Nonmelanoma skin cancer (NMSC) is the most common human cancer, and despite growing public awareness of the harmful effects of sun exposure, its incidence continues to rise 1,2. NMSC refers mainly to basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) 3. In NMSC, keratinocytes can hyperproliferate and continually regenerate through regulated proliferation followed by differentiation into the suprabasal epidermal cell types 4. This process is essential for the protective barrier function of the skin. Skin disorders including NMSC can result from dysregulation in the homeostasis of keratinocyte function 5. Keratoacanthoma (KA) is a rapidly growing skin tumor, which is characterized by a distinct keratin-filled, cup-shaped appearance. In addition, the histopathological findings of KA are occasionally indistinguishable from those of well-differentiated SCC, leading some authors to believe that KA is a subtype of SCC 6. Seborrheic keratosis (SK) is a noncancerous (benign) skin growth that some people develop as they age. They grow slowly, in groups or singly 7. Psoriasis is a multifactorial disease involving both genetic predisposition and external triggers, resulting in epidermal and immune dysfunction with epidermal barrier function disturbance, which has been suggested to contribute to cutaneous inflammation as well as to disease exacerbation 8.
Aquaporins (AQPs) are a family of small, integral membrane proteins that transport water and in some cases solutes, such as glycerol, termed aquaglyceroporins (AQPs 3, 7, and 9). AQP-3 is one such water-transporting/glycerol-transporting protein 9. It is expressed strongly in plasma membranes of basal epidermal cells in skin as well as the suprabasal layer 10. AQP-3 and glycerol channels interact functionally in epidermal keratinocytes of the skin to inhibit their proliferation. Others have suggested that AQP-3 is proproliferative in keratinocytes and is upregulated in NMSC. Results suggest that abnormalities in AQP-3 expression correlate with hyperproliferation in NMSC 11.
Little is known about the distribution of AQP-3 in normal epidermis and NMSC; therefore, there is a need to study the expression and localization of AQP-3 in normal skin and detect its value in the pathogenesis of NMSC such as BCC and SCC as well as inflammatory proliferative skin disorders such as psoriasis.
Patients and methods
The present study included 186 samples representing 33 BCCs (group I), 30 SCCs (group II), 33 SKs (group III), 30 KAs (group IV), 30 psoriasis cases (group V), and 30 normal skin samples (group VI, control group), which were obtained during plastic surgical operations. The samples were obtained from 99 (33 from each of BCC and SK, 3 of SCC, and 30 of plaque psoriasis) patients presenting to the Dermatology and Venereology, Plastic Surgery, and Oncology Departments of Tanta University Hospitals during the period from January 2013 to May 2014. Approval for the study was obtained from the research ethics committee of the hospital (code no. 623/01/13), and written informed consent was obtained from each participant. A total of 57 (27 of SCC and 30 of KA) archival paraffin blocks were prepared at the Pathology Department of Tanta University Hospitals. All patients had their clinical diagnosis on the basis of typical appearance of skin lesions confirmed by typical histopathological examination. The available clinical data of the blocks were collected from pathology requests with regard to age and sex.
Patients who agreed to join the study gave their informed consent, and were not suffering from any other dermatological or systemic diseases. Patients with psoriasis who did not apply any topical or systemic treatment on the affected site for 1 month were included in this study. Psoriasis area and severity index (PASI) score calculations were carried out to determine the severity of psoriasis 12.
After obtaining informed written consent, excisional skin biopsy specimens were obtained from patients undergoing plastic surgery, and punch biopsy samples were obtained from psoriatic patients.
The biopsy samples were immediately fixed in 10% formalin and embedded in paraffin.
All paraffin blocks (from archives, patient specimens, and controls) were cut into 3–5-m-thick slices using an ordinary microtone on glass slides. The samples were then stained with hematoxylin and eosin for light microscopy to assess the general histopathological pictures of the studied tumors and psoriasis samples. Next, SCC samples were graded according to the Broder classification 13. Immunohistochemical analysis was carried out using primary antibody rabbit polyclonal antihuman AQP-3 (1 : 300 dilution; Novus, Littleton, Colorado, USA) to detect AQP-3 expression.
Procedures of staining 11
Five-micrometer-thick sections were cut from the paraffin blocks on positively charged adhesive slides that were deparaffinized in xylene and rehydrated in graded descending alcohol. For antigen retrieval, sections were boiled in 10-mm citrate buffer, pH 6.0 (Neomarkers Inc.; Fremont, California, USA) for 20 min and then washed in phosphate buffered saline (PBS), pH 7.3. Blocking of endogenous peroxidase activity by 6% H2O2 in methanol was carried out. The slides were then incubated overnight with the primary antibody in a humidified chamber at 4°C, washed in PBS, pH 7.3, incubated with a secondary antibody (Biotinylated Goat antipolyvalent, ready to use; Lab Vision Corporation, Fremont, California, USA) for 10 min at room temperature, and again washed in PBS, pH 7.3. Diaminobenzidine was used as a chromogen. Slides were finally counterstained with Mayer’s hematoxylin.
Evaluation of immunostaining 11,14
Positivity was evaluated in lesion cells, and we assessed both the localization of the stain (membranous or cytoplasmic) and the intensity of staining and percentage of expression. The intensity was evaluated using the positivity of the sebaceous gland and/or sweat gland as an internal landmark for intermediate or moderate staining intensity (positive reaction). Intensity was assessed in lesional cells as follows: negative (0), less than 25% of cells showed positive staining; weak or mild (+1), 25–50% of cells showed positive staining; moderate (+2), 50–75% of cells showed positive staining; and marked or strong (+3), more than 75% of cells showed positive staining.
Data were fed to a computer and analyzed using IBM SPSS software package, version 20.0 (Chicago, Illinois, USA). Qualitative data were described using numbers and percentages. Quantitative data were described using ranges (minimum and maximum), mean, SD, and median. Comparison between different groups regarding categorical variables was tested using the χ2-test. When more than 20% of the cells had expected count less than 5, correction for χ2 was conducted using Fisher’s exact test or Monte Carlo correction. Significant test results are quoted as two-tailed probabilities. Statistical significance was determined at a level of P value of up to 0.05 15.
Clinical results are presented in Table 1.
Regarding the histological subtypes of different tumor groups, in BCC [12 (36.4%) nodular, 12 (36.4%) adenocystic, and 9 (27.3%) superficial], in SCC [9 (30%) well-differentiated gradeI, 12 (40%) moderately differentiated grade II and 9 (30%) poor differentiated grade III], in SK [18 (54.5%) acanthotic, 12 (36.4%) pigmented, and 3 (9.1%) adenoid], in KA [15 (50%) proliferating, 9 (30%) well developed mature, and 6 (20%) involuting].
The positivity of AQP-3 expression in the studied groups was membranous–cytoplasmic.
The normal epidermis showed membranous expression of AQP-3 in the basal layers more than the suprabasal layers, and was absent from the stratum corneum (SC). BCC showed diffuse patterns of AQP-3 in the normal overlying epidermis and was downregulated in the tumor cells of BCC. In SCC, a patchy pattern was detected, with some areas of the lesion showing marked staining for AQP-3 and the others showed weak or no staining. Decreased or absent expression of AQP-3 was found in keratin pearls. In SK, AQP-3 expression was diffuse and was decreased or absent in the horn cyst. AQP-3 expression in KA showed marked staining in some regions and weak or no staining in other regions. In the psoriasis group, AQP-3 showed mainly a granular cytoplasmic pattern together with mild staining of the plasma membranes in some cases, especially in the basal layers and to a lesser extent in the suprabasal layers. In psoriasis, nuclear staining was nonspecific and was found in all cases, and thus it was neglected during interpretation.
The differences in the percentage of its expression between lesional skin of the disease groups compared with normal skin of the control group are shown in Figs 1–6b. The expression of AQP-3 in the different disease groups showed significant downregulation compared with the control group (P≤0.001), except for BCC, where there was no significant downregulation. The highest mean value of the studied groups was control (2.40±0.52) followed by BCC (2.36±0.67), psoriasis (2.33±0.66), KA (1.40±0.52), SK (1.30±0.82), and then SCC (1.18±0.87). The expression of AQP-3 between the different studied groups showed significant differences (BCC vs. either SCC, SK, or KA) (psoriasis vs. either SCC, SK, or KA) (P≤0.001) (Table 2).
With regard to the percentage of AQP-3 expression, there was no relationship with age of patients and duration of disease. There was a statistically significant positive correlation with the PASI score in psoriatic patients (P=0.001) (Fig. 7).
Localization of AQP-3 in the different studied groups was mainly cytoplasmic. There was a significant difference between the disease groups and controls (Table 3).
The AQP-3 plays a role in cutaneous hydration, elasticity, cell proliferation, and migration 16. The present study detected the membranous expression of AQP-3 in normal skin of the control group, AQP-3 staining was present in the basal cell layer and to a lesser extent in the suprabasal layers, whereas in the disease groups the same pattern was observed in the normal-appearing overlying epidermis, and cellular localization of AQP-3 in tumor cells was mainly membranous–cytoplasmic. The membranous pattern of AQP-3 is in agreement with the fact that AQP-3 is an integral membrane protein, which is localized mainly to the plasma membrane, particularly in suprabasal layers 17. Cytoplasmic staining in basal keratinocytes of normal human skin has also been reported by Sougrat et al.18. In the present study, the normal epidermis showed the presence of AQP-3 in the basal layers more than the suprabasal layers and its absence from the SC; this result was also reported by Olsson et al.19. They stated that this pattern creates a gradient of AQP-3 that may lead to a corresponding water gradient in the epidermis, with a sharp decrease of water content in the SC. This maintains optimal hydration of the epidermis while preventing excessive water loss at the SC. The present study detected the expression of AQP-3 in normal skin and in tumor cells. There was a statistically significant downregulation of AQP-3 expression in all the tumor groups compared with the control group, except for BCC where there was no significant downregulation. In this study, BCC showed a diffuse pattern of AQP-3 expression, which was found in the normal overlying epidermis and was downregulated in the tumor cells of BCC; however, in SCC, a patchy pattern was detected, with some areas of the lesion showing marked staining for AQP-3 and others showing weak or no staining. Decreased or absent expression of AQP-3 was detected in keratin pearls. In agreement with the results of the present study, Voss et al.11 and Seleit et al.20 showed downregulation of AQP-3 in both tumors. They suggested that downregulation of AQP-3 in the epidermis may be a marker of tumorigenic potential. Qin et al.21 showed that the decreased AQP-3 level in NMSC is associated with keratinocyte proliferation in tumorigenesis. Similarly, Hara-Chikuma and Verkman 16 demonstrated that loss of AQP-3 inhibits human keratinocyte proliferation, and adenovirus-mediated AQP-3 re-expression returns proliferation values to normal. On the other hand, Kusayama et al.22 reported increased expression of AQP-3 in SCC and suggested a potentially important role of AQP-3 in maintaining cell-to-cell adhesion and also reported that its suppression results in inhibition of cell growth – namely, anoikis, which is a form of apoptosis. Thus, AQP-3 helps maintain a dynamic balance between cell turnover and survival, which is the core of regulation of cellular proliferation. To the best of our knowledge, no study thus far has commented on the expression of AQP-3 in SK and KA. Regarding SK, in the present study, a significant downregulation was found between normal controls and SK, and a significant difference was also found between SK and BCC. In SK, AQP-3 expression in the present study was diffuse and decreased or was absent in the horn cyst. This can be matched with the fact that SK is a noncancerous, wart-like growth on the surface of the skin, which is a benign form of keratinocyte tumor. In the present study, AQP-3 expression in KA showed marked staining in some regions of the lesion and others showed weak or no staining. A significant downregulation was found between normal controls and KA, with mean values higher than SCC, but with no statistically significant difference, which may be because of the small number of patients. Martorell-Calatayud et al.23 considered that KA is a variant of well-differentiated SCC, and Voss et al.11, who found that AQP-3 protein expression within the epidermis of SCC appeared to be excluded from the atypical keratinocytes corresponding to cells on the inner edge of the epidermis, suggest that AQP-3 may be associated with differentiation. They suggested that downregulation of AQP-3 in both BCC and SCC can be an attempt to compensate for the excessive growth seen in hyperproliferative disorders, which can also be applied to other hyperproliferative disorders including KA and SK, thus explaining its possible downregulation in those tumors. This means that AQP-3 may be considered an antihyperproliferative marker. With regard to differentiation of tumors, the highest mean value in the studied tumor groups was for BCC followed by KA and SK; SCC had the lowest mean value. This order suggests that AQP-3 may be associated with differentiation. In addition, in the present study, AQP-3 expression decreased in poorly differentiated SCC more than in well-differentiated SCC, and this was in agreement with Shen et al.24 who underscored the suggested role of AQP-3 in cell proliferation and survival. Psoriasis is a common disabling hyperproliferative skin disorder in which keratinocytes fail to differentiate and proliferate excessively 25. In the psoriasis group, this study revealed that AQP-3 showed mainly a granular cytoplasmic pattern together with mild staining of the plasma membranes in some cases, mainly in the basal layers and to a lesser extent in the suprabasal layers. In comparison with the control group, this study reported downregulation of AQP-3 in the psoriasis group with no statistically significant difference, whereas the present study reported a statistically significant positive correlation with the PASI score and with different degrees of cytoplasmic expression of AQP-3 in psoriasis patients, which strongly increased with psoriasis severity. AQP-3 tends to change its normal localization from a membranous to a cytoplasmic pattern. Consistent with this study’s findings, Voss et al.11 and Lee et al.26 reported the same cytoplasmic pattern of AQP-3 staining in psoriasis, with downregulation of AQP-3 in psoriatic samples when compared with normal controls. They also stated that as AQP-3 is a glycerol water channel and has an important role in regulating the hydration status of the epidermis, its abnormal localization (mainly cytoplasmic pattern in psoriasis) and downregulation can explain the dryness of the psoriatic lesions. AQP-3 helps maintain a dynamic balance between cell turnover and survival, which is the core of regulation of cellular proliferation, which can explain that AQP-3 downregulation in psoriasis may be a cofactor in the imbalanced epidermal cell turnover rate in this disease 21. Tutuncu and Kavanaugh 27 and Krueger and Bowcock 28 found that tumor necrosis factor-α is present in high levels in the skin of psoriasis patients and that it promotes psoriasis development by increasing proliferation of keratinocytes. In addition, Horie et al.29,30 suggested that tumor necrosis factor-α decreases AQP-3 expression in keratinocytes, and this reduction affects water permeability in the plasma membrane. Therefore, impaired SC hydration due to skin inflammation may be caused by a decrease in AQP-3 expression in keratinocytes.
The AQP-3 may have a role in differentiation of these tumors more than in proliferation, and may have a role as an antihyperproliferative prodifferentiative marker. This may help in understanding the effect of AQP-3 expression on keratinocytes proliferation and differentiation in common hyperproliferative disorders. In addition, this may help in discovering new therapeutic modalities of treatment for these diseases.
Conflicts of interest
There are no conflicts of interest.
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Keywords:© 2017 Egyptian Women's Dermatologic Society
aquaporins; nonmelanoma skin cancer; psoriasis