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Clinical Cancer Research Vol. 9, 4891-4896, October 15, 2003
© 2003 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

The Role of Gelatinase in Hepatic Metastasis of Colorectal Cancer

Yu-Wen Tien, Po-Huang Lee1, Rey-Heng Hu, Su-Ming Hsu and King-Jen Chang

Department of Surgery [Y-W. T., P-H. L., R-H. H., K-J. C.], and Pathology [S-M. H.], National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan 10002, Republic of China


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: To determine the role of gelatinase in the hepatic metastatic process of colorectal cancer, we correlated gelatinolytic activity in colorectal tumor tissue both to the presence of intravasated colorectal epithelial cells and to the formation of liver metastasis.

Experimental Design: The gelatinolytic activity was analyzed in tumor tissue samples from 68 colorectal cancer patients by using gelatin substrate zymography. The presence of intravasated colorectal epithelial cells was defined as detection of guanylyl cyclase C mRNA in blood sampled from drainage vein of a tumor-bearing colorectal segment.

Results: Forty of 68 patients were noted to have guanylyl cyclase C mRNA expression in their drainage venous blood. Fifteen patients were noted to have liver metastasis at the time of surgery, and another 15 patients developed liver metastasis during median follow-up period of 53 months. Either individual or total gelatinolytic activity in colorectal tumor tissue failed to predict the presence of intravasated colorectal epithelial cells in the drainage venous blood or formation of liver metastasis. Presence of both intravasated colorectal epithelial cells and high total gelatinolytic activity in colorectal tumor tissue, however, is a strong predictor of liver metastasis (P = 0.004).

Conclusion: Our data suggested that gelatinolytic activity in colorectal tumor tissues may facilitate the hepatic metastatic process in the steps after intravasation but not during or before intravasation.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MMPs,2 especially MMP-2 (gelatinase A) and MMP-9 (gelatinase B), and type IV collagenase have long been associated with metastasis because of their ability to degrade proteins that make up basement membrane, the first barrier for an invading epithelial tumor, thus facilitating invasion through connective tissue and blood vessel wall (intravasation and extravasation; Refs. 1, 2, 3 ). MMPs and their inhibitors, however, are also noted to be important regulators of the growth of tumors, both at the primary site and as metastases (4, 5, 6) . Thus, MMPs may also facilitate metastasis in steps other than intravasation and extravasation.

Most studies about the mechanistic role of MMPs in metastasis used end point assays in which the state of the MMP in the primary tumor and outcome (survival rate and ratio of metastasis) were known. The mechanisms by which the MMP resulted in the outcome were derived from inference rather than from direct observation (3 , 7, 8, 9, 10, 11) . In this study, in contrast to those studies using formation of metastasis as end point, we attempt to dissect the metastatic process into two parts: before intravasation (from primary tumor to entry into circulation) and after intravasation (from intravasated tumor cells to formation of metastatic lesion). In other words, with the presence of intravasated tumor cells used as end points, positive correlation between gelatinolytic activity in the tumor tissue and the presence of intravasated tumor cells should imply that gelatinase plays a role in the metastatic process at steps before or during intravasation. On the contrary, if the gelatinolytic activity is not correlated to the presence of intravasated tumor cells but positively correlated to the formation of metastasis, gelatinase must play its role at metastatic steps after intravasation. By correlating the gelatinolytic activity in colorectal tumor tissue both to the presence of intravasated colorectal epithelial cells (detected by GCC RT-PCR) and to the formation of liver metastasis, we attempt to determine the role of gelatinase in the metastatic process.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients.
From January 1997 to January 1998, 69 consecutive patients with histologically confirmed colorectal adenocarcinoma operated by the same surgeon at our institution were included in this study. The study protocol was approved by the ethics committee of the National Taiwan University, and informed consent was obtained from all of the patients. Thirty-three patients had colon carcinoma, and 36 patients had rectal carcinoma. Patients with prior malignancy were excluded, as were patients with previous chemotherapy or radiation therapy for colorectal adenocarcinoma. Fifty-four patients underwent potentially curative resections, whereas 15 patients underwent palliative resections for liver metastases. The distribution of clinical and pathological data for the entire population, except 1 patient who died from stroke at 7 months after the surgery, was listed in Table 1Citation .


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  		Table 1 Breakdown of 68 patients with colorectal carcinoma

 
Patients were followed every 3 months during year 1, every 4 months during year 2, and ever 6 months thereafter in the outpatient clinic. History and physical examinations were performed at each follow-up visit. Additionally, hemography, liver panel, CEA level, chest radiographs, and abdominal ultrasonography were obtained. Colonoscopy was performed at 24 and 48 weeks, and then annually. Abdominal computerized tomography was performed in those patients with an elevated CEA level or abnormal abdominal ultrasonography. Patients who failed to attend clinics as scheduled were contacted telephonically. In the event of death, the date and cause of death were recorded.

Blood and Tumor Sample Collection.
Immediately after entering the peritoneal cavity, before manipulation of the tumor, 10 ml of blood was collected from the drainage vein of tumor-bearing colorectal segment as portal venous sample. A piece (at least 2 cm3) of freshly harvested tumor tissue from each resected specimen was snap frozen in liquid nitrogen at the time of operation and stored at -70°C.

In addition, peripheral blood samples from 11 healthy volunteers were obtained as control.

RNA Extraction and Nested Duplex RT-PCR.
RNA extraction from mononuclear blood cells and frozen tissue, and GCC RT-PCR were performed as described previously (12) . In brief, cDNA was synthesized in a 20-µl reaction mixture containing 2 µg of total RNA. For the first round of the nested PCR, 20-µl reactions were prepared with 4 µl of the cDNA preparation and out primer (antisense, nucleotides 1197–1218; and sense, nucleotides 685–708). The second PCR was performed with 10 µl of this reaction mixture and antisense (nucleotides 1000–1021) and sense (nucleotides 759–781) primers. RT-PCR products were analyzed by agarose gel (2.5%) electrophoresis and visualized by UV transillumination after staining with ethidium bromide (0.5 µg/ml). The nested GCC PCR yielded a 262-bp product. The amplified products were sequenced using the ABI Model 373A DNA Sequencer (Perkin-Elmer Biosystems) as specified by the manufacturer. The DNA sequences were aligned and analyzed using an Acer computer.

Sensitivity of GCC RT-PCR.
The sensitivity of the GCC RT-PCR assay was determined in cell spiking experiments as described previously, allowing the detection of 10 CCL-220 cells in 10 ml of blood (12) .

Tissue Samples.
Up to four 10-µm sections from each cryopreserved tumor (between 10 and 20 mg of tissue) were homogenized in protein extraction buffer (500 µl). Ten min later the sample was centrifuged at 4°C at maximum rpm for 10 min, and the supernatant was collected and stored in -20°C until assayed for protein. Using a Bio-Rad protein assay reagent, the protein content of each sample was measured against BSA (13) .

Gelatin Zymography.
Gelatin zymography was performed according to the method described by Parsons et al. (14) . Briefly, each sample (20 µg of extracted protein) was run in parallel with a molecular weight marker and 20 µg of extracted protein from patient 1 was included as an internal standard on SDS-polyacrylamide gels (7.5%) containing 0.1% gelatin as the substrate. This method can detect the inactive proforms of collagenases because SDS causes activation of the enzymes without proteolytic cleavage of the inhibitory NH2-terminal sequence (15) . Western blotting using monoclonal antibodies for latent MMP-9, active MMP-9, latent MMP-2, and active MMP-2 was performed to verify that the bands seen on zymography were as described.

Control Gels for MMPs.
Control gels contained the MMP inhibitor EDTA in the MMP incubation buffer to confirm that the lysis bands were because of MMPs.

Quantitation of the Gels.
Quantification was performed using laser densitometry and Quantity One software (Discovery Series; Pharmacia Biotech, Buckinghamshire, United Kingdom). The relative gelatinolytic activity was determined for each proteinase by multiplying the area of each band by its absorbance. The following four lysis bands were observed on the gelatin zymography in all of the patient samples: Mr 92,000 corresponding to latent MMP-9 (gelatinase B); Mr 82,000, active MMP-9; Mr 72,000, latent MMP-2 (gelatinase A); and finally Mr 62,000, active MMP-2 (Fig. 1)Citation . Because the object of this study is to determine the role of gelatinolytic activity in primary colorectal cancer tissue in the process of liver metastasis, total gelatinolytic activity of each sample (expressed in arbitrary units/20 µg of protein) was also obtained by summing the activities of all of the four lysis bands (the Mr 92,000 latent MMP-9, Mr 82,000 active MMP-9, Mr 72,000 latent MMP-2, and Mr 62,000 active MMP-2 bands). To correct the variation in background staining of the gel (intergel variation), the total gelatinolytic activity of 20 µg of protein from patient 1 on each gel was defined as 20 arbitrary units of gelatinolytic activity and served as internal standard. Each type (active or latent gelatinase A or B) and total gelatinolytic activity of each specimen on the same gel were then expressed in arbitrary units/20 µg of protein.



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  		Fig. 1. Representative zymogram of specimens from patients with colorectal carcinomas. Lane 1, sample from patient 1; Lanes 2–6, samples from other patients. The arrows label the bands that were measured. These were, from the top, latent MMP-9 (Mr 92,000), active MMP-9 (Mr 82,000), inactive MMP-2 (Mr 72,000) and active MMP-2 (Mr 62,000).

 
Reproducibility of Zymography.
Samples from different parts of the block, at least six sections away from each other, were assayed to assess reproducibility. We expected the levels of the enzyme to vary with the ratio between stromal cells, the extracellular matrix, and the tumor cell component of the tissue in each section. However, we found a good concordance between samples from the same block. Typical data on five proteins are shown in Table 2Citation .


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  		Table 2 Reproducibility of gelatin zymography assay: samples from different parts of block, at least six sections apart, were subjected to quantitative zymography

 
Statistical Analysis.
Because the outcome of interest in our study, intravasation or liver metastasis, was either present or absent, we used logistic regression. We looked for associations between the presence of intravasated tumor cells (or liver metastasis) and potential prognostic factors, such as tumor size, tumor grade, tumor stage, gelatinolytic activity of each type, or total gelatinolytic activity. This method provides odds ratios or estimates of the relative risk of the presence of intravasated colorectal epithelial cells or liver metastasis. We first examined univariate relations to determine which factors were related to intravasation or liver metastasis, and next performed stepwise multivariate logistic regression to determine whether some combination of variables provided a better estimate of the relative risk of intravasation or liver metastasis than any single variable.

Probability values <0.05 were considered significant; all of the reported Ps are two-sided.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Clinical Outcomes.
At the time of this analysis, the median follow-up time for the 51 survivors was 53 months, ranging from 46 to 58 months; the remaining 17 patients died between 7 and 53 months after surgery. During the follow-up period, 15 of 53 patients with non-stage D tumors developed liver metastasis. Along with the previous 15 patients with liver metastases found at the time of operation, liver metastases were found in a total of 30 patients.

Sensitivity and Specificity of GCC RT-PCR.
As shown in our previous study, a 262-bp PCR product was detected in samples containing >=10 CCL-220 cells in 10 ml of blood. This fragment had the expected size delineated from the position of the chosen primers.

The 262-bp products from 2 tumor, 2 portal venous blood samples, and 1 cell line specimen were sequenced and were identical with the GCC sequences published by Carrithers et al. (16) . GCC transcripts were detected in all of the tumor specimens and, thus, confirmed the presence of GCC-expressing cells in all of the tumors.

None of the peripheral blood samples from 11 healthy volunteers or portal venous blood samples from 6 patients undergoing colorectal resection for benign disease were positive for GCC mRNA.

Correlation between Gelatinolytic Activity in Colorectal Tumor Tissue and Expression of GCC mRNA in Portal Venous Blood.
Sixty-eight patients were classified into one of two groups according to low or high gelatinolytic activity (individual type or total) in their colorectal cancer tissue. The cutoff level corresponded to the median value of the entire population for this classification scheme.

GCC transcripts were detected in portal venous blood in 40 of 68 patients (59%). There was no significant difference in GCC RT-PCR positive expression among stage A (1 of 2; 50%), stage B (12 of 23; 52%), and stage C (13 of 28; 46%) patients. However, there was a significant difference in GCC RT-PCR positive expression between patients with stage D tumors (14 of 15; 93%) and non-stage D tumors (26 of 53; 49%; P = 0.004). There was no significant difference in patient age, sex, tumor size, lymph node metastasis, lymphatic invasion, vascular invasion, CEA level in peripheral blood, or gelatinolytic activity (either individual type or total) between patients with and without GCC mRNA detected in drainage venous blood (Table 3)Citation .


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  		Table 3 Association of various factors with rate of detection of GCC mRNA in portal venous blood as determined logistic regression

 
Correlation between the Gelatinolytic Activity in Colorectal Tumor Tissue and Formation of Liver Metastasis.
There was no significant difference in developing liver metastasis between patients with (21 of 40; 53%) and without (9 of 28; 32%) GCC mRNA detected in the drainage venous blood (P = 0.099; Table 3Citation ). High gelatinolytic activity (either individual type or total) in colorectal tumor tissue also failed to predict the formation of liver metastasis (Table 3)Citation . However, of the 40 patients with GCC mRNA expression in portal venous blood, liver metastasis developed more frequently in patients with tumors of high total gelatinolytic activity (16 of 23 patients) than in patients with tumors of low total gelatinolytic activity (5 of 17 patients; P = 0.012). In stepwise multivariate logistic regression, the combination of GCC mRNA present in portal venous blood and high total gelatinolytic activity also provided the best estimate of the relative risk of developing liver metastasis (Table 3Citation ; P = 0.004; odds ratio, 5.061).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Most clinical studies about MMPs were focused on detection of MMP protein or mRNA (3 , 5, 6) . As a matter of fact, the MMP activity is highly regulated at many levels (4) . Thus, detection of MMP mRNA does not mean that these detected mRNAs will be translated into MMP protein. Likewise, detection of MMP protein does not mean that these detected MMP proteins are activated, and even if activated, that these MMP proteins are not inhibited by other proteinase inhibitors. In contrast to detection of MMP protein or mRNA, substrate zymography measures the proteolytic activity in tumor tissue for specific substrate and may more faithfully represent the functional MMP activity in the tumor tissue. However, most studies about activity of gelatinase in benign and malignant colorectal disease have focused on comparison of individual latent or active MMP2 or MMP9 activity (shown by gelatin zymography) between normal colorectal mucosa and colorectal cancer (7, 8, 9) . As shown in these studies, a colorectal tumor from 1 patient, when compared with one from another patient, may have higher MMP2 but lower MMP9 activity (7, 8, 9) . Thus, it is difficult to assess the biological effect of gelatinolytic activity in tumor tissue on clinical outcome. Besides, comparison of gelatinolytic activity between normal and cancer tissue will be more suitable for study of tumorigenicity than for study of metastasis. Because the interest of this study is metastasis, we tried to obtain the total gelatinolytic activity by summing up the active and latent MMP2 and MMP9 activities shown on gelatin zymography and used this total as a measure of total gelatinolytic activity in colorectal tumor tissue. Both individual type and total gelatinolytic activity were used to predict the presence of intravasated GCC mRNA positive cells or formation of liver metastasis.

PCR-based assays of mutated DNA or tissue-specific RNA are highly sensitive methods for detecting intravasated tumor cells. DNA-targeted PCR assay may detect DNA derived from degraded instead of viable tumor cells (17) . Therefore, an RNA-targeted PCR assay was selected as the most appropriate assay to detect intravasated viable tumor cells with metastatic potential. Detection of cytokeratin-20 mRNA (18 , 19) , CEA mRNA (20 , 21) , and GCC mRNA (16) have been used for the detection of circulating viable colorectal tumor cells. Because GCC transcripts have greater specificity than transcripts of cytokeratin 20 or of CEA, we chose to detect them by RT-PCR in circulating colorectal epithelial cells (22) .

Specificity is a major concern in the RT-PCR system for detecting intravasated tumor cells because of the detection of tissue-specific mRNA instead of tumor cell-specific mRNA. However, normal colon epithelia or liver cells rapidly underwent anoikosis after having been released into the circulation (23) . Besides, we sampled blood by direct puncture of drainage vein of tumor-bearing segment to minimize contamination. With this technique, 40 of 68 patients were found to have GCC mRNA expression in their portal venous blood. To our surprise, either high individual or high total gelatinolytic activity in colorectal cancer tissue did not increase the chance of finding GCC mRNA-positive cells in the drainage venous blood. This implied that either high individual or high total gelatinolytic activity in colorectal tumor tissue does not facilitate the first part of metastatic process (from primary tumor to entry into the circulation).

In our patients, both the presence of GCC mRNA in portal venous blood and high gelatinolytic activity (either individual type or total) in colorectal tumor tissue failed to predict the formation of liver metastasis (Table 3)Citation . The fact that 19 of 40 patients with GCC mRNA detected in the drainage venous blood did not develop subsequent liver metastasis during median follow-up period of 53 months implies that intravasated tumor cells in these cases failed to fulfill the subsequent metastatic steps after intravasation (extravasation and growth at target organ). However, in multivariate analysis, the combination of high gelatinolytic activity (either latent MMP2 or active MMP9) and the presence of GCC mRNA in drainage venous blood showed borderline significance in prediction of liver metastasis (for latent MMP2: 95% CI, 0.984–8.854, P = 0.053; for active MMP9: 95% CI, 1–7.946, P = 0.05; Table 4Citation ). The correlation became more prominent when combination of high total gelatinolytic activity and presence of intravasated tumor cells was used to predict the formation of liver metastasis (95% CI, 1.702–15.047; P = 0.004). This implies that despite not contributing to intravasation, high total gelatinolytic activity in primary colorectal tumor tissue does increase the risk of developing liver metastasis in patients with GCC mRNA-positive cells in their drainage venous blood. Thus, gelatinases in colorectal tumor tissue may facilitate the second part of hepatic tumor metastatic process (from intravasated tumor cells to formation of liver metastasis). Because liver metastasis from colorectal tumor comprises many steps controlled by different factors, a combination of factors responsible for different metastatic factors may provide a better predictor of liver metastasis than any single factor does. As shown in our studies, the combination of high gelatinolytic activity and the presence of intravasated tumor cells in drainage venous blood provide the best prediction of formation of liver metastasis.


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  		Table 4 Association of various factors with rate of formation of liver metastasis as determined by logistic regression analysis

 
MMPs and their inhibitors have been reported to be important regulators of the growth of tumors, both at the primary site and as metastases (4, 5, 6) . This may account for the result of our studies that total gelatinolytic activity in colorectal tumor tissue may contribute to hepatic metastatic process at steps other than intravasation, for example, growth of extravasated tumor cells. Possible mechanisms of this growth regulation include: (a) change in the growth environment by regulating access to growth factors obtained from the extracellular matrix surrounding the growing tumor cells; and (b) regulation of angiogenesis (4 , 24) . Angiogenesis is required for growth of both primary tumors and metastases, beyond small size, and MMPs play a contributory role in regulation of angiogenesis (24) . An understanding of the molecular role of MMPs at each of the sequential steps required to produce clinically evident metastases will be important in the design and appropriate use of novel therapeutics designed to combat metastasis.

In conclusion, either individual or total gelatinolytic activity in colorectal tumor tissue could not predict the presence of GCC mRNA positive cells in blood sampled from the drainage vein of a tumor-bearing colorectal segment. Liver metastasis could not be predicted either by the presence of GCC mRNA-positive cells in drainage venous blood or by high individual or total gelatinolytic activity in the colorectal tumor tissue. However, presence of both GCC mRNA-positive cells in drainage venous blood and high total gelatinolytic activity in tumor tissue suggests high risk of formation of liver metastasis. Gelatinolytic activity in colorectal tumor tissue may contribute to the hepatic metastatic process in steps after intravasation, but not during or before intravasation.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a grant from the National Taiwan University Hospital (88N115).

1 To whom requests for reprints should be addressed, at Department of Surgery, National Taiwan University Hospital, 7 Chung-Shan South Road, Taipei 10002, Taiwan, Republic of China. Phone: 886-2-23123456, extension 5106; Fax: 886-2-23568810; E-mail: ywt5106{at}ha.mc.ntu.edu.tw Back

2 The abbreviations used are: MMP, matrix metalloproteinase; GCC, guanylyl cyclase C; CEA, carcinoembryonic antigen; RT-PCR, reverse transcription-PCR; CI, confidence interval. Back

Received 12/19/02; revised 5/30/03; accepted 6/24/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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