Synthesis of some potentially cytotoxic nitrogen mustards and studies on alkylation reactions of related compounds

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Epithelial-mesenchymal plasticity determines estrogen receptor positive breast cancer dormancy and epithelial reconversion drives recurrence

More than 70% of human breast cancers (BCs) are estrogen receptor α-positive (ER+). A clinical challenge of ER+ BC is that they can recur decades after initial treatments. Mechanisms governing latent disease remain elusive due to lack of adequate in vivo models. We compare intraductal xenografts of ER+ and triple-negative (TN) BC cells and demonstrate that disseminated TNBC cells proliferate similarly as TNBC cells at the primary site whereas disseminated ER+ BC cells proliferate slower, they decrease CDH1 and increase ZEB1,2 expressions, and exhibit characteristics of epithelial-mesenchymal plasticity (EMP) and dormancy. Forced E-cadherin expression overcomes ER+ BC dormancy. Cytokine signalings are enriched in more active versus inactive disseminated tumour cells, suggesting microenvironmental triggers for awakening. We conclude that intraductal xenografts model ER + BC dormancy and reveal that EMP is essential for the generation of a dormant cell state and that targeting exit from EMP has therapeutic potential

Atlas of Lobular Breast Cancer Models: Challenges and Strategic Directions

Invasive lobular carcinoma (ILC) accounts for up to 15% of all breast cancer (BC) cases and responds well to endocrine treatment when estrogen receptor α-positive (ER+) yet differs in many biological aspects from other ER+ BC subtypes. Up to 30% of patients with ILC will develop late-onset metastatic disease up to ten years after initial tumor diagnosis and may experience failure of systemic therapy. Unfortunately, preclinical models to study ILC progression and predict the efficacy of novel therapeutics are scarce. Here, we review the current advances in ILC modeling, including cell lines and organotypic models, genetically engineered mouse models, and patient-derived xenografts. We also underscore four critical challenges that can be addressed using ILC models: drug resistance, lobular tumor microenvironment, tumor dormancy, and metastasis. Finally, we highlight the advantages of shared experimental ILC resources and provide essential considerations from the perspective of the European Lobular Breast Cancer Consortium (ELBCC), which is devoted to better understanding and translating the molecular cues that underpin ILC to clinical diagnosis and intervention. This review will guide investigators who are considering the implementation of ILC models in their research programs

Oestrogen receptor α AF-1 and AF-2 domains have cell population-specific functions in the mammary epithelium.

Oestrogen receptor α (ERα) is a transcription factor with ligand-independent and ligand-dependent activation functions (AF)-1 and -2. Oestrogens control postnatal mammary gland development acting on a subset of mammary epithelial cells (MECs), termed sensor cells, which are ERα-positive by immunohistochemistry (IHC) and secrete paracrine factors, which stimulate ERα-negative responder cells. Here we show that deletion of AF-1 or AF-2 blocks pubertal ductal growth and subsequent development because both are required for expression of essential paracrine mediators. Thirty percent of the luminal cells are ERα-negative by IHC but express Esr1 transcripts. This low level ERα expression through AF-2 is essential for cell expansion during puberty and growth-inhibitory during pregnancy. Cell-intrinsic ERα is not required for cell proliferation nor for secretory differentiation but controls transcript levels of cell motility and cell adhesion genes and a stem cell and epithelial mesenchymal transition (EMT) signature identifying ERα as a key regulator of mammary epithelial cell plasticity

Patient-derived xenograft (PDX) models in basic and translational breast cancer research

Patient-derived xenograft (PDX) models of a growing spectrum of cancers are rapidly supplanting long-established traditional cell lines as preferred models for conducting basic and translational preclinical research. In breast cancer, to complement the now curated collection of approximately 45 long-established human breast cancer cell lines, a newly formed consortium of academic laboratories, currently from Europe, Australia, and North America, herein summarizes data on over 500 stably transplantable PDX models representing all three clinical subtypes of breast cancer (ER+, HER2+, and "Triple-negative" (TNBC)). Many of these models are well-characterized with respect to genomic, transcriptomic, and proteomic features, metastatic behavior, and treatment response to a variety of standard-of-care and experimental therapeutics. These stably transplantable PDX lines are generally available for dissemination to laboratories conducting translational research, and contact information for each collection is provided. This review summarizes current experiences related to PDX generation across participating groups, efforts to develop data standards for annotation and dissemination of patient clinical information that does not compromise patient privacy, efforts to develop complementary data standards for annotation of PDX characteristics and biology, and progress toward "credentialing" of PDX models as surrogates to represent individual patients for use in preclinical and co-clinical translational research. In addition, this review highlights important unresolved questions, as well as current limitations, that have hampered more efficient generation of PDX lines and more rapid adoption of PDX use in translational breast cancer research

Signal transduction by TGFβ/activin A and early endosomes: role of SARA protein

Members of the TGFβ/Activin A family transduce their signals via serine/threonine transmembrane receptors type I and II. Constitutively active type II receptors phosphorylate type I receptors, which in turn activate RSMADs (SMAD2 and SMAD3) at their C-terminal SSXS motifs. The R-Smads, in turn, oligomerize with the common partner SMAD4 and translocate to the nucleus where they bind to the promoters of a large variety of target genes and regulate their expression in a positive or negative manner. SARA (SMAD Anchor for Receptor Activation) plays an important role in this process, recruiting SMAD2/3 to the vicinity of the receptor for phosphorylation. SARA harbours a FYVE domain (Fab1, YOTB/ZK632.12, Vac1, και EEA1), which targets the protein to the endocytic compartment via binding to PI(3)P, which is enriched in the early endosome. In the present study, using yeast-two hybrid system, we identified ERBIN (ERBB2 Interacting Protein) as a novel SARA interacting protein. The interaction was confirmed using biochemical approaches, such as GST pull down assays, immunoprecipitation experiments and sucrose flotation experiments. Moreover, using confocal microscopy, we found that overexpression of GFP-SARA recruits ERBIN to the early endocytic compartment. To define the domains of SARA and ERBIN responsible for the interaction, we generated a series of deletion mutants of both proteins and tested their interaction. To this end, we found that SARA interacts with ERBIN via a domain, which we called ERBID (ERBIN Binding Domain). ERBIN associated with SARA via a 57 aa domain. ERBIN was recently shown to bind and segregate phosphorylated SMAD2/3 in the cytoplasm, thereby inhibiting SMAD2/3-dependent transcription. We further mapped the domain of ERBIN, which interacted with SMAD2/3 and found that SMAD2/3 and SARA bind to the same domain of ERBIN, an area we called SSID (SARA and SMAD Interacting Domain). Moreover, we found that ERBIN interacts with non-phosphorylated and phosphorylated SMAD2/3 proteins. Thus, ERBIN may retain phosphorylated SMAD2/3 in the cytoplasm inhibiting the translocation to the nucleus and regulating transcription of target genes. Due to the fact that SARA and SMAD2/3 bind to the same domain of ERBIN, we addressed whether this binding was competitive. We showed that SARA and SMAD2/3 compete with each other for their binding to the SSID domain of ERBIN. In accordance, SARA or ERBID overexpression, reverts the inhibitory effect of ERBIN on SMAD2/3 dependent translation. In conclusion, we have identified ERBIN as a novel SARA-interacting protein that can be recruited by the latter to early endosomes, where SARA predominantly resides. SARA binds to ERBIN using a novel domain, which we have called ERBID. ERBIN interacts with SARA via a domain that interacts also with SMAD2 and SMAD3 (aa 1208-1265, SSID). As a consequence, SARA competes with SMAD2/3 for binding to ERBIN. As ERBIN binds and segregates phosphorylated SMAD2/3 in the cytoplasm, thereby inhibiting SMAD2/3-dependent transcription, over-expression of SARA or ERBID reverses the inhibitory effect of ERBIN on SMAD2/3-dependent transcription. Thus, SARA not only ensures proper presentation of SMAD2/3 for phosphorylation by TGFβ/Activιν A receptors, but also facilitates the nuclear transfer of phosphorylated SMAD2/3 by out-competing their cytoplasmic segregation by ERBIN. Our data suggest that the response of cells to TGFβ/Activin A might be regulated by a complex interplay between the relative concentrations of SARA, ERBIN, and SMAD2/3, as well as their binding affinities.Μέλη της οικογένειας TGFβ/Ακτιβίνης Α μετάγουν σήματα μέσω διαμεμβρανικών υποδοχέων τύπου Ι και ΙΙ με ενεργότητα σερίνης/θρεονίνης. Ο συνεχώς ενεργός υποδοχέας τύπου II φωσφορυλιώνει τον τύπου Ι υποδοχέα ο οποίος στη συνέχεια ενεργοποιεί τους διαμεσολαβητές SMAD2 και SMAD3. Οι τελευταίοι συνδέονται με τη SMAD4 και το σύμπλοκο που δημιουργείται μετατοπίζεται στο πυρήνα του κυττάρου όπου ρυθμίζει τη μεταγραφή γονιδίων στόχων. Η πρωτεΐνη SARA (SMAD Anchor for Receptor Activation) διαδραματίζει σημαντικό ρόλο στη διαδικασία αυτή αφού στρατολογεί και παρουσιάζει τη SMAD2 και τη SMAD3 στους τύπου Ι υποδοχείς για φωσφορυλίωση. Η SARA όχι μόνο εντοπίζεται στα πρώιμα ενδοσώματα, αλλά και η περιοχή FYVE (Fab1, ΥΟΤΒ/ΖΚ632.12, Vac1, και ΕΕΑ1), μέσω της σύνδεσης της με το λιπίδιο ΡΙ(3)Ρ που είναι εμπλουτισμένο στο διαμέρισμα αυτό, είναι αρκετή για τη στόχευσή της στις μεμβράνες των ενσωμάτων. Στην παρούσα εργασία, χρησιμοποιώντας το σύστημα δύο υβριδίων, ταυτοποιήσαμε την ERBIN (ERBB2 interacting Protein) ως μια πρωτεΐνη που αλληλεπιδρά με τη SARA. Η αλληλεπίδραση αυτή επιβεβαιώθηκε με βιοχημικές τεχνικές, όπως δοκιμασίες καταβύθισης, πειράματα ανοσοκατακρήμνισης και επίπλευση μεμβρανικών κλασμάτων σε βαθμίδωση σουκρόζης. Επιπλέον, χρησιμοποιώντας συνεστιακή μικροσκοπία, βρήκαμε ότι η υπερέκφραση της GFP-SARA είναι ικανή να στρατολογήσει την ERBIN στα πρώιμα ενδοσώματα, αφού οδήγησε σε σημαντικό βαθμό συνεντοπισμού μεταξύ των δύο πρωτεϊνών. Στη συνέχεια, εστιάσαμε την προσοχή μας στη μοριακή χαρτογράφηση, δηλαδή στην ανεύρεση των ελάχιστων πρωτεϊνικών περιοχών που είναι υπεύθυνες για την αλληλεπίδραση. Για το σκοπό αυτό, δημιουργήσαμε αλληλεπικαλυπτόμενες πλασμιδιακές κατασκευές που χρησιμοποιήθηκαν σε μια σειρά πειραμάτων καταβύθισης από τα οποία προκύπτει ότι η πρωτεΐνη SARA αλληλεπιδρά με την ERBIN χρησιμοποιώντας μια περιοχή, την οποία την ονομάσαμε ERBID (ERBIN Binding Domain) και η οποία για πρώτη φορά ταυτοποιείται να αλληλεπιδρά με τη SARA. Από την άλλη πλευρά, η πρωτεΐνη ERBIN συνδέεται με τη SARA μέσω της ίδιας περιοχής (aa 1208-1265) με την οποία αλληλεπιδρούν επίσης οι R-SMADs (SMAD2 και SMAD3). Την παραπάνω περιοχή την ονομάσαμε SSID (SARA and SMAD Interacting Domain). Επιπλέον, διαλευκάναμε ότι η ERBIN μπορεί να αλληλεπιδρά όχι μόνο με τις μη-φωσφορυλιωμένες, αλλά και με τις φωσφορυλιωμένες μορφές των SMAD2/3 πρωτεϊνών, μορφές με τις οποίες δεν αλληλεπιδρά η SARA. Έτσι, η ERBIN είναι σε θέση να κατακρατεί τις φωσφορυλιωμένες SMAD2/3 στο κυτταρόπλασμα αναστέλλοντας τη μετακίνηση τους στον πυρήνα και τη ρύθμιση της μεταγραφής των γονιδίων στόχων. Με βάση το γεγονός ότι η SARA και οι SΜAD2/3 δεσμεύονται στην ίδια περιοχή της ERBIN, διερευνήσαμε αν αυτή η σύνδεση είναι ανταγωνιστική. Δείξαμε λοιπόν, ότι οι SMAD2/3 πρωτεΐνες και η SARA, που αλληλεπιδρούν με την ERBIN ανεξάρτητα η μια από την άλλη, ανταγωνίζονται μεταξύ τους για την πρόσδεση στην περιοχή SSID της ERBIN. Σε συμφωνία, η υπερέκφραση της SARA ή του πολυπεπτιδίου ERBID αντιστρέφει την ανασταλτική επίδραση της ERBIN στην από τις πρωτεΐνες SMAD2/3-εξαρτώμενη μεταγραφή. Εν κατακλείδι, τα αποτελέσματα της παρούσας μελέτης προτείνουν ότι η SARA, έκτος από την παρουσίαση των SMAD2/3 για φωσφορυλίωση από τους υποδοχείς του TGFβ ή της Ακτιβίνης Α, διευκολύνει επίσης τη μετακίνηση των φωσφορυλιωμένων SΜAD2/3 στον πυρήνα δρώντας ανταγωνιστικά ως προς την κατακράτηση στο κυτταρόπλασμα των φωσφορυλιωμένων SMAD2/3 πρωτεϊνών που προκαλεί η παρουσία της ERBIN. Φαίνεται, λοιπόν, ότι η απόκριση των κυττάρων στον TGFβ ή την Ακτιβίνη Α μπορεί να ρυθμίζεται από μια πολύπλοκη διασύνδεση που εξαρτάται από τις σχετικές συγκεντρώσεις της SARA, της ERBIN και των SMAD2/3 πρωτεϊνών, καθώς επίσης και των σταθερών διάστασης των αλληλεπιδράσεών τους

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens

The challenges of modeling hormone receptor-positive breast cancer in mice.

Estrogen receptor-positive (ER+) tumors account for 70-80% of all breast cancer (BC) cases and are characterized by estrogen dependency for their growth. Endocrine therapies using estrogen receptor antagonists or aromatase inhibitors represent a key component of the standard of care for these tumors. The occurrence of de novo or acquired resistance to estrogen withdrawal represents an important clinical problem, impacting on patient survival. In addition, despite an initially favorable outcome, a part of ER+ BC patients present with disease recurrence locally or at distant sites years or even decades after apparent remission. <i>In vivo</i> models that closely mimic human disease are urgently needed to study the biology of these tumors, investigate the molecular mechanisms underlying endocrine resistance and identify patients at risk of recurrence. Despite the similarities in the overall hormonal regulation of mammary gland development between mice and humans, the majority of the mammary carcinomas occurring in genetically engineered mouse models (GEMMs) are ER negative and most xenograft models are based on few ER+ cancer cell lines. We recently showed that the microenvironment is critical for ER+ cancer cells and discuss in this review the potential of intraductal xenograft model for basic and preclinical research

The histone chaperone SET/TAF-Ibeta interacts functionally with the CREB-binding protein

he oncoprotein SET/TAF-Ibeta is a histone chaperone which is involved in cell-cycle control and chromatin remodeling. Confocal laser scanning microscopy reveals that SET is localized in distinct foci of variable size throughout the nucleoplasm of interphase cells. We report here that SET interacts directly with the acetyltransferase CREB-binding protein (CBP) and enhances the transactivation potential of the transcription coactivator. Our data suggest that the histone chaperone SET regulates the CBP-mediated transcription and may indicate a general principle by which transcriptional regulators cooperate with histone chaperones for gene activation