Dissecting the roles of viral T-antigens in Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare but aggressive form of skin malignancy with increasing incidence. Merkel cell polyomavirus (MCPyV) is associated with ~80% of MCCs, and a minority is caused by ultra-violet (UV)-induced DNA damage. The MCPyV T-antigens are major drivers for MCC oncogenesis. However, the mechanism how these viral oncoproteins initiate tumorigenesis and affect tumor progression remains largely unknown. Furthermore, the effective treatment options for advanced MCC are limited. Conventional therapies, such as chemotherapy, have shown limited effect on patient survival and nondurable response in patients with advanced disease. For the past few years, the use of immune checkpoint inhibitors (ICI) has changed the treatment paradigm for advanced MCC. However, half of the patients are non-responsive to these treatment and development of acquired resistance are major challenges for improving patient outcome. This thesis work aimed to dissect the roles of MCPyV T-antigens in autophagy regulation and their relationship with immunotherapy response.

In Paper I, we demonstrated that MCPyV T-antigens post-transcriptionally up-regulate miR-375, miR-30a-3p and miR-30a-5p expressions, where the HSC70 binding site of viral Tantigen is required for the induction of miRNAs. These miRNAs suppressed autophagy by targeting the key autophagy genes ATG7, SQSTM1 and BECN1. Concordantly, low expressions of ATG7 and SQSTM1 were lower in virus-positive tumors. In addition, treatment with Torin-1 (mTOR inhibitor) induced cell death, which could be rescued by blocking autophagy. These findings highlight the importance of autophagy suppression in cell viability in MCC.

In Paper II, we addressed the mechanisms how MCPyV T-antigens regulate miRNAs. We found that DICER1, one of the key miRNA processors, mRNA and protein expressions were induced by MCPyV T-antigen through HSC70. Further investigation revealed that HSC70 directly interacted with DICER1 mRNA in the AU-rich elements (AREs) in the presence of MCPyV T-antigen. Importantly, the LT-HSC70 interaction could induce luciferase activity of synthetic ARE-containing reporter, as well as mRNA stability and protein expression of multiple ARE-containing RNAs. This study highlights a new role for the LTHSC70 interaction in mRNA regulation, particularly for DICER1.

In Paper III, we employed spatially resolved transcriptomic and described the spatial immune heterogeneity in MCC tumors. We found that tumor immunity inside the tumor region was highly downregulated in the ICI non-responder, especially for antigen processing/presentation and T cell related immune process. Furthermore, the ICI resistant tumor also showed higher expressions of multiple inhibitory immune checkpoint molecules, which might contribute to immune suppression. Correlation analysis also highlighted the link between MCPyV T-antigen and tumor immune response, suggesting its role in immune evasion. This study provides a starting point for further understanding the molecular mechanisms of immune evasion in MCC.

In Paper IV, we observed a paranuclear dot-like expression pattern of c-KIT in most MCPyV-positive tumors. Mechanistically, we demonstrated that truncated LT caused retention of c-KIT to a paranuclear compartment via its Vam6p binding site. Consequently, c-Kit directly bound to Beclin-1 that suppresses autophagy by reinforcing the Beclin-1/Bcl2 interaction. Further investigation revealed that MCPyV LT was degraded by autophagy, which highlights the effect of autophagy repression in MCC for sustaining the expression of viral oncoprotein.

Collectively, this thesis work has provided novel insights into the roles of MCPyV Tantigens in autophagy regulation and its relationship with immune response, which may contribute to oncogenesis and immunotherapy response in MCC.

List of scientific papers

I. Satendra Kumar*, Hong Xie*, Hao Shi, Jiwei Gao, Carl Christofer Juhlin, Viveca Björnhagen, Anders Höög, Linkiat Lee, Catharina Larsson and 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

II. Jiwei Gao, Hao Shi, Carl Christofer Juhlin, Catharina Larsson and Weng-Onn Lui#. Merkel cell polyomavirus T-antigens regulate DICER1 mRNA stability and translation through HSC70. iScience. 2021. 24(11):103264. #Corresponding author.
https://doi.org/10.1016/j.isci.2021.103264

III. Jiwei Gao#, Yajie Yang, Hao Shi, Hannah Björn Andtback, Carl Christofer Juhlin, Catharina Larsson and Weng-Onn Lui#. Spatial immune heterogeneity in Merkel cell carcinoma. #Corresponding author. [Manuscript]

IV. Hao Shi#, Jiwei Gao, Satendra Kumar, Hong Xie, Ziqing Chen, Harri Sihto, Virve Koljonen, Vladana Vukojevic, Filip Farnebo, Viveca Björnhagen, Anders Höög, Carl 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. #Corresponding author. [Manuscript]