bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2295: AMP-activated protein kinase (AMPK) signaling in the context of gain-of-function mutant p53 in vivo
Authors: Jo Se-Young , Moon Hye-Min , Lee ChuHee , Jang Se Jin , Suh Young-Ah
Published: August 2015

There is growing evidence that tumor cells depend on unique metabolism for their continued growth and survival, and that cancer cells are peculiarly addicted to the rapacious uptake of glucose and glutamine. AMP-activated protein kinase (AMPK) is one of the central regulators of cellular and organismal metabolism in eukaryotes, playing critical roles in regulating cell growth and reprogramming metabolism. Several of the downstream effects of AMPK on metabolic adaptation can be attributed to the AMPK-dependent activation of the p53 tumor suppressor, resulting in its transcriptional activation and initiating a metabolic cell cycle checkpoint. However, in the absence of p53, treatment with the blood glucose-lowering drug metformin delays tumor progression, suggesting that the AMPK-p53 signaling axis responds in diverse manner depending on genetic alteration. Due to the gain-of-function, mutant p53 protein contributes to tumor metastasis and shortening the latency of tumor progression. Thereby activated K-ras/mutant p53 mouse is preferentially used as lung adenocarcinoma preclinical model. Here, we have established mouse tumor cells derived from activated K-ras/mutant p53 mouse to generate syngeneic lung cancer mouse model, allowing in vivo imaging. We asked whether the gain-of-function metastatic phenotype observed in the mouse is responsible for unique metabolic conditions resulting in the formation of lung tumors. We have evaluated tumor metabolic environment of this model and examined AMPK signaling pathways.

Citation Format: Se-Young Jo, Hye-Min Moon, ChuHee Lee, Se Jin Jang, Young-Ah Suh. AMP-activated protein kinase (AMPK) signaling in the context of gain-of-function mutant p53 in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2295. doi:10.1158/1538-7445.AM2015-2295
Abstract 23: Nuclear receptor PPARα activation triggers hepatic cell death in Ikkβ-deficient mice
Authors: Kim Jung-Hwan , Kim Taehyeong , Qu Aijuan , Gonzalez Frank J.
Published: August 2015

Peroxisome proliferator-activated receptor α (PPARα) plays a critical role in lipid metabolism, inflammation, carcinogenesis, and cell survival. Recent study showed that PPARα agonist dramatically induced the cell cycle arrest genes, such as p21 and Gadd45b, in the liver of mice. However, the inductions of these genes were not lead to hepatic cell death. Thus, here, we investigated the Ikkb gene whether this can affect the hepatic cell death or survival after induction of p21 and Gadd45b. To exam the function of Ikkb after PPARα activation, the Wild-type (IkkbF/F) and liver-specific Ikkb-null (IkkbF/F-AlbCre) mice were treated with the PPARα agonist Wy-14,643 for two weeks. As results, Hepatic cell death was undergone to apoptosis by increases of cleaved-caspase-3 and cleaved poly (ADP-ribose) polymerase only in the liver of IkkbF/F-AlbCre mice by Wy-14,643 and this was also supported by TUNEL staining. Moreover, Wy-14,643 treatment elevated phosphorylation of c-Jun N-terminal kinase only in the liver of IkkbF/F-AlbCre mice. Liver histology showed notable feature of hepatitis only in the IkkbF/F-AlbCre mice treated with Wy-14,643. And marked increase Tnfa mRNA was followed in Wy-14,643-treated IkkbF/F-AlbCre mice. However, expression of the gene related to lipid metabolism such as Ppara and Acox1 were not significantly changed by Wy-14,163 in both IkkbF/F and IkkbF/F-AlbCre mice. These studies suggest that Ikkb gene may play a critical role in hepatic cell survival against PPARα activation.

Citation Format: Jung-Hwan Kim, Taehyeong Kim, Aijuan Qu, Frank J. Gonzalez. Nuclear receptor PPARα activation triggers hepatic cell death in Ikkβ-deficient mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 23. doi:10.1158/1538-7445.AM2015-23
Abstract 2304: The role of methyltransferase, enhancer of zeste homolog 2 (EZH2) in mouse hepatocyte and human hepatocellular carcinoma
Authors: Bae Woo Kyun , Shim Hyun Jeong , Cho Sang Hee , Chung Ik-Joo , Park In-Kyu , Hennighausen Lothar
Published: August 2015

To investigate the role of EZH1 and EZH2 in liver homeostasis, mice were generated that carried Ezh1-/- and EZH2fl/fl alleles and an Alb-Cre transgene. Only combined loss of EZH1 and EZH2 in mouse hepatocytes caused a depletion of global H3K27me3 marks and the specific loss over ∼1900 genes at 3 months of age. Ezh1-/-,Ezh2fl/flAlb-Cre mice exhibited progressive liver abnormalities manifested by development of regenerative nodules and concomitant periportal fibrosis, inflammatory infiltration and activation of A6-positive hepatic progenitor cells at 8 months of age. In response to chronic treatment with CCl4, all experimental mice but none of the controls (n = 27 each) showed increased hepatic degeneration associated with liver dysfunction and reduced ability to proliferate. After 2/3ds partial hepatectomy, mutant mice (n = 5) displayed increased liver injury and a blunted regenerative response. Genome-wide analyses at 3-months of age identified 51 genes that had lost H3K27me3 marks and their expression was significantly increased. These genes were involved in regulation of cell survival, fibrosis, and proliferation. H3K27me3 levels and liver physiology were unaffected in mice lacking either EZH1 globally or EZH2 specifically in hepatocytes. This work demonstrates a critical redundancy of EZH1 and EZH2 in maintaining hepatic homeostasis and regeneration. Next, we checked the expression EZH2 and H3K27me3 status in 67 human hepatocellular carcinoma (HCC) samples. We also checked the gene expression by the next-generation sequencing based on EZH2 expression. This work demonstrates candidate EZH2 target genes in human HCC.

Citation Format: Woo Kyun Bae, Hyun Jeong Shim, Sang Hee Cho, Ik-Joo Chung, In-Kyu Park, Lothar Hennighausen. The role of methyltransferase, enhancer of zeste homolog 2 (EZH2) in mouse hepatocyte and human hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2304. doi:10.1158/1538-7445.AM2015-2304
Abstract 2309: Murine model of dual mTORC kinase inhibition identifies CDK6 as a synergistic target in T-ALL
Authors: Gary Joy M. , Xu Jinfei , Simmons John , Zhang Shuling , Gamache Benjamin , Zhang Ke , Kovalchuk Alexander , Michalowski Aleksandra , Chen Jin-Qiu , Herrmann Michelle , Dubois Wendy , Testa Joseph , Mock Beverly A.
Published: August 2015

The PI3K/AKT/mTOR pathway is frequently hyperactivated in T- cell acute lymphoblastic leukemia (T-ALL). To model inhibition of this pathway in lymphoma, mice with constitutively-active AKT specific to T-lymphocytes (Lck-MyrAkt2) were crossed with mice that have genetically-reduced mTOR expression (mTOR knock-down, KD). Lck-MyrAkt2 mice with mTOR KD had altered thymic T-lymphocyte development; these mice also had delayed thymic pre-T cell lymphoblastic leukemia/lymphoma (pre-T LBL) progression and increased survival relative to wild type (WT) mTOR/ Lck-MyrAkt2 mice (average survival of 24 versus 14 weeks, respectively). Delayed pre-T LBL formation was also observed when WT mTOR/Lck-MyrAkt2 mice were treated for 8 weeks with the rapamycin analog, everolimus, an inhibitor of the mTOR TORC1 complex. Transcriptional profiling of thymic lymphomas from the WT vs KD mTOR/ Lck-MyrAkt2 mice revealed that mTOR KD was associated with decreased expression of Cdk6, a critical proliferative control node in T- cell development and oncogenic transformation. Pharmacologic inhibition of mTOR in tumor cells also decreased CDK6. Tumor cells from WT mTOR/Lck-MyrAkt2 mice were more sensitive to CDK4/6 inhibitors than tumor cells from KD mTOR/Lck-MyrAkt2 mice. Combining an mTOR inhibitor (rapamycin) with a CDK4/6 inhibitor (PD-0332991) synergistically inhibited the viability and decreased downstream signaling in both mouse lymphoma cells and human T-cell acute lymphoblastic leukemia (T-ALL) cell lines. Combining a dual mTORC inhibitor (PP242) with a CDK4/6 inhibitor decreased downstream signaling to an even greater extent in human T-ALL cell lines. Our results suggest that an mTORi/CDKi combination may be beneficial in the treatment of human T-ALL.

Citation Format: Joy M. Gary, Jinfei Xu, John Simmons, Shuling Zhang, Benjamin Gamache, Ke Zhang, Alexander Kovalchuk, Aleksandra Michalowski, Jin-Qiu Chen, Michelle Herrmann, Wendy Dubois, Joseph Testa, Beverly A. Mock. Murine model of dual mTORC kinase inhibition identifies CDK6 as a synergistic target in T-ALL. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2309. doi:10.1158/1538-7445.AM2015-2309
Abstract 2314: d16HER2 splice variant regulates the activity of HER2-positive breast cancer-initiating cells
Authors: Castagnoli Lorenzo , Koschorke Ada , Ghedini Gaia C. , Galvani Lorenzo , Ciravolo Valentina , Ghirelli Cristina , Palladini Arianna , Lamolinara Alessia , Iezzi Manuela , Lollini Pier Luigi , Triulzi Tiziana , Nanni Patrizia , Tagliabue Elda , Pupa Serenella M.
Published: August 2015

The transmembrane tyrosine kinase receptor HER2, overexpressed in ∼20% of human breast cancers (BCs), identifies an aggressive tumor subtype and is reportedly an important regulator of breast cancer-initiating cell activity (BCIC). We found that ∼90% of HER2+ BC patients constitutively express a splice isoform of wild-type HER2 (WTHER2) gene characterized by the lack of exon 16 (d16HER2), a deletion that promotes the generation of a particularly aggressive HER2 isoform and that forms stable and constitutively activated d16HER2 homodimers. Our comparison of the tumorigenic potential of the human d16HER2 and WTHER2 genes in the corresponding transgenic mouse models revealed a significantly shorter tumor latency period (p< 0.001) and a higher tumor incidence in the d16HER2 mice (p<0.001), suggesting a role for this variant in HER2-driven activation of BCICs. In this context, our preliminary analyses of HER2-positive primary mammary tumor cell lines MI6 and MI7 derived from spontaneous transgenic d16HER2 mice showed a significantly higher mammosphere-forming efficiency and higher levels of stem cell marker transcripts, including CD44, Wnt, Notch and Bmi1, compared to transgenic mouse WTHER2 tumor cells (WTHER2_1 and WTHER2_2). Mammospheres generated from human HER2-overexpressing breast cancer cell lines BT474 and MDA-MB-361, which also express the d16HER2 variant, exhibited an increase in the relative abundance of d16HER2 mRNA compared with that in the same parental cells cultured in adhesion conditions, as indicated by qPCR analyses. Experiments in mice injected into the mammary fat pad with the d16HER2- and WTHER2-positive cell lines at different serial dilutions indicate a consistently higher “stemness potential” of MI6 and MI7 cells compared to WTHER2_1 and WTHER2_2 cells, strongly suggesting that the d16HER2 variant plays a greater role than WTHER2 in regulating BCICs of HER2-driven mammary tumors.

Supported by Minister of Health and AIRC

Citation Format: Lorenzo Castagnoli, Ada Koschorke, Gaia C. Ghedini, Lorenzo Galvani, Valentina Ciravolo, Cristina Ghirelli, Arianna Palladini, Alessia Lamolinara, Manuela Iezzi, Pier Luigi Lollini, Tiziana Triulzi, Patrizia Nanni, Elda Tagliabue, Serenella M. Pupa. d16HER2 splice variant regulates the activity of HER2-positive breast cancer-initiating cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2314. doi:10.1158/1538-7445.AM2015-2314
Abstract 2318: Genetic and functional diversity of propagating cells in glioblastoma
Authors: Piccirillo Sara Grazia Maria , Colman Sue , Potter Nicola E. , van Delft Frederik W. , Lillis Suzanne , Carnicer Maria-Jose , Kearney Lyndal , Watts Colin , Greaves Mel
Published: August 2015

Glioblastoma (GBM) is a lethal malignancy whose clinical intransigence has been linked to extensive intraclonal genetic and phenotypic diversity and the common emergence of therapeutic resistance. This interpretation embodies the implicit assumption that cancer stem cells or tumor-propagating cells are themselves genetically and functionally diverse. To test this, we screened primary GBM tumors by SNP array to identify copy number alterations (a minimum of three) that could be visualized in single cells by multicolor fluorescence in situ hybridization. Interrogation of neurosphere-derived cells (from four patients) and cells derived from secondary transplants of these same cells in NOD-SCID mice allowed us to infer the clonal and phylogenetic architectures. Whole-exome sequencing and single-cell genetic analysis in one case revealed a more complex clonal structure.This proof-of-principle experiment revealed that subclones in each GBM had variable regenerative or stem cell activity, and highlighted genetic alterations associated with more competitive propagating activity in vivo.

Citation Format: Sara Grazia Maria Piccirillo, Sue Colman, Nicola E. Potter, Frederik W. van Delft, Suzanne Lillis, Maria-Jose Carnicer, Lyndal Kearney, Colin Watts, Mel Greaves. Genetic and functional diversity of propagating cells in glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2318. doi:10.1158/1538-7445.AM2015-2318
Abstract 2325: Activated glucocorticoid signaling in pancreatitis contributes to acinar-to-ductal metaplasia and KrasG12D-driven tumorigenesis
Authors: Lin Pei-Yu , Su Chien-Chang , Hsieh Chi-Che , Shen Chia-Ning
Published: August 2015

Acinar-to-ductal metaplasia (ADM), an inflammatory lesion associated with replacement of acinar cells by tubular complexes, is thought to represent a condition with increased risk of neoplasia. However, the direct evidence linking acinar-to-ductal metaplasia and development of pancreatic neoplasm is still lacking. Glucocorticoid-mediated signaling pathway is known to be a part of the feedback control machinery of immune system that acts to suppress excessive immune activity. In the present study, we explored the role of GR in development of ADM and pancreatic intraepithelial neoplasia (PanIN) in mice with pancreatitis. We initially identified that evaluated levels of activated glucocorticoid receptors (GR) were observed in patients of chronic pancreatitis and pancreatic ductal adenocarcinomas (PDAC). Utilizing mice conditionally expressed KrasG12D in acinar cell with or without loss of GR, we demonstrated that GR activation was required for generation of metaplastic ductal lesion and development of pancreatic intraepithelial neoplasia (PanIN) in mice with experimental pancreatitis. Administration of dexamethasone, a potent synthetic glucocorticoid, was sufficient to induce development of PanINs and PDACs in KrasG12D mice that harbored single-allele deletion of p53. We further revealed that non-genomic activation of GR was a key action that led to evaluate c-Src signaling and trigger a pathological threshold of Ras activity necessary for neoplastic transformation. In contrast, treatment of dasatinib, a Src kinase inhibitor, suppressed development of PanINs and PDACs induced by dexamethasone. Importantly, Cre-lox based lineage-tracing mice was applied and identified that GR activation directly transformed GFP-tagged KrasG12D-expressing acinar cells to tumorigenic cells. The findings explained why inflammatory ductal reprogramming predisposes to development of pancreatic neoplasm.

Citation Format: Pei-Yu Lin, Chien-Chang Su, Chi-Che Hsieh, Chia-Ning Shen. Activated glucocorticoid signaling in pancreatitis contributes to acinar-to-ductal metaplasia and KrasG12D-driven tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2325. doi:10.1158/1538-7445.AM2015-2325
Abstract 2326: Sox10 promotes both stem-like and EMT properties in mammary cells
Authors: Dravis Christopher , Wahl Geoffrey M.
Published: August 2015

Molecular pathways that guide cell proliferation and programming events during embryonic development may be aberrantly re-activated in the cancer state to drive tumor progression. To identify mechanisms that drive progression of aggressive breast cancers, we identified molecules that are present in the fetal mammary stem cells (fMaSCs) that drive early development of the mammary gland. These analyses revealed that Sox10 is one of the most highly expressed transcription factors within fMaSCs, and prompted us to investigate the function of Sox10 in the mammary gland and in subtypes of cancer originating from it. Our studies described here indicate that Sox10 is specifically expressed in mammary cells that exhibit higher levels of stem or progenitor cell functions using in vitro or in vivo assays. This includes fetal and adult mammary cells in vivo, as well as mammary cells cultured in vitro as organoids, and for the first time, male fetal mammary stem cells. Sox10 appears to contribute to these stem/progenitor behaviors, as the genetic deletion of Sox10 limits stem/progenitor activities, while the overexpression of Sox10 significantly expands clonogenic behavior. Intriguingly, ectopic Sox10 expression is also able to elicit an EMT-like response in mammary organoids. Sox10 thus represents a single molecule capable of directly mediating both stem-like and EMT-like properties in mammary cells. Furthermore, by modulating Sox10 levels in 3D mammary organoids, we are able to induce cells into sequential motile and stem/progenitor states. This provides a striking in vitro surrogate for metastatic behavior. Consistent with these findings, we find that Sox10 is highly expressed in basal-like and claudin-low subtypes of breast cancer, which are the most stem-like and EMT-like manifestations of the disease, respectively. Finally, we show that Sox10 expression is regulated through a feedback loop involving FGF signaling, and that inhibition of FGF signaling can block EMT-like cell behaviors mediated by Sox10. These findings have important implications in how stem-like and metastatic properties may be specified in mammary cells.

Citation Format: Christopher Dravis, Geoffrey M. Wahl. Sox10 promotes both stem-like and EMT properties in mammary cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2326. doi:10.1158/1538-7445.AM2015-2326
Abstract 2345: The collagen-dense tumor microenvironment increases neutrophil recruitment in mouse mammary carcinoma
Authors: Garcia Mendoza Maria Gracia , Inman David , Ponik Suzanne M. , Keely Patricia J.
Published: August 2015

The breast tumor microenvironment (TME) is a diverse area where complex interactions between the extra-cellular matrix (ECM) and multiple cell types (tumor epithelial, endothelial, and inflammatory cells, fibroblasts, and adipocytes) occur resulting in tumor promotion, invasion and metastasis. Changes in breast tissue density have been correlated with a 4-6-fold increased risk for developing breast cancer, and are associated with increased stromal deposition of the ECM protein, collagen I. In a transgenic mouse model of increased collagen (Col1A1Tm1Jae), there is a 3-fold increase in MMTV-PyMT tumor formation and lung metastases compared to control mice. Of the non-malignant cells in the TME, tumor-associated macrophages (TAMs) and tumor-associated neutrophils (TANs) have been shown by several investigators to alter the TME and ECM, enhance tumor cell migration and invasion, stimulate angiogenesis, and suppress antitumor immunity. The direct interaction of both increased collagen and immune (myeloid) cell recruitment leading to increased cancer progression and metastasis has not been characterized. We hypothesized that a collagen-dense tumor microenvironment recruits an abnormal population of myeloid cells, which promotes tumor progression and metastasis. Flow cytometry analysis of cells from collagen-dense mouse tumors showed an increase in Ly6G+ TAN recruitment compared to wild type tumors, while TAM recruitment did not seem to be affected. The increased Ly6G+ neutrophil recruitment is a global immune response also observed in the spleens and circulating blood. Using a cytokine/chemokine ELISA plate array, we found that the collagen-dense TME cytokine cross talk supports neutrophil and monocyte recruitment and maturation. In addition, many classic inflammatory signals that support lymphocyte recruitment and activation, B and T-cell proliferation, and natural killer cell and monocyte activation are downregulated in the collagen-dense TME. Further experiments will determine the time point where neutrophil recruitment is critical for tumor progression. Experiments are underway to study the possible suppression of natural killer cell activity in the collagen-dense TME.

This work is being supported by NIH grants (NIH-CA-114462 and NIH-U54-CA163131) to P.J.K. and NSF Graduate Research Fellowship (2011129283) to M.G.G.M.

Citation Format: Maria Gracia Garcia Mendoza, David Inman, Suzanne M. Ponik, Patricia J. Keely. The collagen-dense tumor microenvironment increases neutrophil recruitment in mouse mammary carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2345. doi:10.1158/1538-7445.AM2015-2345
Abstract 2347: Multiplexed quantitative assessment and prognostic value of TIL subtypes in non-small cell lung cancer
Authors: Schalper Kurt A. , Hoyt Cliff , Wang Chichung , Rimm David
Published: August 2015

Background: The presence of high levels of tumor-infiltrating lymphocytes (TILs) is associated with favorable outcome in diverse human neoplasms including non-small cell lung cancer (NSCLC). Previous studies suggest that different TIL subtypes have distinct biological roles in cancer progression and their contribution to prognosis remains unclear. Here, we simultaneously quantified TIL subtypes using 2 different automated, objective methods in human NSCLC and determined their association with tumor characteristics and outcome.

Methods: Using a sequential multiplexed immunofluorescence staining protocol with tyramide signal amplification and heat-inactivation steps, we simultaneously visualized all cell nuclei (DAPI), epithelial cells (Cytokeratin AE1/AE3, Dako), T helper cells (CD4-368, Leica), Cytotoxic T cells (CD8, C8/144B, Dako), B-lymphocytes (CD20, L26, Leica) and Regulatory T cells [Tregs], (Foxp3, D608R, CST) in 202 stages I-IV NSCLC represented in tissue microarray format. The preparations were digitalized and the number of each TIL subpopulation was counted using multispectral fluorescence analysis and a trainable tissue/cell image segmentation algorithm with the inForm software (PerkinElmer). Markers were also measured using the AQUA method of quantitative immunofluorescence (QIF).

Results: NSCLC samples show a highly variable TIL content ranging from 0 to 2717 lymphocytes per tumor histospot. The median TIL content was 23.9 T-helper cells, 55.8 Cytotoxic T cells, 59.7 B-lymphocytes and 6.1 Tregs. Although the amount of CD4+, CD8+ and CD20+ cells was comparable between the histological NSCLC subtypes, the average number of Tregs (CD4+/Foxp3+ cells) was significantly higher in squamous cell carcinomas than in adenocarcinomas (10.4 and 4.9 cells per histospot, respectively, P = 0.03). Increased numbers of CD8+ or CD20+ TILs were significantly associated with longer overall survival (log-rank P<0.05), but the amount of CD4+ and Foxp3+ cells were not. Correlations were seen between multiplexed counting methods and QIF.

Conclusions: This strategy allows simultaneous and objective quantification of diverse TIL subpopulations in lung tumor samples. Our results indicate that increased Cytotoxic T cells and B-lymphocytes, but not T helper cells or Tregs have prognostic value in NSCLC. Accurate quantification of TIL subpopulations in the tumor microenvironment could be useful to predict response and study the local effects of novel anti-cancer immunostimulatory therapies.

Citation Format: Kurt A. Schalper, Cliff Hoyt, Chichung Wang, David Rimm. Multiplexed quantitative assessment and prognostic value of TIL subtypes in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2347. doi:10.1158/1538-7445.AM2015-2347
Abstract 2349: EGFR is transferred from triple negative breast cancer cells to immune cells via trogocytosis and expression of EGFR on immune cells is associated with high tumor grade of triple negative breast cancer patients
Authors: Suzuki Eiji , Yamaguchi Ayane , Kataoka Tatsuki R. , Hirata Masahiro , Kawaguchi Kosuke , Nishie Mariko , Toi Masakazu
Published: August 2015

We and others previously reported that HER2 was transferred from HER2 overexpressing breast cancer cells to CD14+ monocytes via trastuzumab dependent trogocytosis. In the current study, we report that EGFR of MDA-MB-231 (triple negative human breast cancer cell line) is also transferred to immune cells via trogocytosis and those immune cells express EGFR on the cell surface without antibody administration. Interestingly, the EGFR-trogocytosed MDA-MB-231 cells reduced the expression of EGFR and changed their morphology to the spindle shape (well known as mesenchymal transition) and acquired greater cell motility. These cancer cell properties are representative as higher malignant potentials in breast cancer patients. Thus, we hypothesized that EGFR expression on tumor infiltrating lymphocytes (TILs), which probably is caused via EGFR trogocytosis by TILs, is correlated with higher tumor grade. We evaluated the EGFR expression of TILs in 36 triple negative breast cancer specimens and found the correlation between EGFR expression status on TILs and higher tumor grade (P = 0.02). These findings may explain the reason why triple negative breast cancer with greater proportion of TILs exhibit higher tumor grade and poor prognosis.

Citation Format: Eiji Suzuki, Ayane Yamaguchi, Tatsuki R. Kataoka, Masahiro Hirata, Kosuke Kawaguchi, Mariko Nishie, Masakazu Toi. EGFR is transferred from triple negative breast cancer cells to immune cells via trogocytosis and expression of EGFR on immune cells is associated with high tumor grade of triple negative breast cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2349. doi:10.1158/1538-7445.AM2015-2349
Abstract 2374: Mir-29/Hsp47 regulate breast cancer progression by modulating the ECM transcription network
Authors: Zhu Jieqing , Xiong Gaofeng , Xu Ren
Published: August 2015

The extracellular matrix (ECM), an essential component of tumor microenvironment, play crucial roles in cancer development and progression. Using gene co-expression network analysis, we have identified an ECM transcription network from hundreds of human breast cancer. In this report, we show how the molecular chaperone protein Hsp47 functions as a nodal hub in regulating an ECM gene transcription network. We found that Hsp47 expression was activated during breast cancer development and progression. Hsp47 silencing reprogrammed human breast cancer cells to form growth-arrested and/or non-invasive structures in 3D cultures, and to limit tumor growth in xenograft assays by reducing deposition of collagen and fibronectin. Co-expression network analysis also showed that levels of microRNA-29b and 29c were inversely correlated with expression of Hsp47 and ECM network genes in human breast cancer tissues. We found that miR-29 repressed expression of Hsp47 along with multiple ECM network genes. Ectopic expression of miR-29b suppressed malignant phenotypes of breast cancer cells in 3D culture. Clinically, increased expression of Hsp47 and reduced levels of miR-29b and 29c were associated with poor survival outcomes in breast cancer patients. Our results show that Hsp47 is regulated by miR-29 during breast cancer development and progression, and that increased Hsp47 expression promotes cancer progression in part by enhancing deposition of ECM proteins.

Citation Format: Jieqing Zhu, Gaofeng Xiong, Ren Xu. Mir-29/Hsp47 regulate breast cancer progression by modulating the ECM transcription network. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2374. doi:10.1158/1538-7445.AM2015-2374
Abstract 2380: Integrin αv is necessary for skin tissue generation and SCC tumor invasion, but dispensable for tissue maintenance
Authors: Duperret Elizabeth K. , Dahal Ankit , Ridky Todd W.
Published: August 2015

Integrin heterodimers play crucial roles in epithelial adhesion, proliferation, differentiation, wound healing and tumorigenesis. However, the effects of acute loss of many of the 26 integrin subunits in epidermis have not been determined. This information is critical since many inhibitors for specific integrin heterodimers are entering clinical trials for various cancers. We performed an shRNA-based multiplexed screen to define roles for each integrin subunit in a 3-dimensional in vitro organotypic skin model. In this screen, we transduced individual populations of human keratinocytes with shRNAs against each integrin subunit. We then quantitatively determined the fitness of each cell population in a competition assay in reconstituted human skin. This screen identified the integrin αv class of heterodimers as essential for generation of human skin tissue: the population of keratinocytes lacking αv was selected against 125-fold compared to non-silencing control cells (p<0.001). An identical screen in human melanocytes showed no change in fitness upon loss of integrin αv, indicating cell-intrinsic functions for this subunit. Loss of integrin αv alone in keratinocytes leads to cell cycle arrest with a 4-5 fold increase in the G1/S ratio. Surprisingly, we found that integrin αv is not localized within focal adhesions in human keratinocytes and does not control focal adhesion dynamics in this cell type. Instead, it forms an independent signaling complex at the cellular membrane that controls keratinocyte growth through a FAK c-myc signaling axis. Both FAK and c-myc total protein levels are lost upon αv knock down, and re-expression of c-myc in αv-null keratinocytes is sufficient to completely rescue the cell cycle defects and skin tissue phenotypes. The effects of integrin αv loss are dependent on αv's binding partners β5 and β6, but not β1 or β8. Using a doxycycline-inducible system, we show that αv loss plays a crucial role in tissue generation and tumor invasion, but is not necessary for epidermal tissue maintenance. Furthermore, both β5 and β6 binding partners are required for tumor invasion, thus identifying αvβ5 and αvβ6 as therapeutic targets for metastatic squamous cell carcinoma.

Citation Format: Elizabeth K. Duperret, Ankit Dahal, Todd W. Ridky. Integrin αv is necessary for skin tissue generation and SCC tumor invasion, but dispensable for tissue maintenance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2380. doi:10.1158/1538-7445.AM2015-2380
Abstract 2391: Microglia-stimulated glioblastoma cell invasion is dependent on the EGFR ligand amphiregulin
Authors: Desai Bhavika , Rath Uttama , Segall Jeffrey E. , Coniglio Salvatore J.
Published: August 2015

Glioblastoma Multiforme (GBM) is one of the deadliest human cancers with a median survival rate of only one year following diagnosis. The highly infiltrative nature of GBM is one of the main reasons this cancer is so difficult to completely remove surgically. Therefore increasing our knowledge of the mechanism GBM cells use to invade normal brain is of critical importance in designing novel therapies. We recently showed that tumor associated microglia stimulate GBM cell invasion and this process is mediated by CSF-1R signaling. In this study, we seek to identify factors that are upregulated in microglia in a CSF-1R-dependent manner and ascertain whether they are involved in mediating invasion of GBM cells. We assayed cDNA and protein from microglia treated with conditioned media from the murine GBM cell line GL261, and discovered that the EGFR ligand amphiregulin (AREG) is strongly upregulated. This upregulation is blocked by addition of a pharmacological CSF-1R inhibitor. Using RNA interference, we show that microglia depleted of AREG can not promote invasion of GL261 cells into matrigel-coated invasion chambers as effectively. In addition, an AREG blocking antibody strongly attenuates the ability of THP1 macrophages to activate human GBM cell line U87 invasion. Furthermore, we have identified a signaling pathway which involves CSF-1R signaling through MAPK ERK to upregulate AREG expression in microglia and interfering with ERK using pharmacological inhibitors also prevents microglia-stimulated GL261 invasion. Interestingly, microglia that have been activated by GBM cells appear to require cell-cell contact in order to promote invasion. This data highlights AREG as a key factor in produced by tumor associated microglia in promoting glioblastoma invasion.

Citation Format: Bhavika Desai, Uttama Rath, Jeffrey E. Segall, Salvatore J. Coniglio. Microglia-stimulated glioblastoma cell invasion is dependent on the EGFR ligand amphiregulin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2391. doi:10.1158/1538-7445.AM2015-2391
Abstract 2400: Circulating tumor DNA as a non-invasive substitute to metastasis biopsy for targeted sequencing in a prospective randomized trial for personalized treatment in all tumor type: The SHIVA study
Authors: Pierga Jean-Yves , Decraene Charles , Bernard Virginie , Kamal Maud , Blin Anthony , Leroy Quentin , Rio Frio Thomas , Pierron Gaëlle , Callens Céline , Bieche Ivan , Saliou Adrien , Madic Jordan , Rouleau Etienne , Bidard François-Clément , Lantz Olivier , Stern Marc-Henri , Lebofsky Ronald , Le Tourneau Christophe
Published: August 2015

Rationale: The noninvasive biomarker circulating cell-free tumor DNA (ctDNA) has the potential to enable noninvasive diagnostic tests for personalized medicine in providing similar molecular information as that derived from invasive tumor biopsies. The ongoing large multicentric randomized histology-independent phase II trial (SHIVA, NCT01771458) compares molecularly targeted therapy based on tumor molecular profiling versus conventional therapy in patients with any type of refractory cancer. The screening phase consists of the invasive biopsy of metastatic tumor tissue and downstream analysis using Ion Torrent's Ampliseq hotspot cancer panel. We evaluated whether ctDNA analysis would identify the same mutations as those obtained through invasive biopsy.

Patients and methods: de novo detection of somatic mutations using ctDNA in 34 patients covering 18 different tumor types, scanning ∼50 genes and more than 2500 mutations with a multiplexed next generation sequencing panel.

Results: In 27 patients, 27 of 28 actionable mutations identified in metastasis biopsies (96%) were detected in matched ctDNA. Among these 27 patients, two additional mutations were found in ctDNA only. In the seven other patients, mutation detection from metastasis biopsy failed due to inadequate biopsy material, but was successful in all plasma DNA samples providing three more potential actionable mutations. Conclusion; These results suggest that ctDNA analysis is a cheaper, safer and quicker alternative to invasive biopsy of metastasis, irrespective of cancer type and metastatic site, for multiplexed mutation detection in selecting personalized therapies based on the patient's tumor genetic content.

Citation Format: Jean-Yves Pierga, Charles Decraene, Virginie Bernard, Maud Kamal, Anthony Blin, Quentin Leroy, Thomas Rio Frio, Gaëlle Pierron, Céline Callens, Ivan Bieche, Adrien Saliou, Jordan Madic, Etienne Rouleau, François-Clément Bidard, Olivier Lantz, Marc-Henri Stern, Ronald Lebofsky, Christophe Le Tourneau. Circulating tumor DNA as a non-invasive substitute to metastasis biopsy for targeted sequencing in a prospective randomized trial for personalized treatment in all tumor type: The SHIVA study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2400. doi:10.1158/1538-7445.AM2015-2400
Abstract 2408: A novel multiplex droplet digital PCR approach to KRAS mutation detection in circulating tumor DNA
Authors: Pender Alexandra , Garcia-Murillas Isaac , Rana Sareena , de Castro David Gonzalez , Turner Nicholas , Popat Sanjay , Downward Julian
Published: August 2015

Microdroplet digital PCR can be used to detect low frequency mutations in oncogene-driven lung cancer. Detection of circulating tumour DNA in plasma can be limited by paucity of material, particularly during response to anti-cancer therapy. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach for efficient mutation detection in circulating DNA.

We designed and optimised 3 droplet digital PCR multiplex assays investigating 9 different KRAS mutations using PrimePCR™ ddPCR™ Mutation Assays and the Bio-Rad QX100 system. DNA was extracted from FFPE tissue and plasma from patients with advanced lung adenocarcinoma and was analysed to assess for the presence of these KRAS activating mutations.

Multiple sub-clonal KRAS mutant populations were detectable in both tissue and circulating tumour DNA at low frequency (up to 0.1%). Similar KRAS allele frequency was observed using multiplex or duplex assays (r2 = 0.99). Sub-clonal population representation varied over the disease course.

Multiplex microdroplet digital PCR assays can be adapted to be used to efficiently identify tissue and circulating KRAS mutations in advanced lung adenocarcinoma. Variation in sub-clonal populations between tissue and plasma, and at different time points over the disease course, illustrates the potential use of circulating DNA analysis as a more readily accessible, non-invasive and accurate representation of the molecular profile of NSCLC throughout treatment.

Citation Format: Alexandra Pender, Isaac Garcia-Murillas, Sareena Rana, David Gonzalez de Castro, Nicholas Turner, Sanjay Popat, Julian Downward. A novel multiplex droplet digital PCR approach to KRAS mutation detection in circulating tumor DNA. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2408. doi:10.1158/1538-7445.AM2015-2408
Abstract 2415: Development of a clinical cell-free DNA assay for cancer molecular profiling
Authors: Clark Travis , Kennedy Mark , Young Geneva , Young Lauren , He Jie , Yelensky Roman , Ali Siraj , Otto Geoff , Lipson Doron , Miller Vince , Stephens Phil
Published: August 2015

Background:

Profiling cell-free circulating tumor DNA (ctDNA) for the genomic alterations which drive oncogenesis in patients with cancer promises to provide information important for understanding cancer biology, informing therapy selection when conventional biopsies are unobtainable and monitoring response to therapy. Facile collection of ctDNA samples could enable recurrent molecular profiling of an individual patient's disease with lower cost, discomfort and risk as compared to conventional biopsies. To assess the clinical validity of profiling cancer genomics in ctDNA, a highly accurate targeted NGS-based assay was developed and ctDNA results were compared to patient-matched FFPE tumor biopsies characterized by comprehensive genomic profiling using the FoundationOne assay from over 150 patients with lung, breast and other cancer types.

Methods:

To ensure robust performance, the ctDNA assay was developed as part of an integrated workflow including primary sample collection, storage and transport, and ctDNA purification, followed by optimized construction of adaptor-ligated sequencing libraries and enrichment by solution hybridization and then sequencing to high depth (Illumina HiSeq). Computational methodologies were developed to enable sensitive and specific detection of base substitutions, indels, genomic rearrangements and high-level amplificagtions from ctDNA. Accuracy and reproducibility was assessed using cell-line mixtures designed to simulate the limited DNA inputs and low tumor purity expected in routine clinical samples.

Results:

The ctDNA assay enabled accurate detection of most genomic alterations in cell-line mixtures. Short variants (base substitutions and indels) and genomic rearrangements in ALK and RET were detected with high sensitivity and specificity at <1% tumor. Comparative results from patient-matched ctDNA isolated from plasma and FFPE biopsies across more than 150 lung, breast and other cancer samples will be presented.

Conclusions:

Accurate profiling of ctDNA can enable detection of biologically and clinically relevant genomic alterations in clinical plasma samples. These results demonstrate the potential utility of ctDNA molecular profiling for the management of patients with cancer, but prior to integration into routine practice, extensive rigorous tumor-type specific studies of patient-matched ctDNA and solid biopsy specimens are required.

Citation Format: Travis Clark, Mark Kennedy, Geneva Young, Lauren Young, Jie He, Roman Yelensky, Siraj Ali, Geoff Otto, Doron Lipson, Vince Miller, Phil Stephens. Development of a clinical cell-free DNA assay for cancer molecular profiling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2415. doi:10.1158/1538-7445.AM2015-2415
Abstract 2419: Predicting clinical response based on ex vivo drug response in renal cell carcinoma using kinase activity profiling
Authors: Ruijtenbeek Rob , Houkes-van Kerkhoff Liesbeth , Hilhorst Maria , Mulders Peter , Oosterwijk-Wakka Jeannette , Kiemeney Lambertus , Oosterwijk Egbert
Published: August 2015

Introduction: Sunitinib, a potent multitargeted receptor tyrosine kinase inhibitor, is the first line treatment for metastatic renal cell carcinoma (mRCC). Because sunitinib responses and toxicity are highly variable, there is a need for biomarkers predicting sunitinib response or predicting the optimal sequence preference when using alternative tyrosine kinase inhibitors. The aim of this study was to investigate the correlation between ex vivo drug response and clinical response in renal cell carcinoma (RCC) and to explore alternative treatment options. This study has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no 259939.

Methods: Protein tyrosine kinase activity profiles were generated on PamChip® peptide microarrays of lysed tumor resection tissues (1-5mm3) from 22 mRCC patients. The ex vivo effect of kinase inhibitors (sunitinib, axitinib, sorafenib, pazopanib, erlotinib and crenolanib) was determined and analyzed with Bionavigator software. A two-group (sunitinib responders versus non-responders) comparison applied on the inhibition ratios identified the significantly different peptide phosphorylations. Peptides were clustered according to their correlation with clinical response.

Results: As little as 5 μg protein input (0.05 mm3 tissue) was used per kinase activity profile. The ex vivo sunitinib effect positively correlated with clinical responses especially in the subgroup which received sunitinib as 1st line treatment (4 responders vs. 4 non-responders). 18 of the 105 peptides were significantly (p<0.05) less phosphorylated upon ex vivo sunitinib treatment in 4 responders compared to 4 non-responders. Pathway analysis of the peptide phosphorylation patterns revealed sunitinib target involvement. Ex vivo erlotinib, axitinib or crenolanib showed reversed inhibition patterns i.e. more inhibition in the non-responder than in the responder group.

In summary, we have shown that predicting clinical response to sunitinib based on the ex vivo response to sunitinib is feasible. This requires further investigation with a larger sample set. Furthermore, the ex vivo response to a drug panel suggests that identification of novel treatment options for non-responders might be feasible.

Citation Format: Rob Ruijtenbeek, Liesbeth Houkes-van Kerkhoff, Maria Hilhorst, Peter Mulders, Jeannette Oosterwijk-Wakka, Lambertus Kiemeney, Egbert Oosterwijk. Predicting clinical response based on ex vivo drug response in renal cell carcinoma using kinase activity profiling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2419. doi:10.1158/1538-7445.AM2015-2419
Abstract 2432: A technology platform for personalized medicine in melanoma
Authors: Girotti Maria R. , Rothwell Dominic , Viros Amaya , Mandal Amit Kumar , Lim Kok Haw J. , Gremel Gabriela , Furney Simon , Pedersen Malin , Rogan Jane , Swan Jacqueline , Fusi Alberto , Brady Ged , Lorigan Paul , Dive Caroline , Marais Richard
Published: August 2015

We developed patient derived xenografts (PDX), performed whole exome sequencing (WES) and analyzed circulating tumor DNA (ctDNA) to complement clinical management of four melanoma patients. Synchronous treatment of patients and mice showed that PDX responses mirrored those of the patients’ tumor to BRAF inhibitors. PDXs were used to test second-line treatments in relapsed patients and to validate WES-based therapies in tumors that did not have a BRAF mutation. Longitudinal analysis of ctDNA was predictive of responses to targeted and immunotherapies, and to determine mechanisms of resistance. Thus, we describe a powerful combination of techniques for personalized medicine in melanoma and discuss the challenges and limitations of implementing these novel technologies in patient management.

Citation Format: Maria R. Girotti, Dominic Rothwell, Amaya Viros, Amit Kumar Mandal, Kok Haw J. Lim, Gabriela Gremel, Simon Furney, Malin Pedersen, Jane Rogan, Jacqueline Swan, Alberto Fusi, Ged Brady, Paul Lorigan, Caroline Dive, Richard Marais. A technology platform for personalized medicine in melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2432. doi:10.1158/1538-7445.AM2015-2432