bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1191: The RNA binding protein, HuR, regulates pancreatic cancer cell metabolism
Authors: Blanco Fernando F. , Zarei Mahsa , Brody Jonathan R. , Boros Laszlo G. , Winter Jordan M.
Published: August 2015

Introduction: Few proteins have been found to be master-regulators of pancreatic ductal adenocarcinoma (PDA) cell metabolism (e.g., LKB1, KRAS). We recently demonstrated that the regulatory RNA binding protein, HuR, binds to numerous metabolic mRNA transcripts and regulates their expression. Additionally, HuR silencing with small interfering RNAs(siRNAs) inhibited growth of PDA cells in vitro under glucose deprivation. Herein, we manipulated HuR expression levels in PDA cells and performed isotope tracer fate association studies to better understand HuR's role in metabolic reprogramming.

Methods: We used 13C-labeled nutrients to map carbon flux in BxPC3 pancreatic cancer cells that were either transiently transfected with siRNA oligos against HuR (siHuR) or scrambled control (siCTRL). Cells were incubated with standard DMEM or mild glucose deprivation (5 mM glucose) for 48 hours, and media were supplemented with [1,2-13C2]-D-glucose or [U-13C5]-L-glutamine tracers for the final 24 hours. A total of 24 samples were snap frozen and prepared for GC-MS isotopomer analysis (3 replicates X 2 different siRNA oligos X 2 isotope tracers X 2 glucose concentrations). Transfection experiments were validated for reduced HuR expression (>70% reduction) and reduced protein expression of at least one metabolic and established HuR target (e.g., IDH1) by immunoblot.

Results: HuR silencing directly impaired fatty acid (Table, line 1-3), ribose (line 5) and glycogen synthesis (line 6). Reductive carboxylation of glutamine-derived isocitrate was impaired and futile carbon exchange fluxes were prevalent (not shown).

Conclusions: HuR enhances metabolic efficiency in PDA cells by directly regulating multiple metabolic pathways. Ongoing microarray studies will highlight which metabolic transcripts are post-transcriptionally regulated by HuR, resulting in the observed phenotype.

Altered metabolites due to HuR silencing in PDA cells ([1,2-13C2]-D-glucose tracer)PathwayMetabolitesiHuR 25mM glucosesiHuR 5 mM glucoseCorrelation1Myristate (C:14) Intracell13C enrichment70.271.01.02Myristate (C:14) intracellFNS (direct)56.060.41.03Oleate (C:18-1) IntracellIndirect synthes-m187.689.51.04Glutam extracell [C2-C5]13C-m1 (m/z198)97.497.91.05RNA-ribose Intracell [C1-C4]13C-m3(m/z242)99.399.10.9846Glucose intracell [C3-C6]Peak area, Glycog-gluc74.180.10.983

Citation Format: Fernando F. Blanco, Mahsa Zarei, Jonathan R. Brody, Laszlo G. Boros, Jordan M. Winter. The RNA binding protein, HuR, regulates pancreatic cancer cell metabolism. [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 1191. doi:10.1158/1538-7445.AM2015-1191
Abstract 1192: Glutamic acid and glutaminolysis mark aggressive tumorigenesis in head and neck squamous cell carcinoma
Authors: Banda Adam D. , Bermudez Cortes Mercedes , Kamarajan Pachiyappan , Rajendiran Thekkenlnaycke , Chinnaiyan Arul M. , Kapila Yvonne L.
Published: August 2015

The purpose of this study was to identify markers of aggressive tumor behavior to inform processes underlying tumor aggressivity in head and neck squamous cell carcinoma (HNSCC). To this end, metabolomic profiling and validation of biomarkers of aggressive HNSCC tumorigenesis were assessed in HNSCC human specimens and cell lines. Using LG/GC-MS, we found that glutamic acid is one of the most highly differentially elevated metabolites in HNSCC specimens. In subsequent validation studies, where we incorporated metastatic tissues, glutamic acid was further verified as differentially elevated in metastatic tissues compared to primary HNSCC and normal adjacent tissues. In addition, significantly elevated expression levels of c-Myc and glutaminase, the enzyme that regulates glutamic acid levels, were highest in metastatic tissues compared to primary HNSCC and normal tissues. Parallel changes in c-Myc and glutaminase expression in HNSCC tissues suggest a link between c-Myc regulation and glutamine metabolism. Furthermore, Western Blot analyses show similar expression patterns for c-Myc and glutaminase in oral cancer cell lines. Inhibiting glutamic acid levels by stably suppressing glutaminase expression in HNSCC cells, led to suppression of aggressive tumorigenesis in vitro, whereas enrichment with external glutamine or glutamic acid further enhanced tumorigenesis. Together, this data suggest that oncogenic levels of c-Myc may induce a transcriptional program that promotes elevated levels of glutamic acid and glutaminolysis via glutaminase to promote tumor aggressivity in head and neck cancer.

Citation Format: Adam D. Banda, Mercedes Bermudez Cortes, Pachiyappan Kamarajan, Thekkenlnaycke Rajendiran, Arul M. Chinnaiyan, Yvonne L. Kapila. Glutamic acid and glutaminolysis mark aggressive tumorigenesis in head and neck squamous cell 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 1192. doi:10.1158/1538-7445.AM2015-1192
Abstract 1212: Mutant p53 stabilizes and protects the transcription factor ETS2 from proteasomal degradation by the ubiquitin ligase COP1/DET1
Authors: Carrero Zunamys I. , Kollareddy Madhusudhan , Chauhan Krishna M. , Ramakrishnan Gopalakrishnan , Martinez Luis A.
Published: August 2015

Genetic mutations of the tumor suppressor gene TP53 contribute to a great majority of human cancers. One mechanism by which mutant TP53 (mtp53) acts is through the interaction with other transcription factors, which can either enhance or repress the transcription of their target genes. Mtp53 preferentially interacts with ETS2, an ETS transcription factor, and increases its stability. This results in an increase of the expression of ETS target genes that are associated with the transition of normal cells into tumor cells. Previous studies have shown that the ubiquitin-proteasomal pathway regulates ETS2 stability. To study the mechanism of ETS2 ubiquitin-mediated degradation, we used siRNA to knockdown ubiquitin ligases known to interact with ETS2. A significant increase in ETS2 stability was observed when the ubiquitin ligase COP1 and its binding partner DET1 were knocked down. We find that ectopically expressed COP1/DET1 leads to ETS2 ubiquitination and degradation, and that a COP1 without an intact RING domain fails to degrade ETS2. Mutation of COP1 putative binding sites in ETS2 also prevented its degradation. Given that mtp53 interacts with ETS2, we assessed mtp53's role in ETS2 stability by conducting ubiquitination assays. Our data showed that mtp53 prevents COP1/DET1 from ubiquitinating ETS2, thus preventing it from being degraded. We are currently studying the mechanism of COP1/DET1 binding to ETS2 and how mtp53 interferes with this process, thus rescuing ETS2. We surmise that mtp53 disrupts the binding of COP1/DET1 to ETS2, leading to its stabilization. Through this mechanism, mtp53 can alter the balance of ETS proteins in normal cells, which results in an increased ETS2 transcriptional program that drives cancer cells to be more invasive and metastatic.

Citation Format: Zunamys I. Carrero, Madhusudhan Kollareddy, Krishna M. Chauhan, Gopalakrishnan Ramakrishnan, Luis A. Martinez. Mutant p53 stabilizes and protects the transcription factor ETS2 from proteasomal degradation by the ubiquitin ligase COP1/DET1. [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 1212. doi:10.1158/1538-7445.AM2015-1212
Abstract 1218: A biological crosstalk between p53 and MYCN/MYC in neuroblastoma
Authors: Ikegaki Naohiko , Tang Xao
Published: August 2015

We previously showed that TSA (an HDAC inhibitor) and Epoxomycin (a proteasome inhibitor) as single agents and in combination significantly suppressed growth of MYCN-amplified neuroblastoma cells. However, these compounds had contrasting effects on MYCN expression. TSA down-regulated MYCN expression, but Epoxomycin and the TSA/Epoxomycin combination led to MYCN hyper-expression (defined as markedly increased expression beyond that observed in the untreated cells). The expression of p53 was also increased in MYCN-amplified cells treated with Epoxomycin or the TSA/Epoxomycin combination. In an independent study, we also found that treatment of neuroblastoma cells (MYCN-amplified or non-MYCN amplified) with 17-DMAG and S(+) Ibuprofen resulted in an increase in p53 expression and a reduction in MYCN or MYC expression. In this study, we examined (i) the pattern of gene expression induced by MYCN hyper-expression in MYCN-amplified cells, and (ii) a potential functional relationship between p53 and MYCN/MYC in neuroblastoma. Transient transfection of MYCN and TP53 into neuroblastoma cells was done by electroporation. Gene expression profiling, TaqMan real-time PCR, and Western blot assays were used to detect expression patterns of genes and proteins. It was observed that ectopic over MYCN expression in MYCN-amplified IMR5 cells resulted in growth suppression. Gene expression profiling analysis revealed that the hyper-expression of MYCN in the MYCN-transfected IMR5 cells led to an increased expression of genes involved in growth suppression and apoptosis, including EGR1, EPHA2, KLF2, PERP and SEL1L. The expression of PERP and EPHA2 was confirmed by TaqMan real-time PCR and Western blot assay, respectively. Interestingly, co-transfection of TP53 and MYCN in IMR5 cells led to high p53 expression but a reduction in MYCN expression (below the levels of endogenous MYCN). Transfection of TP53 into IMR5, SY5Y, and SKNAS reduced endogenous MYCN and MYC expression in these cells. Consistent with these observations, treatment of IMR5 and SY5Y cells with known p53-inducers, Doxorubicin and CoCl2, resulted in an increased p53 expression and a reduction of MYCN and MYC expression. Although high MYCN expression sustains growth of MYCN-amplified neuroblastoma, the hyper-expression of MYCN is deleterious to survival of these cells. In addition, augmented p53 expression may elicit a negative feedback regulation of MYCN/MYC expression in neuroblastoma.

Citation Format: Naohiko Ikegaki, Xao Tang. A biological crosstalk between p53 and MYCN/MYC in neuroblastoma. [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 1218. doi:10.1158/1538-7445.AM2015-1218
Abstract 122: PARK2 is a negative regulator of Wnt and EGFR pathways in glioma
Authors: Xu Liang , Lin De-Chen , Chen Ye , Yan Haiyan , Hazawa Masaharu , Doan Ngan , Said Jonathan W. , Ding Ling-Wen , Liu Li-Zhen , Yang Henry , Yu Shi-zhu , Kahn Michael , Yin Dong , Koeffler Phillip
Published: August 2015

PARK2 is an E3 ubiquitin ligase whose dysfunction has been associated with the progression of autosomal recessive juvenile Parkinson's disease and human malignancies. However, its role in cancer remains to be explored. In this study, we report that PARK2 is frequently deleted and underexpressed in human glioma, and low PARK2 expression is associated with poor survival in cohorts of both low-grade glioma and glioblastoma multiforme (GBM). Functional studies revealed a tumor-suppressive role of PARK2 in GBM cells. Restoration of PARK2 significantly inhibited glioma cell growth both in vitro and in vivo, while depletion of endogenous PARK2 promoted cell proliferation. cDNA microarray analysis showed that PARK2 expression strongly altered the activity of both the Wnt and EGFR pathways. Immunoblot analysis confirmed that ectopic expression of PARK2 suppressed the intracellular levels of β-catenin, EGFR, as well as their down-stream targets including Cyclin D1, TCF4, c-Myc, p-AKT and p-S6K, etc. Notably, PARK2 physically interacted with both β-catenin and EGFR, and promoted their ubiquitination in an E3-ligase dependent manner. Similar to the ligase-dead PARK2 mutant (C431S), three PARK2 mutants harboring cancer-derived somatic mutations (T173A, T240M and P294S) showed decreased ability to ubiquitinate either β-catenin or EGFR proteins. We further found that PARK2 attenuated the cellular response to both Wnt3a and EGF stimulation, suggesting PARK2 is a negative regulator of both the Wnt and EGFR pathways. Lastly, inspired by these newly identified functions of PARK2, we tested and proved that the combination of small-molecule inhibitors targeting both Wnt-β-catenin and EGFR-AKT pathways synergistically impaired glioma cell viability. In aggregate, our findings uncover novel, cancer-associated functions of PARK2 and provide a potential therapeutic approach to treat glioma.

Citation Format: Liang Xu, De-Chen Lin, Ye Chen, Haiyan Yan, Masaharu Hazawa, Ngan Doan, Jonathan W. Said, Ling-Wen Ding, Li-Zhen Liu, Henry Yang, Shi-zhu Yu, Michael Kahn, Dong Yin, Phillip Koeffler. PARK2 is a negative regulator of Wnt and EGFR pathways in glioma. [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 122. doi:10.1158/1538-7445.AM2015-122
Abstract 1223: LIF is a novel negative regulator of p53 in colorectal cancers
Authors: Hu Wenwei , Yu Haiyang , Yue Xuetian , Zhao Yuhan , Zhang Cen , Young Ken , Liu Juan , Feng Zhaohui
Published: August 2015

Leukemia inhibitory factor (LIF), a cytokine of the interleukin 6 superfamily,is involved in multiple biological functions. Recently, we identified that LIF is a p53 target gene which mediates the role of p53 in maternal implantation under normal physiological conditions in both mice and humans. However, to date, the role of LIF in tumorigenesis, especially colorectal cancers, is poorly understood. Here, we report that LIF is a negative regulator of p53; LIF downregulates p53 protein levels and function in transcriptional activity, apoptosis and senescence in human colorectal cancer cells. The downregulation of p53 by LIF is mediated by the activation of Stat3; blocking the Stat3 pathway largely abolishes the inhibitory effect of LIF on p53. We further identified that Stat3 transcriptionally induces ID1, the helix-loop-helix (HLH) protein inhibitor of differentiation and DNA binding to mediate its role in the negative regulation of p53. ID1 upregulates MDM2, a key negative regulator of p53, and promotes MDM2-mediated p53 protein degradation. We found that LIF is overexpressed in a large percentage of colorectal cancers. In turn, LIF overexpression promotes cellular resistance towards chemotherapeutic agents in cultured colorectal cancer cells and colorectal xenograft tumors in a largely p53-dependent manner. Furthermore, we found that overexpression of LIF is associated with a poor prognosis in colorectal patients. Taken together, LIF is a novel negative regulator of p53, overexpression of LIF is an important mechanism for the attenuation of p53, which promotes chemoresistance in colorectal cancers. Targeting LIF, Stat3 as well as ID1 to reactivate p53 is a potential therapeutic strategy to enhance chemosensitivity in colorectal cancer, especially in tumors with LIF overexpression.

Citation Format: Wenwei Hu, Haiyang Yu, Xuetian Yue, Yuhan Zhao, Cen Zhang, Ken Young, Juan Liu, Zhaohui Feng. LIF is a novel negative regulator of p53 in colorectal cancers. [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 1223. doi:10.1158/1538-7445.AM2015-1223
Abstract 1227: Differential efficacy of p53 restoration in induction and maintenance of senescence in premalignant and malignant cells
Authors: Harajly Mohammad , Zalzali Hassan , Ghayad Sandra , Ghamloush Farrah , Jabbour Mark , Basma Hussein , Tawil Ayman , Nawaz Zafar , Saab Raya H.
Published: August 2015

Restoration of p53 in tumors has been suggested as a possible therapeutic approach, based on preclinical in vivo and in vitro evidence of possible efficacy. However, the relationship between the timing of p53 restoration and its efficacy is still unclear. We now show that restoration of p53 in murine pre-malignant proliferating pineal tumors results in effective cell cycle exit and induction of cellular senescence, while p53 reactivation in established malignant pineal tumors does not impact cell proliferation, unless paired with DNA damaging therapies. This differential effect was not related to levels of p19Arf expression, as its expression was more pronounced in malignant tumors, nor to MAPK pathway activity. Interestingly, in premalignant lesions induced to senesce by p53 restoration, inactivation of p53 after senescence resulted in re-entry into the cell cycle, and rapid tumor progression. These findings were also validated in cell culture models of Cyclin D1-induced and RasV12-induced senescence in murine pineal cells and mouse embryo fibroblasts, respectively. Evaluation of a panel of human supratentorial primitive neuroectodermal tumors (sPNET), of which pineoblastoma is a subtype, showed low activity of the p53 pathway, while only one of 6 tumors had p53 deletion or mutation. Together, this data shows that restoration of the p53 pathway has different effects in premalignant versus invasive pineal tumors, where it may need to be paired with DNA damaging agents, to engage it in tumor suppression. Furthermore, p53 activation may need to be continually sustained for effective tumor suppression in such lesions undergoing senescence as a tumor suppressor mechanism. Finally, p53 restoration approaches may be worth exploring in sPNET, where the p53 gene seems to be intact but the pathway inactive in the majority of examined tumors.

Citation Format: Mohammad Harajly, Hassan Zalzali, Sandra Ghayad, Farrah Ghamloush, Mark Jabbour, Hussein Basma, Ayman Tawil, Zafar Nawaz, Raya H. Saab. Differential efficacy of p53 restoration in induction and maintenance of senescence in premalignant and malignant 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 1227. doi:10.1158/1538-7445.AM2015-1227
Abstract 1254: Mitochondrial SOD2 regulates EMT and cancer stem cell-like cell populations
Authors: Whelan Kelly A. , Kinugasa Hideaki , Tanaka Koji , Srinivasan Satish , Guha Manti , St. Clair Daret , Klein-Szanto Andres , Avadhani Narayan , Diehl Alan , Rustgi Anil , Nakagawa Hiroshi
Published: August 2015

Epithelial-mesenchymal transition (EMT) is involved in cancer cell invasion, metastasis and treatment failure. EMT may promote conversion of a subset of cancer cells defined by low expression of CD44 (CD44L) to those with high expression of CD44 (CD44H), the latter associated with increased malignant properties of cancer cells. Triggered by a variety of stimuli such as transforming growth factor (TGF)-β, hypoxia and chemoradiation, EMT may be activated in cancer cells via stress-induced reactive oxygen species (ROS) in the tumor microenvironment. Excessive ROS may cause cell death or senescence; however, cells undergoing EMT may require a physiological level of ROS. It remains elusive as to how cellular antioxidant capabilities may influence EMT. Mitochondrial superoxide dismutase 2 (SOD2) is frequently overexpressed in oral and esophageal cancers. Here, we investigated mechanisms of SOD2 transcriptional regulation in EMT as well as the functional role of this antioxidant in EMT. Using well-characterized genetically engineered oral and esophageal human epithelial cell lines coupled with RNA interference (RNAi) and flow cytometric approaches, we find that TGF-β stimulates EMT, resulting in conversion of CD44L to CD44H cells, the latter of which display SOD2 upregulation. SOD2 induction in transformed cells was concurrent with suppression of TGF-β-mediated induction of both ROS and senescence. SOD2 gene expression appeared to be transcriptionally regulated by NF-κB and ZEB2, but not ZEB1. Moreover, SOD2-mediated antioxidant activity may restrict conversion of CD44L cells to CD44H cells at the early stages of EMT. In aggregate, this study provides novel mechanistic insights into the dynamic expression of SOD2 during EMT and delineates a functional role for SOD2 in EMT, influencing distinct CD44L and CD44H subsets of cancer cells that have been implicated in oral and esophageal tumor biology.

Note: This abstract was not presented at the meeting.

Citation Format: Kelly A. Whelan, Hideaki Kinugasa, Koji Tanaka, Satish Srinivasan, Manti Guha, Daret St. Clair, Andres Klein-Szanto, Narayan Avadhani, Alan Diehl, Anil Rustgi, Hiroshi Nakagawa. Mitochondrial SOD2 regulates EMT and cancer stem cell-like cell populations. [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 1254. doi:10.1158/1538-7445.AM2015-1254
Abstract 1275: Chemotactic factors underlying tumor infiltration by immunocompetent cells in colorectal cancer
Authors: Cremonesi Eleonora , Amicarella Francesca , Padovan Elisabetta , Muraro Manuele G. , Mele Valentina , Zajac Paul , Hirt Christian , Droeser Raoul , Oertli Daniel , Zuber Markus , Adamina Michel , Rosso Raffaele , Garzon Jesus G. , Borsig Lubor , Spagnoli Giulio C. , Iezzi Giandomenica
Published: August 2015

Colorectal cancer (CRC) is a leading cause of cancer related death. Several studies have convincingly showed that CRC infiltration by immunocompetent cells and, in particular, cytotoxic CD8+ T cells, IFN-γ-producing cells, Foxp3+ regulatory T cells and CD16+ MPO+ neutrophils, is significantly associated with prolonged patient survival. However, the factors driving these cell populations into the tumor site remain to be elucidated. We are investigating the chemokine/chemokine receptor network promoting CRC infiltration by immune cells associated to favorable prognosis.

We have analysed the expression of a panel of genes encoding for 39 chemokines and for 7 markers identifying specific immune cell types in 58 freshly excised specimens of CRC and corresponding control colonic tissues. In parallel, chemokine receptor profiles on clinically relevant immunocompetent cell populations have been characterized by flow cytometry.

Our results suggest that tumor infiltration by immunocompetent cells associated to favorable prognosis is linked to the expression of three main chemokine patterns: 1) CCL3, CCL5, CXCL9, and CXCL10 capable of recruiting cytotoxic CD8+ and IFN-γ producing T cells; 2) CCL22 and CXCL12 attracting Foxp3+ regulatory T cells; 3) CXCL2, CXCL5 and CXCL8 inducing migration of CD16+ MPO+ neutrophils. We have started to investigate the chemokine sources. CRC cells purified from tumor specimens were found to express chemokines potentially recruiting T and myeloid cells, including CCL3, CCL5, CXCL9, CXCL10, CXCL12, and CXCL2, CXCL5, CXCL8. The stimuli driving chemokine expression by tumor cells remain however to be clarified and are currently under investigation.

Citation Format: Eleonora Cremonesi, Francesca Amicarella, Elisabetta Padovan, Manuele G. Muraro, Valentina Mele, Paul Zajac, Christian Hirt, Raoul Droeser, Daniel Oertli, Markus Zuber, Michel Adamina, Raffaele Rosso, Jesus G. Garzon, Lubor Borsig, Giulio C. Spagnoli, Giandomenica Iezzi. Chemotactic factors underlying tumor infiltration by immunocompetent cells in colorectal 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 1275. doi:10.1158/1538-7445.AM2015-1275
Abstract 1279: Expression of B7-H5 in human pancreatic caner
Authors: Zhu Yuwen , Byers Joshua , Paniccia Alessandro , Kaplan Jeffrey , Koenig Michelle , Schulick Richard , Edil Barish
Published: August 2015

The B7-H5/CD28H pathway is the most recently identified costimulatory pathway of the B7 family. Here we investigated how the ligand B7-H5 is expressed in normal human pancreas tissues, as well as its expression changes in pancreas cancer. By immunohistochemical (IHC) staining of frozen specimens from patients undergoing pancreatic resection, we found that B7-H5 protein was constitutively expressed on normal ductal epithelium within the pancreas. Other cell types from normal pancreas, like acinar cells or islet cells, did not express B7-H5. In adenocarcinoma, B7-H5 staining was decreased or absent, though normal ducts adjacent to tumors were highly B7-H5-positive. No B7-H5 protein was found on other cancer types such as neuroendocrine tumors. Our study suggests that loss of the B7-H5 signal might contribute to immune evasion of pancreatic ductal adenocarcinoma.

Citation Format: Yuwen Zhu, Joshua Byers, Alessandro Paniccia, Jeffrey Kaplan, Michelle Koenig, Richard Schulick, Barish Edil. Expression of B7-H5 in human pancreatic caner. [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 1279. doi:10.1158/1538-7445.AM2015-1279
Abstract 1289: Renal cell carcinoma patients have altered HLA expression patterns which show correlations to clinical parameter
Authors: Goebel Steffen , Bluemke-Anbau Karen , Altermann Wolfgang , Bilkenroth Udo , Meye Axel , Fuessel Susanne , Kehlen Astrid , Lautenschlaeger Christine , Melchior Andres , Fornara Paolo , Taubert Helge
Published: August 2015

The aim of this study was to evaluate altered HLA (Human Leukocyte Antigen) expression patterns of renal cell carcinoma (RCC) patients and healthy control persons (blood donors).

Peripheral blood samples from RCC patients and from healthy controls were investigated for the incidence of HLA groups. The frequency of HLA groups was analyzed statistically and correlated with relevant clinical data. In addition, HLA subtypes were analyzed for RCC patients and for healthy blood donors. A multidimensional statistical comparison was performed. An update of the follow up for RCC patients was conducted in 2014.

After following the CD45 autoMACS depletion protocol, we identified CTCs in 96 out of 233 peripheral blood samples (41%), which originated from 81 out of 154 (53%) RCC patients. We detected two HLA groups, HLA-Cw*07 and HLA-DRB1*04 that were significantly higher in healthy controls than in RCC patients (p < 0.05), respectively. In a Kaplan-Meier survival analysis these results were confirmed (p < 0.05). On the one hand, RCC patients had less alleles HLA-Cw*07 (p < 0.05; χ2-test) and HLA-DRB1*04 (p < 0.05; χ2-test) compared to healthy blood donors. On the other hand, RCC patients showed an increased occurrence of the allele HLA-Cw*12 compared to healthy blood donors.

There are HLA groups that occur less often in RCC patients than in healthy controls. But it needs larger study populations and further immunological studies to investigate if special HLA-types can have a protective effect against RCC carcinogenesis. Occurrence of the alleles HLA-Cw*07, and HLA-DRB1*04 was significantly altered in RCC patients compared to healthy blood donors and may change the risk to suffer from a RCC.

The integration of our results in systematic functional studies will help to earlier identify persons with an increased risk of RCC development and may have an impact in future patient care.

Citation Format: Steffen Goebel, Karen Bluemke-Anbau, Wolfgang Altermann, Udo Bilkenroth, Axel Meye, Susanne Fuessel, Astrid Kehlen, Christine Lautenschlaeger, Andres Melchior, Paolo Fornara, Helge Taubert. Renal cell carcinoma patients have altered HLA expression patterns which show correlations to clinical parameter. [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 1289. doi:10.1158/1538-7445.AM2015-1289
Abstract 1294: Identification of tumor-specific antigens associated with RET/PTC3 expression
Authors: Sponton Laura , Cosma Gabriela , Mendonca Mark , Giovarelli Mirella , Eisenlohr Laurence Crane
Published: August 2015

Relocated in transformation/papillary thyroid carcinoma, RET/PTC3 (RP3) is a fusion oncogene that causes a form of papillary thyroid cancer (PTC). In addition to driving transformation, the constitutively active kinase precociously phosphorylates itself as well as other intracellular proteins, thereby providing tumor-specific targets for the adaptive immune system.

PTCs are known to have the ability to escape the immune system driving a dysregulation of the activity of several cell populations involved in the immune response. Indeed we found that mice immunized with RP3+/MHC class II+ cells produce dramatically fewer IFNg-secreting CD4+ T cells compared to mice immunized with RP3-/MHC class II+ counterparts as measured in the spleens.

We demonstrated the immunogenicity of RP3 with reactivity in both BALB/c and C57BL/6 mice to RP3/IEd- and RP3/CIITA-expressing cells. Also, we observed immunogenicity of RP3-derived phosphopeptides in ELISpot assays, supporting the hypothesis that the aberrant autophosphorylation of RP3 is a source of tumor-specific immunogenicity.

Finally, utilizing a RP3-expressing vaccinia vector for immunization of C57BL/6 mice, we identified peptide sequences that appear to be immunogenic on the basis of unique conformation of the fusion protein that impacts antigen processing.

Taken together these results could pave the way for better vaccine approaches without accompanying autoimmunity.

Citation Format: Laura Sponton, Gabriela Cosma, Mark Mendonca, Mirella Giovarelli, Laurence Crane Eisenlohr. Identification of tumor-specific antigens associated with RET/PTC3 expression. [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 1294. doi:10.1158/1538-7445.AM2015-1294
Abstract 1297: Increases in the numbers of myeloid-derived suppressor cells in Egyptian children with acute lymphoid leukemia
Authors: Salem Mohamed L.
Published: August 2015

Background: Acute lymphoblastic leukemia (ALL) or acute lymphoid leukemia is an acute form of leukemia. Although the mechanism behind the relapse in ALL is not clearly known, one possible mechanism could be the accumulation of immune regulatory cells, in particular myeloid-derived suppressor cells (MDSCs). We and others have reported that MDSCs cause suppression of host immune responses through several mechanisms. Aim: The main goal of this study was to determine whether these cells accumulate in ALL patients and whether they are affected by chemotherapy. Materials and Methods: Upon informed consent, patients with B-Linage Acute Lymphoblastic Leukemia “B-ALL” (n = 20) were recruited in this study along with healthy volunteers (n = 20). Patients included in this study were risk stratified according to risk classification system and treated according to treatment protocols including high risk pre B-ALL protocol, standard risk pre B-ALL. MDSCs were characterized as HLA-DR-Lin-CD33+CD11b+ cells. Results: Our preliminary results showed significant increases in the numbers of myeloid population with MDSC-like phenotype in B-ALL patients at different stages of chemotherapy treatment when compared with healthy control volunteers (Mean = 1.01%). The increases in the numbers of these cells were higher after treatment of the patients with induction chemotherapy. Further, the increased numbers of MDSCs associated with high numbers of circulating leukemic stem cells (LSCs). Conclusion: Our preliminary data suggest that cells with MDSCs like-phenotype emerge in ALL patients and possibly play a critical role in establishing an immune suppressive environment in children with ALL. Further research is needed to determine if circulating MDSCs like-phenotype populations are a immunosuppressive and whether they can be modulated.

Acknowledgment:

This work has been supported by a grant (ID# 5245) funded from the Science and Technology Development Fund (STDF), Ministry of Scientific Research and Technology, Egypt to Mohamed L. Salem, the Principal investigator of this project.

Note: This abstract was not presented at the meeting.

Citation Format: Mohamed L. Salem. Increases in the numbers of myeloid-derived suppressor cells in Egyptian children with acute lymphoid leukemia. [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 1297. doi:10.1158/1538-7445.AM2015-1297
Abstract 1317: Biomarker evaluation for PD-1 targeted therapies in non-small cell lung cancer (NSCLC) patients
Authors: Kamphorst Alice O. , Pillai Rathi N. , Yang Shu , Akondy Rama , Koenig Lydia , Yu Ke , McCausland Megan , Sica Gabriel , Khuri Fadlo R. , Owonikoko Taofeek K. , Ramalingam Suresh S. , Ahmed Rafi
Published: August 2015

Sustained expression of programmed cell death (PD)-1 inhibitory receptor is a hallmark of exhausted T cells during chronic infections and cancer. PD-1 signaling inhibits T cell activation, and maintains T cells in a dysfunctional state. Notably, inhibition of PD-1 interactions with PD-L1 is able to restore function in exhausted T cells. Several clinical trials have now shown that PD-1 blockade therapy results in durable clinical benefits in advanced stage cancer patients refractory to other treatments, and efficacy has been reported in multiple tumor types. Nonetheless many patients do not respond to PD-1 targeted therapies, hence a better understanding of the immunological responses induced by PD-1 pathway blockade and identification of predictive biomarkers is imperative. In this ongoing study we have analyzed peripheral blood mononuclear cells (PBMC) from advanced stage NSCLC patients, before and during the course of PD-1 pathway blockade therapy. We find that CD8 T cell activation is observed in some patients following therapy, but it is not consistently associated with clinical response. Interestingly, treatment-induced CD8 T cell proliferation is not sustained in some patients even with continuation of therapy. After PD-1 pathway blockade, we find co-expression of CTLA-4 on proliferating PD-1+ CD8 T cells in the blood. Accordingly, we found that tumor infiltrating CD8 T cells expressing high levels of PD-1 also co-express CTLA-4 (and Tim-3) in surgical specimens of patients with NSCLC. We will present additional data on CD8 and CD4 T cells in NSCLC patients enrolled in PD-1 targeted therapies clinical trials and discuss potential biomarkers. We conclude that peripheral blood analysis can provide valuable insights into anti-cancer effects of PD-1 targeted therapy.

Citation Format: Alice O. Kamphorst, Rathi N. Pillai, Shu Yang, Rama Akondy, Lydia Koenig, Ke Yu, Megan McCausland, Gabriel Sica, Fadlo R. Khuri, Taofeek K. Owonikoko, Suresh S. Ramalingam, Rafi Ahmed. Biomarker evaluation for PD-1 targeted therapies in non-small cell lung cancer (NSCLC) 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 1317. doi:10.1158/1538-7445.AM2015-1317
Abstract 1323: Both programmed cell death protein 1 and programmed death-ligand 1 molecules can be expressed on the cell surface of small-cell lung cancer
Authors: Yamane Hiromichi , Isozaki Hideko , Ochi Nobuaki , Kudo Kenichiro , Honda Yoshihiro , Yamagishi Tomoko , Kubo Toshio , Kiura Katsuyuki , Takigawa Nagio
Published: August 2015

Background: Programmed cell death protein-1 (PD-1) and programmed death ligand-1 (PD-L1) play a major role in suppressing the immune system by forming the PD-1/PD-L1 complex, which transmits an inhibitory signal to reduce T-cell activity. PD-L1 is often expressed in various malignant tumors. On the other hand, PD-1 is generally observed in activated lymphocytes and myeloid-derived dendritic cells. Of the malignant cells, only Jurkat cells (under special conditions) and angioimmunoblastic T-cell lymphoma tissue cells express PD-1 on their surface.

Purpose: To clarify whether PD-1/PD-L1 complex participates in the immunotolerance on small-cell lung cancer (SCLC) cells.

Materials and methods: We examined the expression levels of PD-1 and PD-L1 on the cell surface of SCLC cell lines using flowcytometry and reverse transcription polymerase chain reaction. Subsequently, the soluble PD-L1 (sPD-L1) concentration was measured using enzyme-linked immunosorbent assay, and the cell growth inhibitory effect of IFN-γ was determined by direct cell counting using a hemocytometer and Trypan blue staining.

Results: Among the four SCLC cell lines examined, only SBC-3 cells expressed both PD-1 and PD-L1. The sPD-L1 concentrations in culture medium gradually increased according to cell growth. Although IFN-γ alone inhibited the growth of SBC-3 cells, PD-L1 expression on the cell surface was not induced by IFN-γ.

Conclusions: We demonstrated that both PD-1 and PD-L1 molecules are co-expressed on the surface of SCLC cells. Although the biological implications of this remain unclear, we speculate that PD-1 and its ligand on the SCLC cells may participate in the growth inhibition of tumor cells as is reported in cytotoxic T cells.

Citation Format: Hiromichi Yamane, Hideko Isozaki, Nobuaki Ochi, Kenichiro Kudo, Yoshihiro Honda, Tomoko Yamagishi, Toshio Kubo, Katsuyuki Kiura, Nagio Takigawa. Both programmed cell death protein 1 and programmed death-ligand 1 molecules can be expressed on the cell surface of 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 1323. doi:10.1158/1538-7445.AM2015-1323
Abstract 1334: Chemotherapy regulated microRNA-125/HER2 pathway as a novel therapeutic target for trastuzumab-mediated cellular cytotoxicity in small-cell lung cancer
Authors: Yagishita Shigehiro , Fujita Yu , Fujimoto Yuichi , Takahashi Fumiyuki , Takahashi Kazuhisa , Tamura Tomohide , Koizumi Fumiaki
Published: August 2015

Small-cell lung cancer (SCLC) accounts for 15% of all lung cancer cases and is a highly lethal disease. For the last several decades, the standard treatment for SCLC has been deadlocked, and new therapeutic strategies are urgently needed. Human epidermal growth factor receptor 2 (HER2) is a member of the HER family and has been reported to be overexpressed in 30% of SCLC cases with poor prognosis. However, the clinical relevance of HER2-targeted therapy for SCLC remains unclear. Here, we firstly identify that cytotoxic drugs induce significant HER2 overexpression through microRNA-125a (miR-125a) and miR-125b downregulation, which in turn act as a novel therapeutic target for trastuzumab-mediated cellular cytotoxicity in SCLC. In this study, we showed that treatment of the HER2-positive SCLC cells, SBC-3 and SBC-5, with cytotoxic drugs induced a significant upregulation of HER2. Cisplatin (CDDP) treatment of SCLC cells resulted in a significant downregulation of miR-125a and miR-125b. We confirmed that miR-125a and miR-125b bound to the 3′-untranslated regions of HER2 mRNA, and that downregulation of miR-125a and miR-125b resulted in upregulation of HER2 in SCLC cells, suggesting a relationship between cytotoxic drug exposure and miR-125/HER2 dysregulation. Furthermore, using a calcein assay we demonstrated a significant synergistic cytotoxic effect of CDDP and trastuzumab that was mediated via antibody-dependent cellular cytotoxicity. Finally, we clearly demonstrated the synergistic anti-tumor effect of these agents in an orthotopic lung cancer model in vivo. Our result offer a novel therapeutic strategy for HER2-positive SCLC by using trastuzumab combined with cytotoxic drugs.

Citation Format: Shigehiro Yagishita, Yu Fujita, Yuichi Fujimoto, Fumiyuki Takahashi, Kazuhisa Takahashi, Tomohide Tamura, Fumiaki Koizumi. Chemotherapy regulated microRNA-125/HER2 pathway as a novel therapeutic target for trastuzumab-mediated cellular cytotoxicity in 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 1334. doi:10.1158/1538-7445.AM2015-1334
Abstract 1337: Up-regulation of mannose 6-phosphate receptor induced by BRAF inhibitor PLX4720 is linked to phosphatidylserine externalization and shows positive effects for combination therapies
Authors: Lavilla-Alonso Sergio , Brekken Rolf , Gabrilovich Dmitry
Published: August 2015

New therapies of metastatic melanoma, including BRAF inhibitors, check-point blockade or adoptive T-cell transfer, hold high promise. However, their efficacy remains unsatisfactory. Combination of different modalities offers an opportunity to enhance their effect. We studied mannose 6-phosphate receptor (MPR) previously implicated in synergistic effect of cancer chemotherapy and immunotherapy. Approximately 50% of human melanomas bear the activating mutation V600E in BRAF that can be selectively targeted with BRAF inhibitor PLX4720. We have observed that in vitro treatment of human V600E-positive melanoma cells 451Lu and WM983B leads to a dose-dependent up-regulation of mannose 6-phosphate receptor (MPR) in the cell surface. An increase in cell surface MPR, enhanced recombinant Granzyme B uptake, a requirements for effective combination with immunotherapy. This effect of PLX4720 on MPR expression was reproduced in a mouse model featuring xenograft 451Lu-derived subcutaneous tumors. This effect was limited only to sensitive cell lines, disappearing completely after resistance had been induced by extended treatment with growing doses of the drug. We have previously determined that autophagy has a key role in MPR up-regulation after chemotherapy or radiation therapy. However, in the case of PLX4720, autophagy occurs 1-2 days after the start of the treatment while membrane MPR was up-regulated not early than 3-4 and 10-14 days after the start of the treatment in 451Lu and WM983B cells respectively. In addition, autophagy was present independently of whether the cells were resistant to PLX4720 or not. These data indicate that autophagy was probably not responsible in PLX4720 inducible up-regulation of MPR. We observed a parallel changes in MPR up-regulation and phosphatidylserine (PS) externalization in live melanoma cells. PS and MPR co-localized on cell surface after the treatment with PLX4720. Co-incubation of PLX4720 with PS-specific antibody resulted in an ablation of the subsequent membrane MPR up-regulation. These observations help to explain the synergism between immunotherapy and targeted therapy of melanoma and to design clinical trials.

Citation Format: Sergio Lavilla-Alonso, Rolf Brekken, Dmitry Gabrilovich. Up-regulation of mannose 6-phosphate receptor induced by BRAF inhibitor PLX4720 is linked to phosphatidylserine externalization and shows positive effects for combination therapies. [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 1337. doi:10.1158/1538-7445.AM2015-1337
Abstract 134: CCN5 down regulates HER2/ neu expression in HER2 positive breast cancer cells
Authors: Sarkar Sandipto , Maity Gargi , Das Amlan , Majumder Monami , Banerjee Snigdha , Banerjee Sushanta
Published: August 2015

Breast cancers are broadly classified into four different subtypes which are Luminal A, Luminal B, Basal type and Her2 positive cancers. The cells in Her2 positive tumor lesions overexpress the tyrosine kinase receptor ErbB2 or Her2 which serve as the major oncoprotein and drives the unrestrained proliferation of the cells which is one of the major hallmarks of cancer. About 25% of the breast cancer overexpress Her2 (ErbB2) proto-oncogene resulting in aggressive tumor phenotype and is associated in poor prognosis in patients. Though Her2 targeted therapies are being used hugely in clinical practice, a significant fraction of the Her2 positive tumors develop mechanisms to evade the targeted therapies of Her2 inhibition. CCN5 or Wisp2 is a matricellular protein which is now considered as an ‘anti-invasive gene’. It has been already that established from multiple evidences that CCN5 protein inhibits progression of triple negative breast cancer. The goal of the study is to investigate if CCN5 can regulate Her2 in Her2 positive breast cancer. We have found that CCN5 treatment also negatively regulates expression and activity of Her2 receptor in Her2 overexpressed breast cancer cells. It has been indicated from Her2 promoter assays that Wisp2/CCN5 negatively regulates the expression of Her2 in a transcriptional level. Wisp2 /CCN5 treatment of Her2 overexpressed breast cancer cell lines, SKBR3 and Bt474, exerts a negative impact on the migration and proliferative properties of the cancer cells. Thus it can be concluded that our initial studies on effect of CCN5 on Her2 positive cells can pave the way for a novel approach of therapy to control Her2 overexpressed tumor cell growth.

Citation Format: Sandipto Sarkar, Gargi Maity, Amlan Das, Monami Majumder, Snigdha Banerjee, Sushanta Banerjee. CCN5 down regulates HER2/ neu expression in HER2 positive breast cancer 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 134. doi:10.1158/1538-7445.AM2015-134