Merkel Cell Polyomavirus targets SET/PP2A complex to promote cellular proliferation and migration

Abstract

Merkel Cell Carcinoma (MCC) is a rare neuroendocrine skin cancer. In our previous work, we decoded genes specifically deregulated by MCPyV early genes as opposed to other polyomaviruses and established functional importance of NDRG1 in inhibiting cellular proliferation and migration in MCC. In the present work, we found the SET protein, (I2PP2A, intrinsic inhibitor of PP2A) upstream of NDRG1 which was modulated by MCPyV early genes, both in hTERT-HK-MCPyV and MCPyV-positive (+) MCC cell lines. Additionally, MCC dermal tumour nodule tissues showed strong SET expression. Inhibition of the SET-PP2A interaction in hTERT-HK-MCPyV using the small molecule inhibitor, FTY720, increased NDRG1 expression and inhibited cell cycle regulators, cyclinD1 and CDK2. SET inhibition by shRNA and FTY720 also decreased cell proliferation and colony formation in MCPyV(+) MCC cells. Overall, these results pave a path for use of drugs targeting SET protein for the treatment of MCC.

Keywords: FTY720; Merkel Cell Polyomavirus; NDRG1; PP2A; SET.

Fig. 1.. Expression of SET protein regulated by MCPyV.

Total protein lysates isolated from hTERT-HK stably expressing MCPyV early genes (A) or Merkel positive cell lines transduced with shpanT and shctrl (B) were subjected to immunoblotting and expression of indicated proteins were analyzed in these samples. (C) TOP left: HE panoramic image where MCC appears as a dermal tumor nodule. TOP right: HE 20X where the tumor cells are small blue cells with basophilic nuclei and minimal cytoplasm. MIDDLE: HE 40X shows mitosis is frequent and the apoptosis index is high. Cytoplasm staining for synaptophysin. Paranuclear dot-like pattern for CK 20. LOWER: Panoramic image shows strong expression of SET in MCC. 10X, 20X and 40X images showing expression of SET in MCC which appears to be strongly nuclear. The results (±SD) are representative of those from at least three independent experiments. *, P < 0.05 and P > 0.01; **, P < 0.01 and P > 0.001; ***, P < 0.001 and P > 0.0001; ****, P < 0.0001; ns = non-significant.

Fig. 2.

Pathway analysis upstream of NDRG1. (A) hTERT-HK cells expressing early region of MCPyV were plated on coverslips and after 24 h were treated with FTY720 (SET inhibitor) (5 μM) for 24 h and were probed for SET using anti-SET monoclonal antibody followed by secondary Alexa488-conjugated antibody. Nuclei were stained with DAPI (pseudocoloured red) and cells were analyzed under microscope. Images were merged using ImageJ software. (B) As before, hTERT-HK cells expressing early region of MCPyV was treated with FTY720 (SET inhibitor) (5 μM) for 24 h and whole cell lysates were subjected to immunoprecipitation with anti-PP2A antibody and immunoblotted using SET or PP2A antibodies. (C–D) hTERT-HK cells expressing early region of MCPyV was treated with FTY720 (SET inhibitor) (5 μM) for 24 h and whole cell lysates were subjected to immunoblotting using indicated antibodies. The results (±SD) are representative of those from at least three independent experiments. *, P < 0.05 and P > 0.01; **, P < 0.01 and P > 0.001; ns = non-significant.

Fig. 3.

Expression and subcellular distribution of β-catenin. Total protein lysates isolated from hTERT HK stably expressing MCPyV early genes (A) and treated with FTY720 (SET inhibitor) (5 μM) for 24 h (B) were subjected to immunoblotting. (C) For subcellular fractionation, cells were processed as mentioned in Materials and methods. Individual fractions were loaded on SDS-PAGE gel and purity of the fractions was analyzed using specific organelle marker: β-tubulin for cytosol, HDAC1 for nucleus and EGFR for membrane fraction along with protein of interest β-catenin. (D) pLXSN control or MCPyV ER gene transduced cells treated or not with FTY720 were plated on coverslips and after 24 h were probed for β-catenin using anti-β-catenin monoclonal antibody followed by secondary Alexa488-conjugated antibody. Nuclei were stained with DAPI (psedocoloured red) and cells were analyzed under microscope. Images were merged using ImageJ software. The results (±SD) are representative of those from at least three independent experiments. *, P < 0.05 and P > 0.01; **, P < 0.01 and P > 0.001.

Fig. 4.

Effect of SET inhibition on hTERT-HK stably expressing MCPyV early genes. (A) Cells treated with FTY720 (SET inhibitor) (5 μM) for 24 h were plated in 6-well plates at ratio 1:10, 1:100 or 1:1000 for 7–8 days as described in Materials and Methods section. Representative image shows colony formation at 1:100 dilution. (B) hTERT-HK-MCPyV keratinocytes were seeded in 6 well plates and treated with FTY720 for 24 h. After this, a scratch was introduced using a pipette tip and imaged every 24 h as mentioned in Materials and Methods. The results are representative of those from at least three independent experiments.

Fig. 5.

SET is required for MCPyV(+) MCC tumorigenesis. Merkel positive cell lines MKL-1 and MKL-2 were transduced with two shRNAs that target SET gene (shSET1 and shSET2). Cell lysates were subjected to immunoblotting to confirm SET knockdown (A), and the cells with SET knockdown were assessed in cell proliferation assays (B). (C) MKL-1 or MKL-2 cells were seeded in 96 well plates and treated with different doses of FTY720 and cell viability measured using resazurin. (D) Cells treated with FTY720 (SET inhibitor) (5 μM) for 24 h were plated in 6-well plates at ratio 1:10, 1:100 or 1:1000 for 21 days as described in the Materials and Methods section. Representative image shows colony formation at 1:100 dilution. The results (±SD) are representative of those from at least three independent experiments.

Fig. 6.

Merkel Cell Polyomavirus mediated NDRG1 regulation. Stable expression of Merkel Cell Polyomavirus early genes leads to strong expression of SET protein, also known as IPP2A or intrinsic inhibitor of PP2A. Increased SET expression leads to increased SET and PP2A interaction. As a result PP2A is no longer able to cause β-catenin degradation. Accumulation of β-catenin, activates c-myc which is a negative regulator of NDRG1. NDRG1 inactivation helps cell proliferation and migration in Merkel Cells. Treatment with FTY720, an inhibitor of SET protein, led to disruption of SET-PP2A interaction, causing β-catenin degradation with decrease in c-myc expression. This led to overall increase in NDRG1 expression with decrease in cell proliferation and migration ability.