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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.

As part of the Reproducibility Project: Cancer Biology we published a Registered Report (Khan et al., 2015), that described how we intended to replicate selected experiments from the paper "A coding-independent function of gene and pseudogene mRNAs regulates tumour biology" (Poliseno et al., 2010). Here we report the results. We found PTEN depletion in the prostate cancer cell line DU145 did not detectably impact expression of the corresponding pseudogene PTENP1. Similarly, depletion of PTENP1 did not impact PTEN mRNA levels. The original study reported PTEN or PTENP1 depletion statistically reduced the corresponding pseudogene or gene (Figure 2G; Poliseno et al., 2010). PTEN and/or PTENP1 depletion in DU145 cells decreased PTEN protein expression, which was similar to the original study (Figure 2H; Poliseno et al., 2010). Further, depletion of PTEN and/or PTENP1 increased DU145 proliferation compared to non-targeting siRNA, which was in the same direction as the original study (Figure 2F; Poliseno et al., 2010), but not statistically significant. We found PTEN 3'UTR overexpression in DU145 cells did not impact PTENP1 expression, while the original study reported PTEN 3'UTR increased PTENP1 levels (Figure 4A; Poliseno et al., 2010). Overexpression of PTEN 3'UTR also statistically decreased DU145 proliferation compared to controls, which was similar to the findings reported in the original study (Figure 4A; Poliseno et al., 2010). Differences between the original study and this replication attempt, such as level of knockdown efficiency and cellular confluence, are factors that might have influenced the results. Finally, where possible, 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.

As part of the Reproducibility Project: Cancer Biology we published a Registered Report (Evans et al., 2015), that described how we intended to replicate selected experiments from the paper ‘Wnt activity defines colon cancer stem cells and is regulated by the microenvironment’ (Vermeulen et al., 2010). Here, we report the results. Using three independent primary spheroidal colon cancer cultures that expressed a Wnt reporter construct we observed high Wnt activity was associated with the cell surface markers CD133, CD166, and CD29, but not CD24 and CD44, while the original study found all five markers were correlated with high Wnt activity (Figure 2F; Vermeulen et al., 2010). Clonogenicity was highest in cells with high Wnt activity and clonogenic potential of cells with low Wnt activity were increased by myofibroblast-secreted factors, including HGF. While the effects were in the same direction as the original study (Figure 6D; Vermeulen et al., 2010) whether statistical significance was reached among the different conditions varied. When tested in vivo, we did not find a difference in tumorigenicity between high and low Wnt activity, while the original study found cells with high Wnt activity were more effective in inducing tumors (Figure 7E; Vermeulen et al., 2010). Tumorigenicity, however, was increased with myofibroblast-secreted factors, which was in the same direction as the original study (Figure 7E; Vermeulen et al., 2010), but not statistically significant. Finally, we report meta-analyses for each results where possible.

As part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Li et al., 2015), that described how we intended to replicate selected experiments from the paper ‘The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44’ (Liu et al., 2011). Here we report the results. We found the microRNA, miR-34a, was expressed at twice the level in CD44+ prostate cancer cells purified from xenograft tumors (LAPC4 cells) compared to CD44- LAPC4 cells, whereas the original study reported miR-34a was underexpressed in CD44+ LAPC4 cells (Figure 1B; Liu et al., 2011). When LAPC4 cells engineered to express miR-34a were injected into mice, we did not observe changes in tumor growth or CD44 expression; however, unexpectedly miR-34a expression was lost in vivo. In the original study, LAPC4 cells expressing miR-34a had a statistically significant reduction in tumor regeneration and reduced CD44 expression compared to control (Figure 4A and Supplemental Figures 4A,B and 5C; Liu et al., 2011). Furthermore, when we tested if miR-34a regulated CD44 through binding sites in the 3’UTR we did not find a statistically significant difference, whereas the original study reported miR-34a decreased CD44 expression that was partially abrogated by mutation of the binding sites in the CD44 3’UTR (Figure 4D; Liu et al., 2011). Finally, where possible, we report meta-analyses for each result.

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.

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 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.

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