Phd in MORPHOGENESIS AND TISSUE ENGINEERING

Thesis title: miRNAs-enriched Trout Embryonic Extracts promote the reversion of the malignant phenotype in liver tumor cells.

ABSTRACT - RATIONALE/BACKGROUND: Hepatoblastoma (HB) and Hepatocellular Carcinoma (HCC) are the most common liver tumors found in pediatric and adult patients, respectively. The research for novel therapeutic regimens aligned with the principles of personalized medicine has been ongoing for decades and has intensified since the early 21st century. Indeed, there is a pressing need to improve and refine current gold-standard treatments to reduce toxicity and enhance efficacy. In this context, one of the most promising strategies is the inhibition of recurrence and metastasis formation from the time of diagnosis to support the effectiveness of pharmacological and surgical interventions. As a matter of fact, the epithelial-mesenchymal transition (EMT), a key cellular process in the development of metastases, is frequently and precociously activated in both hepatoblastoma and hepatocellular carcinoma. Fish embryonic extracts have been shown to enhance therapeutic efficacy in patients with hepatocellular carcinoma and to inhibit EMT in vitro. It appears that miRNAs from specific embryonic stages of fish represent the active components responsible for these effects. - GENERAL OBJECTIVES: the objectives of the project are focused on identifying embryonic stages of rainbow trout (Oncorhynchus mykiss) that exert antitumor effects on hepatocellular carcinoma cells. This will be followed by mapping a molecular network to define the factors involved in the cellular response to treatment. Finally, the role of miRNAs contained in the extract will be elucidated in relation to the effects observed on cancer cells. - EXPERIMENTAL DESIGN AND METHODS: nine miRNA-enriched embryonic extracts from rainbow trout were tested at three concentrations on HepG2 cells (human hepatoblastoma) using proliferation, wound healing, migration, and transwell invasion assays. qPCR and Western blot analyses were subsequently performed to characterize the molecular effects of the most active extracts, with a focus on EMT-related pathways, apoptotic regulators, and cellular metabolism. The study was further extended to assess invasive behavior in a 3D model using the Hep3B hepatocellular carcinoma cell line. Expression profiling of selected oncogenic and tumor-suppressor miRNAs relevant to liver cancer was conducted. Finally, active miRNAs within the extracts were characterized to identify potential effectors of the observed antitumor activity. - RESULTS: embryonic stages of O. mykiss spanning gastrulation and neurulation exhibited significant efficacy in inhibiting cell motility, migration, and invasion in both 2D and 3D models of hepatoblastoma and hepatocellular carcinoma. Consistently, treated cells displayed reduced gene expression and protein levels of PI3K, as well as members of the SNAIL, TWIST, and ZEB families, and the mesenchymal marker N-Cadherin, along with a concomitant upregulation of GSK-3β protein levels. Furthermore, an enhancement of tight junctions was observed, evidenced by increased E-cadherin protein levels and its colocalization with β-catenin at the plasma membrane. In addition, HIF1α gene expression was downregulated, and p53 protein levels were upregulated. Ongoing analyses suggest that miRNA modulation occurs in hepatocarcinoma cells exposed to the active embryonic stages and aim to elucidate the role of extract-derived miRNAs in relation to the observed effects. - CONCLUSIONS: Embryonic stages of O. mykiss spanning gastrulation and neurulation exhibit significant efficacy in reprogramming hepatocarcinoma cells in 2D and 3D models, with effects varying according to the specific developmental stage. This study highlights the ability of fish embryonic extracts to inhibit EMT in neoplastic cells and lays the groundwork for the characterization of their active components, aiming to further elucidate the molecular mechanisms underlying previously reported in vitro and clinical observations. Thus, fish embryonic extracts may hold potential as adjunctive therapeutic agents in tailored treatment strategies for hepatocellular carcinoma to enhance therapeutic efficacy.