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Supplementary Figures 1-7 from FGFR1 Amplification Drives Endocrine Therapy Resistance and Is a Therapeutic Target in Breast Cancer

Supplementary Methods, Figure Legends 1-7 from FGFR1 Amplification Drives Endocrine Therapy Resistance and Is a Therapeutic Target in Breast Cancer

As part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Bhargava et al., 2016) that described how we intended to replicate selected experiments from the paper “RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth” (Hatzivassiliou et al., 2010). Here we report the results. We found two unrelated RAF inhibitors, PLX4720 or GDC-0879, selectively inhibited BRAF(V600E) cell proliferation, while the MEK inhibitor, PD0325901, inhibited BRAF(V600E), wild-type RAF/RAS, and mutant RAS cancer cell proliferation, similar to the original study (Figure 1A; Hatzivassiliou et al., 2010). We found knockdown of CRAF, but not BRAF, in mutant RAS cells attenuated the phospho-MEK induction observed after PLX4720 treatment, similar to the original study (Figure 2B; Hatzivassiliou et al., 2010). The original study reported analogous results with GDC-0879, which was not observed in this replication, although unexpected control results confound the interpretation. We also attempted a replication of an assay with recombinant proteins to test the differential effect of RAF inhibitors on BRAF-CRAF heterodimerization (Figure 4A; Hatzivassiliou et al., 2010). Although we were unable to conduct the experiment as planned, we observed differential binding of BRAF by RAF inhibitors; however, it was between BRAF and beads, independent of CRAF. While these data were unable to address whether, under the conditions of the original study, the same observations could be observed, we discuss key differences between the original study and this replication that are important to consider for further experiments. Finally, where possible, we report meta-analyses for each result.

As part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Phelps et al., 2016) that described how we intended to replicate selected experiments from the paper ‘Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs’ (Tay et al., 2011). Here, we report the results. We found depletion of putative PTEN competing endogenous mRNAs (ceRNAs) in DU145 cells did not impact PTEN 3’UTR regulation using a reporter, while the original study reported decreased activity when SERINC1, VAPA, and CNOT6L were depleted (Figure 3C; Tay et al., 2011). Using the same reporter, we found decreased activity when ceRNA 3’UTRs were overexpressed, while the original study reported increased activity (Figure 3D; Tay et al., 2011). In HCT116 cells, ceRNA depletion resulted in decreased PTEN protein levels, a result similar to the findings reported in the original study (Figure 3G,H; Tay et al., 2011); however, while the original study reported an attenuated ceRNA effect in microRNA deficient (DicerEx5) HCT116 cells, we observed increased PTEN protein levels. Further, we found depletion of the ceRNAs VAPA or CNOT6L did not statistically impact DU145, wild-type HCT116, or DicerEx5 HCT116 cell proliferation. The original study reported increased DU145 and wild-type HCT116 cell proliferation when these ceRNAs were depleted, which was attenuated in the DicerEx5 HCT116 cells (Figure 5B; Tay et al., 2011). Differences between the original study and this replication attempt, such as variance between biological repeats, are factors that might have influenced the results. Finally, we report meta-analyses for each result.

BACKGROUND: Metastasis represents a critical turning point from curable to incurable breast cancer. Alterations in gene expression underlie aspects of this transition and are coordinated both genetically and epigenetically. HYPOTHESIS: Epigenetically controlled microRNA-mRNA regulatory networks underlie the ability of breast cancer cells to metastasize and acquire treatment resistance. METHODS: Human MDA-MB-231 and MDA-MB-436 breast cancer xenograft primary tumors and corresponding lymph node, liver, lung and diaphragm metastases were generated by inoculation of cell lines into the mammary fat pad of NSG mice. Human-specific gene expression profiling was performed using microarrays with qPCR validation. MicroRNA targets were validated by 3'UTR mutagenesis-luciferase assays. Lentiviral shRNA and miRNA mimic constructs were stably transduced into the same cell lines and growth properties were assessed in vitro and in vivo. Mice were euthanized when tumors achieved 10% of body weight or at day 100, whichever came first. RESULTS: A common set of 18 mRNAs was differentially regulated at all 4 metastatic sites relative to primary tumors. Of these, 17 were downregulated, and 13 of these were strikingly enriched for microRNA binding sites. In the same tissues, we identified 21 microRNAs that underwent downregulation during primary tumor growth relative to parental cell cultures, but were highly upregulated at all 4 metastatic sites. 18 of these had at least one target among the downregulated genes, and 6 had multiple targets in the same 13 mRNA 3' UTRs. One, miR-203, previously described as a tumor suppressor, directly targeted two of the repressed genes, TWF1 and APBB2. MiR-203 levels positively correlated with primary tumor size in METABRIC (p=0.0096). MiR-203 overexpression blunted in vitro tumorigenic properties, modestly reduced primary tumor growth rates, and prevented metastasis formation. Remarkably, knockdown of miR-203 also reversed multiple tumorigenic properties of both cell lines, including proliferation, migration, and growth in soft agar, and conferred a fibroblastic morphology on MDA-MB-231 cells. In addition, primary miR-203KD xenograft tumors grew extremely slowly and underwent partial involution between days 80-100. Residual miR-203KD tumors and metastases at day 100 demonstrated re-gain of miR-203 expression through loss of the shRNA silencing vector. CONCLUSIONS: A small group of microRNAs displays biphasic expression during cancer growth. At least one of these, microRNA-203, is required for metastatic growth, but also inhibits metastasis formation when expressed at high levels. Our findings suggest opposing roles for the same microRNA at different stages of breast cancer progression, and support the existence of dynamic, context-sensitive epigenetic mechanisms that adapt breast cancer cells to thrive at remote sites. These mechanisms may serve as targets for intervention during the evolution of metastatic disease. Citation Format: Bishopric NH, Speransky S, Kajan D, Laderian B, Iorns E, Clarke J, Lippman ME. Dynamic regulation of a microRNA-mRNA network during breast cancer metastasis reveals an essential tumor-promoting role for miR-203 [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-07-03.

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.

Cancer is caused by mutations in oncogenes and tumor suppressor genes, resulting in the deregulation of processes fundamental to the normal behavior of cells. The identification and characterization of oncogenes and tumor suppressors has led to new treatment strategies that have significantly improved cancer outcome. The advent of next generation sequencing has allowed the elucidation of the fine structure of cancer genomes, however, the identification of pathogenic changes is complicated by the inherent genomic instability of cancer cells. Therefore, functional approaches for the identification of novel genes involved in the initiation and development of tumors are critical. Here we report the first whole human genome in vivo RNA interference screen to identify functionally important tumor suppressor genes. Using our novel approach, we identify previously validated tumor suppressor genes including TP53 and MNT, as well as several novel candidate tumor suppressor genes including leukemia inhibitory factor receptor (LIFR). We show that LIFR is a key novel tumor suppressor, whose deregulation may drive the transformation of a significant proportion of human breast cancers. These results demonstrate the power of genome wide in vivo RNAi screens as a method for identifying novel genes regulating tumorigenesis.

Background: Overexpression of the epidermal growth factor receptor ERBB2 (HER2) is found in 20% of human breast cancers. Therapies targeting ERBB2 including trastuzumab and lapatinib have significantly improved the outlook for women with ERBB2+ breast cancer. However, resistance to these agents occurs frequently and remains a significant clinical problem. In the case of lapatinib resistance, the mechanism(s) of resistance remain poorly understood, since the current proposed rationale thought to limit lapatinib's anti-tumor effects has been difficult to reconcile with clinical data. Therefore, we hypothesize that novel mechanisms of resistance could be identified by mapping genomic variations in ERBB2+ cells with acquired resistance to lapatinib. The identification of such mutations may provide insights into mechanisms of resistance and may indicate therapeutic strategies to overcome lapatinib resistance in ERBB2+ breast cancer. Material and Methods: SKBR3 breast cancer cells resistant to lapatinib were generated through serial passage by exposure of drug sensitive parental SKBR3 cells to increasing concentrations of lapatinib up to the peak plasma concentration observed in human subjects (2.6 uM (SK-lapR)). Multiple signaling pathways in lapatinib sensitive and resistant cells were interrogated by Reverse Phase Protein Array (RPPA) and western blot analysis. To identify genome wide somatic mutations, the Exome of lapatinib resistant and sensitive SKBR3 cells was sequenced utilizing next generation deep sequencing. Following exclusion of germline variants, the acquired gene mutations in lapatinib resistant SKBR3 cells were confirmed by DNA re-sequencing of PCR amplified DNA segments. Results and Discussion: Analysis of activated signaling pathways in lapatinib resistant and sensitive SKBR3 cells did not confirm any of the previously proposed mechanisms of resistance. In particular, these cells show no activation of AKT or alternative receptor tyrosine kinases such as IGF-IR, ERBB3 or c-Met. However they exhibit sustained activation of mTORC1 and ERK1/2, as well as phosphorylation of STAT3, STAT5, rpS6 and CREB. Initial sequence analysis of exome and transcriptome reveals the presence of 76 single nucleotide variants/Indels differing between sensitive and resistant cells with 34/76 validated as true mutations present in the genome of lapatinib resistant SKBR3 cells, including mutations in LATS2, MAP3K5, SMAD3 and PDGFRA. This is the first exome sequence analysis to be reported which defines a drug resistant phenotype in ERBB2+ breast cancer. Ongoing work includes investigation of mutations as drug resistance mediators and analysis of copy number variations and gene fusions/translocations to systematically search for molecular alterations, with the goal of providing a rationale for the design of new combination therapies aimed at lapatinib resistance for ERBB2+ breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD01-09.

Identifying the gene expression alterations that occur in both the tumor and stroma is essential to understanding tumor biology. We have developed a dual-species microarray analysis method that allows the dissection of both tumor and stromal gene expression profiles from xenograft models, based on limited interspecies cross-hybridization on Illumina gene expression beadchips. This methodology allows for simultaneous genome-wide analysis of gene expression profiles of both tumor cells and the associated stromal tissue.

In 2015, as part of the Prostate Cancer Foundation–Movember Foundation Reproducibility Initiative, we published a Registered Report (Shan et al., 2015) that described how we intended to replicate selected experiments from the paper “Androgen Receptor Splice Variants Determine Taxane Sensitivity in Prostate Cancer” (Thadani-Mulero et al., 2014). Here we report the results of those experiments. Growth of tumor xenografts from two prostate cancer xenograft lines, LuCaP 86.2, which expresses wild-type androgen receptor (AR) and AR variant 567, and LuCaP 23.1, which expresses wild-type AR and AR variant 7, were not affected by docetaxel treatment. The LuCaP 23.1 tumor xenografts grew slower than in the original study. This result is different from the original study, which reported significant reduction of tumor growth in the LuCaP 86.2. Furthermore, we were unable to detect ARv7 in the LuCaP 23.1, although we used the antibody as stated in the original study and ensured that it was detecting ARv7 via a known positive control (22rv1, Hörnberg et al., 2011). Finally, we report a meta-analysis of the result.

Tamoxifen is the most commonly used drug to treat breast cancer and acts by blocking ERα (oestrogen receptor α) signalling. Although highly effective, its usefulness is limited by the development of resistance. Given this, strategies that limit resistance by sensitizing cells to tamoxifen may be of use in the clinic. To gain insight into how this might be achieved, we used chemical and genetic screens to identify targets and small-molecule inhibitors that cause tamoxifen sensitization. A high-throughput genetic screen, using an RNA interference library targeting 779 kinases and related proteins, identified the PDK1 (phosphoinositide-dependent kinase 1) signalling pathway as a strong determinant of sensitivity to multiple ERα antagonists, including tamoxifen. A chemical screen using existing drugs and known kinase inhibitors also identified inhibitors of the PDK1 pathway, including triciribine and tetrandrine. Aside from identifying novel agents and targets for tamoxifen sensitization, this approach also provides evidence that performing chemical and genetic screens in parallel may be useful.

Drugs that target the reliance of tumor cells upon estrogen signalling have revolutionised the treatment of breast cancer. Despite this, resistance to these endocrine therapies limits their utility. While the study of individual genes has contributed greatly to understanding drug resistance, relatively unbiased screening approaches may also be illuminating. The results of a high-throughput RNA interference screen identifying novel determinants of tamoxifen resistance support this conjecture and demonstrate that such approaches can identify clinically relevant genes, such as CDK10.

SummaryTherapies that target estrogen signaling have transformed the treatment of breast cancer. However, the effectiveness of these agents is limited by the development of resistance. Here, an RNAi screen was used to identify modifiers of tamoxifen sensitivity. We demonstrate that CDK10 is an important determinant of resistance to endocrine therapies and show that CDK10 silencing increases ETS2-driven transcription of c-RAF, resulting in MAPK pathway activation and loss of tumor cell reliance upon estrogen signaling. Patients with ERα-positive tumors that express low levels of CDK10 relapse early on tamoxifen, demonstrating the clinical significance of these observations. The association of low levels of CDK10 with methylation of the CDK10 promoter suggests a mechanism by which CDK10 expression is reduced in tumors.

With the development of RNA interference (RNAi) libraries, systematic and cost-effective genome-wide loss-of-function screens can now be carried out with the aim of assessing the role of specific genes in neoplastic phenotypes, and the rapid identification of novel drug targets. Here, we discuss the existing applications of RNAi in cancer drug discovery and highlight areas in this process that may benefit from this technology in the future.

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.

Ribonucleic acid interference (RNAi) screening has emerged as an indispensable genetic research tool, allowing determination of phenotypic effects after silencing entire suites of genes. As the catalog of fully sequenced genomes and transcriptomes grows, production of small interfering/short-hairpin RNA libraries that target every gene in a particular cell, tissue, or organism is achievable, allowing high-throughput “genome-wide” RNAi screening. This technology has been embraced by cancer biologists and has been used to analyze a myriad of phenotypic effects of genetic loss of function in human cancers.

Truncated-ERBB2 isoforms (t-ERBB2s), resulting from receptor proteolysis or alternative translation of the ERBB2 mRNA, exist in a subset of human breast tumors. t-ERBB2s lack the receptor extracellular domain targeted by therapeutic anti-ERBB2 antibodies and antibody–drug conjugates, including trastuzumab, trastuzumab-DM1 and pertuzumab. In clinical studies, expression of t-ERBB2 in breast tumors correlates with metastasis as well as trastuzumab resistance. By using a novel immuno-microarray method, we detect a significant t-ERBB2 fraction in 18 of 31 (58%) of immunohistochemistry (IHC)3+ ERBB2+ human tumor specimens, and further show that t-ERBB2 isoforms are phosphorylated in a subset of IHC3+ samples (10 of 31, 32%). We investigated t-ERBB2 biological activity via engineered expression of full-length and truncated ERBB2 isoforms in human mammary epithelial cells (HMECs), including HMEC and MCF10A cells. Expression of p110 t-ERBB2, but not p95m (m=membrane, also 648CTF) or intracellular ERBB2s, significantly enhanced cell migration and invasion in multiple cell types. In addition, only expression of the p110 isoform led to human breast epithelial cell (HMLE) xenograft formation in vivo. Expression of t-ERBB2s did not result in hyperactivation of the phosphoinositide kinase-3/AKT or mitogen-activated protein kinase signaling pathways in these cells; rather, phosphoproteomic array profiling revealed attenuation of phosphorylated signal transducer and activator of transcription 5 (STAT5) in p110-t-ERBB2-expressing cells compared to controls. Short hairpin-mediated silencing of STAT5 phenocopied p110-t-ERBB2-driven cell migration and invasion, while expression of constitutively active STAT5 reversed these effects. Thus, we provide novel evidence that (1) expression of p110 t-ERBB2 is sufficient for full transformation of HMEC, yielding in vivo xenograft formation, and (2) truncated p110 t-ERBB2 expression is associated with decreased phosphorylation of STAT5.

Cancer is caused by mutations in oncogenes and tumor suppressor genes, resulting in the deregulation of processes fundamental to the normal behavior of cells. The identification and characterization of oncogenes and tumor suppressors has led to new treatment strategies that have significantly improved cancer outcome. The advent of next generation sequencing has allowed the elucidation of the fine structure of cancer genomes, however, the identification of pathogenic changes is complicated by the inherent genomic instability of cancer cells. Therefore, functional approaches for the identification of novel genes involved in the initiation and development of tumors are critical. During this research period we conducted the first whole human genome in vivo RNA interference screen to identify functionally important tumor suppressor genes. Using our novel approach, we identified previously validated tumor suppressor genes including TP53 and MNT, as well as several novel candidate tumor suppressor genes including leukemia inhibitory factor receptor (LIFR). These results demonstrate the power of genome wide in vivo RNAi screens as a method for identifying novel genes regulating tumorigenesis.

Amplification of fibroblast growth factor receptor 1 (FGFR1) occurs in ∼10% of breast cancers and is associated with poor prognosis. However, it is uncertain whether overexpression of FGFR1 is causally linked to the poor prognosis of amplified cancers. Here, we show that FGFR1 overexpression is robustly associated with FGFR1 amplification in two independent series of breast cancers. Breast cancer cell lines with FGFR1 overexpression and amplification show enhanced ligand-dependent signaling, with increased activation of the mitogen-activated protein kinase and phosphoinositide 3-kinase–AKT signaling pathways in response to FGF2, but also show basal ligand-independent signaling, and are dependent on FGFR signaling for anchorage-independent growth. FGFR1-amplified cell lines show resistance to 4-hydroxytamoxifen, which is reversed by small interfering RNA silencing of FGFR1, suggesting that FGFR1 overexpression also promotes endocrine therapy resistance. FGFR1 signaling suppresses progesterone receptor (PR) expression in vitro, and likewise, amplified cancers are frequently PR negative, identifying a potential biomarker for FGFR1 activity. Furthermore, we show that amplified cancers have a high proliferative rate assessed by Ki67 staining and that FGFR1 amplification is found in 16% to 27% of luminal B–type breast cancers. Our data suggest that amplification and overexpression of FGFR1 may be a major contributor to poor prognosis in luminal-type breast cancers, driving anchorage-independent proliferation and endocrine therapy resistance. Cancer Res; 70(5); 2085–94

Endocrine therapies, which inhibit estrogen receptor (ERα) signalling, are the most common and effective treatments for ERα positive breast cancer. However, the utility of these agents is limited by the frequent development of resistance. The precise mechanisms underlying endocrine therapy resistance remain incompletely understood. In our laboratory, an RNA interference (RNAi) screen was used to identify modifiers of sensitivity to the most commonly used endocrine therapy, tamoxifen. The cyclin-dependent kinase 10 (CDK10) gene was identified as an important determinant of resistance and the mechanism whereby this gene modulates sensitivity to tamoxifen was investigated further. Silencing of CDK10 gene expression was shown to activate the MAPK signalling pathway, circumventing the reliance of breast cancer cells upon estrogen signalling. Patients with ERα positive breast tumours that express low levels of CDK10 were shown to relapse early on tamoxifen and methylation of the CDK10 gene promoter was observed in a significant proportion of patients, suggesting a mechanism for loss of CDK10 expression in tamoxifen resistant tumours. By suppressing gene expression RNAi, to a certain extent, models the pharmacological inhibition of a target protein. We performed parallel small molecule screens alongside the RNAi screen to identify compounds that sensitise to tamoxifen. Both the RNAi and small molecule screens identified the PDK1 pathway as a potential target for sensitisation to inhibit the development of endocrine therapy resistance.

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.

More than half of biomedical research cannot be replicated. We urgently need to do something about it, says Elizabeth Iorns

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

Background: Truncated ERBB2 receptors are present in a subset of human ERBB2+ amplified/overexpressing breast tumors, and are associated with trastuzumab resistance, metastasis, and poor clinical prognosis. However, whether truncated ERBB2 receptors are drivers of metastasis has not been well defined. In this study, we examined effects of full-length (p185) and truncated (p110) ERBB2 on the migration and invasion of human mammary epithelial cells, including HMLE and MCF10A cells. Material and Methods: Recombinant p185 and p110 ERBB2 were stably expressed in human mammary epithelial cells (HMLE) and MCF10A cells via retroviral vector. Expression of comparable levels of p185 and p110 in cells was confirmed by western blot. The phosphorylation states of downstream signaling proteins including STAT5 were assayed via phosphoproteomics and Collaborative Enzyme Enhanced Reactive (CEER™) immunoassay. The effects of the p110 constructs on cell migration and invasion were investigated by transwell assays. shRNA-encoding lentivirus was used for specific silencing of STAT5b in HMLE cells, and STAT5b silencing was confirmed at the protein level using western blot. Results and Discussion: Expression of p110 ERBB2 increased cell migration (HMLE, p = 0.04; MCF10A, p< 0.01) and invasion (HMLE, p= 0.03) when compared to expression of p185. Furthermore, expression of p110 in HMLE cells was associated with reduced phosphorylation of STAT5b. shRNA mediated silencing of STAT5b was sufficient to increase the migration (p < 0.01) and invasion of HMLE cells, phenocopying the p110 driven effects on HMLE cells. In clinical studies, loss of activated STAT5 protein correlates with breast cancer progression and is a negative predictor of survival. By analyzing publicly available gene expression datasets, we found that STAT5b mRNA expression is also significantly decreased in breast cancer compared to normal breast tissues in several studies, as well as in ERBB2 amplified vs. nonamplified samples. To our knowledge, this is the first reported perturbation of STAT signaling by truncated ERBB2 receptor, and suggests a mechanism by which truncated p110 ERBB2 (CTF611) increases migration and invasion of breast epithelial cells. This study extends the available data regarding STAT5 loss in breast cancer progression. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-25.

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

Background: Cancer is caused by mutations in oncogenes and tumor suppressor genes resulting in the deregulation of processes fundamental to the normal behavior of cells. The identification and characterization of oncogenes and tumor suppressors has led to new treatment strategies that have significantly improved cancer outcome. The advent of next generation sequencing has allowed the elucidation of the fine structure of cancer genomes, however, the identification of pathogenic changes is complicated by the inherent genomic instability of cancer cells. Therefore, functional approaches for the identification of novel genes involved in the initiation and development of tumors are critical. Methods: In order to identify functionally important tumor suppressor genes we have conducted the first human whole genome in vivo RNA interference (RNAi) screen. Partially transformed human mammary epithelial cells (HMLEs), which do not form tumors in immunodeficient mice, were infected with the Expression Arrest™ GIPZ lentiviral shRNA library consisting of 62,000 shRNAs targeting the whole human genome, and injected into the mammary fat pad of immunodeficient mice. shRNAs that silenced tumor suppressor genes fully transformed the mammary epithelial cells resulting in tumor formation. Candidate tumor suppressor genes were identified by PCR amplification and sequencing of tumor integrated shRNAs. For validation, candidate tumor suppressor genes were silenced in HMLEs and ectopically expressed in fully transformed breast cancer cells. The effect of modifying gene expression on the transformed phenotype was assessed using soft agar colony formation assays. Clinical significance was determined by comparing expression in normal and cancerous human breast tissue using Oncomine Research. Results and Discussion: Using our novel approach, we identify previously validated tumor suppressor genes including TP53 and MNT, as well as several novel candidate tumor suppressor genes including leukemia inhibitory factor receptor (LIFR). Silencing LIFR expression with multiple shRNA constructs fully transformed human mammary epithelial cells resulting in enhanced colony formation in soft agar (P Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-05-02.

Background:The cancer stem cell model suggests that tumors arise from and are maintained by a relatively small proportion of cells, termed cancer stem cells. According to this model, only these cells have the capacity for self-renewal and the ability to repopulate the tumor bulk by giving rise to the phenotypically heterogeneous populations of cells that comprise the primary tumor. Evidence to support the cancer stem cell model has been provided by isolation of subpopulations of cells on the basis of differential expression of cell surface markers, followed by functional transplantation into animal models. These studies reveal that only a small subset of CD44+/CD24-/low cancer cells retain the ability to form new tumors after transplantation in immunodeficient mice, consistent with the cancer stem cell model. These results indicate that it may be necessary to target and eliminate cancer stem cells in order to eradicate tumors.Material and Methods:Previous studies have suggested that only breast stem cells have the capacity to proliferate in suspension as spherical colonies termed mammospheres, and that mammosphere derived breast cancer cells are CD44+/CD24-/low and able to form tumors that reproduce the heterogeneity of the original primary tumor when injected into the mammary fat pad of immunodeficient mice. This indicates that mammospheres are comprised of breast cancer stem cells. To identify and characterize breast cancer stem cells more completely, the mammosphere culture system was used to isolate and propagate breast cancer stem cells from three basal breast cancer cell lines (BT549, MDA231, MDA436) and three luminal breast cancer cell lines (MCF7, SKBR3, T47D). The tumorigenicity of the cultured mammospheres and parental cell lines was examined using dilution studies in immunodeficient mice and gene expression profiles were studied by microarray analysis.Results and Discussion:No significant differences in tumorigenicity were observed between breast cancer cells cultured as mammospheres versus standard culture conditions. Furthermore, no consistent differences in gene expression profiles were observed between mammosphere and parental cells across the panel of cancer cell lines. These results suggest that mammosphere culture is not capable of significantly enriching for a more tumorigenic breast cancer stem cell population from established breast cancer cell lines. This may indicate that cancer subpopulations that exist in primary tumors are lost during the long term culture of breast cancer cell lines and that these systems may not be a suitable model for breast cancer stem cell studies. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 502.

Background:Objective clinical response to trastuzumab monotherapy in erbB2-amplified first line metastatic breast cancer is 34% (Vogel, et al., JCO 20: 719-26, 2002). Amongst patients who respond, most develop resistance (defined by disease progression on trastuzumab). One proposed mechanism of trastuzumab resistance is proteolytic cleavage of erbB2 receptor from its full-length (p185) form into truncated, constitutively active p95. Increased expression of p95erbB2 correlates with increased nodal involvement and poor clinical outcome. Because p95erbB2 lacks the trastuzumab binding epitope, expression may designate patients who would be suitable for treatment with erbB2 kinase inhibitors.Materials and Methods:Recombinant p185erbB2 and p95erbB2 constructs were stably expressed in several cell types via retroviral vector. Additionally, an intracellular form of p95erbB2 that arises via alternative translation and an intracellular p95erbB2 construct containing two copies of a nuclear localization sequence were also expressed. Expression and proper subcellular localization of constructs were confirmed by cell fractionation, western blot analysis and confocal microscopy. Transformation of human mammary epithelial (HMEC) and NIH3T3 cells by p185erbB2 and p95erbB2 isoforms was evaluated by anchorage independent growth using a quantitative fluorescent soft agar assay, and effects on migration and invasion of these cells were investigated by wound-healing and transwell assays. Cells transfected with oncogenic Ras or empty vector were used as positive and negative controls in these experiments.Results and Discussion:Recombinant p185erbB2 and p95erbB2 constructs were stably expressed in HMEC and NIH3T3 cells, and were correctly directed to the cell membrane; nuclear targeted intracellular p95erbB2 was correctly localized to the cell nucleus. Both p185 erbB2 and membrane-bound truncated p95erbB2 were sufficient to transform HMEC cells as compared to empty vector control transfected cells [mean fluorescence intensity empty vector control 2480 ± 464 (1 standard deviation); mean fluorescence intensity p185erbB2 9208 ± 2528, p= 0.0106; mean fluorescence intensity p95erbB2 6615 ± 1588, p= 0.0124)] as was the positive control oncogenic Ras (mean fluorescence intensity 4350 ± 433, p=0.0069). Interestingly, nuclear-targeted p95erbB2 was also sufficient to transform HMEC cells (mean fluorescence intensity 6492 ± 818, p=0.0018). These data support the hypothesis that truncated p95erbB2 species may be major pathogenic drivers in erbB2-amplified cancers. P95erbB2 therefore represents an attractive target for diagnosis and treatment of erbB2+ breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3136.