Dual Role of Pituitary adenylate cyclase activating polypeptide (PACAP) in Melanoma: Autocrine and Paracrine Mechanisms on Tumor and Immune cells
This thesis is based on investigation of PACAP and PAC1R expression and functional roles of PACAP in human melanoma as well as immune cells. In the first part, the study demonstrates for the first time the expression of PACAP in primary and metastatic melanoma tissues. PACAP is distributed in two...
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|This thesis is based on investigation of PACAP and PAC1R expression and functional roles of PACAP in human melanoma as well as immune cells.
In the first part, the study demonstrates for the first time the expression of PACAP in primary and metastatic melanoma tissues. PACAP is distributed in two distinct subtypes, PACAP 27 and PACAP 38. PACAP 38 expression seems to be more prevalent in primary melanomas but metastatic melanomas profoundly produce PACAP 27. Another very interesting result is the predominant expression of high affinity PACAP receptor, PAC1R, in metastatic melanoma but not in primary melanoma. Co-expression of PACAP 27 and PAC1R can be also detected in metastatic melanoma cell lines. This finding let us consider that PACAP may regulate melanoma cells in an autocrine manner.
PACAP function has been investigated on cell survival, cell growth, and cytokine production. Two melanoma lines were selected, SK-Mel 37 and NW-Mel 450, which express similar tumor-antigens, such as NY-ESO/LAGE-1, and MAGE 3, and have different morphological features and size. PACAP supplement achieved a positive regulation on survival or cell growth in SK-Mel 37, which is clearly susceptible to serum-starvation for reduced survival, in contrast to NW-Mel 450. Gene or protein expressions related to cell survival (BCL-2) and proliferation (Ki67) were induced by PACAP. Moreover, PACAP positively modulated protein expression of cell differentiation (i.e. MITF) and of chemokine receptor (i.e. CCR7) in melanomas. These factors are considered as important checkpoints for malignancy of melanoma.
In addition, the spontaneous cytokine production of both melanoma lines is different. SK-Mel 37 releases IL-10, an immunosuppressive cytokine, while NW-Mel 450 produces IL-6, a cytokine associated with tumor growth and angiogenesis. Our results show that PACAP up-regulates IL-6 release in NW-Mel 450 but may not change IL-10 production in SK-Mel 37. These observations clarify, that PACAP induced pro-tumor features by different mechanisms.
In the second part of the thesis PACAP-treated immune cells were investigated showing noticeable differences from untreated cells in the aspect of suppressive regulation. Two separated T cell subsets, CD4+CD25+CD127low/- T cells (regulatory T cells, Treg) and CD4+CD25- T cells (effector or responder T cells, Teff or Tresp) were studied. Both T cell subsets following PACAP supplement showed not only elevated gene transcripts of hallmarks for Treg, FoxP3 and CTLA-4, and of anti-inflammatory cytokines, IL-10 and TGF-β, but also significantly increased chemokine receptors, CCR4 and CCR7, being responsible for migration into dedicated tissue sites. Interestingly, a stronger effectiveness of PACAP on RNA level is observable in Treg cells compared to Teff/Tresp cells. This activity of PACAP may be predominantly mediated by the type II receptor, VPAC1R, which is present on CD4+ T cells. Our study, however, shows for the first time the expression of type I receptor, PAC1R, on T cells following activation with α-CD3/CD28 and particularly PACAP supplement. This finding let us consider, that perpetually provided PACAP by tumor cells may induce PAC1R expression on T cells. In addition in Treg functional assay a stronger suppression of cell growth of Teff/Tresp cells was observed, when co-culturing in the presence of PACAP.
Taken together, for the first time our results show the expression of PACAP and high affinity PACAP receptor, PAC1R, in melanoma and the ability of PACAP to influence not only the cellular activation in cell growth and cytokine production of melanoma but also to enhance the characteristic features of Treg. Therefore, reducing the PACAP effect in melanoma may become valuable to influence the course of the disease. Further investigation of the cellular mechanism triggered by PACAP in melanoma and the PACAP-mediated suppressive regulation of Treg through PAC1R will be required.