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The study of early stage and metastatic breast cancer relies heavily on the use of established cell lines often derived from metastatic lesions rather than from primary tumors, limiting our understanding of primary tumor development. Study of the mechanisms governing the metastatic process in its entirety is not always feasible with these types of cellular and xenograft models, as often the primary tumor needs to be excised, or breast cancer cells are introduced into circulation of host mice. While this approach has allowed a better understanding of metastasis, it may unintentionally enhance the colonization efficiency and pass over important events occurring in the tumor microenvironment. In order to provide a cellular model that more closely recapitulates breast cancer development and progression, we generated breast cancer cellular cultures by dissociating specimens of human estrogen receptor-negative primary breast tumors and placing them in culture. Within our group of dissociated tumor (DT) cell cultures, we had shown that five cultures are formed by breast cancer cells and these were classified by PAM50 predictor analysis as belonging to the ER−/PR−/Her2+ and triple negative subtypes. DT cells are tumorigenic in NOD/Scid and NOD/Scid gamma (NSG) mice. Our first aim was to assess the metastatic potential of the DT cells in two mouse models. We show that 3 of the 5 cultures are metastatic to lymph nodes, liver and or lungs; common sites of metastasis in breast cancer patients. Spontaneous metastases were observed without the requirement of primary tumor excision. The metastatic frequency ranged from 25–90% in NOD/Scid mice, and from 80–100% in NSG mice. Moreover, expression of key proteins such as epidermal growth factor receptor (EGFR) detected in primary tumors was also found in the metastatic lesions. Because the DT cells have been isolated from primary breast tumors they represent a clinically relevant and valuable model to study breast cancer and metastasis. A critical factor influencing tumor progression and metastasis is the infiltration of immune cells including myeloid-derived suppressor cells (MDSCs), which have been shown to be increased in a tumor-dependent manner in xenograft models of breast cancer. Previous work from our lab has shown that stroma of tumor-bearing mice exhibited gene expression changes, including increased expression of calcium binding proteins S100A8 and S100A9, that are consistent with myeloid immune cell infiltration. Our second aim was thus to analyze the recruitment of S100A8+ MDSCs by xenografted DT tumors. We show that the recruitment of S100A8+ MDSCs is detected in DT tumors and sites of metastasis, and more importantly, their presence was significantly greater in the tumors that metastasized compared to those that did not. These data strongly suggest that the metastatic potential of breast cancer cells may be driven at least in part by the recruitment of S100A8+ MDSCs. Taken together, our observations and our closer-to-primary xenograft models may lead to a better understanding of metastatic disease, as well as to the development and pre-clinical screening of targeted breast cancer therapeutics, particularly in the metastatic setting. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-12.

Therapies targeting the ERBB2 receptor, including the kinase inhibitor lapatinib (Tykerb, GlaxoSmithKline), have improved clinical outcome for women with ERBB2-amplified breast cancer. However, acquired resistance to lapatinib remains a significant clinical problem, and the mechanisms governing resistance remain poorly understood. We sought to define molecular alterations that confer an acquired lapatinib resistance phenotype in ER-/ERBB2+ human breast cancer cells. ERBB2-amplified SKBR3 breast cancer cells were rendered resistant to lapatinib via culture in increasing concentrations of the drug, and molecular changes associated with a resistant phenotype were interrogated using a collaborative enzyme-enhanced immunoassay platform and immunoblotting techniques for detection of phosphorylated signaling cascade proteins. Interestingly, despite apparent inactivation of the PI3K/AKT signaling pathway, resistant cells exhibited constitutive activation of mammalian target of rapamycin complex 1 (mTORC1) and were highly sensitive to mTOR inhibition with rapamycin and the dual PI3K/mTOR inhibitor NVP-BEZ235. These data demonstrate a role for downstream activation of mTORC1 in the absence of molecular alterations leading to PI3K/AKT hyperactivation as a potential mechanism of lapatinib resistance in this model of ERBB2+ breast cancer and support the rationale of combination or sequential therapy using ERBB2 and mTOR-targeting molecules to prevent or target resistance to lapatinib. Moreover, our data suggest that assessment of mTOR substrate phosphorylation (i.e., S6) may serve as a more robust biomarker to predict sensitivity to mTOR inhibitors in the context of lapatinib resistance than PI3K mutations, loss of PTEN and p-AKT levels.

The Prostate Cancer Foundation-Movember Foundation Reproducibility Initiative (PCFMFRI) seeks to address growing concerns about reproducibility in scientific research by conducting replications of recent papers in the field of prostate cancer. This Registered Report describes the proposed replication plan of key experiments from "The Androgen Receptor Induces a Distinct Transcriptional Program in Castration-Resistant Prostate Cancer in Man" by Sharma and colleagues (2013), published in Cancer Cell in 2013. Of thousands of targets for the androgen receptor (AR), the authors elucidated a subset of 16 core genes that were consistently downregulated with castration and re-emerged with castration resistance. These 16 AR binding sites were distinct from those observed in cells in culture. The authors suggested that cellular context can have dramatic effects on downstream transcriptional regulation of AR binding sites. The present study will attempt to replicate Fig. 7C by comparing gene expression of the 16 core genes identified by Sharma and colleagues in xenograft tumor tissue compared to androgen treated LNCaP cells in vitro. The Prostate Cancer Foundation-Movember Foundation Reproducibility Initiative is a collaboration between the Prostate Cancer Foundation, the Movember Initiative, and Science Exchange, and the results of the replications will be published by PeerJ.

The mechanisms by which breast cancer (BrC) can successfully metastasize are complex and not yet fully understood. Our goal was to identify tumor-induced stromal changes that influence metastatic cell behavior, and may serve as better targets for therapy. To identify stromal changes in cancer-bearing tissue, dual-species gene expression analysis was performed for three different metastatic BrC xenograft models. Results were confirmed by immunohistochemistry, flow cytometry, and protein knockdown. These results were validated in human clinical samples at the mRNA and protein level by retrospective analysis of cohorts of human BrC specimens. In pre-clinical models of BrC, systemic recruitment of S100A8+ myeloid cells-including myeloid-derived suppressor cells (MDSCs)-was promoted by tumor-derived factors. Recruitment of S100A8+ myeloid cells was diminished by inhibition of tumor-derived factors or depletion of MDSCs, resulting in fewer metastases and smaller primary tumors. Importantly, these MDSCs retain their ability to suppress T cell proliferation upon co-culture. Secretion of macrophage inhibitory factor (MIF) activated the recruitment of S100A8+ myeloid cells systemically. Inhibition of MIF, or depletion of MDSCs resulted in delayed tumor growth and lower metastatic burden. In human BrC specimens, increased mRNA and protein levels of S100A8+ infiltrating cells are highly associated with poor overall survival and shorter metastasis free survival of BrC patients, respectively. Furthermore, analysis of nine different human gene expression datasets confirms the association of increased levels of S100A8 transcripts with an increased risk of death. Recruitment of S100A8+ myeloid cells to primary tumors and secondary sites in xenograft models of BrC enhances cancer progression independent of their suppressive activity on T cells. In clinical samples, infiltrating S100A8+ cells are associated with poor overall survival. Targeting these molecules or associated pathways in cells of the tumor microenvironment may translate into novel therapeutic interventions and benefit patient outcome.

Recent attempts at replicating highly-cited peer-reviewed studies demonstrate that the “reproducibility crisis” is indeed upon us. However, punitive measures against individuals committing research misconduct are neither sufficient nor useful because this is a systemic issue stemming from a lack of positive incentive. As an alternative approach, here we propose a system of checks and balances for the publishing process that involves 1) technical review of methodology by publishers, and 2) incentivizing direct replication of key experimental results. Together, these actions will help restore the self-correcting nature of scientific discovery.

Breast cancer is the most common cancer in women, and this prevalence has a major impact on health worldwide. Localized breast cancer has an excellent prognosis, with a 5-year relative survival rate of 85%. However, the survival rate drops to only 23% for women with distant metastases. To date, the study of breast cancer metastasis has been hampered by a lack of reliable metastatic models. Here we describe a novel in vivo model using human breast cancer xenografts in NOD scid gamma (NSG) mice; in this model human breast cancer cells reliably metastasize to distant organs from primary tumors grown within the mammary fat pad. This model enables the study of the entire metastatic process from the proper anatomical site, providing an important new approach to examine the mechanisms underlying breast cancer metastasis. We used this model to identify gene expression changes that occur at metastatic sites relative to the primary mammary fat pad tumor. By comparing multiple metastatic sites and independent cell lines, we have identified several gene expression changes that may be important for tumor growth at distant sites.

The Prostate Cancer Foundation-Movember Foundation Reproducibility Initiative seeks to address growing concerns about reproducibility in scientific research by conducting replications of recent papers in the field of prostate cancer. This Registered Report describes the proposed replication plan of key experiments from "Androgen Receptor Splice Variants Determine Taxane Sensitivity in Prostate Cancer" by Thadani-Mulero and colleagues (2014) published in Cancer Research in 2014. The experiment that will be replicated is reported in Fig. 6A. Thadani-Mulero and colleagues generated xenografts from two prostate cancer cell lines; LuCaP 86.2, which expresses predominantly the ARv567 splice variant of the androgen receptor (AR), and LuCaP 23.1, which expresses the full length AR as well as the ARv7 variant. Treatment of the tumors with the taxane docetaxel showed that the drug inhibited tumor growth of the LuCaP 86.2 cells but not of the LuCaP 23.1 cells, indicating that expression of splice variants of the AR can affect sensitivity to docetaxel. The Prostate Cancer Foundation-Movember Foundation Reproducibility Initiative is a collaboration between the Prostate Cancer Foundation, the Movember Foundation and Science Exchange, and the results of the replications will be published by PeerJ.

The lack of reproducibility of preclinical research is a significant and growing problem which slows basic research and leads to fruitless clinical trials. An increasing number of reports have found discrepancies in published preclinical studies across scientific disciplines. For example: * Amgen found that 47 of 53 “landmark” oncology publications could not be reproduced. * Bayer found that their internal results contradicted academic publications in 43 of 67 oncology and cardiovascular projects. * Dr. John Ioannidis and his colleagues found that of 432 publications purporting sex differences in hypertension, multiple sclerosis, or lung cancer, only one data set was reproducible. These studies, and the many others that report similar results, highlight a significant problem in the development of new therapies to treat disease. With increasing reports of discrepancies in preclinical publications, pharmaceutical companies are being forced to re-evaluate their reliance on academic research. In fact, Bayer recently decided to halt nearly two-thirds of target-validation projects.In this session, we'll report the work we have done to address this issue. We will bring together researchers and representatives from funders and publishers to discuss the issue of reproducibility. We will share funder and publisher initiatives to promote reproducibility and also discuss research practices that lead to more reproducible research such as using proper statistical tests, reporting all experimental data, experimental blinding, and identification and validation of research reagents. We'll also share preliminary results from an Arnold Foundation-funded study to replicate the 50 most high impact cancer biology studies from 2010-1012. Reproducibility ResearchAmgen47 of 53 “landmark” oncology publications could not be reproducedBayerinternal results contradicted academic publications in 43 of 67 oncology and cardiovascular projectsDr. John Ioannidis and colleagues432 publications purporting sex differences in hypertension, multiple sclerosis, or lung cancer, only one data set was reproducible Note: This abstract was not presented at the meeting. Citation Format: Gunn William, Elizabeth Iorns, Elizabeth Silva, Brian Nosek. Preliminary results from the Reproducibility Project: Cancer Biology - a replication of 50 high-impact cancer cell biology papers from 2010-2012. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5053. doi:10.1158/1538-7445.AM2014-5053

Background SATB1 has been previously proposed as a key protein that controls the development and progression of breast cancer. We explored the potential of the SATB1 protein as a therapeutic target and prognostic marker for human breast cancer. Methods We used aggressive (MDA-MB-231 and BT549) and nonaggressive (SKBR3 and MCF7) breast cancer cell lines to investigate the potential of SATB1 as a therapeutic target. SATB1 mRNA expression was silenced in aggressive cells by use of short hairpin RNAs against SATB1. SATB1 was overexpressed in nonaggressive cells by use of SATB1 expression vectors. We assessed the effect of modifying SATB1 expression on the transformed phenotype by examining anchorage-independent cell proliferation, acinar morphology on matrigel, and migration by wound healing in cultured cells. We examined tumor formation and metastasis, respectively, by use of orthotopic mammary fat pad and tail vein xenograft mouse models (mice were used in groups of six, and in total, 96 mice were used). SATB1 mRNA expression was compared with outcome for patients with primary breast cancer from six previous microarray studies that included a total of 1170 patients. All statistical tests were two-sided. Results The transformed phenotype was not suppressed by SATB1 silencing in aggressive cells and was not enhanced by ectopic expression of SATB1 in nonaggressive cells. Modifying SATB1 expression did not alter anchorage-independent cell proliferation, invasive acinar morphology, or cell migration in cultured cells and did not affect tumor formation or metastasis in xenograft mouse models. In addition, SATB1 expression was not associated with decreased overall survival of patients with primary breast cancer in six previous independent microarray studies (overall odds ratio = 0.80, 95% confidence interval = 0.62 to 1.03, P = .10). Conclusion In contrast to previous studies, we found that SATB1 expression did not promote breast cancer progression and was not associated with breast cancer outcome.

Introduction: Scientific research is becoming increasingly specialized, necessitating greater use of collaborations and outsourcing to draw on experimental expertise. To facilitate this, many research institutions have established core facilities to help reduce the inefficiencies associated with the historical system of ‘bartering’ to gain access to specialized equipment and expertise. However, the current core facility system is fragmented: researchers have varying degrees of access to services, quality is hard to evaluate and pricing is not transparent. Researchers who need to collaborate outside of their institution lack an easy way to find providers, evaluate them, coordinate logistics and pay for work. Similarly, core facilities lack a robust mechanism for promoting their expertise and services to relevant researchers outside their institution. Methods: Science Exchange, an online marketplace accessible at www.ScienceExchange.com, was launched in August 2011 to address these issues. Science Exchange works by signing up core facilities as “providers” of different experiment types. Researchers can search for an experiment type they wish to outsource and choose a facility to perform the work, or post an open project and receive bids from qualified facilities. Science Exchange acts as a centralized hub for provider information and reviews, and assists with project management, including billing and payment. The goal of Science Exchange is to create an integrated and accessible marketplace for experimental services. Results: As of October 2011 more than 3,000 scientists from over 500 research institutions have registered with Science Exchange. The top 10 most searched for experiment types are, in descending order: DNA sequencing, Bioinformatics, Microarray, DNA analysis, electron microscopy, protein extraction/ purification, confocal microscopy, realtime qPCR, microRNA microarray, and NMR. On average, experiments outsourced through the platform have saved users 54% of the value of their experiment ($4,664 per experiment). Conclusions: Science Exchange has begun to address the barriers to effective experiment outsourcing. As with any marketplace, awareness and participation by key players is essential to increase utility. Experience also provides data necessary to make outsourcing as frictionless as possible for the particulars of each experiment type. Our team has an enduring commitment to making the platform as useful for researchers and providers as possible. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3986. doi:1538-7445.AM2012-3986

The full-length p185 ERBB2 (HER2) receptor tyrosine kinase is a validated target for breast cancer (BC) therapy. It is now appreciated that a portion of ERBB2+ BC expresses truncated isoforms of the receptor, including the membrane localized forms p95 and p110. Importantly, t-ERBB2s lack the extracellular domain epitope of trastuzumab and act as a resistance mechanism to therapy. Data from transgenic mouse models implicate expression of t-ERBB2s in tumor formation and metastasis. Furthermore, human clinical studies have revealed that expression of t-ERBB2s correlates with poor patient outcome, increased nodal involvement, and increased metastasis. In this study, we used partially transformed breast epithelial cells (HMLEs) to extend the studies of t-ERBB2 expression in human cells. Stable expression of cDNAs coding for p110 t-ERBB2 led to increased migration (79 cells/ well versus 39, p = 0.04), and invasion (583 cell/ well vs. 290) p = 0.03) of p110 versus p185 expressing HMLE cells, respectively. Importantly, expression of p110 t-ERBB2, but not other isoforms, led to xenograft formation by HMLE cells in NOD/SCID mice (p110, 10 of 12 possible xenografts formed, p185, no xenografts formed). Accurate clinical profiling of t-ERBB2 expression may aid in selection of ERBB2-targeted therapy. To this end, we used a novel proximity-mediated immunoassay (CoPIA) to detect and quantify t-ERBB2 receptors in patient samples. Analysis of a cohort of 74 primary breast tumors revealed that 52% of breast tumor samples with a high ERBB2-IHC score (3+) contained t-ERBB2, with 32% containing phosphorylated t-ERBB2. In several samples t-ERBB2 accounted for 10-20% of the total ERBB2 receptor population in tumor cells. Finally, fine-needle aspirates of ERBB2+ metastases also revealed expression and phosphorylation of t-ERBB2s. In addition to its role in prognosis and therapeutic guidance, p110 t-ERBB2 represents an attractive target for therapy. We have generated several mouse monoclonal antibodies targeting the N-termini of p110 t-ERBB2s. Binding of p110 on the surface of living cells was assayed by flow cytometry, and the biological activity of these antibodies is currently being assessed in vitro and in vivo. Together, these data imply a functional role for the p110 t-ERBB2 isoform in tumorigenesis, invasion, and migration. Furthermore, expression of t-ERBB2 in the majority of IHC 3+ tumor samples reveals a significant incidence of t-ERBB2 in human cancers. Targeting of p110 t-ERBB2 may allow for novel therapeutic intervention in ERBB2+ primary and metastatic breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1211. doi:10.1158/1538-7445.AM2011-1211