Uncovering metastasis biomarkers and molecular mechanisms in Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare but highly aggressive skin cancer with high metastatic potential and poor prognosis. Due to its nonspecific symptoms, MCC is often misdiagnosed or diagnosed late. At diagnosis, ~40% of patients present with regional or distant metastasis. These patients have poor 5-year overall survival (OS) rates, 35%-63% for regional and only 14-24% for distant metastases, compared to 51%-75% for those with localized disease. This thesis aims to deepen our understanding of MCC by characterizing molecular differences between primary MCC tumors and metastases, identifying metastasis biomarkers and potential treatment targets, and exploring the role of Merkel cell polyomavirus (MCPyV) in MCC. In Paper I, we investigated the role of Insulin-like growth factor 2 mRNA- binding protein 3 (IGF2BP3) in MCC and found its expression elevated in metastases compared to primary tumors. High levels of IGF2BP3 were associated with shorter MCC-specific survival. A xenograft model confirmed increased IGF2BP3 expression in lung metastases. Functional studies demonstrated that IGF2BP3 promotes cell migration and invasion. Through RNA immunoprecipitation, we identified 281 direct RNA targets of IGF2BP3, many of which are associated with metastasis-related processes and overlap with genes differentially expressed between primary MCC tumors and metastases. These findings suggest that IGF2BP3 and its targets contribute to tumor progression. Furthermore, we found that inhibiting or silencing the transcriptional coactivator bromodomain- containing protein 4 (BRD4) reduced IGF2BP3 expression, suggesting BRD4 as a potential regulator of IGF2BP3. Our results support IGF2BP3 as both a prognostic biomarker and a metastasis driver in MCC. In Paper II, we performed a combined transcriptomic analysis of publicly available bulk and single-cell RNA sequencing (RNA-seq) data from treatment- naïve, immunocompetent MCC patients. Bulk RNA-seq identified 708 differentially expressed genes (DEGs; fold change ≥ 2, adjusted p < 0.05), with enrichment of immune- and progression-associated pathways (such as IFN-Y/a response, KRAS signaling, epithelial-mesenchymal transition (EMT), allograft rejection and complement) in primary tumors. Pseudobulk analysis of tumor cells from the scRNA-seq dataset identified 165 DEGs and confirmed similar pathway enrichments. Single-sample GSEA (ssGSEA) further supported increased IFN-Y response and EMT activity in primary tumors, particularly in early-stage tumors. These findings suggest that primary MCC tumors exhibit a more immunologically active and mesenchymal phenotypes, which declines during tumor progression. In Paper III, we examined the role of KIT and revealed its direct role in autophagy regulation. We showed that truncated large T antigen (LT) of MCPyV stabilized and sequestered KIT in the paranuclear compartment via VPS39 binding. KIT interacts with phosphorylated BECN1, facilitating its binding to BCL2 while reducing its interaction with the PIK3C3 complex. This shift suppresses autophagy. Depletion of KIT induced both autophagy and apoptosis, and reduced LT expression. Conversely, inhibition of autophagy in KIT-depleted cells restored LT levels and rescued cells from apoptosis. In vivo, autophagy induction increased tumor cell death and significantly reduced tumor growth in MCC xenograft mouse models. These results identify KIT-mediated autophagy suppression as a key survival mechanism in MCC and suggest that autophagy-inducing agents hold therapeutic potential for advanced MCPyV-positive MCC. Together, this thesis advances our understanding of MCC biology by identifying IGF2BP3 as a key regulator of metastasis, elucidating molecular differences between primary tumors and metastases, and uncovering a viral strategy for autophagy suppression that is critical for tumor cell survival. These insights open new avenues for developing biomarkers, improving prognostic stratification, and advancing targeted therapies for patients with advanced MCC. List of scientific papers This thesis is based on the following papers, which will be referred to in the text by their Roman numerals: I. Yajie Yang*, Jiwei Gao, Hao Shi, Harri Sihto, Sami Kilpinen, François Vilcot, Libuse Janská, Jakob Jeschonneck, Todor Cvetanovic, Anders Hoog, Jan Siarov, John Paoli, C. Christofer Juhlin, Lisa Villabona, Catharina Larsson and Weng-Onn Lui *. IGF2BP3 as a prognostic biomarker and regulator of metastasis in Merkel cell carcinoma. JID Innovations. 2025 Feb 12; 5(3):100355. https://doi.org/10.1016/j.xjidi.2025.100355 II. Yajie Yang*, Sami Kilpinen, Harri Sihto, Libuse Janská, Xiaohao Wang, Hao Shi, Lisa Villabona, Catharina Larsson and Weng-Onn Lui *. Discovery of prognostic biomarkers through transcriptomic differences between metastases and primary tumors in Merkel cell carcinoma. [Manuscript] III. Hao Shi*, Yajie Yang, Jiwei Gao, Satendra Kumar, Hong Xie, Ziqing Chen, Jiawen Lyu, Harri Sihto, Virve Koljonen, Silvia Vega-Rubin-de- Celis, Vladana Vukojevic, Filip Farnebo, Viveca Björnhagen, Anders Höög, C. Christofer Juhlin, Linkiat Lee, Malin Wickström, Jürgen C. Becker, John Inge Johnsen, Catharina Larsson and Weng-Onn Lui *. KIT mediated autophagy suppression driven by a viral oncoprotein emerges as a crucial survival mechanism in Merkel cell carcinoma. Autophagy. 2025 Jul; 21(7):1523-1543. https://doi.org/10.1080/15548627.2025.2477385 *Corresponding author