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While several resources exist that interpret therapeutic significance of genomic alterations in cancer, many regional real-world issues limit access to drugs. There is a need for a pragmatic, evidence-based, context-adapted tool to guide clinical management based on molecular biomarkers. To this end, we have structured a compendium of approved and experimental therapies with associated biomarkers following a survey of drug regulatory databases, existing knowledge bases, and published literature. Each biomarker-disease-therapy triplet was categorised using a tiering system reflective of key therapeutic considerations: approved and reimbursed therapies with respect to a jurisdiction (Tier 1), evidence of efficacy or approval in another jurisdiction (Tier 2), evidence of antitumour activity (Tier 3), and plausible biological rationale (Tier 4). Two resistance categories were defined: lack of efficacy (Tier R1) or antitumor activity (Tier R2). Based on this framework, we curated a digital resource focused on drugs relevant in the Australian healthcare system (TOPOGRAPH: Therapy Oriented Precision Oncology Guidelines for Recommending Anticancer Pharmaceuticals). As of November 2020, TOPOGRAPH comprised 2810 biomarker-disease-therapy triplets in 989 expert-appraised entries, including 373 therapies, 199 biomarkers, and 106 cancer types. In the 345 therapies catalogued, 84 (24%) and 65 (19%) were designated Tiers 1 and 2, respectively, while 271 (79%) therapies were supported by preclinical studies, early clinical trials, retrospective studies, or case series (Tiers 3 and 4). A companion algorithm was also developed to support rational, context-appropriate treatment selection informed by molecular biomarkers. This framework can be readily adapted to build similar resources in other jurisdictions to support therapeutic decision-making.

Purpose: Although enzalutamide (ENZ) has been widely used to treat de novo or castration-resistant metastatic prostate cancer, resistance develops and disease progression is ultimately inevitable. There are currently no approved targeted drugs to specifically delay or overcome ENZ resistance. Experimental Design: We selected several ENZ-resistant cell lines that replicated clinical characteristics of the majority of patients with ENZ-resistant disease. A high-throughput pharmacologic screen was utilized to identify compounds with greater cytotoxic effect for ENZ-resistant cell lines, compared with parental ENZ-sensitive cells. We validated the potential hits in vitro and in vivo, and used knockdown and overexpression assays to study the dependencies in ENZ-resistant prostate cancer. Results: ABT199 (BCL-2 inhibitor) and IMD0354 (IKKB inhibitor) were identified as potent and selective inhibitors of cell viability in ENZ-resistant cell lines in vitro and in vivo which were further validated using loss-of-function assays of BCL-2 and IKKB. Notably, we observed that overexpression of BCL-2 and IKKB in ENZ-sensitive cell lines was sufficient for the emergence of ENZ resistance. In addition, we confirmed that BCL-2 or IKKB inhibitors suppressed the development of ENZ resistance in xenografts. However, validation of both BCL-2 and IKKB in matched castration-sensitive/resistant clinical samples showed that, concurrent with the development of ENZ/abiraterone resistance in patients, only the protein levels of IKKB were increased. Conclusions: Our findings identify BCL-2 and IKKB dependencies in clinically relevant ENZ-resistant prostate cancer cells in vitro and in vivo, but indicate that IKKB upregulation appears to have greater relevance to the progression of human castrate-resistant prostate cancer.

BackgroundThe interferon response can influence the primary and metastatic activity of breast cancers and can interact with checkpoint immunotherapy to modulate its effects. UsingN-ethyl-N-nitrosourea mutagenesis, we found a mouse with an activating mutation in oligoadenylate synthetase 2 (Oas2), a sensor of viral double stranded RNA, that resulted in an interferon response and prevented lactation in otherwise healthy mice.MethodsTo determine if sole activation ofOas2could alter the course of mammary cancer, we combined theOas2mutation with theMMTV-PyMToncogene model of breast cancer and examined disease progression and the effects of checkpoint immunotherapy using Kaplan–Meier survival analysis with immunohistochemistry and flow cytometry.ResultsOas2mutation prevented pregnancy from increasing metastases to lung. Checkpoint immunotherapy with antibodies against programmed death-ligand 1 was more effective when theOas2mutation was present.ConclusionsThese data establish OAS2 as a therapeutic target for agents designed to reduce metastases and increase the effectiveness of checkpoint immunotherapy.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies. It is phenotypically heterogeneous with a highly unstable genome and provides few common therapeutic targets. We found that MCL1, Cofilin1 (CFL1) and SRC mRNA were highly expressed by a wide range of these cancers, suggesting that a strategy of dual MCL-1 and SRC inhibition might be efficacious for many patients. Immunohistochemistry revealed that MCL-1 protein was present at high levels in 94.7% of patients in a cohort of PDACs from Australian Pancreatic Genome Initiative (APGI). High MCL1 and Cofilin1 mRNA expression was also strongly predictive of poor outcome in the TCGA dataset and in the APGI cohort. In culture, MCL-1 antagonism reduced the level of the cytoskeletal remodeling protein Cofilin1 and phosphorylated SRC on the active Y416 residue, suggestive of reduced invasive capacity. The MCL-1 antagonist S63845 synergized with the SRC kinase inhibitor dasatinib to reduce cell viability and invasiveness through 3D-organotypic matrices. In preclinical murine models, this combination reduced primary tumor growth and liver metastasis of pancreatic cancer xenografts. These data suggest that MCL-1 antagonism, while reducing cell viability, may have an additional benefit in increasing the antimetastatic efficacy of dasatinib for the treatment of PDAC.

BACKGROUND: The D-type cyclin and cyclin dependent kinase 4/6 (CDK) complex phosphorylates retinoblastoma protein, thereby driving cell cycle progression. This process is blocked by inhibitors of CDK4/6. As part of the Molecular Screening and Therapeutics program, this phase IIa trial tested the clinical activity of CDK4/6 inhibitor monotherapy in tumors with cell cycle pathway alterations.PATIENTS AND METHODS: Eligible patients ≥ 18 years old, with advanced or metastatic solid cancers, along with amplification of CDK4/6, CCND1/2/3, or loss of function alterations in CDKN2A were recruited. The primary objective of this signal-seeking trial was to evaluate the clinical activity of palbociclib – a composite of objective responses and the ratio of time to progression (TTP) on palbociclib, to TTP on treatment preceding trial entry.RESULTS: Ten patients had CDK4/CDK6 or Cyclin D1 amplifications, six patients had CDKN2A deletions. After a median follow-up of 35 months, there were no objective responses. Seven patients had stable disease and one had non-complete response/non-progressive disease (non-CR/non-PD) based on evaluation of a non-target lesion. Two of these seven patients maintained stable disease for at least 6 months, as did the patient with non-CR/non-PD. Median PFS and OS were 3.5 and 11.0 months respectively. No unexpected toxicities were observed. Translational correlates yielded strategies for targeting cell cycle interactions with other molecular pathways and the immune system.CONCLUSION: Palbociclib monotherapy was not associated with any objective responses, but stable disease lasting at least 6 months was observed in 19% of patients. There was no clear relationship with alteration type or histotype. The signals of immune activation provide insights into the design of future trials, with a combination approach adding checkpoint blockade to CDK4/6 inhibition.

BACKGROUNDWhile several key resources exist that interpret therapeutic significance of genomic alterations in cancer, many regional real-world issues limit access to drugs. There is a need for a pragmatic, evidence-based, context-adapted tool to guide clinical management based on molecular biomarkers.METHODSA compendium of approved and experimental therapies with associated biomarkers was built following a survey of drug regulatory databases, existing knowledge bases, and published literature. Each biomarker-disease-therapy triplet was then categorized using a tiering system reflective of key therapeutic considerations: approved and reimbursed standard-of-care therapies with respect to a jurisdiction (Tier 1), evidence of efficacy or approval in another jurisdiction (Tier 2), evidence of antitumour activity (Tier 3), and plausible biological rationale (Tier 4). Two resistance categories were defined: lack of efficacy (Tier R1), and lack of antitumor activity (Tier R2).RESULTSFollowing comprehensive literature review and appraisal, we developed a curated knowledge base focused on drugs relevant and accessible in the Australian healthcare system (TOPOGRAPH: Therapy Oriented Precision Oncology Guidelines for Recommending Anticancer Pharmaceuticals). As of November 2020, TOPOGRAPH comprised 2810 biomarker-disease-therapy triplets in 989 expert-appraised entries, including 373 therapies, 199 predictive biomarkers, and 106 cancer types. In the 345 biomarker-linked therapies catalogued, 84 (24%) and 65 (19%) therapies in contexts of different cancer types have Tier 1 and 2 designations respectively, while 271 (79%) therapies were supported by preclinical studies, early clinical trials, retrospective studies, or case series (Tiers 3 and 4). A total of 119 of 373 (33%) therapies associated with biomarkers of resistance were also catalogued. A clinical algorithm was also developed to support therapeutic decision-making using predictive biomarkers. This resource is accessible online at https://topograph.info/.CONCLUSIONTOPOGRAPH is intended to support oncologists with context-appropriate clinical decision-making– optimising selection and accessibility of the most appropriate targeted therapy for any given genomic biomarker. Our approach can be readily adapted to build jurisdiction-specific resources to standardise decision-making in precision oncology.

Acquired resistance to endocrine therapy is responsible for half of the therapeutic failures in the treatment of breast cancer. Recent findings have implicated increased expression of the ETS transcription factor ELF5 as a potential modulator of estrogen action and driver of endocrine resistance, and here we provide the first insight into the mechanisms by which ELF5 modulates estrogen sensitivity. Using chromatin immunoprecipitation sequencing we found that ELF5 binding overlapped with FOXA1 and ER at super enhancers, enhancers and promoters, and when elevated, caused FOXA1 and ER to bind to new regions of the genome, in a pattern that replicated the alterations to the ER/FOXA1 cistrome caused by the acquisition of resistance to endocrine therapy. RNA sequencing demonstrated that these changes altered estrogen-driven patterns of gene expression, the expression of ER transcription-complex members, and 6 genes known to be involved in driving the acquisition of endocrine resistance. Using rapid immunoprecipitation mass spectrometry of endogenous proteins, and proximity ligation assays, we found that ELF5 interacted physically with members of the ER transcription complex, such as DNA-PKcs. We found 2 cases of endocrine-resistant brain metastases where ELF5 levels were greatly increased and ELF5 patterns of gene expression were enriched, compared to the matched primary tumour. Thus ELF5 alters ER-driven gene expression by modulating the ER/FOXA1 cistrome, by interacting with it, and by modulating the expression of members of the ER transcriptional complex, providing multiple mechanisms by which ELF5 can drive endocrine resistance., Author summary Two thirds of breast cancers are initially treated with endocrine therapy because they are likely to rely on estrogen for their proliferation. Understanding why this therapy ultimately fails in 2/3 of cases offers the chance of durable treatment. In 2012 we hypothesised that normal developmental cell fate decisions taken by mammary progenitor cells persist in tumours that are maintained by instances of a cancerous progenitor, and that a change in the relative influence of the two major transcription factors that drive progenitor cell fate, ER to specify the hormone sensing lineage, and ELF5 to specify the ER- alveolar lineage, may allow a cancer to shift control of proliferation from estrogen to ELF5. Here we show that these transcription factors are often co located at super enhancers, enhancers and at the promoters of differentially regulated genes. When the levels of ELF5 were increased this caused ER and its pioneer factor FOXA1 to move to new regions of the genome associated with resistance to hormonal therapy. We also showed that ELF both regulated and bound directly to members of the ER-transcriptional complex. These findings provide the first indication of the mechanisms by which an increase in ELF5 could drive the acquisition of endocrine resistance.

Myeloid cell leukemia-1 (MCL-1), closely related to B-cell lymphoma 2 (BCL-2), has a well-established role in cell survival and has emerged as an important target for cancer therapeutics. We have demonstrated that inhibiting MCL-1 is efficacious in suppressing tumour progression in pre-clinical models of breast cancer and revealed that in addition to its role in cell survival, MCL-1 modulated cellular invasion. Utilizing a MCL-1-specific genetic antagonist, we found two possible mechanisms; firstly MCL-1 directly binds to and alters the phosphorylation of the cytoskeletal remodeling protein, Cofilin, a protein important for cytoskeletal remodeling during invasion, and secondly MCL-1 modulates the levels SRC family kinases (SFKs) and their targets. These data provide evidence that MCL-1 activities are not limited to endpoints of extracellular and intracellular signaling culminating in cell survival as previously thought, but can directly modulate the output of SRC family kinases signaling during cellular invasion. Here we review the pleotropic roles of MCL-1 and discuss the implications of this newly discovered effect on protein kinase signaling for the development of cancer therapeutics.

Basal-like breast cancer (BLBC) is an aggressive molecular subtype that represents up to 15% of breast cancers. It occurs in younger patients, and typically shows rapid development of locoregional and distant metastasis, resulting in a relatively high mortality rate. Its defining features are that it is positive for basal cytokeratins and, epidermal growth factor receptor and/or c-Kit. Problematically, it is typically negative for the estrogen receptor and human epidermal growth factor receptor 2 (HER2), which means that it is unsuitable for either hormone therapy or targeted HER2 therapy. As a result, there are few therapeutic options for BLBC, and a major priority is to define molecular subgroups of BLBC that could be targeted therapeutically. In this review, we focus on the highly proliferative and anti-apoptotic phenotype of BLBC with the goal of defining potential therapeutic avenues, which could take advantage of these aspects of tumor development.

Contains fulltext : 240100.pdf (Publisher’s version ) (Open Access) Basal breast cancer is associated with younger age, early relapse, and a high mortality rate. Here, we use unbiased droplet-based single-cell RNA sequencing (RNA-seq) to elucidate the cellular basis of tumor progression during the specification of the basal breast cancer subtype from the luminal progenitor population in the MMTV-PyMT (mouse mammary tumor virus-polyoma middle tumor-antigen) mammary tumor model. We find that basal-like cancer cells resemble the alveolar lineage that is specified upon pregnancy and encompass the acquisition of an aberrant post-lactation developmental program of involution that triggers remodeling of the tumor microenvironment and metastatic dissemination. This involution mimicry is characterized by a highly interactive multicellular network, with involution cancer-associated fibroblasts playing a pivotal role in extracellular matrix remodeling and immunosuppression. Our results may partially explain the increased risk and poor prognosis of breast cancer associated with childbirth.

Cancer is a heterogeneous and complex disease. Tumors are formed by cancer cells and a myriad of non-cancerous cell types that together with the extracellular matrix form the tumor microenvironment. These cancer-associated cells and components contribute to shape the progression of cancer and are deeply involved in patient outcome. The immune system is an essential part of the tumor microenvironment, and induction of cancer immunotolerance is a necessary step involved in tumor formation and growth. Immune mechanisms are intimately associated with cancer progression, invasion, and metastasis; as well as to tumor dormancy and modulation of sensitivity to drug therapy. Transcriptome analyses have been extensively used to understand the heterogeneity of tumors, classifying tumors into molecular subtypes and establishing signatures that predict response to therapy and patient outcomes. However, the classification of the tumor cell diversity and specially the identification of rare populations has been limited in these transcriptomic analyses of bulk tumor cell populations. Massively-parallel single-cell RNAseq analysis has emerged as a powerful method to unravel heterogeneity and to study rare cell populations in cancer, through unsupervised sampling and modeling of transcriptional states in single cells. In this context, the study of the role of the immune system in cancer would benefit from single cell approaches, as it will enable the characterization and/or discovery of the cell types and pathways involved in cancer immunotolerance otherwise missed in bulk transcriptomic information. Thus, the analysis of gene expression patterns at single cell resolution holds the potential to provide key information to develop precise and personalized cancer treatment including immunotherapy. This review is focused on the latest single-cell RNAseq methodologies able to agnostically study thousands of tumor cells as well as targeted single-cell RNAseq to study rare populations within tumors. In particular, we will discuss methods to study the immune system in cancer. We will also discuss the current challenges to the study of cancer at the single cell level and the potential solutions to the current approaches.

Quantification of cellular antigens and their interactions via antibody-based detection methods are widely used in scientific research. Accurate high-throughput quantitation of these assays using general image analysis software can be time consuming and challenging, particularly when attempted by users with limited image processing and analysis knowledge. To overcome this, we have designed Andy’s Algorithms, a series of automated image analysis pipelines for FIJI, that permits rapid, accurate and reproducible batch-processing of 3,3′-diaminobenzidine (DAB) immunohistochemistry, proximity ligation assays (PLAs) and other common assays. Andy’s Algorithms incorporates a step-by-step tutorial and optimization pipeline to make batch image analysis simple for the untrained user and adaptable across laboratories. Andy’s algorithms provide a simpler, faster, standardized work flow compared to existing programs, while offering equivalent performance and additional features, in a free to use open-source application of FIJI. Andy’s Algorithms are available at GitHub, publicly accessed at https://github.com/andlaw1841/Andy-s-Algorithm.

We identified a non-synonymous mutation in Oas2 (I405N), a sensor of viral double-stranded RNA, from an ENU-mutagenesis screen designed to discover new genes involved in mammary development. The mutation caused post-partum failure of lactation in healthy mice with otherwise normally developed mammary glands, characterized by greatly reduced milk protein synthesis coupled with epithelial cell death, inhibition of proliferation and a robust interferon response. Expression of mutant but not wild type Oas2 in cultured HC-11 or T47D mammary cells recapitulated the phenotypic and transcriptional effects observed in the mouse. The mutation activates the OAS2 pathway, demonstrated by a 34-fold increase in RNase L activity, and its effects were dependent on expression of RNase L and IRF7, proximal and distal pathway members. This is the first report of a viral recognition pathway regulating lactation., Author summary Using ENU-mutagenesis in mice we discovered a pedigree with lactation failure. Mammary development through puberty and pregnancy appeared normal in mutant animals, but the activation of lactation failed in the immediate post partum period and no milk reached the pups. Failure of lactation was accompanied by greatly diminished milk protein synthesis, decreased epithelial cell proliferation, increased epithelial cell death and a robust interferon response. A non-synonymous mutation in Oas2 (I405N) in the viral sensor Oas2 was found and expression of mutant Oas2 in mammary cells recapitulated these phenotypes. RNase L, the most proximal effector of OAS2 action, was activated in the mammary glands of mutant mice and in mammary cells expressing mutant Oas2. Knockdown of RNase L, or the distal pathway member IRF7, prevented these effects, indicating that the mutation in OAS2 caused activation of the viral signaling pathway. These results show that viral detection in the mammary gland can prevent lactation.

Advances in the study of hematopoietic cell maturation have paved the way to a deeper understanding the stem and progenitor cellular hierarchy in the mammary gland. The mammary epithelium, unlike the hematopoietic cellular hierarchy, sits in a complex niche where communication between epithelial cells and signals from the systemic hormonal milieu, as well as from extra-cellular matrix, influence cell fate decisions and contribute to tissue homeostasis. We review the discovery, definition and regulation of the mammary cellular hierarchy and we describe the development of the concepts that have guided our investigations. We outline recent advances in in vivo lineage tracing that is now challenging many of our assumptions regarding the behavior of mammary stem cells, and we show how understanding these cellular lineages has altered our view of breast cancer.

SUMMARY The ETS transcription factor ELF5 specifies the formation of the secretory cell lineage of the mammary gland during pregnancy, by directing cell fate decisions of the mammary progenitor cells. The decision-making activity continues in breast cancer, where in luminal breast cancer cells forced ELF5 expression suppresses estrogen sensitivity and shifts gene expression toward the basal molecular subtype. The development of anti-estrogen resistance in luminal breast cancer is accompanied by increased expression of ELF5 and acquired dependence on ELF5 for continued proliferation, providing a potential new therapeutic target or prognostic marker to improve the treatment of this stage of the disease. Forced ELF5 expression suppresses the mesenchymal phenotype, making cells more epithelial and producing lower rates of invasion and motility. Conversely, loss of ELF5 promotes metastasis, with a clear corollary in the claudin-low subtype of breast cancer, which does not express ELF5 and is highly metastatic, or during the final stages of tumor progression, where loss of ELF5 expression may be involved in the acquisition of the lethal phenotype. In circumstances where ELF5 expression increases in parallel with metastatic potential, such as anti-estrogen resistant luminal breast cancers and basal breast cancer, there is much more to be understood about ELF5 and metastasis.

Background Metastatic disease is largely resistant to therapy and accounts for almost all cancer deaths. Myeloid cell leukemia-1 (MCL-1) is an important regulator of cell survival and chemo-resistance in a wide range of malignancies, and thus its inhibition may prove to be therapeutically useful. Methods To examine whether targeting MCL-1 may provide an effective treatment for breast cancer, we constructed inducible models of BIMs2A expression (a specific MCL-1 inhibitor) in MDA-MB-468 (MDA-MB-468-2A) and MDA-MB-231 (MDA-MB-231-2A) cells. Results MCL-1 inhibition caused apoptosis of basal-like MDA-MB-468-2A cells grown as monolayers, and sensitized them to the BCL-2/BCL-XL inhibitor ABT-263, demonstrating that MCL-1 regulated cell survival. In MDA-MB-231-2A cells, grown in an organotypic model, induction of BIMs2A produced an almost complete suppression of invasion. Apoptosis was induced in such a small proportion of these cells that it could not account for the large decrease in invasion, suggesting that MCL-1 was operating via a previously undetected mechanism. MCL-1 antagonism also suppressed local invasion and distant metastasis to the lung in mouse mammary intraductal xenografts. Kinomic profiling revealed that MCL-1 antagonism modulated Src family kinases and their targets, which suggested that MCL-1 might act as an upstream modulator of invasion via this pathway. Inhibition of MCL-1 in combination with dasatinib suppressed invasion in 3D models of invasion and inhibited the establishment of tumors in vivo. Conclusion These data provide the first evidence that MCL-1 drives breast cancer cell invasion and suggests that MCL-1 antagonists could be used alone or in combination with drugs targeting Src kinases such as dasatinib to suppress metastasis. Electronic supplementary material The online version of this article (doi:10.1186/s13058-016-0781-6) contains supplementary material, which is available to authorized users.

Background E74-like factor 5 (ELF5) is an epithelial-specific member of the E26 transforming sequence (ETS) transcription factor family and a critical regulator of cell fate in the placenta, pulmonary bronchi, and milk-producing alveoli of the mammary gland. ELF5 also plays key roles in malignancy, particularly in basal-like and endocrine-resistant forms of breast cancer. Almost all genes undergo alternative transcription or splicing, which increases the diversity of protein structure and function. Although ELF5 has multiple isoforms, this has not been considered in previous studies of ELF5 function. Methods RNA-sequencing data for 6757 samples from The Cancer Genome Atlas were analyzed to characterize ELF5 isoform expression in multiple normal tissues and cancers. Extensive in vitro analysis of ELF5 isoforms, including a 116-gene quantitative polymerase chain reaction panel, was performed in breast cancer cell lines. Results ELF5 isoform expression was found to be tissue-specific due to alternative promoter use but altered in multiple cancer types. The normal breast expressed one main isoform, while in breast cancer there were subtype-specific alterations in expression. Expression of other ETS factors was also significantly altered in breast cancer, with the basal-like subtype demonstrating a distinct ETS expression profile. In vitro inducible expression of the full-length isoforms 1 and 2, as well as isoform 3 (lacking the Pointed domain) had similar phenotypic and transcriptional effects. Conclusions Alternative promoter use, conferring differential regulatory responses, is the main mechanism governing ELF5 action rather than differential transcriptional activity of the isoforms. This understanding of expression and function at the isoform level is a vital first step in realizing the potential of transcription factors such as ELF5 as prognostic markers or therapeutic targets in cancer. Electronic supplementary material The online version of this article (doi:10.1186/s13058-015-0666-0) contains supplementary material, which is available to authorized users.

SummaryThe recent identification of mouse mammary stem cells (MaSCs) and progenitor subpopulations has enhanced the prospect of investigating the genetic control of their lineage specification and differentiation. Here we have explored the role of the Notch pathway within the mammary epithelial hierarchy. We show that knockdown of the canonical Notch effector Cbf-1 in the MaSC-enriched population results in increased stem cell activity in vivo as well as the formation of aberrant end buds, implying a role for endogenous Notch signaling in restricting MaSC expansion. Conversely, Notch was found to be preferentially activated in the ductal luminal epithelium in vivo and promoted commitment of MaSCs exclusively along the luminal lineage. Notably, constitutive Notch signaling specifically targeted luminal progenitor cells for expansion, leading to hyperplasia and tumorigenesis. These findings reveal key roles for Notch signaling in MaSCs and luminal cell commitment and further suggest that inappropriate Notch activation promotes the self-renewal and transformation of luminal progenitor cells.

Hormonal cues regulate mammary development, but the consequent transcriptional changes and cell fate decisions are largely undefined. We show that knockout of the prolactin-regulated Ets transcription factor Elf5 prevented formation of the secretory epithelium during pregnancy. Conversely, overexpression of Elf5 in an inducible transgenic model caused alveolar differentiation and milk secretion in virgin mice, disrupting ductal morphogenesis. CD61+ luminal progenitor cells accumulated in Elf5-deficient mammary glands and were diminished in glands with Elf5 overexpression. Thus Elf5 specifies the differentiation of CD61+ progenitors to establish the secretory alveolar lineage during pregnancy, providing a link between prolactin, transcriptional events, and alveolar development.

Mammary gland development is coupled to reproductive events by hormonal cues of ovarian and pituitary origin, which activate a genomic regulatory network. Identification of the components and regulatory links that comprise this network will provide the basis for defining the network's dynamic response during normal development and its perturbation during breast carcinogenesis. In this study KIBRA was identified as a transcript showing decreased expression associated with failed mammary gland development in Prlr knockout mammary epithelium. It is strongly up-regulated during pregnancy, falls during lactation and is again up-regulated during involution of the gland at weaning. A bioinformatic approach was undertaken to identify potential binding partners which interact with the WW domains of KIBRA. We show that KIBRA binds to a WW domain binding motif, PPxY, in the tyrosine kinase receptor DDR1, and dissociates upon treatment with the DDR1 ligands collagen type I or IV. In addition we show that KIBRA and DDR1 also interact with PKCz to form a trimeric complex. Finally, overexpression and knockdown studies demonstrate that KIBRA promotes the collagen-stimulated activation of the MAPK cascade. Thus KIBRA may play a role in how the reproductive state influences the mammary epithelial cell to respond to changing cell-context information, such as experienced during the tissue remodeling events of mammary gland development.

Mammary morphogenesis is orchestrated with other reproductive events by pituitary-driven changes to the systemic hormone environment, initiating the formation of a mammary ductal network during puberty and the addition of secretory alveoli during pregnancy. Prolactin is the major driver of development during pregnancy via regulation of ovarian progesterone production (in many species) and direct effects on mammary epithelial cells (in all species). Together these hormones regulate two aspects of development that are the subject of intense interest: (1) a genomic regulatory network that integrates many additional spatial and temporal cues to control gene expression and (2), the activity of a stem and progenitor cell hierarchy. Amalgamation of these two aspects will increase our understanding of cell proliferation and differentiation within the mammary gland, with clear application to our attempts to control breast cancer. Here we focus on providing an over-view of prolactin action during development of the model murine mammary gland.

Prolactin (PRL) is one of a family of related hormones including growth hormone (GH) and placental lactogen (PL) that are hypothesized to have arisen from a common ancestral gene about 500 million years ago. Over 300 different functions of PRL have been reported, highlighting the importance of this pituitary hormone. PRL is also synthesized by a number of extra-pituitary tissues including the mammary gland and the uterus. Most of PRL's actions are mediated by the unmodified 23 kDa peptide, however, PRL may be modified post-translation, thereby altering its biological effects. PRL exerts these effects by binding to its receptor, a member of the class I cytokine receptor super-family. This activates a number of signaling pathways resulting in the transcription of genes necessary for the tissue specific changes induced by PRL. Mouse knockout models of the major forms of the PRL receptor have confirmed the importance of PRLs role in reproduction. Further knockout models have provided insight into the importance of PRL signaling intermediates and the advent of transcript profiling has allowed the elucidation of a number of PRL target genes.

Cumulative exposure to estrogen (E) and progesterone (P) over the menstrual cycle significantly influences the risk of developing breast cancer. Despite the dogma that PR in the breast merely serves as a marker of an active estrogen receptor (ER), and as an inhibitor of the proliferative actions of E, it is now clear that in the breast P increases proliferation independently of E action. We show here that the progesterone receptor (PR) and ER are expressed in different epithelial populations, and target non-overlapping pathways in the normal human breast. In breast cancer, PR becomes highly correlated with ER, and this convergence is associated with signaling pathways predictive of disease metastasis. These data challenge the established paradigm that ER and PR function co-operatively in normal breast, and have significant implications not only for our understanding of normal breast biology, but also for diagnosis, prognosis and/or treatment options in breast cancer patients.

Outcomes have improved significantly for many women diagnosed with breast cancer. For the heterogeneous group of tumours lacking expression of the oestrogen, progesterone and HER2 receptors, 'triple negative' breast cancers (TNBC), the prognosis overall has remained quite poor. When TNBC recurs, there is often little response to chemotherapy, and there are a few treatment options in this setting. Thus, there is an urgent clinical need to identify new therapeutic targets in order to improve the outlook for these patients. This review highlights the most promising therapeutic targets identified through new sequencing technologies, as well as through studies of apoptosis. We also present mounting evidence that the developmental signalling pathways Wnt/β-catenin, NOTCH and Hedgehog play an important role in the pathogenesis and progression of TNBC with new therapeutic approaches inhibiting these pathways in advanced preclinical studies or early clinical trials.

The transcription factor ELF5 is responsible for gene expression patterning underlying molecular subtypes of breast cancer and may mediate acquired resistance to anti-estrogen therapy., We have previously shown that during pregnancy the E-twenty-six (ETS) transcription factor ELF5 directs the differentiation of mammary progenitor cells toward the estrogen receptor (ER)-negative and milk producing cell lineage, raising the possibility that ELF5 may suppress the estrogen sensitivity of breast cancers. To test this we constructed inducible models of ELF5 expression in ER positive luminal breast cancer cells and interrogated them using transcript profiling and chromatin immunoprecipitation of DNA followed by DNA sequencing (ChIP-Seq). ELF5 suppressed ER and FOXA1 expression and broadly suppressed ER-driven patterns of gene expression including sets of genes distinguishing the luminal molecular subtype. Direct transcriptional targets of ELF5, which included FOXA1, EGFR, and MYC, accurately classified a large cohort of breast cancers into their intrinsic molecular subtypes, predicted ER status with high precision, and defined groups with differential prognosis. Knockdown of ELF5 in basal breast cancer cell lines suppressed basal patterns of gene expression and produced a shift in molecular subtype toward the claudin-low and normal-like groups. Luminal breast cancer cells that acquired resistance to the antiestrogen Tamoxifen showed greatly elevated levels of ELF5 and its transcriptional signature, and became dependent on ELF5 for proliferation, compared to the parental cells. Thus ELF5 provides a key transcriptional determinant of breast cancer molecular subtype by suppression of estrogen sensitivity in luminal breast cancer cells and promotion of basal characteristics in basal breast cancer cells, an action that may be utilised to acquire antiestrogen resistance., Author Summary The molecular subtypes of breast cancer are distinguished by their intrinsic patterns of gene expression and can be used to group patients with different prognoses and treatment options. Although molecular subtyping tests are currently under evaluation, some of them are already in use to better tailor therapy for patients; however, the molecular events that are responsible for these different patterns of gene expression in breast cancer are largely undefined. The elucidation of their mechanistic basis would improve our understanding of the disease process and enhance the chances of developing better predictive and prognostic markers, new therapies, and interventions to overcome resistance to existing therapies. Here, we show that the transcription factor ELF5 is responsible for much of the patterning of gene expression that distinguishes the breast cancer subtypes. Additionally, our data suggest that ELF5 may also be involved in the development of resistance to therapies designed to stop estrogen stimulation of breast cancer. These effects of ELF5 appear to represent a partial carryover into breast cancer of its normal role in the mammary gland, where it is responsible for the development of milk-producing structures during pregnancy.

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.

During pregnancy, the ETS transcription factor ELF5 establishes the milk-secreting alveolar cell lineage by driving a cell fate decision of the mammary luminal progenitor cell. In breast cancer, ELF5 is a key transcriptional determinant of tumor subtype and has been implicated in the development of insensitivity to anti-estrogen therapy. In the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT) model of luminal breast cancer, induction of ELF5 levels increased leukocyte infiltration, angiogenesis, and blood vessel permeability in primary tumors and greatly increased the size and number of lung metastasis. Myeloid-derived suppressor cells, a group of immature neutrophils recently identified as mediators of vasculogenesis and metastasis, were recruited to the tumor in response to ELF5. Depletion of these cells using specific Ly6G antibodies prevented ELF5 from driving vasculogenesis and metastasis. Expression signatures in luminal A breast cancers indicated that increased myeloid cell invasion and inflammation were correlated with ELF5 expression, and increased ELF5 immunohistochemical staining predicted much shorter metastasis–free and overall survival of luminal A patients, defining a group who experienced unexpectedly early disease progression. Thus, in the MMTV-PyMT mouse mammary model, increased ELF5 levels drive metastasis by co-opting the innate immune system. As ELF5 has been previously implicated in the development of antiestrogen resistance, this finding implicates ELF5 as a defining factor in the acquisition of the key aspects of the lethal phenotype in luminal A breast cancer., Up-regulation of the transcription factor ELF5 in tumors helps to create a micro-environment that recruits the innate immune system and increases vascular permeability, leading to increased metastasis in luminal breast cancer. Together with its role in anti-estrogen resistance, this suggests that ELF5 is a major driver of a lethal phenotype., Author Summary The transcription factor Elf5 defines hormone-insensitive and endocrine-therapy–resistant breast cancer. In this study, we have discovered that ELF5 drives the spread of tumor cells to the lungs. We demonstrate that the underlying mechanism for this metastatic spread is via recruitment of the innate immune system. Interestingly, this effect is able to overcome the other tumor-suppressive effects of ELF5 on cancer cells, such as reduced proliferation, motility, and invasion. This important finding challenges the more conventional view that the most potent determinant of metastatic activity lies within the cancer cell. We clearly demonstrate that the innate immune system strongly influences the metastatic activity of cancer cells despite their cell-intrinsic spread potential. Our previous work demonstrated that in luminal breast cancer, ELF5 is a key determinant of antiestrogen therapy resistance. Here, we show that the metastatic mechanism driven by ELF5 is most important in luminal breast cancer patients, in whom higher ELF5 expression is associated with low presence of cytotoxic T lymphocytes, an immune cell population responsible for tumor rejection. Thus, we now see that ELF5 may be behind the two most important processes that cause luminal breast cancers to progress towards the lethal phenotype; resistance to antiestrogen therapy and the development of metastatic activity. This understanding could pave the way for new therapeutic strategies to be devised and new predictive tests to be developed.

Zardawi S J, Zardawi I, McNeil C M, Millar E K A, McLeod D, Morey A L, Crea P, Murphy N C, Pinese M, Lopez-Knowles E, Oakes S R, Ormandy C J, Qiu M R, Hamilton A, Spillane A, Soon Lee C, Sutherland R L, Musgrove E A & O’Toole S A (2010) Histopathology56, 286–296 High Notch1 protein expression is an early event in breast cancer development and is associated with the HER-2 molecular subtype Aims: Activation of Notch signalling results in hyperplasia and tumorigenesis in murine mammary epithelium. However, there is little information regarding the expression of Notch1 in premalignant lesions and early breast cancer. We investigated expression of Notch1 in breast cancer development and its association with molecular subtypes. Methods and results: Immunohistochemical expression of Notch1 was determined in a murine model of mammary carcinogenesis and in breast tissue from two cohorts of breast cancer patients, the first (n = 222) comprising a histological progression series and the second an outcome series of 228 patients with operable invasive ductal carcinoma. Enhanced expression of Notch1 protein was an early event in both murine and human breast cancer development with progressive increases in expression with the development of hyperplasia and malignancy. High Notch1 was not prognostic in the outcome cohort. There was, however, a highly significant association of high Notch1 protein with the HER-2 molecular subtype of breast cancer (P = 0.008). Conclusions: These data demonstrate that aberrant Notch regulation is an early event in mammary carcinogenesis and is associated with the HER-2 molecular subtype of breast cancer, and suggest the Notch signalling pathway may be a potential therapeutic target worthy of further investigation.

Massive tissue remodelling occurs within the mammary gland during pregnancy, resulting in the formation of lobuloalveoli that are capable of milk secretion. Endocrine signals generated predominantly by prolactin and progesterone operate the alveolar switch to initiate these developmental events. Here we review the current understanding of the components of the alveolar switch and conclude with an examination of the role of the ets transcription factor Elf5. We propose that Elf5 is a key regulator of the alveolar switch.

The proliferative phase of mammary alveolar morphogenesis is initiated during early pregnancy by rising levels of serum prolactin and progesterone, establishing a program of gene expression that is ultimately responsible for the development of the lobuloalveoli and the onset of lactation. To explore this largely unknown genetic program, we constructed transcript profiles derived from transplanted mammary glands formed by recombination of prolactin receptor (Prlr) knockout or wild-type mammary epithelium with wild-type mammary stroma. Comparison with profiles derived from prolactin-treated Scp2 mammary epithelial cells produced a small set of commonly prolactin-regulated genes that included the negative regulator of cytokine signaling, Socs2 (suppressor of cytokine signaling 2), and the ets transcription factor, E74-like factor 5 (Elf5). Homozygous null mutation of Socs2 rescued the failure of lactation and reduction of mammary signal transducer and activator of transcription 5 phosphorylation that characterizes Prlr heterozygous mice, demonstrating that mammary Socs2 is a key regulator of the prolactin-signaling pathway. Reexpression of Elf5 in Prlr nullizygous mammary epithelium restored lobuloalveolar development and milk production, demonstrating that Elf5 is a transcription factor capable of substituting for prolactin signaling. Thus, Socs2 and Elf5 are key members of the set of prolactin-regulated genes that mediate prolactin-driven mammary development.

The secretory activation stage of mammary gland development occurs after parturition and converts inactive lobuloalveoli to active milk secretion. This process is triggered by progestin withdrawal and depends upon augmented prolactin (Prl) signaling. Little is known about the Prl-induced transcriptional changes that occur in the mammary gland to drive this process. To examine changes in the mammary transcriptome responsible for secretory activation, we have used transcript profiling of three mouse models that exhibit failure of secretory activation: knockout of galanin (a regulator of pituitary Prl production and a mammary cell autonomous modulator of Prl action); treatment with S179D Prl (a phosphoprolactin mimic); and knockout of a single Prl receptor allele. A significant reduction in expression was observed in genes belonging to 46 gene ontologies including those representing milk proteins, metabolism, lipid, cholesterol and fatty acid biosynthetic enzymes, immune response, and key transcription factors. A set of 35 genes, commonly regulated in all three models, was identified and their role in lactogenesis was validated by examining their expression in response to Prl stimulation or signal transducer and activator of transcription 5 knockdown in the HC11 mouse mammary cell culture model. The transcript profiles provided by these experiments identify 35 key genes (many for the first time) involved in the secretory activation phase of mammary gland development, show that S179D acts as an antagonist of Prl action, and provide insight into the partial penetrance of failed lactation in Prl receptor heterozygous females.

Decreased expression of the cyclin-dependent kinase (CDK) inhibitor p27(Kip1) is common in breast cancer and is associated with poor prognosis. p27 is also an important mediator of steroidal regulation of cell cycle progression. We have therefore investigated the role of p27 in mammary epithelial cell proliferation. Examination of the two major functions of p27, assembly of cyclin D1-Cdk4 complexes and inhibition of Cdk2 activity, revealed that cyclin D1-Cdk4 complex formation was not impaired in p27−/− mammary epithelial cells in primary culture. However, cyclin E-Cdk2 activity was increased approximately 3-fold, indicating that the CDK inhibitory function of p27 is important in mammary epithelial cells. Increased epithelial DNA synthesis was observed during pregnancy in p27−/− mammary gland transplants, but this was paralleled by increased apoptosis. During pregnancy and at parturition, development and differentiation of p27+/+ and p27−/− mammary tissue were indistinguishable. These results demonstrate a role for p27 in both the proliferation and survival of mammary epithelial cells. However, the absence of morphological and cellular defects in p27−/− mammary tissue during pregnancy raises the possibility that loss of p27 in breast cancer may not confer an overall growth advantage unless apoptosis is also impaired.

Hyperprolactinemia results in prostatic hypertrophy and hyperplasia, but it is not known whether prolactin plays an essential role in these processes in the prostate. To address this question, we investigated prostate development, gene expression, and simian virus 40 (SV40)T-induced prostate carcinogenesis in prolactin receptor knockout mice. These animals showed a small increase in dorsolateral and ventral prostate weight but no change in the weight of the anterior prostate. The dorsal but not ventral or lateral lobes showed a 12% loss of epithelial cells; all other morphological parameters were normal. The area of SV40T-induced prostate intraepithelial neoplasia was reduced by 28% in the ventral lobe but not the dorsal lobe, and no tumors were seen in 20 prolactin receptor knockout animals, compared with 1 of 11 detected in wild-type and 4 of 21 found in heterozygous animals. Oligonucleotide microarrays were used to identify essential transcriptional roles of prolactin and revealed a small set of genes with decreased expression involved in sperm/oocyte interaction and copulatory plug formation. Infertility or reduced fertility was apparent in these animals. These findings establish essential though subtle roles for prolactin in the regulation of prostate morphology, gene expression, SV40T-induced neoplasia, and reproductive function.

Hypothalamic neuropeptide Y (NPY) is a major central regulator of sexual behavior and reproductive functions. Intracerebroventricular (ICV) administration of NPY to sex steroid-primed ovariectomized (OVX) rats increases secretion of luteinizing hormone (LH), and stimulates secretion of gonadotropin releasing hormone (GnRH) from the median eminence in vitro, functions that probably mediate the preovulatory surge of LH release (Sabatino et al. 1990; Urban et al. 1996; Jain et al. 1999). In contrast, when centrally administered to sex steroid-deficient OVX rats, or to intact male and female rats, NPY markedly inhibits reproductive function (Clark et al. 1985; Reznikov and McCann 1993; Xu et al. 1993; Pierroz et al. 1996). This includes decreased GnRH receptor concentration in the pituitary gland, reduced pituitary weight, and decreased plasma concentrations of prolactin, LH, follicle stimulating hormone (FSH), and testosterone. Testicular and seminal vesicle or ovarian weights are also reduced, and sexual maturation and estrous cyclicity in female rats is disrupted, leading to drastic suppression of male and female copulatory behavior (Clark et al. 1985; Reznikov and McCann 1993; Xu et al. 1993; Pierroz et al. 1996). These inhibitory effects of NPY on reproductive function probably contribute to the decreased fertility observed under conditions of negative energy balance, such as food restriction, heavy exercise, lactation, and insulin-dependent diabetes mellitus, all of which are associated with elevated hypothalamic NPY expression (Aubert et al. 1998; Krysiak et al. 1999). In this way NPY coordinates energy availability with reproduction, inhibiting procreation during unfavorable metabolic conditions. Interestingly, obesity is also associated with reproductive defects and reduced fertility (Caprio et al. 2001). Genetically obese ob/ob mice lack functional leptin, the hormone produced mainly by white adipose tissue. This mutation not only leads to hyperphagia, massive obesity, and the associated hormonometabolic defects (hypercorticosteronemia, hyperinsulinemia, hyperglycemia, and insulin resistance), but also leads to infertility due to insufficient hypothalamo-pituitary-gonadal drive, underdevelopment of reproductive organs, and impaired spermatogenesis (Caprio et al. 2001). In ob/ob mice, the lack of leptin-mediated inhibition of NPY expression and secretion in the hypothalamus (Stephens et al. 1995; Schwartz et al. 1996; Widdowson and Wilding 2000) leads to chronically elevated hypothalamic NPY-ergic activity. This secondary effect of leptin deficiency contributes to many of the associated defects. Indeed, treatment of ob/ob mice with leptin reduces NPY mRNA expression and peptide levels in the hypothalamus, reduces the hyperphagic, obese phenotype, and also restores fertility of male and female mice by improved function of the hypothalamo-pituitary-gonadal axis (Stephens et al. 1995; Chehab et al. 1996; Mounzih et al. 1997). Importantly, food restriction that produced a degree of weight loss similar to that produced by leptin treatment did not reduce central NPY expression and did not restore fertility in ob/ob mice (Mounzih et al. 1997). Further evidence that elevated central NPY-ergic tonus mediates the pathology of leptin deficiency is that most of the defects of ob/ob mice, including infertility, are attenuated or normalized when crossed onto NPY knockout mice (Erickson et al. 1996). The various functions of NPY are mediated by the Y receptor gene family, consisting of at least five distinct members (Y1, Y2, Y4, Y5, and y6) (Blomqvist and Herzog 1997). In addition to their involvement in reproduction and energy homeostasis, these Y-receptors when activated by their ligands, NPY, peptide YY (PYY), and pancreatic polypeptide (PP), can also modulate other important physiological functions, including circadian rhythms, gastrointestinal motility, memory, anxiety, nociception, and blood pressure (Hokfelt et al. 1998; Gehlert 1999; Kalra et al. 1999). Messenger RNAs for Y1, Y2, and Y5 are widely distributed throughout the brain (Naveilhan et al. 1998; Parker and Herzog 1999). In contrast, the Y4 receptor is predominantly expressed in the periphery including tissues such as the pancreas, intestine, colon, heart, and liver (Bard et al. 1995; Lundell et al. 1995). However, significant amounts of Y4 mRNA and specific binding sites have also been found in key areas of the hypothalamus such as the paraventricular nucleus and in certain brainstem nuclei including the area postrema and the nucleus tractus solitarius (Parker and Herzog 1999; Larsen and Kristensen 2000). Y receptors show very low primary amino acid sequence identity, yet surprisingly exhibit very similar pharmacology, with NPY and PYY being equipotent at all receptor subtypes. PP has high affinity only for the Y4 receptor, and in some species it also has moderate affinity for the Y1 and Y5 receptors. Although NPY is known to be involved in numerous physiological and pathophysiological processes, the clarification of the functions of specific Y receptor subtypes has been severely hampered by the lack of subtype-selective agonists and antagonists. The functions of the Y4 receptor subtype and its high-affinity agonist PP are among the less well understood of the Y receptor family. However, it was recently demonstrated that ICV administration of the Y1 antagonist and Y4 agonist 1229U91 (Schober et al. 1998) to estrogen-primed OVX or intact male rats rapidly increased FSH and/or LH secretion (Jain et al. 1999; Raposinho et al. 2000). These effects were attributed to Y4 activation by pharmacological analysis of other partial Y4 agonists (Raposinho et al. 2000), and also because rat PP, which is specific for the rat Y4 receptor, induced a similar profile of LH secretion in estrogen-primed OVX rats (Jain et al. 1999). Since PP is not expressed within the brain and does not cross the blood–brain barrier, it is possible that instead NPY acts as a central, albeit lower-affinity ligand for the hypothalamic Y4 receptor when expression levels are sufficiently high to agonize this receptor. We therefore hypothesized that under conditions of high hypothalamic NPY levels, as in energy deficit or leptin-deficient obesity, NPY could modulate reproductive functions through activation of Y4 receptors. To test this hypothesis we generated Y4 receptor knockout mice and crossed them with the obese ob/ob mutant strain and analyzed the effects on energy homeostasis and fertility.

Background: Impairment of apoptosis is a hallmark of cancer and can result in resistance to chemotherapy. Tumor resistance to apoptosis is frequently acquired through deregulated expression of BCL-2 family members or inactivation of the p53 tumor suppressor pathway. Over-expression of the pro-survival protein BCL-2 is common in breast cancer (where it is readily detected by immunostaining), and has been shown to be an important prognostic marker. A potential role for BCL-2 as a therapeutic target in breast cancer, however, has not been explored. Recently, small molecules termed ‘BH3-mimetics’ have been developed that mimic the action of pro-apoptotic BH3-only proteins. These bind and neutralize pro-survival proteins including BCL-2. Here we have derived a panel of primary breast tumor xenografts (that include basal-like breast tumors) to study the efficacy of the BH3-mimetic ABT-737 combined with docetaxel in targeting BCL-2-positive breast cancer. Methods and Results: We first studied the expression of BCL-2, pro-survival family members BCL-XL and MCL-1, and the pro-apoptotic protein BIM in tissue microarrays of 197 primary breast tumors, which were subtyped on the basis of ER, PR, HER2, CK5/6 and EGFR expression. BCL-2 was overexpressed in luminal (83.3%), HER2−positive (50.0%), basal-like (18.5%) and ‘marker-null’ (41.4%) breast cancers. BCL-2-positive tumors generally co-expressed BCL-XL (96.2%), MCL-1 (94.7%) and BIM (93.5%). To determine whether the BH3-mimetic ABT-737 (which neutralizes BCL-2, BCL-XL and BCL-W) was effective in targeting BCL-2-expressing breast tumors, we generated a panel of 28 primary breast tumor xenografts in immunocompromised NOG mice. Five xenograft lines (838T, 24T, 315T, 13T and 806T) were selected for further analysis. Four were basal-like, and one (315T) was a luminal B tumor, as determined by gene profiling. Mice bearing tumor xenografts were treated with ABT-737 (50 mg/kg i.p. d1-10), docetaxel (10 mg/kg i.p. d1) or a combination in q21d cycles. Tumor response and overall survival were significantly improved by combination therapy, but only for tumors that expressed elevated levels of BCL-2. Treatment with ABT-737 alone was ineffective, suggesting that ABT-737 sensitized tumors to docetaxel. Combination therapy was accompanied by a marked increase in apoptosis and dissociation of BIM from BCL-2, indicating that a perturbation of BIM complexes may contribute to the activation of the apoptotic cascade. Notably, ABT-737 appeared effective in targeting BCL-2-expressing basal-like tumor xenografts (838T and 24T) harboring p53 mutations. Discussion: Primary breast tumor xenograft models that recapitulate the phenotype of the primary tumor have been developed as useful ‘proof-of-principle', pre-clinical models. Here we provide the first in vivo evidence that BH3-mimetics can be used to sensitize primary BCL-2-expressing breast tumors to taxane chemotherapy. Our results suggest that elevated BCL-2 expression constitutes a predictive response marker in breast cancer. These findings provide a rationale for the development of clinical protocols using the oral analogue ABT-263 (navitoclax) as an adjunct to taxane chemotherapy in BCL-2-expressing basal-like and luminal breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD08-02.

Abstract #24 Background: The Hedgehog (Hh) signaling pathway is a highly conserved system which regulates development and cell fate. It also contributes to the pathogenesis of a number of human malignancies, including basal cell, prostate and pancreatic carcinoma as well as medulloblastoma. There is emerging evidence that Hh may play an important role in breast cancer. Methods: We examined the immunohistochemical expression of key Hh proteins, sonic hedgehog (Shh), Patched (Ptch) and Gli1 in 3 cohorts of patients; 2 independent cohorts of the histological progression model of breast cancer and a cohort of 292 patients with a median follow up of 5.5 years. We also investigated expression of Shh in the C3/SV40T mouse model of mammary carcinogenesis. We then investigated the effects of recombinant Shh in the MCF10A in vitro model of early breast cancer development. Finally, a mammary reconstitution graft model was employed where donor mammary epithelial cells were briefly cultured and transduced with a retroviral construct carrying a Shh-GFP construct, then transplanted into the cleared mammary fat pad of a recipient mouse. Results: Overexpression of Shh was an early event in human premalignant lesions, with progressive increases (p Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 24.

Impairment of apoptosis is a hallmark of cancer and can result in resistance to chemotherapy. Tumour resistance to apoptosis is frequently acquired through deregulated expression of BCL-2 family members or inactivation of the p53 tumour suppressor pathway. Over-expression of the pro-survival protein BCL-2 is common in breast cancer (where it is readily detected by immunostaining), and has been shown to be an important prognostic marker. A potential role for BCL-2 as a therapeutic target in breast cancer, however, has not been explored. Recently, small molecules termed ‘BH3-mimetics’ have been developed that mimic the action of pro-apoptotic BH3-only proteins. These bind and neutralize pro-survival proteins including BCL-2. Tissue microarrays containing 197 primary breast tumours were evaluated for the expression of BCL-2, its anti-apoptotic relatives MCL-1 and BCL-XL, and the pro-apoptotic BH3-only ligand BIM. These proteins were co-expressed at relatively high levels in a substantial proportion of heterogeneous breast tumours, 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 tumours, we generated a panel of primary breast tumour xenografts in immunocompromised mice and treated recipients with either ABT-737, docetaxel or a combination. Tumour response and overall survival were significantly improved by combination therapy, but only for tumour xenografts that expressed elevated levels of BCL-2. Treatment with ABT-737 alone was ineffective, suggesting that ABT-737 sensitizes the tumour 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. In summary, primary breast tumour xenograft models that recapitulate the phenotype of the primary tumour have been developed as useful ‘proof-of-principle’, pre-clinical models. Our findings provide the first in vivo evidence that BH3-mimetics can be used to sensitize primary BCL-2-expressing breast tumours to taxane chemotherapy. Our results suggest that elevated BCL-2 expression constitutes a predictive response marker in breast cancer. These findings provide a rationale for the development of clinical protocols using the oral analogue ABT-263 (navitoclax) as an adjunct to taxane chemotherapy in BCL-2-expressing basal-like and luminal breast cancer.