Non-amplified Cells Research Articles

Evaluation of a novel c-MET based circulating tumor cell (CTC) biomarker in patients with gastrointestinal malignancies.

84 Background: Multiple GI malignancies including gastric, pancreatic, and colorectal cancers harbor genetic alterations in the MET oncogene. MET amplification is associated with poor prognosis, and c-MET targeted therapies are in development. We aimed to capture and identify c-MET CTCs using a non-invasive, rapid test. Methods: We modified the CELLSEARCH platform by using nanomagnetic particles conjugated to antibodies against the extracellular domain of c-MET to capture c-MET-expressing CTCs. The method was validated by spiking MET-amplified gastric cancer cells, c-MET-overexpressing, nonamplified cancer cells, and MET-negative cancer cells into peripheral blood from healthy volunteers. Peripheral blood samples were obtained from patients (pts) with refractory metastatic gastric, pancreatic, and colorectal cancers, prepared in duplicate, and characterized by immunofluorescence staining for intracellular c-MET-PE, DAPI, and pan-CK-FITC. CD45 + cells were excluded. CTC enumeration with c-MET capture was compared to EpCAM capture. After CTC isolation, MET DNA FISH was performed. Results: The novel c-MET CTC assay was found to be 80% sensitive for MET-amplified cells, 40-80% sensitive for c-MET-overexpressed cells, and 100% specific for c-MET negative cells and in 10 healthy volunteers. In 7/7 pts with metastatic pancreatic and 8/8 pts with metastatic colorectal cancers, we did not capture c-MET CTCs. Of 3 gastric cancer pts tested thus far, one pt had a significant number of c-MET + CTCs, with 52 CTCs and 90 CTCs in each of the duplicate samples, compared to 20 CTCs with EpCAM capture. CTC FISH demonstrated polysomy 7 and METamplification. This pt had HER2 amplification in primary tumor tissue and had progressed on FOLFOX and trastuzumab at the time of enrollment. Conclusions: We have developed a sensitive and specific c-MET CTC assay. c-MET-expressing CTCs can be detected in gastric cancer but not controls or other GI cancers to date. While not detectable in many pts, high levels in one pt support the importance of MET amplification as a resistance mechanism to HER2 therapy, and suggest that this approach may be useful to identify and follow patients who may be candidates for c-MET directed therapies.

Inhibition of autophagy potentiates the efficacy of Gli inhibitor GANT-61 in MYCN-amplified neuroblastoma cells.

BackgroundAberrant Hedgehog (Hh) signaling is often associated with neuroblastoma (NB), a childhood malignancy with varying clinical outcomes due to different molecular characteristics. Inhibition of Hh signaling with small molecule inhibitors, particularly with GANT-61, significantly suppresses NB growth. However, NB with MYCN amplification is less sensitive to GANT-61 than those without MYCN amplification.MethodsAutophagic process was examined in two MYCN amplified and two MYCN non-amplified NB cells treated with GANT-61. Subsequently, chemical and genetic approaches were applied with GANT-61 together to evaluate the role of autophagy in GANT-61 induced cell death.ResultsHere we show that GANT-61 enhanced autophagy in MYCN amplified NB cells. Both an autophagic inhibitor 3-methyladenine (3-MA) and genetic disruption of ATG5 or ATG7 expression suppressed GANT-61 induced autophagy and significantly increased apoptotic cell death, whereas pre-treatment with an apoptotic inhibitor, Z-VAD-FMK, rescued GANT-61 induced cell death and had no effect on the autophagic process. In the other hand, GANT-61 barely induced autophagy in MYCN non-amplified NB cells, but overexpression of MYCN in MYCN non-amplified NB cells recapitulated GANT-61 induced autophagy seen in MYCN amplified NB cells, suggesting that the level of GANT-61 induced autophagy in NB cells is related to MYCN expression level in cells.ConclusionAberrant Hh signaling activation as an oncogenic driver in NB renders inhibition of Hh signaling an effective measure to suppress NB growth. However, our data suggest that enhanced autophagy concomitant with Hh signaling inhibition acts as a pro-survival factor to maintain cell viability, which reduces GANT-61 efficacy. Besides, MYCN amplification is likely involved in the induction of the pro-survival autophagy. Overall, simultaneous inhibition of both Hh signaling and autophagy could be a better way to treat MYCN amplified NB.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-768) contains supplementary material, which is available to authorized users.

Abstract B61: Targeting glycolysis and compensatory mitochondrial metabolism in neuroblastoma with FDA-approved ritonavir and metformin

Abstract One of the hallmarks of cancer cell development is increased dependence on glucose to fuel aerobic glycolysis for the increased production of cellular metabolites required for rapid cell division. [1] PET with fluorine-18 fluorodeoxyglucose (18F-FDG) exploits this increased aerobic glycolysis of malignant as compared with most normal cells. There is increasing literature to support the utility of PET scans in the diagnosis, staging and monitoring response of neuroblastoma (NBL) to treatment. Glucose is transported via a family of ATP-independent transporters (GLUTs). GLUTs 1, 3 and 4 are high affinity glucose transporters which have been shown to play roles in normal B cell glucose transport [2]. GLUT4 plays a critical role in facilitating glucose transport in hematological malignancies such as multiple myeloma [3]. This study focuses on identifying glucose transporters that may be aberrantly expressed in NBL cells. We have further used HIV protease inhibitor (ritonavir) that has an off-target inhibitory effect on GLUT4 to inhibit glucose transport in these cells. In addition we hypothesized that the cells, which are resistant to ritonavir, are sensitized upon treatment with a complex 1 inhibitor (metformin) that targets compensatory mitochondrial metabolism. Methods: We tested the dependence of NBL cells on glucose by culturing six different cell lines under aerobic conditions in the presence and absence of glucose. These cell lines included both N-myc amplified and non-amplified cell lines. To determine the transporters involved in transport of glucose across the cell membrane we performed PCR and Western Blot for GLUT 1, 4 and 3, the major high affinity glucose transporters in various NBL cell lines. Based on the results of these tests and preliminary data we used lentivirus sh-RNA and knocked down GLUT 4 in the IMR 5 cell line. To further prove our hypothesis, we cultured cell lines in the presence and absence of metformin, ritonavir and both metformin and ritonavir both and determined viability using flow-cytometry analysis. Results: The number of live cells in all the cell lines decreased considerably when deprived of glucose. Western Blot and PCR for various glucose transporters showed that GLUT 4 was highly expressed across all cell lines while the expression of GLUT 1 was variable. On knocking down GLUT 4 using lentivirus sh-RNA in the IMR 5 cell line we observed a large difference between the morphology of the 2 groups ( control sh-RNA and the ones with GLUT 4 knocked down). The cells where GLUT 4 had been knocked down were found to be more vacuolated and failed to form any neural processes which is a characteristic of this cell line at the end of 72 hours. We also observed that the cell line IMR5 (NMYC amplified) showed marked cell death in the presence of metformin and ritonavir compared to SHY5Y ( single copy of NMYC) where the cell death was not so remarkable. Conclusions: NBL cells are highly dependent on glycolysis for their metabolism and to maintain viability. Inhibition of glycolysis through the inhibition of glucose flux into the cells via glucose transporters results in cell death. Further studies to validate this hypothesis and to test in in vivo are underway in our laboratory.

Abstract 805: Disruption of the MDM2-p53 interaction synergizes with MEK inhibition to induce cell death and promote tumor regression in p53 wild-type, Ras or Raf-mutant tumor models

Abstract Disruption of the interaction between p53 and MDM2 with small molecules, and subsequent reactivation of p53, is an attractive treatment strategy for p53 wild-type tumors that has shown striking pre-clinical activity in models exhibiting genomic amplification of the MDM2 gene. However, MDM2 amplified tumors represent only a small proportion of the p53 wild-type tumor population, and single agent regressions may be limited outside of the MDM2 amplified context. In order to increase the potential clinical benefit of MDM2-p53 protein-protein interaction (PPI) inhibitors beyond tumors exhibiting amplification of MDM2, we performed an enhancer library combination screen to identify optimal combination partners for MDM2-p53 PPI inhibitors in p53 wild-type, MDM2 non-amplified tumor cells. The MDM2-p53 PPI inhibitor SAR405838 and a related molecule were examined in combination with an enhancer library of 200 oncology drugs representing a broad molecular diversity of targets across a panel of twenty p53 wild-type cancer cell lines. Analysis results showed that MEK inhibitors were the most statistically significant synergistic combination partners in this screen, with synergy observed in cell lines harboring mutations that activated MAP kinase pathway signaling. These synergy results were validated in independent experiments using the ray design methodology, and extended to multiple K-ras, N-ras and B-raf mutant contexts in vitro. In K-ras, N-ras or B-raf mutant cell lines, the combination of SAR405838 with the MEK inhibitor pimasertib resulted in p53 pathway activation, inhibition of pERK levels, induction of the apoptotic mediators PUMA and BIM, induction of phosphatidyl serine exposure, and induction of caspase activity. In the Ras/Raf wild-type cell line MCF7, no synergy was observed. In cell line and patient-derived xenografts in vivo, the combination of SAR405838 and pimasertib resulted in greater anti-tumor activity than single agents and induced regression in K-ras, N-ras and B-raf mutant tumor models derived from different tissue types, including complete regressions in multiple models. These data indicate that MEK inhibitors synergize with MDM2-p53 PPI inhibitors in tumor models that exhibit activated MAP kinase pathway signaling and p53 wild-type status. Given the relative lack of overlapping clinically significant toxicities of MDM2-p53 PPI inhibitors and MEK inhibitors, such a combination merits further investigation for the treatment of K-ras, N-ras, or B-raf mutant, p53 wild-type tumors, representing a large patient population with significant unmet medical need. Citation Format: Isabelle Meaux, Jean-Paul Nicolas, Steve Rowley, Sukhvinder Sidhu, Francoise Herve, Laurent Dassencourt, Fanny Windenberger, Dimitri Gorge-Bernat, Pascal Pannier, Donald Bergstrom, Laurent Debussche, James Watters. Disruption of the MDM2-p53 interaction synergizes with MEK inhibition to induce cell death and promote tumor regression in p53 wild-type, Ras or Raf-mutant tumor models. [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 805. doi:10.1158/1538-7445.AM2014-805

Abstract 2923: Identification and validation of dysregulated MAPK7 (ERK5) as a novel oncogenic target in squamous cell lung and esophageal carcinoma

Abstract MAPK7/ERK5 (extracellular-signal-regulated kinase 5) is the effector kinase of a canonical three-tiered MAPK (mitogen activated protein kinase) signalling cascade comprising MEK (MAPK/ERK kinase) 5, MEKK(MEK kinase) 2/3 and ERK5 itself(1). Under normal physiological conditions, MEK5 and ERK5 are ubiquitously expressed and are activated by growth factors and cellular stresses to perform roles in blood vessel, cardiac and muscle development(2). Although the MAPK7-axis is the least well studied of the MAPK-signaling pathways, evidence suggests that it plays a role in the pathology of cancer, in particular; breast cancer progression, prostate cancer bone metastases and hepatocellular carcinoma(3-5). Array-based comparative genomic hybridisation profiling (aCGH) of non-small cell lung cancer (NSCLC) patient tumor samples initially identified MAPK7 gene amplification as a novel genetic event. To further explore the role of MAPK7 in human cancer, we assessed the true incidence of MAPK7 gene amplification using fluorescence in situ hybridization (FISH) analysis in a cohort of 80 NSCLC and 95 squamous esophageal cancers (sqEC). High level MAPK7 gene amplification was detected in 4% (3/80) of NSCLC (enriched to 6% (3/49) in squamous cell carcinoma) and 2% (2/95) of sqEC. Immunohistochemical (IHC) analysis of the same tissue samples revealed a good correlation of MAPK7 gene amplification and high level protein expression but importantly, also identified a high prevalence of IHC ‘3+’ MAPK7 protein expression (20% - 15/74 NSCLC) in the absence of gene amplification. To validate MAPK7 as an oncogenic driver of tumor cell proliferation, we performed in vitro siRNA knockdown of MAPK7 in amplified and non-amplified tumor cell lines. Potent anti-proliferative effects were only observed when MAPK7 protein expression was ablated in MAPK7-amplified lines (eg. KYSE30). To support drug discovery efforts, a novel MEK5/MAPK7 co-transfected HEK293 cell line was developed and used for routine cell-based pharmacodynamic screening. In parallel, reverse-phase protein arrays (RPPA) were used to identify novel candidate downstream pharmacodynamic biomarkers of MAPK7 kinase inhibition in tumor cells (pMEF2A and pMEF2C). Finally, a novel MAPK7-amplified esophageal xenograft model was established and used to demonstrate direct pharmacodynamic modulation of pMAPK7 using a commercially available tool compound (XMD8-92). Taken together, these data highlight a broader role for dysregulated MAPK7 in driving tumorigenesis within niche populations of highly prevalent tumor types, and describe current efforts in establishing a robust drug discovery screening cascade. 1. Zhou, G. (1995) J. Biol. Chem. 270, 12665-12669 2. Lochhead PA. (2012) Biochem Soc Trans. 40(1):251-6. 3. Song, H. (2004) Oncogene 23, 8301-8309 4. Mehta, P.B. (2003) Oncogene 22, 1381-1389 5. Zen, K. (2009) Genes Chromosomes Cancer 48,109-120 Citation Format: Paul R. Gavine, Mei Wang, Dehua Yu, Eva Hu, Chunlei Huang, Jenny Xia, Xinying Su, Joan Fan, Tianwei Zhang, Qingqing Ye, Li Zheng, Liang Xie, Luna Han, Guanshan Zhu, Ziliang Qian, Qingquan Luo, Yingyong Hou, Qunsheng Ji. Identification and validation of dysregulated MAPK7 (ERK5) as a novel oncogenic target in squamous cell lung and esophageal carcinoma. [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 2923. doi:10.1158/1538-7445.AM2014-2923

Neratinib shows efficacy in the treatment of HER2/neu amplified uterine serous carcinoma in vitro and in vivo

ObjectivesUterine serous carcinoma (USC) represents an aggressive variant of endometrial cancer and accounts for a large proportion of deaths annually. HER2/neu amplification is associated with USC in approximately 30–35% of cases. The objective of this study was to determine the sensitivity of a panel of primary USC cell lines to the small tyrosine kinase inhibitor neratinib, an ErbB1 and HER2 inhibitor, both in vitro and in vivo. MethodsHER2/neu amplification was determined by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in 24 USC cell lines. Flow cytometry was used to determine the effects of neratinib on cell viability, cell cycle distribution and signaling in vitro. Mice harboring HER2/neu amplified xenografts were treated with neratinib to assess the efficacy of the drug in vivo. ResultsHER2/neu amplification was noted in 8/24 primary cell lines. Data regarding the efficacy of neratinib was determined using 4 HER2 amplified cell lines and 4 non-amplified cell lines with similar growth rates. Data revealed that cell lines with HER2/neu amplification were exquisitely more sensitive to neratinib compared to non-amplified cell lines (mean±SEM IC50: 0.011μM±0.0008 vs. 0.312μM±0.0456 p<0.0001). Neratinib caused arrest in the G0/G1 phase of the cell cycle and resulted in decreased autophosphorylation of HER2 and activation of S6. Neratinib treated mice harboring xenografts of HER2/neu amplified USC showed delayed tumor growth and improved overall survival compared to vehicle (p=0.0019). ConclusionsNeratinib may be a potential treatment option for patients harboring HER2/neu amplified USC. Clinical trials for this subset of endometrial cancer patients are warranted.

Amplification and over‐expression of MAP3K3 gene in human breast cancer promotes formation and survival of breast cancer cells

Gene amplifications in the 17q chromosomal region are observed frequently in breast cancers. An integrative bioinformatics analysis of this region nominated the MAP3K3 gene as a potential therapeutic target in breast cancer. This gene encodes mitogen-activated protein kinase kinase kinase 3 (MAP3K3/MEKK3), which has not yet been reported to be associated with cancer-causing genetic aberrations. We found that MAP3K3 was amplified in approximately 8-20% of breast cancers. Knockdown of MAP3K3 expression significantly inhibited cell proliferation and colony formation in MAP3K3-amplified breast cancer cell lines MCF-7 and MDA-MB-361 but not in MAP3K3 non-amplified breast cancer cells. Knockdown of MAP3K3 expression in MAP3K3-amplified breast cancer cells sensitized breast cancer cells to apoptotic induction by TNFα and TRAIL, as well as doxorubicin, VP-16 and fluorouracil, three commonly used chemotherapeutic drugs for treating breast cancer. In addition, ectopic expression of MAP3K3, in collaboration with Ras, induced colony formation in both primary mouse embryonic fibroblasts and immortalized human breast epithelial cells (MCF-10A). Combined, these results suggest that MAP3K3 contributes to breast carcinogenesis and may endow resistance of breast cancer cells to cytotoxic chemotherapy. Therefore, MAP3K3 may be a valuable therapeutic target in patients with MAP3K3-amplified breast cancers, and blocking MAP3K3 kinase activity with a small molecule inhibitor may sensitize MAP3K3-amplified breast cancer cells to chemotherapy.

Assessment of fine needle aspiration cytology samples for molecular genetic analysis in neuroblastoma

To assess the utility of fine needle aspiration cytology (FNAC) samples for molecular genetic analysis of neuroblastoma. The case files from the pediatric solid tumor clinic were reviewed to identify 20 neuroblastoma patients whose pre-treatment FNAC slides were preserved in the cytology laboratory. The FNAC slides were destained, air dried and hybridisation with fluorescence in situ hybridisation (FISH) probes was performed as per protocol. All slides were screened and analyzed carefully under the fluorescent microscope. Over four-fold increase of the N-myc signal numbers was defined as N-myc amplification. Focal occurrence of cells (at least 50) showing N-myc amplification surrounded by non-amplified tumor cells was defined as focal N-myc amplification. Presence of three or more signals for the long arm of chromosome 17 was defined as 17q gain. FISH analysis gave informative results for all the FNAC smears in our study. FISH analysis of FNAC smears showed N-myc amplification in 5 (25 %) out of 20 patients and 15 (75 %) showed normal N-myc copy number. Three out of these five patients had homogenous amplification and two patients had focal N-myc amplification, indicating tumor heterogeneity. On investigation of chromosome 17q status, 5 (25 %) out of 20 patients demonstrated gain of 17q and 15 (75 %) patients showed normal 17q status. Four out of the five patients with 17q gain also showed N-myc amplification. The current study indicates that FNAC is a rapid and atraumatic diagnostic method for neuroblastoma which provides sufficient material for molecular genetic analyses by means of FISH.

Neratinib overcomes trastuzumab resistance in HER2 amplified breast cancer

Trastuzumab has been shown to improve the survival outcomes of HER2 positive breast cancer patients. However, a significant proportion of HER2-positive patients are either inherently resistant or develop resistance to trastuzumab. We assessed the effects of neratinib, an irreversible panHER inhibitor, in a panel of 36 breast cancer cell lines. We further assessed its effects with or without trastuzumab in several sensitive and resistant breast cancer cells as well as a BT474 xenograft model. We confirmed that neratinib was significantly more active in HER2-amplified than HER2 non-amplified cell lines. Neratinib decreased the activation of the 4 HER receptors and inhibited downstream pathways. However, HER3 and Akt were reactivated at 24 hours, which was prevented by the combination of trastuzumab and neratinib. Neratinib also decreased pHER2 and pHER3 in acquired trastuzumab resistant cells. Neratinib in combination with trastuzumab had a greater growth inhibitory effect than either drug alone in 4 HER2 positive cell lines. Furthermore, trastuzumab in combination with neratinib was growth inhibitory in SKBR3 and BT474 cells which had acquired resistance to trastuzumab as well as in a BT474 xenograft model. Innately trastuzumab resistant cell lines showed sensitivity to neratinib, but the combination did not enhance response compared to neratinib alone. Levels of HER2 and phospho-HER2 showed a direct correlation with sensitivity to neratinib. Our data indicate that neratinib is an effective anti-HER2 therapy and counteracted both innate and acquired trastuzumab resistance in HER2 positive breast cancer. Our results suggest that combined treatment with trastuzumab and neratinib is likely to be more effective than either treatment alone for both trastuzumab-sensitive breast cancer as well as HER2-positive tumors with acquired resistance to trastuzumab.

An siRNA screen identifies the GNAS locus as a driver in 20q amplified breast cancer

Poor prognosis oestrogen receptor positive breast cancer is characterized by the presence of high-level focal amplifications. We utilized a focused siRNA screen in 14 breast cancer cell lines to define genes that were pathogenic in three genomic regions focally amplified in oestrogen receptor positive breast cancer, 8p11-12, 11q13, and 20q. Silencing the GNAS locus, that encodes the G protein alpha stimulatory subunit Gαs, specifically reduced the growth of 20q amplified breast cancer cell lines. Examination of a publically available shRNA data set confirmed GNAS silencing to be selective for 20q amplified cancer cell lines. Cell lines with 20q amplification were found to over-express specifically the extra long Gαs splice variant (XLαs). Over-expression of XLαs induced cAMP levels to a greater extent than Gαs, suggesting that amplification of the GNAS locus, and over-expression of the XLαs variant in particular, enhanced cAMP signalling. GNAS silencing in amplified cell lines reduced ERK1/2 phosphorylation, and conversely over-expression of exogenous XLαs in a non-amplified cell line increased MEK-ERK1/2 phosphorylation, identifying one potential down-stream consequence of enhanced cAMP signalling. Our data indicate that amplification of the GNAS locus may contribute to the pathogenesis of breast cancer, and highlight a previously unrecognized role for the GNAS XLαs variant in cancer.

Genome‐wide identification of genes with amplification and/or fusion in small cell lung cancer

To obtain a landscape of gross genetic alterations in small cell lung cancer (SCLC), genome-wide copy number analysis and whole-transcriptome sequencing were performed in 58 and 42 SCLCs, respectively. Focal amplification of known oncogene loci, MYCL1 (1p34.2), MYCN (2p24.3), and MYC (8q24.21), was frequently and mutually exclusively detected. MYCL1 and MYC were co-amplified with other regions on either the same or the different chromosome in several cases. In addition, the 9p24.1 region was identified as being amplified in SCLCs without amplification of MYC family oncogenes. Notably, expression of the KIAA1432 gene in this region was significantly higher in KIAA1432 amplified cells than in non-amplified cells, and its mRNA expression showed strong correlations with the copy numbers. Thus, KIAA1432 is a novel gene activated by amplification in SCLCs. By whole-transcriptome sequencing, a total of 60 fusion transcripts, transcribed from 95 different genes, were identified as being expressed in SCLC cells. However, no in-frame fusion transcripts were recurrently detected in ≥2 SCLCs, and genes in the amplified regions, such as PVT1 neighboring MYC and RLF in MYCL1 amplicons, were recurrently fused with genes in the same amplicons or with those in different amplicons on either the same or different chromosome. Thus, it was indicated that amplification and fusion of several genes on chromosomes 1 and 8 occur simultaneously but not sequentially through chromothripsis in the development of SCLC, and amplification rather than fusion of genes plays an important role in its development.

Genomic heterogeneity of circulating tumor cells in castration-resistant prostate cancer (CRPC) revealed by single-cell sequencing.

5030 Background: Circulating tumor cells (CTC) provide an opportunity to sample multiple metastatic tumor sites through a single blood draw – a ”fluid biopsy.” NextGen DNA sequencing provides the means to obtain detailed genetic information from captured cells prior to and during treatment. Here we demonstrate the use of DNA sequencing to interrogate genome-wide copy number variations (CNV) at the single-cell level in CTC isolated from pts with CRPC. Methods: Pre- and post-treatment blood samples were obtained from pts treated at MSKCC. EpCAM+ events were collected singly and in groups by cytometric flow sorting and were subjected to DNA amplification and Illumina NextGeneration sequencing. Parallel samples were assayed using the Veridex CellSearch method to ensure the presence of malignant cells. Results: Samples with up to 50 EpCAM+ events analyzed in bulk displayed CNV patterns expected from published CRPC data. Subsequent single cell analyses showed that the method could reliably detect common genomic markers in CRPC, including AR amplification, PTEN and RB1 loss, and the TMPRSS-ERG fusion. Individual genomic CNV profiles obtained from 125 single cells isolated from 15 patients were then analyzed. Using unsupervised clustering, cells from each pt showed a closely related lineage structure, consistent with an evolution from a common ancestor. The degree of genomic heterogeneity within CTC from an individual pt was highly variable, with R2 correlation coefficients ranging from &gt;0.92 (nearly homogeneous) to &lt;0.75 (mixed populations). Two pts harbored separate subpopulations with both amplified AR and non-amplified AR cells and another displayed mixtures of genetic markers that changed over the course of treatment. Conclusions: The observed variation in complexity of CTC populations in CRPC pts underscores the importance of being able to sample and analyze multiple cells from an individual pt on multiple occasions and with real time analytics. Doing so is essential to understand and identify mechanisms of resistance so that they can be targeted effectively. Supported by STARR Cancer Consortium, NCI SPORE in Prostate Cancer; Department of Defense; Prostate Cancer Foundation.

Abstract 1132: Identification of synthetic lethal genes to MYCN-amplified neuroblastoma.

Abstract Purpose. Despite the identification of MYCN amplification as an adverse prognostic marker in neuroblastoma, no drugs that target MYCN have yet been developed, in part due to the inherent difficulty in designing therapeutic molecules that can target transcription factors. An alternative approach is to identify genes that are essential for the growth of MYCN-amplified neuroblastoma. Methods. We have screened for MYCN synthetic lethal genes using a highly efficient microRNA-short-hairpin RNA (miRNA-shRNA) library that contains more than 200,000 shRNAs targeting almost all predicted genes in the human genome. If a shRNA leads to cellular death, this shRNA will diminish in the cell population. The level of each shRNA was measured both by hybridizing genomic PCR products to microarrays that contain complementary sequence to the shRNA region and by direct high-throughput sequencing of the shRNA region. Selected candidates were further validated in MYCN amplified and non-amplified neuroblastoma cell lines. Results. We have performed an shRNA screen utilizing the SHEP-21N cell line, which expresses MYCN under a tetracycline controlled promoter. 396 significantly (P&amp;lt;0.01) depleted or enriched genes by both best shRNA and integrated gene level were identified. Gene set enrichment analysis was performed utilizing the Pathway Commons data set. 25 potential synthetic lethal genes were examined by secondary screening utilizing Dharmacon SMARTpool siRNAs. The potential synthetic lethal gene transcription factor AP-4/activating enhancer binding protein (TFAP4) was further validated in three MYCN amplified cell lines and three MYCN non-amplified cell lines. Knocking down TFAP4 by siRNA inhibited cell growth in MYCN amplified cell lines (NGP, SKNDZ, and Be(2)c) by 30%-50%, 96 hours after transfection, while knocking down TFAP4 in MYCN non-amplified lines (CHP212, SKNSH, and SH-SY5Y) did not affect cell growth. Utilizing a tetracycline inducible shRNA system, knocking down TFAP4 significantly reduced colony formation of NGP by70% and Be(2)c by 25%. Conclusions. By the use of a genome wide RNAi screen, we identified potential synthetic lethal genes to MYCN amplification. The top candidate TFAP4 was validated to inhibit neuroblastoma cell growth and colony formation in a MYCN dependent manner. This may provide novel targets for the treatment of neuroblastoma. Citation Format: Shuobo Zhang, J Yu, N Wei, J Silva, J J. Kandel, D J. Yamashiro. Identification of synthetic lethal genes to MYCN-amplified neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1132. doi:10.1158/1538-7445.AM2013-1132

Abstract 3127: Integrated cistromic and expression analysis of amplified NKX2-1 in lung adenocarcinoma identifies LMO3 as a functional transcriptional target.

Abstract The NKX2-1 transcription factor, a regulator of normal lung development, is the most significantly amplified gene in human lung adenocarcinoma. To better understand how genomic alterations of NKX2-1 drive tumorigenesis, we generated an expression signature associated with NKX2-1 amplification in human lung adenocarcinoma, and analyzed DNA binding sites of NKX2-1 by genome-wide chromatin immunoprecipitation (ChIP-seq) from three NKX2-1-amplified human lung adenocarcinoma cell lines. Integration of these expression and cistromic analyses suggested that aberrant over-expression of NKX2-1 primarily activates the expression of transcriptional target genes whose expression is not trans-activated by NKX2-1 at physiological expression levels, and identified LMO3, itself encoding a transcription regulator, as a candidate direct transcriptional target of NKX2-1. NKX2-1 amplification is most significantly associated with over-expression of the LMO3 gene, a member of the LMO family of oncogenes that are translocated in T-ALL, the same disorder in which NKX2-1 translocation has been observed. We further show that NKX2-1 interacts physically with the LMO3 locus, and activates its expression. RNA interference analysis of NKX2-1-amplified cells compared to non-amplified cells demonstrated that LMO3 mediates cell proliferation downstream of NKX2-1. Further cistromic analyses found that consensus binding motifs including a nuclear hormone receptor signature and a Forkhead box motif, indicating that NKX2-1 might cooperate with Forkhead box FOXA1 and nuclear hormones to regulate gene expression. Genome-wide analysis of FOXA1 occupancy by ChIP-seq in the NCI-H3122 cell line revealed that FOXA1 binds not only to the LMO3 locus upstream of NKX2-1 binding site but also to many NKX2-1 occupied regions (47% of total NKX2-1-bound sites). Our findings provide new insight into the transcriptional regulatory network of NKX2-1 and suggest that LMO3 is a transducer of lineage specific cell survival of NKX2-1-amplified lung adenocarcinomas. Citation Format: Hideo Watanabe, Joshua M. Francis, Michele S. Woo, Banafsheh Etemad, Wenchu Lin, Daniel F. Fries, Shouyong Peng, Eric L. Snyder, Purushothama Rao Tata, Anna C. Schinzel, Jeonghee Cho, Peter S. Hammerman, Roel G. Verhaak, William C. Hahn, Jayaraj Rajagopal, Tyler Jacks, Matthew Meyerson. Integrated cistromic and expression analysis of amplified NKX2-1 in lung adenocarcinoma identifies LMO3 as a functional transcriptional target. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3127. doi:10.1158/1538-7445.AM2013-3127

Abstract 4368: Cathepsin B is a potential therapeutic target in pancreatic and gastric cancer .

Abstract Proteases are critical in tumorigenesis by facilitating rapid cell cycling, mediating local invasion, fueling angiogenesis, and promoting metastasis. Cathepsin B (CTSB), a member of papain subfamily of lysosomal cysteine proteases, is involved in tumor cell invasion, metastasis, and angiogenesis. Emerging evidence indicated that CTSB is up-regulated in many cancer nodes and metastatic lesions and functions to protect tumor cells from apoptosis. Hererin, we have performed microarray-based comparative hybridization (aGCH) to screen gene copy number variations in primary tumor samples and found CTSB gene is amplified in 13% (14/107) of Chinese gastric cancer and 2% (1/54) of Chinese pancreatic samples. Theses amplifications were confirmed by fluorescence in situ hybridization (FISH) analysis. By using siRNA-mediated gene silencing, we demonstrated for the first time a role of CTSB in cancer cell proliferation. Down-regulation of CTSB by siRNA interference resulted in significantly reduced cell proliferation and increased cell death in HupT4, a CTSB amplified human pancreatic cancer cell line, but not in the non-amplified cell lines. Together, our results suggest that CTSB is a potential therapeutic target for pancreatic and gastric cancer with CTSB amplification. Citation Format: Mei Wang, Chunlei Huang, Harvey Dong, Ziliang Qian, Shuqiong Fan, Xinying Su, Li Zheng, Qunsheng Ji, De-Hua Yu. Cathepsin B is a potential therapeutic target in pancreatic and gastric cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4368. doi:10.1158/1538-7445.AM2013-4368

Integrated cistromic and expression analysis of amplified NKX2-1 in lung adenocarcinoma identifies LMO3 as a functional transcriptional target

The NKX2-1 transcription factor, a regulator of normal lung development, is the most significantly amplified gene in human lung adenocarcinoma. To study the transcriptional impact of NKX2-1 amplification, we generated an expression signature associated with NKX2-1 amplification in human lung adenocarcinoma and analyzed DNA-binding sites of NKX2-1 by genome-wide chromatin immunoprecipitation. Integration of these expression and cistromic analyses identified LMO3, itself encoding a transcription regulator, as a candidate direct transcriptional target of NKX2-1. Further cistromic and overexpression analyses indicated that NKX2-1 can cooperate with the forkhead box transcription factor FOXA1 to regulate LMO3 gene expression. RNAi analysis of NKX2-1-amplified cells compared with nonamplified cells demonstrated that LMO3 mediates cell survival downstream from NKX2-1. Our findings provide new insight into the transcriptional regulatory network of NKX2-1 and suggest that LMO3 is a transcriptional signal transducer in NKX2-1-amplified lung adenocarcinomas.

Abstract P1-07-19: Mass Spectrometry Based Quantitative Analysis of the HER Family receptors in FFPE Breast Cancer Tissue

Abstract The human EGF receptor family (HER's) consists of two clinically validated drug targets (EGFR and HER2), a third (HER3) currently under investigation for its possible role in the acquisition of multidrug resistance and a fourth (HER4), the role of which is still matter of debate. Drugs inhibiting EGFR or HER2 show significant antitumor activity in the clinic, however, acquisition of resistance is a hallmark of these and most targeted therapies. In the case of EGFR and HER2, one of the emerging resistance mechanisms is the co-expression of HER3. Indeed, recent reports show that inhibition of the PI3K pathway leads to upregulation of HER3, and subsequent resistance. Clinical analysis of protein levels in formalin fixed paraffin embedded (FFPE) tissues is limited to immunohistochemistry (IHC), which is semi-quantitative and requires significant amounts of tissue. Moreover, the vast majority of research groups consider specific HER3 staining by IHC particularly challenging. However, accurate measurement of these targets is critical both for properly defining treatment groups and predicting patterns of resistance. In order to address these issues, we used trypsin digestion mapping and stable isotope labeled peptides to develop a panel of quantitative mass spectrometric (MS) assays to measure the levels of EGFR, HER2, HER3 and other clinically relevant targets in FFPE breast cancer tissue. These quantitative MS assays were then multiplexed to analyze 1μg of tumor protein. In this study, we multiplexed HER family analysis on 31 HER2 positive breast cancers. Tumor tissue was microdissected from FFPE sections, and subjected to quantitative MS analysis of EGFR, HER2, HER3 as well as IGF1R and cMET. Quantitation of HER2 showed a broad range of HER2 expression in these tissues. The highest expresser measured 26 fmol/ug tumor tissue, representing amplification and massive protein over expression. In contrast, five tissues showed low levels of HER2 expression, below 1 fmol/ug, similar to HER2 non-amplified cell lines. This suggests that MS quantitation can identify patients with low expression of HER2 who are unlikely to respond to trastuzumab therapy. As a matter of fact, 3 of these 5 low expressing patients had outcome data and showed no response to trastuzumab treatment. In 28 of 31 patient tissue samples, HER3 showed low levels of expression (100–300 amol/ug tumor tissue) similar to HER3 expression in cell lines, and comparable to low expressing EGFR and HER2 cell lines. The remaining 3 patients had no detectable HER3. This study demonstrates the feasibility of measuring HER3 in multiplex in FFPE breast cancer tissue. Based on the low but widespread expression of HER3 in this cohort, it may be most useful to assess HER3 expression after initial treatment as a marker of potential resistance to targeted therapies. Taken together, these data demonstrate that a sensitive and quantitative assay to measure oncoproteins in FFPE clinical samples may help stratify patients with variable expression of these targets. Our quantitation of oncogene expression from clinical samples uses a small amount of tissue, is clinically applicable and alleviates the problem of scoring either positive or negative for the expression of a given protein. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-19.

Constitutive autotaxin transcription by Nmyc-amplified and non-amplified neuroblastoma cells is regulated by a novel AP-1 and SP-mediated mechanism and abrogated by curcumin

The motility, angiogenesis and metastasis-stimulating factor Autotaxin (Atx), over expressed by human neuroblastomas (NB), is constitutively expressed by human Nmyc-amplified SK-N-BE and non-Nmyc-amplified SH-SY5Y NB cells. Here, we characterise a novel Atx transcriptional mechanism, utilised by both cell lines, that is restricted to the first 285bp of the Atx promoter and involves AP-1 and SP transcription factors, acting through a CRE/AP-1-like element at position −142 to −149 and a GAbox at position −227 to −235 relative to the Atx translational start site. This novel transcriptional mechanism can be inhibited by internally initiated SP-3 and the natural phenol curcumin.

Trastuzumab induces antibody-dependent cell-mediated cytotoxicity (ADCC) in HER-2-non-amplified breast cancer cell lines

Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by CD56+natural killer (NK) cells may contribute to the activity of trastuzumab in HER-2-amplified tumours. In this study, we investigated the possibility that trastuzumab might induce ADCC against HER-2-non-amplified breast cancer cells. Induction of NK cell-mediated ADCC was examined in trastuzumab-treated HER-2-non-amplified breast cancer cell lines. HER-2 protein levels were also determined in tumour and autologous normal tissue samples from patients with HER-2 negative breast cancer. Trastuzumab induced a significant ADCC response in the HER-2-amplified HCC1954 and SKBR3 cell lines, and in all five of the non-amplified cell lines, which had low levels of detectable HER-2 by western blot (CAL-51, CAMA-1, MCF-7, T47D, and EFM19). Trastuzumab did not induce ADCC in the K562 control cell line or MDA-MB-468, which has very low levels of HER-2 detectable by enzyme-linked immunosorbent assay (ELISA) only. HER-2 protein was detected by ELISA in 14/15 patient tumour samples classified as HER-2-non-amplified. Significantly lower levels of HER-2 were detected in normal autologous tissue compared with tumour samples from the same patients. Our results suggest that HER-2-non-amplified breast cancer cells, with low but detectable levels of HER-2 protein, can bind trastuzumab and initiate ADCC.

Abstract 1716: FGFR1 gene amplification predicts the antitumor response of FGFR inhibitor LY2874455 in human lung cancer cells

Abstract Lung cancer is the leading cause of cancer-related death for both men and women in the world. FGFR receptor tyrosine kinase signaling pathway has been implicated in tumor cell growth, survival, angiogenesis, and metastasis. Activation of the FGFR pathway via FGFR over-expression, and chromosomal amplification and translocation as well as genetic mutation contributes to the tumor progression and the resistance to anti-EGFR and -VEGF treatment in lung cancer. Targeting FGFR pathway is therefore an attractive therapeutic strategy for lung cancer. We have discovered and developed a novel, ATP competitive and orally active small molecule inhibitor of FGFR, LY2874455, currently in clinic development. LY2874455 is a potent inhibitor against four FGFRs, and demonstrates a broad-spectrum of anti-tumor activity in a number of tumor xenograft models. In the current study, we evaluated the anti-tumor activity of LY2874455 in lung tumor cells with or without FGFR1 gene amplification. FGFR1 is known to be amplified in human lung cancer cells NCI-H1581, NCI-H520 and DMS-114, but not in NCI-H460, A549 and NCI-H1299. The mRNA expression levels of FGFR1 are shown to be higher in the FGFR1 amplified cells than those in the FGFR1 non-amplified cells. Consistent with this, the FGFR1 amplified cells exhibited the much more robust b-FGF-induced phosphorylation of FRS2 and ERK, which are also more sensitive to the inhibition by LY2874455 than that in the FGFR1 non-amplified cells. Furthermore, the FGFR1 amplified cells exhibit the better responses to the growth inhibition and apoptosis induction by LY2874455 than those in the FGFR1 non-amplified cells. Therefore, these findings suggest that FGFR1 plays an important role in lung cancer cell growth and survival, and that LY2874455 may provide a promising treatment option for some lung cancer patients whose tumor harbors FGFR1 gene amplification. The in vivo anti-tumor response of LY2874455 is being further investigated in the tumor xenograft models derived from these FGFR1 amplified or non-amplified human lung cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1716. doi:1538-7445.AM2012-1716