Study of oncosuppressive properties of RNASET2, the first human member of the Rh/T2/S family of glycoproteins.

During the three years of my PhD, I have been working on RNASET2, the first human member of the widespread family of T2 ribonucleases. We began to study this gene since it maps in 6q27, a region frequently deleted in many solid neoplasms and haematological malignancies, including ovarian carcinomas, which represent our model. The frequent alteration of this genomic region suggests the presence of one or more tumour suppressor genes. An in-depth study of the promoter, the 5’ and 3’ flanking regions and the coding region showed no inactivating mutations; nevertheless RNASET2 is down-regulated at the RNA level in many primary tumours and cell lines, suggesting a possible role as tumour suppressor gene. RNASET2 showed no influence on cell growth in vitro, but in vivo experimental data revealed a role in the control of tumorigenesis and metastatization for this gene: RNASET2-expressing cells formed smaller tumours in mice respect to the control cells, transfected with the vector only; besides they gave rise to a lower number of metastasis. This tumour suppressive property was demonstrated to be not correlated with the ribonuclease activity of the protein, since tumours derived from cells transfected with a version of the gene mutated at the catalytic site were similar to those obtained with wild-type RNASET2-expressing cells. In order to better characterize the enzyme and its expression, we raised a new polyclonal antibody directed against RNASET2, called anti-hRNASE-2, since the pre-existing antibodies R63 and antihRNASE were not able to recognize the native protein; during this PhD work we set the best conditions for its use in different techniques. We also improved our knowledge about subcellular localization and catalytic activity of the protein: in particular we demonstrated that the proteolytic forms of RNASET2 are located in the lysosomes and are catalytically active, proving that they represent the mature forms of the ribonuclease. Next we started a characterization of RNASET2 in human tissues, performing immunohistochemical analyses on archival specimens, formalin-fixed and paraffin-embedded. We observed a moderate to strong expression of RNASET2 in many tissues, in particular in secretory epithelia of the gastrointestinal tract; besides macrophages, when present, showed a strong positivity in all tissues examined. We also analyzed a panel of ovarian carcinomas, which represents our model, comparing RNASET2 expression with normal and benign specimens from the same histological origin; in fact, we observed a decreasing expression of the protein during grading of ovarian carcinomas, from borderline serous tumours to undifferentiated carcinomas; this trend suggested a possible role of RNASET2 in the evolution of ovarian tumour and in particular we hypothesize its involvement in cell differentiation. In order to understand the possible mechanisms by which RNASET2 exerts its tumour suppressive function, finally we performed a new in vivo experiment in the nude mouse model, inoculating clones from an ovarian highly-metastatic cell line, Hey3MET2, transfected with a control vector or with RNASET2-expressing constructs; this experiment confirmed the role of RNASET2 in control of cell growth in vivo. We progressed performing histological and histochemical analyses on the tumours isolated from athymic mice treated, to highlight possible differences: RNASET2-expressing tumours appeared more differentiated and organized and we observed remarkable differences in the composition and organization of extracellular matrix within the tumours. In conclusion, we bring here evidences about the double life of RNASET2: the ribonuclease activity, due to the proteolytic forms localized to the lysosomes, and the tumour suppressive role, probably carried out by the secreted form. We still have to understand the mechanism by which the protein influences cell growth, but further analyses on tumours derived by the in vivo experiment will help.