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Dear Sir,

PGF is a prostaglandin that binds to the G-protein coupled FP receptor, and is important in parturition (Khan et al., 2008), and is used as an ocular hypotensive in the management of glaucoma. Prostaglandins (PG) have long been implicated in tumor progression, as studies showing that non-steroidal anti-inflammatory agents, which block cyclooxygenase (COX) activity, decrease colorectal carcinoma in men (Fournier and Gordon, 2000). The causative role of PGE2, the major PG released by keratinocytes in response to UVR in non-melanoma skin cancer, is well documented (Muller-Decker and Furstenberger, 2007). The role of PGF in skin cancer is less defined; however, the FP receptor is down-regulated in skin papillomas in mouse carcinogenesis models, and its level of expression is inversely correlated with PGF production, suggesting that PGF regulates levels of the receptor in squamous epithelium (Muller et al., 2000). Melanoma cells express the synthetic enzymes necessary for PG production, and blockade of COX-2 with blocking antibodies has been reported to reduce melanoma proliferation (Scuderi et al., 2008). Analysis of effects of PGF on melanocytes show that PGF stimulates the activation of tyrosinase, the rate limiting step in melanin synthesis, and the formation of dendrites, which facilitate the transfer of melanosomes from melanocytes to keratinocytes (Scott et al., 2004, 2005). It is not known if the FP receptor is expressed by melanoma.

To examine FP receptor expression in melanoma, total cellular lysates of normal human melanocytes and six human melanoma cell lines were blotted for FP receptor, and real time polymerase chain reaction (PCR) was performed on RNA extracted from parallel dishes (Figure 1A, B). Melanocytes and the primary melanoma cell line WM115 expressed both FP receptor protein and FP receptor message; none of the other melanoma cell lines expressed detectable FP protein or message. Melanocytes cultured from lightly pigmented foreskins expressed slightly more FP receptor compared with melanocytes cultured from darkly pigmented foreskins, as shown by Western blotting and real time PCR (Figure S1A, B). To determine if the FP receptor is lost in melanoma in vivo, tissue microarrays (TMA) of benign nevi (n = 26), primary melanoma (n = 27) and metastatic melanoma (n = 24) were stained with antibodies against the FP receptor (Figure 1C). Examination of stained sections of normal skin revealed expression of FP receptor in melanocytes in the epidermis, in endothelial cells of blood vessels, and in smooth muscle of vessels and pilierector muscles. Examination of nevi showed focal positive staining of nevic cells for FP receptor in only 1/26 nevi, whereas none of the primary or metastatic melanomas expressed FP receptor. Type IV cytosolic phospholipase A2 (cPLA2) is the rate limiting step in PG synthesis. Levels of active (phosphorylated) cPLA2 were determined in melanocytes and human melanoma cell lines by Western blotting (Figure 2A). Steady state levels of active cPLA2 were higher in all melanoma cell lines, compared with normal melanocyte. To determine if melanoma cells synthesize PGF, culture supernatants of melanoma cell lines were analyzed for PGF by ELISA assay (Figure 2B). Five of six melanoma cell lines synthesize PGF at levels approximately 2–4-fold higher than normal human melanocytes, which produced 18 pg/ml/cell × 10−6 PGF (Scott et al., 2005). The WM115 melanoma cell line synthesized the most PGF (105 pg/ml/cell × 10−6) with the other cell lines ranging from 38 to 62 pg/ml/cell (×10−6).

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Figure 1.  The FP receptor is lost at the transcriptional level in melanoma and in nevi and melanoma in vivo. Total cell lysates of human melanocytes (MC) and six human melanoma cell lines were blotted with polyclonal antibodies against FP receptor (Cayman Chemicals, Ann Arbor, MI). Melanocytes and the WM115 melanoma cell line, express FP receptor. Real time PCR for FP receptor was performed as previously described (Scott et al., 2005). cDNA from human kidney was used as a positive control (+C). Shown are the amplification curves for FP receptor, each done in triplicate. The WM115 cell line expressed more FP mRNA compared with melanocytes. The other cell lines showed no detectable FP mRNA. Cores from benign nevi and primary and metastatic melanoma were placed in a TMA as previously described (Scott et al., 2008) and stained with antibodies against the FP receptor. Epidermal melanocytes and smooth muscle of pilierector muscle (arrows) express FP receptor. None of the nevi expressed FP receptor with the exception of a single case in which strong staining of nevic cells nest are present (arrow). Dermal macrophages strongly expressed FP receptor (asterisks). None of the primary or metastatic melanomas expressed FP receptor. Bar = 500 microns.

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Figure 2.  Melanoma cells synthesize PGF. Total cell lysates of normal melanocytes (MC) and six human melanoma cell lines were blotted with rabbit polyclonal antibodies against phosphorylated cPLA2 and total cPLA2 (Cell Signaling Technology, Danvers, MA). Steady state levels of P-cPLA2 are higher in the melanoma cell lines, compared with melanocytes. (A) Results are representative of three separate experiments. Cells were cultured for 24 h, and culture supernatant was collected and PGF was assessed by ELISA assay (Cayman Chemicals) and expressed as pg/ml/cell. All melanoma cell lines, with the exception of WM165, produce PGF. Melanocytes also produce PGF. (B) Each bar represents the average of three (melanoma cell lines) or five (melanocytes) separate experiments.

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In an effort to identify additional functions for PGF, we determined the effect of PGF on melanocyte and melanoma migration, and on melanocyte apoptosis in response to ultraviolet irradiation (UVR). PGF did not affect melanocyte migration, even at high doses (Figure S1, C). Similar results were obtained when fluprostenol, a specific FP receptor agonist, was tested (data not shown). PGF also had no effect on migration of WM115 cells, which express the FP receptor (Figure S1, C). To determine if PGF protects melanocytes from apoptosis in response to UVR, melanocytes were pretreated with PGF for 24 h, and then irradiated with 80 mJ/cm2 UVB, and levels of active caspase-3 were measured by fluorescein activated cell sorter (FACS) analysis 24 h later. Because white and black melanocytes express different levels of the FP receptor, pooled cultures of melanocytes isolated from black (n = 4) and white (n = 3) foreskins were tested separately (Table S1). Forty-seven percent (±10% SD) of white melanocytes expressed active caspase-3 following irradiation, compared with 51% (±15% SD) irradiated in the presence of PGF (10 nM), a difference that did not reach statistical significance. As expected, melanocytes cultured from darkly pigmented foreskins were more resistant to apoptosis in response to UVR, with only 25% (±12% SD) expressing active caspase-3 following UVR. The inclusion of PGF decreased active caspase-3 expression in black melanocytes response to UVR (20%±10% SD); however, this did not achieve statistical significance by Student’s t-test (P = 0.067). Active caspase-3 expression in response to PGF did not differ from vehicle treated controls. It is interesting to note that melanocytes cultured from light and darkly pigmented foreskins showed opposing effects of PGF following UVR, with an increase in caspase 3 expression seen in white melanocytes (more apoptosis) and a decrease seen in black melanocytes (less apoptosis). While differences between treated and untreated groups did not reach statistical significance, it is interesting to speculate that PGF may provide a small photoprotective effect for black melanocytes, and may contribute to UVR-dependent apoptosis in white melanocytes.

Our data indicate that the FP receptor is expressed only by melanocytes, but is lost in melanoma and in nevi. A search of the Oncomine database (https://www.oncomine.org) was performed and virtually all melanoma cell lines reported (n = 501) showed loss of the FP receptor (PTGFR) at the message level, consistent with our results on melanoma in vitro and in vivo. We were unable to identify a role for PGF in melanocyte or melanoma migration, or in protection of melanocytes from UVR-induced apoptosis. The significance of loss of FP receptor expression by benign nevi and melanoma remains to be determined. Because nevic cells are generally considered to be modified melanocytes, we were somewhat surprised that nevic cells show essentially total loss of FP receptor expression. While the true nature of nevic cells is still a matter of debate, one generally accepted theory of their ontogenesis is that nevic cells represent a clonal proliferation of melanocytes that acquire the capability to form cohesive nests and traverse the basement membrane. Therefore, loss of FP receptor expression by melanocytes may facilitate, cell–cell adhesion, or invasion through basement membranes. Testing of this notion will require silencing of FP receptor expression in melanocytes followed by analysis for acquisition of a ‘nevic cell’ phenotype. The lack of FP receptor indicates that melanoma will not respond to PGF, indeed, the one cell line (WW115) that expressed FP receptor failed to show changes in migration or proliferation (data not shown) in response to PGF. Our data do indicate that melanomas synthesize abundant amounts of PGF, greater than normal melanocytes (Scott et al., 2005), suggesting that PGF may facilitate melanoma progression through effects on accessory cells, such as fibroblasts and endothelial cells, which express the FP receptor.

Acknowledgement

  1. Top of page
  2. Acknowledgement
  3. References
  4. Supporting Information

This work was supported by 2 RO1 AR45427-04 (Glynis Scott).

References

  1. Top of page
  2. Acknowledgement
  3. References
  4. Supporting Information
  • Fournier, D.B., and Gordon, G.B. (2000). COX-2 and colon cancer: potential targets for chemoprevention. J. Cell. Biochem. Suppl. 34, 97102.
  • Khan, A.H., Carson, R.J., and Nelson, S.M. (2008). Prostaglandins in labor – a translational approach. Front Biosci. 13, 57945809.
  • Muller, K., Krieg, P., Marks, F., and Furstenberger, G. (2000). Expression of PGF(2alpha) receptor mRNA in normal, hyperplastic and neoplastic skin. Carcinogenesis 21, 10631066.
  • Muller-Decker, K., and Furstenberger, G. (2007). The cyclooxygenase-2-mediated prostaglandin signaling is causally related to epithelial carcinogenesis. Mol. Carcinog. 46, 705710.
  • Scott, G., Leopardi, S., Printup, S., Malhi, N., Seiberg, M., and Lapoint, R. (2004). Proteinase-activated receptor-2 stimulates prostaglandin production in keratinocytes: analysis of prostaglandin receptors on human melanocytes and effects of PGE and PGF on melanocyte dendricity. J. Invest. Dermatol. 122, 12141224.
  • Scott, G., Jacobs, S., Leopardi, S., Anthony, F.A., Learn, D., Malaviya, R., Pentland, A. et al. (2005). Effects of PGF(2alpha) on human melanocytes and regulation of the FP receptor by ultraviolet radiation. Exp. Cell Res. 304, 407416.
  • Scott, G.A., McClelland, L.A., Fricke, A.F., and Fender, A. (2008). Plexin C1, a receptor for semaphorin 7A, inactivates cofilin and is a potential tumor suppressor for melanoma progression. J. Invest. Dermatol. 29, 954963.
  • Scuderi, M.R., Anfuso, C.D., Lupo, G., Motta, C., Romeo, L., Guerra, L. et al. (2008). Expression of Ca(2+)-independent and Ca(2+)-dependent phospholipases A(2) and cyclooxygenases in human melanocytes and malignant melanoma cell lines. Biochim. Biophys. Acta 1781, 635642.

Supporting Information

  1. Top of page
  2. Acknowledgement
  3. References
  4. Supporting Information

Figure S1. (A) Lysates from melanocytes (MC) cultured from black (n = 3) and white (n = 6) foreskins were blotted for FP receptor. FP receptor expression was higher in white, compared with black, melanocytes. (B) Quantitative real time PCR show that white melanocytes expressed 1.9 × 10−11 ng/ml (SD ± × 10−12) compared with black melanocytes, which expressed 2.2 × 10−10 ng/ml (SD ± 1.4 × 10−10). (C) The human melanoma cell line WM115, and human melanocytes, were plated in basal media in the top well of a 96 well migration chamber (QCM Chemotaxis Cell Migration kit). PGF2&agr; was placed in the bottom well and twenty-four hours later, cells that had migrated were quantitated as per manufacturer's instructions. PGF2&agr; had no effect on melanoma or melanocyte migration, even at high doses.

Table S1. Lysates from MC cultured from black (pooled cultures from three individual foreskins) and white (pooled cultures from six individual foreskins) foreskins were resolved on 10% SDS-PAGE and blotted for FP receptor.

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FilenameFormatSizeDescription
PCMR_661_sm_figS1AB.tif1273KSupporting info item
PCMR_661_sm_figS1C.tif2059KSupporting info item
PCMR_661_sm_tableS1.tif1561KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.