The high-mobility group A (HMGA) family of proteins in mammals is composed of four
proteins: HMGA1a, HMGA1b, HMGA1c, and HMGA2. The former three proteins are encoded
by a single functional gene, that is, HMGA1 (formerly HMGI(Y)), while the last one
is a product of a separate gene, that is, HMGA2 (formerly HMGI-C) (Manfioletti et
al, 1991; Johnson et al, 1998). High-mobility group A2 has an approximately 50% amino-acid
sequence homology with HMGA1, and features an internal 11 amino-acid deletion that
characterises HMGA1 (Manfioletti et al, 1991; Tallini and Dal Cin, 1999). High-mobility
group A2 proteins bind to the minor groove of AT-rich DNA sequences, thereby inducing
a bend within the DNA (Thanos and Maniatis, 1992). They cannot initiate transcription,
but they can enhance promotor binding of transcription factors (Thanos and Maniatis,
1992; Grosschedl et al, 1994; Mantovani et al, 1998).
High-mobility group A2 has been shown to be expressed abundantly during embryogenesis,
but its expression is either undetectable or remains at low levels in other normal
adult tissues (Manfioletti et al, 1991; Zhou et al, 1995; Rogalla et al, 1996; Rommel
et al, 1997; Hirning-Folz et al, 1998), suggesting that HMGA2 plays an important role
(or roles) in cell proliferation and/or differentiation. Consistent with this, it
has been demonstrated that HMGA proteins are phosphorylated in a cell-cycle-dependent
manner (Reeves et al, 1991). Functionally, knocking out the HMGA2 gene in mice leads
to the pygmy phenotype with characteristic hypoplasia of mesenchymal tissue, thereby
confirming the important role(s) of HMGA2 in mammalian growth and development (Zhou
et al, 1995).
The altered form of the HMGA2 gene, on the other hand, could somehow be related to
the generation of human benign and malignant tumours. Rearrangements of the HMGA2
gene, for example, have been frequently observed in benign tumours of mesenchymal
origin (Ashar et al, 1995; Schoenmarkers et al, 1995). In such cases, the gene rearrangements
were the consequence of chromosomal translocation involving regions 12q13–15, where
the HMGA2 gene is located. The HMGA2 modifications consist of the loss of the carboxyl-terminal
tail and its fusion with ectopic sequences (Ashar et al, 1995; Schoenmarkers et al,
1995). The truncation of HMGA2, rather than its fusion with other genes, has also
been shown to be responsible for cell transformation (Fedele et al, 1998). This was
confirmed in transgenic mice carrying a truncated HMGA2, which developed a giant phenotype
together with a marked expansion of the retroperitoneal and subcutaneous white adipose
tissues (Battista et al, 1999; Arlotta et al, 2000). Interestingly, most of these
tumours related to the alteration in HMGA2 are of nonepithelial origin. In contrast,
only a few data on the expression of HMGA2 in human malignant tumour originating from
epithelial tissue are available (Rogalla et al, 1997). The overexpression of HMGA2
mRNA has been shown to be closely associated with high histologic grade in breast
cancer (Rogalla et al, 1997), suggesting that the expression level of the HMGA2 protein/gene
could be a potential clinicopathological marker with prognostic implications for a
wide range of cancers. To test this possibility, we examined the HMGA2 expression
in pancreatic cancers in the present study, and investigated whether alterations in
HMGA2 are associated with the malignant phenotype of tumours in pancreatic tissue.
To this end, HMGA2 mRNA expression was first analysed by highly sensitive reverse
transcriptase–polymerase chain reaction (RT–PCR) techniques. Immunohistochemical detection
of HMGA2 protein using a specific antibody was also attempted. Although relatively
simple and easy to perform, immunohistochemistry is a potential method of examining
whether the expression of a certain protein is specific to tumour cells, because it
allows precise correlation of the protein expression with the phenotype of the cells
on individual cell basis (Abe et al, 2000). In this sense, immunohistochemistry can
provide more useful information than other assays by which proteins and/or mRNAs are
extracted from tumours; possibly including a mixture of proteins from normal and irrelevant
cells such as acinar cells or islet cells of the pancreas in the analysis (Abe et
al, 2000). Based on the above considerations, we determined HMGA2 protein expression
immunohistochemically on surgically resected specimens, normal pancreatic tissue,
chronic pancreatitis tissue, and carcinomas of the pancreas.
MATERIALS AND METHODS
Tissue samples
The tissue samples were obtained at the time of surgery at the First Department of
Surgery, Kyorin University Hospital, between October 1996 and August 2001. Specimens
from 27 pancreatic carcinomas (20 primary carcinomas, four liver metastases, two peritoneal
metastases, and one lymph node metastasis) and eight non-neoplastic tissues (six normal
pancreatic and two chronic pancreatitis tissues) were obtained. In all, 27 carcinomas
were histologically diagnosed as 12 well-differentiated tubular adenocarcinomas, six
moderately differentiated tubular adenocarcinomas, seven poorly differentiated tubular
adenocarcinomas, and two adenosquamous carcinomas (they were evaluated histologically
according to the criteria of the Japan Pancreas Society). Normal pancreatic tissues
were obtained from either patients who have undergone pancreatectomy due to pancreatic
neoplasms or those with gastric cancer who have undergone pancreatectomy for lymph
node dissection. In either case, specimens were obtained from a macroscopically healthy
region distinct from the neoplastic lesion. All patients gave their informed consent
prior to their inclusion in the study. Among the samples, those from 17 pancreatic
carcinomas and six non-neoplastic pancreatic tissues were frozen on dry ice immediately
after surgical resection for molecular investigation (RT–PCR), and stored at −80°C
until use. All the tissue specimens were fixed for immunohistochemical analysis as
soon as possible after surgical resection in 4% paraformaldehyde in phosphate-buffered
saline (PBS) at 4°C for 14 h, followed by cryoprotection in a graded concentration
series of sucrose in PBS. The specimens were then embedded in the OCT compound, frozen,
and stored at −80°C until analysis. All the tissue specimens were histologically examined
and pathological diagnoses were confirmed.
RT–PCR analysis
Reverse transcriptase–polymerase chain reaction for the HMGA2 expression was performed
using a heminested PCR technique as described previously (Rogalla et al, 1996; Rommel
et al, 1997). Total RNA was extracted by a modified guanidine thiocyanate method as
described previously (Miyatani et al, 1986). cDNA was synthesised using the adapter
primer (AP) AAG GAT CCG TCG ACA TC (T)17 and Superscript II reverse transcriptase
(Gibco BRL, Gaithersburg, MD, USA). For the first and second rounds of the heminested
PCR, the same lower primer (Rev) 5′-TCC TCC TGA GCA GGC TTC-3′ (exon 4/5) was used.
The forward primer 5′-CTT CAG CCC AGG GAC AAC-3′ (exon 1) and the nested primer 5′-CAT
CGC CTC AGA AGA GAG GAC-3′ (exon 1) were used as upper primers. The PCR amplifications
were both performed for 30 cycles (1 min at 94°C, 1 min at 53°C, and 2 min at 72°C).
As a control reaction for intact RNA and cDNA, PCR amplification of the cDNA of the
housekeeping gene β-actin was performed for all samples to exclude false-negative
PCR results. Only those samples positive for β-actin were used for this study. The
resulting PCR products were clearly visualised by gel electrophoresis on a 2% agarose-gel
stained with ethidium bromide as bands at 220 base pairs (bp) for HMGA2 and at 154 bp
for β-actin (Figure 1
Figure 1
Reverse transcriptase–polymerase chain reaction products of HMGA2 after gel electrophoresis
and ethidium bromide staining. Results show specific 220-bp bands. DL, DNA molecular
weight marker; lane 1, positive control (hepatoma cell line HEP3B, which is known
to express high level of HMGA2); lane 2, normal pancreas; lane 3, chronic pancreatitis;
lane 4–11, pancreatic carcinomas.
). The resulting bands were sequenced and their sequences were found to be identical
to that of HMGA2.
Immunohistochemical analysis
Immunohistochemical examinations were performed by the avidin–biotin complex immunoperoxidase
technique using an Avidin-Biotinylated Enzyme Complex kit (Vector Laboratories, Inc.
CA, USA) as described previously (Abe et al, 1999, 2000, 2002). The HMGA2 protein
expression was immunohistochemically analysed on surgically resected specimens, together
with four pancreatic cancer cell lines (PANC-I, MIA PaCa-2, BxPC-3, and AsPc-1) using
HMGA2-specific antibodies, raised in rabbit against the recombinant HMGA2 protein
(Berlingieri et al, 1995). In brief, frozen sections 5 (5 m thick) were prepared,
transferred onto poly-L-lysine-coated slides, air-dried, and then washed in PBS, followed
by quenching of endogeneous peroxidase activity with 0.3% hydrogen peroxide in methanol.
After further rinsing with PBS, the sections were incubated with normal goat serum
for 20 min at room temperature to block nonspecific binding, and then incubated with
the primary anti-HMGA2 antibody (1 : 100 dilution) 14 h at 4°C. After another wash
in 0.2% Triton X in PBS, the sections were further incubated with biotinylated anti-rabbit
IgG for 30 min at room temperature, followed by washes in 0.2% Triton X in PBS. After
the addition of streptavidin–biotin-conjugated peroxidase and incubation for 30 min
at room temperature, the sections were washed in 0.2% Triton X in PBS, and then the
localisation of the HMGA2 protein was visualised by incubating the sections with 3,3′-diaminobenzidine.
The slides were counterstained with Mayer's haematoxylin, dehydrated in a graded alcohol
series, cleared in xylene, and mounted. Negative control staining was carried out
by replacing the primary antibody with normal rabbit serum under the same experimental
conditions. The immunostained slides were evaluated microscopically by a single investigator
(NA) according to the criteria published previously (Abe et al, 1999, 2000) without
prior knowledge of the clinical data for each case. The percentage of HMGA2-positive
cells was scored by counting approximately 300–1000 tumour cells in three randomly
selected fields (Abe et al, 1999, 2000, 2002). The immunohistochemical evaluation
was considered positive when the HMGA2 nuclear immunoreactivity was detected in more
than 20% of the cells according to the criteria published previously (Abe et al, 1999,
2000).
RESULTS
Expression of HMGA2 mRNA determined by RT–PCR
Among the six non-neoplastic tissue samples, five, including three normal tissues
and two chronic pancreatitis tissues, gave rise to detectable HMGA2 bands, while one
normal tissue sample showed no detectable HMGA2 band. The signal intensities of the
HMGA2 band in chronic pancreatitis tissue samples were almost equivalent to those
observed in the normal tissue samples. All the 17 samples of pancreatic carcinomas
also showed HMGA2 bands by RT–PCR (Figure 1, Table 1
Table 1
HMGA2 expression in pancreatic carcinoma
Histological type of pancreatic specimens
No. of positive specimens/no. of specimens analysed by RT–PCR
No. of positivea specimens/no. of specimens analysed by immunohistochemistry
Non-neoplastic tissue
Normal pancreas
3/4 (75%)
0/6 (0%)
Chronic pancreatitis
2/2 (100%)
0/2 (0%)
Duct cell carcinoma
17/17 (100%)
27/27 (100%)
a
The immunostained slides were scored as positive for immunohistochemistry when HMGA2
nuclear immunoreactivity was detected in more than 20% of the cells in the exocrine
region.
). When the signal intensities of these HMGA2 bands were compared between non-neoplastic
and carcinoma samples, the latter showed at least several fold more intense band than
the former (Figure 1). Thus, an increased expression level of the HMGA2 mRNA is a
distinct feature of pancreatic carcinoma.
Expression of HMGA2 protein determined by immunohistochemistry
To determine whether the altered HMGA2 mRNA expression observed in pancreatic carcinoma
is associated with alterations in protein expression, we analysed the expression of
the HMGA2 protein by immunohistochemistry. Its expression was first analysed in four
pancreatic cancer cell lines. Intense multifocal or diffuse HMGA2 nuclear immunoreactivity
was characteristically observed in these cell lines (Figures 2A–D
Figure 2
Immunohistochemical demonstration of the HMGA2 protein expression in pancreatic cancer
cell lines. (A) AsPC-1 (Mayer's haematoxylin; original magnification × 200). (B) PANC-I
(Mayer's haematoxylin; original magnification × 200). (C) MIA PaCa-2 (Mayer's haematoxylin;
original magnification × 100). (D) BxPC-3 (Mayer's haematoxylin; original magnification
× 200). Intense multifocal or diffuse HMGA2 nuclear immunoreactivity (brown colour)
was characteristically observed in cancer cells.
). In both normal pancreas and chronic pancreatitis tissues, acinar cells did not
exhibit any detectable HMGA2 immunoreactivity; however, a small proportion of duct
epithelial cells showed faint HMGA2 immunoreactivity (Figure 3A
Figure 3
Immunohistochemical demonstration of the HMGA2 protein expression in surgically resected
specimens of non-neoplastic pancreatic tissues and pancreatic carcinomas. (A) Non-neoplastic
epithelial cells of the main pancreatic duct. A small proportion of duct epithelial
cells show HMGA2 immunoreactivity (arrows). (Mayer's haematoxylin; original magnification
× 200). (B) Epithelial cells of branch pancreatic duct and islets in chronic pancreatitis
tissue. Islet cells showed intense and diffuse HMGA2 immunoreactivity (arrows), while
epithelial cells of the branch pancreatic duct did not exhibit any detectable HMGA2
immunoreactivity (arrowhead) (Mayer's haematoxylin; original magnification × 100).
(C) Primary pancreatic carcinoma exhibiting well-differentiated tubular adenocarcinoma
(Mayer's haematoxylin; original magnification × 200). (D) Primary pancreatic carcinoma
exhibiting adenosquamous carcinoma (Mayer's haematoxylin; original magnification ×
200). (E) Metastatic lesion in the liver (Mayer's haematoxylin; original magnification
× 200). Intense and multifocal or diffuse HMGA2 immunoreactivity was noted in all
the pancreatic carcinomas (C–E). (F) Section including both carcinoma cells and islet
cells (Mayer's haematoxylin; original magnification × 200). Islet cells showed intense
and diffuse HMGA2 immunoreactivity (arrows), which was almost equivalent to that observed
in carcinoma cells (arrowheads).
). On the other hand, in the endocrine region, islet cells showed intense and diffuse
HMGA2 immunoreactivity (Figures 3B and F). In these cells, although HMGA2 immunoreactivity
was localised mainly in the nuclei, faint staining was also observed within the cytoplasm.
These results clearly indicated that the presence of the HMGA2 gene in non-neoplastic
pancreatic tissue observed in the RT–PCR analysis reflects its expression in islet
cells, together with its focal expression in duct epithelial cells. No significant
difference in immunohistochemical findings was found between normal tissues and chronic
pancreatitis tissues. When the expression of the HMGA2 protein in surgical specimens
of carcinomas was then analysed, multifocally or diffusely distributed intense HMGA2
immunoreactivity was noted in all the pancreatic carcinoma specimens examined (Figures
3C–F). Intense nuclear staining was characteristically observed in the carcinoma cells.
High-mobility group A2-positive carcinoma cells were observed regardless of the degree
of differentiation (well/moderately or poorly differentiated tubular adenocarcinoma),
histology type (tubular adenocarcinoma or adenosquamous carcinoma), or tumour site
(primary or metastatic site). A strong correlation between HMGA2 overexpression and
the diagnosis of carcinoma was noted (Fisher's exact probability, P<0.0001, Table
1).
DISCUSSION
To evaluate the association between HMGA2 expression and the pathological diagnosis
of pancreatic carcinoma, we investigated the expression of HMGA2 gene/protein in duct
cell carcinoma and non-neoplastic tissue of the pancreas. High-mobility group A2 expression
has been shown to be undetectable or to remain at low levels in normal adult tissues
(Manfioletti et al, 1991; Zhou et al, 1995; Rogalla et al, 1996; Rommel et al, 1997;
Hirning-Folz et al, 1998; Gattas et al, 1999). In the present study, however, a highly
sensitive RT–PCR analysis revealed the expression of the HMGA2 gene in non-neoplastic
pancreatic tissue, although its expression level was significantly lower than that
in carcinoma. Immunohistochemical analysis indicated that the presence of the HMGA2
gene in non-neoplastic pancreatic tissue observed in the RT–PCR analysis reflects
its abundant expression in islet cells together with its focal expression in duct
epithelial cells. Thus, this study showed that the HMGA2 gene or protein is present
even in normal pancreatic tissue. In HMGA2 immunohistochemical analysis, while only
a small proportion of duct epithelial cells in the non-neoplastic tissue specimens
showed HMGA2 immunoreactivity, a significantly higher proportion of carcinoma cells
showed intense staining. In fact, a strong correlation between HMGA2 overexpression
and the diagnosis of carcinoma was statistically verified. These findings indicate
that an increased expression level of the HMGA2 protein is closely associated with
the malignant phenotype in the pancreatic exocrine system, and accordingly, HMGA2
could serve as a potential diagnostic molecular marker for distinguishing pancreatic
malignant cells from non-neoplastic pancreatic exocrine cells. A possible application
of the results of the present study would be the determination of the HMGA2 gene and/or
protein expression level in pancreatic juice collected at the time of endoscopic retrograde
pancreatography. Using a sensitive and quantitative method such as competitive RT–PCR
or immunoassay, the detection of even a small number of cancer cells could well be
expected.
In order to evaluate the biological significance of the present results, it would
be essential to understand the mechanisms by which the HMGA2 gene is involved in tumorigenesis,
which unfortunately remain largely unclear. A clue to this issue was, however, provided
by a recent report that transgenic mice carrying the HMGA2 gene developed pituitary
adenomas (Fedele et al, 2002). These findings indicate that the high HMGA2 expression
level has a critical role in neoplastic transformation of cells. Another clue was
also demonstrated when antisense HMGA2 RNA was shown to prevent retrovirally induced
neoplastic transformation of rat thyroid cells in vitro (Berlingieri et al, 1995).
The interaction between HMGA2 and the AP-1 transcriptional complex is considered to
be responsible for the activation of genes whose expressions are associated with carcinogenesis
(Vallone et al, 1997), since thyroid neoplastic transformation is associated with
a drastic increase in AP-1 activity. This AP-1 activity is blocked by suppressing
HMGA protein synthesis in vitro (Battista et al, 1998). The absence or decreased AP-1
transcriptional activity, which is directly or indirectly regulated by HMGA proteins,
would inhibit the expression of AP-1-dependent genes, such as those of vascular endothelial
growth factor (VEGF), collagenase I (matrix metalloproteinase-1; MMP-1), and stromelisin
(MMP-3), which are essential for neoplastic transformation of cells (Vallone et al,
1997). In fact, significant downregulations of these mRNA expression levels were demonstrated
in the retrovirally infected thyroid cell lines expressing the antisense HMGA2 (Vallone
et al, 1997). Considering that the overexpression of the AP-1 (Tessari et al, 1999;
Meggiato et al, 2003), VEGF (Seo et al, 2000), MMP-1 (Ito et al, 1999), and MMP-3
(Bramhall et al, 1996) has been demonstrated in human pancreatic cancer, together
with our results, the interactions among these molecules may play an important role
in pancreatic neoplastic transformation in vivo, Further studies, including the determination
of expression levels of these molecules in tissue samples, have yet to be carried
out to further clarify this issue. Conversely, the HMGA2 gene has recently been shown
not to be necessary for the malignant transformation of thyroid cells in vivo (Scala
et al, 2001). This was demonstrated by comparing the frequency of radiation or papilloma
virus E7 gene-induced thyroid carcinomas in mice carrying disrupted HMGA2 (pygmy mice)
and that in mice carrying wild-type HMGA2 (Scala et al, 2001). Pygmy mice developed
thyroid carcinomas with the same frequency as wild-type mice and furthermore, these
two carcinomas generated in different mice showed no significant macroscopic and microscopic
differences, indicating that HMGA2 is not sufficient for in vivo malignant transformation
of thyroid cells (Scala et al, 2001). Several hypotheses could be considered to explain
the discrepancy with the previous in vitro data, showing that HMGA2 is required for
v-mos- and v-ras-Ki-induced cell transformations. One possible explanation would be
that HMGA1, rather than HMGA2, may be required for thyroid cell transformation. This
hypothesis is supported by the evidence that adenovirus carrying the HMGA1 gene in
an antisense orientation induces programmed cell death in carcinoma cell lines derived
from human thyroid, lung, colon, and breast cancers (Scala et al, 2000). We previously
demonstrated that human pancreatic carcinoma expresses high HMGA1 levels (Abe et al,
2000, 2002), indicating that both HMGA2 and HMGA1 are overexpressed in this lesion.
The expression of only one of the HMGA genes may be sufficient to lead epithelial
cells of the pancreatic duct to exhibit the malignant phenotype. Further studies,
such as the generation of HMGA1-knockout mice and subsequent analysis of their susceptibility
to developing malignancies, need to be carried out in order to clarify the role of
a single HMGA gene in carcinogenesis in a wide variety of epithelial tissues.
In conclusion, this study has clearly demonstrated that an increased expression level
of the HMGA2 gene/protein is closely associated with the malignant phenotype in pancreatic
exocrine tissue, suggesting that HMGA2 could play a vital role in tumorigenesis in
the pancreatic exocrine system. The strong correlation between HMGA2 overexpression
and the histological diagnosis of carcinoma indicates that the determination of the
expression level of HMGA2 can be of great value in the diagnosis of pancreatic neoplasms.