Dissertation topics suggested by the teacher.
PROPOSED TOPICS FOR DISSERTATION (Prof. PORCELLI - Dr. IOMMARINI)
HIF1a PHARMACOLOGICAL DESTABILIZATION
The hypoxic inducible factor HIF1 is the key transcriptional regulator of adaptation to conditions of oxygen deficiency (hypoxia). This status occurs in many physiological and pathological conditions, resulting in increased expression, stabilization and consequent activation of HIF1. In particular, the adaptation to hypoxic conditions is a necessary step for the progression of solid tumors towards malignancy because, through the activation of the transcription of specific target genes, it stimulates glycolytic metabolism and neoangiogenesis by supplying the cancer cells with nutrients and oxygen and allowing them to rapid metabolization. Thus the possibility of inhibiting pharmacologically HIF1 is extremely attractive for possible anticancer therapies. The protein levels of the α subunit of HIF1 are regulated at the post-translational level as a function of the presence of oxygen through hydroxylation by a family of enzymes called prolyl-hydroxylase (PHD). PHD hydroxylates HIF1α at the level of two highly conserved proline residues and this reaction directs HIF1α to proteasome-mediated degradation. PHD activity is regulated by the presence of α-ketoglutarate (α-KG), succinate and Fe3 +. In this context, our research group screened the effect on HIF1α of a series of ester derived from α-KG and selected 4 compounds that were able to induce the destabilization of HIF1α under hypoxic conditions in two tumor cell lines without to be cytotoxic for non-tumor cells. In this project we will study these compounds in 3D tumor cell models (spheroids) obtained from different tumor cells and on genetically modified tumor models of Drosophila melanogaster. In these models we will evaluate the stabilization of HIF1α and its transcriptional activity and the effect on tumorigenic invasion and migration properties, as well as the hypoxic state through specific in vitro and in vivo assays.
SURVIVAL MECHANISMS IN TUMOR CELLS DEFECTIVE FOR OXIDATIVE PHOSPHORILATION
Cancer cells are characterized by mechanisms of cellular adaptation to guarantee their survival even in conditions of nutrient and oxygen paucity. In this context, our research group has shown that tumor cells in which the I (CI) chain of the respiratory chain is completely disassembled are not able to adapt to hypoxia because the metabolic changes induced by CI deficiency prevent stabilization of the factor induced by hypoxia (HIF1) even under hypoxic conditions. However, we have observed that the defective cancer cells of the CI injected into immunodeficient mice are nevertheless able to form tumor masses, although much slower than their wild type counterparts and although these tumors are very different morphologically. In fact, from a histological point of view these masses have the characteristics of a particular subgroup of epithelial tumors called oncocytomas. These predominantly benign tumors are characterized by an accumulation of dysfunctional mitochondria because they are generally carriers of somatic mutations that strongly impact on the structure or function of CI. We have previously shown that these mutations significantly slow tumor growth, but they do not allow tumor eradication, highlighting how these cells are still able to adapt and survive. The aim of this project is to study the possible molecular mechanisms that allow the survival of defective tumor cells for CI.
In particular, the following mechanisms will be analyzed: (i) the PI3K/Akt-mediated signaling pathway that is known to trigger survival mechanisms and which we have found activated in cells and in CI-deficient tumors. In these models, this signaling pathway will be studied by analyzing the phosphorylation of several target proteins and the use of specific inhibitors of this intracellular signaling pathway in order to demonstrate that its inactivation leads to the eradication of tumor cells; (ii) autophagy, and in particular mitophagy, a mechanism known to allow cell survival in the case of nutrient deprivation through the recycling of cellular components and which is finely regulated in relation to the availability of nutrients and the intracellular energy charge.
NEW INTERACTORS OF RESPIRATORY COMPLEX I
Respiratory complex I (CI) is the largest multiprotein complex of the OXPHOS system, composed of 45 subunits, encoded by nuclear or mitochondrial DNA. CI can be associated with CIII and CIV, forming "supercomplexes" (SC), but their assembly mechanisms are still controversial. In a previous study, we studied the biogenesis of CI and SCs in cell models lacking two essential CI structural subunits, i.e. the mtDNA encoded ND1, located in the membrane arm, and the NDUFS3 coded nDNA, in the matrix arm. These experiments allowed us to identify small amounts of SC even in cells that lack these subunits known to be necessary for CI assembly. In these models we also performed quantitative differential proteomics experiments using SILAC (Stable Isotope Labeling with Amino Acids in Cell Culture) in order to identify new CI interactors with a possible role as assembly factors in the early stages of CI biogenesis. These candidates are involved in some mitochondrial metabolic pathways. The aim of this project is to validate proteomics data and to understand the role of such interactors in the assembly process of CI and SC.
