The inhibition of aerobic glycolysis as a therapeutic approach to improve cancer chemotherapy

The aim of the research project discussed in this thesis was to study the inhibition of aerobic glycolysis, that is the metabolic pathway exploited by cancer cells for the ATP generation. This observation has led to the evaluation of glycolytic inhibitors as potential anticancer agents. Lactate dehydrogenase (LDH) is the only enzyme whose inhibition should allow a blocking of aerobic glycolysis of tumor cells without damaging the normal cells which, in conditions of normal functional activity and sufficient oxygen supply, do not need this enzyme. In preliminar experiments we demonstrated that oxamic acid and tartronic acid, two LDH competitive inhibitors, impaired aerobic glycolysis and replication of cells from human hepatocellular carcinoma. Therefore, we proposed that the depletion of ATP levels in neoplastic cells, could improved the chemotherapeutic index of associated anticancer drugs; in particular, it was studied the association of oxamic acid and multi-targeted kinase inhibitors. A synergistic effect in combination with sorafenib was observed, and we demonstrated that this was related to the capacity of sorafenib to hinder the oxidative phosphorylation, so that cells were more dependent to aerobic glycolysis. These results linked to LDH blockage encouraged us to search for LDH inhibitors more powerful than oxamic acid; thus, in collaboration with the Department of Pharmaceutical Sciences of Bologna University we identified a new molecule, galloflavin, able to inhibit both A and B isoforms of LDH enzyme. The effects of galloflavin were studied on different human cancer cell lines (hepatocellular carcinoma, breast cancer, Burkitt’s lymphoma). Although exhibiting different power on the tested cell lines, galloflavin was constantly found to inhibit lactate and ATP production and to induce cell death, mainly in the form of apoptosis. Finally, as LDH-A is able to bind single stranded DNA, thus stimulating cell transcription, galloflavin effects were also studied on this other LDH function.

Study of PI3K/Akt signaling pathway as potential molecular target for T-cell acute lymphoblastic leukemia (T-ALL) treatment: pan-inhibition of PI3K catalitic isoforms as better therapeutic approach

Class I phosphatidylinositol 3-kinases (PI3Ks) are heterodimeric lipid kinases consisting of a regulatory subunit and one of four catalytic subunits (p110α, p110β, p110γ or p110δ). p110γ/p110δ PI3Ks are highly enriched in leukocytes. In general, PI3Ks regulate a variety of cellular processes including cell proliferation, survival and metabolism, by generating the second messenger phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). Their activity is tightly regulated by the phosphatase and tensin homolog (PTEN) lipid phosphatase. PI3Ks are widely implicated in human cancers, and in particular are upregulated in T-cell acute lymphoblastic leukemia (T-ALL), mainly due to loss of PTEN function. These observations lend compelling weight to the application of PI3K inhibitors in the therapy of T-ALL. At present different compounds which target single or multiple PI3K isoforms have entered clinical trials. In the present research, it has been analyzed the therapeutic potential of the pan-PI3K inhibitor BKM120, an orally bioavailable 2,6-dimorpholino pyrimidine derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. BKM120 treatment resulted in cell cycle arrest and apoptosis, being cytotoxic to a panel of T-ALL cell lines and patient T-lymphoblasts. Remarkably, BKM120 synergized with chemotherapeutic agents currently used for treating T-ALL patients. BKM120 efficacy was confirmed in in vivo studies to a subcutaneous xenotransplant model of human T-ALL. Because it is still unclear which agents among isoform-specific or pan inhibitors can achieve the greater efficacy, further analyses have been conducted to investigate the effects of PI3K inhibition, in order to elucidate the mechanisms responsible for the proliferative impairment of T-ALL. Overall, these results indicated that BKM120 may be an efficient treatment for T-ALLs that have aberrant up-regulation of the PI3K signaling pathway and strongly support clinical application of pan-class I PI3K rather than single-isoform inhibitors in T-ALL treatment.

Ribosome Biogenesis and cell cycle regulation: Effect of RNA Polymerase III inhibition

In cycling cells positive stimuli like nutrient, growth factors and mitogens increase ribosome biogenesis rate and protein synthesis to ensure both growth and proliferation. In contrast, under stress situation, proliferating cells negatively modulate ribosome production to reduce protein synthesis and block cell cycle progression. The main strategy used by cycling cell to coordinate cell proliferation and ribosome biogenesis is to share regulatory elements, which participate directly in ribosome production and in cell cycle regulation. In fact, there is evidence that stimulation or inhibition of cell proliferation exerts direct effect on activity of the RNA polymerases controlling the ribosome biogenesis, while several alterations in normal ribosome biogenesis cause changes of the expression and the activity of the tumor suppressor p53, the main effector of cell cycle progression inhibition. The available data on the cross-talk between ribosome biogenesis and cell proliferation have been until now obtained in experimental model in which changes in ribosome biogenesis were obtained either by reducing the activity of the RNA polymerase I or by down-regulating the expression of the ribosomal proteins. The molecular pathways involved in the relationship between the effect of the inhibition of RNA polymerase III (Pol III) activity and cell cycle progression have been not yet investigated. In eukaryotes, RNA Polymerase III is responsible for transcription of factors involved both in ribosome assembly (5S rRNA) and rRNA processing (RNAse P and MRP).Thus, the aim of this study is characterize the effects of the down-regulation of RNA Polymerase III activity, or the specific depletion of 5S rRNA. The results that will be obtained might lead to a deeper understanding of the molecular pathway that controls the coordination between ribosome biogenesis and cell cycle, and might give useful information about the possibility to target RNA Polymerase III for cancer treatment.