Molecular and functional characterization of the beta-F1-RNP and its relevance in cancer

Mitochondria play essential roles in cellular physiology participating among otheractivities in the supply of cellular energy, the execution of cell death and calcium and ROS signaling. Mitochondrial impairment has been reported in a growing number of human pathologies, including cancer. Cancer cells display, in the presence of oxygen, an abnormal enhanced glycolytic phenotype. This characteristic of the cancer cell prompted Otto Warburg to suggest that cancer cell mitochondria should have a bioenergetic impairment. In this thesis, we contribute to the characterization of the role of mitochondria in cancer biology. In particular, we have studied the relative expression level of mitochondrial and glycolytic proteins that are markers of the so-called bioenergetic signature. In agreement with previous findings, the bioenergetic signature of skin basocellular carcinomas and adenocarcinomas and squamous carcinomas of the lung is diminished when compared to paired normal tissue samples of the same patients, supporting the original Warburg’s formulation. 18FDG-PET imaging of lung carcinomas revealed that the bioenergetic signature is significantly associated to the rate of glucose uptake by the tumor. Remarkably, wedemonstrate that β-F1-ATPase expression, the standarized glucose uptake valueand tumor stage are independent markers of prognosis.β-F1-ATPase mRNA (β-mRNA) translation is tightly regulated in mammaliandevelopment, during the cell cycle and in hepatocarcinogenesis. Interestingly, weshow that the repression of β-F1-ATPase expression observed in human cancersis apparently exerted at post-transcriptional levels. Control of β-mRNA localization and translation is exerted by a set of presently uncharacterized and tissuespecifically expressed β-mRNA binding proteins (β-mRNABPs). Here, we describe the development and implementation of an affinity chromatography method for the purification of specific β-mRNABPs from mammalian cells. The innovations of the approach are: (i) the recombinant protein used for the purification of the β-mRNAprotein complexes includes an additional affinity binding domain and (ii) the proteins bound to the target RNA used as bait are eluted by RNAse digestion. The application of this technology in combination with the immunological identification of the eluted proteins revealed that HuR interacts both in vitro and in vivo with the regulatory 3’ untranslated region of β-mRNA. HuR is highly increased in breast carcinomas and its expression levels are significantly associated with the progressive alteration of the bioenergetic signature in breast cancer patients. Furthermore, we show that HuR expression provides an independent marker of tumor progression. However, no evidences have been obtained that could support the specific involvement of HuR in β-mRNA post-transcriptional expression in human cells. The application ofthe affinity purification method with standard proteomic approaches (MALDI-TOF, MS/MS) allowed the identification of DHX9, SFPQ, NonO, NCL, NPM, ILF3, IMP1, G3BP and RL8 as proteins that specifically co-purify with β-mRNA and, therefore, tentatively form part of the β-F1-RNP. Interestingly, in vitro assays showed that the recombinant G3BP, IMP1 and NPM specifically inhibited the translation of β-mRNA. Over-expression experiments showed that only G3BP was capable of specifically inhibit the synthesis of β-F1-ATPase, strongly supporting a direct role for this protein in the masking of β-mRNA in cancer. In addition, we have developed a cell line for the purification of β-mRNABPs that participate in vivo in translation repression of β-mRNA. In this system we have been able to visualize the sub-cellular localization of β-mRNA as well as to purify two additional β-mRNABPs of the cytoskeleton (α-actinin and vimentin) that might participate in sorting/localization of β-mRNA in the cell. Finally, we have generated a construct that, when over-expressed in mammalian cells, interferes with the activity of the mitochondrial H+-ATP synthase strongly suggesting its usefulness for the future development of animal models aimed to assess the relevance of the activity of the H+-ATP-synthase in cancer biology