Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 397-405
doi:10.1111/j.1745-7270.2008.00419.x
Differential gene expression profiling of
human epidermal growth factor receptor 2-overexpressing mammary tumor
Yan Wang1#, Haining Peng1#, Yingli Zhong1#, Daiqiang Li2, Mi Tang2, Xiaofeng Ding1, and Jian Zhang1,3*
1
Key Laboratory of
Protein Chemistry and Developmental Biology of the Education Ministry of China,
College of Life Science, Hunan Normal University, Changsha 410081, China
2
Department of Pathology,
the Second Xiangya Hospital, Central South University, Changsha 410011, China
3
Model Organism Division,
E-Institutes of Shanghai Universities, Shanghai Second Medical University,
Shanghai 200025, China
Received: January
11, 2008
Accepted: March 27, 2008This work was
supported in part by the grants from the National Natural Science Foundation of
China (20335020 and 90608006), the Program for Changjiang Scholars and
Innovative Research Team in University (No. IRT0445), and the Science and
Technology Department of Hunan Province (No. 2006JT2008)
#These authors
contributed equally to this work
*Corresponding
author: Tel/Fax, 86-731-8872792; E-mail, zhangjian@ hunnu.edu.cn
Human epidermal growth factor receptor 2
(HER2) is highly expressed in approximately 30% of breast cancer patients, and
substantial evidence supports the relationship between HER2 overexpression and
poor overall survival. However, the biological function of HER2 signal
transduction pathways is not entirely clear. To investigate gene activation
within the pathways, we screened differentially expressed genes in
HER2-positive mouse mammary tumor using two-directional suppression subtractive
hybridization combined with reverse dot-blotting analysis. Forty genes and
expressed sequence tags related to transduction, cell proliferation/growth/apoptosis
and secreted/extracellular matrix proteins were differentially expressed in
HER2-positive mammary tumor tissue. Among these, 19 were already reported to be
differentially expressed in mammary tumor, 11 were first identified to be
differentially expressed in mammary tumor in this study but were already
reported in other tumors, and 10 correlated with other cancers. These genes can
facilitate the understanding of the role of HER2 signaling in breast cancer.
Keywords mammary tumor; HER2; suppression subtractive hybridization
Overexpression of the human epidermal growth factor receptor 2 (HER2)
gene, a breast cancer marker, is associated with rapid tumor growth, increased
risk of recurrence after surgery, poor response to conventional chemotherapy,
and decreased survival [1].The HER2 gene encodes a 185 kDa transmembrane glycoprotein
that belongs to the epidermal growth factor receptor family. This glycoprotein
can bind tightly to epidermal growth factor receptor family members, such as
mitogen-activated protein kinase and phosphatidylinositol-3 kinase (PI3K), and
enhance kinase-mediated activation of downstream signaling pathways [2]. HER2
amplification or overexpression is an extremely relevant genetic aberration
occurring in 30% of breast cancer patients and correlates with poor patient
survival [3,4]. However, the role of HER2 in breast cancer and the genes
related with HER2 signal transduction pathways are poorly understood.
Therefore, it is important to identify additional genes that are differentially
expressed in HER2-positive breast cancer to understand the role of the HER2
pathway in breast tumorigenesis and discover new therapeutic targets.Mammary tissue is heterogeneous that grows and changes cyclically
under hormonal regulation. The histological types of breast carcinoma are
numerous and complex. Usually, more than two histological types are observed in
the same tissue. However, their clinical and radiographic presentations vary
from one patient to another. Hence, it is difficult to investigate breast
tumorigenesis and to develop new diagnostic and therapeutic agents for human
breast cancer [5]. In contrast, using a single animal model as a research tool
is comparatively simple and accurate to help in understanding the biological
mechanisms of human breast cancer.In mouse mammary tumor virus (MMTV)-neu mice (strain of origin FVB),
transgene HER2 expression is lower in the normal mammary epithelium than
in tumor tissues. We are interested in identifying genes associated with HER2
that might contribute to mammary tumor development and progression. We carried
out suppression subtractive hybridization (SSH) to analyze differential gene
expression profiling between MMTV-neu mouse mammary tumor and FVB mouse normal
mammary tissue in the same developmental period, and between MMTV-neu mouse
mammary tumor and MMTV-neu mouse normal mammary tissue in different
developmental periods. Combined with reverse dot-blotting and sequencing, we
generated suppressive subtractive cDNA libraries, including overexpressed and
down-regulated genes in mouse mammary tumors. These differentially expressed
genes might be involved in human breast cancers and could serve as potential
therapeutic and diagnostic targets.
Materials and Methods
Tissue samples
MMTV-neu mice were purchased from Jackson Laboratory (Bar Harbor,
Maine, USA). Mice homozygous for the MMTV-neu (rat) transgene were viable and
fertile. Focal mammary tumors first appeared within 4 months, with a median
incidence of 205 d. FVB mice were used as controls. All animals were housed
under specific pathogen-free conditions.
Tissue preparation and mRNA isolationMammary tumor specimens and normal mammary tissues were obtained
from three 6-month-old MMTV-neu mice, and normal mammary tissues were obtained
from three FVB/NJ mice of the same age or from 2-month-old MMTV-neu mice.
Total RNA and poly(A) mRNA were isolated using Trizol reagent (Invitrogen,
Shanghai, China), and the poly(A) tract mRNA isolation system kit (Promega,
Madison, USA), respectively, according to the manufacturers protocols.
SSH library constructionSubtraction hybridization was carried out using the polymerase chain
reaction (PCR)-select cDNA subtraction kit (Clontech, Palo Alto, USA),
according to the manufacturers protocol. cDNA was synthesized from poly(A)+ RNA. Two-directional subtractions of SSH were then carried out
between MMTV-neu mouse mammary tumor and MMTV-neu mouse normal mammary tissue,
and between MMTV-neu mouse mammary tumor and FVB mouse normal mammary tissue.
Analysis of subtraction efficiency
All four subtracted cDNA products and corresponding unsubtracted
cDNA (as control) were subjected to 18, 23, 28, or 33 cycles of PCR
amplification using the housekeeping gene glyceraldehyde-3-phosphate
dehydrogenase (G3PDH) gene with the primers provided in the PCR-select
cDNA subtractive kit.
Cloning and colony PCR
The resultant cDNA fragments were purified using the QIAquick PCR
purification kit (Qiagen, Hilden, Germany) prior to cloning into the pGEM-T
easy vector (Promega) and transformed into competent Escherichia coli
DH5a
cells. After blue/white screening, white bacterial colonies were selected and
the presence of the insert was tested using the colony PCR method. Samples that
failed to produce amplifications or produced multiple fragments were excluded
from further analysis.
Reverse dot-blotting
Plasmid DNA of each positive clone was purified. PCR was then
carried out to amplify plasmid DNA inserts using nester primers. Next, 5 ml of each PCR
product was denatured with equal volumes of 0.6 M NaOH, spotted onto Hybond N+ membranes (Millipore, Eschborn, Germany), then ultraviolet
cross-linked. Subsequently, cDNA probes were prepared from tester and driver
cDNA using the DIG high prime DNA labeling and detection starter kit II (Roche,
Mannheim, Germany), according to the manufacturers protocol. Immunological
detection was carried out with antidigoxigenin antibodies conjugated to
alkaline phosphatase and a combination of 5-bromo-4-chloro-3-indoyl phosphate
and nitroblue tetrazolium, according to the manufacturers instructions and the
method previously described [6].
Fragment sequencing and analysisAll the plasmids of the positive clone, as assessed by reverse
dot-blotting with an inserted fragment, were sequenced by Shanghai Sangon
Biological Engineering Technology and Services (Shanghai, China). The DNA
sequence was compared with the GenBank database using advanced BLAST.
Quantitative real-time reverse transcription (RT)-PCR analysis
To confirm the results of SSH, the expression level of nine selected
genes was determined by real-time PCR using the ABI 7900HT instrument and SYBR
Green PCR master mix (Applied Biosystems, Foster City, USA). Real-time PCR
transcript quantification was carried out using the nine candidates and the
housekeeping gene b-actin as the endogenous control using
different primers (Table 1). Cycling parameters were 50 ?C for 2 min, 95
?C for 15 s, 40 cycles at 95 ?C for 15 s, and 60 ?C for 1 min. After PCR
amplification, a melting curve was plotted to measure PCR specificity.
Real-time PCR results were analyzed using the DDCt method. The DCt value was determined by subtracting b-actin Ct value from the studying group Ct value of the
normal group from the DCt value of each group. 2-DDCt represented the average relative amount of mRNA for each group [7,8].
Immunohistochemistry
Immunohistochemistry was carried out using Akt1 (2H10) mouse
monoclonal antibody and HER2 (44E7) mouse monoclonal antibody (Cell Signaling
Technology, Oakland, USA). Antibody staining was carried out using
UltraSensitive SP and 3-amino-9-ethylcarbazole detection kits (Maixin-Bio,
Beijing, China). Incubation with Akt1/HER2 antibodies was carried out using a
1:500 dilution of the antibodies (60 min at room temperature). The slides were
counterstained with hematoxylin, according to the manufacturers instructions.
Statistical analysis
The data obtained were analyzed using SPSS (version 13.0) software
package. c2-test for each experiment is reported. P values less than
0.05 were deemed as statistical significance.
Results
Analysis of subtraction efficiency
After PCR amplification, the housekeeping gene G3PDH appeared
at 18 cycles in unsubtraction samples and at 33 cycles in subtraction samples. This
result indicated that G3PDH expressed in both parts have been greatly
decreased through the subtraction method. If five cycles corresponded roughly
to 20-fold cDNA enrichment, G3PDH would have been decreased almost
300-fold. It implied that other genes expressed in both tissues were reduced
the same fold and the specially expressed genes in the test sample were
selected (Fig. 1).
Colony PCR and reverse dot-blotting analysis of differential
expression
Approximately 600 white clones were randomly selected, and the
presence of the insert was detected by the colony PCR method. Subsequently, 476
clones were analyzed using reverse dot-blotting analysis to confirm
differential expression (Fig. 2) and 80 clones were verified.
Fragment sequencing and analysisThese 80 clones were then sequenced, and the expressed sequence tag
(EST) sequences obtained were compared with the GenBank database using blast. Forty genes and ESTs were
differentially expressed in mouse mammary tumor. In the same developmental
period group, 14 genes were up-regulated and 16 genes/ESTs were down-regulated
in the mammary tumor. In the varying developmental period group, nine genes
were up-regulated and one gene was down-regulated in the mammary tumor. Tables
2 and 3 summarize these genes.
Quantitative real-time RT-PCR analysisTo confirm the SSH results, three down-regulated [complement factor
D (CFD), HRAS-like suppressor 3 (Hrasls3), and ubiquitin-fold
modifier conjugating enzyme 1 (Ufc1)] and six up-regulated genes [Akt1,
interferon-induced transmembrane protein 2 (Ifitm2), immediate early response 3 (Ier3),
lipocalin 2 (Lcn2), lymphocyte antigen 6 complex, locus E (Ly6e),
and member of the RAS oncogene family (Rab25)] in mouse mammary tumor
were further analyzed by quantitative real-time RT-PCR (Fig. 3). As
templates, we used the cDNA synthesized from mouse mammary tumor tissue RNA and
normal mammary tissue total RNA. The signals were normalized using the
housekeeping gene b-actin. CFD, Hrasls3, and Ufc1
mRNA levels were lower in the mammary tumor than in the normal mammary tissue.
In contrast, Akt1, IFITM2, Ier3, Lcn2, Ly6e,
and Rab25 mRNA levels increased significantly in the mammary tumor
(Student-Neumann-Keuls test, n=3, *P<0.05). These findings were consisted with the SSH results.
Immunohistochemistry analysis
Akt1 and HER2 immunoreactivities were evaluated in 50 human breast
cancer tissue samples. Among 36 samples that were positive for HER2, 26 cases
were Akt1-positive (72%). Among 14 cases that were negative for HER2, only six
cases were Akt1-positive. The results indicated that Akt1 expression
significantly correlated with HER2 expression in some human malignant breast
tissues, as determined using the ?2-test (P<0.05) (Table 4). However, Akt1 expression can also be activated through an
HER2-independent pathway. An example of breast cancer positive for both HER2
and Akt1 is shown in Fig. 4.
Discussion
Of the many strategies previously used, we selected SSH to identify
genes that were abnormally expressed in only breast cancer tissue. We isolated
both known genes and several unknown genes that were differentially expressed
in HER2-overexpressing mammary tumor tissue. We classified these genes into three classes. The first class
included 19 genes that were previously reported to be differentially expressed
in mammary tumors. It included the three oncogenes Akt1, Lcn2,
and Rab25 and the anti-oncogene Hrasls3. All three oncogenes were
up-regulated in MMTV-neu mouse mammary tumor tissue. Hrasls3 was
up-regulated in FVB mouse normal mammary tissue compared with MMTV-neu mouse
mammary tumor tissue, consistent with previous reports [9–12]. In the
second class, 11 unique genes were found to be differentially expressed. For
example, IFITM2 has been suggested to be a new molecular marker for
human colorectal tumors [13]. The third class included eight unique genes and
two ESTs that were found to be differentially expressed. For example, Ufc1
was not differentially expressed in cancer and was down-regulated in the
mammary tumor. These genes can facilitate the understanding of the molecular
basis of tumorigenesis and serve as potential biomarkers or prognostic markers.HER2 receptor activation and tyrosine phosphorylation activate
specific signal transduction pathways in cancer cells, including the
Ras-Raf-mitogen-activated protein kinase, PI3K-Akt, and phospholipase C-g pathways [14].
Akt1 is a serine-threonine protein kinase that regulates a variety of cellular
functions, including survival, migration, and intermediary metabolism [9,15–18]. Delord et
al showed that the inhibition of the AKT pathway appears to be the major
mechanism contributing to reduced HER2-mediated oncogenesis and prolonged
survival in a murine model of ovarian cancer [19]. It is activated by HER2
through PI3K [20]. We also found that Akt1 was up-regulated in mouse
HER2-overexpressing mammary tumor, and its expression was significantly
correlated with HER2 expression in human malignant breast tissues. Diverse
intracellular signaling pathways ultimately converge on the cell nucleus, where
the expression of genes that regulate cellular proliferation and
differentiation is tightly coordinated. A number of nuclear transcription
factors have been identified as the targets of HER2 signal transduction
pathways. In this study, Zfp313 was the only transcription factor that was
up-regulated in mammary tumor [21]. Thus, Zfp313 is probably a new target of
HER2 signal transduction pathways.HER2 co-activated genes could affect disease progression and the
clinical behavior of HER2-positive tumors [2]. Simultaneously, the inactivated
and down-expressed genes associated with HER2 facilitate growth inhibition and
apoptosis of cancer cells. We identified six HER2 co-amplified (Akt1, Lcn2,
RAB25, Ier3, IFITM2 and Ly6e) and three
down-expressed genes (CFD, Hrasls3, and Ufc1) in the
mammary tumor using quantitative real-time RT-PCR. Lcn2 (NGAL, also referred to as
neu-related lipocalin) is a member of the lipocalin superfamily and is
specifically overexpressed in tumor cells that are induced by HER2 but not by
Ras or in chemical-induced cancers. It is overexpressed in rat and human
mammary carcinomas and is putatively regulated by HER2 [10]. Rab25 has been implicated in many
tumors, including ovarian and breast cancer. Rab25 levels were amplified
in 80% of ovarian cancer samples and 67% of breast cancer patients [11,22].
However, Rab25 expression was lost in the breast cancer cell lines
containing a Ras point mutation and in some breast cancer tissues derived from
human patients. The loss of Rab25 expression is associated with
tumorigenesis in human mammary epithelial cells [23]. Ier3 (IEX-1) enhances tumor
necrosis factor-a-induced hepatocyte apoptosis by inhibiting Akt activation [24].
However, in another study, IEX-1 increased Akt activity in human tumor cells
[25]. Recently, Maroulakou et al found that Akt1 promotes but Akt2
inhibits mammary tumor induction and tumor growth in MMTV-HER2-neu and
MMTV-PyMT transgenic mice [26]. Hence, Ier3 can either activate or inhibit Akt,
that is, Akt activated by Ier3 is Akt1 and that inhibited by Ier3 is Akt2.Andreu et al specified the IFITM family as a new molecular
marker for human colorectal tumors [13]. They found no significant difference
in IFITM mRNA levels between breast cancer and normal breast tissue samples.
However, as the IFITM cDNA probe detected IFITM2, IFITM1, and IFITM3,
differential expression of IFITM2 in breast cancer could not be proved. This
study identified the correlation of IFITM2 to breast cancer. Hrasls3 (H-REV107-1) is a new class II
tumor suppressor, based on its reversible down-regulation and growth-inhibiting
capacity in HRAS-transformed ANR4 hepatoma cells or in FE-8 fibroblasts. It
plays a role in differentiation-related growth arrest [12,27]. It was detected
in only eight of the 27 cell lines derived from mammary carcinoma, lung
carcinoma, and other tumors. The H-REV107-1 protein was not detectable in any
of these tumor cells. The loss of its expression was observed in cultured human
tumor cell lines and primary squamous cell carcinomas [28].Ufc1 conjugates with ubiquitin-fold
modifier 1 through a thioester linkage [29]. The major function of ubiquitin is
to serve as a tag for protein degradation by the proteasome, and the ubiquitin-proteasome
system mediates the regulation of various cellular processes that are relevant
to cancer. Thus, Ufc1 might facilitate the development and progression of
breast cancer. In conclusion, we found 21 genes that were reported for the first
time to be differentially expressed in HER2-positive mammary tumor; of these
genes, 10 were newly found to be correlated with cancer. These genes are likely
to be considered as genes related to the HER2 pathway or markers of breast
cancer. The differential gene expression profiles evaluated by SSH are useful
bases for gaining a better understanding of mammary tumorigenesis associated
with HER2. We obtained the differentially-expressed gene profiles using mouse
tissues, which might not represent well with the human cases. Clearly, further
studies are needed using human mammary cancer tissues to prove our findings on
the mouse tissues, and to determine whether identified differentially-expressed
genes could be used as markers for human breast cancer research.
Acknowledgements
We gratefully acknowledge the Department of
Pathology, the Second Xiangya Hospital, Central South University (Changsha,
China) for their role in obtaining breast cancer tissue samples.
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