Analysis of the function of AP-1 and Ets oncoproteins in the regulation of terget-genes, induced by mitotic signals in normal and cancer cells

c-Fos and c-Jun proteins are members of the AP-1 transcription factor. The present study is divided in two parts, each one referring to the onco-proteins c-Fos and c-Jun, respectively. In the first part of our study, we have tried to investigate the transcriptional activation of the c-fos gene in a multistage mouse skin carcinogenesis model. We have focused at the SRE, which is the main regulatory element of the c-fos promoter and particularly at the SRF transcription factor that binds on the SRE regulatory element. We have studied the expression and activity of the SRF transcription factor, the mechanisms that regulate its activity, and also the role of SRF in the mouse skin multistage carcinogenesis model. In the second part, we have studied the interactions of the c-Jun protein with the basal transcriptional machinery and analyzed the functional significance of these interactions. Serum response factor (SRF) is an important transcriptional regulator of many genes involved in cell proliferation, differentiation and development. One of the SRF targets is c-fos, an immediate early gene, which can be activated by a variety of growth factors and mitogens through several different signaling pathways. Activation by most of these pathways is mediated through the serum response element (SRE), where a protein complex consisting of SRF and ternary complex factors (TCFs) can be formed. Many signaling pathways have been reported to induce transcription through the SRE, being dependent either on the TCFs or the SRF transcription factors. The TCF family of transcription factors is composed of three members, the Elk-1, Sapla and Sap-2/Erp/Net that bind the DNA sequence GAA through their Ets domain and are recruited on the SRE by direct protein-protein interactions with the SRF. TCFs are activated after phosphorylation by ERK, JNK and p38 kinases. The SRF protein, on the other hand, binds to the DNA sequence GG(AT)6CC, as a homodimer. It is mainly phosphorylated by Casein Kinase II (CKII), which has been reported to increase its binding affinity to the DNA. SRF activation also involves the pathway of the Rho family of GTPases. Rho family members regulate diverse processes including cytoskeletal rearrangements, gene transcription, cell-cycle progression, cell transformation and cytokinesis. In mammalian cells, Rho proteins control the organization of the actin cytoskeleton by regulating the formation of stress fibers and focal adhesions. Transformation by oncogenic Ras is accompanied by dramatic alterations in actin cytoskeleton and it is known that GTPases of the Ras subfamily can activate cascades of the Rho family members. Functional RhoA is also required for serum- and LPA-induced activation of the transcription factor SRF. It has been shown that changes in actin dynamics regulated by RhoA and LIM kinase-1 are critical to SRF activation. It has also been proposed that SRF activation is related to the High Mobility Group Proteins (HMG-ls) that increase the ability of SRF to bind the DNA and consequently induce the transactivation through SRF. As mentioned above, in the present study, we have investigated the role of SRF in chemically induced multistage carcinogenesis. One of the best characterized animal models for studying the genetic and biological alterations in tumor initiation, promotion and progression is the mouse skin system. Induction of H-ras gene mutations, after treatment with the chemical carcinogen DMBA, mark the initiation of carcinogenesis. Further genetic alterations that are caused after treatment with the tumor promoter TPA lead to promotion and progression of carcinogenesis. A series of cell lines, representative of different stages of mouse skin carcinogenesis have been used for our studies. C50 is a non-tumorigenic, immortalized cell line. P1 is a papilloma cell line, B9 is a squamous cell carcinoma from a multiple DMBA- treated mouse and A5 is a spindle cell carcinoma, isolated from the same tumor. CarB is a highly aggressive spindle cell carcinoma. C50, P1 and B9 have a typical epithelioid morphology. A5 and CarB have a mesenchymal or fibroblastoid morphology, and give rise to spindle cell carcinomas upon injection into nude mice. It has been previously shown that c-fos is required for the development of malignant tumors in the multistep mouse skin carcinogenesis model. Furthermore, c-fos deficient mice carrying an H-ras transgene fail to undergo malignant progression. For these reasons, we hypothesized that factors regulating c-fos transcription, like SRF and TCFs could be important in tumor progression. Our results show that SRF is overexpressed and highly active only in the cancer cells that have undergone the epithelial-mesenchymal transition and acquired a spindle phenotype. These were observed after Western blot analysis and Electrophoretic mobility shift assays, respectively, using nuclear protein extracts from the different cell lines. On Western blot analysis, using nuclear extracts from cell lines with difference either in their morphology or their content in the H-ras gene, we showed that the increased expression of SRF is mainly related to the spindle phenotype of the cells. The increased SRF activity in the mesenchymal cells is regulated by RhoA signaling. B9 and A5 cells represent the two extreme stages of the epethelial-mesenchymal transition and perform very different expression and DNA binding activity of SRF. For this reason B9 cells were transfected with an activated form of RhoA, whereas A5 cells were transfected with a dominant negative form of RhoA. The DNA binding activity of SRF was increased in the first and decreased in the second case. Also, transient transfections of the same cell lines with the corresponding forms of the RhoA protein, showed that the increased actin stress fiber formation observed in A5 cells was regulated by RhoA. Conclusively, our data link active RhoA with actin polymerisation, SRF activation, and neoplastic transformation. Furthermore, the architectural proteins HMG-ls were overexpressed in the cancer cell lines B9, A5 and CarB. They were also present in the complex of SRF formed in A5 cells and therefore increased the binding of SRF to the SRE, contributing in this way to the SRF activation. These mechanisms that regulate SRF leaded to the increase of transcriptional activation of the SRF target genes, like SRF gene itself, c-fos, vinculin and actin. In the second part of the present study, the mechanism of transcriptional activation through the c-Jun protein, was studied. The interactions of the c- Jun protein with the general transcription factor, TFIID were analyzed. In experiments, using COS cells, it was found that there is physical interaction between c-Jun and hTAFu55 proteins. This interaction was mediated by the N-terminal part of the c-Jun protein and was shown to be independent of c- Jun phosphorylation. After ectopic expression of the two proteins and the appropriate reporters, and subsequent Luciferase assays, it was shown that the two proteins act synergistically in the transcriptional activation of target-genes. This is an observation that can be used in order to explain the transcriptional activation of the c-Jun transcription factor, which regulates different cellular functions. The mechanisms of transcriptional activation of different genes include interactions with other activators, as well as with the basal transcriptional machinery. The investigation of these mechanisms is important for the better understanding of the molecular biology of the cell, and for the treatment of diseases that are closely related to gene expression, like cancer.Η πρωτεΐνη SRF (Serum Response Factor) είναι ένας μεταγραφικός παράγοντας, ο οποίος ρυθμίζει τη μεταγραφή πολλών πρώιμα εκφρασμένων γονιδίων, που σχετίζονται με τον κυτταρικό πολλαπλασιασμό, τη διαφοροποίηση και την ανάπτυξη. Σε αυτή την κατηγορία γονιδίων ανήκει το πρωτο-ογκογονίδιο c-fos. Στον υποκινητή του c-fos και πιο συγκεκριμένα στο στοιχείο SRE (Serum Response Element), που θεωρείται και το σημαντικότερο για τη μεταγραφή του, προσδένονται οι μεταγραφικοί παράγοντες SRF και TCFs (Ternary Complex Factors). Η οικογένεια των μεταγραφικών παραγόντων TCFs περιλαμβάνει τις πρωτεΐνες Elk-1, SAP1a και SAP-2/Erp/Net. Οι πρωτεΐνες αυτές προσδένονται στο DNA στη χαρακτηριστική αλληλουχία ets (GAA), αλληλεπιδρούν με τον SRF και ενεργοποιούνται από το ογκογονίδιο ras μέσω των MAP κινασών. Η πρωτεΐνη SRF προσδένεται στη χαρακτηριστική αλληλουχία GG(AT)6CC και φωσφορυλιώνεται κυρίως από την κασεϊνική κινάση II, γεγονός που αυξάνει τη δυνατότητα πρόσδεσής της στο DNA. Τελευταία έχουν προταθεί διάφοροι μηχανισμοί ενεργοποίησης της πρωτεΐνης SRF, δύο από τους οποίους σχετίζονται είτε με το σήμα της RhoA GΤΡάσης, ή με την ομάδα πρωτεϊνών υψηλής κινητικότητας (High Mobility Group Proteins, HMG-ls), που αυξάνουν την ικανότητα πρόσδεσής του SRF στο DNA και έτσι ενισχύεται η μεταγραφή των γονιδίων-στόχων. Στα πειράματα μας έχει χρησιμοποιηθεί ένα πολυσταδιακό σύστημα καρκινογένεσης της επιδερμίδας του ποντικού. Το σύστημα αυτό αποτελείται από αθανατοποιημένες φυσιολογικές έως πλήρως μεταστατικές κυτταρικές σειρές, οι οποίες έχουν προέλθει από κατεργασία της επιδερμίδας ποντικών με χημικά καρκινογόνο (DMBA, ΤΡΑ). Οι ουσίες αυτές επιφέρουν γενετικές αλλοιώσεις (με πιο χαρακτηριστικές μία σημειακή μετάλλαξη στο ογκογονίδιο ras και απώλεια του ογκοκατασταλτικού γονιδίου p53), που έχουν ως συνέπεια τη δημιουργία πλακώδους (επιθηλιακού) ή ατρακτοειδούς (μεσεγχυματικού) φαινοτύπου στις καρκινικές κυτταρικές σειρές. Σε αυτό το κυτταρικό σύστημα προσπαθήσαμε να διερευνήσουμε το ρόλο του μεταγραφικού παράγοντα SRF κατά την καρκινογένεση. Τα αποτελέσματά μας έδειξαν ότι ο SRF συμμετέχει στην εξέλιξη του καρκίνου και συγκεκριμένα στην επιθηλιακή-μεσεγχυματική μετάβαση (epithelial-mesenchymal transition, ΕΜΤ) των καρκινικών κυττάρων. Βρέθηκε ότι η ενεργότητα του SRF συσχετίζεται με την ενεργοποίηση της RhoA και τον αυξημένο σχηματισμό ινιδίων stress ακτίνης, στα καρκινικά κύτταρα με ατρακτοειδή φαινότυπο. Επίσης, στα καρκινικά κύτταρα, οι δομικές πρωτεΐνες HMG-I αυξάνουν την πρόσδεση του SRF στο SRE, συμμετέχοντας έτσι στην ενεργοποίηση του SRF. Αποτέλεσμα της δράσης αυτών των μηχανισμών ρύθμισης του SRF είναι η αύξηση της επαγωγής της μεταγραφής μέσω του στοιχείου SRE και κατά συνέπεια η μεταγραφική ενεργοποίηση γονιδίων-στόχων του SRF, όπως το c-fos. Επιπλέον, σε αυτή την εργασία εξετάστηκε ο μηχανισμός της μεταγραφικής ενεργοποίησης μέσω της πρωτεΐνης c-Jun. Συγκεκριμένα, έγινε ανάλυση της αλληλεπίδρασης της πρωτεΐνης c-Jun με το γενικό μεταγραφικό παράγοντα TFIID. Με πειράματα σε κύτταρα COS, βρέθηκε ότι υπάρχει φυσική αλληλεπίδραση των πρωτεϊνών c-Jun και hTAFII55 και συνεργασία μεταξύ τους στην μεταγραφική ενεργοποίηση γονιδίων-στόχων