Ol 8 3 985 PDF

Download as pdf or txt
Download as pdf or txt
You are on page 1of 8

ONCOLOGY LETTERS 8: 985-992, 2014

Immunohistochemical expression of four different stem


cell markers in prostate cancer: High expression of
NANOG in conjunction with hypoxia‑inducible factor‑1α
expression is involved in prostate epithelial malignancy
KATSUHITO MIYAZAWA1, TAKUJI TANAKA2, DAN NAKAI1, NOBUYO MORITA1 and KOJI SUZUKI1

1
Department of Urology, Kanazawa Medical University, Uchinada, Ishikawa 920‑0293;
2
Department of Diagnositic Pathology and Research Center of Diagnostic Pathology, Gifu Municipal Hospital,
Gifu, Gifu 500‑8513, Japan

Received August 25, 2013; Accepted May 23, 2014

DOI: 10.3892/ol.2014.2274

Abstract. Cancer stem cells (CSCs) have been identified expressed in prostate cancers. Furthermore, HIF‑1α expression
in a variety of cancer types, including prostate cancer. The may affect NANOG and/or OCT4 expression. The findings
aim of the present study was to evaluate the immunohisto- of the current study suggested that NANOG expression may
chemical expression of NANOG, octamer 4 (OCT4), cluster of be a biomarker for the diagnosis of prostate cancer, and the
differentiation 133 (CD133) and NESTIN, which are all CSC coexpression of NANOG and HIF‑1α may be involved in
markers, and assess their function in prostate carcinogenesis. prostate carcinogenesis.
A total of 114 patients were referred to the Kanazawa Medical
University Hospital (Uchinada, Japan) having presented Introduction
with elevated serum prostate‑specific antigen levels and/or
abnormal digital rectal examinations, and underwent tran- Prostate cancer is the second leading type of malignancy in
srectal ultrasound sonography guided eight core biopsies. The males in North America with an estimated 186,320 new cases
prostate pathological specimens were re‑evaluated for selec- and 28,660 mortalities reported in 2008 (1). The number
tion in this study. When specimens were diagnosed as prostate of patients with prostate cancer has also been increasing in
cancer, immunohistochemical analysis of the four different Japan (2). Alterations in nuclear morphometry, gene and
stem cell markers (NANOG, OCT4, CD133 and NESTIN) and protein expression, gene promoter methylation and angiogen-
hypoxia‑inducible factor (HIF)‑1α was performed. Prostate esis are known to be involved in prostate carcinogenesis and
cancer was found in 38 cases (33.3%), while the other patients contribute to field cancerization in the prostate (3).
had benign prostate hyperplasia with prostatitis. All prostate Our understanding of carcinogenesis has been enhanced
cancers were histopathologically identified as adenocarci- by the recently revived cancer stem cell (CSC) theory. CSCs
nomas of various grades, and cancer cells and intraepithelial have been reported in multiple solid tumors in different tissues,
neoplasia (high grade) were immunohistochemically shown to including the prostate (4‑6). CSCs are endowed with high
express NANOG and OCT4, but not CD133 and NESTIN. The tumorigenic capacity and may drive tumor formation, maintain
intensity of NANOG expression was much greater than that tumor homeostasis and mediate tumor metastasis. A number of
of OCT4, and the positivity and intensity of the four stem cell primary non‑malignant and malignant tumor‑derived human
markers, including NANOG, were elevated with high Gleason prostate epithelial cell lines have been developed using a retro-
scores. A significant correlation was observed between the viral vector encoding human telomerase reverse transcriptase.
NANOG‑ and HIF‑1α‑positive regions. The CSC markers, in These cell lines exhibit the characteristics of stem cells and
particular OCT4 and NANOG, were immunohistochemically express embryonic stem (ES) cell markers, such as NANOG,
octamer 4 (OCT4) and SRY‑box 2 (Sox‑2), as well as the early
progenitor cell markers, cluster of differentiation 133 (CD133),
CD44 and NESTIN (7,8).
The multipotent stem cell marker NANOG was identi-
Correspondence to: Professor Katsuhito Miyazawa, Department
fied in 2003 (9,10). NANOG is specifically expressed in the
of Urology, Kanazawa Medical University, 1‑1 Daigaku, Uchinada,
Ishikawa 920‑0293, Japan human embryonic pluripotent stem cells of embryos prior to
E‑mail: miyazawa@kanazawa-med.ac.jp or following implantation, primordial germ cells, ES cells
cultured in vitro, embryonic germ cells and embryonic carci-
Key words: cancer stem cell, NANOG, OCT4, prostate cancer, noma cells, and functions in the promotion of cell proliferation.
hypoxia‑inducible factor‑1α NANOG is expressed in dysgerminoma and embryonic carci-
noma, but not in immature teratoma, endodermal sinus tumors
986 MIYAZAWA et al: EXPRESSION OF NANOG AND HIF-1α IN PROSTATE CANCER

or choriocarcinoma (11). NANOG can be used to distinguish 114 biopsy specimens obtained from Japanese patients with
between germ cell tumors and non‑germ cell tumors (11). prostate cancer between January 2011 and December 2011. In
NANOG has also been found to be a sensitive and specific addition, the correlation between the expression of these stem
marker of metastatic germ cell tumors (11,12). With regard cell markers in prostate cancer and non‑cancerous tissues was
to prostate cancer, several studies have recently suggested evaluated. Hypoxia has been associated with an aggressive
the positive reaction of adenocarcinoma (ADC) cells against course and poor clinical outcome of cancer (28,29); low oxygen
NANOG (13,14). Therefore, NANOG is an emerging focus in may promote the self‑renewal of CSCs (14,30‑32). Therefore,
developmental biology, due to its importance in the mainte- the immunohistochemical expression of hypoxia‑inducible
nance of self‑renewal and multipotential capacity in a variety factor (HIF)‑1α was also examined.
of malignancies, including prostate cancer. Octamer 4 (OCT4)
belongs to the family of Pit‑Oct‑Unc‑domain transcription Materials and methods
factors and has been found in ES and germ cells (15). A
number of reports have shown that OCT4 is pivotal in main- Study samples. Between October 2010 and September 2011,
taining the self‑renewal and pluripotency of ES cells (16). a total of 114 patients with elevated serum prostate‑specific
Recently, it has also been shown that cancer cells expressing antigen levels of >4 ng/ml and/or abnormal digital rectal
OCT4 and Sox2 may be crucial in cancer development (17). examinations were referred to the Kanazawa Medical
The two genes, Sox2 and OCT4, are part of an important University Hospital (Uchinada, Japan) and underwent tran-
gene regulatory network, and are essential for embryogenesis srectal ultrasound sonography‑guided eight‑core biopsies.
and the pluripotency and self‑renewal of cells (16). Previous Histopathological diagnosis was re‑evaluated by a certified
studies have also suggested that certain cancers, including pathologist on hematoxylin and eosin‑stained sections from the
prostate cancer (14,18), express Sox2 and OCT4 simultane- biopsy samples. Prior to this study, written informed consent
ously (19,20), and their expression has been associated with the was obtained from all patients. The study was approved by
differentiation of tumors (21). These two genes are significant the Ethics Committee of Kanazawa Medical University
for cancer cell survival. CD133 is a transmembrane glycopro- (Uchinada, Japan), and the Declaration of Helsinki regarding
tein that is originally expressed in a subset of stem cells in the the use of human tissue was strictly followed.
hematopoietic system as well as in the solid tumors of other
tissues (22), including the prostate (23). CD133‑positive cancer Immunohistochemistry. Serial sections, 4 µm in thickness,
cells have cancer stem/progenitor cells that exhibit resistance prepared from formalin‑fixed, paraffin‑embedded speci-
to cancer therapies (including radiation and chemotherapy), a mens, were available for immunohistochemical analysis.
greater invasion ability and metastasis in various malignan- Sections were deparaffinized and rehydrated following
cies. Thus, the utility of CD133 expression as a prognostic standard methods. Briefly, the sections were deparaffinized
marker has been suggested (22), as well as in the prostate (23). three times with xylene for 5 mins, and rehydrated in graded
NESTIN is an intermediate filament protein that is known ethanol (80‑100%) for 5 mins. A microwave antigen retrieval
to be important as a neural stem cell marker (24). However, procedure was performed for 20 min in citrate buffer (pH 6.0)
the expression of NESTIN has recently been reported to be and hydrogen peroxide was used to block non‑specific perox-
associated with the proliferation of progenitor cell popula- idase reactions. Following washing with phosphate‑buffered
tions within neoplasms (25). In addition, the upregulation saline (PBS, pH 7.4), sections were incubated with rabbit
of NESTIN has been found to closely correlate with the polyclonal anti‑human NANOG (ab21624; 1:30 dilution;
malignancy and metastasis of a variety of malignancies (25), Abcam, Cambridge, MA, USA), OCT4 (ab18976; 1:100 dilu-
including prostate cancer (26). tion; Abcam), CD133 (ab19898; 1:200 dilution; Abcam)
The expression of NANOG, OCT4, Sox, NESTIN and and NESTIN (ab93666; 1/120 dilution; Abcam), as well as
CD44 has been observed in human prostate ADC cells (7), mouse monoclonal anti‑human HIF‑1α (ab10625; 1:200 dilu-
which suggests the importance of cancer stem and progenitor tion; Abcam). Following washing three times with PBS,
cells in prostate carcinogenesis. However, OCT4A‑expressing sections were incubated at 37˚C with biotin‑conjugated goat
cells have rarely been identified in human benign and malig- anti‑rabbit polyclonal antibody (ab6720; Abcam) for 20 min.
nant prostate glands (27). The number of OCT4A‑expressing Visualization was achieved by incubation with diaminoben-
cells has been shown to increase in prostate ADC with high zidine for 10 min and slides were counterstained with Mayer
Gleason scores (27). OCT4A‑expressing cancer cells have also hematoxylin. Following hydration in graded alcohol and
been shown to coexpress Sox2, an ES cell marker, but did not clearing with xylene, the slides were mounted with neutral
express other putative stem cell markers, such as NANOG and gum. Seminomas obtained from testicular cancer specimens
CD133 (27). The neuroendocrine differentiation markers, chro- of two patients (Kanazawa Medical University Hospital) who
mogranin A and synaptophysin, are also coexpressed by the had undergone surgical resection, which had been confirmed
majority of OCT4A‑expressing cells (27). Thus, discrepancies to overexpress NANOG and OCT4, were selected as the
exist in reports investigating the role and expression of certain appropriate positive controls (33), and paraffin‑embedded
stem and progenitor cell markers in prostate cancer cells. Caco‑2 cells (cat. no. CRL‑2102; American Type Culture
In the current study, in order to determine whether certain Collection, Manassas, VA, USA) and endothelial cells
stem cell markers may be used for the diagnosis of prostate in ADC obtained from colorectal cancer specimens of
cancer, the immunohistochemical expression of NANOG, two patients (Kanazawa Medical University Hospital) who
OCT4, CD133 and NESTIN, which are well‑known stem had undergone surgical resection were used as internal
cell markers, were investigated in 38 cases from a total of positive controls for CD133 and NESTIN (34,35). Negative
ONCOLOGY LETTERS 8: 985-992, 2014 987

controls were prepared by incubating samples without the OCT4, with the strongest staining for CD133 and NESTIN.
primary antibody. The intensity of immunoreactivity against In the non‑cancerous tissue, as shown in Fig. 4, the immuno-
all the primary antibodies used was assessed using a micro- reactivities of NANOG (P<0.001) and OCT4 (P<0.001) were
scope (Olympus BX41; Olympus Optical, Tokyo, Japan). significantly greater than those of CD133 and NESTIN. In
Indices were determined by counting the number of positive prostate cancer, NANOG (P<0.001) immunoreactivity was the
nuclei among ≥300 cells in high‑power fields, and were indi- strongest among the four stem cell markers (Fig. 5).
cated as percentages. Positive cells were evaluated for their The expression score for NANOG in the prostate cancer
intensity of immunoreactivity on a 0 or 3+ scale. The overall cells was significantly greater than that of cells in high‑grade
intensity of the staining reaction was scored as follows: 0, no PIN and the hyperplastic glands (P<0.001 for each comparison;
immunoreactivity and no positive cells; 1 (+/‑), weak expres- Fig. 6). The number of atypical cells in high‑grade PIN was also
sion in <50% cells; 2 (+), moderate expression in ≥50% cells; higher than that in the hyperplastic glands (P<0.001; Fig. 6).
3 (++), moderate to strong expression in 51‑75% cells; and The expression of NANOG, OCT4, CD133 and NESTIN in
4 (+++), strong and diffuse expression in >76% cells. Slides prostate ADCs with high Gleason scores (>3+4) was greater
were reviewed by one pathologist blinded to the clinical data. than that in prostate cancers with low Gleason scores (<3+3),
although this difference was not significant (Fig. 7).
Statistical analysis. Incidences among the groups were HIF‑1α immunohistochemistry revealed that specific
compared using a two‑tailed unpaired t‑test and Bonferroni cancer cell nuclei (Fig. 8A and B), corresponding to their
multiple comparison test (GraphPad InStat version 3.05; Gleason score, as well as a few cell nuclei in high‑grade PIN
GraphPad Software, San Diego, CA, USA). P<0.05 was showed a positive reaction for HIF‑1α (Fig. 8C). However,
considered to indicate a statistically significant difference hyperplastic and normal glandular cells were negative for
between the groups. HIF‑1α (Fig. 8D). The mean score for HIF‑1α with a high
Gleason score was significantly greater than that of HIF‑1α
Results with a low Gleason score (P<0.001; Fig. 7).

General observations. Prostate cancer was found in 38 (33.3%) Discussion


of 114 males who underwent eight core biopsy and were
divided into two subgroups according to the following Gleason Pluripotency‑associated transcription factors, including
scores: 30 cases with <6 (3+3) and eight cases with >7 (3+4), NANOG, Sox2 and OCT4, are known as regulators of cellular
as shown in Fig. 1A‑C. Other specimens were diagnosed as identity in ES cells (36) and have recently been identified in the
benign prostate hyperplasia with marginal prostatitis (Fig. 1D) epithelial malignancies of a variety of tissues (33,37), including
or normal prostate glands (Fig. 1E). prostate cancer (13,14,18). CD133 (23) and NESTIN (26) have
also been reported to be expressed in prostate cancer. However,
Immunohistochemical findings. Of the four stem cell markers, these reports were predominantly from human prostate cancer
ADC cells in all specimens of the 38 cases of prostate ADC cell lines. Consistent with their role in sustaining the stemness
were found to positively express the NANOG (Fig. 2A) and of ES cells, pluripotency‑related factors have been suggested
OCT4 (Fig. 2B) proteins. However, the immunohistochemical to be expressed at a higher frequency in cancer exhibiting
expression of CD133 (Fig. 2C) and NESTIN (Fig. 2D) was lower degrees of differentiation (37). In this study, the immu-
extremely weak or absent in the cancer cells of prostate ADC nohistochemical expression of NANOG was markedly higher
and those of non‑cancerous cells. High‑grade prostate intraep- than that of the other stem cell markers, OCT4, CD133 and
ithelial neoplasia (PIN) was positive for NANOG (Fig. 2E) NESTIN. The reason for the discrepancy between the findings
and OCT4 (Fig. 2F); however, the number of positive cells was of the current study and those reported by others is not known;
fewer than that of prostate cancer. The majority of hyperplastic however, differences between prostate cancer obtained from
glands were negative for NANOG (Fig. 2I) and OCT4 (Fig. 2J) biopsy specimens and human prostate cancer cell lines may
staining. The cells of hyperplastic glands were completely have influenced the stainability of the four different stem cell
negative for CD133 (Fig. 2K) and NESTIN (Fig. 2L). Positive markers, NANOG, OCT4, CD133 and NESTIN. Although
reactions for NANOG and OCT4 were predominantly local- Miki et al (8) observed tumor compartments and high‑grade
ized in the nuclei of cancer cells and the cell nuclei of PIN. The PIN with higher CD133 and an inverse correlation with
staining intensity of NANOG was stronger than that of OCT4. androgen receptor staining, a CD133‑positive reaction was
Fig. 3 shows the scorings for the immunohistochemical not detected in the prostate cancer cells, PIN or hyperplastic
expression of the four different stem cell markers in prostate glandular cells in this study.
cancer and non‑cancerous cells. The immunoreactivities of CSCs comprise of ~0.01% of the tumor cell population. In
NANOG (P<0.001) and OCT4 (P<0.01) in prostate cancer was this study, a large number of strongly positive NANOG and/or
significantly greater than those in the non‑cancerous cells. OCT4 cancer cells were observed. This high level of expres-
No significant differences were identified between the immu- sion is not necessarily associated with stem cell behavior, but
noreactivities of CD133 and NESTIN in the prostate cancer rather to the deregulation proteins that provide some type of
and non‑cancerous cells. Based on the detailed analysis, the growth advantage to cancer cells (38‑41).
scoring data for the four different stem cell markers in the Androgen deprivation‑induced atrophy of the prostate
non‑cancerous and prostate cancer cells are also illustrated in gland and subsequent regeneration following androgen
Figs. 4 and 5, respectively. The immunohistochemical inten- replacement have indicated that the stem cell population may
sity of prostate cancer was weakest for NANOG followed by reside in the adult prostate gland in rodents (42). The origin
988 MIYAZAWA et al: EXPRESSION OF NANOG AND HIF-1α IN PROSTATE CANCER

A B C

D E

Figure 1. Histopathology of biopsy specimen. Prostate biopsy specimen: (A‑C) Adenocarcinoma (magnification, x100), (D) benign prostate (glandular)
hyperplasia and (E) non‑lesional area (magnification, x200). Gleason scores: (A) 2+2; (B) 3+3 and (C) 4+4 (hematoxylin and eosin stain; scale bars, 100 µm).

A B C D

E F G H

I J K L

Figure 2. Immunohistochemistry of four stem cell markers in prostate adenocarcinoma and non‑cancerous epithelium. NANOG and OCT4 proteins were
mainly localized in the nucleus and cytoplasm of the tumor cells of prostate cancer. (A) NANOG expression was strong in cancer cell nuclei, while (E) OCT4
expression was weak (magnification, x200; scale bars, 50 µm). OCT4, octamer 4.

of prostate cancer remains unknown and has given rise to multiple pluripotency markers, such as CD44, CD117 and
a series of hypotheses (43). Prostate ADCs are frequently Oct3/4, have been shown to be expressed in prostate cancer,
multifocal, show the same immunohistochemical profile as indicating that prostate cancer may develop from common
benign glandular cells, and lack basal cell markers, such as stem cell‑like or intermediate cells (8,44). The findings on the
p63 and cytokeratin 34β. This indicates that prostate cancer expression of NANOG described in the current study may also
may develop from altered benign glandular cells. However, support this hypothesis.
ONCOLOGY LETTERS 8: 985-992, 2014 989

Figure 3. Immunohistochemical expression scores of four stem cell markers


in prostate ADC and NE. The scores of NANOG and OCT4 in ADC were Figure 5. Immunohistochemical expression scores of four stem cell markers
significantly higher than that in the NE (P<0.001 and P<0.01, respectively). in prostate adenocarcinoma. The score of NANOG was the highest among
ADC, adenocarcinoma; NE, non‑cancerous epithelium; OCT4, octamer 4. the four stem cell markers, and the value was significantly greater than that
of OCT4 (P<0.001), CD133 (P<0.001) and NESTIN (P<0.001). The score
of OCT4 was significantly higher than that of CD133 (P<0.001) and nestin
(P<0.001). OCT4, octamer 4; CD133, cluster of differentiation 133.

Figure 4. Immunohistochemical expression scores of four stem cell markers


in non‑cancerous epithelium. The scores of NANOG and OCT4 were higher
than those of CD133 and NESTIN (P<0.001). No significant difference was
identified between the scores of NANOG and OCT4. OCT4, octamer 4;
CD133, cluster of differentiation 133. Figure 6. Immunohistochemical scores of four stem cell markers and HIF‑1α
in the hyperplastic glands (benign prostate hyperplasia), high‑grade PIN and
prostatic ADC. The score of NANOG in ADC was significantly greater than
that of the hyperplastic glands (P<0.001) and high‑grade PIN (P<0.001); the
value of high‑grade PIN was significantly higher than that of hyperplastic
NANOG (9,10) is one of the four factors known to repro- glands (P<0.001). The score of OCT4 in ADC was significantly higher than
gram adult cells into germline‑competent induced pluripotent that in the hyperplastic glands (P<0.05), while the scores of CD133 and
stem cells (45). NANOG is also critical in maintaining the NESTIN of three lesions (hyperplastic glands, high‑grade PIN and ADC)
were almost similar. HIF‑1α was expressed in the nuclei of ADC, but not in
self‑renewal and pluripotency of ES cells by regulating the
the hyperplastic glands and high‑grade PIN. HP, hyperplastic; ADC, adeno-
cell fate of the pluripotent inner cell mass (46‑48). Notably, carcinoma; HIF‑1α, hypoxia‑inducible factor‑1α; PIN, prostate intraepithelial
elevated NANOG protein expression in several types of neoplasia; OCT4, octamer 4; CD133, cluster of differentiation 133.
human cancer has been reported, predominantly in germ cell
tumors, as well as the malignancies of non‑germ cells (38),
suggesting the involvement of NANOG in tumorigenesis and
progression. Non‑germ cell tumors, including breast (38) and the regulation of NANOG gene expression via binding with
oral cancer (49), also express NANOG. A systematic study the NANOG proximal promoter (51). TGF‑β functions as a key
using animal models and in vitro cell systems has provided tumor suppressor of the prostate and can also promote malig-
substantial evidence for the key function of NANOG in human nant progression and metastasis of the advanced disease (52).
tumor development (50). A recent study has shown that the Human cultured prostate cancer cells, prostate cancer xeno-
transforming growth factor (TGF)‑β pathway is involved in grafts and primary prostate cancer cells express a functional
990 MIYAZAWA et al: EXPRESSION OF NANOG AND HIF-1α IN PROSTATE CANCER

Figure 7. (A) Immunohistochemical scores of four stem cell markers and HIF‑1α in prostatic ADC with Gleason scores of <3+3 and >3+4. No significant differ-
ences were identified between the scores of NANOG, OCT4, CD133 and NESTIN in the two different Gleason score groups. However, the score of HIF‑1α was
greater in ADC with Gleason scores of >3+4 than that in ADC with Gleason scores of <3+3 (P<0.001). The number of NANOG‑positive cancer cells in (B) high
Gleason score groups (3+4) was greater than that of (C) low Gleason score groups (2+2) (magnification, 200; scale bars, 50 µm). ADC, adenocarcinoma; HIF‑1α,
hypoxia‑inducible factor‑1α; OCT4, octamer 4; CD133, cluster of differentiation 133.

A B C D

Figure 8. HIF‑1α immunohistochemistry in ADC, high‑grade PIN and hyperplastic glands. The number of HIF‑1α‑positive nuclei of (A and B) ADCs was
greater than in (C) high‑grade PIN. The number of HIF‑1α‑positive nuclei of (B) ADCs with Gleason scores of >3+4 was larger than that of (A) ADCs with
Gleason scores of <3+3. No positive cell nuclei for HIF‑1α were identified in the hyperplastic glandular cells (magnification, x200; scale bars, 50 µm). ADC,
adenocarcinoma; HIF‑1α, hypoxia‑inducible factor‑1α.; PIN, prostate intraepithelial neoplasia.

variant of NANOG, NANOG mRNA, in cancer cells (50). This trophectoderm (53). A recent report questioned the function
expression is derived predominantly from a retrogene locus of OCT4 as a pure stem cell marker by showing its expres-
termed NANOGp8 (50). In this study, the NANOG protein was sion in differentiated cells (54). Ugolkov et al (18) reported
detected in the nucleus of cancer cells, but was not expressed that OCT4 nuclear expression was markedly associated with
in hyperplastic glandular cells. These findings suggest that benign prostatic lesions, but not prostate cancer. In the present
NANOG has a particular function in prostate cancer develop- study, OCT4 expression was found in the prostate cancer
ment. In addition, a significant correlation has been reported and non‑cancerous glandular cells; however, differences
between NANOG‑, OCT4‑ and HIF‑1α‑positive regions (31). were observed in its expression between prostate cancer and
Low oxygen levels promote self‑renewal in stem cells and non‑cancerous glands. Although, these differences were not as
hypoxia has been associated with an aggressive disease course marked as those observed in NANOG expression.
and poor clinical outcomes in malignancies, including prostate One notable observation in the current study was that
cancer (28,29). Furthermore, a number of aggressive neoplasms prostate cancer cells expressing NANOG and OCT4 were also
exhibit gene expression signatures characteristic of human ES positive for HIF‑1α reactivity. Hypoxia‑regulated genes are
cells. Thus, HIF may act as a key inducer of a dynamic state of mediated by the HIF‑1 complex composed of a heterodimeric
stemness in pathological conditions. pair of HIF‑1α and ‑1β  (28,29), and HIF‑1α is an important
OCT4 maintains pluripotency in embryogenesis; the transcription factor in prostate carcinogenesis, which suggests
upregulation of OCT4 results in differentiation to the that HIF‑1α may be a potential prognostic biomarker in
primitive endoderm and mesoderm, while downregulation the proteomic assessments of prostate cancers (55,56).
induces a loss of pluripotency and dedifferentiation into the Additionally, HIF‑1α induces human ES cell markers, such
ONCOLOGY LETTERS 8: 985-992, 2014 991

as NANOG (14,30,31), OCT4 (14,30,31) and CD133 (32), in 14. Ma Y, Liang D, Liu J, Axcrona K, Kvalheim G, Stokke T,
Nesland JM and Suo Z: Prostate cancer cell lines under hypoxia
cancer cells. The findings of the current study showing the exhibit greater stem‑like properties. PLoS One 6: e29170, 2011.
coexpression of NANOG, OCT4 and HIF‑1α support these 15. Scholer HR, Hatzopoulos AK, Balling R, Suzuki N and
studies. However, a slightly positive reaction or null of CD133 Gruss P: A family of octamer‑specific proteins present
during mouse embryogenesis: evidence for germline‑specific
in cancer cells was observed. The reason for this was unknown; expression of an Oct factor. EMBO J 8: 2543‑2550, 1989.
although, a strong correlation was identified between NANOG 16. Boia n i M a nd Scholer H R: Regulator y networks in
and HIF‑1α expression, which may suggest that NANOG and embryo‑derived pluripotent stem cells. Nat Rev Mol Cell
Biol 6: 872‑884, 2005.
HIF‑1α co‑operate in prostate carcinogenesis. 17. Monk M and Holding C: Human embryonic genes re‑expressed
The results of this study showed that of the four CSC markers in cancer cells. Oncogene 20: 8085‑8091, 2001.
examined (NANOG, OCT4, CD133 and NESTIN), NANOG 18. Ugolkov AV, Eisengart LJ, Luan C and Yang XJ: Expression
analysis of putative stem cell markers in human benign and
was intensively expressed in prostate cancer. In addition, malignant prostate. Prostate 71: 18‑25, 2011.
HIF‑1α was coexpressed in cancer cells. These findings suggest 19. Mallanna SK and Rizzino A: Systems biology provides new
that NANOG, in conjunction with HIF‑1α, may be important in insights into the molecular mechanisms that control the fate of
embryonic stem cells. J Cell Physiol 227: 27‑34, 2012.
prostate carcinogenesis. In addition, the immunohistochemical 20. Tysnes BB: Tumor‑initiating and ‑propagating cells: cells that
expression of NANOG may present as a biomarker for investi- we would like to identify and control. Neoplasia 12: 506‑515,
gating the pathobiology of prostate cancer. 2010.
21. Till JE: Stem cells in differentiation and neoplasia. J Cell
Physiol Suppl 1: 3‑11, 1982.
Acknowledgements 22. Grosse‑ Gehling P, Fa rgeas CA, Dittfeld C, Garbe Y,
Alison MR, Corbeil D and Kunz‑Schughart LA: CD133 as
a biomarker for putative cancer stem cells in solid tumours:
The authors would like to thank Dr Kohei Kawaguci, Dr Osamu limitations, problems and challenges. J Pathol 229: 355‑378,
Ueki and Mr. Hideaki Nishida for providing the biopsy 2013.
samples. This study was supported, in part, by a grant (2010) 23. Missol‑Kolka E, Karbanova J, Janich P, Haase M, Fargeas CA,
Huttner WB and Corbeil D: Prominin‑1 (CD133) is not
from the Hokkoku Cancer Research Promotion Foundation restricted to stem cells located in the basal compartment of
and a Grant for Alumni Research from the Kanazawa Medical murine and human prostate. Prostate 71: 254‑267, 2011.
University (grant no. AR2012‑01). 24. Lendahl U, Zimmerman LB and McKay RD: CNS stem cells
express a new class of intermediate filament protein. Cell 60:
585‑595, 1990.
References 25. Ishiwata T, Matsuda Y and Naito Z: Nestin in gastrointestinal
and other cancers: effects on cells and tumor angiogenesis.
 1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T and Thun M: World J Gastroenterol 17: 409‑418, 2011.
Cancer statistics, 2008. CA Cancer J Clin 58: 71‑96, 2008. 26. Kleeberger W, Bova GS, Nielsen ME, Herawi M, Chuang AY,
 2. Matsuda T, Marugame T, Kamo K, Katanoda K, Ajiki W and Epstein JI and Berman DM: Roles for the stem cell associated
Sobue T: Cancer incidence and incidence rates in Japan in 2002: intermediate filament Nestin in prostate cancer migration and
based on data from 11 population‑based cancer registries. Jpn J metastasis. Cancer Res 67: 9199‑9206, 2007.
Clin Oncol 38: 641‑648, 2008. 27. Sotomayor P, Godoy A, Smith GJ and Huss WJ: Oct4A is
 3. Nonn L, Ananthanarayanan V and Gann PH: Evidence for field expressed by a subpopulation of prostate neuroendocrine cells.
cancerization of the prostate. Prostate 69: 1470‑1479, 2009. Prostate 69: 401‑410, 2009.
 4. Al‑Hajj M, Wicha MS, Benito‑Hernandez A, Morrison SJ and 28. Kimbro KS and Simons JW: Hypoxia‑inducible factor‑1 in
Clarke MF: Prospective identification of tumorigenic breast cancer human breast and prostate cancer. Endocr Relat Cancer 13:
cells. Proc Natl Acad Sci USA 100: 3983‑3988, 2003. 739‑749, 2006.
 5. Collins AT, Berry PA, Hyde C, Stower MJ and Maitland NJ: 29. Mabjeesh NJ and Amir S: Hypoxia‑inducible factor (HIF) in
Prospective identification of tumorigenic prostate cancer stem human tumorigenesis. Histol Histopathol 22: 559‑572, 2007.
cells. Cancer Res 65: 10946‑10951, 2005. 30. Bao B, Ahmad A, Kong D, Ali S, Azmi AS, Li Y, Banerjee S,
 6. O'Brien CA, Pollett A, Gallinger S and Dick JE: A human colon Padhye S and Sarkar FH: Hypoxia induced aggressiveness of
cancer cell capable of initiating tumour growth in immunodeficient prostate cancer cells is linked with deregulated expression of
mice. Nature 445: 106‑110, 2007. VEGF, IL‑6 and miRNAs that are attenuated by CDF. PLoS
 7. Gu G, Yuan J, Wills M and Kasper S: Prostate cancer cells with One 7: e43726, 2012.
stem cell characteristics reconstitute the original human tumor 31. Mathieu J, Zhang Z, Zhou W, Wang AJ, Heddleston JM,
in vivo. Cancer Res 67: 4807‑4815, 2007. Pinna CM, Hubaud A, Stadler B, Choi M, Bar M, et al: HIF
 8. Miki J, Furusato B, Li H, Gu Y, Takahashi H, Egawa S, induces human embryonic stem cell markers in cancer cells.
Sesterhenn IA, McLeod DG, Srivastava S and Rhim JS: Cancer Res 71: 4640‑4652, 2011.
Identification of putative stem cell markers, CD133 and CXCR4, 32. Salnikov AV, Liu L, Platen M, Gladkich J, Salnikova O,
in hTERT‑immortalized primary nonmalignant and malignant Rysch ich E, Mat ter n J, Molden hauer G, Wer ner J,
tumor‑derived human prostate epithelial cell lines and in prostate Schemme P, et al: Hypoxia induces EMT in low and highly
cancer specimens. Cancer Res 67: 3153‑3161, 2007. aggressive pancreatic tumor cells but only cells with cancer
 9. Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S and stem cell characteristics acquire pronounced migratory
Smith A: Functional expression cloning of Nanog, a pluripotency potential. PLoS One 7: e46391, 2012.
sustaining factor in embryonic stem cells. Cell 113: 643‑655, 2003. 33. Clark AT: The stem cell identity of testicular cancer. Stem Cell
10. Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Rev 3: 49‑59, 2007.
Takahashi K, Maruyama M, Maeda M and Yamanaka S: The 34. Cizkova D, Soukup T and Mokry J: Nestin expression reflects
homeoprotein Nanog is required for maintenance of pluripotency formation, revascularization and reinnervation of new
in mouse epiblast and ES cells. Cell 113: 631‑642, 2003. myofibers in regenerating rat hind limb skeletal muscles. Cells
11. Zhang S, Balch C, Chan MW, Lai HC, Matei D, Schilder JM, Tissues Organs 189: 338‑347, 2009.
Yan PS, Huang TH and Nephew KP: Identification and charac- 35. Horst D, Kriegl L, Engel J, Kirchner T and Jung A: CD133
terization of ovarian cancer‑initiating cells from primary human expression is an independent prognostic marker for low
tumors. Cancer Res 68: 4311‑4320, 2008. survival in colorectal cancer. Br J Cancer 99: 1285‑1289, 2008.
12. Santagata S, Ligon KL and Hornick JL: Embryonic stem cell 36. Chambers I and Tomlinson SR: The transcriptional foundation
transcription factor signatures in the diagnosis of primary and of pluripotency. Development 136: 2311‑2322, 2009.
metastatic germ cell tumors. Am J Surg Pathol 31: 836‑845, 2007. 37. Ben‑Porath I, Thomson MW, Carey VJ, Ge R, Bell GW,
13. Gong C, Liao H, Guo F, Qin L and Qi J: Implication of expression Regev A and Weinberg RA: An embryonic stem cell‑like gene
of Nanog in prostate cancer cells and their stem cells. J Huazhong expression signature in poorly differentiated aggressive human
Univ Sci Technolog Med Sci 32: 242‑246, 2012. tumors. Nat Genet 40: 499‑507, 2008.
992 MIYAZAWA et al: EXPRESSION OF NANOG AND HIF-1α IN PROSTATE CANCER

38. Ezeh UI, Turek PJ, Reijo RA and Clark AT: Human embryonic 49. Chiou SH, Yu CC, Huang CY, Lin SC, Liu CJ, Tsai TH,
stem cell genes OCT4, NANOG, STELLAR, and GDF3 are Chou SH, Chien CS, Ku HH and Lo JF: Positive correlations of
expressed in both seminoma and breast carcinoma. Cancer 104: Oct‑4 and Nanog in oral cancer stem‑like cells and high‑grade
2255‑2265, 2005. oral squamous cell carcinoma. Clin Cancer Res 14: 4085‑4095,
39. Li L, Yu H, Wang X, Zeng J, Li D, Lu J, Wang C, Wang J, Wei J, 2008.
Jiang M and Mo B: Expression of seven stem‑cell‑associated 50. Jeter CR, Badeaux M, Choy G, Chandra D, Patrawala L,
markers in human airway biopsy specimens obtained via Liu C, Calhoun‑Davis T, Zaehres H, Daley GQ and Tang DG:
fiberoptic bronchoscopy. J Exp Clin Cancer Res 32: 28, 2013. Functional evidence that the self‑renewal gene NANOG
40. Luo W, Li S, Peng B, Ye Y, Deng X and Yao K: Embryonic stem regulates human tumor development. Stem Cells 27: 993‑1005,
cells markers SOX2, OCT4 and Nanog expression and their 2009.
correlations with epithelial‑mesenchymal transition in nasopha- 51. Xu RH, Sampsell‑Barron TL, Gu F, Root S, Peck RM, Pan G,
ryngeal carcinoma. PLoS One 8: e56324, 2013. Yu J, Antosiewicz‑Bourget J, Tian S, Stewart R and Thomson JA:
41. Yin X, Li YW, Zhang BH, Ren ZG, Qiu SJ, Yi Y and Fan J: NANOG is a direct target of TGFbeta/activin‑mediated SMAD
Coexpression of stemness factors Oct4 and Nanog predict liver signaling in human ESCs. Cell Stem Cell 3: 196‑206, 2008.
resection. Ann Surg Oncol 19: 2877‑2887, 2012. 52. Danielpour D: Functions and regulation of transforming
42. English HF, Santen RJ and Isaacs JT: Response of glandular growth factor‑beta (TGF‑beta) in the prostate. Eur J Cancer 41:
versus basal rat ventral prostatic epithelial cells to androgen 846‑857, 2005.
withdrawal and replacement. Prostate 11: 229‑242, 1987. 53. Pesce M and Scholer HR: Oct‑4: control of totipotency and
43. Lawson DA and Witte ON: Stem cells in prostate cancer initiation germline determination. Mol Reprod Dev 55: 452‑457, 2000.
and progression. J Clin Invest 117: 2044‑2050, 2007. 54. Zangrossi S, Ma rabese M, Broggini M, Giordano R,
44. Hurt EM, Kawasaki BT, Klarmann GJ, Thomas SB and D'Erasmo M, Montelatici E, Intini D, Neri A, Pesce M,
Farrar WL: CD44+ CD24(‑) prostate cells are early cancer Rebulla P and Lazzari L: Oct‑4 expression in adult human
progenitor/stem cells that provide a model for patients with poor differentiated cells challenges its role as a pure stem cell
prognosis. Br J Cancer 98: 756‑765, 2008. marker. Stem Cells 25: 1675‑1680, 2007.
45. Yu J, Vodyanik MA, Smuga‑Otto K, Antosiewicz‑Bourget J, 55. Lekas A, Lazaris AC, Deliveliotis C, Chrisofos M, Zoubouli C,
Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, et al: Lapas D, Papathomas T, Fokitis I and Nakopoulou L: The
Induced pluripotent stem cell lines derived from human somatic expression of hypoxia‑inducible factor‑1alpha (HIF‑1alpha)
cells. Science 318: 1917‑1920, 2007. and angiogenesis markers in hyperplastic and malignant
46. Pan G and Thomson JA: Nanog and transcriptional networks in prostate tissue. Anticancer Res 26: 2989‑2993, 2006.
embryonic stem cell pluripotency. Cell Res 17: 42‑49, 2007. 56. Makarewicz R, Zyromska A and Andrusewicz H: Comparative
47. Pereira L, Yi F and Merrill BJ: Repression of Nanog gene tran- analysis of biological profiles of benign prostate hyperplasia
scription by Tcf3 limits embryonic stem cell self‑renewal. Mol and prostate cancer as potential diagnostic, prognostic and
Cell Biol 26: 7479‑7491, 2006. predictive indicators. Folia Histochem Cytobiol 49: 452‑457,
48. Suzuki A, Raya A, Kawakami Y, Morita M, Matsui T, 2011.
Na kash ima K, Gage F H, Rod r íguez‑Esteba n C a nd
Izpisúa Belmonte JC: Maintenance of embryonic stem cell pluri-
potency by Nanog‑mediated reversal of mesoderm specification.
Nat Clin Pract Cardiovasc Med 3 (Suppl 1): S114‑S122, 2006.

You might also like