Dissertation committee:
Πατσούρης Ευστράτιος, Ομότιμος Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Λάζαρης Χ. Ανδρέας, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Σαέττα Αγγελική, Αναπλ. Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Νόννη Αφροδίτη, Αναπλ. Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Γακιοπούλου Χαρά, Αναπλ. Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Αγρογιάννης Γεώργιος, Αναπλ. Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Μιχαλόπουλος Νικόλαος, Επικ. Καθηγήτης, Ιατρική Σχολή, ΕΚΠΑ
Summary:
Wnt signaling pathway plays an important role in embryonic development and adult tissue homeostasis, regulating important cell functions such as proliferation and migration, morphogenesis and planar cell polarity. In the mammary gland, Wnt signals are strongly implicated in initial development of the mammary rudiments, and in the ductal branching and alveolar morphogenesis that occurs during pregnancy. In addition crosstalk between the Wnt pathway and growth factor signaling pathway has been observed, such PI3K/AKT pathway. Alterations in Wnt pathway components’ expression are implicated in the pathogenesis of several tumor types, such as breast cancer. Therefore, Wnt pathway constitutes a promising anticancer therapeutic target with several agents currently in clinical trials. One hundred breast carcinomas were analyzed of which 68 were fresh frozen tissues and 32 were RCL2 fixed paraffin-embedded tissues. In addition 10 normal adjacent breast samples were analyzed. The relative mRNA expression levels of WNT2, WNT3, WNT5A, FRIZZLED 4 (FZD4), FRIZZLED 6 (FZD6), FRIZZLED 7 (FZD7), CTNNB1 (encoded β-catenin), GSK3-B, TCF4, LEF1 genes were determined by semi-quantitative Real Time PCR and ΔΔCT. Data were analyzed using the comparative ΔΔCt method and PPIA was used as reference gene. In addition for the determination Relative mRNA expression levels were determined by RT-PCR and analyzed with the ΔΔCt method. Furthermore, for the determination of protein levels of β-cetenin, 31 RCL2 fixed paraffin-embedded breast tissues were analyzed with immunochemistry, while GSK3-β protein expression levels were determined with Western Blot. In addition 41 breast carcinomas were investigated for mutations in PIK3CA gene using HRM-PCR and Sanger sequencing. Statistical analysis between the results and clinicopathological data, was performed by SPSSv2 package. Increased relative mRNA expression levels of WNT2, WNT5A, FZD4, FZD6, FZD7, CTNNB1, GSK3-B, TCF4 and LEF1 genes 6%, 47%, 10%, 0%, 13%, 5%, 21%, 7%, 5%, 31%, were found in of carcinomas, respectively. Furthermore, decreased relative mRNA expression levels were found in 54%, 36%, 57%, 83%, 47%, 73%, 29%, 40%, 80%, 39% in breast carcinomas, respectively, and stable relative mRNA expression levels were observed in 40%, 17%, 33%, 17%, 40%, 22%, 50%, 53%, 15%, 30% in carcinomas, respectively. Statistical significant correlations were established, in breast carcinomas between mRNA expression levels and clinicopathological features. Specially, mRNA relative levels of WNT2 ligand was decreased in samples with the absent of estrogen receptor (p=0,033). A statistically significant correlation emerged between increased mRNA expression levels of WNT3 and low grade tumours (Grade I & II) and decreased mRNA expression levels of WNT3 and pN-category (0,02). Statistical significant correlation was found between decreased expression levels of FZD6 and older patients (>65 years old). Expression levels of FZD7 receptor were significantly correlated with PR negative cases (p=0,02). In addition, statistical significant correlations were established between decreased relative expression levels of β-catenin and high grade tumours (Grade III & IV) (p=0,028) and increased relative expression levels of β-catenin and age of patients below 65 gears old (p=0,007). Expression levels of TCF4 were significantly correlated with Luminal A molecular subtype (p=0,011) of breast tumours. Marginal correlation was found between LEF1 expression levels and HER2 enriched molecular subtype. Statistical significant linear correlations among the expression levels of investigated genes were observed: WNT3/WNT5 p<0,01, WNT5A/FDZ6 p<0,01, WNT3/FZD6 p<0,01, WNT3/β-catenin p<0,01, WNT5A/β-catenin p<0,01, WNT3/GSK3-B p<0,01, WNT5A p<0,01, FZD6/GSK3-B p=0,020, FZD4/GSK3-B p=0,047, β-catenin/GSK3-B p<0,01, FZD4/TCF4 p<0,01, TCF4/LEF1 p=0,017, WNT5A/LEF1 p<0,01, WNT2/LEF1 p=0,017, FZD7/LEF1 p,0,01, FZD6/LEF1 p<0,01, LEF1/GSK3-B p<0,01, LEF1/β-catenin p=0,046. Furthermore, no statistical significant correlation was found in mRNA expression levels of investigated genes and APC methylation. Protein expression levels of GSK3-β showed decreased expression in 78% of the examined samples, while protein expression levels of β-catenin showed increased cytoplasmic and expression and decreased membranous expression in the majority of our samples. Lastly, mutations in PIK3CA gene were found 35% of breast carcinomas. Specially, 20% and 15% of the mutated samples were showed mutations in exon 9 and 20 in PIK3CA gene, respectively. Our results demonstrate the deregulation of Wnt signaling pathway, highlight the complexity of Wnt pathway in breast cancer as well as the emergence of possibly future predictive biomarkers in an effort to maximize the efficacy of Wnt targeted therapies.
