New perspectives on melanoma: The role of PAX3

Background: Cutaneous melanoma is an aggressive form of skin cancer, arising in cutaneous melanocytes. The transcription factor PAX3 is critical for the proper development of neural crest lineages including melanocytes. Melanocytic cells show continued PAX3 expression from melanoblast formation in the neural crest to their differentiation into melanocytes. While many studies clarify the importance of PAX3 in embryonic development of melanocytes, less well understood, and more perplexing, is the continued PAX3 expression in adult skin melanocytes. By contrast PAX3 is frequently found in melanomas and naevi, and its expression correlates with melanoma staging. In this study we explore the multiple roles of PAX3 in melanocyte genesis and melanoma progression. While PAX3 is known to regulate melanocyte differentiation, survival, proliferation and migration during embryonic development, it is not clear if these same functions are maintained in adult melanocytes or melanoma cells. Drawing on evidence from development, we propose here a more encompassing theory that PAX3 is a key regulator of the myriad steps in melanocytic cell determination and function. We discuss the possibility that these roles may be accomplished by differential association with cofactors, via alternate transcripts or posttranslational protein modification(s). Moreover, we consider its possible roles in melanoma and provide a comprehensive consideration of the significance of PAX3 expression in melanoma

Mechanisms Contributing To Differential Regulation of PAX3 Downstream Target Genes in Normal Human Epidermal Melanocytes Versus Melanoma Cells

Melanoma is a highly aggressive and drug resistant form of skin cancer. It arises from melanocytes, the pigment producing cells of the skin. The formation of these melanocytes is driven by the transcription factor PAX3 early during embryonic development. As a result of alternative splicing, the PAX3 gene gives rise to eight different transcripts which encode isoforms that have different structures and activate different downstream target genes involved in pathways of cell proliferation, migration, differentiation and survival. Furthermore, posttranslational modifications have also been shown to alter the functions of PAX3.We previously identified PAX3 downstream target genes in melanocytes and melanoma cells. Here we assessed the effects of PAX3 down-regulation on this panel of target genes in primary melanocytes versus melanoma cells. We show that PAX3 differentially regulates various downstream target genes involved in cell proliferation in melanoma cells compared to melanocytes. To determine mechanisms behind this differential downstream target gene regulation, we performed immunoprecipitation to assess post-translational modifications of the PAX3 protein as well as RNAseq to determine PAX3 transcript expression profiles in melanocytes compared to melanoma cells. Although PAX3 was found to be post-translationally modified, there was no qualitative difference in phosphorylation and ubiquitination between melanocytes and melanoma cells, while acetylation of PAX3 was reduced in melanoma cells. Additionally, there were differences in PAX3 transcript expression profiles between melanocytes and melanoma cells. In particular the PAX3E transcript, responsible for reducing melanocyte proliferation and increasing apoptosis, was found to be down-regulated in melanoma cells compared to melanocytes. These results suggest that alternate transcript expression profiles activate different downstream target genes leading to the melanoma phenotype

Inflamation and oxidative stress : The molecular connectivity between insulin resistance, obesity and Alzheimer\u27s disease

Type 2 diabetes (T 2 DM), Alzheimer’s disease (AD), and insulin resistance are age-related conditions and increased prevalence is of public concern. Recent research has provided evidence that insulin resistance and impaired insulin signalling may be a contributory factor to the progression of diabetes, dementia, and other neurological disorders. Alzheimer’s disease (AD) is the most common subtype of dementia. Reduced release (for T 2 DM) and decreased action of insulin are central to the development and progression of both T 2 DM and AD. A literature search was conducted to identify molecular commonalities between obesity, diabetes, and AD. Insulin resistance affects many tissues and organs, either through impaired insulin signalling or through aberrant changes in both glucose and lipid (cholesterol and triacylglycerol) metabolism and concentrations in the blood. Although epidemiological and biological evidence has highlighted an increased incidence of cognitive decline and AD in patients with T 2 DM, the common molecular basis of cell and tissue dysfunction is rapidly gaining recognition. As a cause or consequence, the chronic in flammatory response and oxidative stress associated with T 2 DM, amyloid- ! (A ! ) protein accumulation, and mitochondrial dysfunction link T 2 DM and AD

PAX3 Expression in Normal Skin Melanocytes and Melanocytic Lesions (Naevi and Melanomas)

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

PAX3 and cutaneous malignant melanoma

Cutaneous Malignant Melanoma (CMM) is the most aggressive form of skin cancer, with high mortality rate in patients with metastatic spread. The focus of recent research has been to find molecular markers of melanoma that can be used to detect metastatic cells in the blood of patients with CMM and to assist in diagnosis and staging as well as to predict the outcome of the disease. PAX3, a transcription factor encoding gene that regulates melanocyte migration, proliferation and differentiation during development is found to be highly expressed in melanomas and melanoma cells, particularly its alternate transcript PAX3d. Therefore, the aim of this study was to establish an assay that can be used to detect occult melanoma cells in peripheral blood of patients with CMM and to assess PAX3d as a melanoma cell marker. We used staged formalin-fixed paraffin-embedded tissue and metastatic lymph node tissue to assess the expression of PAX3 transcripts in primary and metastatic melanoma tissue as well as in naevi and normal skin. Although problematic we were able to establish a technique for isolation of total RNA from archival formalin-fixed tissue slides. Furthermore, we perfected blood collection procedures and methods of total RNA isolation from peripheral blood. We also demonstrated that real time qRT-PCR provides a significantly higher marker detection rate relative to conventional gel-based RT-PCR and therefore suggest its use as a more sensitive and accurate detection method. Analysis of PAX3 expression in peripheral blood of patients diagnosed with CMM showed significantly higher marker expression frequency in patient blood samples in contrast to no expression in blood from healthy volunteers. Results also showed that the alternate transcript PAX3d was detected at significantly higher frequencies in peripheral blood of melanoma patients than the PAX3c alternate transcript. Moreover PAX3d was more frequently expressed in patients diagnosed with metastatic disease compared to those diagnosed with in-situ and invasive melanoma. Frequent expression of PAX3d in patients with in-situ melanoma even after more than one year since diagnosis was interesting, and suggests the presence of circulating melanoma cells even at this early stage of the disease. Since recent studies have found that melanoma cells with high metastatic potential have a different gene expression signature compared to highly proliferative cells with low metastatic potential, it is still to be clarified whether PAX3d is a marker of metastatic cells with low or high metastatic potential. Nevertheless, results presented here show that PAX3d is a sensitive and useful marker of melanoma cells in peripheral blood of melanoma patients

Differential PAX3 functions in normal skin melanocytes and melanoma cells

The PAX3 transcription factor is the key regulator of melanocyte development during embryogenesis and is also frequently found in melanoma cells. While PAX3 is known to regulate melanocyte differentiation, survival, proliferation and migration during development, it is not clear if its function is maintained in adult melanocytes and melanoma cells. To clarify this we have assessed which genes are targeted by PAX3 in these cells. We show here that similar to its roles in development, PAX3 regulates complex differentiation networks in both melanoma cells and melanocytes, in order to maintain cells as “stem” cell-like (via NES and SOX9). We show also that mediators of migration (MCAM and CSPG4) are common to both cell types but more so in melanoma cells. By contrast, PAX3-mediated regulation of melanoma cell proliferation (through TPD52) and survival (via BCL2L1 and PTEN) differs from that in melanocytes. These results suggest that by controlling cell proliferation, survival and migration as well as maintaining a less differentiated “stem” cell like phenotype, PAX3 may contribute to melanoma development and progression

Co-expression analysis of PAX3-positive follicular melanocytes.

<p>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).</p

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

<p>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.</p