Basal cell carcinoma (BCC) is the most common malignancy in humans and accounts for nearly 80% of all nonmelanoma skin cancers. It typically occurs in areas of sun exposure 1. Most BCCs are not invasive, rarely form metastases, or lead to mortality 2. However, delayed diagnosis and resultant local progression of the tumor may lead to involvement of surrounding tissues and severe anatomical deformations, especially in facial lesions3.
Matrix metalloproteinase (MMP) family members are the major enzymes that degrade the components of the extracellular matrix (ECM) 4. They are a group of genetically related enzymes that play a role in several physiological remodeling processes, including tissue repair, morphogenesis, and embryological development 5,6. MMPs are also involved in several pathological conditions including acute and chronic inflammation, skin aging, chronic ulcers as well as cancers, where they play a major role in angiogenesis, cell proliferation, tumor invasion, and metastasis 7,8.
Among the various MMPs, MMP-1, also known as collagenase-1 or interstitial collagenase, is the only enzyme able to initiate breakdown of the interstitial collagens, collagen types I, II, and III 9. It plays an important role in tumor progression and invasion because of its ability to degrade fibrillar collagens and components of the basement membrane. It also cleaves cell surface molecules and other nonmatrix substrates, making MMP-1 a multifunctional protein 10,11.
The activity of MMPs, particularly MMP-1, is considered to be elevated in BCC relative to normal skin 12. Moreover, MMP-1 concentration and expression in stromal cells was found to correlate to the loss of the palisading arrangement in BCC 13. Ultraviolet (UV) irradiation is an important etiological factor for BCC and also stimulates the expression of MMP-1 14. MMP-1 levels may also be elevated in skin with chronological aging 15,16. Whether upregulation of MMP-1 in BCC is merely the tumor’s influence or a result of sun exposure is a point that needs further evaluation 17.
The aim of the present work was to compare MMP-1 expression using immunohistochemistry in BCC-related skin (tumoral, peritumoral, and safety margin) with nontumor associated, sun-exposed skin from the same patient to determine the possible role of MMP-1 in the pathogenesis of BCC and its invasiveness.
Patients and methods
This study included 24 patients with BCC, Patients were recruited from the Outpatient Dermatology Clinic of Ain Shams University and Alhoad Almarsood Hospital. This study was approved by the local ethical committee.
Patients had different clinical and histological subtypes of BCC that was diagnosed by histopathological examination. Patients who received immunosuppressive therapy (e.g. chemotherapy) or those on regular vitamin D intake were excluded as these may affect MMP levels 18,19.
All patients were subjected to a thorough assessment of history: history of present illness, history of excessive sun exposure or exposure to chemicals such as arsenic or presence of BCC-associated syndromes, and past and family history of similar condition or other skin cancers. Dermatological and general examinations were carried out for all patients. An informed consent was obtained from patients and the study was approved by Research Ethics Department in Ain Shams University.
Two skin biopsies were taken from each patient: an excisional biopsy of the tumor and a punch biopsy from normal skin on the dorsum of the hand. Each biopsy specimen was subjected to hematoxylin and eosin (H&E) staining for routine histopathological examination and immunoperoxidase technique to study the expression of MMP-1 by immunohistochemistry.
H&E-stained sections were examined for diagnosis and tumor subtypes were determined according to the definitions provided by Crowson 20.
The immunoperoxidase technique was used to study the expression of MMP-1 by immunohistochemistry using the MMP-1 Ab-6 rabbit polyclonal antibody (manufactured by Thermo Scientific; Waltham, Massachusetts, USA) at a dilution of 1 : 25–50 for 20 min at room temperature using Ultravision LP systems (Waltham, Massachusetts, USA). Staining of formalin-fixed tissues required boiling tissue sections in 10 mM citrate buffer, pH 6.0, for 10–20 min, followed by cooling at room temperature for 20 min.
Assessment of MMP-1 staining was performed according to the number of cells stained in high power field (×400) in four areas: tumoral (cells of the tumor nest), peritumoral (the stromal area surrounding immediately the tumor nests) (Fig. 1), safety margin (area of normal skin surrounding the tumor), and control areas. Five nonoverlapping fields were examined in each area and scores were assigned according to the average number of stained cells: no stained cells=0 score, stained cells less than 10%=+1, stained cells from 10 to 50%=+2, and stained cells more than 50%=+3. Immunostaining detection was performed semiquantitatively according to Prasad et al.21, with some modification.
Software (SPSS 16.0; SPSS Inc., Chicago, Illinois, USA) was used for the data analyses. Descriptive statistics were calculated as mean±SD and range for quantitative data and number (n) and percentage (%) for qualitative data. Statistical analysis was carried out using (a) Student’s paired t-test to assess the statistical significance of the difference between two study group means and (b) analysis of variance test to assess the statistical significance of the difference between the means of more than two study groups.
Probability value: level of significance: P value greater than 0.05 is nonsignificant; P less than or equal to 0.05 is significant.
The study included 24 patients with BCC. The clinical data of patients and histopathological tumor subtypes are summarized in Table 1.
Immunohistochemical staining for MMP-1 showed moderate staining in the basal layer and appendages, which was similar for all specimens. However, there was a noticeable difference in the expression of MMP-1 in the dermis.
Comparison of matrix metalloproteinase-1 expression between the studied areas
There was a statistically significant difference between the four areas examined as determined by one-way analysis of variance (F=2.7, P=0.003) (Table 2).
Comparison of matrix metalloproteinase-1 expression within lesional skin
The peritumoral area showed significantly higher MMP-1 expression compared with the tumoral area (1.83±0.82 vs. 1.04±0.75; P=0.006). Similarly, the peritumoral area showed significantly higher MMP-1 expression compared with safety margin areas (1.83±0.82 vs. 1.33±0.92; P=0.04) (Table 2).
However, no statistically significant difference was found between enzyme expression in tumoral and safety margin areas (1.04±0.75 vs. 1.33±0.92; P=0.183).
Fibroblasts, macrophages, and to a small extent lymphocytes were the cells responsible for the high MMP-1 level in the peritumoral area (Fig. 2).
Comparison of enzyme expression in lesional versus control skin
The peritumoral area showed statistically significantly higher MMP-1 expression compared with the control areas (1.83±0.82 vs. 1.21±0.42; P=0.003). However, no statistically significant difference was found between tumoral and control areas (1.04± 0.75 vs. 1.21±0.42; P=0.356) or between the safety margin and control areas (1.33±0.92 vs. 1.21±0.42; P=0.574) (Table 2).
Comparison of matrix metalloproteinase-1 expression in relation to clinical data and histopathological subtypes
Comparison of enzyme expression in each of the studied areas between men and women
No significant difference was found between men and women in MMP-1 expression in tumoral (P=0.84), peritumoral (P=0.168), safety margin (P=0.83), or control areas (P=0.544) (Table 3).
Comparison of enzyme expression in each of the studied areas between younger (<60 years) and older (≥60 years) age groups
Patients were divided according to age into younger (<60 years) and older (≥60 years) age groups. No significant difference was noted between the two groups in MMP-1 expression in tumoral (P=0.128) and peritumoral (P=0.137) areas. However, MMP-1 expression in the safety margin and control areas from the older age group was statistically significantly higher compared with their counterparts in the younger age group (P=0.049 and 0.0375, respectively) (Table 3). Most of the MMP-1 expression was found in the dermis.
Comparison of enzyme expression in each of the studied areas between patients with and without a history of excessive sun exposure
Patients were divided into two groups according to history of excessive sun exposure (outdoor occupations, e.g. farmers, drivers). No significant difference was detected in MMP-1 expression between patients with and without excessive sun exposure in tumoral (P=0.149), peritumoral (P=0.73), and safety margin areas (P=0.5). However, patients with outdoor occupations showed statistically greater expression of MMP-1 (mainly in the dermis) in control areas in comparison with control skin from patients without excessive sun exposure (P=0.0223) (Table 3).
Comparison of enzyme expression in each of the studied areas between undifferentiated and differentiated tumors
A statistically significant difference in the MMP-1 expression between undifferentiated and differentiated BCC was detected in peritumoral areas (P=0.033), whereas it was insignificant in tumoral (P=0.829), safety margin (P=0.576), or control areas (P=0.466) (Table 3).
Gross appearance, H&E-stained specimens, and immunohistochemical staining for MMP-1 of one of the cases are shown in Fig. 3.
Although MMP-1 expression in BCC has been reported in several studies, it remains uncertain whether this increase is a consequence of UV exposure or an effect of the tumor itself 17. This is because of the fact that appropriate sun-exposed normal skin from the same individuals has not been used as control. To the best of our knowledge, this is the first study comparing MMP-1 expression in BCC-associated tissue (tumoral, peritumoral, safety margin) with nontumor-associated sun-exposed skin from the same patient. This helps to eliminate, primarily, the influence of sun exposure as well as the possible effect of other factors, e.g. age and individual variances on MMP-1 expression.
In the present study, the expression of MMP-1 was found to be significantly higher in the peritumoral (stromal) area in comparison with each of the tomoral, safety margin, and control areas. MMP-1 was mainly expressed by stromal fibroblasts, macrophages, and, to a small extent, lymphocytes. The higher expression of MMP in stromal tissue of BCC has been investigated in several studies 12,17. It has been suggested that tumor cells secrete several factors including interleukins, cytokines, and angiogenic factors that trigger the stromal cells around the cancer cells to produce MMPs 4,17. This may explain our finding that stromal cells showed more staining concentrations (MMP-1 expression) than the tumoral cells.
Our results are in agreement with previous studies17,22–25 that reported that MMP-1 is mainly expressed by stromal cells surrounding malignant cells in BCCs, postulating the role of inflammatory cells in the process of propagation. The expression of MMP-1 was found to be significantly enhanced by fibroblasts at the invasive front of BCC, suggesting its role in the initial steps of tumor proliferation, which is mediated by cleaving the ECM 24. Varani et al.12 reported that much of the MMP activity associated with BCC originates in the stroma, although the tumor cells themselves and the adjacent normal epithelium may also contribute. Interestingly, Boyd et al.24 suggested that abundant peritumoral expression of tissue inhibitors of metalloproteinases (TIMPs) in BCC limits ECM degradation permissive for cancer cell migration, emphasizing the importance of the balance between MMPs and their inhibitors in determining the behavior of the cancer cells.
In the present study, we compared MMP-1 expression between differentiated and undifferentiated lesions. MMP-1 showed significantly higher expression in peritumoral areas of undifferentiated BCCs compared with differentiated lesions, suggesting the relation of MMP-1 to tumor behavior. Overexpression of MMP-1 has been found to correlate positively with tumor aggressiveness and a poor clinical outcome in BCC as well as in several types of other cancers. Agarwal et al.26 reported that MMPs including MMP-1 are potentially associated with poor tumor differentiation in BCC. MMP-1 expressed in stromal cells was associated with the invasiveness of squamous and BCC of the skin, and with the loss of palisading arrangement in BCC 13. In addition, stromal MMP-1 overexpression is a poor prognostic marker in a variety of advanced cancers, including, melanoma, colorectal, breast, and lung carcinomas 22,27,28. In contrast, Zlatarova et al.25 found no relation between the invasiveness and the expression of MMP-1 in BCC, which suggests that many factors (including TIMPs and other MMPs) may be responsible for the behavior of the tumor.
In our study, we examined the expression of MMP-1 in relation to a history of excessive sun exposure. It was found that MMP-1 expression in each of the tumoral, peritumoral, and safety margin areas in patients with a history of excessive sun exposure did not differ significantly from those without a history of excessive sun exposure. A possible explanation is that MMP-1 expression in tumor-associated skin is probably because of the presence of the tumor itself rather than because of sun exposure. However, MMP-1 expression in control areas was significantly higher in those with excessive sun exposure compared with the other group. This is in agreement with other authors who reported that UV-inducible MMPs are constantly elevated in chronic photodamaged skin because of exposure to chronic low-dose UV irradiation 29,30. It is generally accepted that UV irradiation generates severe oxidative stress in skin cells, resulting in increased activator protein-1 and proinflammatory cytokine activity, which increase the synthesis and expression of MMP-1 by dermal fibroblasts 31,32. MMP-1 is the major protease capable of initiating degradation of native fibrillar collagens in human skin. Once cleaved by MMP-1, collagen can be further degraded by other MMPs 29. The elevated levels of MMPs and reduced production of type I collagen are likely responsible for chronic, progressive degradation of the dermal collagenous ECM and loss of collagen in photodamaged human skin 33.
We divided the patients into two age groups to detect the influence of age on the expression of MMP-1 in the examined areas. No difference was noted when the tumoral or peritumoral areas were compared between the two age groups. However, MMP-1 expressions in the safety margin and control areas were significantly higher in the older group in comparison with their counterparts in the younger group. This difference is mostly because of chronological aging as a result of the age difference between the two groups. This is in agreement with Varani et al.15, who reported that during natural or chronological aging of the skin, the same MMPs that are upregulated acutely in response to UV radiation are gradually increased in intact (old vs. young) skin.
Thus, the elevated MMP-1 expression in BCC is mostly dependant on the presence of the tumor. This is based on the finding of significantly elevated MMP-1 in peritumoral areas compared with other areas including control sun-exposed skin from the same patient and is further justified by the absence of a significant difference in peritumoral areas between patients with and without excessive sun exposure. Indeed, if the elevation of MMP-1 in BCC was a result of sun exposure, its expression would have been similar in all areas examined as they are all sun exposed. Furthermore, it would have been higher among patients with excessive sun exposure. The absence of a significant difference in the peritumoral area between young and old age groups shows that elevated MMP-1 in BCC is not because of an age-related process.
MMP-1 expression is elevated in the peritumoral skin of BCC and may play an important role in its invasion. This elevation is probably because of the presence of the tumor itself, rather than other factors such as UV irradiation or aging.
Further studies including a larger number of patients are needed to investigate the significance of the various MMPs in BCC as well as other tumors. Investigating the difference in MMP expression in benign versus invasive tumors, as well as in tumors related and unrelated to sun exposure, will help to further define the role of MMP in various tumors and their invasiveness. In addition, studying the balance between MMPs and their inhibitors (TIMPs) may provide a better understanding of the role of these two important players in the regulation of the tumor microenvironment.
Finally, it is important to note that the identification of the role played by MMP in different pathological processes has opened new horizons for the evolution of MMP inhibitors. Both synthetic and natural MMP inhibitors are being developed and some are already in clinical trials on humans 34. These may be promising agents in a wide range of indications: from combating photoaging, to the prevention and increasing the effectiveness of cancer therapy.
Conflicts of interest
There are no conflicts of interest.
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