Insensitive Cells Research Articles

Abstract 2763: EGFR endocytosis is a rational target in lung cancer with wild-type EGFR

Abstract Oncogenic alterations of epidermal growth factor receptor (EGFR) signaling are frequently observed in lung cancer patients with worse differentiation and poor prognosis. However, the therapeutic efficacy of EGFR-tyrosine kinase inhibitors (TKIs) is currently limited in selected patients with EGFR mutations. Therefore, in this study, we investigated the potential molecular mechanism that contributes to cell viability and the response of gefitinib, one of the EGFR-TKIs, in lung cancer models with wide-type EGFR (wtEGFR). Interestingly, we found that EGF-induced EGFR endocytosis is existed differently between gefitinib-sensitive and -insensitive lung cancer cell lines. Suppressing EGFR endocytos decreased cell viability and increased apoptotic cell death in gefitinib-insensitive lung cancer with wtEGFR in vitro and in vivo. In addition, we found that Rab25 was differentially expressed in between gefitinib-sensitive and -insensitive lung cancer cells. Rab25 knockdown caused the changed EGFR endocytosis and reverted the gefitinib response in gefitinib-sensitive lung cancer with wtEGFR in vitro and in vivo. Taken together, our findings suggest a novel insight that EGFR endocytosis is a rational therapeutic target in lung cancer with wtEGFR, in which the combined efficacy with gefitinib is expected. Furthermore, we demonstrated that Rab25 plays an important role in EGFR endocytosis and gefitinib therapy. Citation Format: Ukhyun Jo, Kyong Hwa Park, Young Mi Whang, Jae Sook Sung, Nam Hee Won, Jong Kuk Park, Yeul Hong Kim. EGFR endocytosis is a rational target in lung cancer with wild-type EGFR. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2763. doi:10.1158/1538-7445.AM2014-2763

Abstract 811: Schweinfurthin activity enhanced by verapamil

Abstract Schweinfurthins are intriguing natural product anti-cancer agents with incompletely defined mechanisms of action. Schweinfurthins increase ER stress and result in apoptosis in vitro. To augment schweinfurthins’ growth inhibitory activity we hypothesized that an inhibitor of P-glycoprotein (Pgp) may increase the intracellular concentration of the schweinfurthins and lead to synergistic growth inhibition. The schweinfurthin sensitive glioblastoma multiforme cell line SF-295 and the relatively schweinfurthin insensitive lung carcinoma cell line A549 were treated with two schweinfurthin isoforms: methyl-G and 3-deoxyschweinfurthin B P-nitro bis stilbene (3dSB-PNBS). Methyl-G and 3dSB-PNBS (1 nM - 1 μM) decreased MTT activity at 48 hours but not at 24-hour incubation in both cell lines. The addition of verapamil (1μM - 1 mM) strongly induced synergy at 24 hours in SF-295 cells, yet verapamil did not greatly potentiate the schweinfurthin effect at 48 hours. This pattern was not evident in A549 cells. Isobologram analysis revealed combination indices of 0.38 and 0.47 at 24 hours in SF-295 cells for methyl-G and 3dSB-PNBS respectively. Fluorescent detection of 3dSB-PNBS demonstrated that verapamil increased the intracellular concentration of 3dSB-PNBS 3-fold in SF-295 and 2-fold in A549 cells. This effect occurred in a Pgp-independent manner as the specific Pgp inhibitor, CP 100356, failed to increase the intracellular concentration of 3dSB-PNBS. Flow cytometric analysis of intracellular calcium release demonstrated that neither methyl-G, verapamil, nor the combination increased intracellular calcium. However, Western blotting revealed that verapamil enhanced the ER stress caused by methyl-G in that there was increased expression of glucose-regulated protein 78 and increased phosphorylation of eukaryotic initiation factor 2α. Pgp expression was increased by verapamil in SF-295 cells, yet this increase was not present when methyl-G was combined with verapamil. Methyl-G also increased the intracellular concentration of a known Pgp substrate, Rhodamine 123 (R123), in SF-295 cells. Pgp requires cholesterol to function properly as a reduction in cellular cholesterol leads to the accumulation of Pgp substrates. Interestingly, since 3dSB depletes intracellular cholesterol and up-regulates the cholesterol efflux pump ABCA1, add-back of cholesterol rescued the methyl-G induced increase in R123 intracellular concentration. These studies demonstrate verapamil is able to potentiate the schweinfurthin growth inhibitory effect by increasing its intracellular concentration and by enhancing schweinfurthin induced ER stress; cholesterol is also identified as a key component of schweinfurthin action. Citation Format: Ryan M. Sheehy, Zoe C. Bachman, Raymond J. Hohl. Schweinfurthin activity enhanced by verapamil. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 811. doi:10.1158/1538-7445.AM2014-811

Abstract 922: Evaluating markers of cisplatin sensitivity and survival in small cell lung cancer

Abstract Background: Molecular characterization of small cell lung cancer (SCLC) can lead to improved outcome through the identification of tumor subtypes with differential clinical outcome and response to treatment. We characterized SCLC cell lines and patient samples by a combination of a genome-wide and a custom expression panel to assess expression changes associated with cisplatin sensitivity, in the case of cell lines, and overall survival, in terms of SCLC patients.Method: SCLC cell lines (H69, H128, H146, H526, H187, H209, D53, DMS153, and DMS114) and 79 archival samples of pulmonary neuroendocrine tumor samples were employed. Sensitivity to cisplatin, and PARP inhibitor, veliparib was determined by MTS assay. In order to identify expression profiles characteristic of sensitive and insensitive cell lines, gene expression for untreated and treated cells was obtained from Illumina microarray gene expression and NanoString platforms. Comparisons of expression were performed between treated cell lines and controls using a combination of ANOVA and empirical variance distributions to assess sensitivity. Prognostic impact of differentially expressed genes in cell lines was assessed in patients using gene expression obtained by NanoString and protein expression by immunohistochemistry (IHC). Kaplan Meier Method, Logrank test, and Cox proportional hazard model were employed for testing differences in survival. Additionally, a quantile survival analyses was performed to identify patient subgroups associated with early and/or late changes in expression.Results: Unsupervised analysis of the Illumina probesets identified a 23 gene panel with differential expression between cell lines with differing platinum sensitivity: Sensitive - GLS, UBEC2, HACL1, MSI2 and LOC100129585; insensitive - CENPE, CRYGS, FAM83D, FLJ44342, GNA12, LOC88523, LRDD, N4BP2L2, SLC35A3, SPC25; shared - AURKA, CENPA, DLGAP5, HMMR, KIF20B, LOC100129585, LOC100131735, RBMX, SFRS3. Unsupervised analysis of expression data from Illumina and nCounter NanoString showed considerable concordance in cluster patterns. The Cox regression models of gene expression on overall survival identified LOC100131735 as a predictive marker and, SLC35A3, SPC25, DLGAP5 and UBE2C genes as prognostic biomarkers. Concomitantly high expression of SLC35A3 and GLS genes characterized SCLC with prolonged survival (Logrank p=0.025) while a trend toward reduced risk of death (HR: 0.798; 95%CI, 0.375-1.700, p=0.559) was observed with high GLS protein expression.Conclusions: Our use of both cell line and patient SCLC data has provided evidence of markers characteristics of cisplatin insensitivity that also show a relation with poor overall survival in patients. Additional studies to fully understand the mechanisms underlying such markers are currently underway. Supported through the Georgia Cancer Coalition Distinguished Scholar Award and NIH 5K23CA164015 grant to TK Owonikoko Citation Format: Taofeek Kunle Owonikoko, Guojing Zhang, Gabriel L. Sica, Zhengjia Chen, Jeffrey M. Switchenko, Sungjin Kim, Anthony A. Gal, Suresh S. Ramalingam, Xingming Deng, Michael R. Rossi, Jeanne Kowalski, Fadlo R. Khuri. Evaluating markers of cisplatin sensitivity and survival in small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 922. doi:10.1158/1538-7445.AM2014-922

Abstract 667: IMGN853, a folate receptor (FR) α-targeting antibody-drug conjugate (ADC), is highly effective against xenograft models with clinically relevant levels of receptor expression

Abstract IMGN853 comprises the anti-FR-α antibody M9346A conjugated to the cytotoxic maytansinoid molecule DM4 via a stable disulfide-containing linker, sulfo-SPDB. IMGN853 is currently in phase I clinical testing; promising clinical results were reported at ASCO 2013. To analyze determinants of the sensitivity of FR-α positive cells to IMGN853, we examined antigen density on the cell surface, internalization and processing of IMGN853, and sensitivity of the cells to IMGN853 and to non-conjugated maytansinoid, S-methyl-DM4, in vitro. Antigen density, internalization and processing were analyzed with 3[H] M9346A. Preliminary experiments demonstrated that 3[H] M9346A is an appropriate surrogate reagent to analyze binding and processing of IMGN853. Sensitivity of the cells to IMGN853 and to the unconjugated maytansinoid was analyzed by a cytotoxicity assay. A panel of seven FR-α positive cell lines was used for the study. Three of the cell lines were sensitive to IMGN853 in an antigen-dependent manner (the cytotoxic effect was blocked in the presence of a saturating excess of the non-conjugated antibody), while four lines were insensitive (the same low cytotoxic activity of IMGN853 was observed with and without the non-conjugated antibody). The proportion of cell surface-bound IMGN853 that was internalized and processed by the sensitive and insensitive cell lines was comparable. The cell lines differed in their FR-α expression, and there was a strong positive correlation between the sensitivity of cells towards IMGN853 and antigen expression on their surface. The cell lines were similar in their sensitivity to S-methyl-DM4. Taken together, the data indicate that the FR-α density on the surface of a cell is a key determinant of its sensitivity to IMGN853. Having observed a correlation between antigen density and sensitivity of the cells to IMGN853, we set out to test IMGN853 activity in xenograft models expressing FR-α at levels comparable to those on patient tumor cells. First, we developed an IHC assay with the use of anti-FR-α antibody BN3.2 (Novocastra Laboratories, UK) that detected various levels of receptor expression in tumor samples. Next, we compared FR-α expression in ovarian carcinoma (OvCa), non-small cell lung carcinoma (NSCLC) samples and in human xenograft models, and examined five models that represented antigen densities of the majority of FR-α positive OvCa and NSCLC patients. We evaluated the anti-tumor activity of IMGN853 in SCID mice bearing these xenograft models after single intravenous injections of 5, 2.5 or 1.2 mg/kg. IMGN853 was highly active with a minimally effective dose from 1.2 to 2.5 mg/kg depending on the model. There was no activity of the conjugate against a FR-α negative xenograft model. Thus, IMGN853 is highly effective against xenograft models with clinically relevant levels of FR-α expression. Citation Format: Olga Ab, Laura M. Bartle, Xiuxia Sun, Rui Wu, Holly A. Johnson, Kathleen R. Whiteman, Alyssa LaBelle, Victor S. Goldmacher. IMGN853, a folate receptor (FR) α-targeting antibody-drug conjugate (ADC), is highly effective against xenograft models with clinically relevant levels of receptor expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 667. doi:10.1158/1538-7445.AM2014-667

Magnolol Causes Alterations in the Cell Cycle in Androgen Insensitive Human Prostate Cancer Cells In Vitro by Affecting Expression of Key Cell Cycle Regulatory Proteins

Prostate cancer, one of the most common cancers in the Western world, affects many men worldwide. This study investigated the effects of magnolol, a compound found in the roots and bark of the magnolia tree Magnolia officinalis, on the behavior of 2 androgen insensitive human prostate cancer cell lines, DU145 and PC3, in vitro. Magnolol, in a 24-h exposure at 40 and 80 μM, was found to be cytotoxic to cells. Magnolol also affected cell cycle progression of DU145 and PC3 cells, resulting in alterations to the cell cycle and subsequently decreasing the proportion of cells entering the G2/M-phase of the cell cycle. Magnolol inhibited the expression of cell cycle regulatory proteins including cyclins A, B1, D1, and E, as well as CDK2 and CDK4. Protein expression levels of pRBp107 decreased and pRBp130 protein expression levels increased in response to magnolol exposure, whereas p16INK4a, p21, and p27 protein expression levels were apparently unchanged post 24-h exposure. Magnolol exposure at 6 h did increase p27 protein expression levels. This study has demonstrated that magnolol can alter the behavior of androgen insensitive human prostate cancer cells in vitro and suggests that magnolol may have potential as a novel anti-prostate cancer agent.

Epibrassinolide-induced apoptosis regardless of p53 expression via activating polyamine catabolic machinery, a common target for androgen sensitive and insensitive prostate cancer cells.

Epibrassinolide (EBR), is a member of the brassinosteroids (BR), has been shown as an apoptotic inducer in different cancer cell lines. We previously showed that EBR induced apoptosis by activating polyamine catabolic pathway, which lead to the accumulation of cytotoxic compounds such as hydrogen peroxide and aldehydes in LNCaP and DU 145 prostate cancer cells. However, we found that LNCaP prostate cancer cells expressing functional androgen receptor (AR) was found more sensitive to EBR than those with non-functional AR (DU 145 cells). To better understand the apoptotic effect of EBR, we aimed to investigate the cellular responses in p53 null, PC3 prostate cancer cells. We showed that EBR induced mitochondria-mediated and caspase-dependent apoptosis in wt and p53 stable transfected PC3 cells, which suggesting that EBR-induced apoptosis regardless of p53 expression. In addition, inhibition of p53 by pifithrin-α orthe activation of Mdm2 by Nutlin-3 co-treatment did not alter EBR induced PARP cleavage. Furthermore, EBR treatment was also induced apoptosis in both LNCaP(wt p53) and DU 145 (mt p53)cells, respectively. These all findings verified that EBR-induced apoptosis regardless of p53 expression. The PA catabolic pathway was also altered in PC3 cells causing the generation of reactive oxygen species (ROS) and intracellular PA pool decrease. However, the silencing of spermidine-spermineacetyltransferase (SSAT), a key enzyme at polyamine catabolic machinery prevented the EBR-induced apoptosis. Therefore, we concluded that EBR-induced apoptosis was mainly related with PA catabolic pathway and independent from p53 expression.

72. Social stress enhances immature neutrophil release from bone marrow in murine Aspergillus fumigatus-induced allergic airway inflammation

It is well established that stress enhances inflammation and exacerbates symptoms in diseases including asthma. Nonetheless, the mechanisms by which stress influences disease progression are unknown. Clinical cases of “severe” asthma have been associated with increased neutrophil trafficking to the lung and increased insensitivity of immune cells to the anti-inflammatory and apoptotic effects of glucocorticoids. We hypothesize that these symptoms can be exacerbated by stress and are potential mechanisms by which patients can become refractory to standard treatment and present with prolonged or dysregulated inflammation. To address this issue, we used a murine repeated social defeat (RSD) paradigm that models stress-induced exacerbation of inflammation and immune cell glucocorticoid insensitivity. For instance, previous studies in an Aspergillus fumigatus (Af) allergic airway inflammation paradigm indicate that RSD increased trafficking of glucocorticoid insensitive immune cells, increased inflammation, and delayed the resolution of inflammation in the lung. Here, we show that RSD increased production of bone marrow-derived granulocytes and increased neutrophil trafficking to the lung. Several distinct populations of neutrophils were quantified including immature neutrophils (CD16int/CD49d-), which were significantly increased in the circulation and lung of RSD and Af challenged mice. Taken together, our data indicate that the stress-induced neutrophilia in the context of allergic airway inflammation may not simply be a marker of disease, but a major factor contributing to RSD exacerbation of pulmonary inflammation and disease pathogenesis.

Антигенные свойства вируса африканской чумы свиней в искусственных и естественных смешанных популяциях

Heterogeneity of African swine fever (ASF) virus is manifested through differences in its physico-chemical, genetic, virulent, haemadsorption and/or antigenic properties. Here some results of studies of ASF virus experimental mixtures and natural isolates are represented based on their haemadsorbing activity and antigen specificity levels which were determined from the data of haemadsorption-inhibition tests (HAIT) and immunological bioassays in pigs. For the investigations, three mixtures of ASFV virulent isolates were prepared: 1) isolates of seroimmunotypes II and IV (Kikassa 74 + Brazil 80), 2) those of seroimmunotypes III and IV (Mozambique 78 + Sao Tome and Principe), and 3) of serotypes I and IV (Diamang + Dnopa Luanda). The mixed in vivo infections were studied in an artificially organized microepizootic outbreak. We found that at infecting animals with mixtures of ASF virus isolates of two different seroimmunotypes, one isolate steadily dominated over the other one for its haemadsorption level, so the typing data for blood samples of pigs contact-infected with mixtures of isolates in HAIT do not correspond to those observed in the immunological bioassays in pigs. The above phenomenon is apparently due to the fact that the biological materials under study contain ASF viruses of seroimmunotypes which are present in the initial mixtures but lost their haemadsorbing properties in the course of the mixed infection of the animals. A principal opportunity of an in vitro recovery of the haemadsorbing potentiality of the non-haemadsorbing variant of the virus present in the mixtures using a method of passage in a porcine bone marrow (PBM) cell culture supplemented with haemadsorption-inhibiting specific sera to isolates that dominated in the mixtures for haemadsoption is shown. At the same time, another approach, namely using alternating passages both in an insensitive cell system (Siberian mountain goat kidney culture) and PBM cells gave no results. Furthermore, we determined that ASF virus can be present in the environment in the form of a population heterogenous with respect to its seroimmunotype. Also, the carried out research work showed that there was a principal opportunity to provide protection against isolate T-67 when immunizing animals with attenuated virus variants prepared from its virulent variants belonging to four seroimmunotypes.

Correlation of Sprouty1 and Jagged1 With Aggressive Prostate Cancer Cells With Different Sensitivities to Androgen Deprivation

Prostate cancer is a heterogeneous disease and thus, it is important to understand whether among the heterogeneous collection of cell types, androgen-deprivation insensitive cells exist prior to hormonal manipulation. We established several LNCaP subclones with distinct insensitivities to androgen deprivation from a parental LNCaP cell line. In the resulting clones, the sensitivity to androgen-deprivation negatively correlated with their PSA expression levels. In two of these clones, an androgen insensitive clone, LNCaP-cl1, and an androgen sensitive clone, LNCaP-cl5, the DNA copy number differed significantly, indicating that these clones contain genetically distinct cells. LNCaP-cl1 had higher PSA expression but lower invasiveness and tumor growth potential than LNCaP-cl5. The expression levels of two genes that are known to be regulated by miR-21, an androgen-regulated microRNA, Sprouty1 (SPRY1) and Jagged1 (JAG1) were significantly lower in LNCaP-cl1 than in LNCaP-cl5. Knocking down SPRY1 in LNCaP cells enhanced PSA expression and cell proliferation. JAG1 administration in LNCaP cells enhanced cell invasion and JAG1 knockdown in PC3 cells suppressed cell invasion and tumor formation. These results indicated that the expression differences in SPRY1 and JAG1 may contribute to the phenotypic differences between the LNCaP-cl1 and LNCaP-cl5 clones. In tissue samples, SPRY1 expression levels were significantly lower in prostate cancer patients with PSA recurrence after surgical treatment (P = 0.0076) and JAG1 expression levels were significantly higher in Gleason sum (GS) 8-9 disease than in GS 5-6 (P = 0.0121). In summary a random population of LNCaP cells comprises a heterogeneous group of cells with different androgen-deprivation sensitivities and potential for invasiveness.

Regulation of GLUT transporters by flavonoids in androgen-sensitive and -insensitive prostate cancer cells.

Cancer cells show different metabolic requirements from normal cells. In prostate cancer, particularly, glycolytic metabolism differs in androgen-responsive and nonresponsive cells. In addition, some natural compounds with antiproliferative activities are able to modify glucose entry into cells by either modulating glucose transporter (GLUT) expression or by altering glucose binding. The aim of this work was to study the regulation of some GLUTs (GLUT1 and GLUT4) in both androgen-sensitive (LNCaP) and -insensitive (PC-3) prostate cancer cells by 4 structurally different flavonoids (ie, genistein, phloretin, apigenin, and daidzein). Glucose uptake was measured using nonradiolabeled 2-deoxyglucose. The evaluation of protein levels as well as subcellular distribution of GLUT1/4 were analyzed by Western blot and immunocytochemistry, respectively. Androgen-insensitive LNCaP-R and androgen-sensitive PC-3-AR cells were used to study the effect of androgen signaling. Additionally, a docking simulation was employed to compare interactions between flavonoids and XylE, a bacterial homolog of GLUT1 to -4. Results show for the first time the presence of functionally relevant GLUT4 in prostate cancer cells. Furthermore, differences in GLUT1 and GLUT4 levels and glucose uptake were found, without differences on subcellular distribution, after incubation with flavonoids. Docking simulation showed that all compounds interact with the same location of transporters. More importantly, differences between androgen-sensitive and -insensitive prostate cancer cells were found in both GLUT protein levels and glucose uptake. Thus, phenotypic characteristics of prostate cancer cells are responsible for the different effects of these flavonoids in glucose uptake and in GLUT expression rather than their structural differences, with the most effective in reducing cell growth being the highest in modifying glucose uptake and GLUT levels.

SU‐E‐J‐31: Biodynamic Imaging of Cancer Tissue and Response to Chemotherapy

Purpose:To measure intracellular motions inside three‐dimensional living cancer tissue samples to establish a novel set of biodynamic biomarkers that assess tissue proliferative activity and sensitivity or resistance to chemotherapy.Methods:Biodynamic imaging (BDI) uses digital holography with low‐coherence low‐intensity light illumination to construct 3D holograms from depths up to a millimeter deep inside cancer tissue models that include multicellular tumor spheroids and ex vivo cancer biopsies from canine non‐Hodgkins lymphoma and epithelial ovarian cancer (EOC) mouse explants. Intracellular motions modulate the holographic intensity with frequencies related to the Doppler effect caused by the motions of a wide variety of intracellular components. These motions are affected by applied therapeutic agents, and BDI produces unique fingerprints of the action of specific drugs on the motions in specific cell types. In this study, chemotherapeutic agents (doxorubicin for canine lymphoma and oxoplatin for ovarian) are applied to the living tissue models and monitored over 10 hours by BDI.Results:Multicellular spheroids and patient biopsies are categorized as either sensitive or insensitive to applied therapeutics depending on the intracellular Doppler signatures of chemotherapy response. For both lymphoma and EOC there is strong specificity to the two types of sensitivities, with sensitive cell lines and biopsies exhibiting a global cessation of proliferation and strong suppression of metabolic activity, while insensitive cell lines and biopsies show moderate activation of Doppler frequencies associated with membrane processes and possible membrane trafficking.Conclusion:This work supports the hypothesis that biodynamic biomarkers from three‐dimensional living tumor tissue, that includes tissue heterogeneity and measured within 24 hours of surgery, is predictive of near‐term patient response to therapy. Future work will correlate biodynamic biomarkers with progression free survival times.This work is supported by NIH 1R01EB016582 and NSF 1263753‐CBET. Nolte, Turek and An have a financial interest in Animated Dynamics, Inc. that will be licensing technology from Purdue University.

P0224 A novel, small molecule inhibitor, MSU-1001, in androgen-sensitive prostate cancer cell lines

Introduction Prostate cancer is one of the leading causes of cancer-related death in men worldwide. Current treatment options are limited to traditional chemotherapy, surgery, radiation, or hormone therapy, which often have unwanted side effects. There is a need to develop safer molecules that attenuate tumour progression without adversely affecting the overall quality of life. We report the in vitro activity of a novel, synthetic, small molecule, with potential to be a lead candidate for the treatment of prostate cancer. Methods A library of novel small molecule inhibitors was developed through rational drug design. A pyrrolo-pyrimidine derivative (MSU-1001) was identified as a potential lead molecule. Prostate and colon cancer cell lines (LNCap, HT-29, and HCT-8) were obtained from the American Type Culture Collection (ATCC). Cells were incubated with various concentrations of MSU-1001 for 72 h. Growth inhibition was determined colorimetrically on a plate-reader using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Findings Colon cancer cell lines HCT-8 and HT-29 were used in conjunction with the androgen-sensitive human prostate adenocarcinoma cell line, LNCaP, to investigate the specificity of MSU-1001 on tumour type. Among the library of compounds tested, MSU-1001 was pursued based on its anti-proliferative activity in LNCaP cells. Addition of MSU-1001 to LNCaP cells resulted in a dose-dependent inhibition of proliferation (GI 50 = 161 nM) with a near-complete inhibition of cell growth noted at 10 μM. By contrast, GI 50 of gemcitabine was more than 6-fold higher (GI 50 = 1.21 μM) in LNCaP cells. Additionally, GI 50 of MSU-1001 in HCT-8 and HT-29 was 1.8 and 8.7 μM, respectively, indicating relative specificity of MSU-1001 against prostate cancer. Interpretation These data show the therapeutic potential of MSU-1001 in prostate cancer. Combination studies of MSU-1001 with testosterone, 17 β -estradiol, and dehydroepiandro-stenone in androgen-sensitive and insensitive prostate cancer cell lines are currently ongoing.

EGFR endocytosis is a novel therapeutic target in lung cancer with wild-type EGFR

Oncogenic alterations of epidermal growth factor receptor (EGFR) signaling are frequently observed in lung cancer patients with worse differentiation and poor prognosis. However, the therapeutic efficacy of EGFR-tyrosine kinase inhibitors (TKIs) is currently limited in selected patients with EGFR mutations. Therefore, in this study, we investigated the potential molecular mechanism that contributes to cell viability and the response of gefitinib, one of the EGFR-TKIs, in lung cancer models with wide-type EGFR (wtEGFR). Interestingly, we found that EGF-induced EGFR endocytosis is existed differently between gefitinib-sensitive and -insensitive lung cancer cell lines. Suppressing EGFR endocytos decreased cell viability and increased apoptotic cell death in gefitinib-insensitive lung cancer with wtEGFR in vitro and in vivo. In addition, we found that Rab25 was differentially expressed in between gefitinib-sensitive and -insensitive lung cancer cells. Rab25 knockdown caused the changed EGFR endocytosis and reverted the gefitinib response in gefitinib-sensitive lung cancer with wtEGFR in vitro and in vivo. Taken together, our findings suggest a novel insight that EGFR endocytosis is a rational therapeutic target in lung cancer with wtEGFR, in which the combined efficacy with gefitinib is expected. Furthermore, we demonstrated that Rab25 plays an important role in EGFR endocytosis and gefitinib therapy.

Comparison of 4-hydroxynonenal-induced p53-mediated apoptosis in prostate cancer cells LNCaP and DU145.

Aim of the studyTo explore the mechanism of oxidative stress in the development of prostate cancer, here we compared 4-hydroxynonenal (4-HNE)- treated LNCaP (hormone-sensitive) and DU145 (hormone insensitive) cells with significant differences in sensitivity to androgen.Material and methodsThe prostate cancer cell line LNCaP and late cell line DU145 were treated with different concentrations of 4-HNE. The cell proliferation, apoptosis and mitochondrial transmembrane potential were detected at different time points, and expression of related molecules in cell proliferation and apoptosis signal pathway was analyzed by Western blot, and the over-expression of glutathione S-transferase (GSTA-4) was used to validate the changes of the effects of 4-HNE on the two kinds of cells.ResultsLNCaP cells showed greater antiproliferative and proapoptotic activities of HNE in a time- and dose-dependent manner corresponding to the activation of p53-mediated intrinsic apoptotic signaling, but JNK activation was not observed. In contrast, HNE-treated DU145 cells showed less apoptosis and proliferation was not inhibited; instead there was sustained activation of JNK, but activation of p53, p-p53, p21, Bax and caspase-3 was not observed. In addition, their effect of induction of apoptosis can be inhibited by overexpression of GSTA-4.ConclusionsThese studies suggest that 4-HNE promotes prostate cancer cell apoptosis through the p53 signaling pathway; the differences of sensitivity to 4-HNE in LNCaP and DU145 cells may be related to the androgen sensitivity of prostate cancer cells; and the 4-HNE-induced p53-mediated apoptosis signal is regulated by GSTA-4.

Pharmacological and genomic profiling identifies NF-κB–targeted treatment strategies for mantle cell lymphoma

Mantle cell lymphoma (MCL) is an aggressive malignancy that is characterized by poor prognosis. Large-scale pharmacological profiling across more than 100 hematological cell line models identified a subset of MCL cell lines that are highly sensitive to the B cell receptor (BCR) signaling inhibitors ibrutinib and sotrastaurin. Sensitive MCL models exhibited chronic activation of the BCR-driven classical nuclear factor-κB (NF-κB) pathway, whereas insensitive cell lines displayed activation of the alternative NF-κB pathway. Transcriptome sequencing revealed genetic lesions in alternative NF-κB pathway signaling components in ibrutinib-insensitive cell lines, and sequencing of 165 samples from patients with MCL identified recurrent mutations in TRAF2 or BIRC3 in 15% of these individuals. Although they are associated with insensitivity to ibrutinib, lesions in the alternative NF-κB pathway conferred dependence on the protein kinase NIK (also called mitogen-activated protein 3 kinase 14 or MAP3K14) both in vitro and in vivo. Thus, NIK is a new therapeutic target for MCL treatment, particularly for lymphomas that are refractory to BCR pathway inhibitors. Our findings reveal a pattern of mutually exclusive activation of the BCR-NF-κB or NIK-NF-κB pathways in MCL and provide critical insights into patient stratification strategies for NF-κB pathway-targeted agents.

Lenalidomide Promotes CRBN-Mediated Ubiquitination and Degradation of IKZF1 and IKZF3

Lenalidomide is a highly effective drug for the treatment of multiple myeloma and has activity in additional B cell lymphomas. Lenalidomide has been shown to bind the CRBN-DDB1 E3 ubiquitin ligase, but it is unknown how lenalidomide alters the activity of this enzyme complex, and how this leads to therapeutic efficacy. We used a combination of quantitative proteomic approaches to demonstrate that lenalidomide acts by a novel mechanism of action for a therapeutic agent: in multiple myeloma cells, lenalidomide increases the binding of two substrates, IKZF1 (Ikaros) and IKZF3 (Aiolos), to the CRBN substrate adaptor; increases the ubiquitination of these substrates; and causes the targeted degradation of these transcription factors that are essential for the differentiation and survival of plasma cells including multiple myeloma cells.To identify targets of the CRBN-DDB1 ubiquitin ligase that are altered by lenalidomide, we applied SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative mass spectrometry studies to globally assess changes in ubiquitination and proteome levels in the multiple myeloma cell line MM1S. Two members of the Ikaros transcription factor family, IKZF1 and IKZF3, were differentially ubiquitinated and decreased after lenalidomide treatment. Subsequent validation experiments in various cell lines demonstrated that lenalidomide, thalidomide, and pomalidomide cause a decrease of endogenous and ectopically expressed IKZF1 and IKZF3 protein levels but not mRNA levels. Furthermore, we confirmed that IKZF1 and IKZF3 bind CRBN in the presence of lenalidomide, supporting CRBN's role as a substrate adaptor. Consistent with this, shRNA mediated knockdown or overexpression of a CRBN mutant (CRBNYWAA) that does not bind lenalidomide abrogated lenalidomide's effect on IKZF1 and IKZF3. Moreover, CRBN promoted IKZF3 ubiquitination in vitro in the presence of lenalidomide, demonstrating that it is an enzymatic substrate.Using deletion mutants of IKZF3 we identified a 58-amino-acid degron in the N-terminal zinc finger domain that is sufficient for lenalidomide-induced degradation. Based on sequence alignment of that region between lenalidomide responding Ikaros proteins IKZF1 and IKZF3 vs. non-responding IKZF2, IKZF4 and IKZF5 we substituted a single amino acid (IKZF3Q147H) that prevented binding of IKZF3 to CRBN and conferred resistance to lenalidomide induced degradation.IKZF1 and IKZF3 are essential transcription factors for terminal B cell differentiation. We evaluated the biological effects of IKZF1 and IKZF3 loss using shRNAs in a variety of cell lines. IKZF1 and IKZF3 specific shRNAs inhibited the growth of multiple myeloma cell lines while lenalidomide insensitive cell lines derived from other hematopoietic neoplasms were unaffected. Similarly, a dominant negative IKZF3 mutant resulted in growth inhibition of MM1S cells. In contrast, expression of IKZF3Q147Hconferred lenalidomide resistance to MM1S cells.Lenalidomide induces IL-2 expression and release in T cells. We found that lenalidomide induced a dose-dependent decrease of IKZF1 and IKZF3 protein levels in primary human T cells. Previous studies have shown that IKZF3 is a transcriptional repressor of IL-2. To further evaluate the effect of IKZF3 loss, we transduced primary human T cells with shRNAs targeting either IKZF3 or control. IL2 RNA levels increased 3.3 fold after lenalidomide treatment in T cells expressing control shRNAs. In contrast, the baseline IL2 RNA level in T cells transduced with IKZF3 specific shRNAs was 3.7 fold higher compared to controls and this effect could not be further stimulated by lenalidomide.In conclusion, selective targeting of two lymphoid transcription factors, IKZF1 and IKZF3, explains lenalidomide's selective growth inhibition in multiple myeloma and likely other B cell lymphomas as well as its immunomodulatory effects in T cells. Furthermore, selective ubiquitination and degradation of specific targets provides a novel mechanism of therapeutic activity for proteins that are not otherwise amenable to small-molecule inhibition. Disclosures:Ebert:Celgene: Membership on an entity's Board of Directors or advisory committees.

Abstract C20: In vitro effects of Debio 1143, a novel oral IAP inhibitor, in human SCCHN cell lines and tumor specimens.

Abstract Background: Resistance to radiotherapy and chemotherapy-induced apoptosis is a hallmark of cancer. Inhibitors of apoptosis proteins (IAPs) are negative modulators of apoptosis frequently expressed in various cancers, and, as such, attractive targets to overcome resistance to cancer therapy. The oral SMAC mimetic Debio 1143 (D1143, a.k.a AT-406), an antagonist of multiple IAPs (cIAP1/2 and XIAP), is currently investigated in a Phase I oncology clinical trial. This study aimed at evaluating D1143 activity as a single agent and in combination with TNF-α, TRAIL, cisplatin or carboplatin, in various SCCHN models. Materials and Methods: The antiproliferative effects of D1143 were evaluated in a panel of 5 human SCCHN cell lines by MTT assay. Baseline and phosphorylated protein levels were detected by Western blot analysis. Tumor samples from SCCHN patients were surgically resected and cut into 300 µm thick slices using a tissue slicer (TIPCAN®). Each slice was exposed to 10 µM D1143 and/or 1 µM of platinum-based drug for 48 hours. Tumor samples were analyzed by immunohistochemistry (IHC) or immunofluorescence to visualize the effects of the treatment on various biomarkers of cell apoptosis, proliferation, and drug target engagement. Results: A panel of 5 SCCHN cell lines was characterized for the expression of c-IAP1/2, XIAP, Bcl2, LRIG1, and other proteins implicated in resistance to cell death. D1143 alone displayed limited antiproliferative activity in only one cell line (Detroit 562). However, two SCCHN cell lines were sensitive to D1143 and TRAIL combined (SQ20B and SCC15), three were sensitive to D1143 and TNF-α combined (SQ20B, SCC61, and Detroit 562), and one was resistant to both combinations (HEP2). In Detroit 562 sensitive cells, 10 µM D1143 induced sustained cIAP1/2 degradation after 15 minutes of exposure. In contrast, in the HEP2 insensitive cell line, D1143 induced slight and transient inhibition of cIAP1/2. IHC analyses revealed that ex vivo exposure to D1143 of tumor explants freshly resected from SCCHN patients decreased cIAP1 staining. Treatment of tumor samples with D1143 combined with cisplatin or carboplatin augmented the cleavage of caspase 3 compared to controls suggesting induction of apoptosis. Conclusion: In 4 out of 5 SCCHN cell lines, D1143 induced cIAP1/2 degradation and potentiated TNF-α or TRAIL-induced antiproliferative effects. D1143 combined with carboplatin and cisplatin in SCCHN patient samples induced caspase 3-dependent apoptosis. D1143 in combination with conventional chemotherapies may be considered as a potential treatment for SCCHN patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C20. Citation Format: Marie Serova, Annemilaï Tijeras-Raballand, Sebastien Albert, Sandrine Faivre, Eric Raymond, Anne Vaslin, Claudio Zanna, Gregoire Vuagniaux, Armand de Gramont. In vitro effects of Debio 1143, a novel oral IAP inhibitor, in human SCCHN cell lines and tumor specimens. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C20.

Establishing a High-Throughput and Automated Cancer Cell Proliferation Panel for Oncology Lead Optimization

Tumor cell proliferation assays are widely used for oncology drug discovery, including target validation, lead compound identification, and optimization, as well as determination of compound off-target activities. Taking advantage of robotic systems to maintain cell culture and perform cell proliferation assays would greatly increase productivity and efficiency. Here we describe the establishment of automated systems for high-throughput cell proliferation assays in a panel of 13 human tumor cell lines. These cell lines were selected from various types of human tumors containing a broad range of well-characterized mutations in multiple cellular signaling pathways. Standard procedures for cell culture and assay performance were developed and optimized in each cell line. Moreover, in-house developed software (i.e., Toolset, Curvemaster, and Biobars) was applied to analyze the data and generate data reports. Using tool compounds, we have shown that results obtained through this panel exhibit high reproducibility over a long period. Furthermore, we have demonstrated that this panel can be used to identify sensitive and insensitive cell lines for specific cancer targets, to drive cellular structure-activity relationships, and to profile compound off-target activities. All those efforts are important for cancer drug discovery lead optimization.

Retargeting T Cells for HER2-Positive Tumor Killing by a Bispecific Fv-Fc Antibody

To exploit the biological and pharmacological properties of immunoglobulin constant domain Fc fragment and increase the killing efficacy of T cells, a single chain variable fragment specific to CD3 was fused with Fcab (Fc antigen binding), a mutant Fc fragment with specificity against Human epidermal growth factor receptor 2 (HER2) developed by F-star. The bispecific fusion named as FcabCD3 was expressed by transient transfection in HEK-293T cells and purified by affinity chromatography. Specific cytolytic activity of retargeted T cells to kill HER2 positive SKBR3 cell line was evaluated in vitro. FcabCD3 was able to retarget T cells to kill both Herceptin insensitive Colo205-luc cell line and HER2 low expression MDA-MB-231-luc cell line. Furthermore, FcabCD3 was effective in eliminating the Colo205 tumor established on BALB/c nu/nu mice.

Metabolism, mechanism of action and sensitivity profile of fluorocyclopentenylcytosine (RX-3117; TV-1360)

A novel cytidine analog fluorocyclopentenylcytosine (RX-3117; TV-1360) was characterized for its cytotoxicity in a 59-cell line panel and further characterized for cytotoxicity, metabolism and mechanism of action in 15 additional cancer cell lines, including gemcitabine-resistant variants. In both panels sensitivity varied 75-fold (IC50: 0.4- > 30 μM RX-3117). RX-3117 showed a different sensitivity profile compared to cyclopentenyl-cytosine (CPEC) and azacytidine, substrates for uridine-cytidine-kinase (UCK). Dipyridamole, an inhibitor of the equilibrative-nucleoside-transporter protected against RX-3117. Uridine and cytidine protected against RX-3117, but deoxycytidine (substrate for deoxycytidine-kinase [dCK]) not, although it protected against gemcitabine, demonstrating that RX-3117 is a substrate for UCK and not for dCK. UCK activity was abundant in all cell lines, including the gemcitabine-resistant variants. RX-3117 was a very poor substrate for cytidine deaminase (66,000-fold less than gemcitabine). RX-3117 was rapidly metabolised to its nucleotides predominantly the triphosphate, which was highest in the most sensitive cells (U937, A2780) and lowest in the least sensitive (CCRF-CEM). RX-3117 did not significantly affect cytidine and uridine nucleotide pools. Incorporation of RX-3117 into RNA and DNA was higher in sensitive A2780 and low in insensitive SW1573 cells. In sensitive U937 cells 1 μM RX-3117 resulted in 90% inhibition of RNA synthesis but 100 μM RX-3117 was required in A2780 and CCRF-CEM cells. RX-3117 at IC50 values did not affect the integrity of RNA. DNA synthesis was completely inhibited in sensitive U937 cells at 1 μM, but in other cells even higher concentrations only resulted in a partial inhibition. At IC50 values RX-3117 downregulated the expression of DNA methyltransferase. In conclusion, RX-3117 showed a completely different sensitivity profile compared to gemcitabine and CPEC, its uptake is transporter dependent and is activated by UCK. RX-3117 is incorporated into RNA and DNA, did not affect RNA integrity, depleted DNA methyltransferase and inhibited RNA and DNA synthesis. Nucleotide formation is related with sensitivity.