INTRODUCTION

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Several in vitro experimental studies and in vivo observations during the past 5 yr have focused on the role of extracellular matrix (ECM) in the process of carcinogenesis and metastasis. Most of these studies have referred to the role of special enzymes, known as metalloproteinases (MPs), that degrade the ECM by participating in its metabolism (1). Increased ECM turnover takes place in many physiologic and pathologic conditions, including tissue remodeling and inflammation, as well as during tumor invasion and metastasis (4, 5). The family of MPs are Zn²⁺- and Ca²⁺-dependent proteinases consisting of the interstitial or specific collagenase, the gelatinases of molecular weights 92 kDa and 72 kDa (collagenases IV), the metalloproteinase putative metalloproteinase-1 (PUMP-1), and the stromelysins-1, -2, and -3 (STR-1, STR-2 and STR-3, respectively) (6, 7). These enzymes have different substrate specificities, and their patterns of expression and function vary under different circumstances. In experiments performed with mice, the secretion of MPs seems to facilitate the infiltrative tendency of neoplastic cells, whereas the action of these enzymes in tissue confines is inhibited by specific tissue inhibitors (inhibitors of metalloproteinases, MIs), which are known in the literature as tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 (8, 9). Cell-culture studies have shown that neoplastic cells of mesenchymal origin more frequently express MPs of the gelatinase group than do cells of epithelial (ectoendodermal) origin (10). It was also found that the presence of gelatinases of molecular weight 92 kDa is associated with increased tumorigenicity (10). Although overexpression of STR-3 is quite rare, an increase in the transcription rate for this enzyme is associated with an infiltrative tendency mainly of undifferentiated neoplastic cells. Expression of the inhibitor TIMP-1 was detected in 50% of human tumor-cell lines studied, whereas expression of TIMP-2 was observed in the majority of cells (10). In another study, expression of 72-kDa gelatinases was observed in human tumorigenic as well as in nontumorigenic cell lines, whereas interstitial collagenase was detected exclusively in tumorigenic cell lines (11). Considerable evidence supports the notion that the balance between the levels of extracellular MPs and TIMPs is the primary determinant of the rate of ECM turnover.

Data relating to the role of MPs and MIs in human tumors are relatively limited, and include studies relating mainly to breast and colon carcinomas and non-Hodgkin's lymphomas (12, 13). Previous publications evaluating the role of MPs in breast carcinomas as well as in benign tumors (fibrous adenomas) point out that most of the MPs are expressed randomly and independently of the pathology of the neoplasm. Exceptions are 92-kDa gelatinases and STR-3, which were expressed predominantly by carcinoma cells (6). In non-Hodgkin's lymphomas, the increased expression of 92-kDa gelatinase is associated with clinical aggressiveness and resistance to therapy. In many cases the increased expression of gelatinase is accompanied by overexpression of the inhibitor TIMP-1 (14). On the other hand, increased expression of the inhibitor TIMP-1 has been observed in colon carcinomas with high infiltrative potential, suggesting that proteolytic activity plays an important role in the degradation of ECM and the invasiveness of cancer cells (15).

In the present study we assessed, in fresh biopsy material from squamous-cell lung carcinomas (SCLCs), the roles of the MPs STR-3, interstitial collagenase, and the gelatinases of 72 kDa and 92 kDa, as well as those of the MP inhibitors TIMP-1 and TIMP-2, as markers of metastatic potential. We used Northern blot analysis and densitometry for semiquantitation of the results, together with immunohistology, and correlated the data thus obtained with pathologic features of the disease, in order to evaluate the different stages in the progress of malignant transformation in SCLC.

	METHODS

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Fresh biopsy samples from 10 SCLCs of high-to-moderate differentiation with no lymph-node metastases, and from 15 infiltrative neoplasms of moderate-to-low differentiation with metastases to either regional or distant lymph nodes, were analyzed. The tissue was obtained from pneumonectomies performed in the 401 Army General Hospital during the 5-yr period 1991 through 1995. Fresh biopsy material from five cases with significant dysplasia of bronchial epithelial cells, selected from sites close to the neoplastic areas, was also used. The mean age of patients was 62 yr (range: 47 to 82 yr); 24 (80%) of the patients were men and 6 (20%) were women. The size of carcinomas varied from 1 cm to 2.5 cm in diameter. The specimens were selected prospectively on the basis of tumor cells comprising more than 90% of the tissue.

Ribonucleic Acid Extraction, Probe Preparation, and Northern Blot Analysis

Total cellular ribonucleic acid (RNA) from tissue samples was extracted according to Chomczynski and Sacchi's method (16). Frozen tissue sections of 50 µm thickness were homogenized with 500 µl of isothiocyanate guanylphenol solution (RNAzol B; Biotecx Labs, Houston, TX), taking all necessary precautions to avoid external contamination. The suspension, which contained total RNA, was transferred to sterile, conical polypropylene tubes to which chloroform was added in a ratio of 1:10 (vol/vol) of the initial solution. The mixture was shaken well and centrifuged at 12,000 × g for 15 min at a temperature of 4° C. Centrifugation yielded two phases: an upper phase in which RNA was concentrated and transferred to a sterile conical Eppendoff tube, and a lower phase that was discarded. An equal volume of isopropanolol was then added, and after centrifugation at 12,000 × g for 15 min at 4° C, the total RNA precipitated at the bottom of the tube. After the supernatant was discarded, the precipitated total RNA was dissolved in alcohol 75% and centrifuged at 7,500 × g for 8 min at 4° C. The supernatant was again discarded and the precipitated RNA was dissolved in 25 to 50 µl of diethylpyrocarbonate (DEPC)-treated water. The concentration of total RNA was estimated photometrically at 260 nm.

Deoxyribonucleic acid (DNA) and RNA probes for interstitial collagenase, TIMP-1 and TIMP-2, STR-3, PUMP-1, and the 72-kDa and 92-kDa gelatinases have been described previously (13, 14). Probing for TIMP-2 revealed two transcripts of different lengths: 3.5 kb (high transcript) and 1 kb (low transcript).

Northern blot analysis was performed according to established protocols (17). Ten micrograms of total RNA from each sample was applied to each well and electrophoresed through formaldehyde-containing agarose gel. The products of electrophoresis were transferred to Duralon membranes and fixed with a UV crosslinker. Afterward, the membranes were hybridized with ³²P-labeled DNA probes. The blots were washed, autoradiographed for 48 h with Kodak XAR-5 film (Kodak, Rochester, NY) at 70° C, and stripped of the labeled probe by incubation with the hybridization solution for 15 min at 80° C.

The measured values of the various enzymes and inhibitors were expressed as the ratio of the densitometrically measured signal from the probed sample to the densitometrically measured signal from the same sample reprobed with an 18S rRNA probe 100X (image analysis system DIS-200; Digital Image Systems, Athens, Greece). The blots were read and interpreted without any knowledge of the tissue type used for extraction.

Immunohistochemical Evaluation

Five-micrometer-thick frozen sections from all tissue samples were stained with the avidin-biotin-complex (ABC) immunoperoxidase technique, as previously described (26). For the detection of STR-3 and TIMP-2, commercially available mAbs (Oncogene Science, Cambridge MA) were used at dilutions suggested by the manufacturer (0.019 mg/ml and 0.027 mg/ml, respectively). Negative controls included substitution of the primary antibody with phosphate-buffered saline (PBS) (pH 7.2 to 7.4) and normal rabbit serum. Immunostaining was repeated on at least one occasion, and the results were assessed by scoring the intensity of staining as follows: negative: , weak staining: +, moderate staining: ++, and strong staining: +++. This system ensured reproducibility of the results and comparison among the different groups.

Statistical Analysis

The values of mRNA transcript expression of the metalloproteinase inhibitors (TIMPs) were treated as continuous variables. Levels of 3.5-kb TIMP-2 mRNA (higher transcripted fragments) were distributed with constant variance, whereas levels of 1-kb TIMP-1 and TIMP-2 (lower transcripted fragments) required a square-root transformation. Transcripts of MPs were analyzed as dichotomous variables with values of 0 (absence) and 1 (presence). Histology was treated as an ordinal-level variable describing the neoplastic transformation of squamous tissue. Codes ranged from 0 for normal mucosa to 6 for carcinoma with a high depth of invasion and nodal metastasis. Mean values for TIMP and MP transcription levels were tested separately, using Pearson's correlation coefficient for TIMPs and Kendall's C for MPs. Overall, statistical evaluation of the results was done through multiple linear regression analysis.

	RESULTS

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A representative Northern blot from our study is shown in Figure 1, and data concerning all seven molecules studied are summarized in Table 1.

View larger version (52K): [in this window] [in a new window]   Figure 1.   TIMP-2, 72-kDa gelatinase, and interstitial collagenase mRNA expression in normal mucosa, dysplastic mucosa, and squamous cell lung carcinomas without and with metastases. All lanes contained 10 µg of total RNA derived from: (1) normal mucosa; (2) carcinoma with metastases; (3) carcinoma with metastases; (4) carcinoma with metastases; (5) normal mucosa; (6) carcinoma without metastases; (7 ) normal mucosa; (8) dysplastic mucosa; (9) normal mucosa; (10) carcinoma with metastases; (11) normal mucosa; and (12) dysplastic mucosa. Size of transcripts is given at the right side. In the upper row the blots were hybridized with 32P nick-translated DNA probes for 72-kDa gelatinase, interstitial collagenase, and TIMP-2. Two TIMP-2 transcripts were observed.

TIMP-2 showed two transcript fragments: one high, of 3.5-kb length, and one low, of 1-kb length (Figure 1). As shown in Table 1, ranges of values for TIMP-1 and TIMP-2 of high and low transcripts varied from 0 to 11.3, 0.7 to 5.2, and 1.6 to 13.9, respectively. Figure 2 provides a graphic display of the distribution of mean values of mRNA for TIMP-1 and TIMP-2. Our data confirm the linear correlation of expression of MIs with the histology of the tissues studied, the higher transcripted fragments of TIMP-2 (3.5 kb) being associated with a low degree of differentiation and enhanced metastic potential.

View larger version (48K): [in this window] [in a new window]   Figure 2.   Mean levels of TIMP-1 and TIMP-2 (3.5 kb) mRNA transcripts.

Figure 3 illustrates the distribution of samples for which measurable levels of MP mRNA were detected. These levels seem to correlate with phenotypic alterations, with STR-3 and interstitial collagenase being the better indicators. Multiple linear regression analysis showed specifically that STR-3 constituted the most reliable index of the neoplastic process.

View larger version (43K): [in this window] [in a new window]   Figure 3.   Percentage of tissue samples with detectable levels of metalloproteinase mRNA transcripts.

Table 2 shows the correlation between STR-3 and the high transcriptional fragment (3.5 kb) of TIMP-2. In the absence of detectable STR-3 mRNA transcripts, the neoplastic process remained benign. Normal tissue expressed lower levels of TIMP-2, with four of the five specimens of normal mucosa having TIMP-2 levels < 3. Detectable levels of the STR-3 mRNA transcript appeared to be necessary for malignant transformation, and 20 of the 25 carcinomas studied had TIMP-2 levels > 3. As shown in Table 2, average levels of STR-3 were lowest for normal and dysplastic mucosa and highest for carcinoma with or without metastases; STR-3 values over 3 were observed mainly in carcinomas.

The expression of TIMP-1, TIMP-2, and MPs did not vary with degree of differentiation. Moreover, their expression in tissues showing early dysplastic changes was not influenced by the coexistence of infiltrative neoplasm at neighboring or distal areas. Immunohistochemistry revealed expression of STR-3 in dysplastic epithelial cells from bronchial mucosa (Figure 4) in two of five cases, in carcinoma cells (Figure 5) in all cases, and in some cells of the stroma (Figure 5), mainly fibroblasts. Expression of TIMP-2 was also observed in cancerous cells (Figure 6), in cells of the stroma, and in cells of normal and dysplastic mucosa (Figure 6).

View larger version (122K): [in this window] [in a new window]   Figure 4.   Dysplastic epithelial cells from bronchial mucosa expressing stromelysin-3. Avidin-biotin-complex immunoperoxidase method; magnification: ×250.

View larger version (130K): [in this window] [in a new window]   Figure 5.   STR-3 cytoplasmic expression in neoplastic cells of a squamous cell lung carcinoma with nodal metastases. Positive staining is also seen in some stromal cells (arrowheads). Avidin-biotin-complex immunoperoxidase method; magnification: ×250.

View larger version (135K): [in this window] [in a new window]   Figure 6.   TIMP-2 membrane and cytoplasmic expression in neoplastic cells of a squamous cell lung carcinoma (large arrows). Small arrow indicates expression of TIMP-2 by a stromal cell (fibroblast). Avidin- biotin-complex immunoperoxidase method; magnification: ×250.

	DISCUSSION

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In this study we performed, for the first time to our knowledge, a combined molecular and immunohistochemical analysis of MPs and their inhibitors in SCLC from the stage of dysplasia to that of carcinoma with and without metastatic infiltration. An effort was also made to correlate phenotypic changes with mRNA expression in order to evaluate the role of these molecules as factors associated with the neoplastic process and biologic behavior of this particular type of tumor. Previous studies of pulmonary carcinomas indicated that proteinases most consistently associated with malignant phenotype include STR-3 and interstitial collagenase (19, 28, 29). The results of our study reveal a strong association of the expression of STR-3 and TIMP-2 with the degree of cellular differentiation, whereas an increase in the expression levels of their mRNA is correlated with nodal metastases.

Thus, the combination of expression of STR-3 and TIMP-2 in neoplastic cells, shown either through Northern blot analysis or immunohistochemistry, is indicative of high metastatic potential, although (as the results of our study show) many other factors contribute to the degradation of the ECM, facilitating the process of cellular spread and metastasis of malignant cells. Detection of STR-3 mRNA transcripts appears to be linked to a diagnosis of malignancy, whereas measuring the levels of TIMP-2 may assist in achieving finer distinctions within the neoplastic continuum. Recently, in another experimental study, Tzunezuka and associates (30) observed that expression of a membrane-type matrix metalloproteinase-1 (MMP-1) (MT1-MMP) enhances pulmonary metastasis. They showed that MT1-MMP has proteolytic activity against components of the ECM and activates progelatinase A (72-kDa Type IV procollagenase/proMMP-2) on the cell surface.

The weak expression of STR-3 observed in dysplastic epithelium is probably due to the decreased transcriptional rate of this enzyme at early stages of carcinogenesis. On the other hand, the expression of STR-3 in differentiated carcinomas was found to be increased, an observation that supports the notion that this enzyme is principally associated with the malignant phenotype. STR-3 expression not only characterizes primary carcinomas, but has also been detected in pulmonary metastases from colonic carcinomas. A review of the literature makes it evident that STR-3 is also expressed by stromal cells (fibroblasts, fibrous cells), mainly at the periphery of the tumor (6, 28). This observation was confirmed in our cases, as is shown in Figure 5. Expression of mRNA for interstitial collagenase was detected in almost all carcinomas, as well as in three cases of dysplastic bronchial epithelium. These findings are in agreement with those of respective studies concerning cancers of the colon and breast (6, 20).

Normal bronchial mucosa expressed both TIMP-1 and TIMP-2. TIMP-2 showed two transcript fragments. Although the presence of these fragments has been described previously, the significance of their presence has not been adequately evaluated (14, 19, 21, 27). Detection of transcripted fragments of the 72-kDa and 92-kDa gelatinases in restricted areas of normal mucosa may indicate that the expression of these enzymes is induced even away from the main tumor.

MPs are enzymes that regulate the metabolism of the ECM (synthesis-degradation) and seem to play an important role in the biologic behavior of cancer cells, intervening mainly to the process of invasion in vitro and in metastasis in vivo (1, 2, 21). Studies of human cell lines have shown that mesenchymal tumor cells express gelatinases more frequently than do epithelial tumor cells, and the 92-kDa gelatinase seems to be associated with increased tumorigenicity. Data on MPs in human tumors are scarce, although a small number of studies of their role in human neoplasias have recently been published (6, 12- 14, 19).

The function of MPs is controlled at many levels, and the transcription of some members of the MP family is strongly influenced by the secretion of cytokines and other mediators (22, 23). The MPs are released from cells as inactive proenzymes that must be activated by proteolysis, often by serine proteinases that function in a cascade mechanism (24). Studies of BEAS-2B pneumonocytes that had the oncogene HA-ras activated have shown the influence of MPs as factors that act in altering the cellular phenotype and the acquisition of metastatic properties. Genomic instabilities and transactivations of chromosomal locations that encode pregelatinase molecules (collagenases IV) are the prerequisites for the activation of a series of biochemical reactions that lead to genetic and phenotypic alterations causing neoplastic transformation (25). During this process, MIs seem to play an important role, probably through deactivation mechanisms. In our study, the finding that MIs were also expressed in stromal cells corroborates the foregoing hypothesis.

In conclusion, molecular analysis and immunohistologic evaluation of enzymes that control the metabolism of ECM in SCLC have shown that: (1) The expression of STR-3 and interstitial collagenase is statistically significantly associated with aggressive biologic tumor behavior, defined as the potential for extensive infiltrative and metastatic activity. Because similar findings were observed in carcinomas of the colon and breast, it is further suggested that these two enzymes contribute to neoplastic progression. (2) Overexpression of TIMPs mRNA is observed mainly in carcinoma cells, rather than in dysplastic epithelium or normal tissue, a finding strongly suggesting the possible involvement of growth factors and cytokines that control their expression.