Notch Lineages and Activity in Intestinal Stem Cells Determined by a New Set of Knock-In Mice

The conserved role of Notch signaling in controlling intestinal cell fate specification and homeostasis has been extensively studied. Nevertheless, the precise identity of the cells in which Notch signaling is active and the role of different Notch receptor paralogues in the intestine remain ambiguous, due to the lack of reliable tools to investigate Notch expression and function in vivo. We generated a new series of transgenic mice that allowed us, by lineage analysis, to formally prove that Notch1 and Notch2 are specifically expressed in crypt stem cells. In addition, a novel Notch reporter mouse, Hes1-EmGFPSAT, demonstrated exclusive Notch activity in crypt stem cells and absorptive progenitors. This roster of knock-in and reporter mice represents a valuable resource to functionally explore the Notch pathway in vivo in virtually all tissues

Interactions of cancer cells with stromal cells during tumorigenesis

Previous evidence had indicated the existence of mutations of p53 in stromal fibroblasts ofhuman tumors. To investigate the possible significance of this deficiency in stromal fibroblasts weperformed an analysis of the latency for tumor onset of xenografts containing wild type or p53 deficientmouse embryonic fibroblasts. My results showed that p53 deficiency increased tumorigenicity butrenders tumors more sensitive to drug treatment. We were able to demonstrate in vitro, that, contraryto their wild type counterparts, during chemotherapy, p53 deficient fibroblasts fail to adequatelysupport the cancer cells. This is due to the failure of p53 deficient fibroblasts to undergo senescence andthus stimulate a paracrine mitogenic program, upon exposure to DNA damaging anti-tumor drugs. Theseresults highlight the importance of considering the effects of treatment on both the malignantcompartment as well as the surrounding stroma and imply a possible therapeutic strategy of blockingsenescence during treatment. Contrary to the p53 gene, the differential genotype for Notch3 in stromalfibroblasts had no effect on tumorigenesis. However, knockout of this gene in the epithelialcompartment results in a marked impediment of tumorigenesis in the MMTV-Neu breast cancer model.Given the implication of Notch3 in basal breast cancer which arises from mammary progenitor cells, weinvestigate the role of Notch3 and the mammary epithelial cells that express it. In order to perform thisanalysis we utilized novel transgenic mice recently developed by Exelixis Inc. in collaboration with Dr.Artavanis-Tsakonas. My results show that Notch3 is expressed in luminal progenitor cells that are able togive cells of all lineages of the luminal compartment, and are able to survive apoptosis during involution.Upon transplantation these cells demonstrate regenerative capacity be regenerating mammary ducts, acapacity previously attributed only to the myoepithelial cells of the mammary gland. When examiningthe morphogenetic integrity of young adult N3KO mammary glands, I observed no striking difference,however transplantation of adult N3KO mammary epithelial cells in the cleared fat pad of a recipientmouse yielded less outgrowths compared to transplanted wild-type cells with an abnormal branchingphenotype. This phenotype indicates deregulation of the normal development of mammary stem cellsand suggests Notch3 is needed for their maintenance after pubertal development. These findingsprovide a link between normal breast physiology and the controversial cell of origin of one of the mostaggressive types of human breast cancer, triple negative breast cancer reinforcing the recent hypothesisthat this type of cancer may arise from a luminal progenitor cell and not basal cell as previously thought.They also provide evidence for a possible diagnostic and therapeutic significance of the Notch3 receptor,which has been found overexpressed in this type of breast cancerΠροηγούμενα αποτελέσματα είχαν δείξει μεταλλαγές του γονιδίου p53 στους ινοβλάστες τουκαρκινικού στρώματος ανθρώπινων καρκίνων. Για μελετήσουμε την επίδραση της έλλειψης αυτής,στην καρκινογένεση αναλύσαμε την επιρροή ινοβλαστών άγριου τύπου αλλά και μεταλλαγμένων γιατα γονίδια p53 και Notch3, στην ανάπτυξη καρκινικών όγκων καθώς και κατά την θεραπεία.Διαπιστώσαμε ότι ενώ οι ινοβλάστες με μεταλλαγμένο p53 προκαλούν ταχύτερη ανάπτυξη όγκων σεσύγκριση με τους ινοβλάστες άγριου τύπου, οι όγκοι αυτοί είναι πιο ευαίσθητοι στη θεραπεία.Περεταίρω διερεύνηση έδειξε ότι ο λόγος για τη ευαισθησία αυτή είναι η αδυναμία των ινοβλαστών μεμεταλλαγμένο p53 να επάγουν senescence, δηλαδή να εισέλθουν σε μια κατάσταση η οποία επάγεταιαπό το κυτταρικό γύρας. Όταν επάγεται senescence στους ινοβλάστες μετά την έκθεση στο γενοτοξικόστρες της αντικαρκινικής θεραπείας αυτοί εκκρίνουν στο άμεσο περιβάλλον αυξητικούς παράγοντες οιοποίοι έχουν ως αποτέλεσμα την επιβίωση των καρκινικών κυττάρων. Τα αποτελέσματα αυτάκαταδεικνύουν την σπουδαιότητα της μελέτης της επιρροής της αντικαρκινικής θεραπείας τόσο στακαρκινικά κύτταρα όσο και στο στρώμα. Επιπλέον αναδεικνύουν την καταστολή του senescence ωςπιθανή αντικαρκινική στρατηγική κατά τη θεραπεία. Σε αντίθεση με το γονίδιο p53, δείξαμε ότι οδιαφορικός γονότυπος των ινοβλαστών για Notch3 δεν επηρεάζει την καρκινογένεση. Ωστόσο ηκαρκινογένεση καθυστερείται σημαντικά όταν το Notch3 απουσιάζει από τα επιθηλιακά κύτταρα τουμαστού σε διαγονιδιακά ποντίκια του μοντέλου MMTV-Neu. Δεδομένου της εμπλοκής του γονιδίουατού στο βασοκυτταρικό καρκίνο του μαστού ο οποίος έχει προταθεί ότι προέρχεται από προγεννητικάκύτταρα του μαστού, διερευνήσαμε το βιολογικό ρόλο του γονιδίου αυτού και των μαζικώνεπιθηλιακών κυττάρων τα οποία το εκφράζουν, στην φυσιολογική ανάπτυξη του μαζικού αδένα. Για ναπραγματοποιήσουμε την ανάλυση αυτή χρησιμοποιήσαμε νέα διαγονιδιακά ποντίκια τα οποίαδημιουργήθηκαν στην Exelixis Inc. σε συνεργασία με το εργαστήριο του Καθηγητή Αρταβάνη-Τσάκωνα.Διαπιστώσαμε ότι το Notch3 εκφράζεται σε προγεννητικά κύτταρα του αυλού των μαζικών πόρων ταοποία έχουν τη δυνατότητα να διαφοροποιηθούν σε όλους τους κυτταρικούς τύπους του αυλού κατάτην φυσιολογική ανάπτυξη. Επιπλέον όταν τα κύτταρα αυτά μεταμοσχεύονται στο ενδογενέςμικροπεριβάλλον στο οποίο βρίσκεται το μαζικό επιθήλιο, έχουν τη δυνατότητα αναγέννησης όλων τωνκυτταρικών τύπων του μαστού. Η ιδιότητα αυτή μέχρι πρότινος είχε αποδοθεί μονάχα σταμυοεπιθηλιακά κύτταρα του μαστού. Τα αποτελέσματα αυτά αναδεικνύουν το γονίδιο Notch3 ωςσημαντή των προγεννητικών κυττάρων του αυλού του μαζικού επιθηλίου και συνδέουν την εμπλοκήτου στο βασοκυτταρικό καρκίνο του μαστού και το κυτταρικό τύπο προέλευσης του καρκίνου αυτο

P53 mutations in stromal fibroblasts sensitize tumors against chemotherapy

The efficacy of chemotherapy is usually viewed as the outcome of cancer-cell-autonomous processes while the contribution of stroma is being overseen. Here we show that p53 mutations in stromal fibroblasts, a genetic lesion that is detectable in primary breast, prostate and probably other cancers, while they accelerate tumorigenesis they also sensitize tumours against conventional chemotherapy by doxorubicin and cis-platinum. The mechanism by which p53 of stromal fibroblasts affects the response of a tumour against chemotherapy is likely to involve the induction of senescence in the fibroblasts which in turns results in the production of growth factors acting onto the cancer cells by paracrine mechanisms. Our findings identify stromal fibroblasts as important modulators of the efficacy of anticancer therapy. (c) 2008 Wiley-Liss, Inc

Notch3 marks clonogenic mammary luminal progenitor cells in vivo

The identity of mammary stem and progenitor cells remains poorly understood, mainly as a result of the lack of robust markers. The Notch signaling pathway has been implicated in mammary gland development as well as in tumorigenesis in this tissue. Elevated expression of the Notch3 receptor has been correlated to the highly aggressive “triple negative” human breast cancer. However, the specific cells expressing this Notch paralogue in the mammary gland remain unknown. Using a conditionally inducible Notch3-CreERT2(SAT) transgenic mouse, we genetically marked Notch3-expressing cells throughout mammary gland development and followed their lineage in vivo. We demonstrate that Notch3 is expressed in a highly clonogenic and transiently quiescent luminal progenitor population that gives rise to a ductal lineage. These cells are capable of surviving multiple successive pregnancies, suggesting a capacity to self-renew. Our results also uncover a role for the Notch3 receptor in restricting the proliferation and consequent clonal expansion of these cells

Luminal progenitors restrict their lineage potential during mammary gland development.

The hierarchical relationships between stem cells and progenitors that guide mammary gland morphogenesis are still poorly defined. While multipotent basal stem cells have been found within the myoepithelial compartment, the in vivo lineage potential of luminal progenitors is unclear. Here we used the expression of the Notch1 receptor, previously implicated in mammary gland development and tumorigenesis, to elucidate the hierarchical organization of mammary stem/progenitor cells by lineage tracing. We found that Notch1 expression identifies multipotent stem cells in the embryonic mammary bud, which progressively restrict their lineage potential during mammary ductal morphogenesis to exclusively generate an ERαneg luminal lineage postnatally. Importantly, our results show that Notch1-labelled cells represent the alveolar progenitors that expand during pregnancy and survive multiple successive involutions. This study reveals that postnatal luminal epithelial cells derive from distinct self-sustained lineages that may represent the cells of origin of different breast cancer subtypes

Essential role of p21/waf1 in the mediation of the anti-proliferative effects of GHRH antagonist JMR-132

GHRH, besides its neuroendocrine action in controlling the release of GH from the pituitary, stimulates the growth of various cancers in vivo and in vitro by direct mechanism(s). However, the molecular mechanism that mediates these proliferative effects of GHRH in extrapituitary tissues remains poorly characterized. In the present study, we investigated whether the tumor suppressor p21/waf1 is involved in the mediation of the proliferative effects of GHRH in A549 human lung cancer epithelial cells. Exposure of A549 cells to the GHRH antagonist JMR-132 caused a significant inhibition in the rate of cell proliferation. In A549 cells, GHRH suppressed while JMR-132 increased the levels of p21 expression in a dose-dependent manner. This suggests that GHRH could regulate p21 levels. We then evaluated whether p21 is required in A549 cells for the regulation of cell proliferation by GHRH. To this end, we knocked-down p21 expression in A549 cells by siRNA and assessed the effects of antagonist JMR-132 on cell proliferation. We found that the loss of p21 expression abolished the anti-proliferative effects of JMR-132. Suppression of p21 expression by siRNA in human HT29 colon cancer cells and non-transformed mouse osteoblasts KS483 also blocked the anti-proliferative effects of JMR-132 suggesting that the regulation of cell proliferation by GHRH is p21 dependent. These results shed light on the molecular mechanism of action of GHRH antagonists in tumor tissues and suggest that the antineoplastic activity of GHRH antagonists could be considered for the treatment of cancers expressing p21

Essential role of p21/waf1 in the mediation of the anti-proliferative effects of GHRH antagonist JMR-132

GHRH, besides its neuroendocrine action in controlling the release of GH from the pituitary, stimulates the growth of various cancers in vivo and in vitro by direct mechanism(s). However, the molecular mechanism that mediates these proliferative effects of GHRH in extrapituitary tissues remains poorly characterized. In the present study, we investigated whether the tumor suppressor p21/waf1 is involved in the mediation of the proliferative effects of GHRH in A549 human lung cancer epithelial cells. Exposure of A549 cells to the GHRH antagonist JMR-132 caused a significant inhibition in the rate of cell proliferation. In A549 cells, GHRH suppressed while JMR-132 increased the levels of p21 expression in a dose-dependent manner. This suggests that GHRH could regulate p21 levels. We then evaluated whether p21 is required in A549 cells for the regulation of cell proliferation by GHRH. To this end, we knocked-down p21 expression in A549 cells by siRNA and assessed the effects of antagonist JMR-132 on cell proliferation. We found that the loss of p21 expression abolished the anti-proliferative effects of JMR-132. Suppression of p21 expression by siRNA in human HT29 colon cancer cells and non-transformed mouse osteoblasts KS483 also blocked the anti-proliferative effects of JMR-132 suggesting that the regulation of cell proliferation by GHRH is p21 dependent. These results shed light on the molecular mechanism of action of GHRH antagonists in tumor tissues and suggest that the antineoplastic activity of GHRH antagonists could be considered for the treatment of cancers expressing p21