Abstract
Aim: To clarify the correlation between morphological features and the mRNA expression of a disintegrin and metalloproteinases (ADAMs) and matrix metalloproteinases (MMPs) in colorectal cancer. Materials and Methods: In 29 colorectal tumors, cancer cells were isolated by laser microdissection, and the mRNA expression of metalloproteinases was compared between the tumor center and the invasive front. Results: Regarding the central region as the standard, the mRNA levels of ADAM-12, MMP-1, -2, -7, and -9 at the invasive front were up-regulated. The degree of up-regulation was significantly higher in solid cancer nest (SCN)-positive tumors than in SCN-negative tumors for the mRNA levels of ADAM-12, MMP-2, -7, and - 9 (SCN-positive, median 5.1-, 3.9-, 9.9-, and 2.7-fold; SCN-negative 2.0-, 1.0-, 2.4-, and 0.8-fold; p<0.05, respectively). Conclusion: Up-regulation of the mRNA expression of particular metalloproteinases was significantly associated with the SCN at the leading edge of colorectal tumors.
The biological activity of colorectal cancer is thought to be more accurately reflected by histological characteristics (such as tumor budding) at the invasive front than in the central area of the tumor (1-3). We have previously shown that the extent of the cancerous area without a gland structure in the invasive front of colorectal cancer is an independent prognosticator, namely that advanced colorectal tumors having few solid cancer nests with no glandular formation (SCN) are associated with a very favorable prognosis. (4) However, the molecular biological characteristics of this heterogeneous morphological feature exclusively observed at the invasive front have remained unclear.
Two metalloproteinase families, matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs), which are regulated by tissue inhibitors of metalloproteinases (TIMPs), have been shown to correlate with tumor invasion. MMPs appear to play an important role in cancer cell proliferation and progression through the process of degradation of the extracellular matrix, processing of growth factors, and activation of cell adhesion molecules (5, 6). ADAMs are members of an MMP-related metalloproteinase family, and are kinds of type I transmembrane proteins that contain a metalloprotease and a disintegrin-like domain (7). Recent reports have shown that by cleaving different components, ADAMs direct cell migration and control various signaling pathways activated in cancer cells. Some ADAM species, including ADAM-10, -12, and -17, are known to be expressed in human colorectal cancer, although the details of their role in the progression of colorectal cancer have not been clarified (7, 8).
The aim of this study was to identify the molecular biological characteristics of this heterogeneous morphological feature at the invasive front of colorectal tumors. First, we examined the pathological significance of the difference in tumor expression of mRNA of ADAMs, MMPs, and TIMPs, between the center of tumor and the invasive front, using laser microdissection and a real-time RT-PCR method. We then analyzed the RNA expression status of these molecules, with special reference to the formation of SCNs at the leading edge of the tumor.
Materials and Methods
Patients. A total of 29 advanced colorectal tumors were examined in this study (Table I). These tumors were derived from patients who consecutively underwent surgical resection of primary colorectal cancer between April and September, 2008, at our Institution. Written informed consent was obtained from every patient according to the institutional regulations. No patient had received chemotherapy or radiotherapy before surgery.
Pathologic evaluation. SCN was defined as a cancer cluster made up of 5 or more cancer cells without a glandular structure (Figure 1A). The tumor was determined to be SCN-positive when it had 10 or more SCN lesions in a microscopic field of ×40 and negative when it had fewer than 10 SCN lesions, where SCN lesions were observed most intensively (4). For tumor budding, an isolated single cancer cell and a cluster composed of fewer than five cancer cells were defined as ‘budding’ foci. The tumor was determined to be high grade when it had 10 or more budding foci in a microscopic field of ×200 and low grade when it had budding foci of fewer than 10 (3). Judgments regarding SCN and tumor budding were made by one of the Authors (YK) with no information regarding the state of nodal involvement or the patient prognostic outcome. The conventional clinicopathological parameters (such as tumor differentiation and lymphovascular invasion) were retrieved from the pathology reports recorded at the time of surgery according to the Japanese Classification of Colorectal Carcinoma (9).
Clinicopathological background of 29 colorectal cancer patients.
Laser microdissection. After finishing resection of the colorectal surgical specimen, full-thickness tumor samples were immediately taken by pathologists. The section was embedded in Tissue Tek OCT mediumTM (Sakura, Tokyo, Japan), frozen in liquid nitrogen, and kept at −80°C. The specimens were cut into serial sections with a thickness of 10 μm, and the sections were fixed in 5% acetic acid in ethanol for 3 minutes. The sections were stained with 0.05% Toluidine Blue Solution™, pH 7.0, (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 30 seconds. The cancer cells at the center of the tumor and at the invasive front, respectively, were microdissected with a laser microdissection system™ (Leica Microsystems, Wetzlar, Germany). When the tumor had SCN lesions, the cancer cells of the SCN lesions were selectively harvested as invasive-front specimens (Figure 1A and B). Additionally, tumor specimens of glandular lesions at the invasive-front of the tumor were also microdissected from the same tumor (Figure 1C and D), to compare the mRNA expression status between the SCN and glandular lesion parts.
Sequences of primers used in this study.
Total RNA extraction and first-strand cDNA synthesis. Total RNA was isolated using an RNeasy mini kit™ (Qiagen K.K., Tokyo, Japan) following the manufacturer's instructions, and all samples were treated with the RNase-free DNase set™ (Qiagen K.K.). The RNA quality control and quantification were carried out on an Agilent 2100 Bioanalyzer using an RNA 6000 Pico LabChip™ (Agilent Technologies, Tokyo, Japan). First-strand cDNA was made from total RNA using the Prime Script RT-reagent Kit™ (Takara Bio Inc, Otsu, Japan), according to the manufacturer's instructions.
Primers. Phosphoglycerate kinase 1 (PGK1), human phosphoribosyl-transferase 1 (HPRT1), and ribosomal protein-large-P2 (RPLP2) were analyzed as reference genes. These three endogenous housekeeping genes were chosen using the geNorm algorithm (10). Briefly, twelve arbitrarily selected colorectal tumors in this study were screened for 15 reference genes (Human Housekeeping Gene Primer Set™; Takara Bio Inc); the algorithm works out the stability of each transcript and sequentially removes the least stable transcript until the three most stable transcripts remain. The primers (Perfect Real Time Support System™; Takara Bio Inc) that had been used for quantitative RT-PCR in this study were as shown in Table II.
Collection of cancer cells at the tumor invasive front using a laser microdissection system. The solid cancer nest (composed of 5 or more cancer cells without glandular structure) (A) and cancer cells of the glandular lesions at the invasive front of colorectal cancer (B) were traced with the computer mouse, as indicated by the line. They were then dissected with an ultraviolet laser (C and D). Bars 200 μm.
Real-time RT-PCR. SYBR green real-time PCR was performed using the Thermal Cycler Dice Real Time System TP800TM (Takara Bio Inc). The PCR reaction mixtures consisted of distilled water, primers, SYBR Premix Ex Taq II™ (Takara Bio Inc), and the cDNA from the dissected colorectal cancer cells. PCR conditions were as follows: 30 seconds at 95°C for the initial denaturation, and then 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. All reactions were run in duplicate. A melting curve was constructed for each primer pair to confirm the product specificity. Real-time PCR data were analyzed by Multiplate RQ software™ (Takara Bio Inc), which is able to treat multiple reference genes. Relative gene expression was determined by a comparative Ct method.
Statistical analysis. The differences between the mRNA levels of the cancer cells at the tumor invasive front and those at the center of the tumor were assessed using the Wilcoxon signed-rank test, and the mRNA expression at the invasive front that was found to be significantly higher than that at the central area was defined as up-regulation. To examine the correlation between the clinicopathological findings (such as tumor differentiation, lymphovascular invasion, tumor budding, and SCN status) and relative mRNA expression at the tumor invasive front, the mRNA levels were assessed using the Mann-Whitney U-test. The differences in mRNA levels of SCN lesions and glandular lesions at the invasive front in the same tumors were evaluated using the Wilcoxon signed-rank test. P-values less than 0.05 were considered to indicate significance. Statistical calculations were performed using Stat View ver. 5.0 software™ (SAS Institute, Cary, NC, USA).
Results
Up-regulation of mRNAs of ADAMs, MMPs, and TIMPs at the tumor invasive front. The ratio of the mRNA expression levels of ADAMs, MMPs, and TIMPs at the invasive front to those at the center of the tumor (F/C ratio) are shown in Table III.
Although the mRNA expression of ADAM-10 and ADAM-17 was similar in the invasive front and the center of the tumor, the ADAM-12 mRNA levels were significantly higher in the cancer cells at the invasive front than at the center of the tumor (median F/C ratio 3.0, p<0.0001). With regard to the MMPs, the expression levels of mRNA at the invasive front were significantly up-regulated for all 5 MMPs examined but one (MMP-3). MMP-7 exhibited a marked up-regulation at the invasive front of the tumor. The median F/C ratio of MMP-7 was 5.4, and the mRNA up-regulation of MMP-7 at the invasive front was observed in 28 out of 29 tumors. With respect to the inhibitors of MMPs, TIMP-1, -2, and -3 also showed up-regulations in the cancer cells at the invasive front of the tumor.
Up-regulation of mRNAs at the tumor invasive front compared to the center of the tumor in colorectal cancer.
Correlation of clinicopathological characters and mRNA expression of up-regulated ADAMs, MMPs, and TIMPs at the tumor invasive front. The correlation between the clinicopathological characters and relative mRNA expressions of up-regulated metalloproteinases and TIMPs at the invasive front are shown in Table IV.
The mRNA expressions of ADAM-12, some MMPs and TIMPs were associated with predominant tumor differentiation and degree of lymphatic invasion. However, venous invasion was not associated with any of these mRNA expressions. The F/C ratios of tumor with nodal involvement for ADAM-12, MMP-2, -7 and -9, TIMP-2 and -3 were significantly higher than those of tumor without nodal involvement. With regard to tumor budding, the tumors with high-grade budding presented significantly higher mRNA levels of all the up-regulated metalloproteinases, except for MMP-1, at the invasive front compared to the tumors with low-grade budding. The F/C ratios of TIMP-2 and -3 mRNA in the tumors which had high grade budding were also significantly higher than those of the tumors which had low-grade budding.
Correlation of SCN status and mRNA expression of ADAMs, MMPs, and TIMPs at the tumor invasive front. Thirteen tumors (44.8%) had SCN lesions at the invasive front of the tumor (SCN-positive), and16 (55.2%) did not (SCN-negative). The F/C ratio was compared, respectively, according to the SCN status (Figure 2). The median F/C ratio of ADAM-12 mRNA was 5.1 (range 0.8-107.0) in the SCN-positive tumors, whereas it was 2.0 (range 0.6-13.4) in the SCN-negative tumors (p=0.029). Similarly, SCN-positive tumors presented significantly higher mRNA levels of MMP-2, -7, and -9 at the invasive front compared to the center of the tumor (median 3.9-, 9.9-, and 2.7-fold, respectively) than the SCN-negative tumors (median 1.0-, 2.4-, and 0.8-fold; p=0.0018, 0.0085, and 0.0022, respectively). Regarding MMP-2 and -9, the mRNA expression was down-regulated at the invasive front in almost half of the SCN-negative tumors, whereas the mRNA expression levels of all 13 SCN-positive tumors but one were up-regulated at the invasive front compared to the center of the tumor. Although, the median F/C ratio of MMP-1 mRNA in all 29 cases was 2.3, no significant difference was observed in the F/C ratio according to the SCN status. With respect to the TIMPs, mRNA up-regulation of TIMP-1, -2, and -3 at the invasive front of the tumor was observed in 12 out of 13 SCN-positive tumors. The median F/C ratios of TIMP-1, -2, and -3 mRNA in the SCN-positive tumors were 2.3, 4.0, and 2.9, significantly higher than those of the SCN-negative tumors (median 1.2, 1.5, and 1.2; p=0.0034, 0.023, and 0.025, respectively).
Differential mRNA expression levels of ADAMs, MMPs, and TIMPs in SCN lesions compared with glandular lesions at the invasive front. In the 13 SCN-positive tumors, the comparisons were performed in terms of mRNA expression of ADAMs, MMPs, and TIMPs between cancer cells of SCN lesions and cancer cells of glandular lesions at the invasive front (Figure 3).
The ADAM-12 mRNA expression levels in SCN lesions were significantly higher than those in glandular lesions at the invasive front (median mRNA expression ratio (SCN lesion/glandular lesion), 2.4 (range 0.6-9.5); p=0.0024). With regard to MMPs, the mRNA expression levels of MMP-1, - 7, and -9 were also significantly higher than those of glandular lesions at the invasive front (median mRNA expression ratio (SCN lesion/glandular lesion) 2.8 (range 0.8-34.5), 4.2 (0.1-16.0), and 2.0 (0.5-6.8); p=0.0046, 0.011, and 0.0076; respectively). With respect to the TIMPs, the mRNA expression levels of TIMP-2 and -3 were constantly higher than those of glandular lesions at the invasive front in all cases examined (median mRNA expression ratio (SCN lesion/glandular lesion): 3.0 (range 1.2-30.5) and 1.5 (1.1-9.3); p=0.0015 in both cases).
Correlation between the clinicopathological characteristics and relative mRNA expressions at the tumor invasive front.
Discussion
In the present study, we revealed that the mRNA expression levels of ADAM-12, MMP-1, -2, -7, -9, TIMP-1, -2, and -3 in the colorectal cancer cells at the invasive front are significantly up-regulated compared with those at the center of the tumor. It has been reported that many kinds of MMPs and TIMPs, except for MMP-7, are predominantly made by stromal cells in colorectal cancer (11-15). However, we used the highly sensitive real-time RT-PCR method to show that the mRNA of several MMPs and TIMPs is expressed in cancer cells. With regard to MMP-7, which is usually synthesized in tumor cells (16), it has been reported that the high expression of MMP-7 protein at the invasive front of colorectal cancer is significantly correlated with the depth of invasion, lymph node metastasis, and poor prognosis (17). In this study, we also revealed that MMP-7 shows marked up-regulation at the invasive front of the tumors at the mRNA level.
Relative mRNA expression levels of ADAMs, MMPs, and TIMPs in cancer cells at the invasive front in comparison to those in the cancer cells at the center of the tumor. SCN: Solid cancer nest; Y-Axis: fold-change as compared to mRNA expression in the cancer cells at the center of tumor. Boxes, first and third quartiles (median inside); bars, 10 and 90 percentiles.
Relative mRNA expression levels of ADAMs, MMPs, and TIMPs in paired solid cancer nest (SCN) and glandular lesions at the invasive front from 13 SCN-positive cases. Y-Axis: Fold-change as compared to mRNA expression in the cancer cells at the center of the tumor.
Although there have been few studies of the mRNA up-regulation of ADAMs, MMPs, and TIMPs at the invasive front of colorectal cancer, Kahlert et al. have observed the invasion front-specific overexpression of MMPs and TIMPs in liver metastases from colorectal cancer by using laser microdissection and Affymetrix microarrays. Their results show that the mRNAs of several MMPs and TIMPs, including MMP-2, -3, -7, -9, -11, -12, -14, -15, -16, -19, and -24, and TIMP-1, -2, and -3, are generally up-regulated in cancer cells at the invasive front of lesions compared with those at the center of lesions in liver metastases (18). With regard to MMPs and TIMPs, the present results are similar to those of Kahlert et al's study. We therefore confirmed that the invasion front-specific overexpression of MMPs and TIMPs also occurs in the cancer cells of primary colorectal cancer. Furthermore, we revealed that the mRNA up-regulation of metalloproteinases and TIMPs are significantly associated with unfavorable morphological features, such as tumor differentiation, lymphatic invasion, and tumor budding.
The SCN was defined as a cancer cluster made up of 5 or more cancer cells without a gland structure, in expectation of its potential contribution to the objective tumor grading. In the previous study, advanced colorectal cancer patients who were classified as SCN-negative, which had been defined in the previous study as grade 1, demonstrated a very favorable prognosis (cancer-related 5-year survival rate of 99.3%) (4). We found that there was a substantial difference in the degree of up-regulation of mRNA levels of all metalloproteinases, except for MMP-1, between SCN-negative tumors and SCN-positive tumors. In particular, the mRNA regulation pattern between SCN-negative and SCN-positive cases differed greatly for MMP-2 and MMP-9, compared to ADAM-12 and MMP-7. Namely, the mRNA expression levels of MMP-2 and -9 at the invasive front were down-regulated in more than half of the SCN-negative tumors, although the mRNA expression of ADAM-12 and MMP-7 in most of the SCN-negative tumors showed at least a little up-regulation at the invasive front. These results show that MMP-2 and -9, both of which are classified as type IV collagenases (i.e. gelatinase), are essential factors associated with the formation of SCN in the invasive front.
Based on the comparison of mRNA expression in cancer cells at the tumor front between SCN and glandular lesions at the invasive front in SCN-positive tumors, it was found that the mRNA expression levels of ADAM-12, MMP-1, -7, -9, TIMP-2, and -3 were significantly higher than those in the glandular lesions. In particular, the mRNA expression levels of TIMP-2 and -3 were consistently higher in SCN than in glandular lesions at the invasive front. In contrast, there was no significant difference between SCN and glandular lesions in the mRNA expression levels of MMP-2 and TIMP-1. Nevertheless, because we did not evaluate the mRNA statuses of stroma cells in this study, we cannot conclude whether the high levels of mRNA expression of several metalloproteinases are a characteristic of cancer cells of the SCN lesion itself or responses that are affected by the stromal reactions around the SCN lesion. To clarify the influence of stromal reactions, further study is needed regarding the expression of metalloproteinases at cancer stroma cells surrounding SCN lesions.
There have recently been several interesting studies of ADAM-10 and -17 in colorectal cancer. The overexpression of ADAM-10 protein was observed in 72-74% of colorectal cancer cases (19, 20) and was linked to a higher tumor stage (19). Moreover, it has been reported that ADAM-10 immunoreactivity is localized at the invasive front of the tumor tissue, and its expression contributes to ectodomain shedding of L1 cell adhesion molecule (20). ADAM-17 (i.e. tumor necrosis factor-α-converting enzyme) controls epidermal growth factor receptor (EGFR) activation through regulated shedding of EGFR ligands. It has been reported that use of a selective ADAM-17 inhibitor results in a concentration-dependent decrease in cell proliferation (21). The overexpression of ADAM-17 mRNA and protein in colorectal cancer has also been reported compared to normal colonic mucosa. (22) However, in the present study, mRNA expression of ADAM-10 and -17 was relatively stable between the invasive front and the center of the tumor, and there was no relation to SCN status at the invasive front.
On the other hand, ADAM-12 (i.e. meltrin α) protein overexpression has been reported to in 50% of colorectal cancer tissue (23), and ADAM-12 expression is associated with tumor aggressiveness and progression in human liver cancer, (24) breast cancer, (25), and bladder cancer (26). Nevertheless, there has been little effort to elucidate the role of ADAM-12 in colorectal cancer. In this study, we revealed that ADAM-12 mRNA expression is significantly up-regulated in cancer cells at the invasive front compared with those at the center of the tumor, similarly to particular types of MMPs and TIMPs, and is related to tumor budding and SCN status at the invasive front. ADAM-12 might play an important role in tumor dedifferentiation at the invasive front of colorectal cancer.
In conclusion, the results of this study highlight the up-regulation of the mRNA expression of particular metalloproteinases was significantly associated with the SCN at the leading edge of colorectal tumors. Further research to disclose the significance of metalloproteinases up-regulation in colorectal cancer is needed.
- Received January 30, 2011.
- Revision received April 25, 2011.
- Accepted April 27, 2011.
- Copyright© 2011 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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