PRMT5 is upregulated in malignant and metastatic melanoma and regulates expression of MITF and p27(Kip1.)

Abstract

Protein arginine methyltransferase-5 (PRMT5) is a Type II arginine methyltransferase that regulates various cellular functions. We hypothesized that PRMT5 plays a role in regulating the growth of human melanoma cells. Immunohistochemical analysis indicated significant upregulation of PRMT5 in human melanocytic nevi, malignant melanomas and metastatic melanomas as compared to normal epidermis. Furthermore, nuclear PRMT5 was significantly decreased in metastatic melanomas as compared to primary cutaneous melanomas. In human metastatic melanoma cell lines, PRMT5 was predominantly cytoplasmic, and associated with its enzymatic cofactor Mep50, but not STAT3 or cyclin D1. However, histologic examination of tumor xenografts from athymic mice revealed heterogeneous nuclear and cytoplasmic PRMT5 expression. Depletion of PRMT5 via siRNA inhibited proliferation in a subset of melanoma cell lines, while it accelerated growth of others. Loss of PRMT5 also led to reduced expression of MITF (microphthalmia-associated transcription factor), a melanocyte-lineage specific oncogene, and increased expression of the cell cycle regulator p27(Kip1). These results are the first to report elevated PRMT5 expression in human melanoma specimens and indicate this protein may regulate MITF and p27(Kip1) expression in human melanoma cells.

Figure 1. PRMT5 is upregulated in human malignant melanoma tumors compared to normal epidermis, and subcellular localization of PRMT5 is heterogeneous amongst patient tumors.

A, Brightfield image of PRMT5 staining in a Stage 2/3 tumor and its adjacent normal epidermis. PRMT5 protein was visualized with Vulcan FastRed label (pink); samples were counterstained with hematoxylin to delineate nuclei (arrows) and connective tissue (blue). B, malignant skin tumor of pathologic stage 4, showing predominantly nuclear PRMT5. C, malignant skin tumor of pathologic stage 4, showing predominantly cytoplasmic PRMT5 (arrows indicate hematoxylin-stained nuclei). D, representative images of PRMT5 staining in patient tumors of pathologic stages I–IV.

Figure 2. PRMT5 is present and displays heterogeneous localization between human cell lines and orthotopic tumors.

A, Detection of PRMT5 in cell lysates from immortalized human epidermal melanocytes (HEM) or a panel of human metastatic melanoma cell lines. β-actin was used as loading control. B, HEM and melanoma cell lines underwent subcellular fractionation and immunoblot analysis for PRMT5, Mep50, or markers for nuclear (lamin B) and cytoplasmic (GAPDH) fractions. C, 1×10⁶ cells from the A375 and Hs294T human melanoma cell lines were implanted into the flank of immunocompromised mice and grown for 21 days. Tumors were removed, fixed in formalin, and analyzed via IHC for PRMT5. Data represent total percentage of cells in each tumor positive for PRMT5 in nucleus, cytoplasm, or both. n≥2 tumors were analyzed.

Figure 3. PRMT5 associates with Mep50, but not cyclin D1 or STAT3 in a subset of melanoma cell lines.

A, HEM and representative human melanoma cell lines were harvested and subjected to total cell lysis followed by immunoprecipitation. 10% of input protein, as well as protein from IgG control and IP directed against Mep50 and cyclin D1, underwent immunoblot analysis for IP target proteins as well as PRMT5. B, IP against STAT3 was performed on the same cell line panel, and subjected to immunoblot analysis using antibodies against STAT3 or PRMT5.

Figure 4. Depletion of PRMT5 via siRNA modulates cell proliferation and expression of MITF and p27^Kip1.

A, Melanoma cell lines were transiently transfected with scrambled siRNA or siRNA targeting PRMT5. Cell growth was measured by MTT assay at 48-transfection. Data represent percent of growth inhibition ± SEM (n≥3). B, Analysis of PRMT5, MITF, p27^KIP1, and β-actin protein expression at 48 hours post-transfection. n≥2 transfections were performed per cell line; representative immunoblots are shown here. The MITF antibody used here for IB predominantly recognizes one of the two known MITF isoforms in human melanocytes.