Collagen Fiber Array of Peritumoral Stroma Influences Epithelial-to-Mesenchymal Transition and Invasive Potential of Mammary Cancer Cells

: Interactions of cancer cells with matrix macromolecules of the surrounding tumor stroma are critical to mediate invasion and metastasis. In this study, we reproduced the collagen mechanical barriers in vitro (i.e., basement membrane, lamina propria under basement membrane, and deeper bundled collagen fibers with different array). These were used in 3D cell cultures to define their effects on morphology and behavior of breast cancer cells with different metastatic potential (MCF-7 and MDA-MB-231) using scanning electron microscope (SEM). We demonstrated that breast cancer cells cultured in 2D and 3D cultures on different collagen substrates show different morphologies: i) a globular/spherical shape, ii) a flattened polygonal shape, and iii) elongated/fusiform and spindle-like shapes. The distribution of different cell shapes changed with the distinct collagen fiber/fibril physical array and size. Dense collagen fibers, parallel to the culture plane, do not allow the invasion of MCF-7 and MDA-MB-231 cells, which, however, show increases of microvilli and microvesicles, respectively. These novel data highlight the regulatory role of different fibrillar collagen arrays in modifying breast cancer cell shape, inducing epithelial-to-mesenchymal transition, changing matrix composition and modulating the production of extracellular vesicles. Further investigation utilizing this in vitro model will help to demonstrate the biological roles of matrix macromolecules in cancer cell invasion in vivo

ESR2 Drives Mesenchymal-to-Epithelial Transition in Triple-Negative Breast Cancer and Tumorigenesis In Vivo

Estrogen receptors (ERs) have pivotal roles in the development and progression of triple-negative breast cancer (TNBC). Interactions among cancer cells and tumor microenvironment are orchestrated by the extracellular matrix that is rapidly emerging as prominent contributor of fundamental processes of breast cancer progression. Early studies have correlated ER beta expression in tumor sites with a more aggressive clinical outcome, however ER beta exact role in the progression of TNBC remains to be elucidated. Herein, we introduce the functional role of ER beta suppression following isolation of monoclonal cell populations of MDA-MB-231 breast cancer cells transfected with shRNA against human ESR2 that permanently resulted in 90% reduction of ER beta mRNA and protein levels. Further, we demonstrate that clone selection results in strongly reduced levels of the aggressive functional properties of MDA-MB-231 cells, by transforming their morphological characteristics, eliminating the mesenchymal-like traits of triple-negative breast cancer cells. Monoclonal populations of shER beta MDA-MB-231 cells undergo universal matrix reorganization and pass on a mesenchymal-to-epithelial transition state. These striking changes are encompassed by the total prevention of tumorigenesis in vivo following ER beta maximum suppression and isolation of monoclonal cell populations in TNBC cells. We propose that these novel findings highlight the promising role of ER beta targeting in future pharmaceutical approaches for managing the metastatic dynamics of TNBC breast cancer

ESR2 Drives Mesenchymal-to-Epithelial Transition in Triple-Negative Breast Cancer and Tumorigenesis In Vivo

Estrogen receptors (ERs) have pivotal roles in the development and progression of triple-negative breast cancer (TNBC). Interactions among cancer cells and tumor microenvironment are orchestrated by the extracellular matrix that is rapidly emerging as prominent contributor of fundamental processes of breast cancer progression. Early studies have correlated ERβ expression in tumor sites with a more aggressive clinical outcome, however ERβ exact role in the progression of TNBC remains to be elucidated. Herein, we introduce the functional role of ERβ suppression following isolation of monoclonal cell populations of MDA-MB-231 breast cancer cells transfected with shRNA against human ESR2 that permanently resulted in 90% reduction of ERβ mRNA and protein levels. Further, we demonstrate that clone selection results in strongly reduced levels of the aggressive functional properties of MDA-MB-231 cells, by transforming their morphological characteristics, eliminating the mesenchymal-like traits of triple-negative breast cancer cells. Monoclonal populations of shERβ MDA-MB-231 cells undergo universal matrix reorganization and pass on a mesenchymal-to-epithelial transition state. These striking changes are encompassed by the total prevention of tumorigenesis in vivo following ERβ maximum suppression and isolation of monoclonal cell populations in TNBC cells. We propose that these novel findings highlight the promising role of ERβ targeting in future pharmaceutical approaches for managing the metastatic dynamics of TNBC breast cancer

The Cytotoxic Effects of Betulin-Conjugated Gold Nanoparticles as Stable Formulations in Normal and Melanoma Cells

Gold nanoparticles are currently investigated as theranostics tools in cancer therapy due to their proper biocompatibility and increased efficacy related to the ease to customize the surface properties and to conjugate other molecules. Betulin, [lup-20(29)-ene-3β, 28-diol], is a pentacyclic triterpene that has raised scientific interest due to its antiproliferative effect on several cancer types. Herein we described the synthesis of surface modified betulin-conjugated gold nanoparticles using a slightly modified Turkevich method. Transmission electron microscopy (TEM) imaging, dynamic light scattering (DLS), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) were used for the characterization of obtained gold nanoparticles. Cytotoxic activity and apoptosis assessment were carried out using the MTT and Annexin V/PI apoptosis assays. The results showed that betulin coated gold nanoparticles presented a dose-dependent cytotoxic effect and induced apoptosis in all tested cell lines

Evaluation of the regulatory mechanisms governing biomolecules expression, functional properties and morphological characteristics of breast cancer cells

Estrogen receptors (ERs) have pivotal roles in breast cancer growth and progression. Even though the contribution of ERα in the modulation of breast cancer cells' behavior is thoroughly studied, the biological functions of its isoform, ERβ, are less elucidated. In the present doctoral thesis, we demonstrated that ERβ suppression in the highly aggressive, ERβ-positive MDA-MB-231 breast cancer cells (shERβ MDA-MB-231) resulted in profound phenotypic changes, inhibition of EMT process and major changes in the properties as well as in gene and protein expression levels of certain functional matrix components of breast cancer cells in a 17-β-estradiol (E2)-independent manner. As observed by scanning electron microscopy, ERβ suppression strongly affects the morphology of shERβ MDA-MB-231 cells, which is followed by downregulated expression levels of the mesenchymal markers fibronectin and vimentin, whereas it increases the expression levels of epithelial marker E-cadherin and cell-cell junctions. These alterations are followed by reduced levels of cell functional properties that promote the aggressiveness of these cells, such as proliferation, migration, spreading capacity, invasion and adhesion. Notably, ERβ suppression reduces the migration of MDA-MB-231 breast cancer cells via EGFR/IGF-IR and JAK/STAT signaling pathways. Moreover, our findings revealed that ERβ has a crucial role in modulation of mRNA levels and protein expression of several matrix mediators, including the transmembrane syndecans and intracellular serglycin, several MMPs, plasminogen activation system components and receptor tyrosine kinases. These data clearly demonstrate that ERβ plays a crucial role in mediating cell behavior and ECM composition of the highly aggressive MDA-MB-231 cells and it opens a new area of research to further understand its role and to improve pharmaceutical targeting of the non-hormone-dependent breast cancer.The epigenetic alterations are responsible for the ability of the tumor cells to metastasize. In the present study, we demonstrated that ER status is associated with distinct miRNA expression profiles in MCF-7 and MDA-MB-231 breast cancer cells, and that mainly miR-10b (oncogenic miRNA) and miR-200b (EMT inhibitor) are the key regulators of MDA-MB-231 cell behavior. Notably, the expression profiles of these miRNAs are mediated through EGFR/IGF-IR crosstalk with E2. Moreover, growing ERα-positive, MCF-7, and ERβ-positive, MDA-MB-231, cells in estrogen-free medium resulted in a diverse impact on miRNA expression and the behavior of these cells, suggesting the specific effect of E2 on the miRNAs expression profile, depending on the ER status of breast cancer cells. Specifically, ERβ suppression in MDA-MB-231 breast cancer cells results in significant changes in the expression profiles of specific miRNAs that regulate breast cancer progression, including miR-10b, miR-200b and miR-145 (tumor-suppressive miRNA). Enhanced miR-10b expression or miR-145 silencing in shERβ MDA-MB-231 cells revealed that these miRNAs can regulate the functional properties, EMT program and expression of major ECM components known as modulators of breast cancer aggressiveness. Our data pinpointed that miR-10b is strongly implicated in the regulation of functional properties, expression of EMT markers and ERK1/2 signaling in shERβ MDA-MB-231 cells, thus affecting ECM composition and subsequently increasing the aggressiveness of these cells. Syndecan-1 and the proteolytic milieu macromolecules, especially MMP2, MMP7 and MMP9, are the most affected among ECM macromolecules. Accordingly, the inhibition of miR-145 expression significantly increased the aggressiveness of shERβ MDA-MB-231 cells and induced EMT. Furthermore, miR-145 silencing resulted in striking changes in mRNA levels and protein expression of major ECM mediators, such as HER2 and several MMPs, whereas it significantly increased the phosphorylated levels of ERK1/2 kinases in these cells, suggesting the crucial role of miR-145 in this signaling pathway.In conclusion, these novel data suggest that the alterations in cell behavior and in ECM composition caused by ERβ suppression in MDA-MB-231 cells are closely related to certain epigenetic miRNA-induced alterations. Targeting the ERα/β-regulated miR-10b, miR-200b and miR-145 serves as a promising tool for early diagnosis and pharmaceutical targeting in aggressive, non-hormone-dependent breast cancer.Οι οιστρογονοϋποδοχείς (ERs) κατέχουν κεντρικό ρόλο στην ανάπτυξη και την εξέλιξη του καρκίνου του μαστού. Παρόλο που η συνεισφορά του ERα στη ρύθμιση της συμπεριφοράς των καρκινικών κυττάρων μαστού έχει μελετηθεί εκτενώς, οι βιολογικές δράσεις της ισομορφής του, ERβ, δεν είναι απολύτως αποσαφηνισμένες. Στην παρούσα διδακτορική διατριβή, αποδείχθηκε ότι η καταστολή του ERβ στα επιθετικά, ERβ-θετικά, MDA-MB-231 καρκινικά κύτταρα μαστού (shERβ MDA-MB-231) οδήγησε σε αξιοσημείωτες φαινοτυπικές αλλαγές, καταστολή της διαδικασίας μετασχηματισμού από επιθηλιακό σε μεσεγχυματικό φαινότυπο (ΕΜΤ), καθώς και σε σημαντικές διαφοροποιήσεις στις ιδιότητες και στα επίπεδα γονιδιακής και πρωτεϊνικής έκφρασης λειτουργικών συστατικών του εξωκυττάριου χώρου (ECM) των καρκινικών κυττάρων μαστού. Όπως παρατηρήθηκε από ηλεκτρονική μικροσκοπία σάρωσης, η καταστολή του ERβ επηρεάζει σημαντικά τα μορφολογικά χαρακτηριστικά των shERβ MDA-MB-231 κυττάρων. Επιπλέον, η καταστολή του ERβ μειώνει την έκφραση των μεσεγχυματικών δεικτών fibronectin και vimentin, ενώ αυξάνει τα επίπεδα έκφρασης του επιθηλιακού δείκτη Ε-cadherin και τις κυτταρικές διεπαφές. Οι συγκεκριμένες αλλαγές ακολουθούνται από μειωμένα επίπεδα των λειτουργικών ιδιοτήτων που προάγουν την επιθετικότητα των συγκεκριμένων καρκινικών κυττάρων, όπως ο πολλαπλασιασμός, η μετανάστευση, η διήθηση και η προσκόλληση. Αξίζει να σημειωθεί ότι η καταστολή του ERβ μειώνει τη μετανάστευση των MDA-MB-231 καρκινικών κυττάρων μαστού μέσω των σηματοδοτικών μονοπατιών EGFR/IGF-IR και JAK/STAT. Επιπλέον, τα δεδομένα μας αποκάλυψαν ότι ο ERβ κατέχει κρίσιμο ρόλο στη ρύθμιση της μεταγραφικής και πρωτεϊνικής έκφρασης σημαντικών τελεστών του ECM, όπως των μεμβρανικών συνδεκανών και της ενδοκυττάριας σεργλυκίνης, πολλών ΜΜΡs, συστατικών του συστήματος ενεργοποίησης του πλασμινογόνου και υποδοχέων κινάσης τυροσίνης. Αυτά τα δεδομένα δείχνουν ξεκάθαρα ότι ο ERβ αποτελεί σημαντικό ρυθμιστή της κυτταρικής συμπεριφοράς και της σύστασης του ECM των υψηλού μεταστατικού δυναμικού, MDA-MB-231 καρκινικών κυττάρων μαστού, ανοίγοντας ένα νέο πεδίο έρευνας για την κατανόηση του ρόλου του και τη βελτίωση της φαρμακολογικής στόχευσης του μη-ορμονοεξαρτώμενου, επιθετικού καρκίνου του μαστού.Oι επιγενετικές αλλαγές είναι υπεύθυνες για την ικανότητα των καρκινικών κυττάρων να μεταναστεύουν. Στην παρούσα διδακτορική διατριβή, αποδείξαμε ότι η έκφραση των ERs συνδέεται με τη διαφορική έκφραση πολλών miRNAs που σχετίζονται με την εξέλιξη του καρκίνου του μαστού, στα MCF-7 και MDA-MB-231 καρκινικά κύτταρα μαστού, ενώ κυρίως τα miR-10b (ογκογενετικό miRNA) και miR-200b (αναστολέας του ΕΜΤ) φαίνεται να είναι εκείνα που εμπλέκονται στη ρύθμιση της επιθετικής συμπεριφοράς των MDA-MB-231 κυττάρων. Μάλιστα η έκφρασή τους διαμεσολαβείται από τη διεπικοινωνία των σηματοδοτικών μονοπατιών EGFR/IGF-IR με τη 17β-οιστραδιόλη (E2). Επιπλέον, η καλλιέργεια των ERα-θετικών, MCF-7, και ERβ-θετικών, MDA-MB-231, κυττάρων σε μέσο καλλιέργειας απουσία οιστρογόνων, έχει διαφορετικό αντίκτυπο στην έκφραση των παραπάνω miRNAs και στη συμπεριφορά αυτών των κυττάρων, γεγονός που εξηγεί τη στοχευμένη επίδραση της E2 στην έκφραση των miRNAs, αναλόγως της έκφρασης των ERs στα καρκινικά κύτταρα μαστού. Παράλληλα, η καταστολή του ERβ στα MDA-MB-231 κύτταρα οδήγησε σε σημαντικές αλλαγές στα προφίλ έκφρασης συγκεκριμένων miRNAs, συμπεριλαμβανομένων των miR-10b, miR-200b και miR-145 (ογκοκατασταλτικό miRNA). Η υπερέκφραση του miR-10b είτε η αποσιώπηση του miR-145 στα shERβ MDA-MB-231 κύτταρα, αποκάλυψαν ότι τα συγκεκριμένα miRNAs ρυθμίζουν τις λειτουργικές ιδιότητες, το πρόγραμμα ΕΜΤ και την έκφραση σημαντικών τελεστών του ECM που είναι γνωστοί για την εμπλοκή τους στην επιθετικότητα του καρκίνου του μαστού. Τα δεδομένα έδειξαν ότι το miR-10b συμμετέχει ενεργά στη ρύθμιση των ιδιοτήτων, στην έκφραση δεικτών του ΕΜΤ και στη σηματοδότηση ERK1/2 στα shERβ MDA-MB-231 καρκινικά κύτταρα, επηρεάζοντας έτσι τη σύσταση του ECM, με αποτέλεσμα την ενίσχυση της επιθετικότητας των κυττάρων αυτών. Σημαντικότερη επίδραση παρουσιάζεται στη συνδεκάνη-1 και σε μόρια του πρωτεολυτικού καταρράκτη, κυρίως στις ΜΜΡ2, ΜΜΡ7, ΜΜΡ9. Αντίστοιχα, η καταστολή του miR-145 επήγαγε σημαντικά την επιθετικότητα των shERβ MDA-MB-231 κυττάρων καθώς και τη διαδικασία ΕΜΤ. Επιπλέον, η καταστολή της έκφρασης του συγκεκριμένου miRNA οδήγησε σε σημαντικές μεταγραφικές και πρωτεϊνικές αλλαγές των ρυθμιστών του ECM, όπως του HER2 και πολλών MMPs, ενώ επήγαγε τα επίπεδα φωσφορυλίωσης των ERK1/2 κινασών σε αυτά τα κύτταρα, υποδηλώνοντας τον κρίσιμο ρόλο του miR-145 στην ενεργοποίηση του συγκεκριμένου σηματοδοτικού μονοπατιού.Συμπερασματικά, τα αποτελέσματα της παρούσας μελέτης αποδεικνύουν ότι οι αλλαγές που παρατηρούνται στην κυτταρική συμπεριφορά και στη σύσταση του ECM από την καταστολή του ERβ στα MDA-MB-231 καρκινικά κύτταρα μαστού, είναι στενά συνδεδεμένες με συγκεκριμένες επιγενετικές τροποποιήσεις σε επίπεδο miRNAs. Καταλήγοντας, η στόχευση των ERα/β-ρυθμιζόμενων miR-10b, miR-200b και miR-145 αποτελεί ένα πολλά υποσχόμενο εργαλείο για την έγκαιρη διάγνωση και την αποτελεσματική φαρμακολογική στόχευση του επιθετικού, μη-ορμονοεξαρτώμενου καρκίνου του μαστού

Matrix Effectors and Cancer

Extracellular matrices (ECMs) are highly dynamic three-dimensional structural meshworks composed of macromolecules, such as proteoglycans/glycosaminoglycans (PGs/GAGs), collagens, laminins, elastin, (glyco)proteins, and matrix-degrading enzymes, such as proteases and glycosidases [...

Estrogen receptor beta as epigenetic mediator of miR-10b and miR-145 in mammary cancer

Even though the role of estrogen receptor alpha (ER\u3b1) in the modulation of breast cancer cells' behavior is thoroughly studied, the biological functions of its isoform, ER\u3b2, are less elucidated. The suppression of ER\u3b2 in the aggressive ER\u3b1-negative MDA-MB-231 breast cancer cells resulted in the inhibition of epithelial to mesenchymal transition (EMT) and major changes in the basic functional properties and expression levels of certain matrix components of breast cancer cells. This arrest in metastatic potential of breast cancer cells suggests the contribution of ER\u3b2 in the induction of a more aggressive phenotype in MDA-MB-231 breast cancer cells. The epigenetic alterations are responsible for the ability of the tumor cells to metastasize. Here, we report for the first time that the suppression of ER\u3b2 in MDA-MB-231 breast cancer cells leads to significant changes in the expression profiles of specific microRNAs, including miR-10b, miR-200b and miR-145. Growth of MCF-7 and MDA-MB-231 cells in estrogen-free medium has a diverse impact on miRNA expression and the behavior of these cells, suggesting the specific effect of estradiol on the miRNA expression profile depending on the ER status of breast cancer cells. Enhanced miR-10b expression or silencing of miR-145 clearly revealed that these microRNAs can regulate the functional properties, EMT program and the expression of major matrix components known to be implicated in breast cancer aggressiveness. Our data revealed that miR-10b is strongly implicated in the regulation of functional properties, EMT program and Erk1/2 signaling in shER\u3b2 MDA-MB-231 cells, thus affecting the extracellular matrix (ECM) composition, including syndecan-1, proteolytic behavior, especially MMP2, MMP7 and MMP9 expression and subsequently the aggressiveness of these cells. Accordingly, the inhibition of miR-145 expression significantly increased the aggressiveness of shER\u3b2 MDA-MB-231 cells and induced EMT. Moreover, miR-145 inhibition resulted in important changes in the gene and protein levels of ECM mediators, such as HER2 and several MMPs, whereas it significantly increased the phosphorylated levels of Erk1/2 kinases in these cells, suggesting the crucial role of miR-145 in this signaling pathway. These novel results suggest that the alterations in cell behavior and in ECM composition caused by the suppression of ER\u3b2 in MDA-MB-231 cells are closely related to certain epigenetic miRNA-induced alterations. Targeting the ER\u3b2-regulated miR-10b and miR-145 is a promising tool for diagnosis and pharmaceutical targeting in breast cancer

Extracellular Matrix-Mediated Breast Cancer Cells Morphological Alterations, Invasiveness, and Microvesicles/Exosomes Release

Breast cancer is a leading disease in women. Several studies are focused to evaluate the critical role of extracellular matrix (ECM) in various biochemical and molecular aspects but also in terms of its effect on cancer cell morphology and therefore on cancer cell invasion and metastatic potential. ECM fibrillar components, such as collagen and fibronectin, affect cell behavior and properties of mammary cancer cells. The aim of this study was to investigate using the scanning electron microscopy (SEM) how the highly invasive MDA-MB-231 breast cancer cells, interplaying with ECM substrates during cell migration/invasion, modify their morphological characteristics and cytoplasmic processes in relation to their invasive potential. In particular we reproduced and analyzed how natural structural barriers to cancer cell invasion, such as the basement membrane (Matrigel) and fibrillar components of dermis (fibronectin as well as the different concentrations/array of type I collagen), could induce morphological changes in 3D cultures. Interestingly, we demonstrate that, even with different effects, all collagen concentrations/arrays lead to morphological alterations of breast cancer cells. Intriguingly, the elongated mesenchymal shaped cells were more prominent in 3D cultures with a dense and thick substrate (thick Matrigel, high concentrated collagen network, and densely packed collagen fibers), even though cells with different shape produced and released microvesicles and exosomes as well. It is therefore evident that the peri-tumoral collagen network may act not only as a barrier but also as a dynamic scaffold which stimulates the morphological changes of cancer cells, and modulates tumor development and metastatic potential in breast cancer

Altered Adipokine Expression in Tumor Microenvironment Promotes Development of Triple Negative Breast Cancer

Obesity is a remarkably important factor for breast carcinogenesis and aggressiveness. The implication of increased BMI in triple negative breast cancer (TNBC) development is also well established. A malignancy-promoting role of the adipose tissue has been supposed, where the adipocytes that constitute the majority of stromal cells release pro-inflammatory cytokines and growth factors. Alterations in adipokines and their receptors play significant roles in breast cancer initiation, progression, metastasis, and drug response. Classic adipokines, such as leptin, adiponectin, and resistin, have been extensively studied in breast cancer and connected with breast cancer risk and progression. Notably, new molecules are constantly being discovered and the list is continuously growing. Additionally, substantial progress has been made concerning their differential expression in association with clinical and pathological parameters of tumors and the prognostic and predictive value of their dysregulation in breast cancer carcinogenesis. However, evidence regarding the mechanisms by which adipose tissue is involved in the development of TNBC is lacking. In the present article we comment on current data on the suggested involvement of these mediators in breast cancer development and progression, with particular emphasis on TNBC, to draw attention to the design of novel targeted therapies and biomarkers