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Published online before print March 18, 2008
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From the Division of Gastroenterology* and the Department of Gastroenterological Surgery, Tohoku University Graduate School of Medicine, Sendai City, Miyagi, Japan; Advanced Industrial Science and Technology, Koto-ku, Tokyo, Japan; and the Mammary Biology and Tumorigenesis Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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On the other hand, enhanced levels of transcripts for MSX2, the human homologue of Msx2 (HOX-8), have been shown in a variety of carcinoma cell lines of epithelial origin compared to their corresponding normal tissues. However, this enhanced expression is not found in hematopoietic tumor cells, suggesting that MSX2 plays a more important role in tumors of epithelial origin than in those of hematopoietic origin.10 Expression of endogenous MSX2 also is up-regulated in v-Ki-ras-transfected NIH3T3 cells.11 Although MSX2 itself failed to confer a transformed phenotype, antisense MSX2 cDNA as well as truncated MSX2 cDNA interfered with the transforming activities of both the v-K-ras and v-raf oncogene.11 These findings indicated that MSX2 might be an important downstream target for the Ras signaling pathway. In addition, MSX2 activates cyclin D1 expression and inhibits cellular differentiation as shown in the mouse myogenic cell line C2C12,12 suggesting a relation of MSX2 with tumorigenesis since cyclin D1 overexpression is found in various carcinomas such as breast13,14 and pancreatic cancer.15
Pancreatic cancer is one of the most malignant gastrointestinal tumors. Once pancreatic cancer is clinically evident, it progresses rapidly to develop metastatic lesions, frequently by the time of diagnosis. Furthermore, these tumors are usually resistant to conventional chemotherapy and radiation therapy. The pathogenic mechanisms that regulate the aggressive behavior of this cancer still remain to be clarified. Although most pancreatic cancers (more than 90%) contain a K-ras gene mutation at codon 12,16,17 little is known about the expression or function of MSX2 as a candidate downstream gene of K-ras in pancreatic cancer. Therefore, we tested whether the presence of MSX2 would correlate with the malignant behavior of pancreatic cancer cells. Here we clearly show that MSX2-transfected pancreatic cancer cells demonstrate an enhanced malignant phenotype in vitro and in vivo, and that intense expression of this gene is frequently found in human pancreatic cancer tissues.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Four pancreatic cancer cell lines (AsPC-1, BxPC3, Panc-1, and MIAPaca2) were purchased from American Type Culture Collection (Manassas, VA), routinely grown in modified Eagle’s medium (Invitrogen, Grand Island, NY) containing 10% fetal bovine serum (Miles, Kankakee, IL) were maintained at 37°C in 5% CO2 in a humidified environment.
Human pancreatic stellate cells were isolated from the surgically resected normal pancreas tissues of patients with pancreatic cancer, under the approval by the Ethics Committee of Tohoku University School of Medicine. The cells were maintained in Ham’s F-12/Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal bovine serum (ICN Biomedicals, Aurora, OH), penicillin sodium, and streptomycin sulfate. Human umbilical vein endothelial cells and their optimized culture medium were purchased from Clonetics (San Diego, CA). Human umbilical vein endothelial cells were grown on 0.2% gelatin-coated tissue culture dishes (Corning, Corning, NY).
For cell RNA, total RNA was prepared using the RNeasy kit (QIAGEN, Hilden, Germany) with DNase treatment to eliminate DNA contamination according to the protocol provided by the manufacturer. First-strand cDNA was generated from 1 µg of total RNA using RETROscript (Ambion, Austin, TX) in a total volume of 20 µl according to the manufacturer’s protocol. PCR was performed on 2 µl of RT product in a 25 µl of reaction mixture using Ex Taq polymerase (Takara, Ohtsu, Japan) with 3' and 5' primer concentration of 10 µmol/L each. Gene expression was normalized to respective glyceraldehyde-3-phosphate dehydrogenase (GAPDH) level. The PCR conditions for our cDNA templates were optimized to ensure replication is in the linear phase for each primer set used. To quantify the gene expression level, we also exploited quantitative real-time RT-PCR using LightCycler and LightCycler–FastStart DNA Master SYBR Green I (Roche Diagnostics, Basel, Switzerland). All reactions were performed according to the manufacturer’s protocol. The annealing temperature for these primer sets was 60°C. The specificity of each PCR reaction was confirmed by melting curve analyses. The level of target gene expression in each sample was normalized to the respective GAPDH expression level. Each experiment was repeated at least three times, and representative data are shown. The primer pairs used were: MSX2, forward 5'-GGAGCGGCGTGGATGCAGGAA-3' and reverse 5'-AAGCACAGGTCTATGGAACGG-3', which span the approximately 3.5 kbp intron18,19 ; GAPDH, forward 5'-GGGAAGGTGAAGGTCGGAG-3' and reverse 5'-GAGGGGGCAGAGATGATGA-3'; and Twist 1, forward 5'-CACTGAAAGGAAAGGCATCA-3' and reverse 5'-GGCCAGTTTGATCCCAGTAT-3'.20
Generation of MSX2 Overexpressing Pancreatic Cancer Cell Lines
The PCR-amplified coding region of human MSX2 (808 bp, containing amino acids 1-267) using full-length human cDNA (provided by Dr. Takahashi, Kyoto University, Japan) as a template was subcloned into the pCDNA3.1 v5 vector (Invitrogen Life Technologies, Carlsbad, CA) in the sense orientation. Transfection of cells with expression vectors (MSX2 cDNA or vector alone) was performed using FuGENE 6 (Roche, Indianapolis, IN) as recommended by the supplier and cell lines were selected with 800 µg/ml G418 (Invitrogen). After G418 selection, clones were subjected to Western blot analyses with a specific antibody against v5 (Invitrogen) to confirm MSX2 expression. After establishment of empty vector (EV) or MSX2-transfected clonal cell lines, the same passages were used for each experiment.
RNA Interference
The small interfering RNA for MSX2 (MSX2 siRNA) expressing vector was generated by cloning the following annealed and BamHI and HindIII digested oligonucleotides into pBAsi-U6 Neo DNA vector (Takara Bio Inc., Ohtu, Japan): 5'-GATCCACACAAGACCAATCGGAAGTTCAAGAGACTTCCGATTGGTCTTGTGTTTTTTTA-3' and 5'-AGCTTAAAAAAACACAAGACCAATCGGAAGTCTCTTGAACTTCCGATTGGTCTTGTGTG-3'. This generates siRNA directed against the sequence 5'-ACACAAGACCAATCGGAAG-3', corresponding to nucleotide human MSX2 at 409 to 428 (NCBI access number NM_002449) under the control of the human U6 promoter. The MSX2si expression vector or empty vector was transfected into Panc-1 cells using FuGENE 6 (Roche) as recommended by the supplier. After G418 selection, clones were subjected to RT-PCR to confirm MSX2 expression.
Western Blot Analysis
For whole-cell protein extraction, cells were lysed by the addition of lysis buffer (50 mmol/L Tris-Hcl, pH 7.4, 1% Nonidet P40, 0.5% sodium deoxycholate). Nuclear protein was extracted with nuclear and cytoplasmic extraction reagents (Pierce Biotechnology Inc., Rockford, IL) according to the manufacturer’s recommendations. Cytosolic and membrane protein were extracted using a cell compartment kit (Qiagen) according to the manufacturer’s protocol. Protein concentration in each sample was determined using the Bradford assay kit (Dojin, Kumamoto, Japan). After addition of 5X sample buffer (1 mol/L Tris-HCl, pH 6.8, sodium dodecyl sulfate, glycerol, and bromphenol blue) the aliquots were boiled for 5 minutes and subjected to 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After blocking for 1 hour at room temperature in a buffer containing 10 mmol/L Tris-HCl (pH 7.5), 100 mmol/L NaCl, 0.1% Tween 20, and 5% dry milk, nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA) were incubated either with monoclonal mouse v5 antibody (Invitrogen), monoclonal mouse monoclonal mouse β-catenin antibody (BD Transduction Laboratories, Lexington, KY), polyclonal rabbit Twist 1 antibody (Santa Cruz Biotechnology, Inc. Santa Cruz, CA), polyclonal lamin B1 antibody (Santa Cruz Biotechnology, Inc.), polyclonal GAPDH antibody (Trevigen, Gaithersburg, MD), polyclonal TIMM23 antibody (ProteinTech Group, Inc, Chicago, IL), or monoclonal mouse 
-tubulin (Santa Cruz Biotechnology) antibody overnight at 4°C. The membranes were then washed with a buffer containing 10 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, 0.1% Tween 20 and incubated with anti-goat (Zymed Laboratories, South San Francisco, CA), anti-rabbit, or mouse-IgG coupled to peroxidase (Amersham Biosciences, Buckinghamshire, UK) for 1 hour at room temperature. Reactive bands were detected using ECL chemiluminescence reagent (Amersham Biosciences). The obtained bands were subjected to densitometry analysis by using Scion Image Software (Scion Corporation, Frederick, MD).
Fluorescence Immunohistochemistry
MSX2- or EV-transfected BxPC3 cells and EV- or MSX2si-transfected Panc-1 cells were grown to subconfluence on BD Falcon culture slides (BD Biosciences, San Jose, CA) and fixed with ice-cold methanol (Wako, Osaka, Japan). After blocking with normal goat serum, cells were incubated with mouse monoclonal E-cadherin antibody (Santa Cruz Biotechnology) or monoclonal mouse β-catenin antibody (BD Transduction Laboratories) overnight at 4°C, and then slides were incubated with fluorescein-conjugated goat anti- mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, CA). Cells were then incubated with propidium iodide (Wako) for nuclear staining and mounted with Vectashield (Vector Laboratories, Inc.). Cells were then incubated with propidium iodide (Wako) for nuclear staining and mounted with Vectashield (Vector Laboratories, Inc.). For double staining for MSX2 and Twist-1, cells were fixed with ice-cold methanol. After blocking with 3% bovine serum albumin in phosphate-buffered saline, cells were incubated with goat polyclonal anti-MSX2 antibody (Santa Cruz Biotechnology, Inc.) and rabbit polyclonal Twist-1 antibody (Santa Cruz Biotechnology, Inc.) overnight at 4°C, and then slides were incubated with Alexa Fluor 546 donkey anti-goat IgG (Molecular Probes, Eugene, OR) and Alexa Fluor 488 donkey anti-rabbit IgG (Molecular Probes), respectively, and mounted with Vectashield (Vector Laboratories, Inc). Cells were visualized for immunofluorescence with a confocal TIRF-C1 microscope (Nikon Instech Co., Ltd, Kawasaki, Japan).
Cell Growth Assays
For the cell growth assay, 6000 MSX2-transfected cells, MSX2 antisense-transfected cells, or EV cells were seeded per well in 96-well plates (Corning Incorporated, Corning, NY) in normal cell growth media. The 5-bromo-2-deoxyuridine assay was performed after 24 hours and 72 hours of incubation using a kit (Roche) according to the manufacturer’s protocol. For each cell line the proliferation index was evaluated and the absorbance at 72 hours normalized to that at 24 hours.
Soft Agar Assay
For soft agar assay, 4 x 104 transfected BXPC3 cells were suspended in 0.3% Bacto agar (BD Falcon) supplemented with Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and layered over 1 ml of an 0.8% agar medium base layer in six-well plates. After 21 days, the cells were stained with nitroblue tetrazolium (Roche), and anchorage-independent growth was estimated by counting the number of colonies using a microscope in high-power view.
Scrape Motility Assay
Pancreatic cancer cells were grown to confluence in 24-well culture dishes (BD Falcon) with normal growth media. The cell monolayer was mechanically scarred with a sterile pipette tip, and the plates were incubated with serum-free Dulbecco’s modified Eagle’s medium for an additional 2 to 4 days. Cells were visualized with an Olympus model CK2 inverted microscope using a 10X objective. Images were captured in a time-lapse manner with an Olympus C2000 digital camera. The scratched area covered by migrated cells was measured in three independent wells and normalized to initial scratched area using Scion Image Software (Scion Corporation).
Two-Chamber Migration Assays
Cell invasion was also determined by using a modified two-chamber migration assay (8-mm pore size, BD Biosciences) according to the manufacturer’s instructions. A total of 1 x 104 cells were seeded in serum-free medium in the upper chamber, and migration during 24 hours toward the lower chamber that contained 10% fetal bovine serum as a chemoattractant was evaluated. Cells in the upper chamber were carefully removed using a cotton bud, and cells at the bottom of the membrane were fixed and stained with Diff-Quick (International Reagents Corp., Kobe, Japan). Quantification was performed by directly counting in random 5 high-power fields after 24 hours of incubation.
Tumor Growth in Nude Mice
Tumor formation in vivo was assayed in female athymic nude mice by subcutaneously injecting each of 2 x 106 cells suspended in 200 µl of sterile phosphate-buffered saline. Tumor volume was measured every week after the first incidence of tumor formation. Volume was determined by the equation V = L x W2 x 0.5, where V is volume, L is length, and W is width. The mice were sacrificed 7 weeks after injection and confirmed the histology confirmed by hematoxylin and eosin staining.
Orthotopic Implantation
To assess metastasis formation, MSX2-, MSX2si-, and empty vector-transfected pancreatic cancer cells (1.5 x 106 cells suspended in 50 µl) were injected into the pancreatic tails of female athymic nude mice. The mice were sacrificed 7 weeks after injection and tumor progression was confirmed. Histology was evaluated by hematoxylin and eosin staining.
Microarray
CodeLink Whole Human Genome Expression Bioarray (Amersham Biosciences), representing approximately 55,000 of the most well annotated human genes published in public databases, was used for cDNA microarray analysis. The platform employs single-color detection rather than a dual-color detection system, where multiple experiment comparisons are possible without replicating the reference sample. Procedures were performed according to the manufacturer’s protocol, and all reagents were provided in the CodeLink Expression Assay Kit (Amersham Biosciences). In brief, 10-µg aliquots of total RNA was fragmented at 94°C for 20 minutes in the presence of magnesium. The fragmented RNA was hybridized to Uniset Human Whole Genome Expression Bioarray slides in hybridization buffer at 37°C for 24 hours in an INNOVA 4080 shaking incubator (New Brunswick Scientific, Edison, NJ) at 300 rpm. After hybridization, the arrays were washed in 0.75X TNT buffer [1X TNT: 0.1 mol/L Tris-HCl (pH 7.6), 0.15 mol/L NaCl, and 0.05% Tween 20] at 46°C for 1 hour followed by incubation with Cy5-streptavidin at room temperature for 30 minutes in the dark. Arrays were then washed in 1X TNT four times for 5 minutes each followed by a rinse in 0.1X standard saline citrate/0.05% Tween 20 in water. The slides were then dried by centrifugation and kept in the dark until scanning.
Gene Expression Data Analysis
Array slides were scanned using an Array WoRx (GE Healthcare Bio-Sciences Corp., Piscataway, NJ), and expression values were measured and manipulated subsequently by CodeLink Expression Analysis version 4.0 software (Amersham Biosciences). Each array contains a total of 55,776 spots, of which 54,840 are for human (nonbacterial) genes. Among the 54,840 gene expression values, low-intensity spots whose values were less than the detection threshold were all adjusted to the threshold. All good quality spots indicated by "G" flag as well as the low-intensity spots indicated by "L" were further processed for the statistical analysis. Those spots with poor quality were excluded from the analysis. Statistically, 54,530 spots were detected as either "G" or "L" flag in both experiments (B3-EV versus B7), thus most of genes are subsequently used in the following analysis.
To normalize data we compared the gene expression values from the two experiments and drew an intensity-ratio plot (MA plot). For each data point, an intensity (A) dependent normalization method (LOWESS)21 is applied for adjusting the ratio (M) value. In LOWESS, we used the simple adjusting method that is log2M – c(A), where c(A) is the mean ratio of the nearest 10,000 data points surrounding the current data point. After the calibration of M values, P values for evaluating how significantly those M values were apart from the mean were obtained under a cumulative normal distribution model whose variance is calculated with the same 10,000 data points. The array data were deposited in the National Center for Biotechnology Information Gene Expression Omnibus database (GSE6585).
Tissues, Immunohistochemistry, and Fluorescence Immunohistochemistry
Pancreatic cancer tissues were obtained from patients who underwent surgical operations for the tumors. The tissues collected at the time of surgery were immediately embedded in Tissue-Tek O.C.T. compound medium (Sakura, Tokyo, Japan), frozen in liquid nitrogen, and stored at –80°C or fixed in 10% paraformaldehyde overnight and embedded in paraffin wax. Thirty two pancreatic cancer tissues were used for the immunohistochemistry. The grade of differentiation and the stage of pancreatic cancer were determined according to methods described previously.22,23 Informed consent was obtained from all patients before surgery.
Localization of MSX2 and Twist 1 in human pancreatic tissues was investigated by immunohistochemistry. The tissue sections were deparaffinized and antigens were retrieved by boiling the sections in Target Retrieval Solution (Dako, Carpinteria, CA) in the microwave oven. Then the sections were incubated in methanol with 0.3% hydrogen peroxide for 30 minutes to block the endogenous peroxidase activity. Thereafter, the Histofine kit (Nichirei, Tokyo, Japan) for MSX2 (Santa Cruz Biotechnology, Inc.) or the Santa Cruz staining kit for Twist 1 (Santa Cruz Biotechnology, Inc.) was used. Visualization of the immunoreaction was performed in 0.06 mmol/L 3,3'-diaminobenzidine tetrahydrochloride (Dojin, Kumamoto, Japan) containing 2 mmol/L hydrogen peroxide in phosphate-buffered saline for several minutes at room temperature. For the negative control, the immunostaining processes were performed by replacing the primary antibody with phosphate-buffered saline. The negative control sections showed no specific immunoreactivity. In addition, the specificity of antibody was determined in an absorption test using an excess amount of blocking peptide for MSX2 antibody (sc-17729 P, Santa Cruz Biotechnology, Inc.). Fluorescence immunohistochemistry was performed described above using 8-µm sections from the frozen tissues.
The degree of immunostaining for MSX2 was evaluated as follows: negative, less than 5% positive cells found; weak, 5 to 30% positive cells observed; moderate, 30 to 75% positive cells observed; intense, more than 75% immunoreactive cells observed in most areas of the tissue sections. The immunostaining for Twist 1 was judged positive when more than 10% of positive nuclear cells was observed. The evaluation of immunostaining was done independently by two observers (K.S. and A.K.) who had not been informed of the histological diagnosis.
Statistical Analysis
The computer software StatView for Macintosh (Abacus Concepts, Berkeley, CA) was used for all statistical analyses. The correlation of MSX2 expression with the patient’s clinicopathological variables and the correlation between orthotopic injected mice and metastasis or dissemination were analyzed by the 
2
test. The differences among the cells for proliferation and anchorage-independent growth were statistically analyzed by analysis of variance. The difference between two groups was statistically analyzed by unpaired t-test or Mann-Whitney U-test. A P value of <0.05 was regarded as statistically significant.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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First, we examined MSX2 expression in pancreatic cancer cell lines and compared their expression level to pancreatic stellate cells or human umbilical vein endothelial cells. The quantitative real-time RT-PCR showed a difference in the MSX2 expression level of each cell line and revealed higher expression in carcinoma cells than in normal cultured cells (pancreatic stellate cells or human umbilical vein endothelial cells (Table 1)
. MSX2 expression was intense in Panc-1 and ASPC-1 cells, weak in MIAPaCa2, and very faint in BxPC-3 cells. Interestingly, the expression level of MSX2 was higher in K-ras gene-activated cell lines than in wild-type cell line BxPC324
(Table 1)
. The expression level of MSX2 in BxPC3 cells was similar to that of normal cultured cells, suggesting that this did not function as a carcinoma-related gene in this cell line. Therefore, we chose BxPC3 cells to confirm the effect of MSX2 in the gain-of-function manner.
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The MSX2- or MSX2si-transfected cells were cloned and subjected to Western blot analysis or quantitative real-time RT-PCR to confirm the expression of v5-tagged MSX2 protein or MSX2 RNA, respectively. We generated several clones of BxPC3 stably overexpressing MSX2 and Panc-1 stably expressing MSX2si; representative clones are shown in Figure 1
. A significant morphological difference was observed between MSX2-transfected cells (B21 and B7) and control cells (parental BxPC3 and B3-EV). As shown in Figure 1B
, B21 and B7 cells showed loose cell junctions and scattered morphology relative to control cells. The MSX2-expressing cell lines, B21 and B7, demonstrated a more fibroblast-like appearance compared to parental and B-3EV cells. This alteration of morphology resembled a mesenchymal phenotype rather than the usual phenotype of BxPC3, indicating that the cells were undergoing epithelial to mesenchymal transition (EMT) by MSX2 overexpression. A similar morphological change was observed between MSX2-expressing and down-regulated Panc-1 cells. As shown in Figure 1D
, MSX2-expressing parental Panc-1 and EV cells showed loose cell junctions and scattered morphology, whereas the MSX2 down-regulated cell lines, si201 and si215, demonstrated a cobblestone-like phenotype.
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, BxPC3 cells transfected with MSX2 exhibited weakly diffuse distribution of E-cadherin and β-catenin in the cytoplasm, whereas control cells (B3-EV) showed dominant membrane-bound staining. Consistently, Western blotting showed that the expression of E-cadherin was decreased and that nuclear and cytosolic expression of β-catenin was increased, whereas membranous β-catenin expression was reduced in MSX2-expressing cells (Figure 2, C and D)
. These molecular changes in MSX2-expressing cells are consistent with EMT. On the other hand, fluorescence immunostaining and Western blotting demonstrated that the membranous expression of E-cadherin and β-catenin was increased in MSX2si cells, whereas cytoplasmic or nuclear expression of these proteins was up-regulated in control cells (Figure 2, B–D)
.
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To assess the effects of MSX2 on pancreatic cancer cell proliferation, a 5-bromo-2-deoxyuridine assay was used. B21 and B7 cells showed significant induction of proliferation after 72 hours of culture with normal medium compared to control cells (BxPC3 versus B7, P = 0.032; BxPC3 versus B21, P = 0.032; EV versus B7, P = 0.003; EV versus B21, P = 0.006) (Figure 3A)
. To elucidate the functions of MSX2 in anchorage-independent growth of pancreatic cancer cells, we used the soft agar assay. BXPC-3 cells overexpressing MSX2 showed a large number of colonies on soft agar, whereas parental and B-3EV cells showed very few colonies after 3 weeks of culture on soft agar (Figure 3B
and Supplementary Figure S1A at http://ajp.amjpathol.org). Fourfold and sixfold more colonies were seen with B21 and B7 cells, respectively, compared to control cells (P < 0.05).
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We next examined the cell migration ability of MSX2-expressing and down-regulated pancreatic cancer cells by a wound-healing scratch assay and two-chamber migration assay. Because serum has been shown to activate mitogen-activated protein kinase,25
the wound healing scratch assay was performed under serum-starved conditions. As shown in Supplementary Figure S1B (http://ajp.amjpathol.org) and Figure 3C
, MSX2-expressing pancreatic cancer cells covered the scratched area with migrating cells in serum-starved conditions but B-3EV cells did not. The bar graph clearly shows that the number of migrated cells increased in a MSX2-dependent manner, indicating that MSX2 promoted cell migration (Figure 3C)
. In contrast, fewer MSX2 down-regulated cells (si201 and si215) migrated into the cell-free zone compared to the vector control cells (EV) (Figure 3C
and Supplementary Figure S1C at http://ajp.amjpathol.org), indicating that down-regulation of MSX2 is associated with suppression of cell migration. To exclude the effect of proliferation to covered area in the scratch assay, we further examined the cell migration by the Transwell assay. As shown in Figure 3D
, MSX2-expressing cells (B21 and B7 in upper panel, and EV and Panc-1 in lower panel) showed the large number of cells compared to MSX2 down-regulated (si201 and si215 in lower panel) or lower-expressing cells (EV and BxPC3 in upper panel).
MSX2 Promoted Cell Growth in Nude Mice
To determine whether MSX2 cells also promote tumor growth in vivo, 2 x 106 of EV cells or MSX2-expressing cells (B7) were injected subcutaneously into the dorsal flanks of nude mice. One week after injection, tumors began to appear in all B7-injected and some of the EV-injected nude mice. MSX2 derived tumors showed significantly faster growth and formed large tumors relative to those arising from EV cells (Figure 4A
and Supplementary Figure S2A at http://ajp.amjpathol.org).
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To assess whether MSX2 expression also promotes cell migration or metastasis formation in an orthotopic environment, 1.5 x 106 EV cells, MSX2-expressing cells (B7), or MSX2si cells (si215) were injected into the pancreas of nude mice. Tumors were observed in the pancreas of mice implanted with all MSX2-expressing or MSX2si cells and control cells. MSX2-expressing cells frequently showed metastases to the liver (3/5, P < 0.05) and peritoneal dissemination (5/5, P < 0.01) while control cells demonstrated no liver metastasis or only one peritoneal invasion (Table 2
and Supplementary Figure S2, B–D, at http://ajp.amjpathol.org). In addition, histological examination revealed that MSX2 cells exhibited increased intercellular separation and loss of cell polarity (Figure 4B, d, f, and g)
compared to those produced by EV cells (Figure 4B, b and e)
. On the other hand, the metastasis to the liver (0/5) and peritoneal dissemination were suppressed (0/5) in mice injected with MSX2 down-regulated cells, whereas mice implanted with control cells showed frequent liver metastasis (4/5, P < 0.005) and peritoneal dissemination (3/5, P < 0.05) (Table 2
and Supplementary Figure S2, E and F, at http://ajp.amjpathol.org). Tumors formed by control cells showed poorly differentiated fibroblast-like cells by histological examination (Figure 4C
, left panel), while MSX2-inactivated cells formed mixed phenotype tumors where tubular type carcinoma cells were occasionally found (Figure 4C
, right panel), indicating that MSX2 inactivation resulted in a reversal of the state of EMT.
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To better understand the mechanisms underlying the effect of MSX2 in pancreatic carcinoma cells, we searched for differentially expressed genes in EV and B7 cells by cDNA microarray analysis. Among the genes significantly up-regulated by MSX2, we found Twist 1 as one of the most strongly induced genes in B7 compared to EV cells (Figure 5A)
. To confirm the result from the microarrays, RT-PCR and Western blotting were employed, using specific primers and antibody for Twist 1, respectively. As shown in Figure 5B
, Twist 1 expression was induced in MSX2-expressing cells (B7 and B8, which expressed MSX2 weakly as shown in the upper panel in Figure 5B
) compared to EV cells. In addition, the level of Twist 1 expression was consistent with that of MSX2. In addition, we examined double-fluorescence immunostaining to assess whether Twist 1 and MSX2 are coexpressed in pancreatic cancer cells. As shown in Figure 5C
, MSX2-transfected BxPC3 showed simultaneous expression of these proteins in cancer cell nuclei; no coexpression was seen in empty vector-transfected BxPC3. Similarly, empty vector-transfected Panc-1 cells showed simultaneous expression of these proteins in cancer cell nuclei, but no coexpression was seen in MSX2 down-regulated Panc-1 cells (Figure 5D)
. Western blot also showed that nuclear Twist 1 expression was not detectable in MSX2si cells while clearly detectable in control cells (Figure 5E)
.
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We next investigated MSX2 protein expression in human pancreatic cancer tissues by immunohistochemistry using a specific antibody and examined the association of its expression with clinicopathological features. Protein expression was found in cancer cell nuclei and occasionally in stromal cells neighboring the carcinoma cells (Figure 6A, a)
while no or weak staining was seen in normal duct or acinar cells. Nuclear expression of MSX2 was found in 23 of 32 (71.8%) pancreatic cancer tissues. Among 32 cases of pancreatic carcinoma tissues, 10 cases (31.2%) were classified as intensely stained for MSX2, 8 cases (25%) were moderately stained, 5 cases (15.6%) were weakly stained, and 9 cases (28.1%) were negative for staining. Significant correlation was found between MSX2 expression and histological differentiation (P = 0.004) and vascular invasion (P = 0.00003) (Figure 6A
and Table 3
). However, there was no association of MSX2 expression with stage and T classification. The results of immunohistochemistry for MSX2 are summarized in Table 3
.
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. We evaluated its expression as positive when intense nuclear or cytoplasmic with nuclear staining was detected (Figure 6B, a and d)
, since weak cytoplasmic expression without nuclear staining was found in normal ducts. Positive Twist 1 expression was found in 14 of 32 (43.7%) cases of pancreatic cancer tissues. Double-fluorescence immunohistochemistry revealed that Twist 1 expression was observed in the carcinoma cells where MSX2 was expressed (Figure 6B, a–c)
and was significantly associated with increased expression of MSX2 (P = 0.0009, Table 4
).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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To our knowledge, the involvement of MSX2 in pancreatic cancer has not been clarified previously. MSX2 expression was more intense in pancreatic cancer cell lines examined than normal cells and found in more than 70% of human pancreatic carcinoma tissues, whereas no or very weak expression was detected in normal pancreatic ducts. Interestingly, this expression was associated with less differentiation of carcinoma cells, suggesting that MSX2 is involved mainly in pancreatic carcinoma progression rather than carcinogenesis. MSX2 has been suggested to act to stimulate proliferation and inhibit differentiation of osteoprogenitors.35
MSX2 also caused an increase in the number of proliferative osteoblasts in the osteogenic front of the skulls of postnatal mice.36
In addition, MSX2 stimulates branching morphogenesis of mouse mammary ducts,37,38
indicating that this gene function is associated with the regulation of the differentiation and/or proliferation of epithelial cells as well as osteogenic cells. Furthermore, MSX2 has been shown to be up-regulated in adult pancreas in interferon-
transgenic mice in which aggressive growth of pancreatic ducts and the continuous differentiation of new endocrine cells were observed,39
suggesting that MSX2 promotes the growth of duct cells that are the origin of pancreatic cancer. These observations, together with the fact that MSX2 stimulates pancreatic cancer cell proliferation in vitro, suggest that MSX2 contributes the development of pancreatic carcinoma by promoting cell proliferation and regulating cellular differentiation.
To clarify the molecular mechanism for poor prognosis of pancreatic cancer patients, we have examined the expression of c-erb B-2,40 gelatinase A,41 ROCK-1,42 and survivin43 and demonstrated that their expression was correlated with the invasiveness and/or the frequency of metastasis in pancreatic cancer. In addition to these factors, we have shown in the current study that MSX2 expression is correlated with biological aggressiveness of human pancreatic cancer. Although up-regulation of MSX2 in carcinoma of epithelial origin has been demonstrated, there was no investigation of the association of MSX2 expression and clinicopathological features of any type of carcinomas. Thus, the current results are the first demonstration that increased MSX2 expression is involved in poor differentiation of carcinoma cells. Although poor differentiation of pancreatic carcinoma is associated with short survival time,44 further studies in this area are required.
Twist 1 was initially identified as a crucial regulator of embryonic morphogenesis in Drosophila.45 Recent studies reveal that Twist 1 expression is associated with invasion and/or metastasis in breast and nasopharyngeal cancer.46 Ectopic expression of Twist 1 resulted in loss of E-cadherin-mediated cell adhesion and induction of cell motility, suggesting that this gene promotes an EMT. In pancreatic cancer cells, this gene is shown to be induced when cancer cells are undergoing EMT after vascular endothelial growth factor stimulation.47 Twist 1 and MSX2 have been reported to control the differentiation and proliferation cooperatively in frontal bone skeletogenic mesenchyme.48 Although the authors hypothesized either gene could regulate the expression of the other, their results obtained by in situ hybridization showed that MSX2 and Twist 1 do not regulate each other’s activity at the level of mRNA abundance. Our cDNA array clearly revealed the significant induction of Twist 1 in MSX2 overexpressing BxPC3 cells, and this induction was confirmed by semiquantitative RT-PCR and Western blotting. Conversely, nuclear expression of Twist 1 disappeared when MSX2 was down-regulated in Panc-1 cells. Finally, immunohistochemical analyses revealed that Twist 1 expression was correlated with MSX2 expression in human pancreatic carcinoma tissues, and colocalization of these proteins was demonstrated by double staining of fluorescence immunohistochemistry, indicating that Twist 1 was a target gene of MSX2. Consistent with these findings, MSX2 appears to function in leading the pancreatic cancer cells to the state of EMT and an enhanced malignant phenotype through up-regulation of Twist 1.
Among approximately 55,000 genes, Twist 1 was identified as the gene most up-regulated by MSX2. Although we focused on Twist 1 in this study, since it is an EMT-related gene, we could also identify other candidate target genes for MSX2 by this method. These include the ATP-binding cassette, subfamily G, member 2 (ABCG2),49 and synuclein gamma,50 which have been reported to be associated with resistance to chemotherapy and carcinoma development, respectively. This suggests that MSX2 also functions to enhance the biological aggressiveness of pancreatic cancer cells through pathways in addition to EMT, since many factors other than EMT contribute to the malignant phenotype of pancreatic cancer. On the other hand, the array analysis also revealed that the expression of snail, which is also a key regulator of EMT through reduced E-cadherin expression,51 was higher in MSX2-expressing cells compared to controls. In addition, we recently revealed that MSX2 itself was indispensable for bone morphogenetic protein 4 (BMP4) induced EMT in pancreatic cancer cells.52 In this context, MSX2 itself as well as many molecules or downstream pathways is likely to be involved in EMT in pancreatic carcinoma cells. Therefore, in addition to Twist 1 or other molecules as stated above, we are investigating the MSX2-mediated molecules as candidate therapeutic targets in pancreatic cancer.
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Supported in part by grants-in-aid 17390213 and 19590745 from the Ministry of Education, Science, Sports and Culture of Japan.
Supplemental material for this article can be found on http://ajp. amjpathol.org.
Accepted for publication January 15, 2008.
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