Elevated insulin-like growth factor 1 receptor signaling induces antiestrogen resistance through the MAPK/ERK and PI3K/Akt signaling routes

INTRODUCTION: Insulin-like growth factor 1 (IGF-1) receptor (IGF-1R) is phosphorylated in all breast cancer subtypes. Past findings have shown that IGF-1R mediates antiestrogen resistance through cross-talk with estrogen receptor (ER) signaling and via its action upstream of the epidermal growth factor receptor and human epidermal growth factor receptor 2. Yet, the direct role of IGF-1R signaling itself in antiestrogen resistance remains obscure. In the present study, we sought to elucidate whether antiestrogen resistance is induced directly by IGF-1R signaling in response to its ligand IGF-1 stimulation.METHODS: A breast cancer cell line ectopically expressing human wild-type IGF-1R, MCF7/IGF-1R, was established by retroviral transduction and colony selection. Cellular antiestrogen sensitivity was evaluated under estrogen-depleted two-dimensional (2D) and 3D culture conditions. Functional activities of the key IGF-1R signaling components in antiestrogen resistance were assessed by specific kinase inhibitor compounds and small interfering RNA.RESULTS: Ectopic expression of IGF-1R in ER-positive MCF7 human breast cancer cells enhanced IGF-1R tyrosine kinase signaling in response to IGF-1 ligand stimulation. The elevated IGF-1R signaling rendered MCF7/IGF-1R cells highly resistant to the antiestrogens tamoxifen and fulvestrant. This antiestrogen-resistant phenotype involved mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol 3-kinase/protein kinase B pathways downstream of the IGF-1R signaling hub and was independent of ER signaling. Intriguingly, a MAPK/ERK-dependent agonistic behavior of tamoxifen at low doses was triggered in the presence of IGF-1, showing a mild promitogenic effect and increasing ER transcriptional activity.CONCLUSIONS: Our data provide evidence that the IGF-1/IGF-1R signaling axis may play a causal role in antiestrogen resistance of breast cancer cells, despite continuous suppression of ER transcriptional function by antiestrogens

Automated Whole Animal Bio-Imaging Assay for Human Cancer Dissemination

A quantitative bio-imaging platform is developed for analysis of human cancer dissemination in a short-term vertebrate xenotransplantation assay. Six days after implantation of cancer cells in zebrafish embryos, automated imaging in 96 well plates coupled to image analysis algorithms quantifies spreading throughout the host. Findings in this model correlate with behavior in long-term rodent xenograft models for panels of poorly- versus highly malignant cell lines derived from breast, colorectal, and prostate cancer. In addition, cancer cells with scattered mesenchymal characteristics show higher dissemination capacity than cell types with epithelial appearance. Moreover, RNA interference establishes the metastasis-suppressor role for E-cadherin in this model. This automated quantitative whole animal bio-imaging assay can serve as a first-line in vivo screening step in the anti-cancer drug target discovery pipeline

Quantitative phosphoproteomics to unravel the cellular response to chemical stressors with different modes of action

Damage to cellular macromolecules and organelles by chemical exposure evokes activation of various stress response pathways. To what extent different chemical stressors activate common and stressor-specific pathways is largely unknown. Here, we used quantitative phosphoproteomics to compare the signaling events induced by four stressors with different modes of action: the DNA damaging agent: cisplatin (CDDP), the topoisomerase II inhibitor: etoposide (ETO), the pro-oxidant: diethyl maleate (DEM) and the immunosuppressant: cyclosporine A (CsA) administered at an equitoxic dose to mouse embryonic stem cells. We observed major differences between the stressors in the number and identity of responsive phosphosites and the amplitude of phosphorylation. Kinase motif and pathway analyses indicated that the DNA damage response (DDR) activation by CDDP occurs predominantly through the replication-stress-related Atr kinase, whereas ETO triggers the DDR through Atr as well as the DNA double-strand-break-associated Atm kinase. CsA shares with ETO activation of CK2 kinase. Congruent with their known modes of action, CsA-mediated signaling is related to down-regulation of pathways that control hematopoietic differentiation and immunity, whereas oxidative stress is the most prominent initiator of DEM-modulated stress signaling. This study shows that even at equitoxic doses, different stressors induce distinctive and complex phosphorylation signaling cascades.Toxicolog

Current approaches and future role of high content imaging in safety sciences and drug discovery

High content imaging combines automated microscopy with image analysis approaches to simultaneously quantify multiple phenotypic and/or functional parameters in biological systems. The technology has become an important tool in the fields of safety sciences and drug discovery, because it can be used for mode-of-action identification, determination of hazard potency and the discovery of toxicity targets and biomarkers. In contrast to conventional biochemical endpoints, high content imaging provides insight into the spatial distribution and dynamics of responses in biological systems. This allows the identification of signaling pathways underlying cell defense, adaptation, toxicity and death. Therefore high content imaging is considered a promising technology to address the challenges for the Toxicity testing in the 21st century approach. Currently high content imaging technologies are frequently applied in academia for mechanistic toxicity studies and in pharmaceutical industry for the ranking and selection of lead drug compounds or to identify/confirm mechanisms underlying effects observed in vivo. A recent workshop gathered scientists working on high content imaging in academia, pharmaceutical industry and regulatory bodies with the objective to compile the state-of-the-art of the technology in the different institutions. They defined technical and methodological gaps, addressed the need for quality control, suggested control compounds and acceptance criteria, highlighted cell sources and new readouts and discussed future requirements for regulatory implementation. This review summarizes the discussion, proposed solutions and recommendations of the specialists contributing to the workshop.JRC.I.5-Systems Toxicolog

Integrative analysis of genomic amplification-dependent expression and loss-of-function screen identifies ASAP1 as a driver gene in triple-negative breast cancer progression

The genetically heterogeneous triple-negative breast cancer (TNBC) continues to be an intractable disease, due to lack of effective targeted therapies. Gene amplification is a major event in tumorigenesis. Genes with amplification-dependent expression are being explored as therapeutic targets for cancer treatment. In this study, we have applied Analytical Multi-scale Identification of Recurring Events analysis and transcript quantification in the TNBC genome across 222 TNBC tumors and identified 138 candidate genes with positive correlation in copy number gain (CNG) and gene expression. siRNA-based loss-of-function screen of the candidate genes has validated EGFR, MYC, ASAP1, IRF2BP2, and CCT5 genes as drivers promoting proliferation in different TNBC cells. MYC, ASAP1, IRF2BP2, and CCT5 display frequent CNG and concurrent expression over 2173 breast cancer tumors (cBioPortal dataset). More frequently are MYC and ASAP1 amplified in TNBC tumors (>30%, n = 320). In particular, high expression of ASAP1, the ADP-ribosylation factor GTPase-activating protein, is significantly related to poor metastatic relapse-free survival of TNBC patients (n = 257, bc-GenExMiner). Furthermore, we have revealed that silencing of ASAP1 modulates numerous cytokine and apoptosis signaling components, such as IL1B, TRAF1, AIFM2, and MAP3K11 that are clinically relevant to survival outcomes of TNBC patients. ASAP1 has been reported to promote invasion and metastasis in various cancer cells. Our findings that ASAP1 is an amplification-dependent TNBC driver gene promoting TNBC cell proliferation, functioning upstream apoptosis components, and correlating to clinical outcomes of TNBC patients, support ASAP1 as a potential actionable target for TNBC treatment

Investigation of Nrf2, AhR and ATF4 Activation in Toxicogenomic Databases

Toxicological responses to chemical insult are largely regulated by transcriptionally activated pathways that may be independent, correlated and partially or fully overlapping. Investigating the dynamics of the interactions between stress responsive transcription factors from toxicogenomic data and defining the signature of each of them is an additional step toward a system level understanding of perturbation driven mechanisms. To this end, we investigated the segregation of the genes belonging to the three following transcriptionally regulated pathways: the AhR pathway, the Nrf2 pathway and the ATF4 pathway. Toxicogenomic datasets from three projects (carcinoGENOMICS, Predict-IV and TG-GATEs) obtained in various experimental conditions (in human and rat in vitro liver and kidney models and rat in vivo, with bolus administration and with repeated doses) were combined and consolidated where overlaps between datasets existed. A bioinformatic analysis was performed to refine pathways' signatures and to create chemical activation capacity scores to classify chemicals by their potency and selectivity of activation of each pathway. With some refinement such an approach may improve chemical safety classification and allow biological read across on a pathway level

An increased cell cycle gene network determines MEK and Akt inhibitor double resistance in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with poor clinical prognosis and limited targeted treatment strategies. Kinase inhibitor screening of a panel of 20 TNBC cell lines uncovered three critical TNBC subgroups: 1) sensitive to only MEK inhibitors; 2) sensitive to only Akt inhibitors; 3) resistant to both MEK/Akt inhibitors. Using genomic, transcriptomic and proteomic datasets of these TNBC cell lines we unravelled molecular features associated with the MEK and Akt drug resistance. MEK inhibitor-resistant TNBC cell lines were discriminated from Akt inhibitor-resistant lines by the presence of PIK3CA/PIK3R1/PTEN mutations, high p-Akt and low p-MEK levels, yet these features could not distinguish double-resistant cells. Gene set enrichment analyses of transcriptomic and proteomic data of the MEK and Akt inhibitor response groups revealed a set of cell cycle-related genes associated with the double-resistant phenotype; these genes were overexpressed in a subset of breast cancer patients. CDK inhibitors targeting the cell cycle programme could overcome the Akt and MEK inhibitor double-resistance. In conclusion, we uncovered molecular features and alternative treatment strategies for TNBC that are double-resistant to Akt and MEK inhibitors

Multi-targeted kinase inhibition alleviates mTOR inhibitor resistance in triple-negative breast cancer

Purpose: Owing to its genetic heterogeneity and acquired resistance, triple-negative breast cancer (TNBC) is not responsive to single-targeted therapy, causing disproportional cancer-related death worldwide. Combined targeted therapy strategies to block interactive oncogenic signaling networks are being explored for effective treatment of the refractory TNBC subtype. Methods: A broad kinase inhibitor screen was applied to profile the proliferative responses of TNBC cells, revealing resistance of TNBC cells to inhibition of the mammalian target of rapamycin (mTOR). A systematic drug combination screen was subsequently performed to identify that AEE788, an inhibitor targeting multiple receptor tyrosine kinases (RTKs) EGFR/HER2 and VEGFR, synergizes with selective mTOR inhibitor rapamycin as well as its analogs (rapalogs) temsirolimus and everolimus to inhibit TNBC cell proliferation. Results: The combination treatment with AEE788 and rapalog effectively inhibits phosphorylation of mTOR and 4EBP1, relieves mTOR inhibition-mediated upregulation of cyclin D1, and maintains suppression of AKT and ERK signaling, thereby sensitizing TNBC cells to the rapalogs. siRNA validation of cheminformatics-based predicted AEE788 targets has further revealed the mTOR interactive RPS6K members (RPS6KA3, RPS6KA6, RPS6KB1, and RPS6KL1) as synthetic lethal targets for rapalog combination treatment. Conclusions: mTOR signaling is highly activated in TNBC tumors. As single rapalog treatment is insufficient to block mTOR signaling in rapalog-resistant TNBC cells, our results thus provide a potential multi-kinase inhibitor combinatorial strategy to overcome mTOR-targeted therapy resistance in TNBC cells

Uncovering the Signaling Landscape Controlling Breast Cancer Cell Migration Identifies Novel Metastasis Driver Genes

Ttriple-negative breast cancer (TNBC) is an aggressive and highly metastatic breast cancer subtype. Enhanced TNBC cell motility is a prerequisite of TNBC cell dissemination. Here, we apply an imaging-based RNAi phenotypic cell migration screen using two highly motile TNBC cell lines (Hs578T and MDA-MB-231) to provide a repository of signaling determinants that functionally drive TNBC cell motility. We have screened ~4,200 target genes individually and discovered 133 and 113 migratory modulators of Hs578T and MDA-MB-231, respectively, which are linked to signaling networks predictive for breast cancer progression. The splicing factors PRPF4B and BUD31 and the transcription factor BPTF are essential for cancer cell migration, amplified in human primary breast tumors and associated with metastasis-free survival. Depletion of PRPF4B, BUD31 and BPTF causes primarily down regulation of genes involved in focal adhesion and ECM-interaction pathways. PRPF4B is essential for TNBC metastasis formation in vivo, making PRPF4B a candidate for further drug developmen

IGF1R signaling drives antiestrogen resistance through PAK2/PIX activation in luminal breast cancer

Antiestrogen resistance in estrogen receptor positive (ER+) breast cancer is associated with increased expression and activity of insulin-like growth factor 1 receptor (IGF1R). Here, a kinome siRNA screen has identified 10 regulators of IGF1R-mediated antiestrogen with clinical significance. These include the tamoxifen resistance suppressors BMPR1B, CDK10, CDK5, EIF2AK1, and MAP2K5, and the tamoxifen resistance inducers CHEK1, PAK2, RPS6KC1, TTK, and TXK. The p21-activated kinase 2, PAK2, is the strongest resistance inducer. Silencing of the tamoxifen resistance inducing genes, particularly PAK2, attenuates IGF1R-mediated resistance to tamoxifen and fulvestrant. High expression of PAK2 in ER+ metastatic breast cancer patients is correlated with unfavorable outcome after first-line tamoxifen monotherapy. Phospho-proteomics has defined PAK2 and the PAK-interacting exchange factors PIXα/β as downstream targets of IGF1R signaling, which are independent from PI3K/ATK and MAPK/ERK pathways. PAK2 and PIXα/β modulate IGF1R signaling-driven cell scattering. Targeting PIXα/β entirely mimics the effect of PAK2 silencing on antiestrogen re-sensitization. These data indicate PAK2/PIX as an effector pathway in IGF1R-mediated antiestrogen resistance