Connecting viral oncoproteins to microRNA, autophagy and metabolism in Merkel cell carcinoma

Merkel cell carcinoma (MCC) is an aggressive type of skin cancer. Around 80% of MCCs harbor an integrated Merkel cell polyomavirus (MCPyV) genome with a mutation in the large T antigen (LT) gene, leading to expression of truncated LT. It is evident that the viral truncated LT and small T antigen (sT) play important roles in MCC tumorigenesis. Yet, the molecular mechanisms how these viral oncoproteins contribute to MCC development remain unclear. The studies presented in this thesis aim to further our understanding of the functional role of these MCPyV oncoproteins in MCC tumorigenesis.

In Paper I, we demonstrated that MCPyV T-antigens induce miR-375, miR-30a-3p and miR-30a-5p through the DnaJ domain of the viral T-antigen. These miRNAs could target multiple autophagy genes (ATG7, SQSTM1 and BECN1) and suppress autophagy in MCC cells. Additionally, we showed that both sT and truncated LT also suppress autophagy, but not the full-length LT. Inhibition of autophagy, but not pan-caspases, could rescue cell death induced by the mTOR inhibitor Torin-1, suggesting that suppression of autophagy is crucial for cell survival in MCC. In Paper II, we found paranuclear dot-like staining of c-KIT in MCPyV positive (MCPyV+) MCC cell lines and tumor samples. Mechanistically, we showed that MCPyV truncated LT induces paranuclear retention of c-KIT through its Vam6p binding site. Our results also revealed that c-KIT interacts with BECN1. Silencing of c-KIT increased autophagy and apoptosis, and decreased LT expression. Inhibition of autophagic degradation in c-KIT depleted cells restored the LT expression, suggesting the importance of autophagy suppression to sustain the expression of viral oncoprotein and cell survival. In Paper III, we showed that overexpression of miR-375 suppressed cell growth and migration in MCPyV-MCC cell lines, while suppression of miR-375 decreased cell growth and increased apoptosis in MCPyV+ MCC cell lines. The expression of LDHB, a target of miR-375, was inversely correlated with miR-375. Silencing of LDHB decreased cell growth in MCPyV-MCC cells, but rescued cell growth suppression resulted from miR-375 inhibition in MCPyV+ MCC cells. Our findings suggest that miR-375 regulation of LDHB plays distinct roles in MCPyV+ and MCPyV-MCC. In Paper IV, we demonstrated that MCPyV T-antigens reduce LDHB expression and promote glycolysis in MCC. Overexpression of LDHB reduced cell viability and increased apoptosis in MCPyV+ MCC cells. Ectopic expression of LDHB reversed the growth-promoting effect of MCPyV oncoproteins. Inhibition of glycolysis reduced cell growth in MCPyV+ MCC cells, while inhibition of mitochondrial respiratory activity inhibited cell growth in MCPyV-MCC cells. Our findings suggest that MCPyV+ and MCPyV-MCC cells are dependent on different energy metabolism for cell growth. Overall, this thesis work highlights the diverse functions of MCPyV oncoproteins and their involvement in regulating miRNA expression, autophagy and energy metabolism.

List of scientific papers

I. Satendra Kumar, Hong Xie, Hao Shi, Jiwei Gao, Carl Christofer Juhlin, Viveca Björnhagen, Anders Höög, Linkiat Lee, Catharina Larsson, Weng-Onn Lui. Merkel cell polyomavirus oncoproteins induce microRNAs that suppress multiple autophagy genes. Int J Cancer. 2019 Jun 10.
https://doi.org/10.1002/ijc.32503

II. Hao Shi, Linkiat Lee, Jiwei Gao, Satendra Kumar, Hong Xie, Vladana Vukojevic, Filip Farnebo, Viveca Björnhagen, Anders Höög, Carl Christofer Juhlin, Catharina Larsson, Weng-Onn Lui. Merkel cell polyomavirus oncoprotein induces paranuclear retention of c-KIT suppressing autophagy through interaction with Beclin-1. [Manuscript]

III. Satendra Kumar, Hong Xie, Patrick Scicluna, Linkiat Lee, Viveca Björnhagen, Anders Höög, Catharina Larsson, Weng-Onn Lui. MiR-375 regulation of LDHB plays distinct roles in polyomavirus-positive and -negative Merkel cell carcinoma. Cancers (Basel). 2018 Nov 14, 10: E443.
https://doi.org/10.3390/cancers10110443

IV. Satendra Kumar, Patrick Scicluna, Hao Shi, Jiwei Gao, Viveca Björnhagen, Anders Höög, Catharina Larsson, Weng-Onn Lui. Merkel cell polyomavirus oncoproteins suppress LDHB and promote glycolysis for cell growth. [Manuscript]