Regulation of cellular degradation pathways by viral oncoproteins and microRNAs

The cellular degradation system actively participates in cell homeostasis. Despite ongoing efforts and much progress in recent years, the underlying mechanisms of these pathways are not entirely understood. This thesis aims to contribute further insights how microRNAs and viral oncoproteins regulate key genes involved in cellular degradation pathways.

In Paper I, we found overexpression of miR-223-3p in testicular germ cell tumors (TGCTs), in which its expression was negatively correlated with the mRNA level of the FBXW7 ubiquitin E3 ligase. Overexpression of miR-223-3p suppressed, while its inhibition increased, FBXW7 protein level in TGCT cell lines, suggesting FBXW7 as a target of miR-223-3p. Using both gain- and loss-of-function experiments, we showed that miR-223-3p induced cell growth and reduced apoptosis. Ectopic expression of the FBXW7 open reading frame could reverse the effect of miR-223-3p. In conclusion, we suggest the oncogenic role of miR-223-3p – FBXW7 regulation in TGCT. In Paper II, we demonstrated that miR-375, miR-30a-3p and miR-30a-5p are regulated by Merkel cell polyomavirus (MCPyV) T-antigens through the DnaJ domain. These miRNAs could suppress autophagy and target ATG7, SQSTM1 (p62) and BECN1. Lower protein levels of ATG7 and p62 are associated with MCPyV-positive MCC tumors. Additionally, we showed that ectopic expression of MCPyV oncoproteins could suppress autophagy and blockage of autophagy rescued Torin-1 mediated cytotoxicity in MCC cells. This study provides evidence that MCPyV oncoproteins induce microRNAs and suppress autophagy in MCC, suggesting the importance of autophagy suppression in protecting MCC cell survival. In Paper III, we uncover a function and mechanism of MCPyV oncoprotein in autophagy evasion through c-KIT receptor tyrosine kinase. We show that the viral oncoprotein promotes c-KIT retention in late endosomes through its Vam6p binding site, which promotes c-KIT binding to Beclin-1 and enhances Beclin-1-BCL2 interaction. Silencing of c-KIT induces autophagy, which leads to degradation of the viral oncoprotein. Thus, MCPyV has developed a strategy to hijack cellular degradation system to sustain the viral oncoprotein expression. In Paper IV, we identify CK20 paranuclear dot as a part of aggressome in MCC, in which these structures are associated with MCPyV status, localized at microtubule-organizing center and dependent on BAG3 expression and dynein-mediated microtubule transport. Additionally, we show that the MCPyV truncated large T-antigen promotes aggresome formation through its Vam6p binding site. This study suggests a model of BAG3-dependent aggresome formation contributed by viral oncoprotein in MCC.

Overall, this thesis work has demonstrated the involvement of microRNAs and viral oncoproteins in regulation of cellular degradation pathways, which contributes to the understanding of the molecular interplays between cellular degradation system and cancer development in TGCT and MCC (as illustrated in the Graphical Abstract).

List of scientific papers

I. Jikai Liu#, Hao Shi, Xidan Li, Gang Chen, Catharina Larsson, and Weng- Onn Lui#. miR-223-3p regulates cell growth and apoptosis via FBXW7 suggesting an oncogenic role in human testicular germ cell tumors. International journal of oncology. 2017, 50(2): 356-364. #Corresponding author.
https://doi.org/10.3892/ijo.2016.3807

II. 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. International journal of cancer. 2020, 146(6): 1652-1666. *Equal contribution, #Corresponding author.
https://doi.org/10.1002/ijc.32503

III. Hao Shi, Jiwei Gao, Satendra Kumar, Hong Xie, Ziqing Chen, Harri Sihto, Virve Koljonen, Vladana Vukojevic, Filip Farnebo, Viveca Björnhagen, Anders Höög, C. Christofer Juhlin, Linkiat Lee, Catharina Larsson, and Weng-Onn Lui#. Merkel cell polyomavirus oncoprotein induces paranuclear retention of c-KIT suppressing autophagy through interaction with Beclin-1. [Submitted]

IV. Hao Shi#, Jiwei Gao, Linkiat Lee, Hong Xie, Harri Sihto, Catharina Larsson, Weng-Onn Lui#. Merkel cell polyomavirus oncoprotein promotes BAG3- mediated aggresome formation. #Corresponding author. [Manuscript]