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.

FRET biosensor-based kinase inhibitor screen for ERK and AKT activity reveals differential kinase dependencies for proliferation in TNBC cells.

Enhanced expression and activity of protein kinases are critical in tumor cell proliferation and cancer progression. These various cancer-related kinases form intricate interdependent signaling networks. Evaluation of the effect of various kinase inhibitors on these networks is critical to understand kinase inhibitor efficacy in cancer therapy. The dynamic activation of some kinases can be monitored by fluorescence resonance energy transfer (FRET) biosensors with high temporal resolution. Here, we established a FRET biosensor-based high throughput imaging approach to determine ERK and AKT activity in two triple negative breast cancer (TNBC) cell lines HCC1806 and Hs578T. FRET functionality was systematically evaluated using EGF stimulation and different MEK and AKT inhibitors, respectively. Next, we assessed the effect of a kinase inhibitor library containing >350 different kinase inhibitors (KIs) on ERK and AKT kinase activity using a FRET high-throughput screening setting. Suppression of FRET-ERK activity was generally positively correlated with the proliferation phenotype against inhibitors targeting MAPK signaling in both cell lines containing FRET-ERK reporter. AKT inhibitor (AKTi) resistant HCC1806 showed decreased proliferation associated with downregulated dynamics of FRET-ERK when treated with KIs targeting protein receptor tyrosine kinase (RTK). Yet, MEK inhibitor (MEKi) resistant Hs578T showed positively correlated FRET-AKT and proliferative responses against different PI3K and AKT inhibitors. Altogether, our data demonstrate the feasibility to integrate high throughput imaging-based screening of intracellular kinase activity using FRET-based biosensors in assessing kinase specificity and possible signaling crosstalk in direct relation to therapeutic outcome.

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.

A kinase inhibitor screen identifies a dual cdc7/CDK9 inhibitor to sensitise triple-negative breast cancer to EGFR-targeted therapy.

BACKGROUND: The effective treatment of triple-negative breast cancer (TNBC) remains a profound clinical challenge. Despite frequent epidermal growth factor receptor (EGFR) overexpression and reliance on downstream signalling pathways in TNBC, resistance to EGFR-tyrosine kinase inhibitors (TKIs) remains endemic. Therefore, the identification of targeted agents, which synergise with current therapeutic options, is paramount. METHODS: Compound-based, high-throughput, proliferation screening was used to profile the response of TNBC cell lines to EGFR-TKIs, western blotting and siRNA transfection being used to examine the effect of inhibitors on EGFR-mediated signal transduction and cellular dependence on such pathways, respectively. A kinase inhibitor combination screen was used to identify compounds that synergised with EGFR-TKIs in TNBC, utilising sulphorhodamine B (SRB) assay as read-out for proliferation. The impact of drug combinations on cell cycle arrest, apoptosis and signal transduction was assessed using flow cytometry, automated live-cell imaging and western blotting, respectively. RNA sequencing was employed to unravel transcriptomic changes elicited by this synergistic combination and to permit identification of the signalling networks most sensitive to co-inhibition. RESULTS: We demonstrate that a dual cdc7/CDK9 inhibitor, PHA-767491, synergises with multiple EGFR-TKIs (lapatinib, erlotinib and gefitinib) to overcome resistance to EGFR-targeted therapy in various TNBC cell lines. Combined inhibition of EGFR and cdc7/CDK9 resulted in reduced cell proliferation, accompanied by induction of apoptosis, G2-M cell cycle arrest, inhibition of DNA replication and abrogation of CDK9-mediated transcriptional elongation, in contrast to mono-inhibition. Moreover, high expression of cdc7 and RNA polymerase II Subunit A (POLR2A), the direct target of CDK9, is significantly correlated with poor metastasis-free survival in a cohort of breast cancer patients. RNA sequencing revealed marked downregulation of pathways governing proliferation, transcription and cell survival in TNBC cells treated with the combination of an EGFR-TKI and a dual cdc7/CDK9 inhibitor. A number of genes enriched in these downregulated pathways are associated with poor metastasis-free survival in TNBC. CONCLUSIONS: Our results highlight that dual inhibition of cdc7 and CDK9 by PHA-767491 is a potential strategy for targeting TNBC resistant to EGFR-TKIs.

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/ß as downstream targets of IGF1R signaling, which are independent from PI3K/ATK and MAPK/ERK pathways. PAK2 and PIXα/ß modulate IGF1R signaling-driven cell scattering. Targeting PIXα/ß 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.

Nonparametric Tree-Based Predictive Modeling of Storm Outages on an Electric Distribution Network.

This article compares two nonparametric tree-based models, quantile regression forests (QRF) and Bayesian additive regression trees (BART), for predicting storm outages on an electric distribution network in Connecticut, USA. We evaluated point estimates and prediction intervals of outage predictions for both models using high-resolution weather, infrastructure, and land use data for 89 storm events (including hurricanes, blizzards, and thunderstorms). We found that spatially BART predicted more accurate point estimates than QRF. However, QRF produced better prediction intervals for high spatial resolutions (2-km grid cells and towns), while BART predictions aggregated to coarser resolutions (divisions and service territory) more effectively. We also found that the predictive accuracy was dependent on the season (e.g., tree-leaf condition, storm characteristics), and that the predictions were most accurate for winter storms. Given the merits of each individual model, we suggest that BART and QRF be implemented together to show the complete picture of a storm's potential impact on the electric distribution network, which would allow for a utility to make better decisions about allocating prestorm resources.

Shenmai injection enhances the cytotoxicity of chemotherapeutic drugs against colorectal cancers via improving their subcellular distribution.

Shenmai injection (SMI) is a Chinese patent-protected injection, which was mainly made of Red Ginseng and Radix Ophiopogonis and widely used for treating coronary heart disease and tumors by boosting Qi and nourishing Yin. In this study we examined whether SMI could produce direct synergetic effects on the cytoxicity of adriamycin (ADR) and paclitaxel (PTX) in colorectal cancers in vivo and in vitro, and explored the underlying pharmacokinetic mechanisms. BALB/c nude mice with LoVo colon cancer xenografts were intraperitoneally injected with ADR (2 mg·kg-1·3d-1) or PTX (7.5 mg·kg-1·3d-1) with or without SMI (0.01 mL·g-1·d-1) for 13 d. Co-administration of SMI significantly enhanced the chemotherapeutic efficacy of ADR and PTX, whereas administration of SMI alone at the given dosage did not produce visible anti-cancer effects, The chemosensitizing action of SMI was associated with increased concentrations of ADR and PTX in the plasma and tumors. In Caco-2 and LoVo cells in vitro, co-treatment with SMI (2 µL/mL) significantly enhanced the cytotoxicity of ADR and PTX, and resulted in some favorable pharmacokinetic changes in the subcellular distribution of ADR and PTX. In addition, SMI-induced intracellular accumulation of ADR was closely correlated with the increased expression levels of P-glycoprotein in 4 colon cancer cell lines (r2=+0.8558). SMI enhances the anti-cancer effects of ADR and PTX in colon cancers in vivo and in vitro by improving the subcellular distributions of ADR and PTX.

Non-antibiotic agent ginsenoside 20(S)-Rh2 enhanced the antibacterial effects of ciprofloxacin in vitro and in vivo as a potential NorA inhibitor.

The aim of this study is to explore the potential enhancing effect of ginsenoside 20(S)-Rh2 (Rh2) towards ciprofloxacin (CIP) against Staphylococcus aureus (S. aureus) infection in vitro and in vivo, and analyze the possible mechanisms through NorA inhibition from a target cellular pharmacokinetic view. In combination with non-toxic dosage of Rh2, the susceptibilities of S. aureus strains to CIP were significantly augmented, and the antibacterial kinetics of CIP in the S. aureus strains were markedly promoted. This enhancing effect of Rh2 towards CIP was also observed in S. aureus infected peritonitis mice, with elevated survival rate and reduced bacteria counts in blood. However, Rh2 did not influence the plasma concentrations of CIP. Further analysis indicated that Rh2 significantly promoted the accumulations of CIP in S. aureus, and inhibited the NorA mediated efflux of pyronin Y. The expressions of NorA gene on S. aureus were positively correlated with the enhancing effect of Rh2 with CIP. This is the first report of the enhancing effect of Rh2 with CIP for S. aureus infection in vitro and in vivo, of which it is probably that Rh2 inhibited NorA-mediated efflux and promoted the accumulation of CIP in the bacteria.

Liquid chromatography/tandem mass spectrometry method for quantification of trans-stilbene glycoside in rat plasma and its pharmacokinetic application.

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of trans-stilbene glycoside (SG) in rat plasma. As trans-SG can be rapidly isomerized under light exposure, trans-SG plasma samples were prepared in the dark and assayed immediately. Trans-SG and internal standard were extracted by protein precipitation. Chromatographic separation was achieved on a C(18) column with a gradient elution program. The detection of analytes was performed by negative ion via multiple reaction monitoring mode. The precursor-to-product ions of m/z 405.1 → 242.9 for trans-SG and m/z 389.1 → 226.9 for polydatin (internal standard) were monitored. No interference of endogenous components was observed for any plasma samples that we studied.The method was linear over the concentration range of 1.0-1000.0 ng/mL with a good correlation coefficient. The lower limit of quantification was 1.0 ng/mL for trans-SG. The intra and inter-batch accuracy for trans-SG in stable rat plasma samples ranged from 93.3 to 102.7% and the variation was less than 8.1%. The extraction recoveries of trans-SG in rat plasma were from 102.8 to 112.4% and the matrix effects were also acceptable. This method was successfully applied to pharmacokinetic study of trans-SG in rats after intravenous administration.