CellTiter Blue Research Articles

Abstract 2105: Single cell RNA sequencing reveals multiple adaptive resistance mechanisms to regorafenib in colon cancer

Abstract Relapses in cancer therapy occur due to the presence of sub-populations that exhibit intrinsic or acquired resistance to treatment. Acquired resistance can arise via non-mutational adaptive mechanisms in response to a perturbation. Identification of these heterogenous mechanisms mediated via stochastic changes in cellular signaling is challenging using traditional bulk methods that average signals at a cellular population level. We harnessed the power of single-cell RNA sequencing (scRNA-seq) to identify the adaptive resistance mechanisms to regorafenib, a clinically approved multi-tyrosine kinase inhibitor, in a colon cancer model. HCT116 cells were treated with an IC50 dose of regorafenib or vehicle control for 1 hour, 3 days, 7 days or 12 days and subjected to scRNA-seq using the 10X Genomics platform. Cells were sequenced at an average depth of 50000 reads/cell using Illumina sequencing. We controlled for batch effects by incorporating independent biological replicates and multiplexing using cell “hashing” with barcoded antibodies (BD Biosciences). Graph-based clustering, differential expression analysis and cell cycle phase assignment was performed using the Seurat algorithm. Proliferation was assessed using PI staining for cell cycle status, CellTiter Blue viability assay and clonogenic assays. We analyzed a total of 19,879 single cells following a time-course of regorafenib or vehicle treatment. Regorafenib treatment at one hour resulted in no significant transcriptional changes. At all further time points, we could confirm the down-regulation of MAPK signaling, reflecting the mechanism of action of regorafenib. At 72 hours, we detected sub-populations with varying activation levels of ER stress response and increased lipid metabolism, together with upregulation of transcription factors SOX4 and ID3. These features continued to be detected till 7 and 12 days after treatment. By 12 days, we observed upregulation of keratins 7, 8, 10, 18, 19 together with markers of G2M/S phase progression. Survival of clonal cell populations at 12 days was confirmed by crystal violet staining. Small molecule-based inhibition of ER stress (azoramide) and lipid synthesis (fatostain) was synergistic with regorafenib at day 3 and successfully inhibited the emergence of these resistant clones by day 12. Our results reveal multiple signaling events that lead to a resistance phenotype characterized by increased proliferation and keratin upregulation that can be counteracted by combination therapies. For future studies we will continue to dissect the molecular networks underlying these events by gene silencing and protein over-expression studies. Citation Format: Anuja Sathe, Billy T. Lau, Sue Grimes, Stephanie Greer, Hanlee Ji. Single cell RNA sequencing reveals multiple adaptive resistance mechanisms to regorafenib in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2105.

Abstract 1254: Predictive models for tumor cell targeting with plinabulin, derived from in vitro screening and Affymetrix mRNA expression data

Abstract Tubulin binding drugs are approved for the treatment of many cancer types, without the use of molecular markers to select patients likely to respond. Plinabulin binds β-tubulin in a differentiated pocket and is being tested in a Phase 3 clinical study for the treatment of NSCLC. Additional indications are being considered for plinabulin and an algorithm for selecting especially responsive cancers and patient subgroups would be of significant value. With this in mind, Affymetrix HG-U133 Plus 2.0 array mRNA expression data for 43 human breast, lung, prostate, ovarian or CNS cancer cell lines were utilized to develop mathematical models to predict in vitro plinabulin potency against the same cell lines. Cells were treated for only 24 hours with plinabulin and then cultured for another 48 hours without plinabulin. Viable cell number was then measured with a Cell Titer-Blue Assay, and the plinabulin concentration causing a 70% reduction in viable cells (IC70) versus vehicle treated controls was derived. Cell lines were clearly separable into plinabulin Active (21 cell lines with IC70<1.0 μM) and Inactive (IC70>9.5 μM) groups. Log2 transformed Affymetrix gene probeset signal values, preprocessed with the GeneChip robust multi-array average analysis algorithm, were selected and ranked as predictors of plinabulin activity with a bootstrap forest partitioning technique, utilizing JMP 14.1 statistical software. 56 HIT probesets were identified that also had significantly different expression in responding versus non-responding cell lines (p<0.01, uncorrected t-test). For probesets with gene annotation, only the probeset for each gene with the highest Jetset score was utilized (Li et al., 2011). Top HIT predictor genes include CTNNB1 (β-catenin; oncogene), CALD1 (caldesmon; inhibits myosin ATPase activity), ERI1 (RNA processing), LGR5 (adult stem cell biomarker), SECISBP2L (SLAN; prolongs mitosis), and TRAK1 (mitochondrial/endosome trafficking). Models were constructed from HIT gene probesets in JMP to identify plinabulin responding cell lines, utilizing either one-layer TanH multimode fit neural networks or binary logistic regression. Surprisingly, models incorporating approximately 4-10 probeset values were derived that perfectly predicted plinabulin activity. Importantly, the cell lines tested in the above analyses were not those known to express high levels of multi-drug resistant (MDR) transporters. The importance of MDR status was therefore evaluated separately. Plinabulin activity, unlike that of taxanes, was not significantly affected by the MDR transporter inhibitor verapamil (10 μM), in ovarian cancer cell lines with a known MDR phenotype. To conclude, our work provides novel algorithms that may be of value in selecting cancer patients with tumor cells that are particularly susceptible to the direct cytotoxic effects of plinabulin. Citation Format: James R. Tonra, Hagen Klett, Chenghao Shen, Gerhard Kelter, Ramon W. Mohanlal, G. Kenneth Lloyd, Lan Huang. Predictive models for tumor cell targeting with plinabulin, derived from in vitro screening and Affymetrix mRNA expression data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1254.

A new free light chain immunoassay shows better agreement with the quantification of the serum protein electrophoresis M-protein compared to a nephelometric assay in patients with multiple myeloma

The continuing development of nanotechnology necessitates the reliable assessment of potential adverse health consequences associated with human exposures. The physicochemical properties of nanomaterials can be responsible for unexpected interactions with components of classical toxicity assays, which may generate erroneous interpretations.In this paper, we describe how particle interference can be observed in in vitro toxicity tests (CellTiter Blue, CyQUANT, WST-1 and CellTiter-Glo assay) and in cell biology tests using flow cytometry (cell cycle analysis). We used cobalt oxide (Co3O4) particles as an example, but these assays can be performed, in principle, regardless of the nanoparticle considered. We have shown that cobalt particles interfere with most of these tests. We adapted the protocol of the CellTiter-Glo assay to circumvent this interference and demonstrated that, using this protocol, the toxicity level is consistent with results obtained using the clonogenic assay, which is considered to be the reference test.Before assessing particle toxicity using in vitro toxicity tests, interference testing should be performed to avoid false interpretations. Furthermore, in some cases of interference, protocol adaptation can be considered to allow the reliable use of these quick and convenient in vitro tests.

Fabrication and evaluation of poly(lactic acid), chitosan, and tricalcium phosphate biocomposites for guided bone regeneration

ABSTRACTThis study presents and evaluates an approach for fabricating poly(lactic acid) (PLA)/chitosan (CS)/tricalcium phosphate (TCP) electrospun scaffolds for guided bone regeneration, a dental procedure that uses membranes to direct and delineate regions of osteogenesis. Biomaterials were pre‐processed using cryomilling, a solid‐state grinding technique that facilitates the generation of powdered biocomposites conducive to electrospinning. X‐ray diffraction (XRD) confirmed the generation of cryomilled blends consisting of PLA, CS, and TCP. Results from the differential scanning calorimetry showed an upward shift in glass transition temperature and an increase in crystallinity with the inclusion of TCP reinforcing the observations from XRD. Murine macrophages were used to confirm the biocompatibility of the cryomilled powders and was evaluated using CellTiter‐Blue (CTB) cell viability assay and brightfield microscopy. Scanning electron microscopy was used to examine the morphology of the fibers produced via electrospinning, while Raman spectroscopy confirmed material homogeneity.In vitrostudies with MG‐63 cells validated the capacity of composite scaffolds to encourage proliferation, while Coherent anti‐Stokes Raman scattering and fluorescence microscopies provided visual evidence of cell proliferation. CTB assay revealed that cells maintain viability and metabolic activity at 3 and 7 days after seeding, demonstrating the potential of the biocomposite membranes. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci.2018,135, 46692.

A matrix-assisted laser desorption ionization-time-of-flight-time-of-flight-mass spectrometry-based toxicoproteomic screening method to assess in vitro particle potencies.

Knowledge of biological reactivity and underlying toxicity mechanisms of airborne particulate matter (PM) is central to the characterization of the risk associated with these pollutants. An integrated screening platform consisting of protein profiling of cellular responses and cytotoxic analysis was developed in this study for the estimation of PM potencies. Mouse macrophage (J774A.1) and human lung epithelial cells (A549) were exposed in vitro to Ottawa urban particles (EHC6802) and two reference mineral particles (TiO2 and SiO2). Samples from the in vitro exposure experiment were tested following an integrated classical cytotoxicity/toxicoproteomic assessment approach for cellular viability (CellTiter Blue®, lactate dehydrogenase) and proteomic analyses. Cellular proteins were pre‐fractionated by molecular weight cut‐off filtration, digested enzymatically and were analyzed by matrix‐assisted laser desorption ionization–time‐of‐flight–time‐of‐flight–mass spectrometry for protein profiling and identification. Optimization of detergent removal, pre‐fractionation strategies and enzymatic digestion procedures led to increased tryptic peptide (m/z) signals with reduced sample processing times, for small total protein contents. Proteomic analyses using this optimized procedure identified statistically significant (P < 0.05) PM dose‐dependent changes at the molecular level. Ranking of PM potencies based on toxicoproteomic analysis were in line with classical cytotoxicity potency‐based ranking. The high content toxicoproteomic approach exhibited the potential to add value to risk characterization of environmental PM exposures by complementing and validating existing cytotoxicity testing strategies.

Abstract P5-21-15: The synergistic antitumor activity of entinostat (MS-275) in combination with palbociclib (PD 0332991) in estrogen receptor-positive and triple-negative breast cancer

Abstract BACKGROUND: CDK4/6 regulates the G1-S phase transition by phosphorylating the retinoblastoma protein (Rb). Given their potent clinical efficacy, CDK4/6 inhibitors used in combination with hormone receptor (HR) blockade (with an aromatase inhibitor or fulvestrant) are emerging as the standard of care for patients with metastatic HR-positive breast cancers. The CDK4/6 inhibitors palbociclib and ribociclib are FDA-approved for use in HR-positive breast cancer patients, and abemaciclib is currently in phase III trials. We observed that approximately 74% (25/34) of breast cancer cell lines had high phosphorylated Rb (phospho-Rb) expression levels and that triple-negative breast cancer (TNBC) cell lines often expressed phospho-Rb, suggesting that targeting phospho-Rb via CDK4/6 inhibition may be effective against TNBC. The histone deacetylase (HDAC) inhibitors increase p21Cip1 levels, promoting proteasomal degradation of cyclin B1 and resulting in G2/M arrest. Entinostat is an oral, class 1, selective HDAC inhibitor currently in phase III testing in HR-positive breast cancer. Preclinical and clinical data demonstrate that entinostat, in combination with HR blockade, has anticancer activity. Our group recently reported that entinostat combined with other anticancer drugs induced apoptosis via induction of proapoptotic proteins such as Noxa and Bim in breast cancer cell lines. Based on these findings, we hypothesized that entinostat-induced apoptosis and palbociclib-induced cell cycle arrest synergize to produce enhanced antitumor effects in estrogen receptor (ER)-positive breast cancer and TNBC cell lines with high phospho-Rb expression levels. METHODS: We assessed the combination antitumor effects and their mechanisms via CellTiter Blue and sulforhodamine B assays, flow cytometry, apoptosis (caspase 3/7) assays, anchorage-independent growth assays, Western blotting, reverse phase protein array (RPPA), and mammary fat pad xenograft mouse models. RESULTS: RPPA data showed that ER-positive and TNBC cell lines more often expressed phospho-Rb than did other breast cancer cell subtypes (7/10 and 8/17 cell lines, respectively). We found that the combination of entinostat and palbociclib synergistically inhibited tumor cell proliferation (combinational index less than 1.0), reduced in vitro colony formation (P &amp;lt; 0.05), inhibited in vivo tumor growth in ER-positive MCF-7 breast cancer cells (P &amp;lt; 0.05), and inhibited tumor growth in TNBC xenograft mouse models (MDA-MB-231) more effectively than did either drug alone. CONCLUSION: Taken together, our data provide evidence that combining entinostat with palbociclib enhances the antitumor effects of these drugs. Along with our continued effort to determine predictive biomarkers, our findings justify conducting a clinical trial of combination treatment with entinostat and palbociclib in patients with ER-positive breast cancer or TNBC. Citation Format: Lee J, Lim B, Pearson T, Tripathy D, Ordentlich P, Ueno NT. The synergistic antitumor activity of entinostat (MS-275) in combination with palbociclib (PD 0332991) in estrogen receptor-positive and triple-negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-21-15.

Screening the ToxCast phase II libraries for alterations in network function using cortical neurons grown on multi-well microelectrode array (mwMEA) plates.

Methods are needed for rapid screening of environmental compounds for neurotoxicity, particularly ones that assess function. To demonstrate the utility of microelectrode array (MEA)-based approaches as a rapid neurotoxicity screening tool, 1055 chemicals from EPA's phase II ToxCast library were evaluated for effects on neural function and cell health. Primary cortical networks were grown on multi-well microelectrode array (mwMEA) plates. On day in vitro 13, baseline activity (40min) was recorded prior to exposure to each compound (40µM). Changes in spontaneous network activity [mean firing rate (MFR)] and cell viability (lactate dehydrogenase and CellTiter Blue) were assessed within the same well following compound exposure. Following exposure, 326 compounds altered (increased or decreased) normalized MFR beyond hit thresholds based on 2× the median absolute deviation of DMSO-treated wells. Pharmaceuticals, pesticides, fungicides, chemical intermediates, and herbicides accounted for 86% of the hits. Further, changes in MFR occurred in the absence of cytotoxicity, as only eight compounds decreased cell viability. ToxPrint chemotype analysis identified several structural domains (e.g., biphenyls and alkyl phenols) significantly enriched with MEA actives relative to the total test set. The top 5 enriched ToxPrint chemotypes were represented in 26% of the MEA hits, whereas the top 11 ToxPrints were represented in 34% of MEA hits. These results demonstrate that large-scale functional screening using neural networks on MEAs can fill a critical gap in assessment of neurotoxicity potential in ToxCast assay results. Further, a data-mining approach identified ToxPrint chemotypes enriched in the MEA-hit subset, which define initial structure-activity relationship inferences, establish potential mechanistic associations to other ToxCast assay endpoints, and provide working hypotheses for future studies.

Abstract 1236: Novel androgen receptor inhibitors in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC), which lacks expression of hormone receptors and human epidermal growth factor receptor 2, has the worst prognosis of all breast cancer subtypes, due to inherent chemo-resistance and a lack of therapeutic targets. A subset of TNBC has been shown to express androgen receptor (AR) but despite promising pre-clinical data, clinical trials evaluating the use of approved anti-androgens (bicalutamide, enzalutamide) in AR-positive TNBC has shown modest efficacy to date. In prostate cancer, bicalutamide binds to AR with low affinity and in the presence of AR gene amplification or mutation, can exhibit partial agonist activity leading to resistance. In order to increase affinity for AR and to avoid AR antagonist to agonist conversion synthesized 7-substituted umbelliferones (UMB) derivatives with a distinct scaffold modified from available AR antagonists (Kandil et al Bioorganic &amp; Medicinal Chemistry Letters, 2016). These agents exhibited inhibitory activity in human prostate cancer cells lines at sub-micro molar level, a 50-fold and 30-fold improvement over bicalutamide and enzalutamide, respectively. We hypothesized that these novel AR antagonists would be effective in inhibiting the growth of AR-positive TNBC cells. Methods: TNBC AR negative mesenchymal MDA-MB-231 (as control) and AR positive MDA-MB-231 cells were utilized for determination of the efficacy of the UMB derivatives. For proliferation assays, viability of the cells was monitored over a period of 72 hours with redox dye Cell Titer-Blue. Standard Western blotting techniques were used for protein detection. For apoptosis analysis, standard flow cytometry reagents, FITC labelled Annexin V and Propidium Iodine(PI) mix were utilized. Results: UMB derivatives decreased the expression of AR in AR-positive MDA-MB-453 in a dose dependent manner. UMB derivatives, at micro Molar concentration decreased cell proliferation of AR-positive and interestingly AR-negative cell lines by 20 to 50%, also in a dose dependent manner. UMB derivatives induced apoptosis in both cell lines. Interestingly, UMB derivatives induced a change from a mesenchymal to epithelial phenotype in AR-negative MDA-MB-231 cells, which was confirmed by decreased expression of vimentin and other mesenchymal markers. Conclusions: Novel UMB derivatives inhibit growth and cause apoptosis in TNBC cells and induce mesenchymal-to-epithelial transition in an AR-negative mesenchymal TNBC cell line. Targeting AR with novel AR antagonists seem to be a promising therapeutic approach for AR-positive TNBC and further evaluation of AR antagonists in AR-negative mesenchymal TNBC is warranted. Citation Format: Saswati Bhattacharya, Yuanqi Cai, Steven Kurina, Pamela Westmark, Benjamin Hokanson, Matthew Anderson, Ruth O’Regan. Novel androgen receptor inhibitors in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1236. doi:10.1158/1538-7445.AM2017-1236

HOXB7 accelerates the malignant progression of hepatocellular carcinoma by promoting stemness and epithelial-mesenchymal transition

BackgroundHomeobox B7 (HOXB7) has been identified associated with poor prognosis of hepatocellular carcinoma (HCC). However, the specific mechanism by which HOXB7 promotes the malignant progression of HCC remains to be determined.MethodsImmunohistochemistry (IHC) was used to detect the expression level of HOXB7 in 77-paired HCC tissue samples, and the correlation between HOXB7 and HCC prognosis was assessed. The location of HOXB7 was confirmed by immunofluorescence. Cell Titer-Blue assay was used to assess the proliferation of hepatoma cells. The stem-like properties of hepatoma cells were analysed by sphere formation and clone formation assays. The effect of HOXB7 on expression of cancer stem cell markers was evaluated. Transwell and wound-healing assays were performed to estimate the invasion and migration abilities of hepatoma cells. A xenograft tumor model was established in nude mice to assess the role of HOXB7 in tumor growth. Bioluminescence imaging was used to survey the effect of HOXB7 on the metastatic ability of hepatoma cells in vivo.ResultsHigher expression of HOXB7 was detected in HCC tissues compared with noncancerous tissues and significantly associated with poor prognosis of HCC. In addition, HOXB7 knockdown suppressed the cell proliferation, clone formation, sphere formation, invasion and migration of hepatoma cells in vitro; conversely, these biological abilities of hepatoma cells were enhanced by HOXB7 overexpression. Moreover, the cancer stem cell markers EPCAM and NANOG were up-regulated by HOXB7. The role of HOXB7 in promoting tumor growth and metastasis was verified in vivo. Further investigation revealed that c-Myc and Slug expression was elevated by HOXB7 and the AKT pathway was activated.ConclusionOverexpression of HOXB7 was significantly correlated with poor prognosis of HCC. HOXB7 up-regulated c-Myc and Slug expression via the AKT pathway to promote the acquisition of stem-like properties and facilitate epithelial-mesenchymal transition of hepatoma cells, accelerating the malignant progression of HCC.

In-vitro Assessment of the Antiproliferative and Apoptotic Potential of the Ethyl acetate Extract of Peltophorumafricanum on Different Cancer Cell Lines.

We evaluated the in-vitro antiproliferative and apoptotic potential of the ethyl acetate extract (EAE) of Peltophorum africanum, a member of the family Fabaceae (Sond) in order to validate its pharmacological use. Antiproliferation of human breast (MCF-7), colon (HT-29) and cervical (HeLa) cancer cell lines by EAE was investigated using the Cell Titer-Blue viability assay and the mechanism of action delineated using the Nucleic Acid and Protein Purification Nucleospin® Tissue Kit, Scanning Electron Microscope (SEM), propidium iodide (PI) and acridine orange (AO) double-staining techniques. We observed a significant reduction in cell viability of the MCF-7 cells from 100% (untreated) to 54.33 ± 1.84% after 72 h of treatment with 5 µg/mL of EAE (P. value < 0.05). Internucleosomal DNA of MCF-7, HT-29 and HeLa cells was randomly fragmented into an uninterrupted spectrum of sizes, complemented by the intercalation of nucleic acid-specific fluorochromes by PI and AO spotting two phases of apoptosis; early (EA) and late (LA) apoptosis. Distinctive ultramorphological changes observed included; cell shrinkage, membrane blebbing, and typical cell induced death. The ethyl acetate extract of P.africanum has the potential to induce apoptosis and is undergoing further studies in-vivo as a likely template for new anticancer therapy.

Epigenetic pathway inhibitors represent potential drugs for treating pancreatic and bronchial neuroendocrine tumors

Cancer is associated with alterations in epigenetic mechanisms such as histone modifications and methylation of DNA, and inhibitors targeting epigenetic mechanisms represent a novel class of anti-cancer drugs. Neuroendocrine tumors (NETs) of the pancreas (PNETs) and bronchus (BNETs), which may have 5-year survivals of <50% and as low as 5%, respectively, represent targets for such drugs, as >40% of PNETs and ~35% of BNETs have mutations of the multiple endocrine neoplasia type 1 (MEN1) gene, which encodes menin that modifies histones by interacting with histone methyltransferases. We assessed 9 inhibitors of epigenetic pathways, for their effects on proliferation, by CellTiter Blue assay, and apoptosis, by CaspaseGlo assay, using 1 PNET and 2 BNET cell lines. Two inhibitors, referred to as (+)-JQ1 (JQ1) and PFI-1, targeting the bromo and extra terminal (BET) protein family which bind acetylated histone residues, were most effective in decreasing proliferation (by 40–85%, P<0.001) and increasing apoptosis (by 2–3.6 fold, P<0.001) in all 3 NET cell lines. The anti-proliferative effects of JQ1 and PFI-1 remained present for at least 48 hours after removal of the compound. JQ1, but not PFI-1, had cell cycle effects, assessed by propidium iodide staining and flow cytometry, resulting in increased and decreased proportions of NET cells in G1, and S and G2 phases, respectively. RNA Sequencing analysis revealed that these JQ1 effects were associated with increased histone 2B expression, and likely mediated through altered activity of bromodomain-containing (Brd) proteins. Assessment of JQ1 in vivo, using a pancreatic beta cell-specific conditional Men1 knockout mouse model that develops PNETs, revealed that JQ1 significantly reduced proliferation (by ~50%, P<0.0005), assessed by bromodeoxyuridine incorporation, and increased apoptosis (by ~3 fold, P<0.0005), assessed by terminal deoxynucleotidyl transferase dUTP nick end labelling, of PNETs. Thus, our studies demonstrate that BET protein inhibitors may provide new treatments for NETs.

Abstract P1-05-10: MLL3 mutation in ER+ breast cancers leads to dramatic shift in ER binding locations and consequentially alters transcriptional programs

Abstract Hypothesis: Endocrine therapy (ET) is an effective treatment for estrogen-receptor positive (ER+) breast cancer; however, more than 80% of women will develop endocrine resistance. ER ChIP-seq studies in clinical samples show tumors likely to relapse have a unique set of ER binding sites that correlate with gene signatures predicting clinical outcome. Understanding how several factors, including transcriptional co-activator-complexes, influence ER binding location is crucial to improving treatment. MLL3, which is recurrently mutated in ~7% of ER+ cases, is a member of one such complex that interacts with ER. It is a histone methyltransferase that marks active enhancers. We hypothesized that mutation of MLL3 may change the genomic landscape of enhancers and thus change binding patterns of ER, altering response to ET. Methods: To elucidate transcriptional consequences of MLL3 mutation in ER+ breast cancers, we leveraged TCGA ER+ breast cancer RNA-seq data. We used a gene-by-gene multivariate linear model to identify differentially expressed genes (DEGs) between MLL3-mutant and wild-type (WT) samples. This data was compared to DEG found with in-house RNA-seq data for MLL3-mutant MCF7 cells, MLL3-WT ZR751 cells, and MLL3-knockdown (KD) ZR751 cells to identify a testable MLL3-mutant signature. ChIP-seq data for ER and H3K4me1, the histone mark made by MLL3, was then produced using these cell lines in order to pinpoint genes with changes in both H3K4me1 and ER-binding upon MLL3 mutation/KD. This gene list was compared to DEG found through RNA-Seq. To illuminate inherent differences between MLL3-WT and mutant/KD cells in response to ET, Cell-Titer Blue, Cell-Titer Glo, and Caspase-Glo assays were performed using DMSO, 4-OHT, and Fulvestrant. Results: TCGA RNA-seq data revealed MLL3-mutant ER+ breast cancers have differentially expressed transcriptional regulators, including ER itself. iRegulon analysis showed enrichment for DEG-regulated by ER-tethering factor SP1, a phenomenon suggested to be associated with more aggressive behavior. ChIP-seq data revealed substantial shifts in both ER-binding locations and H3K4me1 marks upon MLL3 mutation/KD. Interestingly, annotated peaks for MLL3-KD ZR751 showed overlap with those of MLL3-mutant MCF7, and GREAT analysis of new ER binding sites in MLL3-KD ZR751 showed an up-regulation of signatures associated with endocrine resistance. Indeed, MLL3-KD ZR751 cells were more resistant to 4-OHT/Fulvestrant than MLL3-WT ZR751. Conclusions: Loss of MLL3 function leads to a massive shift in H3K4me1, indicating a shift in the genomic landscape of enhancers. This shift is associated with a shift in ER binding as well as alterations in gene expression. Pathway analysis of these genes suggest that loss of MLL3 function may increase endocrine resistance, and indeed, cell lines with loss of MLL3 function are more resistant to ER inhibition than MLL3-WT cell lines. This work demonstrates that loss of MLL3 function leads to shifts in the enhancer landscape, alterations in ER binding and regulation of gene expression, and contributes to more aggressive tumor behavior. Citation Format: Stauffer K, Stricker T. MLL3 mutation in ER+ breast cancers leads to dramatic shift in ER binding locations and consequentially alters transcriptional programs [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 P1-05-10.

Relationship between TRAF6 and deterioration of HCC: an immunohistochemical and in vitro study

ObjectiveTo explore the relationship between tumor necrosis factor receptor-associated factor 6 (TRAF6) and the clinicopathological features in HCC as well as its biological function.MethodsTotally, 412 liver tissues were collected, including 171 hepatocellular carcinoma (HCC) and their corresponding non-tumor tissues, 37 cirrhosis and 33 normal liver tissues. The expression of TRAF6 was assessed by immunohistochemistry. Then, analysis of the correlations between TRAF6 expression and clinicopathological parameters in HCC was conducted. Furtherer, in vitro experiments on HepG2 and Hep3B cells were performed to validate the biological function of TRAF6 on HCC cells. TRAF6 siRNA was transfected into HepG2 and Hep3B cell lines and TRAF6 expression was evaluated with RT-qPCR and western blot. The assays of cell viability, proliferation, apoptosis and caspase-3/7 activity were carried out to investigate the effects of TRAF6 on HCC cells with RNA interference. Cell viability was assessed with Cell Titer-Blue kit. Cell proliferation was tested with MTS kit. Cell apoptosis was checked through morphologic detection with fluorescence microscope, as well as caspase-3/7 activity was measured with fluorogenic substrate detection.ResultsThe positive expression rate of TRAF6 protein was 49.7 % in HCC, significantly higher than that of normal liver (12.1 %), cirrhosis (21.6 %) and adjacent non-cancerous tissues (36.3 %, all P < 0.05). Upregulated TRAF6 was detected in groups with metastasis (Z = −2.058, P = 0.04) and with low micro-vessel density (MVD) expression (Z = −2.813, P = 0.005). Spearman correlation analysis further showed that the expression of TRAF6 was positively correlated with distant metastasis (r = 0.158, P = 0.039) and negatively associated with MVD (r = −0.249, P = 0.004). Besides, knock-down of TRAF6 mRNA in HCC cell lines HepG2 and Hep3B both resulted in cell viability and proliferation inhibition, also cell apoptosis induction and caspase-3/7 activity activation.ConclusionsTRAF6 may contribute to metastasis and deterioration of the HCC via influencing cell growth and apoptosis. Thus, TRAF6 might become a predictive and therapeutic biomarker for HCC.

Abstract 545: Differential regulation of human T-cells by TGR-1202, a novel PI3Kδ inhibitor

Abstract INTRODUCTION: TGR-1202 is a novel, next-generation PI3Kδ inhibitor presenting significant structural and pharmacological differences from prior small-molecule PI3Kδ inhibitors. TGR-1202 has high clinical efficacy in treatment of B cell malignancies with a substantially differentiated adverse event profile compared to previous PI3Kδ inhibitors, specifically concerning hepatotoxicity or colitis which have been minimal or non-existent. It has been postulated that these effects may be due to T cell immune-mediated mechanisms. We hypothesized that TGR-1202 preserves the function of the regulatory T cell (Treg) population, translating to decreased immune-mediated side effects after treatment. Here, we aimed to compare effects of clinically available PI3Kδ inhibitors on T cells with an emphasis on Tregs. METHODS: We compared activity of idelalisib, duvelisib, and TGR-1202 as single agents in vitro in isolated human T cells. Viability, apoptosis, and proliferation were determined using CellTiter Blue®, annexin V/PI and CFSE staining. CBA and qRT PCR were used to measure cytokines and transcription factors. Flow cytometry was used to detect subset ratios and surface marker expression. RESULTS: First, we observed comparable dose-dependent increases in cytotoxicity beginning at 25uM following treatment of isolated T cell populations with idelalisib, duvelisib, or TGR-1202. At this dose, apoptosis was induced between 48 and 72h. Second, all inhibitors reduced cytokine production in CD3+ T cells upon stimulation. Particularly, IFN-γ, IL-10 and IL-17a reduction was less pronounced after TGR-1202 treatment, indicating relative conservation of T cell response. All inhibitors lowered mRNA expression of T-bet (Th1), GATA-3 (Th2) and FoxP3 (Treg), however FoxP3 levels were consistently higher in TGR-1202 treated T cells. Third, we detected normal CD4:CD8 ratio and unaffected proliferative capacity of CD4+ and CD8+ subsets after drug treatment. Finally, all inhibitors decreased total percent of Tregs following stimulation (CD4+ CD25HI FoxP3+) accompanied by decreased expression of co-inhibitory molecules CTLA-4 and PD-1 on the Tregs. Interestingly, TGR-1202 significantly preserved the percent of Tregs closer to normal as well as surface expression of CTLA-4 and PD-1 on Tregs, indicating greater retention of immune checkpoint blockade and suppressive phenotype. CONCLUSIONS: We report herein that TGR-1202 affects human T cells differently than idelalisib and duvelisib. TGR-1202 sustains IL-10 production, FoxP3 mRNA expression, and maintains Treg percentage and expression of immune checkpoint molecules, suggesting relative preservation of numbers and function of Tregs. Data presented begin to provide novel insight into immune mediated cellular mechanisms responsible for lack of side effects in clinical trials of TGR-1202. In vivo models to further characterize effects on the T cell compartment are ongoing. Citation Format: Kamira K. Maharaj, John Powers, Renee Fonseca, Hari Miskin, Dave Maryanski, Eva Sahakian, Javier Pinilla-Ibarz. Differential regulation of human T-cells by TGR-1202, a novel PI3Kδ inhibitor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 545.

Abstract 1589: Identification of EMT-selective cytotoxic compounds

Abstract Background: Epithelial to mesenchymal transition (EMT) is a cellular phenotype which may contribute to metastasis and therapeutic resistance in cancer. Therapeutic targeting of EMT-like phenotypes in cancer cells, thus may afford an approach to treating metastatic disease and/or thwarting therapeutic resistance. Methods: We have developed a cell-based high-throughput screening protocol using the high content Operetta imaging platform to identify EMT cytotoxic compounds. This screen was performed on PC3E and TEM 4-18 cell lines which were originally isolated form a parental PC3 prostate cancer cell line based on their differential ability to invade an endothelial monolayer. A library of 2,640 compounds (Microsource) was screened on a co-culture of epithelial cells (PC3E GFP cells) and EMT-like cells (TEM 4-18 mCherry cells). After 72h exposure to compounds, relative numbers of GFP- and mCherry-positive cells were quantitated using the Operetta system. Dose-response curves were established for each compound exhibiting a greater toxicity against EMT-like cells. The results were validated on PC3E and TEM 4-18 cell lines cultured separately with the CellTiter Blue viability assay. Results: Among the 2,640 compounds tested, monensin and salinomycin, both monovalent cation ionophores, exhibited a relatively greater toxicity against EMT-like cells. Nigericin, compound closely related to monensin but absent from the Microsource library, was also evaluated. The highest potency and selectivity for the EMT-like cells was obtained with nigericin (IC50 357nM for PC3E vs 7nM for TEM 4-18), followed by monensin (IC50 457nM for PC3E vs 11nM for TEM 4-18) and salinomycin (IC50 1687nM for PC3E vs 278nM for TEM 4-18). Monensin and nigericin induced apoptosis, a cell cycle arrest, and an increase in ROS production in TEM 4-18 without apparent mitochondrial disruption. In addition, monensin rapidly induced swelling of golgi apparatus likely resulting in a blockage of intracellular protein trafficking and consequently cell death. To extend our findings, we evaluated the toxicity of these three ionophores in 15 cancer cell lines from different origins and classified them as resistant or sensitive. We analyzed publically available gene expression data for these cell lines and performed a Gene Set Enrichment Analysis to identify the gene sets differentially represented in the 2 groups. Supporting our hypothesis, the gene set involving EMT was the top gene sets enriched in the sensitive group. Conclusion: In this study we identified three chemically-related compounds (monensin, salinomycin and nigericin) exhibiting relatively greater toxicity against EMT-like cells. These compounds may serve as chemical probes for exploring the biology of EMT-like cells and point to strategies for selectively targeting these cells to disrupt metastasis and/or therapeutic resistance. Citation Format: Marion Vanneste, Qin Huang, Meng Wu, Michael D. Henry. Identification of EMT-selective cytotoxic compounds. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1589.

MiR-1908 Overexpression Inhibits Proliferation, Changing Akt Activity and p53 Expression in Hypoxic NSCLC Cells.

The ribosomal protein (RP)–p53 pathway has been shown to play a key role in apoptosis and senescence of cancer cells. miR-1908 is a newly found miRNA that was reported to have prognostic potential in melanoma. However, its role and mechanism in the progression of non-small cell lung cancer (NSCLC) are largely unknown. In this study, we found that expression of miR-1908 was significantly downregulated in human NSCLC cell lines, including SK-MES-1, A549, and NCI-H460. Then the role of miR-1908 in NSCLC cell proliferation was explored. The miR-1908 mimic was transfected into NSCLC cell lines, and their proliferation was detected. MTT and Cell Titer-Blue H analyses showed that the cell proliferation was notably reduced by the miR-1908 mimic transfection. Moreover, we found the RP–p53 pathway was activated by miR-1908 mimic. Moreover, the miR-1908 inhibitor transfection had a completely opposite effect on the NSCLC cell proliferation than that of miR-1908 mimic. To explore the underlying mechanism of that, TargetScan bioinformatics server and 3′-UTR luciferase reporter assay were applied to identify the targets of miR-1908. Our results showed that AKT1 substrate 1 (AKT1S1), a newly proven suppressor of the RP–p53 pathway, was a target of miR-1908, suggesting a probable mechanism for miR-191 suppressing NSCLC cell proliferation. Our findings provide a novel molecular target for the regulation of NSCLC cell proliferation.

Cytotoxicity assessment of porous silicon microparticles for ocular drug delivery

Porous silicon (PSi) is a promising material for the delivery and sustained release of therapeutic molecules in various tissues. Due to the constant rinsing of cornea by tear solution as well as the short half-life of intravitreal drugs, the eye is an attractive target for controlled drug delivery systems, such as PSi microparticles. Inherent barriers ensure that PSi particles are retained in the eye, releasing drugs at the desired speed until they slowly break down into harmless silicic acid. Here, we have examined the in vitro cytotoxicity of positively and negatively charged thermally oxidized (TOPSi) and thermally carbonized (TCPSi) porous silicon microparticles on human corneal epithelial (HCE) and retinal pigment epithelial (ARPE-19) cells. In addition to ocular assessment under an inverted microscope, cellular viability was evaluated using the CellTiter Blue™, CellTiter Fluor™, and lactate dehydrogenase (LDH) assays. CellTiter Fluor proved to be a suitable assay but due to non-specific and interfering responses, neither CellTiter Blue nor LDH assays should be used when evaluating PSi particles. Our results suggest that the toxicity of PSi particles is concentration-dependent, but at least at concentrations less than 200μg/ml, both positively and negatively charged PSi particles are well tolerated by human corneal and retinal epithelial cells and therefore applicable for delivering drug molecules into ocular tissues.

Abstract P6-14-01: A class I histone deacetylase inhibitor, entinostat, enhances lapatinib efficacy in both HER2-overexpressing inflammatory and non-inflammatory breast cancer cells through FOXO3-mediated Bim1 expression

Abstract Background: Human epidermal growth factor receptor 2 (HER2) overexpression has been reported in 15%-20% of breast cancers and is associated with shorter survival and worse clinical outcome. Inflammatory breast cancer (IBC), a very aggressive subtype of advanced breast cancer, accounts for approximately 2% of all breast cancers and 8%-10% of all breast cancer-related deaths in the United States. Recent study represented that about 60% of IBCs overexpress HER2. Although there are effective HER2-targeted agents, patients with HER2+ breast cancer often have intrinsic and acquired resistance to the anti-HER2 agents that are currently approved by the U.S. Food and Drug Administration. Therefore, novel combination strategies are needed to treat HER2+ breast cancers that develop drug resistance. To this end, we investigated the combinational effect of entinostat, an oral isoform-selective histone deacetylase type I inhibitor, and lapatinib, a HER2/epidermal growth factor receptor dual tyrosine kinase inhibitor, in HER2+ inflammatory breast cancer (IBC) and non-IBC breast cancer cells. Methods: We assessed the combinational synergistic effect and its mechanism via CellTiter Blue assay, flow cytometry, anchorage-independent growth, quantitative real-time polymerase chain reaction, small interfering RNA, Western blotting, and mammary fat pad xenograft mouse models. Results: We found that compared with entinostat or lapatinib alone, the two drugs in combination synergistically inhibited tumor cell proliferation (P &amp;lt; 0.001), reduced in vitro colony formation (P &amp;lt; 0.05), and resulted in significant in vivo tumor shrinkage or growth inhibition in both IBC and non-IBC xenograft mouse models (SUM190 and BT474, P &amp;lt; 0.001). The synergistic antitumor activity of the entinostat-lapatinib combination was due to downregulation of phosphorylated Akt, which induced the transcriptional activity of FOXO3, resulting in the induction of Bim1 (a BH3 domain-containing pro-apoptotic protein). Furthermore, entinostat sensitized trastuzumab/lapatinib-resistant HER2+ cells to the trastuzumab-lapatinib combination and enhanced the anti-proliferation effect compared with single-agent with lapatinib or combination treatment with lapatinib and trastuzumab. Conclusion: Taken together, our data provide evidence that entinostat has enhanced antitumor effect in combination with the HER2-targeted reagent lapatinib and results in the induction of apoptosis by FOXO3-mediated Bim1 expression. Our findings justify conducting a clinical trial (clinicaltrial.gov, NCT01434303) of combinational treatment with entinostat, lapatinib, and trastuzumab in patients with HER2+ IBC or non-IBC that is resistant to trastuzumab-based treatment. Citation Format: Jangsoon Lee, Chandra Bartholomeusz, Gabriel N Hortobagyi, Peter Ordentlich, Naoto T Ueno. A class I histone deacetylase inhibitor, entinostat, enhances lapatinib efficacy in both HER2-overexpressing inflammatory and non-inflammatory breast cancer cells through FOXO3-mediated Bim1 expression [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-14-01.

A class I histone deacetylase inhibitor, entinostat, enhances lapatinib efficacy in HER2-overexpressing breast cancer cells through FOXO3-mediated Bim1 expression.

Although there are effective HER2-targeted agents, novel combination strategies in HER2-overexpressing breast cancers are needed for patients whose tumors develop drug resistance. To develop new therapeutic strategy, we investigated the combinational effect of entinostat, an oral isoform-selective histone deacetylase type I inhibitor, and lapatinib, a HER2/EGFR dual tyrosine kinase inhibitor, in HER2+ breast cancer cells. We assessed the combinational synergistic effect and its mechanism by CellTiter Blue assay, flow cytometry, anchorage-independent growth, quantitative real-time PCR, small interfering RNA, Western blotting, and mammary fat pad xenograft mouse models. We found that compared with entinostat or lapatinib alone, the two drugs in combination synergistically inhibited proliferation (P < 0.001), reduced in vitro colony formation (P < 0.05), and resulted in significant in vivo tumor shrinkage or growth inhibition in two xenograft mouse models (BT474 and SUM190, P < 0.001). The synergistic anti-tumor activity of the entinostat/lapatinib combination was due to downregulation of phosphorylated Akt, which activated transcriptional activity of FOXO3, resulting in induction of Bim1 (a BH3 domain-containing pro-apoptotic protein). Furthermore, entinostat sensitized trastuzumab/lapatinib-resistance-HER2-overexpressing cells to the trastuzumab/lapatinib combination and enhanced the anti-proliferation effect compare with single or double combination treatment. This study provides evidence that entinostat has enhanced anti-tumor effect in combination with HER2-targeted reagent, lapatinib, and resulting in induction of apoptosis by FOXO3-mediated Bim1 expression. Our finding justifies for conducting a clinical trial of combinational treatment with entinostat, lapatinib, and trastuzumab in patients with HER2-overexpressing breast cancer resistant to trastuzumab-based treatment.

Identification and characterisation of a novel heat shock protein 90 inhibitor ONO4140

Heat shock protein (Hsp) 90 is a key component of the super-chaperone complex that maintains functionally active conformation of various client proteins. Many of these client proteins regulate important nodal points in multiple signalling pathways that promote cancer cell growth and survival. Inhibitors of Hsp90, therefore, have the potential of functioning as anti-cancer agents with pleiotropic effects. Identification of novel Hsp90 inhibitors with more favourable pharmacological properties is a priority in cancer therapy. To achieve this goal, we screened a compound library using a biochemical assay based on refolding of denatured firefly luciferase. The assay revealed high sensitivity, reliability and reproducibility with a Z-factor of 0.81±0.17. Six Hsp90 inhibitory compounds identified by this screening with IC50 values between 1.0 and 6μM were further characterised for anti-proliferative activity by Cell Titer-Blue Cell Viability Assay using multiple tumour cell lines. Of particular interest was ONO4140 with lowest GI50 values in three different cancer cell lines viz; DU-145, BT-474 and K562 cell lines. This study also revealed that short-term exposure of tumour cells with ONO4140 is sufficient to inhibit the catalytic activity of Hsp90, evaluated through disruption of Hsp90-p23 association by immunoprecipitation. This short term exposure appears to initiate events like degradation of Hsp90 client proteins such as ErbB2/Her-2 and Akt with concomitant inhibition of survival signalling leading to the apoptotic death of tumour cells as seen by western blotting and Caspase Glow-3,7 assay. The study also reveals that apoptosis following Hsp90 inhibition with ONO4140 occurs via Caspase9–Caspase3 intrinsic apoptotic pathway, a process that is likely triggered by inactivation of Akt. In conclusion, we have identified a novel class of synthetic compounds which show potent Hsp90 inhibitory action in preclinical studies. The discovery of this novel class of synthetic Hsp90 inhibitors with simple chemical backbone allows us to conduct further structural modifications to improve their potency and pharmacokinetic properties for use in cancer therapy.