Basal cell carcinoma (BCC) is a malignant skin tumor composed of cells similar to those in the basal layer of the epidermis and its appendages 1. Although BCC is a malignant tumor, it is generally only locally invasive and rarely metastasized, but if neglected, it may cause destruction of the nose and eye and may lead to death 2. Aggressive histologic variants with a high risk of recurrence include the micronodular, infiltrative, basosquamous, morpheaform, and mixed subtypes 3. Squamous cell carcinoma (SCC) is a malignant tumor of epithelial keratinocytes. In contrast to BCC, SCC arises from more superficial layers of the epidermis and may be more aggressive as it can invade tissue beneath the skin and spread to lymph nodes or distant parts of the body 4. Seborrheic keratoses (SKs) are noncancerous (benign) skin growths that some individuals develop as they age. SKs grow slowly in groups or singly 5,6.
Proteases are defined as hydrolytic enzymes acting on peptide bonds in a process termed proteolysis. Proteases, like all enzymes, are very specific; thus, they recognize side chains to know where to cleave 7. Proteases are classified, on the basis of their catalytic mechanisms, into aspartic, metallo, cysteine, glutamic, threonine, and serine proteases 8. Cathepsins are lysosomal proteolytic enzymes subdivided according to their active site amino acids, which confer catalytic activity in cysteine (cathepsins B, C, F, H, K, L, N, O, S, T, U, W, and X), serine (cathepsins A and G), and aspartic cathepsins (cathepsins D and E) 9,10. Cathepsin D potentially activates transglutaminase that cross-links the precursor proteins involucrin, loricrin, keratolinin, and cornifin and catalyzes the production of cornfield envelope. Therefore, cathepsin D acts by two mechanisms: by causing an imbalance between proteinases and their inhibitors and by inducing the activation of transglutaminase involved in abnormal keratinization of SCC 11.
Matrix metalloproteinases (MMPs), also called matrixins 12, are a family of zinc-dependent proteolytic enzymes, which, as their name implies, are associated with degradation of the extracellular matrix (ECM) including the basement membrane (BM) as well as nonmatrix proteins. MMPs can be divided into subgroups according to their structure and substrate specificity 13, which include collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs, and others 14. They mediate tumor angiogenesis, malignant conversion, proliferation, and apoptosis by degrading BM, cell attachment proteins, and various matrix components, as well as by activating growth factors. Metalloproteinase-9 (MMP-9) is one of the gelatinases subgroup (gelatinase B). It plays an important role in angiogenesis as well as tumor invasion and metastasis, and has frequently been associated with a poor prognosis 15.
A concurrent comparison of the enzyme activities in various kinds of tumors was may be beneficial to determine the biochemical aspects of malignant tumors, which may be lead to the possibility of good therapeutic options 16.
Therefore, the aim of this work was to explore the immunohistochemical expression of cathepsin D and MMP-9 in SCC, BCC, and SK compared with normal skin of controls.
Patients and methods
The current retrospective case–control study was carried out on 45 patients with available paraffin blocks and clinical data collected from the archives of the Pathology Department, Faculty of Medicine, Tanta University Hospitals. The blocks were divided into groups as follows: group I included 15 blocks of SKs, group II included 15 blocks of BCCs, group III included 15 blocks of SCCs, and group IV included 10 blocks of normal skin as a control group. The control group was age and sex matched to the cases whose paraffin blocks were examined in the current study. Specimens of the control group were obtained from normal skin specimens received from head and neck plastic operations. All blocks were sectioned and stained with hematoxylin and eosin stain for the detection of general histopathological criteria of the tumors and immunohistochemistry for cathepsin D and MMP-9, in the same way, by the following steps.
Three to 5 μm sections were deparaffinized in xylene and then rehydrated in a descending concentration of alcohols (ethanol). Sections were incubated in 0.3% H2O2 for 30 min to block endogenous peroxidases. Slides were then washed with PBS and heated in an 830-W microwave oven for at least 15 min in 10 mmol/l sodium citrate buffer (pH 6.0) for antigen retrieval. Sections were incubated with the two primary antibodies (cathepsin D: rabbit, 1 : 50 anti-human polyclonal antibody, catalog #A0032; ScyTek, Logan, Utah, USA) and [MMP-9 (Ab-9), and rabbit anti-human polyclonal antibody, catalog #RB-9234-P; Lab Vision, Fremont, California, USA] overnight at 4°C. For the negative control, the primary antibody was replaced with PBS. Rabbit anti-mouse horseradish peroxidase-conjugated secondary antibody was added, followed by incubation for 40 min at room temperature. The color was developed using diaminobenzidine (DAB) as a chromogen. Slides were washed extensively with PBS after each step. Finally, they were counterstained with Meyer’s hematoxylin. The internal positive control was the sweat glands in each section. Both markers showed cytoplasmic positivity. Assessment of immunoreactivity was performed individually by two independent pathologists and then a consensus was achieved when any discrepancy was found. The stained sections were scanned using the ×4 objective lens to allocate the areas of highest positivity for the epithelial cells of each lesion and the stromal cells (including the inflammatory cells and the stromal fibroblasts). The percentage of positive cells in each of the two components was determined in 10 high-power fields and the mean percentage for each type of cells was recorded separately in each section. Then the extent of positivity for both components were scored as follow: 0; no immunoreactivity −1; less than 10% of the cells positive −2; 10–25% of cells stained −3: 25–50% of cells stained −4; more than 50% of cells stained. The overall intensity of the stain in each section was assessed subjectively, in relation to the positivity of the sweat glands. In healthy controls, the immunoreactivity was limited to sweat glands, which was considered a nonspecific reaction and was used as a reference. The investigated cells were considered positive if the intensity of their staining was higher than the intensity of the sweat glands 17. The intensity of the stain was classified into negative (score zero = the intensity of the sweat gland staining), mild (score 1), moderate (score 2) and intense (score 3). Score 1, 2, 3 were higher than the sweat gland staining intensity.
An immunoreactivity index was obtained by multiplying the extent by the intensity of staining and the range of score was as follows: negative 0–2, mild >2–5, moderate >5–8, and intense >8 17.
All data were transferred to the statistical package for the social sciences, version 15 (IBM Co., New York, New York, USA) for analysis. Data were summarized using mean±SD for quantitative variables and number and percentage for qualitative variables. Comparisons between nonparametric quantitative variables were made using Mann–Whitney U-test and Kruskal–Wallis test. The correlation between ordinal variables was assessed using Spearman’s rank correlation. Statistical significance was determined at a level of P value less than 0.05 and high significance was considered at a level of P value of 0.001 or less.
The demographic, clinical, and histopathological criteria of the groups studied are presented in Table 1.
In healthy controls, immunoreactivity was limited to sweat glands’ epithelium, which was considered a nonspecific negative reaction in both markers (Fig. 1). Positivity was found in both epithelial cells of the lesion and the stromal inflammatory and fibroblastic cells (Figs 2–8). The expression of both markers was distributed homogenously in SK (Figs 2 and 3), whereas positivity was highest in the malignant lesions (Figs 9 and 10).
Cathepsin D epithelial and stromal scores showed statistically significant differences between the four groups with the highest values in SCC. The epithelial cathepsin D score was significantly higher than the stromal score in SK (P=0.001) and BCC (P=0.036) (Table 2). MMP-9 epithelial and stromal scores also showed statistically significant differences between the four groups with the highest level of expression in SCC. The expression of MMP-9 in the stromal cells was higher than its expression in the epithelial cells in all groups, except in the normal control group. The difference between the epithelial and stromal score was statistically significant in SK (P=0.018) and BCC (P=0.03) (Table 3). The expression of both markers in both the epithelial and the stromal compartments showed a statistically significant increase in BCC with a high risk of recurrence [four cases (26.7%) morphea-like and three cases (20.0%) micronodular type] than those with a low risk of recurrence [two cases (13.3%) pigmented type, four cases (26.7%) nodular type, and two cases (13.3%) adenoid type] (Table 4). The expressions of both markers in epithelial and stromal compartments were significantly positively correlated with advancement in the histological grade of SCC (Table 5 and Fig. 11). There was a significant positive correlation between cathepsin D and MMP-9 in both the epithelial and the stromal compartment (Table 6 and Fig. 12).
Tumors derived from keratinocyte such as SCC, BCC, and SK have different clinical characteristics on the basis of invasive and metastatic potentials. In the present study, cathepsin D epithelial and stromal scores were statistically significantly elevated in all the groups studied compared with the normal control group with the highest values in SCC. This result was in agreement with that of Tsuboi et al. 18, who found high cathepsin D activity in the same investigated tumors, but with no significant difference, and suggested that no difference was found in the lysosomal functions of the tumors. The present study was also in agreement with a study carried out by Fröhlich et al. 19, who found that in BCCs, cathepsins B, L, and D were increased. In addition, Kawada et al. 20 found that cathepsin D was highly expressed in all lesions of SCC as cathepsin D is important in the invasion and metastasis of SCC.
Kawada et al. 21 noted that half of SCC cases showed enhanced cathepsin D expression, but Bowen’s disease, SK, or BCC did not show any staining for cathepsin D. They concluded that upward production of cathepsin D might be associated with malignancy of keratinocytes. It has also been suggested that the catalytic activity of secreted cathepsin D may be implicated in releasing growth factors, such as fibroblast growth factor-2, from the ECM 22. Cathepsin D has been shown to play a crucial paracrine role in the tumor microenvironment by stimulating fibroblast outgrowth and tumor angiogenesis, and inhibiting antitumor responses. In the mean time, endothelial cells have been shown to secrete procathepsin D through the action of inflammatory cytokines, producing a vicious circle 23.
In the present study, the expressions of cathepsin D in both the epithelial and the stromal compartments were significantly higher in BCC types with a high risk of recurrence than the low-risk types and were significantly positively correlated with advancement in the histological grade in SCC. Our results were in agreement with those of Fröhlich et al. 19, who detected correlations between cathepsin D levels and tumor size, grade, aggressiveness, incidence of metastasis, prognosis, and a degree of chemoresistance in BCC. Also, Kawada et al. 20 and Goldmann et al. 24 suggested that cathepsin D may be a prognostic factor of SCCs.
In the present study, MMP-9 epithelial and stromal scores showed a significant increase in all the groups studied compared with the normal control group with the highest values in SCC. The present results were in agreement with those of the study carried out by O’Grady et al. 17, who reported that MMP-9 stromal expression was recorded in all SCCs and BCCs studied. In the current study, the expression of MMP-9 in the stromal cells was higher than its expression in the epithelial cells in all groups. O’Grady et al. 17 observed that MMP-9 expression was more extensive in the stroma than in the epithelium of SCC and BCC. Kerkelä et al. 25,26 previously reported that stromal cells are the major source of MMPs. Boyd et al. 27 reported that MMP-9 was not detected in the BCC epithelium, but in neutrophils and macrophages surrounding it. Whether this strong stromal positivity for MMP-9 is a result of the inflammatory response around the tumors rather than specific tumor activation is still unknown 17.
In the present study, the expression of MMP-9 in both the epithelial and the stromal compartments was significantly higher in BCC with a high risk of recurrence than those with a low risk of recurrence. This result was also in agreement with that of Boyd et al. 27. In the current study, there was a statistically significant relationship between MMP-9 expression and the histopathological grade of SCC cases. This was in agreement with the results of other authors 28,29, who reported that the expression was more frequent in poorly differentiated tumors when compared with less aggressive SCCs. Moreover, O’Grady et al. 17 found that the intensity and extent of MMP-9 expression appeared to be related to the degree of cellular atypia and tumor progression in SCC cases. Recently, Zhu et al. 30 reported that MMP-9 expression was detected in 11/15 of primary SCC, 11/11 of metastatic SCC cases but not in normal keratinocytes, all SK and BCC specimens.
Dumas et al. 28 reported that cell–matrix interactions are important upregulators and downregulators for MMPs. The changes in the compositions of different BM components may influence MMP expression. Type VII and IV collagens were expressed less in SCC than BCC. Type IV collagen is the major component of BM and represents the first barrier in tumor cell invasion. Thus, reduced expression of collagen IV, combined with an increased expression of MMP-9, could account for the increased invasive potential of SCC versus BCC.
Interactions between stromal and epithelial cells are important in cancer progression and metastasis. Stromal and tumor cells can exchange numerous tumor-promoting factors, such as growth factors, cytokines, and proteases, to activate the adjacent ECM and, in turn, stimulate migration and invasion and promote the proliferation and survival of tumor cells 30,31. Carcinoma, from its earliest stages, seems to depend on the ability of transformed epithelial cells to first recruit and then subvert a variety of stromal cells originating from adjacent normal tissue to become infiltrating or invading 32.
To the best of our knowledge, this is the first study to discuss the expression of both cathepsin D and MMP-9 in epidermal tumors. It can be concluded that both proteases’ immunoreactivity could be useful predictors of tumor progression and to recognize patient groups that need more aggressive treatment. Moreover, inhibitors of both enzymes could be studied as therapeutic options to limit the invasiveness of SCC and recurrence in BCC.
Conflicts of interest
There are no conflicts of interest.
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Keywords:© 2014 Egyptian Women's Dermatologic Society
cathepsin D; epidermal tumors; matrix metalloproteinase-9; proteases