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Wnt signalizacija regulirana je na više razina, velikim brojem efektora. Modulirajuće molekule funkcioniraju kao agonisti ili antagonisti. Fina uštimanost njihova odnosa je od velike važnosti za homeostazu stanice i normalno funkcioniranje cjelokupnog organizma. Predmet istraživanja u ovom radu bila je razina ekspresije proteina sFRP3 kao jednog od antagonističkih Wnt molekula, u astrocitomima različitih histopatoloških gradusa ( I, II, III i glioblastoma ). Metodom imunohistokemije određena je razina ekspresije sFRP3 proteina.Na uzorku 200 stanica po preparatu utvrđena je ekspresija proteina sFRP3 s lokalizacijom u jezgrama i citoplazmi stanica astrocitoma. Razina ekspresija u jezgri stanice ukazuje da je smanjen broj stanica u kategorijama umjerene i visoke razine ekspresije za sve graduse astrocitoma, uz dodatni nalaz statistički značajnog smanjenja medijana stanica za više graduse što je u skladu s porastom malignosti astrocitoma, s najvišim vrijednostima kod astrocitoma I i II – visoka (25 odnosno 26.5 stanica), umjerena (14 odnosno 13.5 stanica ), a za visoke graduse broj stanica s visokom i umjerenom razinom ekspresije iznosi za astrocitome III 3 stanice s umjerenom, a 1 stanica s visokom ekspresijom, dok kod glioblastoma nalazimo medijan od 2.5 stanice za umjerenu ekspresiju odnosno 2 stanice za visoku ekspresiju. Analizom razine ekspresije proteina sFRP3 u citoplazmi utvrđen je pad broja stanica u kategorijama umjerene i visoke ekspresije za sve graduse astrocitoma. Uočeno je da broj stanica s visokom razinom ekspresije u malignijih gradusa veći ( glioblastomi – 51 stanica , astrocitomi 42) u odnosu na broj stanica kod nižih gradusa ( astrocitomi II – 26.5 stanica, astrocitomi I – 25 stanica). Prema našim rezultatima zaključujemo da je sniženje ekspresije proteina sFRP3 u jezgri u pozitivnoj korelaciji s porastom gradusa astrocitoma, dok povišenje ekspresije proteina sFRP3 u citoplazmi viših gradusa astrocitoma sugerira dvojnu prirodu sFRP3 u kojoj je u nekim slučajevima antagonist, dok u drugim agonist signalnog puta Wnt., Wnt signaling is regulated at several levels, by a large number of effectors. Modulating molecules function as antagonist or agonist. Fine tuning of their relationship is of great importance for the cell homeostasis and normal function of the entire organism. The subject of this research was the expression level of sFRP3 as one of the antagonistic Wnt molecules, in astrocytomas of different histopathological grade (I, II, III, and glioblastoma). Immunohistochemistry was the method of choice for determining the levels of sFRP3 expression. On a sample of 200 cells per slide we determined that the expression of sFRP was localized in the nuclei and cytoplasm of astrocytic tumor cells. The level of expression in the cell nucleus indicates a decrease of the number of cells with moderate and high levels of expression in all astrocytomic grades, with the additional statistically significant finding that shows a decrease of the median in higher grades which is in accordance with the increase of malignancy in this grades, with the highest values in astrocytomas I and II - high (25 or 26.5 cells), moderate (14 or 13.5 cells) and in the high astrocytoma grades the number of cells with high and moderate levels of expression amounts to astrocytoma III 3 cells with moderate, and 1 cells with high expression, whereas we find in glioblastomas median of 2.5 cells for the expression of moderate or 2 cells for high expression. By analyzing the expression level of sFRP3 in the cytoplasm we determined a decrease in the number of cells in categories of moderate and high expression for all grades of astrocytomas. It was observed that the number of cells with high levels of expression is higher in higher malignant grades (glioblastomas - 51 cells, astrocytoma III - 42) in relation to the number of cells at lower grades ( astrocytomas II - 26.5 cells, astrocytoma I - 25 cells). According to our results, we conclude that the decrease of sFRP3 expression in the nucleus is positively correlated with increasing grade of astrocytomas, whereas an increase in protein expression of sFRP3 in the cytoplasm of higher grade astrocytomas suggests a dual nature sFRP3 where in some cases it acts like an antagonist, while in other cases like an agonist of Wnt signaling pathway.

Molecular and genetic landscapes of human astrocytic brain tumors still need elucidation. In the present study key players of Wnt signaling, beta-catenin (CTNNB1), TCF1 and LEF1, adenomatous polyposis coli (APC), AXIN1, SFRP3 and dishevelleds 1, 2 and 3 (DVL1, DVL2, DVL3) were investigated in human astrocytic tumors. Genetic changes were tested by PCR/loss of heterozygosity (LOH), microsatellite instability (MSI) and heteroduplex methods. Protein expressions were investigated by immunohistochemistry.

In the present study the expression intensities and localizations of SFRP3 protein across different histopathological grades of astrocytic brain tumors were investigated by immunohistochemistry, digital scanning and image analysis. Our results demonstrated that the differences between expression levels and malignancy grades were statistically significant. Moderate (P=0, 014) and strong (P=0, 028) nuclear expression levels were significantly different with pilocytic and diffuse astrocytomas showed higher expression values. When we divided our sample into two groups, both moderate and high cytoplasmic expression levels were significantly higher in glioblastomas than in the group comprising astrocytomas II and III. We also showed that high grade tumors had lower values of moderate (P=0, 002) and strong nuclear (P=0, 018) expression comparing to low grade tumors. Analysis of cytoplasmic staining showed that strong cytoplasmic expression was significantly higher in the astrocytomas III and glioblastoma group than in astrocytomas I and II group (P=0, 048). Moreover, we found that lower grade astrocytomas had fewer membranous SFRP3 stain than higher grade astrocytomas and that this difference was significant (P=0.036). Our results demonstrated that SFRP3 protein expression levels decreased when located in the nucleus in higher astrocytoma grades, indicating expected behavior as an antagonist of Wnt signaling, whereas when located in the cytoplasm an increase in SFRP3 expression was noted in high grade as compared to low grade astrocytomas. This may suggest that SFRP3 can also act as an agonist of Wnt signaling promoting invasive behavior.

Recently it has become evident that human astrocytic tumors’ growth is driven by complex signaling networks. Wnt signaling is a crucial factor in the development of many cancers and we believe that aberrant Wnt signaling has an important role in the development of astrocytic brain tumors, too. Since molecular and genetic landscapes of human astrocytic brain tumors still need elucidation we decided to study key players of Wnt signaling, dishevelleds (DVL1, DVL2, DVL3), TCF1 and LEF1 transcription factors, and SFRP3, an antagonist of Wnt signaling, in the set of human astrocytic brain tumors. Genetic changes were analyzed by polymerase chain reaction/loss of heterozygosity (LOH) and protein expressions by immunohistochemistry.

A crosstalk between Wnt and p53 signaling pathways in cancer has long been suggested. In the present study we wanted to investigate the involvement of the two pathways in meningioma by analyzing their main effector molecules, beta-catenin for Wnt and p53 for p53 signaling. Genetic changes were analyzed by polymerase chain reaction/loss of heterozygosity (LOH) and protein expressions by immunohistochemistry.