Estudio de PARP-1 en células tumorales y células madre de cáncer de páncreas. Implicación en la resistencia tumoral y uso como diana terapéutica

Resum

Gastrointestinal tumors represent a heterogeneous group of diseases that generate 4,8 million cases and 3,4 million deaths per year, accounting for a quarter of the incidence and 35% of cancer deaths worldwide. Among these, pancreatic cancer is the twelfth most diagnosed cancer and the seventh leading cause of death, with a high lethality rate in developed countries. One of its main problems is the absence of specific symptomatology, which leads to late diagnosis when the patient is in advanced stages and metastasis has already occurred. These reasons, together with the phenomenon of resistance to the drugs used, mean that current therapy is ineffective in this type of tumor, making it necessary to discover new avenues of treatment. DNA damage repair is one of the relevant processes in this resistance and the PARP family of enzymes is of great importance in this repair. Its main member (PARP-1) acts as a molecular sensor in several repair pathways such as homologous recombination or single-strand break repair (SSBR). Its function is performed through the activation of proteins involved in these processes by PARylation, which is a process where this enzyme modifies proteins by adding ADP-ribose residues. In addition, it has been observed that PARP-1 is involved in tumorigenesis and tumor progression, and it has been shown that the use of specific inhibitors against it sensitized different types of cancer against chemotherapy drugs, thus increasing their antitumoral effect. The main objective of this PhD thesis work was to study the role of PARP-1 expression in tumor cells derived from gastrointestinal tumors (specifically, pancreatic cancer), and in different subpopulations such as tumor stem cells, and how PARP-1 inhibition, both genetically (with RNAi) and pharmacologically with Olaparib (OLA), can improve chemoresistance processes. Our results showed that tumor lines of GI tumors overexpress PARP-1 and that subpopulations within these more drug-resistant tumor lines have a higher expression of this enzyme compared to non-tumor tissues. Furthermore, it was shown that the drugs Doxorubicin and Gemcitabine (GEM) were able to produce PARP-1 activation in pancreatic cancer lines. By studying PARP-1 gene silencing in a modified line that had inhibited PARP-1 expression (6-fold lower), the results showed that the modified line (Panc02-L) had increased sensitivity to several of the cytotoxic drugs tested (Gemcitabine, Doxorubicin and Paclitaxel). Additionally, pharmacological inhibition of PARP-1 using Olaparib generated synergy with the drug Gemcitabine, which was manifested through increased cell apoptosis in the combination of both treatments. In addition, the combination of Olaparib with irradiation showed a sensitizing effect of the cells to the drug Gemcitabine. The different studies we carried out on the involvement of PARP-1 in processes related to tumor aggressiveness showed that pharmacological or stable inhibition of PARP-1 in the Panc02 line reduced tumor progression, showing reduced cell clonogenicity, migration and angiogenesis. In addition, cells with this PARP-1 repression had lower expression of characteristic tumor stem cell markers such as DNMT1 and SOX2 and were more sensitive to gemcitabine treatment in three-dimensional tumorosphere formation assays. These interesting results obtained in vitro were replicated in in vivo experiments with murine models of Panc02-derived subcutaneous pancreatic tumors, where it was shown that simultaneous treatment with Gemcitabine and Olaparib reduced tumor volume by 86%, a significantly better result than that obtained by the drugs alone (70% and 64%, respectively). In addition, tumors induced from the Panc02-L line had 62% lower growth compared to the baseline. Regarding the relative survival of these mice, there were no significant differences between the experimental groups, although none of the mice induced from the Panc02-L line died. Analysis of the tumors showed an induction of early apoptosis after treatment of Panc02 tumors with Olaparib or in tumors derived from the Panc02-L line. This interesting antitumor effect caused by the inhibition of PARP-1 by Olaparib encouraged us to try to enhance the effect of Olaparib using nanotechnology. To do this, Olaparib was encapsulated in calcium phosphate nanoformulations and combined with ascorbic acid (AA), a vitamin that had shown synergistic effects with PARP-1 inhibitors in other types of cancer. Cytotoxicity results showed that nanoformulations coencapsulating Olaparib and ascorbic acid (NP-ACP-OLA-AA) had a superior cytotoxic effect on three pancreatic adenocarcinoma lines tested: Panc02, PANC-1 and MIA PaCa- 2. These nanoformulations produced IC50 reductions of 50, 56 and 28% in the cell lines versus free drug. Subsequently, we found that the combination of ascorbic acid at nontoxic doses and Olaparib was synergistic, which may explain the interesting results obtained in toxicity studies. In addition, treatments with the free and nanoencapsulated drug were shown to reduce protein expression of the proliferation marker Ki67, decreasing cell growth. The deaths produced in the tumor cells by the nanoparticle and the free drug were generated by apoptosis, this effect being 4 times greater in the former. This effect was produced through the greater genotoxicity induced by the nanoparticle, being 50% higher than that of the free drug after 24 hours of treatment. These findings were tested in vivo in immunosuppressed NOD-SCID mice generated with PANC-1 cells. The results showed that the nanoformulations reduced tumor volume by 59.1% compared to the control and increased the effect of the free drug, its inhibition being 31% greater. This effect could be justified by the synergistic action of Olaparib together with ascorbic acid and by the EPR effect (enhanced permeability and retention effect), which allows a greater accumulation of nanoparticles in the tumor. Histological analysis by hematoxylin-eosin and alizarin red showed that the NPs reached the tissue correctly, producing voids in the tumor sinus and generating a greater apoptotic induction compared to the free drug (proven by a TUNEL assay). Based on all these results, we can conclude that this doctoral thesis work advances the study of PARP-1 as a new target for antitumor treatment, demonstrating that PARP-1 is overexpressed in tumor lines of gastrointestinal tumors, such as pancreatic cancer, and that its expression is linked to chemoresistance and cellular malignancy processes both in vitro and in vivo. By studying the effects of its modulation, both stable inhibition (through viral vectors) and pharmacological inhibition are shown to be an effective therapy to allow the sensitization of pancreatic tumor cells against the drug Gemcitabine. Finally, the generation of Olaparib nanoformulations in combination with ascorbic acid allows the synergy shown by both drugs to enhance the cytotoxic effect of the free drug both in vitro and in vivo. In conclusion, PARP-1 inhibition, alone or in combination with Gemcitabine, may become an interesting therapeutic strategy for the treatment of pancreatic tumors, as it reduces tumor aggressiveness and allows evading chemotherapy resistance mechanisms. Furthermore, the new nanoformulations tested could improve this treatment strategy by enhancing the effect of free Olaparib.