Publications by authors named "Jane E Visvader"

132 Publications

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

EMBO J 2021 May 5: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
May 2021

The Cellular Organization of the Mammary Gland: Insights From Microscopy.

J Mammary Gland Biol Neoplasia 2021 Apr 9. Epub 2021 Apr 9.

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

Despite rapid advances in our knowledge of the cellular heterogeneity and molecular regulation of the mammary gland, how these relate to 3D cellular organization remains unclear. In addition to hormonal regulation, mammary gland development and function is directed by para- and juxtacrine signaling among diverse cell-types, particularly the immune and mesenchymal populations. Precise mapping of the cellular landscape of the breast will help to decipher this complex coordination. Imaging of thin tissue sections has provided foundational information about cell positioning in the mammary gland and now technological advances in tissue clearing and subcellular-resolution 3D imaging are painting a more complete picture. In particular, confocal, light-sheet and multiphoton microscopy applied to intact tissue can fully capture cell morphology, position and interactions, and have the power to identify spatially rare events. This review will summarize our current understanding of mammary gland cellular organization as revealed by microscopy. We focus on the mouse mammary gland and cover a broad range of immune and stromal cell types at major developmental stages and give insights into important tissue niches and cellular interactions.
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http://dx.doi.org/10.1007/s10911-021-09483-6DOI Listing
April 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 Mar 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

Long-term culture, genetic manipulation and xenotransplantation of human normal and breast cancer organoids.

Nat Protoc 2021 04 10;16(4):1936-1965. Epub 2021 Mar 10.

Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands.

Organoid technology has revolutionized the study of human organ development, disease and therapy response tailored to the individual. Although detailed protocols are available for the generation and long-term propagation of human organoids from various organs, such methods are lacking for breast tissue. Here we provide an optimized, highly versatile protocol for long-term culture of organoids derived from either normal human breast tissues or breast cancer (BC) tissues, as well as culturing conditions for a panel of 45 biobanked samples, including BC organoids covering all major disease subtypes (triple-negative, estrogen receptor-positive/progesterone receptor-positive and human epidermal growth receptor 2-positive). Additionally, we provide methods for genetic manipulation by Lipofectamine 2000, electroporation or lentivirus and subsequent organoid selection and clonal culture. Finally, we introduce an optimized method for orthotopic organoid transplantation in mice, which includes injection of organoids and estrogen pellets without the need for surgery. Organoid derivation from tissue fragments until the first split takes 7-21 d; generation of genetically manipulated clonal organoid cultures takes 14-21 d; and organoid expansion for xenotransplantation takes >4 weeks.
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http://dx.doi.org/10.1038/s41596-020-00474-1DOI Listing
April 2021

Intravital microscopy of dynamic single-cell behavior in mouse mammary tissue.

Nat Protoc 2021 04 24;16(4):1907-1935. Epub 2021 Feb 24.

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

Multiphoton intravital imaging is essential for understanding cellular behavior and function in vivo. The adipose-rich environment of the mammary gland poses a unique challenge to in vivo microscopy due to light scattering that impedes high-resolution imaging. Here we provide a protocol for high-quality, six-color 3D intravital imaging of regions across the entire mouse mammary gland and associated tissues for several hours while maintaining tissue access for microdissection and labeling. An incision at the ventral midline and along the right hind leg creates a skin flap that is then secured to a raised platform skin side down. This allows for fluorescence-guided microdissection of connective tissue to provide unimpeded imaging of mammary ducts. A sealed imaging chamber over the skin flap creates a stable environment while maintaining access to large tissue regions for imaging with an upright microscope. We provide a strategy for imaging single cells and the tissue microenvironment utilizing multicolor Confetti lineage-tracing and additional dyes using custom-designed filters and sequential excitation with dual multiphoton lasers. Furthermore, we describe a strategy for simultaneous imaging and photomanipulation of single cells using the Olympus SIM scanner and provide steps for 3D video processing, visualization and high-dimensional analysis of single-cell behavior. We then provide steps for multiplexing intravital imaging with fixation, immunostaining, tissue clearing and 3D confocal imaging to associate cell behavior with protein expression. The skin-flap surgery and chamber preparation take 1.5 h, followed by up to 12 h of imaging. Applications range from basic filming in 1 d to 5 d for multiplexing and complex analysis.
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http://dx.doi.org/10.1038/s41596-020-00473-2DOI Listing
April 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

Halting triple negative breast cancer by targeting PROCR.

Cell Res 2019 11;29(11):875-876

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

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http://dx.doi.org/10.1038/s41422-019-0245-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6889165PMC
November 2019

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

Stem Cells and the Differentiation Hierarchy in Mammary Gland Development.

Physiol Rev 2020 04 20;100(2):489-523. Epub 2019 Sep 20.

Cancer and Stem Cell Biology Program, Duke-NUS Medical School, Singapore, Singapore; Tumour-Host Interaction Laboratory, Francis Crick Institute, London, United Kingdom; Cancer Biology and Stem Cells Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia; Department of Medicine, The University of Melbourne, Parkville, Victoria, Australia; Royal Melbourne Hospital and Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia; and Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia.

The mammary gland is a highly dynamic organ that undergoes profound changes within its epithelium during puberty and the reproductive cycle. These changes are fueled by dedicated stem and progenitor cells. Both short- and long-lived lineage-restricted progenitors have been identified in adult tissue as well as a small pool of multipotent mammary stem cells (MaSCs), reflecting intrinsic complexity within the epithelial hierarchy. While unipotent progenitor cells predominantly execute day-to-day homeostasis and postnatal morphogenesis during puberty and pregnancy, multipotent MaSCs have been implicated in coordinating alveologenesis and long-term ductal maintenance. Nonetheless, the multipotency of stem cells in the adult remains controversial. The advent of large-scale single-cell molecular profiling has revealed striking changes in the gene expression landscape through ontogeny and the presence of transient intermediate populations. An increasing number of lineage cell-fate determination factors and potential niche regulators have now been mapped along the hierarchy, with many implicated in breast carcinogenesis. The emerging diversity among stem and progenitor populations of the mammary epithelium is likely to underpin the heterogeneity that characterizes breast cancer.
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http://dx.doi.org/10.1152/physrev.00040.2018DOI Listing
April 2020

Lgr5 pericentral hepatocytes are self-maintained in normal liver regeneration and susceptible to hepatocarcinogenesis.

Proc Natl Acad Sci U S A 2019 09 5;116(39):19530-19540. Epub 2019 Sep 5.

Cancer and Stem Cell Biology Program, Duke-NUS Medical School, 169857 Singapore;

Emerging evidence suggests that hepatocytes are primarily maintained by self-renewal during normal liver homeostasis, as well as in response to a wide variety of hepatic injuries. However, how hepatocytes in distinct anatomic locations within the liver lobule are replenished under homeostasis and injury-induced regeneration remains elusive. Using a newly developed bacterial artificial chromosome (BAC)-transgenic mouse model, we demonstrate that expression in the liver is restricted to a unique subset of hepatocytes most adjacent to the central veins. Genetic lineage tracing revealed that pericentral Lgr5 hepatocytes have a long lifespan and mainly contribute to their own lineage maintenance during postnatal liver development and homeostasis. Remarkably, these hepatocytes also fuel the regeneration of their own lineage during the massive and rapid regeneration process following two-thirds partial hepatectomy. Moreover, Lgr5 hepatocytes are found to be the main cellular origin of diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) and are highly susceptible to neoplastic transformation triggered by activation of Erbb pathway. Our findings establish an unexpected self-maintaining mode for a defined subset of hepatocytes during liver homeostasis and regeneration, and identify Lgr5 pericentral hepatocytes as major cells of origin in HCC development.
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http://dx.doi.org/10.1073/pnas.1908099116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6765306PMC
September 2019

High-resolution 3D imaging of fixed and cleared organoids.

Nat Protoc 2019 06 3;14(6):1756-1771. Epub 2019 May 3.

Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands.

In vitro 3D organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of organoids and demonstrated organoids' phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor organoids, as well as on mouse mammary gland organoids. It includes a simple clearing method utilizing a homemade fructose-glycerol clearing agent that captures 3D organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From organoid harvest to image analysis, the protocol takes 3 d.
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http://dx.doi.org/10.1038/s41596-019-0160-8DOI Listing
June 2019

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

Isolation and Propagation of Mammary Epithelial Stem and Progenitor Cells.

Methods Mol Biol 2019 ;1940:217-229

Department of Medical Biology, University of Melbourne, Melbourne, VIC, Australia.

Several methods of mammary gland dissociation have been described that utilize a combined strategy of mechanical and enzymatic dissociation to isolate mammary epithelial cells (MECs) from intact tissue (Smalley et al., J Mammary Gland Biol Neoplasia 17:91-97, 2012). Here we detail a robust method that enables the isolation of all major stem and progenitor MEC populations, which has been successfully used to study stem cell behavior when coupled with transplantation and in vitro assays (Shackleton et al., Nature 439:84-88, 2006; Bouras et al., Cell Stem Cell 3:429-441, 2008; Sheridan et al., BMC Cancer 15:221, 2015; Jamieson et al., Development 144:1065-1071, 2017). Furthermore, we outline two prominent methods for culturing MECs for the purposes of ex vivo manipulation or study: 2D feeder layer cultures and 3D Matrigel colony assays. Importantly, all outlined methods retain stem and progenitor cell behaviors and can be used in combination with downstream in vivo, in vitro, or in silico analyses.
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http://dx.doi.org/10.1007/978-1-4939-9086-3_16DOI Listing
July 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

Differential methylation analysis of reduced representation bisulfite sequencing experiments using edgeR.

F1000Res 2017;6:2055. Epub 2017 Nov 28.

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

Cytosine methylation is an important DNA epigenetic modification. In vertebrates, methylation occurs at CpG sites, which are dinucleotides where a cytosine is immediately followed by a guanine in the DNA sequence from 5' to 3'. When located in the promoter region of a gene, DNA methylation is often associated with transcriptional silencing of the gene. Aberrant DNA methylation is associated with the development of various diseases such as cancer. Bisulfite sequencing (BS-seq) is the current "gold-standard" technology for high-resolution profiling of DNA methylation. Reduced representation bisulfite sequencing (RRBS) is an efficient form of BS-seq that targets CpG-rich DNA regions in order to save sequencing costs. A typical bioinformatics aim is to identify CpGs that are differentially methylated (DM) between experimental conditions. This workflow demonstrates that differential methylation analysis of RRBS data can be conducted using software and methodology originally developed for RNA-seq data. The RNA-seq pipeline is adapted to methylation by adding extra columns to the design matrix to account for read coverage at each CpG, after which the RRBS and RNA-seq pipelines are almost identical. This approach is statistically natural and gives analysts access to a rich collection of analysis tools including generalized linear models, gene set testing and pathway analysis. The article presents a complete start to finish case study analysis of RRBS profiles of different cell populations from the mouse mammary gland using the Bioconductor package edgeR. We show that lineage-committed cells are typically hyper-methylated compared to progenitor cells and this is true on all the autosomes but not the sex chromosomes. We demonstrate a strong negative correlation between methylation of promoter regions and gene expression as measured by RNA-seq for the same cell types, showing that methylation is a regulatory mechanism involved in epithelial linear commitment.
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http://dx.doi.org/10.12688/f1000research.13196.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5747346PMC
November 2017

Canonical PRC2 function is essential for mammary gland development and affects chromatin compaction in mammary organoids.

PLoS Biol 2018 08 6;16(8):e2004986. Epub 2018 Aug 6.

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

Distinct transcriptional states are maintained through organization of chromatin, resulting from the sum of numerous repressive and active histone modifications, into tightly packaged heterochromatin versus more accessible euchromatin. Polycomb repressive complex 2 (PRC2) is the main mammalian complex responsible for histone 3 lysine 27 trimethylation (H3K27me3) and is integral to chromatin organization. Using in vitro and in vivo studies, we show that deletion of Suz12, a core component of all PRC2 complexes, results in loss of H3K27me3 and H3K27 dimethylation (H3K27me2), completely blocks normal mammary gland development, and profoundly curtails progenitor activity in 3D organoid cultures. Through the application of mammary organoids to bypass the severe phenotype associated with Suz12 loss in vivo, we have explored gene expression and chromatin structure in wild-type and Suz12-deleted basal-derived organoids. Analysis of organoids led to the identification of lineage-specific changes in gene expression and chromatin structure, inferring cell type-specific PRC2-mediated gene silencing of the chromatin state. These expression changes were accompanied by cell cycle arrest but not lineage infidelity. Together, these data indicate that canonical PRC2 function is essential for development of the mammary gland through the repression of alternate transcription programs and maintenance of chromatin states.
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http://dx.doi.org/10.1371/journal.pbio.2004986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6095611PMC
August 2018

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

A critical epithelial survival axis regulated by MCL-1 maintains thymic function in mice.

Blood 2017 12 29;130(23):2504-2515. Epub 2017 Sep 29.

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

T-cell differentiation is governed by interactions with thymic epithelial cells (TECs) and defects in this process undermine immune function and tolerance. To uncover new strategies to restore thymic function and adaptive immunity in immunodeficiency, we sought to determine the molecular mechanisms that control life and death decisions in TECs. Guided by gene expression profiling, we created mouse models that specifically deleted prosurvival genes in TECs. We found that although BCL-2 and BCL-XL were dispensable for TEC homeostasis, MCL-1 deficiency impacted on TECs as early as embryonic day 15.5, resulting in early thymic atrophy and T-cell lymphopenia, with near complete loss of thymic tissue by 2 months of age. MCL-1 was not necessary for TEC differentiation but was continually required for the survival of mature cortical and medullary TECs and the maintenance of thymic architecture. A screen of TEC trophic factors in organ cultures showed that epidermal growth factor upregulated MCL-1 via MAPK/ERK kinase activity, providing a molecular mechanism for the support of TEC survival. This signaling axis governing TEC survival and thymic function represents a new target for strategies for thymic protection and regeneration.
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http://dx.doi.org/10.1182/blood-2017-03-771576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6207344PMC
December 2017

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

Elf5 is a principal cell lineage specific transcription factor in the kidney that contributes to Aqp2 and Avpr2 gene expression.

Dev Biol 2017 04 17;424(1):77-89. Epub 2017 Feb 17.

Sanford Children's Health Research Center, Sanford Research, 2301 East 60(th) Street North, Sioux Falls, SD 57104, USA; Department of Pediatrics, Sanford School of Medicine, Sioux Falls, SD 57104, USA. Electronic address:

The mammalian kidney collecting ducts are critical for water, electrolyte and acid-base homeostasis and develop as a branched network of tubular structures composed of principal cells intermingled with intercalated cells. The intermingled nature of the different collecting duct cell types has made it challenging to identify unique and critical factors that mark and/or regulate the development of the different collecting duct cell lineages. Here we report that the canonical Notch signaling pathway components, RBPJ and Presinilin1 and 2, are involved in patterning the mouse collecting duct cell fates by maintaining a balance between principal cell and intercalated cell fates. The relatively reduced number of principal cells in Notch-signaling-deficient kidneys offered a unique genetic leverage to identify critical principal cell-enriched factors by transcriptional profiling. Elf5, which codes for an ETS transcription factor, is one such gene that is down-regulated in kidneys with Notch-signaling-deficient collecting ducts. Additionally, Elf5 is among the earliest genes up regulated by ectopic expression of activated Notch1 in the developing collecting ducts. In the kidney, Elf5 is first expressed early within developing collecting ducts and remains on in mature principal cells. Lineage tracing of Elf5-expressing cells revealed that they are committed to the principal cell lineage by as early as E16.5. Over-expression of ETS Class IIa transcription factors, including Elf5, Elf3 and Ehf, increase the transcriptional activity of the proximal promoters of Aqp2 and Avpr2 in cultured ureteric duct cell lines. Conditional inactivation of Elf5 in the developing collecting ducts results in a small but significant reduction in the expression levels of Aqp2 and Avpr2 genes. We have identified Elf5 as an early maker of the principal cell lineage that contributes to the expression of principal cell specific genes.
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http://dx.doi.org/10.1016/j.ydbio.2017.02.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5382981PMC
April 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

Out-RANKing BRCA1 in Mutation Carriers.

Cancer Res 2017 02 19;77(3):595-600. Epub 2017 Jan 19.

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

Beyond prophylactic mastectomy, there are currently very few options available to BRCA1 mutation carriers to help reduce their risk of developing breast cancer. An effective prevention therapy therefore remains a pressing area of need. Accumulating evidence points to amplification of the progesterone signaling axis in precancerous tissue from BRCA1 mutation carriers. Given that RANKL is an important paracrine mediator of hormonal signaling in breast tissue, there has been considerable interest in exploring a potential role for this pathway in oncogenesis. Recent findings indicate that the RANK and NF-κB pathways are aberrantly activated in luminal progenitor cells resident in preneoplastic BRCA1 breast tissue. The augmented proliferation of these cells and their predilection for DNA damage suggest that they are prime cellular targets for basal-like cancers arising in BRCA1 mutation carriers. The end result is a hyperactive pathway, initiated by progesterone and amplified by DNA damage-induced NF-κB signaling, that likely accounts for the susceptibility of BRCA1 luminal progenitor cells to oncogenesis and tissue specificity. Specific targeting of this progenitor subset has revealed a compelling new prevention strategy for these and possibly other high-risk women. Cancer Res; 77(3); 595-600. ©2017 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-16-2025DOI Listing
February 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