Role of transportome in chemoresistance and chemosensitization in hepatoblastoma

Zusammenfassung

Hepatoblastoma (HB) is the most common liver cancer in children. Despite the relative success rate of conventional chemotherapy based on the combined administration of cisplatin and doxorubicin, approximately 20% of patients do not respond favorably and have a poor prognosis. This lack of response could be due to the presence of mechanisms of chemoresistance (MOCs), among them is the reduction of the amount of intracellular drug due to changes in the expression and/or function of plasma membrane solute carriers (SLCs) involved in drug uptake and/or of the ATP-binding cassette (ABC) superfamily of proteins involved in drug export. Thus, the overall aim of this Thesis was to characterize the role of the transportome in the lack of response of HB to chemotherapy and subsequently design pharmacological strategies to overcome chemoresistance using them as targets. Our studies using patient¿s samples revealed that downregulation of the leading drug uptake transporters and high expression of drug export pumps may lead to a reduction in intracellular drug concentrations and, therefore, play a role in reducing the response of HB to the pharmacological treatment. Notably, despite the existence of marked interindividual heterogeneity, MRP2 expression levels in hepatoblastoma are associated with a worse response to chemotherapy. Owing to the difficulty of obtaining tumor samples before and after treatment of children with HB, we used both classical (HepG2 and HuH6) and patient-derived xenograft (PDX) cell lines to study the impact on transportome-dependent chemoresistance of this cancer. First, we characterized the expression profile of genes involved in drug uptake and export and found similarities with tumors. Then, we studied the effect of first-line drugs used to treat these patients on the transportome in HB cells, after both short-term and long-term exposure to pharmacological stress. Our results showed an upregulation of some ABC pumps, such as MDR1, MRP1, and MRP2, induced in vitro by doxorubicin or cisplatin after 72 h exposure and in chemoresistant HB sublines generated in this Thesis. Furthermore, we used these chemotherapy-refractory cell lines to search for alternative antitumor pharmacological strategies. Although we found cross-resistance to a panel of anticancer drugs, cabozantinib was an effective antitumor drug even in these resistant cells. Given these results, we selected three ABC proteins as targets to design chemosensitizing strategies based on their inhibition. MDR1 was chosen because it transports doxorubicin, was highly expressed in patients with HB, and its overexpression was the most remarkable change observed among the MOC genes studied in the doxorubicin-resistant cell subline. MRP1 was chosen because it also transports doxorubicin and was upregulated in HB. MRP2 was included because tumors had high expression of this pump, which can transport both doxorubicin and cisplatin, and was found to be upregulated in resistant HB cell sublines. Indeed, we sensitized HB cells, even refractory sublines, by manipulating MDR1 function. Thus, zosuquidar, a third generation MDR1 inhibitor, markedly enhanced the sensitivity of HB cells to doxorubicin, a substrate of this pump, both in vitro and in vivo. Besides, CCL-17 and CCL-24, two drugs that inhibit tyrosine kinase receptors, reduced cell viability and inhibited MDR1-mediated doxorubicin export, acting synergistically when combined. Moreover, we identified several novel MRP1 and MRP2 inhibitors using molecular docking by in silico screening a large dataset of natural and semi-synthetic compounds, followed by in vitro confirmatory transport studies. One of them, the non-toxic flavonoid CCL-45, significantly sensitized hepatoblastoma cells to antitumor MRP1 and MRP2 substrates, such as doxorubicin and cisplatin.