The devil is in the methods: lineage tracing, functional screens and sequencing, hormones, tumour-stroma interactions, and expansion of human breast tumours as xenografts

The meeting of the European Network for Breast Development and Cancer (ENBDC) on 'Methods in Mammary Gland Development and Cancer' has become an annual international rendezvous for scientists with interests in the normal and neoplastic breast. The third meeting in this series, held in April-May 2011 in Weggis, Switzerland, focussed on functional screens and sequencing, hormones, lineage tracing, tumor-stroma interactions and the expansion of human breast tumours as xenografts.


Introduction
Th e third international meeting of the European Network for Breast Development and Cancer (ENBDC) again promoted the sharing of protocols and ideas between groups working on breast development and cancer. Graduate students, postdocs and research associates were encouraged to attend. Th e following topics were covered in depth: functional screens and sequencing, hormones, lineage tracing, the propagation of human breast tumours as xenografts and tumour-stroma interactions.

Functional screens and sequencing (Chair: Momo Bentires-Alj)
Chris Lord from the Breakthrough Breast Cancer Research Centre at the Institute of Cancer Research in London presented examples of the power of genomewide functional screens. First, his group combined tamoxifen treatment of breast cancer cells in vitro and a small interfering RNA (siRNA) screen for kinases in an approach to identify events leading to tamoxifen sensitivity and tamoxifen resistance. Th ey identifi ed low cyclin-dependent kinase (CDK)10 expression as an important mediator of resistance to endocrine therapy in breast cancer. Knockdown of CDK10 blocked its inhibitory eff ect on ETS2, which in turn induced transcription of c-Raf and led to activation of the ERK/mitogenactivated protein kinase (MAPK) pathway and resistance to tamoxifen [1]. Th ey also used a pooled genome-wide small hairpin RNA (shRNA) screen in the presence or absence of tamoxifen, coupled with massively parallel sequencing, and identifi ed groups of genes the silencing of which increased sensitivity (for example C10orf72, C15orf55/NUT, EDF1, ING5, KRAS) or resistance (for example, BAP1, CLPP, GPRC5D, NAE1, NF1) to tamoxifen [2]. Second, they used a synthetic lethal unbiased shRNA screen for identifying sensitizers to a poly (ADP-ribose) polymerase (PARP) inhibitor in BRCA1 wild-type breast cancer cells. Knockdown of RAD51D, a novel ovarian cancer susceptibility gene, dramatically increased cell death upon PARP inhibition [3]. Th ird, they used a kinome-wide siRNA screen to identify genetic dependencies of breast cancer cell lines. Characterization of the genetic dependencies of breast tumour cell lines with diff erent tumorigenic mutations demonstrated a synthetic lethality between loss of PTEN and the inhibition of the mitotic checkpoint kinase TTK/ MPS1, which suggests a novel therapeutic approach [4]. Th ese studies warrant further in-depth validation and mechanistic studies, which could reveal clinically relevant combined therapies or novel targets for cancer.
David Adams from the Wellcome Trust Sanger Institute described the eff orts made by his team to fi nd driver mutations in mouse models of cancer. First, they carried out insertion mutagenesis in Apc fl x/+ /Ah-Cre mice using Sleeping Beauty insertional mutagenesis technology, followed by deep sequencing of insertion sites to identify genes collaborating with loss of the tumour

Abstract
The meeting of the European Network for Breast Development and Cancer (ENBDC) on 'Methods in Mammary Gland Development and Cancer' has become an annual international rendezvous for scientists with interests in the normal and neoplastic breast. The third meeting in this series, held in April-May 2011 in Weggis, Switzerland, focussed on functional screens and sequencing, hormones, lineage tracing, tumor-stroma interactions and the expansion of human breast tumours as xenografts.

© 2010 BioMed Central Ltd
The devil is in the methods: lineage tracing, functional screens and sequencing, hormones, tumour-stroma interactions, and expansion of human breast tumours as xenografts suppressor Apc in bowel cancer and driving tumour formation. Th ey found that inactivation of the H3K4 methyltransferase Mll3 induces the Wnt pathway and increases tumorigenesis, either alone or in concert with loss of Apc [5]. Second, he summarized their eff orts to sequence the genomes of mouse models of breast cancer (including models of lobular and basal-like breast cancer), which led to the identifi cation of patterns of rearrangement in the diff erent models and to candidate driver cancer genes. Preliminary data on these studies have recently been published [6].

Hormones (Chairs: María dM Vivanco and Momo Bentires-Alj)
Cathrin Brisken (Swiss Institute for Experimental Cancer Research/EPFL Lausanne) spoke on the 'Genetic dissection of hormonal control in mammary gland development' . Tissue recombination techniques have shown that while oestrogen targets the mammary epithelium to invoke ductal elongation, progesterone is required for ductal side branching. Results from the Brisken lab showed that progesterone drives proliferation in two waves: fi rst, cyclin D1 is required for the proliferation of some progesterone receptor (PR) + cells; second, PRcells in particular proliferate through a Receptor activator of nuclear factor kappa-B ligand (RANKL)-dependent mechanism [7]. Th ese results highlight the relevance of RANKL in the mediation of progesterone function, since it rescues the PR -/phenotype. Importantly, they demonstrate a key role of progesterone in inducing proliferation by a paracrine mechanism. In addition, by modulating the expression levels of DeltaNp63 in primary human breast epithelial cells, they showed that this transcription factor is required to confer a basal cell phenotype. Notably, Notch, which reduces DeltaNp63 expression, can shift the balance towards a luminal phenotype [8]. Furthermore, in vivo ablation of Notch signalling demonstrated that it is not possible to establish and/or maintain luminal cells in the absence of Notch. Th ese fi ndings reveal antagonistic eff ects of Notch and p63 on cell fate determination in the mammary epithelium.
Maria Vivanco from CIC bioGUNE in Bilbao spoke on 'Hormonal regulation in human breast stem cells' focusing on the eff ects of oestrogen on breast stem cells. She showed that expression of the stem cell markers Nanog, Oct4 and Sox2 is higher in stem cells isolated from normal or breast tumour tissue and can be reduced by serum-induced diff erentiation or treatment with oestrogen, whilst tamoxifen had the opposite eff ect. Vivanco further demonstrated that overexpression of each individual stem cell gene is suffi cient to increase both the number of stem cells, as measured by mammosphere formation, aldehyde dehydrogenase activity and the cell-surface phenotypes EMA + CALLA + and CD44 + CD24 -/low , and their invasion capacity; these are characteristics of tumourigenesis and poor prognosis [9]. Interestingly, this led to reduced oestrogen receptor (ER) expression, which is in agreement with the reported absence or low expression of ER in breast stem cells. Th e results indicate that oestrogen reduces the pool of breast stem cells, providing a plausible explanation for several clinical observations, namely that ER + tumours coincide with a better prognosis than ERtumours, and that women on hormone-replacement therapy using oestrogen alone have a lower risk of breast cancer than those taking oestrogen plus progestin, where the risk is elevated. Finally, this report emphasised that future therapies should combine treatments directed at diminishing tumour bulk with new strategies to eliminate cancer stem cells whilst sparing normal stem cells [10].

Lineage tracing (Chair: John Stingl)
Nick Barker from the Institute of Medical Biology in Singapore summarized his work and that of his former colleagues in the Hans Clever's lab on characterizing mouse intestinal stem cells. Th ey demonstrated the importance of Wnt as a key regulator of intestinal epithelial cell homeostasis. Wnt factors, which are produced by epithelial cells in the intestinal crypt, are essential for Paneth cell diff erentiation, which in turn function as the stem cell niche and also have antimicrobial properties. Wnt is also a mitogen for transitamplifying (TA) cells and for maintaining the intestinal stem cell population. Lgr5 (also known as Gpr49), which is an orphan G protein-coupled receptor, was identifi ed as a Wnt target gene and found to have a unique distribution within the epithelium, with expression restricted to basal columnar cells of the crypt. Elegant genetic lineage tracing experiments in mice targeting Lgr5 + cells revealed the capacity of these cells to maintain all of the diff erent lineages of the intestinal epithelium in the long-term, thus fulfi lling the functional requirements of intestinal epithelial stem cells. Fate mapping of individual stem cells using multicolour reporter mice revealed that intestinal stem cells predominantly divide symmetrically and that daughter cells stochastically adopt stem or TA cell fates. Selective deletion of the adenomatous polyposis coli (APC) gene in Lgr5 + cells demonstrated that aberrant activation of Wnt signalling in these cells causes aggressive adenoma growth throughout the intestine. In contrast, loss of APC in TA cells resulted only in the generation of microadenomas, whose growth rapidly stalled. Th ese results demonstrate the likely stem cell origin of colorectal cancer in this mouse model. Interestingly, mouse intestinal tumours also contain a minor population of Lgr5 + cells that reside in a Paneth cell niche, although it is not clear at this stage whether these cells function as colon cancer stem cells. Barker also presented data demonstrating that clonal outgrowths can be generated and passaged in Matrigel cultures in the absence of a cellular stromal niche, and that cells from these structures have the potential to contribute to the colonic epithelium when introduced into suitable recipient mice.

Propagation of human breast tumours as xenografts (Chair: John Stingl)
Th e participants of the meeting were invited to take part in a group discussion on the xenotransplantation of human breast tumours into immunodefi cient mice. Th e ability to propagate primary human tumours in mice is a powerful tool for studying breast cancer stem cells, as well as for breast tumour modelling and other aspects of breast cancer cell biology. Unfortunately, human breast tumours, particularly those that are ER + , are notoriously diffi cult to engraft into mice. During this group discussion, participants shared their experiences of the dissociation and transplantation of human breast tumour cells into mice and several factors critical for successful engraftment were identifi ed. Th ese include (1) minimal tissue dissociation in order to minimize the toxicity of collagenase over-digestion, (2) the preferred use of NOD scid gamma (NSG; NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ JAX) mice, (3) co-implantation of cells with Matrigel, and (4) patience, since tumour formation may take up to 6 months.

Tumour-stroma interactions (Chair: Rob Clarke)
Akira Orimo (Paterson Institute for Cancer Research, University of Manchester, UK) discussed a method that allows the experimental generation of cancer-associated fi broblasts (exp-CAFs) from human mammary fi broblasts using a co-implantation tumour xenograft model. Human mammary fi broblasts, co-implanted with breast carcinoma cells into immunodefi cient nude mice, were extracted from breast tumour xenografts. Th ese fi broblasts were shown to increasingly acquire the ability to promote carcinoma growth during tumour progression. Exp-CAFs also included large numbers of myofi broblasts, which are a hallmark of activated fi broblasts present in wound healing and chronic fi brosis. Taken together, these fi ndings suggested that exp-CAFs in their tumourpromoting myofi broblastic phenotypes resemble primary CAFs prepared from breast carcinomas dissected from breast cancer patients [11]. Moreover, Orimo demonstrated that the establishment of autocrine signalling loops mediated by transforming growth factor-β and stromal cell-derived factor-1 cytokines is responsible for the induction and maintenance of CAFs' tumourpromoting myofi broblastic ability, thereby indicating the evolution of resident mammary fi broblasts to tumourpromoting CAFs during the course of tumour progression. Th e roles of exp-CAFs in promoting tumour metastasis were also investigated.
Clare Isacke (Breakthrough Breast Cancer Centre, London, UK) described approaches to high-throughput in vivo RNA interference screens for identifying novel determinants of the breast cancer metastatic process. Th e pros and cons of diff erent biological systems in which to conduct a screen, of RNA interference libraries, of the methodologies for read-out and analysis, and of diff erent approaches to validate potential hits were described.
Th e third annual ENBDC methods meeting served as a valuable international forum for exchanging protocols and ideas relevant to mammary gland development and cancer. Th e 2012 ENBDC methods meeting will be held in Weggis on April 13-15 [12].