Resumen de Regulation of miRNA expression during Sorafenib treatment in hepatocellular carcinoma. Development of a new therapeutic approach
Hepatocellular carcinoma (HCC) is one of the most frequent neoplasias and one of the major causes of cancer-related death worldwide. It usually arises upon chronic liver damage in cirrhotic livers. Several etiologies with different incidence increase the risk of HCC development, including hepatitis B and C virus infection, non-alcoholic liver disease (NAFLD) and alcohol abuse, among others. In the last years, there has been a shift in epidemiology from viral hepatitis to NAFLD and alcoholic liver disease (ALD). Treatment and patient prognosis are stablished according to the Barcelona Clinic Liver Cancer Staging System (BCLC). While early stages might receive curative treatments, intermediate and advanced stages of the disease rely on palliative treatments. The standard of care for intermediate patients at BCLC-B stages is transartherial chemoembolization (TACE), that consists in the embolization of the arteries that irrigate the tumor and the delivery of doxorubicin eluting beads. The landscape of treatments for advanced stages (BCLC-C) and patients at BCLC-B with untreatable progressive disease has profoundly changed. Nowadays, the combined treatment of Atezolizumab + Bevacizumab is the first line treatment. For patients in whom immunotherapy is not feasible, the tyrosine kinase inhibitors (TKIs) Sorafenib or Lenvatinib are still suitable therapeutic options. Regorafenib might be used upon Sorafenib progression in Sorafenib tolerant patients, and Cabozantinib has shown to be similar to Regorafenib. Given the limited efficacy of Sorafenib and other tyrosine kinase inhibitors (TKIs) in the clinical setting, new biomarkers of patient prognosis are urgently needed. In particular, liquid biopsy such as circulating tumor cells (CTCs), miRNAs, and extracellular vesicles (EVs). Therefore, the current thesis focuses on the impact of CTCs, EVs and miRNAs in the prognosis of patients with HCC at intermediate or advanced stages treated with TACE, Sorafenib or Lenvatinib. Circulating epithelial cells (CECs) were used as surrogate biomarkers of CTCs. Results were expressed as the percentage of cytokeratin positive cells in total enriched nucleated cells. Herein we demonstrated that CECs might be potential diagnostic liquid biopsy biomarkers of HCC, as CECs ≥ 5.21 (%) and clustered CECs ≥ 0.285 (%) correlated with HCC. High counts of CECs were related to worse prognosis in HCC patients treated with TACE. Baseline counts of CECs ≥ 15.315 (%) were related to increased risk of death in BCLC-A and BCLC-B patients treated with TACE. In BCLC-B stages, baseline levels of CECs (%), clusters ≥ 0.48 (%) and clustered CECs ≥ 1.395 (%) were associated with higher risk of progression. In more advanced tumor stages, time-dependent values of CECs after 1 month of Sorafenib treatment were related to increased risk of progression in BCLC-B and BCLC-C patients. CECs displayed differential proliferative and apoptotic features in response to Sorafenib, Regorafenib, Lenvatinib, Cabozantinib and Doxorubicin treatment ex vivo, supporting personalized medicine approaches. In liver cancer cells, Sorafenib exerted anti-tumoral properties in HepG2 cells by downregulating proliferation, migration and invasiveness, and upregulating apoptosis and autophagy. Sorafenib was the TKI with the most anti-tumoral effect, compared to Lenvatinib, Regorafenib or Cabozantinib. Sorafenib altered the expression of 11 miRNAs. At 6 hours it induced the downregulation of miR-551a, and upregulation of miR-122-5p, miR-200c-3p and miR-505-5p. At 24 hours of Sorafenib treatment, miR-148b-3p, miR-194-5p, miR-222-5p and miR-512-3p were downregulated, and miR-27a-3p, miR-193b-3p and miR-375 were upregulated. From these, miR-27a-3p and miR-200c-3p exerted clear anti-tumoral properties, while miR-122-5p, miR-148b-3p, miR-194-5p, miR- 505-5p and miR-512-3p displayed unequivocal pro-tumoral functions. miR-551a, miR-193b-3p and miR-222-5p showed both anti- and pro-tumorigenic properties. miR-375 did not show any effect. Transcriptomic profiles suggested that miRNAs regulate Sorafenib response by controlling apoptosis, proliferation, damage response, oxidation-reduction processes, mitochondria, cell cycle, and metabolism, among others. The anti-tumoral effect of Sorafenib was related to increased release of apoptotic bodies and decreased secretion of exosomes. Regorafenib and Cabozantinib also induced the secretion of apoptotic bodies, but to a lesser extent than Sorafenib. Lenvatinib induced the release of exosomes. In particular, Sorafenib induced the secretion of miR-122-5p, miR-200c-3p, miR-27a-3p, miR-148b-3p, miR-193b-3p, miR-194-5p and miR-375 in the exosomal fraction. The usage of this miRNA signature was tested as circulating biomarkers of HCC in two independent cohorts of patients with advanced HCC treated with Sorafenib. Baseline levels of miR-200c-3p were related to increased survival, whereas time-dependent values of miR-222-5p and miR-512-3p at 1 month of Sorafenib treatment were related to bad prognosis. These miRNAs were related to cell dedifferentiation and aggressiveness. Thanks to the development of these new liquid biomarkers and given the limited effectiveness of Sorafenib treatment in patients, we developed a new therapeutic strategy for the delivery of anti-tumoral miR-200c-3p to tumor cells. We were able to encapsulate miR-200c-3p mimics in β-cyclodextrins AMC6 and ADM70. AMC6 CDplexes delivering 15 nM of miRNA mimics were engulfed by liver cancer cells by endocytic mechanisms to efficiently increase miR-200c-3p levels. In summary, this project found liquid biopsy biomarkers of therapy response in HCC. CTCs were explored as diagnostic and prognostic markers in intermediate and advanced HCC patients treated with TACE and Sorafenib. Also, the secretion of a specific EVs pattern was related to Sorafenib effectiveness. Circulating levels of miR-200c-3p, miR-222-5p and miR-512-3p were related to Sorafenib response in patients with advanced HCC. Lastly, β-cyclodextrins nanovectors were constructed to efficiently deliver the anti-tumoral miR-200c-3p to liver cancer cells.
