Investigation of the involvement of the polyadenylation factors in breast cancer development (Doctoral thesis)

Κομίνη, Χρυσούλα

The formation of the 3΄ end of mRNA involves the selection of the poly(A) site and the addition of the poly (A) tail which plays a role in translocation to the cytoplasm, stability and translation. The selection of non-canonical poly (A) signals appears to be a regulated process associated with the increased expression of oncogenes (which possess multiple poly(A) signals) and with highly proliferating cells. Poly(A) polymerase alpha (PAPOLA) is one of the entities responsible for mRNA polyadenylation and 3΄ end formation. Since high levels of poly(A) polymerase activity have been proposed as an independent unfavorable prognostic factor in node-negative breast cancer patients, we aimed to investigate any possible contribution of PAPOLA to the malignant phenotype of breast cancer and to elucidate the underlying mechanisms through which PAPOLA may promote carcinogenesis. PAPOLA expression levels were evaluated by immunohistochemical staining of breast cancer tissues. PAPOLA expression strongly correlated with sporadic rather than familial breast cancer, triple negative breast cancer, lobular histological type and progesterone receptor negative tumors. Furthermore, within the well differentiated, Grade I tumor category, PAPOLA was associated with adverse prognostic factors (tumor’s size, advanced TNM stage, survival status).Silencing PAPOLA in MCF-7 and MDA-MB 231 breast cancer cells resulted in reduction of proliferation and a higher fraction of cells in G0/G1 phase of cell cycle. Moreover, the lack of PAPOLA reduced anchorage independent growth. Also, preliminary experiment showed that silencing PAPOLA increased breast cancer cells chemosensitivity to Paclitaxel. Surprisingly, overexpressing PAPOLA in MCF-7 and HEK 293 cells resulted in inhibition of proliferation and eventually cell death. Furthermore, an increase in the levels of the tumor suppressor p53 and the 89KD fragment of PARP implied the initiation of the apoptotic process. On the other hand, overexpressing PAPOLA in MDA-MB 231 cells, which carry a mutant p53, promoted cell proliferation as well a dramatic increase of the size of their colonies in soft agar.Silencing PAPOLA in MCF-7 and MDA-MB 231 cells led to reduced cyclin D1 (CCND1) levels, both in terms of mRNA and protein, whereas overexpression resulted in up regulation in the cells that could tolerate it. Furthermore, PAPOLA promoted the alternative polyadenylation of the corresponding mRNA since low expression levels of PAPOLA resulted in higher representation of the isoform with the longer 3΄ UTR of CCND1 mRNA, whereas overexpression of the enzyme had the opposite effect. No changes in the poly(A) tail length were observed in MCF-7 cells lacking PAPOLA, whereas high levels of the enzyme in MDA-MB 231 cells resulted in the lengthening of the poly(A) tail of both the mRNA isoforms. The poly(A) tail analysis also revealed that long transcripts acquire a shorter poly(A) tail compared to alternative polyadenylated counterparts. In conclusion it appears that overexpression of PAPOLA in breast cancer contributes to the malignant phenotype by lengthening the poly(A) tail and promoting alternative polyadenylation of mRNAs and thus increasing the expression of genes such as CCND1.
Institution and School/Department of submitter: Πανεπιστήμιο Ιωαννίνων. Σχολή Επιστημών Υγείας. Τμήμα Βιολογικών Εφαρμογών και Τεχνολογιών
Subject classification: Μαστός -- Καρκίνος
Keywords: Μετά - μεταγραφική ρύθμιση,Πολυαδενυλίωση,Εναλλακτική πολυαδενυλίωση,Πολυ(Α) πολυμεράση α,Πολυ(Α) ουρά,Κυκλίνη D1,CCND1,PAPOLA,Post-transcriptional regulation,Polyadenylation,Alternative polyadenylation,Poly(A) polymerase α,Poly(A) tail,Cyclin D1
Appears in Collections:Διδακτορικές Διατριβές

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