PAX3 expression in normal skin melanocytes and melanocytic lesions (naevi and melanomas)

Abstract

Background: Cutaneous Malignant Melanoma is an aggressive form of skin cancer, arising in cutaneous melanocytes. The transcription factor PAX3 regulates melanocyte specification from neural crest cells during development but expression in differentiated melanocytes is uncertain. By contrast it is frequently found in melanomas and naevi and is a marker for melanoma staging and detection. In this study we analysed the expression of PAX3 across the spectrum of melanocytic cells, from normal melanocytes to cells of benign and malignant lesions to better assess its function in these various tissues. Pax3 and PAX3 (italicized) refer to the mouse and human gene, respectively; whereas Pax3 and PAX3 (non-italicized) refer to the corresponding mouse and human protein.

Methodology and principal findings: PAX3 expression was analysed by immunohistochemistry and qRT-PCR. Immunofluorescence was used for co-expression with differentiation, migration and survival markers. As expected PAX3 expression was observed in naevi and melanoma cells. It was also found in melanocytes of normal skin where it co-expressed with melanocyte markers, MITF and MLANA. Co-expression with its downstream target, antiapoptotic factor BCL2L1 confirms PAX3 as a cell survival regulator. PAX3 was also co-expressed with melanoma cell migration marker MCAM in dermal naevi and melanoma cell nests, but this downstream target of PAX3 was not present in normal epidermal melanocytes, suggesting differential roles for PAX3 in normal epidermal melanocytes and melanoma cells. Most interestingly, a proportion of PAX3-positive epidermal melanocytes in normal skin show HES1 and Ki67 co-expression, indicating their less differentiated proliferative phenotype.

Conclusions and significance: Our results suggest that a previously identified role for PAX3, that of regulator of an undifferentiated plastic state, may operate in melanocytes of normal skin. This role, possibly required for cellular response to environmental stimuli, may contribute to formation and development of melanocytic lesions in which PAX3 expression is prominent.

Figure 1. PAX3 expression in melanocytic and melanoma cells.

A) Immunohistochemistry shows PAX3 expression (top panel) in representative samples of normal skin, naevus, primary melanoma and melanoma metastasis, compared to MITF expression in adjacent sections (bottom panel). B–D) Show the distribution of PAX3-positive melanocytes (arrowheads) in normal skin: along the hair follicle (early (B) and late (C) anagen); and in the epidermis (D). Arrows in (D) point to the cytoplasmic melanin deposit distinguishable from the nuclear PAX3 staining (arrowheads). Asterisk in (B) and (C) marks a dermal papilla. PAX3 was labelled with mouse monoclonal antibody (DSHB). E) PAX3 expression was analysed by RT-PCR and the graph shows the mean fold increase of PAX3 expression in naevi, primary melanomas and lymph node metastases normalised to the expression in normal skin.

Figure 2. Co-expression of PAX3 and MITF in melanocytic and melanoma cells.

A–C) Double immunofluorescent staining showing co-expression of PAX3 and MITF in: (A) the epidermal melanocytes of normal skin; (B) primary melanoma; and (C) in an A2058 metastatic melanoma cell line. Arrowheads in (A) show PAX3-positive normal epidermal melanocytes. Lines in (A) and (B) demarcate epidermal-dermal border (EDB). The variable PAX3 expression was clearly visible in the A2058 cell line (C). For all these experiments depicted in the figure, PAX3 was labelled with rabbit polyclonal antibody (Invitrogen). D) Graph showing the overall number of MITF and PAX3 double labelled cells in normal skins, naevi, primary melanomas and melanoma metastases. Each column represents a percentage of MITF-positive cells that are also PAX3-positive averaged across all samples. E) Double immunofluorescent staining shows PAX3 and MITF co-expressing melanocytes (yellow-orange) in the bulb of the hair follicle of normal skin. Note the single MITF-labelled melanocytes (green) at the base of the hair.

Figure 3. Co-expression analysis of PAX3-positive epidermal melanocytes of normal skin.

Epidermal melanocytes of normal skin show a variable differentiation status: less differentiated melanocytes co-expressing PAX3 (mouse monoclonal antibody, DSHB) and HES1 (A); more differentiated co-expressing PAX3 (rabbit polyclonal, Invitrogen) and MLANA (B); and mature melanocytes expressing only MLANA (B). Single HES1-labelled cells in the epidermis are keratinocytes (A). C) PAX3 (mouse monoclonal antibody, DSHB) and Ki67 co-expressing melanocytes are also observed in the epidermis of sun-exposed skin. Lines in (A), (B) and (C) demarcate the epidermal-dermal border (EDB) or epidermal surface (ES). D) Graph shows the distribution of differentiation marker expression in normal skin melanocytes with respect to melanocyte location (in epidermis, outer root sheath (ORS), or hair follicle bulb).

Figure 4. Co-expression analysis of PAX3-positive follicular melanocytes.

A) The transverse section of the hair follicle shows both PAX3 and HES1 co-expressing (enlarged in B), and single PAX3-expressing (enlarged in C), melanocytes in the outer root sheath (ORS). The line circumscribes the hair follicle. PAX3 was labelled with mouse monoclonal antibody (DSHB). D) The longitudinal section of the hair follicle shows PAX3 and MLANA co-expressing (enlarged in the insert on the left) and single PAX3-expressing (enlarged in the insert on the right) melanocytes in the outer root sheath (ORS). E) Single PAX3-expresing (arrows) and PAX3 and MLANA co-expressing (arrowheads) melanocytes in the matrix of the hair bulb. PAX3 was labelled with rabbit polyclonal antibody (Invitrogen).

Figure 5. Co-expression of PAX3 with markers of cell survival and migration in melanocytic and melanoma cells.

A) Double immunofluorescent staining showing PAX3 (mouse monoclonal antibody, DSHB) and BCL2L1 co-expression in representative samples of normal skin, naevus, primary melanoma and melanoma metastasis. B) PAX3 (mouse monoclonal antibody, DSHB) and MCAM co-expression in normal skin (epidermal melanocytes), naevus, primary melanoma and melanoma metastasis. Lines in (A) and (B) demarcate the epidermal-dermal border (EDB) or epidermal surface (ES). C) In contrast to the epidermal melanocytes, some PAX3-positive melanocytes in the outer root sheath (ORS) co-express MCAM. D) Graph showing the overall number of PAX3, BCL2L1 double-labelled cells in normal skins, naevi, primary melanomas and melanoma metastases. Each column represents a percentage of PAX3-positive cells that are also BCL2L1-positive, averaged across all samples. E) Graph showing the overall number of PAX3, MCAM double-labelled cells in normal skins, naevi, primary melanomas and melanoma metastases. Each column represents a percentage of PAX3-positive cells that are also MCAM-positive, averaged across all samples.