Publications by authors named "François Vaillant"

48 Publications

A single-cell RNA expression atlas of normal, preneoplastic and tumorigenic states in the human breast.

EMBO J 2021 Jun 5;40(11):e107333. Epub 2021 May 5.

ACRF Cancer Biology and Stem Cells Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic, Australia.

To examine global changes in breast heterogeneity across different states, we determined the single-cell transcriptomes of > 340,000 cells encompassing normal breast, preneoplastic BRCA1 tissue, the major breast cancer subtypes, and pairs of tumors and involved lymph nodes. Elucidation of the normal breast microenvironment revealed striking changes in the stroma of post-menopausal women. Single-cell profiling of 34 treatment-naive primary tumors, including estrogen receptor (ER) , HER2 , and triple-negative breast cancers, revealed comparable diversity among cancer cells and a discrete subset of cycling cells. The transcriptomes of preneoplastic BRCA1 tissue versus tumors highlighted global changes in the immune microenvironment. Within the tumor immune landscape, proliferative CD8 T cells characterized triple-negative and HER2 cancers but not ER tumors, while all subtypes comprised cycling tumor-associated macrophages, thus invoking potentially different immunotherapy targets. Copy number analysis of paired ER tumors and lymph nodes indicated seeding by genetically distinct clones or mass migration of primary tumor cells into axillary lymph nodes. This large-scale integration of patient samples provides a high-resolution map of cell diversity in normal and cancerous human breast.
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http://dx.doi.org/10.15252/embj.2020107333DOI Listing
June 2021

Preclinical small molecule WEHI-7326 overcomes drug resistance and elicits response in patient-derived xenograft models of human treatment-refractory tumors.

Cell Death Dis 2021 03 12;12(3):268. Epub 2021 Mar 12.

Walter and Eliza Hall Institute, Parkville, VIC, 3052, Australia.

Targeting cell division by chemotherapy is a highly effective strategy to treat a wide range of cancers. However, there are limitations of many standard-of-care chemotherapies: undesirable drug toxicity, side-effects, resistance and high cost. New small molecules which kill a wide range of cancer subtypes, with good therapeutic window in vivo, have the potential to complement the current arsenal of anti-cancer agents and deliver improved safety profiles for cancer patients. We describe results with a new anti-cancer small molecule, WEHI-7326, which causes cell cycle arrest in G2/M, cell death in vitro, and displays efficacious anti-tumor activity in vivo. WEHI-7326 induces cell death in a broad range of cancer cell lines, including taxane-resistant cells, and inhibits growth of human colon, brain, lung, prostate and breast tumors in mice xenografts. Importantly, the compound elicits tumor responses as a single agent in patient-derived xenografts of clinically aggressive, treatment-refractory neuroblastoma, breast, lung and ovarian cancer. In combination with standard-of-care, WEHI-7326 induces a remarkable complete response in a mouse model of high-risk neuroblastoma. WEHI-7326 is mechanistically distinct from known microtubule-targeting agents and blocks cells early in mitosis to inhibit cell division, ultimately leading to apoptotic cell death. The compound is simple to produce and possesses favorable pharmacokinetic and toxicity profiles in rodents. It represents a novel class of anti-cancer therapeutics with excellent potential for further development due to the ease of synthesis, simple formulation, moderate side effects and potent in vivo activity. WEHI-7326 has the potential to complement current frontline anti-cancer drugs and to overcome drug resistance in a wide range of cancers.
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http://dx.doi.org/10.1038/s41419-020-03269-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955127PMC
March 2021

Tissue-resident ductal macrophages survey the mammary epithelium and facilitate tissue remodelling.

Nat Cell Biol 2020 05 27;22(5):546-558. Epub 2020 Apr 27.

Cancer Biology and Stem Cells Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.

Macrophages are diverse immune cells that reside in all tissues. Although macrophages have been implicated in mammary-gland function, their diversity has not been fully addressed. By exploiting high-resolution three-dimensional imaging and flow cytometry, we identified a unique population of tissue-resident ductal macrophages that form a contiguous network between the luminal and basal layers of the epithelial tree throughout postnatal development. Ductal macrophages are long lived and constantly survey the epithelium through dendrite movement, revealed via advanced intravital imaging. Although initially originating from embryonic precursors, ductal macrophages derive from circulating monocytes as they expand during puberty. Moreover, they undergo proliferation in pregnancy to maintain complete coverage of the epithelium in lactation, when they are poised to phagocytose milk-producing cells post-lactation and facilitate remodelling. Interestingly, ductal macrophages strongly resemble mammary tumour macrophages and form a network that pervades the tumour. Thus, the mammary epithelium programs specialized resident macrophages in both physiological and tumorigenic contexts.
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http://dx.doi.org/10.1038/s41556-020-0505-0DOI Listing
May 2020

Targeting triple-negative breast cancers with the Smac-mimetic birinapant.

Cell Death Differ 2020 10 27;27(10):2768-2780. Epub 2020 Apr 27.

The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia.

Smac mimetics target inhibitor of apoptosis (IAP) proteins, thereby suppressing their function to facilitate tumor cell death. Here we have evaluated the efficacy of the preclinical Smac-mimetic compound A and the clinical lead birinapant on breast cancer cells. Both exhibited potent in vitro activity in triple-negative breast cancer (TNBC) cells, including those from patient-derived xenograft (PDX) models. Birinapant was further studied using in vivo PDX models of TNBC and estrogen receptor-positive (ER) breast cancer. Birinapant exhibited single agent activity in all TNBC PDX models and augmented response to docetaxel, the latter through induction of TNF. Transcriptomic analysis of TCGA datasets revealed that genes encoding mediators of Smac-mimetic-induced cell death were expressed at higher levels in TNBC compared with ER breast cancer, resulting in a molecular signature associated with responsiveness to Smac mimetics. In addition, the cell death complex was preferentially formed in TNBCs versus ER cells in response to Smac mimetics. Taken together, our findings provide a rationale for prospectively selecting patients whose breast tumors contain a competent death receptor signaling pathway for the further evaluation of birinapant in the clinic.
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http://dx.doi.org/10.1038/s41418-020-0541-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7492458PMC
October 2020

Dual Targeting of CDK4/6 and BCL2 Pathways Augments Tumor Response in Estrogen Receptor-Positive Breast Cancer.

Clin Cancer Res 2020 08 3;26(15):4120-4134. Epub 2020 Apr 3.

Cancer Biology and Stem Cells Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.

Purpose: Although cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors significantly extend tumor response in patients with metastatic estrogen receptor-positive (ER) breast cancer, relapse is almost inevitable. This may, in part, reflect the failure of CDK4/6 inhibitors to induce apoptotic cell death. We therefore evaluated combination therapy with ABT-199 (venetoclax), a potent and selective BCL2 inhibitor.

Experimental Design: BCL2 family member expression was assessed following treatment with endocrine therapy and the CDK4/6 inhibitor palbociclib. Functional assays were used to determine the impact of adding ABT-199 to fulvestrant and palbociclib in ER breast cancer cell lines, patient-derived organoid (PDO), and patient-derived xenograft (PDX) models. A syngeneic ER mouse mammary tumor model was used to study the effect of combination therapy on the immune system.

Results: Triple therapy was well tolerated and produced a superior and more durable tumor response compared with single or doublet therapy. This was associated with marked apoptosis, including of senescent cells, indicative of senolysis. Unexpectedly, ABT-199 resulted in Rb dephosphorylation and reduced G-S cyclins, most notably at high doses, thereby intensifying the fulvestrant/palbociclib-induced cell-cycle arrest. Interestingly, a CRISPR/Cas9 screen suggested that ABT-199 could mitigate loss of Rb (and potentially other mechanisms of acquired resistance) to palbociclib. ABT-199 did not abrogate the favorable immunomodulatory effects of palbociclib in a syngeneic ER mammary tumor model and extended tumor response when combined with anti-PD1 therapy.

Conclusions: This study illustrates the potential for targeting BCL2 in combination with CDK4/6 inhibitors and supports investigation of combination therapy in ER breast cancer.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-1872DOI Listing
August 2020

Modeling Breast Cancer Using CRISPR-Cas9-Mediated Engineering of Human Breast Organoids.

J Natl Cancer Inst 2020 05;112(5):540-544

ACRF Cancer Biology and Stem Cells Division.

Breast cancer is characterized by histological and functional heterogeneity, posing a clinical challenge for patient treatment. Emerging evidence suggests that the distinct subtypes reflect the repertoire of genetic alterations and the target cell. However, the precise initiating events that predispose normal epithelium to neoplasia are poorly understood. Here, we demonstrate that breast epithelial organoids can be generated from human reduction mammoplasties (12 out of 12 donors), thus creating a tool to study the clonal evolution of breast cancer. To recapitulate de novo oncogenesis, we exploited clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 for targeted knockout of four breast cancer-associated tumor suppressor genes (P53, PTEN, RB1, NF1) in mammary progenitor cells from six donors. Mutant organoids gained long-term culturing capacity and formed estrogen-receptor positive luminal tumors on transplantation into mice for one out of six P53/PTEN/RB1-mutated and three out of six P53/PTEN/RB1/NF1-mutated lines. These organoids responded to endocrine therapy or chemotherapy, supporting the potential utility of this model to enhance our understanding of the molecular events that culminate in specific subtypes of breast cancer.
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http://dx.doi.org/10.1093/jnci/djz196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225674PMC
May 2020

Intraclonal Plasticity in Mammary Tumors Revealed through Large-Scale Single-Cell Resolution 3D Imaging.

Cancer Cell 2019 04 28;35(4):618-632.e6. Epub 2019 Mar 28.

Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Department of Medical Biology, The University of Melbourne, Parkville, VIC 3010, Australia. Electronic address:

Breast tumors are inherently heterogeneous, but the evolving cellular organization through neoplastic progression is poorly understood. Here we report a rapid, large-scale single-cell resolution 3D imaging protocol based on a one-step clearing agent that allows visualization of normal tissue architecture and entire tumors at cellular resolution. Imaging of multicolor lineage-tracing models of breast cancer targeted to either basal or luminal progenitor cells revealed profound clonal restriction during progression. Expression profiling of clones arising in Pten/Trp53-deficient tumors identified distinct molecular signatures. Strikingly, most clones harbored cells that had undergone an epithelial-to-mesenchymal transition, indicating widespread, inherent plasticity. Hence, an integrative pipeline that combines lineage tracing, 3D imaging, and clonal RNA sequencing technologies offers a comprehensive path for studying mechanisms underlying heterogeneity in whole tumors.
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http://dx.doi.org/10.1016/j.ccell.2019.02.010DOI Listing
April 2019

Comparative oncogenomics identifies combinations of driver genes and drug targets in BRCA1-mutated breast cancer.

Nat Commun 2019 01 23;10(1):397. Epub 2019 Jan 23.

Division of Molecular Pathology, The Netherlands Cancer Institute, 1066 CX, Amsterdam, The Netherlands.

BRCA1-mutated breast cancer is primarily driven by DNA copy-number alterations (CNAs) containing large numbers of candidate driver genes. Validation of these candidates requires novel approaches for high-throughput in vivo perturbation of gene function. Here we develop genetically engineered mouse models (GEMMs) of BRCA1-deficient breast cancer that permit rapid introduction of putative drivers by either retargeting of GEMM-derived embryonic stem cells, lentivirus-mediated somatic overexpression or in situ CRISPR/Cas9-mediated gene disruption. We use these approaches to validate Myc, Met, Pten and Rb1 as bona fide drivers in BRCA1-associated mammary tumorigenesis. Iterative mouse modeling and comparative oncogenomics analysis show that MYC-overexpression strongly reshapes the CNA landscape of BRCA1-deficient mammary tumors and identify MCL1 as a collaborating driver in these tumors. Moreover, MCL1 inhibition potentiates the in vivo efficacy of PARP inhibition (PARPi), underscoring the therapeutic potential of this combination for treatment of BRCA1-mutated cancer patients with poor response to PARPi monotherapy.
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http://dx.doi.org/10.1038/s41467-019-08301-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344487PMC
January 2019

Foxp1 Is Indispensable for Ductal Morphogenesis and Controls the Exit of Mammary Stem Cells from Quiescence.

Dev Cell 2018 12 25;47(5):629-644.e8. Epub 2018 Oct 25.

Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Department of Medical Biology, The University of Melbourne, Parkville, VIC 3010, Australia. Electronic address:

Long-lived quiescent mammary stem cells (MaSCs) are presumed to coordinate the dramatic expansion of ductal epithelium that occurs through the different phases of postnatal development, but little is known about the molecular regulators that underpin their activation. We show that ablation of the transcription factor Foxp1 in the mammary gland profoundly impairs ductal morphogenesis, resulting in a rudimentary tree throughout life. Foxp1-deficient glands were highly enriched for quiescent Tspan8 MaSCs, which failed to become activated even in competitive transplantation assays, thus highlighting a cell-intrinsic defect. Foxp1 deletion also resulted in aberrant expression of basal genes in luminal cells, inferring a role in cell-fate decisions. Notably, Foxp1 was uncovered as a direct repressor of Tspan8 in basal cells, and deletion of Tspan8 rescued the defects in ductal morphogenesis elicited by Foxp1 loss. Thus, a single transcriptional regulator Foxp1 can control the exit of MaSCs from dormancy to orchestrate differentiation and development.
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http://dx.doi.org/10.1016/j.devcel.2018.10.001DOI Listing
December 2018

A Phase Ib Dose-Escalation and Expansion Study of the BCL2 Inhibitor Venetoclax Combined with Tamoxifen in ER and BCL2-Positive Metastatic Breast Cancer.

Cancer Discov 2019 03 5;9(3):354-369. Epub 2018 Dec 5.

The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia.

Venetoclax, a potent and selective BCL2 inhibitor, synergizes with endocrine therapy in preclinical models of ER-positive breast cancer. Using a phase Ib 3 + 3 dose-escalation and expansion study design, 33 patients with ER and BCL2-positive metastatic disease (mean prior regimens, 2; range, 0-8) were treated with daily tamoxifen (20 mg) and venetoclax (200-800 mg). Apart from uncomplicated "on-target" lymphopenia, no dose-limiting toxicities or high-grade adverse events were observed in the escalation phase (15 patients), and 800 mg was selected as the recommended phase II dose (RP2D). In the expansion phase (18 patients), few high-grade treatment-related adverse events were observed. For 24 patients treated at the RP2D, the confirmed radiologic response rate was 54% and the clinical benefit rate was 75%. Treatment responses were preempted by metabolic responses (FDG-PET) at 4 weeks and correlated with serial changes in circulating tumor DNA. Radiologic responses (40%) and clinical benefit (70%) were observed in 10 patients with plasma-detected mutations. SIGNIFICANCE: In the first clinical study to evaluate venetoclax in a solid tumor, we demonstrate that combining venetoclax with endocrine therapy has a tolerable safety profile and elicits notable activity in ER and BCL2-positive metastatic breast cancer. These findings support further investigation of combination therapy for patients with BCL2-positive tumors...
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http://dx.doi.org/10.1158/2159-8290.CD-18-1151DOI Listing
March 2019

Construction of developmental lineage relationships in the mouse mammary gland by single-cell RNA profiling.

Nat Commun 2017 11 20;8(1):1627. Epub 2017 Nov 20.

ACRF Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia.

The mammary epithelium comprises two primary cellular lineages, but the degree of heterogeneity within these compartments and their lineage relationships during development remain an open question. Here we report single-cell RNA profiling of mouse mammary epithelial cells spanning four developmental stages in the post-natal gland. Notably, the epithelium undergoes a large-scale shift in gene expression from a relatively homogeneous basal-like program in pre-puberty to distinct lineage-restricted programs in puberty. Interrogation of single-cell transcriptomes reveals different levels of diversity within the luminal and basal compartments, and identifies an early progenitor subset marked by CD55. Moreover, we uncover a luminal transit population and a rare mixed-lineage cluster amongst basal cells in the adult mammary gland. Together these findings point to a developmental hierarchy in which a basal-like gene expression program prevails in the early post-natal gland prior to the specification of distinct lineage signatures, and the presence of cellular intermediates that may serve as transit or lineage-primed cells.
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http://dx.doi.org/10.1038/s41467-017-01560-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5696379PMC
November 2017

Characterization of the Human Pancreas Side Population as a Potential Reservoir of Adult Stem Cells.

Pancreas 2018 01;47(1):25-34

Objectives: The side population (SP) contains cells with stem cell/progenitor properties. Previously, we observed that the mouse pancreas SP expanded after pancreatic injury. We aimed to characterize the SP in human pancreas as a potential source of stem cells.

Methods: Human organ donor pancreata were fractionated into islets and exocrine tissue, enriched by tissue culture and dispersed into single cells. Cells were phenotyped by flow cytometry, and the SP was defined by efflux of fluorescent dye Hoechst 33342 visualized by ultraviolet excitation. Cells were flow sorted, and their colony-forming potential measured on feeder cells in culture.

Results: An SP was identified in islet and exocrine cells from human organ donors: 2 with type 1 diabetes, 3 with type 2 diabetes, and 28 without diabetes. Phenotyping revealed that exocrine SP cells had an epithelial origin, were enriched for carbohydrate antigen 19-9 ductal cells expressing stem cell markers CD133 and CD26, and had greater colony-forming potential than non-SP cells. The exocrine SP was increased in a young adult with type 1 diabetes and ongoing islet autoimmunity.

Conclusions: The pancreatic exocrine SP is a potential reservoir of adult stem/progenitor cells, consistent with previous evidence that such cells are duct-derived and express CD133.
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http://dx.doi.org/10.1097/MPA.0000000000000950DOI Listing
January 2018

Synergistic action of the MCL-1 inhibitor S63845 with current therapies in preclinical models of triple-negative and HER2-amplified breast cancer.

Sci Transl Med 2017 Aug;9(401)

ACRF Stem Cells and Cancer Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia.

The development of BH3 mimetics, which antagonize prosurvival proteins of the BCL-2 family, represents a potential breakthrough in cancer therapy. Targeting the prosurvival member MCL-1 has been an area of intense interest because it is frequently deregulated in cancer. In breast cancer, MCL-1 is often amplified, and high expression predicts poor patient outcome. We tested the MCL-1 inhibitor S63845 in breast cancer cell lines and patient-derived xenografts with high expression of MCL-1. S63845 displayed synergistic activity with docetaxel in triple-negative breast cancer and with trastuzumab or lapatinib in HER2-amplified breast cancer. Using S63845-resistant cells combined with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9) technology, we identified deletion of BAK and up-regulation of prosurvival proteins as potential mechanisms that confer resistance to S63845 in breast cancer. Collectively, our findings provide a strong rationale for the clinical evaluation of MCL-1 inhibitors in breast cancer.
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http://dx.doi.org/10.1126/scitranslmed.aam7049DOI Listing
August 2017

Combined immune checkpoint blockade as a therapeutic strategy for -mutated breast cancer.

Sci Transl Med 2017 06;9(393)

Stem Cells and Cancer Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.

Immune checkpoint inhibitors have emerged as a potent new class of anticancer therapy. They have changed the treatment landscape for a range of tumors, particularly those with a high mutational load. To date, however, modest results have been observed in breast cancer, where tumors are rarely hypermutated. Because -associated tumors frequently exhibit a triple-negative phenotype with extensive lymphocyte infiltration, we explored their mutational load, immune profile, and response to checkpoint inhibition in a -deficient tumor model. -mutated triple-negative breast cancers (TNBCs) exhibited an increased somatic mutational load and greater numbers of tumor-infiltrating lymphocytes, with increased expression of immunomodulatory genes including () and , when compared to TNBCs from -wild-type patients. Cisplatin treatment combined with dual anti-programmed death-1 and anti-cytotoxic T lymphocyte-associated antigen 4 therapy substantially augmented antitumor immunity in -deficient mice, resulting in an avid systemic and intratumoral immune response. This response involved enhanced dendritic cell activation, reduced suppressive FOXP3 regulatory T cells, and concomitant increase in the activation of tumor-infiltrating cytotoxic CD8 and CD4 T cells, characterized by the induction of polyfunctional cytokine-producing T cells. Dual (but not single) checkpoint blockade together with cisplatin profoundly attenuated the growth of -deficient tumors in vivo and improved survival. These findings provide a rationale for clinical studies of combined immune checkpoint blockade in -associated TNBC.
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http://dx.doi.org/10.1126/scitranslmed.aal4922DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5822709PMC
June 2017

RE: Bilateral Oophorectomy and Breast Cancer Risk in BRCA1 and BRCA2 Mutation Carriers.

J Natl Cancer Inst 2017 10;109(10)

Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research; Department of Medical Biology, and Department of Medicine, University of Melbourne, Melbourne, VIC, Australia; Parkville Familial Cancer Centre, The Royal Melbourne Hospital and Peter MacCallum Cancer Centre, Parkville, VIC, Australia.

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http://dx.doi.org/10.1093/jnci/djx038DOI Listing
October 2017

Identification of quiescent and spatially restricted mammary stem cells that are hormone responsive.

Nat Cell Biol 2017 03 13;19(3):164-176. Epub 2017 Feb 13.

ACRF Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia.

Despite accumulating evidence for a mammary differentiation hierarchy, the basal compartment comprising stem cells remains poorly characterized. Through gene expression profiling of Lgr5 basal epithelial cells, we identify a new marker, Tetraspanin8 (Tspan8). Fractionation based on Tspan8 and Lgr5 expression uncovered three distinct mammary stem cell (MaSC) subsets in the adult mammary gland. These exist in a largely quiescent state but differ in their reconstituting ability, spatial localization, and their molecular and epigenetic signatures. Interestingly, the deeply quiescent MaSC subset (Lgr5Tspan8) resides within the proximal region throughout life, and has a transcriptome strikingly similar to that of claudin-low tumours. Lgr5Tspan8 cells appear to originate from the embryonic mammary primordia before switching to a quiescent state postnatally but can be activated by ovarian hormones. Our findings reveal an unexpected degree of complexity within the adult MaSC compartment and identify a dormant subset poised for activation in response to physiological stimuli.
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http://dx.doi.org/10.1038/ncb3471DOI Listing
March 2017

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

Cancer Metastasis Rev 2016 12;35(4):547-573

Huntsman Cancer Institute, University of Utah, 2000 Circle of Hope, Salt Lake City, UT, 84112, USA.

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.
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http://dx.doi.org/10.1007/s10555-016-9653-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5396460PMC
December 2016

RANK ligand as a potential target for breast cancer prevention in BRCA1-mutation carriers.

Nat Med 2016 08 20;22(8):933-9. Epub 2016 Jun 20.

ACRF Stem Cells and Cancer Division, Walter and Eliza Hall Institute of Medical Research (WEHI), Parkville, Victoria, Australia.

Individuals who have mutations in the breast-cancer-susceptibility gene BRCA1 (hereafter referred to as BRCA1-mutation carriers) frequently undergo prophylactic mastectomy to minimize their risk of breast cancer. The identification of an effective prevention therapy therefore remains a 'holy grail' for the field. Precancerous BRCA1(mut/+) tissue harbors an aberrant population of luminal progenitor cells, and deregulated progesterone signaling has been implicated in BRCA1-associated oncogenesis. Coupled with the findings that tumor necrosis factor superfamily member 11 (TNFSF11; also known as RANKL) is a key paracrine effector of progesterone signaling and that RANKL and its receptor TNFRSF11A (also known as RANK) contribute to mammary tumorigenesis, we investigated a role for this pathway in the pre-neoplastic phase of BRCA1-mutation carriers. We identified two subsets of luminal progenitors (RANK(+) and RANK(-)) in histologically normal tissue of BRCA1-mutation carriers and showed that RANK(+) cells are highly proliferative, have grossly aberrant DNA repair and bear a molecular signature similar to that of basal-like breast cancer. These data suggest that RANK(+) and not RANK(-) progenitors are a key target population in these women. Inhibition of RANKL signaling by treatment with denosumab in three-dimensional breast organoids derived from pre-neoplastic BRCA1(mut/+) tissue attenuated progesterone-induced proliferation. Notably, proliferation was markedly reduced in breast biopsies from BRCA1-mutation carriers who were treated with denosumab. Furthermore, inhibition of RANKL in a Brca1-deficient mouse model substantially curtailed mammary tumorigenesis. Taken together, these findings identify a targetable pathway in a putative cell-of-origin population in BRCA1-mutation carriers and implicate RANKL blockade as a promising strategy in the prevention of breast cancer.
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http://dx.doi.org/10.1038/nm.4118DOI Listing
August 2016

A pooled shRNA screen for regulators of primary mammary stem and progenitor cells identifies roles for Asap1 and Prox1.

BMC Cancer 2015 Apr 3;15:221. Epub 2015 Apr 3.

ACRF Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, 3052, Australia.

Background: The molecular regulators that orchestrate stem cell renewal, proliferation and differentiation along the mammary epithelial hierarchy remain poorly understood. Here we have performed a large-scale pooled RNAi screen in primary mouse mammary stem cell (MaSC)-enriched basal cells using 1295 shRNAs against genes principally involved in transcriptional regulation.

Methods: MaSC-enriched basal cells transduced with lentivirus pools carrying shRNAs were maintained as non-adherent mammospheres, a system known to support stem and progenitor cells. Integrated shRNAs that altered culture kinetics were identified by next generation sequencing as relative frequency changes over time. RNA-seq-based expression profiling coupled with in vitro progenitor and in vivo transplantation assays was used to confirm a role for candidate genes in mammary stem and/or progenitor cells.

Results: Utilizing a mammosphere-based assay, the screen identified several candidate regulators. Although some genes had been previously implicated in mammary gland development, the vast majority of genes uncovered have no known function within the mammary gland. RNA-seq analysis of freshly purified primary mammary epithelial populations and short-term cultured mammospheres was used to confirm the expression of candidate regulators. Two genes, Asap1 and Prox1, respectively implicated in breast cancer metastasis and progenitor cell function in other systems, were selected for further analysis as their roles in the normal mammary gland were unknown. Both Prox1 and Asap1 were shown to act as negative regulators of progenitor activity in vitro, and Asap1 knock-down led to a marked increase in repopulating activity in vivo, implying a role in stem cell activity.

Conclusions: This study has revealed a number of novel genes that influence the activity or survival of mammary stem and/or progenitor cells. Amongst these, we demonstrate that Prox1 and Asap1 behave as negative regulators of mammary stem/progenitor function. Both of these genes have also been implicated in oncogenesis. Our findings provide proof of principle for the use of short-term cultured primary MaSC/basal cells in functional RNAi screens.
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http://dx.doi.org/10.1186/s12885-015-1187-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4399223PMC
April 2015

EGF-mediated induction of Mcl-1 at the switch to lactation is essential for alveolar cell survival.

Nat Cell Biol 2015 Apr 2;17(4):365-75. Epub 2015 Mar 2.

1] ACRF Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia [2] Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia.

Expansion and remodelling of the mammary epithelium requires a tight balance between cellular proliferation, differentiation and death. To explore cell survival versus cell death decisions in this organ, we deleted the pro-survival gene Mcl-1 in the mammary epithelium. Mcl-1 was found to be essential at multiple developmental stages including morphogenesis in puberty and alveologenesis in pregnancy. Moreover, Mcl-1-deficient basal cells were virtually devoid of repopulating activity, suggesting that this gene is required for stem cell function. Profound upregulation of the Mcl-1 protein was evident in alveolar cells at the switch to lactation, and Mcl-1 deficiency impaired lactation. Interestingly, EGF was identified as one of the most highly upregulated genes on lactogenesis and inhibition of EGF or mTOR signalling markedly impaired lactation, with concomitant decreases in Mcl-1 and phosphorylated ribosomal protein S6. These data demonstrate that Mcl-1 is essential for mammopoiesis and identify EGF as a critical trigger of Mcl-1 translation to ensure survival of milk-producing alveolar cells.
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http://dx.doi.org/10.1038/ncb3117DOI Listing
April 2015

Dual roles for Id4 in the regulation of estrogen signaling in the mammary gland and ovary.

Development 2014 Aug 18;141(16):3159-64. Epub 2014 Jul 18.

ACRF Stem Cells and Cancer Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia

The HLH transcriptional regulator Id4 exerts important roles in different organs, including the neural compartment, where Id4 loss usually results in early lethality. To explore the role of this basally restricted transcription factor in the mammary gland, we generated a cre-inducible mouse model. MMTV- or K14-cre-mediated deletion of Id4 led to a delay in ductal morphogenesis, consistent with previous findings using a germ-line knockout mouse model. A striking increase in the expression of ERα (Esr1), PR and FoxA1 was observed in both the basal and luminal cellular subsets of Id4-deficient mammary glands. Together with chromatin immunoprecipitation of Id4 on the Esr1 and Foxa1 promoter regions, these data imply that Id4 is a negative regulator of the ERα signaling axis. Unexpectedly, examination of the ovaries of targeted mice revealed significantly increased numbers of secondary and antral follicles, and reduced Id4 expression in the granulosa cells. Moreover, expression of the cascade of enzymes that are crucial for estrogen biosynthesis in the ovary was decreased in Id4-deficient females and uterine weights were considerably lower, indicating impaired estrogen production. Thus, compromised ovarian function and decreased circulating estrogen likely contribute to the mammary ductal defects evident in Id4-deficient mice. Collectively, these data identify Id4 as a novel regulator of estrogen signaling, where Id4 restrains ERα expression in the basal and luminal cellular compartments of the mammary gland and regulates estrogen biosynthesis in the ovary.
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http://dx.doi.org/10.1242/dev.108498DOI Listing
August 2014

Targeting BCL-2 with the BH3 mimetic ABT-199 in estrogen receptor-positive breast cancer.

Cancer Cell 2013 Jul;24(1):120-9

ACRF Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia.

The prosurvival protein BCL-2 is frequently overexpressed in estrogen receptor (ER)-positive breast cancer. We have generated ER-positive primary breast tumor xenografts that recapitulate the primary tumors and demonstrate that the BH3 mimetic ABT-737 markedly improves tumor response to the antiestrogen tamoxifen. Despite abundant BCL-XL expression, similar efficacy was observed with the BCL-2 selective inhibitor ABT-199, revealing that BCL-2 is a crucial target. Unexpectedly, BH3 mimetics were found to counteract the side effect of tamoxifen-induced endometrial hyperplasia. Moreover, BH3 mimetics synergized with phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitors in eliciting apoptosis. Importantly, these two classes of inhibitor further enhanced tumor response in combination therapy with tamoxifen. Collectively, our findings provide a rationale for the clinical evaluation of BH3 mimetics in therapy for breast cancer.
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http://dx.doi.org/10.1016/j.ccr.2013.06.002DOI Listing
July 2013

Global changes in the mammary epigenome are induced by hormonal cues and coordinated by Ezh2.

Cell Rep 2013 Feb 31;3(2):411-26. Epub 2013 Jan 31.

ACRF Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville VIC 3052, Australia.

The mammary epithelium is a dynamic, highly hormone-responsive tissue. To explore chromatin modifications underlying its lineage specification and hormone responsiveness, we determined genome-wide histone methylation profiles of mammary epithelial subpopulations in different states. The marked differences in H3K27 trimethylation between subpopulations in the adult gland suggest that epithelial cell-fate decisions are orchestrated by polycomb-complex-mediated repression. Remarkably, the mammary epigenome underwent highly specific changes in different hormonal contexts, with a profound change being observed in the global H3K27me3 map of luminal cells during pregnancy. We therefore examined the role of the key H3K27 methyltransferase Ezh2 in mammary physiology. Its expression and phosphorylation coincided with H3K27me3 modifications and peaked during pregnancy, driven in part by progesterone. Targeted deletion of Ezh2 impaired alveologenesis during pregnancy, preventing lactation, and drastically reduced stem/progenitor cell numbers. Taken together, these findings reveal that Ezh2 couples hormonal stimuli to epigenetic changes that underpin progenitor activity, lineage specificity, and alveolar expansion in the mammary gland.
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http://dx.doi.org/10.1016/j.celrep.2012.12.020DOI Listing
February 2013

Aldehyde dehydrogenase activity is a biomarker of primitive normal human mammary luminal cells.

Stem Cells 2012 Feb;30(2):344-8

Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada.

Elevated aldehyde dehydrogenase (ALDH) expression/activity has been identified as an important biomarker of primitive cells in various normal and malignant human tissues. Here we examined the level and type of ALDH expression and activity in different subsets of phenotypically and functionally defined normal human mammary cells. We find that the most primitive human mammary stem and progenitor cell types with bilineage differentiation potential show low ALDH activity but undergo a marked, selective, and transient upregulation of ALDH activity at the point of commitment to the luminal lineage. This mirrors a corresponding change in transcripts and protein levels of ALDH1A3, an enzyme involved in retinoic acid synthesis and the most highly expressed ALDH gene in normal human mammary tissue. In contrast, ALDH1A1 is expressed at low levels in all mammary epithelial cells. These findings raise interesting questions about the reported association of ALDH activity with breast cancer stem cells and breast cancer prognosis.
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http://dx.doi.org/10.1002/stem.1001DOI Listing
February 2012

Gata-3 negatively regulates the tumor-initiating capacity of mammary luminal progenitor cells and targets the putative tumor suppressor caspase-14.

Mol Cell Biol 2011 Nov 19;31(22):4609-22. Epub 2011 Sep 19.

The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.

The transcription factor Gata-3 is a definitive marker of luminal breast cancers and a key regulator of mammary morphogenesis. Here we have explored a role for Gata-3 in tumor initiation and the underlying cellular mechanisms using a mouse model of "luminal-like" cancer. Loss of a single Gata-3 allele markedly accelerated tumor progression in mice carrying the mouse mammary tumor virus promoter-driven polyomavirus middle T antigen (MMTV-PyMT mice), while overexpression of Gata-3 curtailed tumorigenesis. Through the identification of two distinct luminal progenitor cells in the mammary gland, we demonstrate that Gata-3 haplo-insufficiency increases the tumor-initiating capacity of these progenitors but not the stem cell-enriched population. Overexpression of a conditional Gata-3 transgene in the PyMT model promoted cellular differentiation and led to reduced tumor-initiating capacity as well as diminished angiogenesis. Transcript profiling studies identified caspase-14 as a novel downstream target of Gata-3, in keeping with its roles in differentiation and tumorigenesis. A strong association was evident between GATA-3 and caspase-14 expression in preinvasive ductal carcinoma in situ samples, where GATA-3 also displayed prognostic significance. Overall, these studies identify GATA-3 as an important regulator of tumor initiation through its ability to promote the differentiation of committed luminal progenitor cells.
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http://dx.doi.org/10.1128/MCB.05766-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3209243PMC
November 2011

Proteomic profiling of secretome and adherent plasma membranes from distinct mammary epithelial cell subpopulations.

Proteomics 2011 Oct 8;11(20):4029-39. Epub 2011 Sep 8.

Ludwig Institute for Cancer Research, Parkville, Victoria, Australia.

The stem cell niche comprises stem cells (SCs), stromal cells, soluble factors, extracellular matrix constituents and vascular networks. The ability to identify signals that regulate SC self-renewal and differentiation is confounded by the difficulty in isolating pure SC niche components in sufficient quantities to enable their biochemical characterisation. Here, we report the extracellular (secretome) and adherent plasma membrane proteomes of three distinct epithelial cell subpopulations isolated and immortalized from the mouse mammary gland--basal and mammary stem cell (basal/MaSC), luminal progenitor (LP) and mature luminal (ML) cell lines. GeLC-MS/MS-based proteomic profiling revealed a distinct switch in components modulating Wnt and ephrin signalling, and integrin-mediated interactions amongst the three cell subpopulations. For example, expression of ephrin B2, ephrin receptors A1, and A2, as well as integrins α2β1 and α6β4 were shown to be enriched in basal/MaSCs, relative to LP and ML cells. Conspicuously, Wnt10a was uniquely detected in basal/MaSCs, and may modulate the canonical Wnt signalling pathway to maintain basal/MaSC activity. By contrast, non-canonical Wnt signalling might be elevated in ML cells, as evidenced by the high expression levels of Wnt5a, Wnt5b, and the transmembrane tyrosine kinase Ror2.
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http://dx.doi.org/10.1002/pmic.201100102DOI Listing
October 2011

Sensitization of BCL-2-expressing breast tumors to chemotherapy by the BH3 mimetic ABT-737.

Proc Natl Acad Sci U S A 2012 Feb 18;109(8):2766-71. Epub 2011 Jul 18.

The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia.

Overexpression of the prosurvival protein BCL-2 is common in breast cancer. Here we have explored its role as a potential therapeutic target in this disease. BCL-2, its anti-apoptotic relatives MCL-1 and BCL-XL, and the proapoptotic BH3-only ligand BIM were found to be coexpressed at relatively high levels in a substantial proportion of heterogeneous breast tumors, including clinically aggressive basal-like cancers. To determine whether the BH3 mimetic ABT-737 that neutralizes BCL-2, BCL-XL, and BCL-W had potential efficacy in targeting BCL-2-expressing basal-like triple-negative tumors, we generated a panel of primary breast tumor xenografts in immunocompromised mice and treated recipients with either ABT-737, docetaxel, or a combination. Tumor response and overall survival were significantly improved by combination therapy, but only for tumor xenografts that expressed elevated levels of BCL-2. Treatment with ABT-737 alone was ineffective, suggesting that ABT-737 sensitizes the tumor cells to docetaxel. Combination therapy was accompanied by a marked increase in apoptosis and dissociation of BIM from BCL-2. Notably, BH3 mimetics also appeared effective in BCL-2-expressing xenograft lines that harbored p53 mutations. Our findings provide in vivo evidence that BH3 mimetics can be used to sensitize primary breast tumors to chemotherapy and further suggest that elevated BCL-2 expression constitutes a predictive response marker in breast cancer.
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http://dx.doi.org/10.1073/pnas.1104778108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3286989PMC
February 2012

Jekyll or Hyde: does Matrigel provide a more or less physiological environment in mammary repopulating assays?

Breast Cancer Res 2011 May 25;13(3):108. Epub 2011 May 25.

In vivo transplantation is the current 'gold-standard' assay for evaluating mammary stem cell (MaSC) function. Matrigel, a reconstituted extracellular matrix derived from a mouse sarcoma line, is increasingly being utilized for mammary repopulating assays, although original studies were carried out in its absence. This matrix has also been shown to enhance tumor-initiating capacity. Whilst Matrigel increases the rate of engraftment by MaSCs, it also appears to promote progenitor activity that is distinct from bona fide stem cell activity. This caveat should be considered when interpreting mammary reconstitution assays that incorporate Matrigel, particularly when transplanting high cell numbers.
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http://dx.doi.org/10.1186/bcr2851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3218928PMC
May 2011

ROAST: rotation gene set tests for complex microarray experiments.

Bioinformatics 2010 Sep 7;26(17):2176-82. Epub 2010 Jul 7.

The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria, Australia.

Motivation: A gene set test is a differential expression analysis in which a P-value is assigned to a set of genes as a unit. Gene set tests are valuable for increasing statistical power, organizing and interpreting results and for relating expression patterns across different experiments. Existing methods are based on permutation. Methods that rely on permutation of probes unrealistically assume independence of genes, while those that rely on permutation of sample are suitable only for two-group comparisons with a good number of replicates in each group.

Results: We present ROAST, a statistically rigorous gene set test that allows for gene-wise correlation while being applicable to almost any experimental design. Instead of permutation, ROAST uses rotation, a Monte Carlo technology for multivariate regression. Since the number of rotations does not depend on sample size, ROAST gives useful results even for experiments with minimal replication. ROAST allows for any experimental design that can be expressed as a linear model, and can also incorporate array weights and correlated samples. ROAST can be tuned for situations in which only a subset of the genes in the set are actively involved in the molecular pathway. ROAST can test for uni- or bi-direction regulation. Probes can also be weighted to allow for prior importance. The power and size of the ROAST procedure is demonstrated in a simulation study, and compared to that of a representative permutation method. Finally, ROAST is used to test the degree of transcriptional conservation between human and mouse mammary stems.

Availability: ROAST is implemented as a function in the Bioconductor package limma available from www.bioconductor.org.
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http://dx.doi.org/10.1093/bioinformatics/btq401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2922896PMC
September 2010