, while the percentage of ER� and
PR� tumors was respecti�ely 24% and 32%. To study
the relationship of markers expression and expression
of NF�κB, we di�ided patients into 3 groups: I — with
high le�els of p50 and p65 nuclear expression; II —
with high p50 and low p65 le�els; III — with low le�els
of p50 and p65. The in�erse correlation between ex�
pression of NF�κB and receptors of steroid hormones
(ER: r=�1.0, p<0.01; PR: r=�0.999, p<0.05); and direct
correlation between p53 accumulation and NF�κB ex��κB ex�κB ex�
pression (r=1.0, p<0.01) were found (Fig. 2).
Also the relation between NF�κB, p53, Ki67 ex��κB, p53, Ki67 ex�κB, p53, Ki67 ex�
pression and molecular profile of tumors was ana�
lyzed. Increase of NF�κB expression (p50: r=0.917,
p<0.05; p65: r=0.974, p<0.05) in the direction of:
Hybrid → Luminal → Basal�like → Her2/neu+ subtype
was found (Fig. 3). These results are in contradiction
to literature data [27, 30–32], according to which the
lowest expression of NF�kB was obser�ed in luminal
subtype, but this could be explained by a small cohort
studied.
0
20
40
60
80
100
120
140
160
ER РR P53
Ex
pr
es
si
on
o
f E
R,
P
R,
a
nd
p
53
p65+/p50+
p65-/p50+
p65-/p50-
Fig. 2. Correlation between expression of NF�kB and ER, PR and
p53 in BC
Ki�67 proliferation index and p53 accumulation
increased in this direction: Luminal → Hybrid → Basal�
like → Her2/neu+ subtype (Ki�67: r=0.928, p<0.05;
p53: r=0.956, p<0.05) (Fig. 3).
0
20
40
60
80
100
120
140
160
180
200
Hybrid Luminal Basal-like Her2/neu+
Ex
pr
es
si
on
o
f p
65
a
nd
р
50
(H
)
р65
р50
0
0,5
1
1,5
2
2,5
3
Luminal Hybrid Basal-like Her2/neu+
Ex
pr
es
si
on
o
f К
і-6
7
an
d
р5
3
Ki-67
p53
a
b
Fig. 3. Correlation between molecular subtype of BC and ex�
pression of p50 and p65 (a), and Ki67 and p53 (b).
So, summing up the results, we can say that NF�
kB expression is correlated with expression of markers
Table 3. Molecular subtypes of breast cancer
Molecular subtype Criteria used for proposed categories Number of patients (%)
Luminal [15] Luminal А [14] ER 3+; Her2 0, 1+ 20 (32) 25 (40)
Luminal B [14] ER 1+, 2+; Her2 0, 1+; або ER 0; PR ≥ 1+; Her2 0, 1+ 5 (8)
Basal-like [15] Triple-negative basal [14] ER (H<30); PR (H<30); Her2 0, 1+; CK5/6 + 8 (13) 12 (20)
Triple-negative non-basal [14]2 ER (H<30); PR (H<30); Her2 0, 1+; CK5/6 - 4 (7)
Her2/neu+ [14, 15] ER (H<30); PR (H<30); Her2 2+, 3+ 7 (11)
Luminal-Her2/neu+
hybrid [15]
Luminal A-Her2/neu+ hybrid [14] ER 3+; Her2 2+, 3+ 12 (19) 18 (29)
Luminal B-Her2/neu+ hybrid [14] ER 1+, 2+; Her2 2+,3+; або ER 0; PR ≥1+; Her2 2+,3+ 6 (10)
362 Experimental Oncology 34, 358–363, 2012 (December)
(ER, PR, p53, molecular profile of tumors) that predict
the poor BC prognosis.
DISCUSSION
In this study we ha�e in�estigated the expression
le�el of NF�κB subunits, and its relationship with clini�
cal and morphological parameters and the expression
of other molecular markers. Being initially described
as a crucial element in the formation of immune re�
sponse, presently NF�κB is considered also as a po�
tential target for cancer treatment. The possibility
of this application of NF�κB is based on the fact that
this transcription factor can inhibit apoptosis, stimulate
cell proliferation, promote drug and radioresistance
of cells. Howe�er, the effects of NF�κB acti�ation
depend on many factors, including the expression
of other proteins that can regulate its functional acti�ity.
Another important point to consider in the study
of NF�κB is that high expression of this factor is not
always indicating its acti�ation. This comes up from the
fact that normally NF�κB is located in the cytoplasm
in an inacti�e, associated with the I�κB state, and only
after acti�ation mo�es to the nucleus, where affects the
transcription of target genes. In this work the expres�
sion le�el of NF�κB was determined by immunohisto�
chemical method, which allows taking into account
location of protein in the cell with a high probability
to speak about his acti�ation. Immunohistochemical
study is also quite reliable method for detection the
mutations in the p53 gene. According to the literature,
positi�e p53 nuclear reaction in 90–100% of cases
corresponds to missense�mutations in the p53 gene,
which determines mutant immunophenotype [36].
The half�life of “wild type” p53 is up to 30 min due
to rapid utilization in proteosome system, whereas
mutant p53 has a lower affinity for proteins of this
system, what increases its half�life to se�eral hours,
and concentration to a le�el that can be determined
by immunohistochemistry.
Summarizing the results we can say that the higher
tumor grade correlates with a decrease of ER and
PR expression, increase of proliferation index (Ki�67)
and p53 accumulation. As for relationship between
expression of NF�κB and other molecular, clinical and
morphological features of BC, se�eral reported data
are contradictory. According to our results, the highest
le�els of NF�κB, Ki�67 and p53 were found in Her2/
neu+ and basal�like subtype of tumors that are associ�
ated with poor prognosis. The increase of nuclear ex�
pression of NF�κB correlates with a decrease of ER and
PR expression and increase of p53 accumulation, that
also worsens BC prognosis. These results are in agree�
ment with literature data about the drug resistance
of tumors with positi�e p65 status [37].
Increased NF�κB expression is associated with
molecular and physiological changes that contribute
to its acti�ation. So, Her2/neu + and basal�like tumor
subtypes generally ha�e a solid growth pattern and
are associated with an increased inflammatory re�
sponse, which leads to increased le�els of hypoxia.
The normal cellular response to hypoxia is go�erned
by two dimeric transcription factors, hypoxia inducible
factor�1 (HIF�1) and NF�kB. One of the effects of hy�
poxia is the degradation of I�kB and NF�kB acti�ation.
NF�kB acti�ates signaling pathways that promote cell
sur�i�al and angiogenesis. Regulator of these pro�
cesses is p53, which inhibits the acti�ation of HIF�1 and
NF�kB. Mutations in the p53 gene leads to the loss
of their ability to regulate the acti�ity of these factors
that promote angiogenesis and tumor progression
[38]. Using this logic, the inhibition of HIF�1 and/or NF�
kB can inhibit the angiogenic acti�ity of tumors, thereby
curtailing their growth and metastases.
In conclusion, we ha�e re�eale correlation between
expression le�els of ER, PR, Ki�67, p53 and BC grade,
correlation between expression profile of NF�κB and
expression le�el of ER, PR and p53. The highest le�els
of NF�κB, Ki�67 and p53 were detected in Her2/neu+ and
basal�like subtype of tumors. Our data allow to propose
that acti�ated NF�κB in BC may indicate poor prognosis
and de�elopment of drug resistance, howe�er, further
research and retrospecti�e analysis performed on larger
number of patients is required.
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