Mελέτη της δυναμικής των κυτταροσκελετικών συμπλόκων in vivo και in vitro κυττάρων πορογενούς καρκινώματος μαστού με ανoσοϊστοχημικές τεχνικές: Ο ρόλος του συγκαρκινογόνου ΡΜΑ στην μεταγωγή σήματος μέσω της πρωτεїνικής κινάσης C

Abstract

Breast cancer is the most common form of cancer and is the second leading cause of cancer death among women in the United States and North Europe. According to epidemiological studies, infiltrating ductal breast carcinoma (IDBC) is the 65 to 80% of mammary carcinomas. IDBC is characterized by a high degree of morphological heterogeneity, which suggests significant biochemical and functional diversity. Interactions between tumor cells and proteins of the extacellular matrix strongly influence tumor development, affecting cell proliferation and survival, as well as their ability to migrate beyond the original location into other tissues to form metastases. Many of these interactions are mediated by integrins, a ubiquitously expressed family of adhesion receptors. By regulating the adhesive capacity of tumor cells, integrins control the formation of metastatic tumors. Migration, growth and development, susceptibility to an internal cell death program, and the formation of new blood vessels to provide nutrients for the growing tumor are all influenced by integrins. Gene expression of integrins is controlled by a cytosplasmic family of proteins known as protein kinase C. The latter consists of a family of 12 different PKC isoforms which play essential role in many signal transduction pathways controlling cell growth, transformation and differentiation. The PKCα, one of the classical isoforms, is widely expressed in tissues and abnormal levels have been found in many transformed cell lines and in several human tumours. The PKCα is implicated in a variety of biological functions such as cell proliferation, apoptosis, and angiogenesis, also it is implicated in malignant phenotypes of several tumours and it plays an important role in cell differentiation. The PKCα plays important roles in breast cancer and increased in the activity of this enzyme was seen in breast cancer. Expression of PKCα has been associated with transformed breast cell lines, tumour cell proliferation, multidrug resistance and alteration of the estrogen status in the breast cancer cell lines. Correlation between the catalytic activity of PKCα and metastatic phenotype in breast cancer cell line was found. The purpose of the present study was to investigate the role of PKCα in invasiveness and motility of breast cancer cells of human biopsies, and for this purpose a model system of primary breast cancer and benign tissues were developed using the explant technique, in which small fragments of minced tissue were allowed to attach onto substrate without any previous trypsinization. Given that the most preclinical breast cancer studies are based on established cell lines which, however, may not reflect the primary tumor of origin due to genotypic changes, the developed model system of primary breast cancer cell culture consists a reliable experimental tool for the assessment of tumor cell behavior. PMA (phorbol 12-myristate-13-acetate) is general tumour promoter for PKC, which induce activation the diacylglycerol-dependent PKC after short period of exposure to the PMA, while induce down regulation for the PKC after prolonged exposure to PMA. It was used in the present study to modulate the PKCα in epithelial breast cancer cells and fibroadenomas cells and we studied the result in the F-actin organization and in integrin αvβ3 distribution in these cells. The study concerned in the distribution of PKCα in epithelial-like cells of primary cultures of breast cancer biopsies and of biopsies of fibroadenomas with immunofluorescence labeling. However, the main purpose was the study of the changes in the organization of the F-actin cytoskeleton protein with the application of fluorescent techniques, and the changes in the distribution of integrin αvβ3 with immunofluorescence labeling after treatment with 10-7 M PMA for 1h, the period for activation the PKCα and 5h of 10⁻⁷ M PMA treatment which induce down regulation of PKCα. The results showed an increase in the expression of PKCα in the malignant cells than in benign ones. The immunofluorescent staining of PKCα in the cytoplasm of most of the fibroadenomas cells was uniformly diffused; the nuclei were visualized with no staining, while the staining pattern of PKCα was variable among the malignant cells; the immunofluorecent intensity was somewhat heterogeneous among the cells cytoplasm as well as among the nuclei. After 1h exposure to 10⁻⁷ M PMA, an increase in immunostaining intensity in the cytoplasmic PKCα in the most of epithelial-like cells of primary cultures of fibroadenomas biopsies and malignant cells which indicated activation of the enzyme after 1h of treatment with PMA. Translocation of cytoplasmic PKCα to the nucleus or to the perinucleur region after activation was observed in the most of epithelial cancer cells. After 5h exposure to 10⁻⁷ M PMA decreased in immunostaining intensity in the cytoplasmic PKCα in the most of epithelial-like cells of primary cultures of malignant biopsies and fibroadenomal cells which indicated down regulation of the protein after long-term exposure to PMA. After 1h exposure 10⁻⁷ M PMA, which is a period for activation the PKCα, the actin bundles were thicker and more organized across the most epithelial cells of benign and cancer cells, while disruption of the actin organization across the cells was observed after 5h treatment with 10⁻⁷ PMA, a period for down regulation of PKCα. By means of epifluorescent microscopy, in the fibroadenomas cells, the immunofluorescent staining of integrin αvβ3 showed a slightly diffused pattern at the ventral cell surface, but mostly an intense distribution of integrin αvβ3 at the perinuclear area. The pattern of integrin αvβ3 immunofluorescence in the most malignant epithelial cells was mainly observed at the marginal region of the cells at the sites of focal contacts, contributing to the adherence of the cancer cells onto the substrate. Thus, integrin αvβ3 immunofluorescence revealed a pattern of bright aggregations (or clustering) distributed along the periphery of the ventral surface of the cells or at the leading edge of the advancing lamellipodium. The distribution of αvβ3 in the breast cancer epithelial cells after activation of PKCα with 100nM of PMA for 1h was changed. Increase bright aggregations (clustering) that distributed along the ventral cell surface, especially at the leading edge of the advancing lamellipodium indicating the increase in internalization of αvβ3 integrin in the cell membrane. Other cells showed increase immunostaining intensity along the cell periphery after 1h of PMA treatment. When the cells treated with 100nM of PMA for 5h, the staining pattern of the αvβ3 was changed as compared with the control cells (non-treated cells), decrease in the immunostaining aggregation in the cell body as well as in the cell periphery indicating the redistribution of the integrin αvβ3 from the cell periphery to the cell surface after down regulation of the PKCα by PMA. The results showed that the expression of PKCα in the breast cancer cells may be useful to use as a clinical marker for breast cancer cells and the expression of PKCα in the malignant cells plays an important role in invasiveness and cell motility of human breast cancer by affect the integrin αvβ3 expression and concomitant F-actin cytoskeletal reorganization. Modulation of PKCα lead to conclude that this enzyme interacts with αvβ3 integrin to promote movement of receptor to the cell surface and that PKCα activity is then responsible for integrin internalization in the cell membrane as well as in regulation of actin cytoskeleton, since integrin clustering and stress fiber formation consist an essential part of a mechanism inducing firm cell adhesion and invasion. In breast cancers, it might be essential to inhibit PKCα; however, it is not clear whether PKCα is the exclusive target of anti-tumor drugs that found until now, a major target or only a minor target. In addition to be a direct target of anti-tumor treatment, PKCα has been reported to be involved in anti-tumor drug resistance and in particular in MDR1-mediated resistance. PKCα modulators have been shown to enhance the antitumor activity of established anti-tumor drugs. However, the results of investigations concerning PKCα and anti-tumor drug resistance are also contradictory. According to them, the expression of PKCα did not corresponds to the expression of MDR1 gene or to the drug-sensitivity and multidrug resistant. The relevant efforts of basic research to identify the key and selective molecular alterations of PKCα and integrin αvβ3 which sustain breast cancer growth and metastasis open the possibility to develop a specific molecular target diagnosis and therapy. Since the integrin expression on breast cancer cells could be regulated through therapeutic strategies targeted to PKCα. These findings present opportunities for developing specific targeted therapies, which hold great promise as the next generation of anticancer therapeutics.

[…] Σκοπός της παρούσας διατριβής ήταν να διερευνηθεί μέσα από βιοψίες η συμμετοχή της PKCα στη μετάσταση και την κινητικότητα των ανθρώπινων καρκινοκυττάρων του μαστού, και γι’ αυτό το σκοπό αναπτύχθηκε ένα μοντέλο καλλιέργειας κυττάρων πρωτοπαθούς καρκίνου του μαστού έτσι ώστε να αντικατοπτρίζεται ο πρωτοπαθής αρχικός όγκος σε μια προσπάθεια να διατηρηθεί η μεγαλύτερη δυνατή ομοιότητα με το περιβάλλον των αρχικών όγκων προέλευση διότι οι προκλινικές μελέτες για τον καρκίνο του μαστού βασίζονται σε συγκεκριμένες κυτταρικές γραμμές οι οποίες μπορεί και να μην είναι αντιπροσωπευτικές της κυτταρικής συμπεριφοράς και του φαινοτύπου του πρωτοπαθούς, αρχικού όγκου λόγω προηγούμενων γονοτυπικών ή φαινοτυπικών αλλαγών. Χρησιμοποιώντας το παραπάνω μοντέλο ως πειραματικό εργαλείο εστιάσαμε τη μελέτη στα ακόλουθα σημεία: α) Στις μορφολογικές αλλαγές που παρατηρούνται στα κύτταρα του μαστού μετά από επώαση σε 10⁻⁷ M PMA για 1 h και 5h αντίστοιχα κι εξέταση υπό το ανάστροφο μικροσκόπιο. β) Στις αλλαγές στην έκφραση της PKC στα καρκινικά κύτταρα του μαστού κατόπιν επώασης σε 10⁻⁷ M PMA για 1h και 5h και εξέταση με την τεχνική του ανοσοφθορισμού. γ) Στις αλλαγές στον κυτταροσκελετό της F-ακτίνης μετά από επώαση σε 10⁻⁷ M PMA για 1h και 5h και εξέταση με τη χρήση φθορισμού. δ) Στις αλλαγές στην κατανομή της ιντεγκρίνης ανβ3 μετά την ενεργοποίηση και τη μειορρύθμιση της PKCα με τη χρήση PMA και την επώαση σε αυτό για 1h και 5h αντιστοίχως. Η τροποποίηση της PKCα μας οδήγησε στο συμπέρασμα ότι αυτή η πρωτεΐνη αλληλεπιδρά φυσικά με την ιντεγκρίνη ανβ3 με στόχο την προώθηση της μετακίνησης του υποδοχέα στην κυτταρική επιφάνεια και ότι η δραστηριότητα της PKCα είναι κατόπιν αυτή που ευθύνεται για την εσωτερίκευση της ιντεγκρίνης στην κυτταρική μεμβράνη καθώς επίσης και τη ρύθμιση του κυτταροσκελετού της ακτίνης. Μπορεί λοιπόν η έκφραση της ιντεγκρίνης στα καρκινικά κύτταρα του μαστού να ρυθμιστεί μέσω θεραπευτικών στρατηγικών που θα στοχεύουν στην PKCα. Αυτά τα ευρήματα αποτελούν ευκαιρία για την ανάπτυξη ειδικά στοχευμένων προσεγγίσεων, οι οποίες και θα είναι εξαιρετικά ελπιδοφόρες ως η επόμενη γενεά αντικαρκινικών θεραπειών. Οι σχετικές προσπάθειες της βασικής έρευνας να ταυτοποιήσει τις κύριες και επιλεκτικές μοριακές αλλαγές της PKCα και της ιντεγκρίνης ανβ3, οι οποίες και συντηρούν τις μεταστάσεις στον καρκίνο του μαστού, ανοίγουν το δρόμο για την ανάπτυξη ειδικών, μοριακά στοχευμένων διαγνώσεων και θεραπειών λόγω του ότι η έκφραση της ιντεγκρίνης στα καρκινικά κύτταρα του μαστού μπορεί να ρυθμιστεί μέσω θεραπευτικών στρατηγικών που έχουν ως στόχο την PKCα. […]