bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4281: Nedd4 controls cell migration by regulating PDGF receptor signaling.
Authors: Tanaka Nobuyuki , Oyama Kyoko , Tamai Keiichi
Published: August 2014

The ubiquitin ligase Nedd4 is an ubuiquitin ligase (E3) which belongs to the HECT family. Like other member of this family members, such as Itch and Smurf, Nedd4 has been proposed to regulate a number of signaling pathways, as well as the traffic of several cell surface molecules, however, its physiological role in mammals has not been well characterized. Former reports identified reduction in the mitogenic activity and increase of the adaptor protein Grb10, aberrant IGF-1, VEGF and FGF receptor localization and / or signaling. In the present study, we performed an analysis of Nedd4-null murine embryonal fibroblastoid cells (MEFs) to show that loss of Nedd4 may affect any additional growth factor signaling. Surface expression of PDGF receptor was significantly increased in homozygous mutant mouse embryonic fibroblasts. Accordingly, PDGF-BB stimulation induced enhanced signaling as judged by MAP kinase p42/44 and Akt phosphorylation. Knockdown of Nedd4 in cell lines also induced similar phenotype. Nedd4, when transiently expressed, ubiquitinated PDGF receptor, which lead to degradation. Under the presence of lysosome inhibitors, PDGF receptor down regulation was at least partially blocked, suggesting its lysosome-dependent degradation. Subcellular localization of the PDGF receptor was also analyzed, but apparent difference was not observed. In the scratch assay, Nedd4 knockout cells showed enhanced migration activity, suggesting their active movement. Thus, Nedd4 appears to negatively control PDGF signaling partly through the regulation of its lysosome dependent degradation.

Citation Format: Nobuyuki Tanaka, Kyoko Oyama, Keiichi Tamai. Nedd4 controls cell migration by regulating PDGF receptor signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4281. doi:10.1158/1538-7445.AM2013-4281
Abstract 4283: Src phosphorylates splicing factor 45 (SPF45) and regulates SPF45 nucleocytoplasmic shuttling, cell migration and invasion.
Authors: Liu Yuying , Conaway Lashardai Neniara , Bethard Jennifer Rutherford , Bradley Amber Thompson , Eblen Scott
Published: August 2014

Src phosphorylates splicing factor 45 (SPF45) and regulates SPF45 nucleocytoplasmic shuttling, cell migration and invasion

Yuying Liu1, LaShardai Conaway1, Jennifer Rutherford Bethard1, Amber Thompson Bradley1 and Scott T. Eblen1,2

From Department of Cell and Molecular Pharmacology and Experimental Therapeutics1 and the Hollings Cancer Center2

Medical University of South Carolina, Charleston, SC, 29425

SPF45 is a bifunctional protein involved in splicing and DNA repair and is overexpressed in several forms of cancer. Previously, we have shown that SPF45 overexpression regulates ovarian cancer cell proliferation and adhesion in a MAP kinase phosphorylation-dependent manner. In this study, we investigated novel roles of SPF45 and their regulation by the tyrosine kinase Src. We show by cell fractionation that endogenous SPF45 localizes in both the nucleus and cytoplasm and that exposure of cells to UV irradiation or osmotic shock increased SPF45 cytoplasmic accumulation. Consistent with this result, heterokaryon assays using SKOV-3 cells stably expressing Myc-SPF45 showed that SPF45 shuttles between the nucleus and cytoplasm and that pretreatment of the cells with the Src family kinase inhibitor PP2 inhibited nucleocytoplasmic shuttling. Src strongly induced SPF45 tyrosine phosphorylation and co-immunoprecipitated with SPF45 after co-transfection into COS-1 cells. Src phosphorylated SPF45 in vitro on five tyrosine residues (Y100, Y108, Y194, Y214 and Y246), as determined by mass spectrometry. Sequential mutation of these sites to phenylalanine inhibited SPF45 tyrosine phosphorylation in cells co-transfected with Src, with mutation of all 5 sites (Myc-SPF45-Y5F) completely inhibiting SPF45 tyrosine phosphorylation. Myc-SPF45-Y5F stably expressed in SKOV-3 cells showed reduced nucleocytoplasmic shuttling compared to wild type Myc-SPF45. SKOV-3-Myc-SPF45 cells demonstrated enhanced migration and invasion compared to vector control cells, while SKOV-3-Myc-SPF45-Y5F cells migrated significantly less than SKOV-3-Myc-SPF45 cells. These data are the first to show that SPF45 is a shuttling protein, a Src substrate, and that Src phosphorylation of SPF45 regulates nucleocytoplasmic shuttling and SPF45-induced cell migration and invasion.

Citation Format: Yuying Liu, Lashardai Neniara Conaway, Jennifer Rutherford Bethard, Amber Thompson Bradley, Scott Eblen. Src phosphorylates splicing factor 45 (SPF45) and regulates SPF45 nucleocytoplasmic shuttling, cell migration and invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4283. doi:10.1158/1538-7445.AM2013-4283
Abstract 4286: Hepatocyte growth factor-mediated gastrin-releasing peptide induces IL-8 expression through Ets-1 in gastric cancer cells.
Authors: Koh Sung Ae , Lee Kyung Hee , Choi Eun Young , Kim Min Kyung , Jang Byung Ik , Kim Se Won , Kim Sang Woon , Song Sun Kyo , Kim Jae Ryong
Published: August 2014

Gastric cancer cells secrete a variety of pro-angiogenic molecules, including IL-8 and VEGF. However, factors regulating the expression of pro-angiogenic genes for gastric cancer remain largely undefined. We investigated the role of HGF-induced activation of GRP and Ets-1 transcription factor in expression of the pro-angioigenic factor, IL-8.

The genes associated with angiogenesis induced by HGF were screened using cDNA microarray technology in two gastric cancer cell lines (NUGC-3 and MKN-28). First, GRP RNA and protein were confirmed to be up-regulated. Then, expression of GRP, Ets-1, and IL-8 were further estimated by Western blot analysis. A role for Ets-1 in HGF-induced up-regulation of IL-8 was determined by knockdown of Ets-1 with Ets-1 sh-RNA and a chromatin immune precipitation assay.

The levels of GRP, Ets-1, and IL-8 were up-regulated in cells treated with HGF in a dose-dependent manner. HGF-induced expression of Ets-1 and IL-8 was increased more by GRP treatment, and inhibited by pre-treatment with an ERK 1/2 inhibitor (PD098059). HGF-induced up-regulation of IL-8 was repressed by Ets-1 knockdown. HGF enhanced the binding activity of Ets-1 to the IL-8 promoter in control cells, but not in the Ets-1 shRNA cells. We confirmed the functional role of HGF-induced Ets-1 in activation of the IL-8 promoter by the reporter gene assay. Down-regulation of IL-8 also decreased in vitro cell invasion.

In conclusion, HGF mediated the GRP induction of IL-8 expression through Ets-1, which thus might serve as a promising target for gastric cancer therapy.

Key words: HGF, GRP, Ets-1, IL-8

Citation Format: Sung Ae Koh, Kyung Hee Lee, Eun Young Choi, Min Kyung Kim, Byung Ik Jang, Se Won Kim, Sang Woon Kim, Sun Kyo Song, Jae Ryong Kim. Hepatocyte growth factor-mediated gastrin-releasing peptide induces IL-8 expression through Ets-1 in gastric cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4286. doi:10.1158/1538-7445.AM2013-4286
Abstract 4287: Intracellular trafficking of androgen receptor splice variant AR8.
Authors: John Ciny , Lu Michael L.
Published: August 2014

In a classic paradigm, the intracellular trafficking of a ligand-stimulated androgen receptor involves receptor dimerization, dissociating from chaperones (such as HSPs), interact with nuclear membrane importin and Ran-GTPase to gain access to nucleus; once there, it complexes with transcriptional regulators and binding to target sites. Despite the compelling evidence for AR non-genomic signaling events, the molecular mechanism of how does AR signaling from cytoplasmic or membrane remains undetermined. Several recent studies suggested that membrane association of AR is mediated by palmitoylation of AR at cysteine-806. Two plamitoylation enzymes, DHHC7 and DHHC21, have been implicated in the process of AR palmitoylation. In the current study, we determined the intracellular trafficking of a newly identified AR isoform, AR8. AR8 is a splice variant of the original full-length AR. AR8 was demonstrated to be up-regulated in castration-resistant prostate cancer cells. Structurally, AR8 protein sequence is different from other AR variants with a substitution of all the amino acids after N-terminal domain (NTD) of a conventional AR with an unique C-terminal sequence. Coincidentally, two cysteine residues within the unique c-terminus, positions 558 and 560, were identified to be palmitoylated. The palmitoylation was determined to be responsible for the localization of AR8 to plasma membrane. Using a fluorescent protein mCherry-tagged AR8 and confocal microscopy, we confirmed the plasma membrane localization of AR8 as previously described. We determined that AR8 co-localized with a Golgi marker GM-130 at peri-nuclear regions by immunofluorescence microscopy. The result was further substantiated by co-localizing mCherry-AR8 with galactosyltransferase (GalT) golgi-targeting sequence-fused GFP (GalT-GFP; green fluorescent protein) at Golgi compartments. We also demonstrated that mCherry-AR8 co-localized with EGFR-GFP (epidermal growth factor receptor-GFP) on the plasma membrane within in the cholesterol rich raft domain. Using time-lapse confocal microscopy, we observed that AR8 co-migrated with EGFR in a retrograde fashion and become associated with endosomal compartments at the onset of EGF (5ng/ml) stimulation. Part of these endosomes was later fused with lysosomes as evident from co-localizations of mCherry-AR8 with GFP-LAMP-1. Although the significance of AR8 expression in the progression of prostate cancer remains to be determined, our observations underline a potential intracellular trafficking pathway associated with AR8 are consistent with the original report that EGF stimulation promotes the interactions between AR8 and EGFR.

Citation Format: Ciny John, Michael L. Lu. Intracellular trafficking of androgen receptor splice variant AR8. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4287. doi:10.1158/1538-7445.AM2013-4287
Abstract 429: Damage-induced apoptosis in primary human lymphocytes is a genetic program modified by gender and age.
Authors: Applebaum Mark A. , Skol Andrew , Bond Gareth , Onel Kenan
Published: August 2014

Apoptosis is a core pathway deregulated in hematologic and other cancers. Although there are considerable functional and genetic data on the regulation of apoptosis in vitro and in model organisms, little is known about how these pathways are modified in primary cells freshly isolated from people, who are continually subjected to a variety of different and often unquantified environmental stimuli. We hypothesize that these exposures may have important and as yet unappreciated modifying effects on apoptosis that may have implications for cancer risk or response to therapy. To test this, we isolated peripheral blood mononuclear cells (PBMCs) from 90 normal healthy individuals and treated them with 5 Gy ionizing radiation (IR). Damage-induced apoptosis was measured at 24 hours after IR by TUNEL. We observed surprising heterogeneity in apoptosis among individuals, ranging from 21-65 percent. To determine whether this diversity in response was programmed or stochastic, we repeated the experiment 3 times in 10 individuals spanning the entire IR-induced apoptotic range and found results to be completely reproducible within individuals, suggesting that it resulted from a genetic program. We then assessed the association with individual characteristics. Whereas neither race nor age was associated with IR-induced apoptosis, gender was significantly associated (Mean: 44.8% vs 32.9%; p = 1.02 x 10-8). Moreover, when stratified by gender, age was significantly associated with IR-induced apoptosis in males (p = 0.026) but not females (p = 0.94). Thus, we conclude that IR-induced apoptosis in freshly isolated human PBMCs results from an intrinsic genetic program modified by age, but only within males. Genetic models to explain this will be discussed.

Citation Format: Mark A. Applebaum, Andrew Skol, Gareth Bond, Kenan Onel. Damage-induced apoptosis in primary human lymphocytes is a genetic program modified by gender and age. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 429. doi:10.1158/1538-7445.AM2013-429
Abstract 4292: RasGRP3 contributes to Ras signaling, growth, and migration of human lung cancer cell lines.
Authors: Yang Jing , Esch Timothy , Yang Dazhi , Blumberg Peter
Published: August 2014

Elevated levels of Ras activity are a prominent feature in human lung cancer. In approximately 20% of lung cancer cases, Ras activation is achieved by mutation of the Ras proto-oncogene. Here, we explore the possible contribution of RasGRP3 to Ras signaling in lung cancer. RasGRP3 is a Ras guanyl nucleotide exchange factor which has been shown to be expressed in prostate tumor and melanoma cell lines, contributing to the level of Ras activation, cell proliferation, and tumor xenograft growth. Here, we report that RasGRP3 is expressed in multiple human lung cancer cell lines but not in normal human bronchial epithelial cells. Likewise, initial analysis reveals its expression in human lung tumors but not in normal lung tissue. Suppression of endogenous RasGRP3 expression in lung cancer cell lines showing reduced H-Ras-GTP and K-Ras-GTP formation as well as Akt phosphorylation downstream from epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA) stimulation. Additionally, suppression of RasGRP3 inhibited translocation of the NF-kB p50 and p65 subunits from the cytoplasm to the nuclear fraction and modulated several other transcriptional factors as well. Conversely, expression of exogenous RasGRP3 elevated H-Ras-GTP and K-Ras-GTP and activated AKT phosphorylation in the lung cancer H496 cell line, which itself only expresses RasGRP3 at low levels. These changes in signaling were mirrored at the cellular level. Downregulation of endogenous RasGRP3 inhibited cell proliferation, impeded cell migration and reduced colony formations in soft agar. Our results suggest that RasGRP3 may provide an addition targetable signaling element in lung cancer.

Citation Format: Jing Yang, Timothy Esch, Dazhi Yang, Peter Blumberg. RasGRP3 contributes to Ras signaling, growth, and migration of human lung cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4292. doi:10.1158/1538-7445.AM2013-4292
Abstract 4295: PHLPP suppresses epithelial-mesenchymal transition and cancer cell invasion by negatively regulating RAF1 activity.
Authors: Li Xin , Stevens Payton D. , Gao Tianyan
Published: August 2014

PHLPP belongs to a family of protein phosphatases that have previously been shown to inhibit Akt-mediated cell proliferation and survival signaling in cancers. Here, we demonstrate that PHLPP exerts its tumor suppressor function by negatively regulating the RAF/MEK/ERK pathway to inhibit cell migration and invasion. Specifically, we identify the oncogenic protein kinase RAF1 as an interacting protein and a novel substrate of PHLPP. PHLPP dephosphorylates the S338 site of RAF1 and inactivates the kinase both in vitro and in cells. Loss of PHLPP expression largely increases the amplitude and duration of RAF/MEK/ERK signaling in cells, whereas overexpression of PHLPP inhibits the pathway. Functionally, knockdown of PHLPP induces epithelial-mesenchymal transition (EMT)-like phenotype, which coincides with a significant increase in migration and invasion in colon cancer cells. Furthermore, we show that upregulation of transcription factors ZEB1 and ZEB2 is responsible for increased invasiveness observed in PHLPP knockdown cells. Taken together, we have identified a novel role of PHLPP in suppressing EMT and cell motility in colon cancer cells by directly antagonizing RAF1 function.

Citation Format: Xin Li, Payton D. Stevens, Tianyan Gao. PHLPP suppresses epithelial-mesenchymal transition and cancer cell invasion by negatively regulating RAF1 activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4295. doi:10.1158/1538-7445.AM2013-4295
Abstract 4301: Transformation by the D816V mutant of c-Kit is dependent on the PI3-kinase isoform p110δ but independent of its lipid kinase activi.
Authors: Sun Jianmin , Mohlin Sofie , Lundby Alicia , Hellman Ulf , Påhlman Sven , Olsen Jesper V. , Rönnstrand Lars
Published: August 2014

Gain-of-function mutations of the receptor tyrosine kinase c-Kit are frequently seen in several malignanices with the D816V mutation being the most common. In this study we demonstrate that a mutant of c-Kit/D816V that is deficient in binding of the p85 subunit of PI3-kinase (M724A) has lost its ability to induce ligand-independent cell survival and proliferation while c-Kit/D816V induced PI3-kinases activity was only marginally reduced. Furthermore, inhibitors of PI3-kinase were unable to block c-Kit/D816V-induced cell survival. These data suggest that PI3-kinase plays a role in c-Kit/D816V induced cell transformation which is independent of its lipid kinase activity. By mass spectrometry, we identified a heavily tyrosine-phosphorylated 110 kDa protein associated with p85 to be the p110δ subunit of PI3-kinase and the site of phosphorylation as Tyr 523, and the phosphorylation is not dependent on the lipid kinase activity of PI3-kinase. Knockdown of p110δ in c-Kit/D816V expressing Ba/F3 cells led to reduced cell survival and proliferation while the PI3-kinase activity remained almost intact. The c-Kit/D816V expressing Ba/F3 cells lost their ability to form colonies in semi-solid culture if p110δ was knocked down. Additionally, knockdown of p110δ λεδ τo ρεδυχεδ τυμoρ σιζε, τυμoρ ωειγητ ανδ blood vessel formation ωηεν c-Kit/D816V expressing Ba/F3 cells were subcutaneously injected into nude mice. Taken together, these results suggest a kinase independent role of p110δ in the D816Vmutant of c-Kit mediated cell transformation and suggest that p110δ could potentially be a selective therapeutic target for c-Kit/D816V expressing tumors.

Citation Format: Jianmin Sun, Sofie Mohlin, Alicia Lundby, Ulf Hellman, Sven Påhlman, Jesper V. Olsen, Lars Rönnstrand. Transformation by the D816V mutant of c-Kit is dependent on the PI3-kinase isoform p110δ but independent of its lipid kinase activi. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4301. doi:10.1158/1538-7445.AM2013-4301
Abstract 4307: Paradoxical activation of the MEK/ERK pathway promotes melanoma invasion and metastasis.
Authors: Sanchez-Laorden Berta , Viros Amaya , Girotti Maria Romina , Martin Matthew , Pedersen Malin , Zambon Alfonso , Niculescu-Duvaz Dan , Springer Caroline , Marais Richard
Published: August 2014

Melanoma is the most deadly form of skin cancer. An important feature of melanoma is its ability to metastasize and this contributes to its poor prognosis. RAS is mutated in ∼20% of melanomas and its downstream kinase BRAF is mutated in a further 50% of cases, these oncogenes regulate proliferation and survival through the MEK-ERK cascade. Selective inhibitors of BRAF such as vemurafenib have a remarkable clinical activity in patients with BRAF mutant melanomas with response rates of approximately 60-80%. In cells expressing activating BRAF mutations, vemurafenib blocks activation of the MAPK pathway. However, in cells expressing wild type BRAF and constitutively active upstream components that activate the MAPK pathway, BRAF inhibitors drive the paradoxical activation of this pathway. Here we show that kinase-dead BRAF promotes melanoma cell invasion and metastasis in the presence of oncogenic RAS. Notably, drugs that target BRAF mimic these effects and also induce RAS mutant melanoma cell invasion. The underlying mechanism is driven by paradoxical activation of the MEK-ERK pathway, resulting in increased secretion of the chemokine IL8 and subsequent secretion of extracellular matrix proteases, which induce invasion. We also show that BRAF inhibitors promote melanoma metastasis in an allograft model and that BRAF inhibitors induce invasion of drug-resistant BRAF mutant melanoma cells. We conclude that paradoxical activation of the MAPK pathway by BRAF inhibitors can drive melanoma invasion and metastasis in RAS mutant melanoma and in drug-resistant BRAF mutant melanoma cells.

Citation Format: Berta Sanchez-Laorden, Amaya Viros, Maria Romina Girotti, Matthew Martin, Malin Pedersen, Alfonso Zambon, Dan Niculescu-Duvaz, Caroline Springer, Richard Marais. Paradoxical activation of the MEK/ERK pathway promotes melanoma invasion and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4307. doi:10.1158/1538-7445.AM2013-4307
Abstract 431: Nuclear WRAP53 is associated with positive response to radiotherapyand improved overall survival in head and neck squamous cell carcinoma.
Authors: Roberg Karin B. , Garvin Stina , Tiefenböck Katharina , Farnebo Lovisa , Farnebo Marianne
Published: August 2014

Purpose: Today there are no reliable predictive markers for radiotherapy response in head and neck squamous cell carcinoma (HNSCC), leading to both under-and over-treatment of patients, personal suffering and negative socio-economic effects. There is therefore a great need to develop new markers to provide patients with these tumors individualized and more effective treatment. WRAP53 is a natural antisense transcript to p53 that regulates levels of endogenous p53 mRNA and the induction of p53 in response to DNA damage. In addition, WRAP53 protein has been shown to be an essential component of nuclear organelles called Cajal bodies and involved in a variety of vital cellular functions such as splicing and telomerase activity. The aim of this study was to investigate WRAP53 as a potential predictive marker of radiotherapy response in head and neck squamous cell carcinoma (HNSCC) patients.

Materials and methods: Investigations were performed on tumor biopisies from patients classified as responders or non-responders to radiotherapy. Protein expression was examined using immunohistochemistry. After downregulation of WRAP53 by siRNA, assessment of intrinsic radiosensitivity was performed on two HNSCC cell lines.

Results: Nuclear WRAP53 expression was significantly associated with better response to radiotherapy (binary regression analysis, p = 0.029) and patients with nuclear WRAP53 had a significantly better survival (p = 0.014). Downregulation of WRAP53 with siRNA in vitro increased resistance to radiation (p < 0.001).

Conclusions: Our study suggests that the nuclear location of WRAP53 is of importance in mediating tumor response to radiotherapy. Further studies are needed to investigate nuclear WRAP53 as a potential predictive marker of radiotherapy response in HNSCC.

Citation Format: Karin B. Roberg, Stina Garvin, Katharina Tiefenböck, Lovisa Farnebo, Marianne Farnebo. Nuclear WRAP53 is associated with positive response to radiotherapyand improved overall survival in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 431. doi:10.1158/1538-7445.AM2013-431
Abstract 4319: Lupus antibody-based cancer therapy.
Authors: Hansen James E. , Chan Grace , Liu Yanfeng , Hegan Denise C. , Dalal Shibani , Dray Eloise , Kwon Youngho , Xu Yuanyuan , Xu Xiaohua , Peterson-Roth Elizabeth , Geiger Erik , Liu Yilun , Gera Joseph , Sweasy Joann B. , Sung Patrick , Rockwell Sara , Nishimura Robert N. , Weisbart Richard H. , Glazer Peter M.
Published: August 2014

A subset of autoantibodies produced by patients with systemic lupus erythematosus (SLE) penetrates into cell nuclei and binds DNA, and we believe that such antibodies may have applications in cancer therapy. We have discovered that the cell-penetrating, nuclear-localizing anti-DNA lupus antibody 3E10 inhibits key steps in DNA single- and double-strand break repair and has potential for development as a targeted therapy for tumors harboring deficiencies in DNA repair. 3E10 preferentially binds DNA substrates with free single-strand tails and interferes with both base excision repair and homology-directed repair (HDR) in vitro, and HDR efficiency is reduced in cells treated with 3E10 as measured in the chromosome-based DR-GFP fluorescent reporter assay. The binding of 3E10 to DNA can be directly visualized under electron microscopy (EM), and EM studies confirmed that 3E10 interferes with RAD51 filament formation, which is a critical step in HDR. The 3E10 single chain variable fragment penetrates into human tumor xenografts in nude mice, and 3E10 sensitizes cancer cells and tumors to DNA-damaging therapy. In addition, 3E10, by itself, is toxic to BRCA2-deficient cancer cells but not to repair-proficient cells, and when combined with a DNA-damaging agent, 3E10 has a very large cytotoxic effect on BRCA2-deficient cancer cells. The synthetically lethal effect of 3E10 on BRCA2-deficient cancer cells is consistent with our finding that 3E10 inhibits DNA repair, and it suggests that 3E10 has potential as a targeted therapy for tumors harboring deficiencies in DNA repair, such as certain breast, ovarian, and prostate cancers. Of note, patients with SLE have lower than expected incidence rates of breast, ovarian, and prostate cancers, and it is tempting to speculate that the circulating cell-penetrating anti-DNA autoantibodies provide patients with SLE some protection against the development of DNA repair-deficient tumors. In summary, our work with the 3E10 antibody has provided proof of principle for the development of a lupus antibody as a cancer therapy and opened up new avenues for exploration into the biology of lupus antibodies.

Citation Format: James E. Hansen, Grace Chan, Yanfeng Liu, Denise C. Hegan, Shibani Dalal, Eloise Dray, Youngho Kwon, Yuanyuan Xu, Xiaohua Xu, Elizabeth Peterson-Roth, Erik Geiger, Yilun Liu, Joseph Gera, Joann B. Sweasy, Patrick Sung, Sara Rockwell, Robert N. Nishimura, Richard H. Weisbart, Peter M. Glazer. Lupus antibody-based cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4319. doi:10.1158/1538-7445.AM2013-4319
Abstract 4321: Development of pyrrolobenzodiazepine-based antibody-drug conjugates for cancer.
Authors: Jeffrey Scott C. , Burke Patrick J. , Sutherland May K. , Meyer David W. , Lyon Robert P. , McEarchern Julie A. , Law Che-Leung , Senter Peter D.
Published: August 2014

Pyrrolobenzodiazepine (PBD) dimers are highly cytotoxic minor groove binding DNA crosslinking agents derived from the anthramycin class of natural products. In our efforts to develop highly effective and well-tolerated treatment options for cancer patients, we have investigated several antibody-drug conjugates (ADCs) based on a PBD dimer. The fully synthetic PBD-linker, comprised of a protease-cleavable val-ala sequence and a reactive maleimide group, was conjugated via engineered cysteine residues to humanized monoclonal antibodies specific for their binding to the well-characterized cancer antigen targets, CD33 and CD70. The resulting ADCs were well-defined, monomeric and showed pronounced in vitro and in vivo activity against both hematologic and solid tumor cancer models. These included models of acute myelogenous leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma and renal cell carcinoma. The ADCs were immunologically specific, and showed potent activity in MDR-positive models, including models resistant to other ADCs. These results underscore the importance of the drug component in developing highly efficacious ADCs for cancer therapy.

Citation Format: Scott C. Jeffrey, Patrick J. Burke, May K. Sutherland, David W. Meyer, Robert P. Lyon, Julie A. McEarchern, Che-Leung Law, Peter D. Senter. Development of pyrrolobenzodiazepine-based antibody-drug conjugates for cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4321. doi:10.1158/1538-7445.AM2013-4321
Abstract 4323: Antibodies against TfR inhibit growth of tumor cells both in vitro and in vivo.
Authors: Zhang Lilin , Ukai Yoshinori , Nomura Fumiko , Aikawa Youichi , Kayukawa Yoko , Mitomo Katsuyuki , Kouda Katsushi , Kodaka Romi , Kurosawa Gene , Kurosawa Yoshikazu , Morishita Kazuhiro , Sudo Yukio
Published: August 2014

Transferrin receptor (TfR) is a type II transmembrane glycoprotein regulating intracellular uptake of iron, and is therefore involved in cell survival and proliferation. TfR has been reported to be more abundantly expressed in dividing cells, e.g. malignant cells, than in quiescent cells, as in most normal cells. Therefore, it is regarded as a potential target for usage in cancer therapy. In the present study, we generated a panel of human anti-TfR antibodies and evaluated its anti-tumor effect both in vitro and in vivo. We found that several antibodies inhibited cell proliferation in various tumor cells, and elicited antibody dependent cellular cytotoxicity (ADCC) activity. Among these antibodies, PPMXT001 and PPMXT002 exerted a significant anti-tumor activity in several xenograft models. PPMXT001 completely suppressed tumor growth in a pancreatic cancer model (MIAPaCa2). PPMXT002 also inhibited tumor growth by 60∼80% in several solid cancer xenograft models including bladder cancer, colon cancer, prostate cancer, and pancreatic cancer. These findings showed that PPMXT001 and PPMXT002 could be potent candidates for developing antibody therapeutics to treat cancer.

Citation Format: Lilin Zhang, Yoshinori Ukai, Fumiko Nomura, Youichi Aikawa, Yoko Kayukawa, Katsuyuki Mitomo, Katsushi Kouda, Romi Kodaka, Gene Kurosawa, Yoshikazu Kurosawa, Kazuhiro Morishita, Yukio Sudo. Antibodies against TfR inhibit growth of tumor cells both in vitro and in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4323. doi:10.1158/1538-7445.AM2013-4323
Abstract 4327: A novel protease resistant antibody platform with selectable cell killing functions.
Authors: Kinder Michelle , Grugan Katharine , Soring Keri , Strohl William , Jordan Robert , Brezski Randall
Published: August 2014

Invasive cancers express matrix metalloproteases, such as MMP-3 and MMP-7, which have the ability to cleave human antibodies in the lower hinge region. The human IgG lower hinge region contains two heavy chains, and cleavage occurs in a two-step process. First, one heavy chain is cleaved, resulting in a singly-cleaved intermediate. Cleavage of the second heavy chain liberates the Fc region from the antigen-binding region, resulting in an Fc fragment and a F(ab’)2 fragment. Previous work has demonstrated that the singly-cleaved intermediate accumulates on the surface of IgG1-opsonzied tumor cells in the presence of MMPs. The singly-cleaved intermediate is indistinguishable from the intact IgG counterpart when assessed by analytical techniques under native conditions and maintains normal antigen binding. However, the singly-cleaved intermediate displays a profound loss of Fc-mediated cell-killing functions both in vitro (e.g. ADCC and CDC) and with in vivo cell clearance, presumably because the lower hinge contains key recognition points for both FcγRs and the C1q component of complement. Singly-cleaved IgGs are of particular concern with regards to human IgG1 monoclonal therapeutic antibodies, because the proposed mechanisms of action for several anti-tumor mAbs in the clinic may be partially dependent on Fc effector functions, such as ADCC and CDC. For this reason, our group and others have proposed that cleavage and the subsequent inactivation of Fc-effector functions of human IgG antibodies may function as an immune evasion mechanism. To overcome the loss-of-function associated with human IgG1 cleavage, we aimed to develop a protease-resistant IgG1 platform which would still maintain Fc-mediated cell-killing functions. Mutation of the lower hinge of IgG1 provided protease-resistance, but also resulted in a loss of Fc-effector functions. Through specific mutations of amino acids in the CH2 region to enhance complement or Fcγ receptor binding, we were able restore or even enhance cell-killing functions selectively while maintaining protease-resistance. Therefore, we were able to generate protease-resistant antibodies with the ability to mediate either complement-dependent and/or antibody-dependent cellular cytotoxicity, which were also significantly resistant to inactivation by proteases associated with many invasive cancers.

Citation Format: Michelle Kinder, Katharine Grugan, Keri Soring, William Strohl, Robert Jordan, Randall Brezski. A novel protease resistant antibody platform with selectable cell killing functions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4327. doi:10.1158/1538-7445.AM2013-4327
Abstract 4332: Development of AGS15E, a novel antibody drug conjugate targeting SLITRK6 for the treatment of bladder cancer.
Authors: Morrison Kendall
Published: August 2014

Agensys (an affiliate of Astellas Pharma), Santa Monica, CA Abstract: SLITRK6 is a member of the SLITRK family of neuronal transmembrane proteins involved in neurite outgrowth. It was discovered by Agensys using suppressive subtractive hybridization on biopsies from bladder cancer patients. Expression in bladder cancer was confirmed by RNA and immunohistochemical analysis. These studies showed SLITRK6 to be highly expressed in bladder carcinoma in situ as well as in invasive and metastatic bladder cancer. In addition, SLITRK6 was shown to be expressed in a subset of lung cancer, breast cancer and glioblastoma specimens.

Agensys has generated a novel antibody drug conjugate (ADC), targeting SLITRK6, which is being developed for the treatment of advanced bladder cancer. This Monomethyl Auristatin E based ADC binds to cell surface SLITRK6 with high affinity and cross reacts with the cynomolgus monkey orthologue. Selection and development of this ADC will be discussed including antibody choice, drug platform choice, conjugation development and other aspects of pre-clinical evaluation.

Citation Format: Kendall Morrison. Development of AGS15E, a novel antibody drug conjugate targeting SLITRK6 for the treatment of bladder cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4332. doi:10.1158/1538-7445.AM2013-4332
Abstract 4340: Formulation of oral microparticulate drug delivery system for cancer.
Authors: Patel Jay V. , Desai Kunal P. , Do Duc P.
Published: August 2014

The development of oral controlled-release cancer drug formulations with enhanced bioavailability and efficacy is highly desired. Our research aim to develop controlled-release capsule formulations containing sulforaphane-encapsulated in a biodegradable polymeric microparticulate system. Sulforaphane, a histone deacetylase inhibitor (HDAC), was investigated as this therapeutic agent reverses aberrant epigenetic changes in cancer cells and the effects of this drug are reversible. Biodegradable polymeric microspheres were prepared by a microencapsulation method, and prepared microspheres were characterized for various properties, including encapsulation efficiency, percent yield, particle size, size distribution, surface morphology, and Zeta potential. Dissolution studies were carried out to analyze the release profiles of the microspheres and capsule-containing microsphere formulations. Data indicated that microspheres were less than 2-μm in diameter size. It has been shown that particles within this size range are effectively taken in by the cells. The prepared microsphere formulations had Zeta potential values of about -30 mV, which suggest that they are stable. Furthermore, microsphere formulations were shown to provide controlled-release of the drug over 36 hours. Capsule formulations containing microspheres exhibited similar drug release characteristics; however, the lag time for drug release was longer. Thus, oral gelatin capsules containing sulforaphane-encapsulated biodegradable microspheres show potential for effective delivery of epigenetic agents.

Citation Format: Jay V. Patel, Kunal P. Desai, Duc P. Do. Formulation of oral microparticulate drug delivery system for cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4340. doi:10.1158/1538-7445.AM2013-4340
Abstract 4344: Intraperitoneal administration of docetaxel loaded in thermo-responsive conjugated linoleic acid-incorporated poloxamer hydrogel for the suppression of peritoneal dissemination of gastric cancer.
Authors: Bae Woo Kyun , Hennighausen Lothar , Lee Ji Hee , Kim Dae Eun , Hwang Jun Eul , Shim Hyun Jeong , Cho Sang Hee , Park In-Kyu , Chung Ik-Joo
Published: August 2014

Thermo-responsive hydrogel consisting of conjugated linoleic acid-coupled Pluronic F127 (Plu-CLA) is an optimized drug delivery system with anti-cancer activity for controlled release of the hydrophobic drug, docetaxel. In this study, we evaluated the effect of Plu-CLA as a potential new intraperitoneal docetaxel delivery system for peritoneal dissemination of gastric cancer. We have established a peritoneal metastasis models using BALB/c mice by injection with TMK-1 human gastric cancer cells. Mice were injected intraperitoneally with docetaxel alone and docetaxel loaded Plu-CLA at 1 week after TMK1 cells were injected. Tumor progression and response to therapy were monitored by micro-positron emission tomography. The total number of peritoneal tumors and ascites volume were also measured. Compared to docetaxel alone, the combination of docetaxel and Plu-CLA significantly and synergistically reduced cell survival. The combination of docetaxel and Plu-CLA showed excellent anti-tumor activity inducing stronger apoptosis than docetaxel alone. The combination of docetaxel and Plu-CLA also significantly reduced number of peritoneal metastatic nodules and increased the survival in the peritoneal gastric cancer xenograft model. Our results showed that intraperitoneal administration of docetaxel combined with Plu-CLA synergistically inhibited peritoneal metastasis and prolonged the survival in peritoneal gastric cancer model. Therefore, Plu-CLA could be a potential intraperitoneal carrier for hydrophobic docetaxel for the effective treatment of peritoneal metastatic gastric cancer.

Citation Format: Woo Kyun Bae, Lothar Hennighausen, Ji Hee Lee, Dae Eun Kim, Jun Eul Hwang, Hyun Jeong Shim, Sang Hee Cho, In-Kyu Park, Ik-Joo Chung. Intraperitoneal administration of docetaxel loaded in thermo-responsive conjugated linoleic acid-incorporated poloxamer hydrogel for the suppression of peritoneal dissemination of gastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4344. doi:10.1158/1538-7445.AM2013-4344
Abstract 435: The role of HIF-1 and Notch pathway in radio-resistance of non small lung cancer cell lines.
Authors: Ikezawa Yasuyuki , Sakakibara-Konishi Jun , Mizugaki Hidenori , Ooizumi Satoshi , Nishimura Masaharu
Published: August 2014

Background: Notch pathway regulates cell-fate determination in multi-cellular organisms and aberrant activation of Notch pathway has been reported in tumorigenesis of many cancers. We previously reported that Notch3 and its downstream effector, HEY1, were upregulated by radiation in non-small cell lung cancer (NSCLC) cell lines. Furthermore, we demonstrated that g-secretase-inhibitor (GSI), when combined with radiation therapy significantly suppressed the growth of lung cancer cells compared to either GSI or radiation alone by a potential mechanism that GSI suppressed radiation-induced Notch activation and its resultant radio-resistance. Since the expression of Notch was reported to be activated via HIF under hypoxia, we, in this study, hypothesized that HIF would be involved in radiation-induced Notch activation in NSCLC and thus HIF inhibitor (YC-1) would have an anti-tumor effect when combined with radiation therapy as is the case with GSI.

Materials and Methods: Two Notch-expressing lung cancer cell lines (H460 and H1793) were used. We first examined, by Western Blotting (WB) and real time RT-PCR, the changes in expressions of HIF-1 and Notch family after treatment by radiation under hypoxia. We then repeated the same experiment while using SiRNA targeting HIF-1, in an attempt to examine the role of HIF-1 on Notch pathway and its downstream proteins. Finally, we investigated an antitumor effect of YC-1, using a MTT proliferation assay and a clonogenic assay, when combined with radiation treatment.

Results: Under hypoxia, treatment by radiation up-regulated protein expression of HIF-1 at 6 hours and that of Notch3 as well at 24 hours, but not of other Notch family. As expected, specific suppression of HIF-1 by siRNA down-regulated Notch3 activation associated with radiation; however, did not affect other Notch family and its downstream effectors, including HES1. An anti-tumor effect of YC-1 was evident only when radiation treatment was concurrently applied, but not when sequentially applied, in the growth of lung cancer cells and also colony formation.

Conclusions: Radiation-induced up-regulations of HIF-1 and Notch pathway may be therapeutic targets for more effective radiation therapy in NSCLC.

Citation Format: Yasuyuki Ikezawa, Jun Sakakibara-Konishi, Hidenori Mizugaki, Satoshi Ooizumi, Masaharu Nishimura. The role of HIF-1 and Notch pathway in radio-resistance of non small lung cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 435. doi:10.1158/1538-7445.AM2013-435
Abstract 4353: Cycloxygenase-2 inhibitor regulates p73 isoform expression pattern, inhibit growth, and induce apoptosis in NHL cell line model with 1p36 chromosomal disruption.
Authors: Hassan Hesham M. , Varney Michelle , Joshi Shantaram S. , Singh Rakesh K. , Dave Bhavana J.
Published: August 2014

Diclofenac is a cycloxynase-2 (COX-2) inhibitor that is used in clinics as an analgesic anti-inflammatory drug and has been shown to increase TP73 gene signature in multiple tumor cell lines. TP73, a gene at 1p36 locus, is controlled by a balance between two isoforms with opposing functions, the full length TAp73 protein (pro-apoptotic) and the NH2-terminally truncated ΔNp73 protein (anti-apoptotic through antagonizing both TAp73 and p53). Our previous work has shown a differential ΔNp73 up-regulation among NHL cases with 1p36 chromosomal disruption correlates with decreased apoptosis, increased cellular proliferation and increased angiogenesis. Modulation of ΔNp73:TAp73 ratio using TAp73 expression vector in Granta-519 (NHL cell line model that harbor disrupted 1p36 and express high levels of ΔNp73) resulted in growth inhibition and increased response to chemotherapy. In this study, we hypothesized that diclofenac treatment will modulate the expression profile of p73 isoforms and alter the behavior of the NHL cells with 1p36 disruption. Diclofenac treatment of Granta-519 cells resulted in a decrease in ΔNp73, increase in TAp73 mRNA expression. MTT assay showed a concentration- and duration-dependent cell growth inhibition. Decreased expression of the proliferation marker ki-67 and cell cycle arrest as indicated by flow cytometric cell cycle analysis; were evident following diclofenac treatment. Furthermore, Annexin-V-Flous flow cytometric analysis showed increased number of apoptotic cells that was associated with increased cleaved caspase-3 following diclofenec treatment. Moreover, pro-apoptotic target genes expression was induced by diclofenac treatment including p21, PUMA, NOXA, and BIM. Next we tested the effect of diclofenac treatment on standard CHOP-resistant Granta-519 sub-clones, that resulted in down-regulation of ΔNp73 mRNA expression and a significant dose-dependent growth inhibitory effect. Together, our data demonstrate that diclofenac treatment caused p73 isoforms reconstitution (↑TAp73 and/or ↓ΔNp73), inhibited growth of NHL cells with 1p36 abnormality and highlights the potential of diclofenac as a potential new adjuvant therapy in NHL with disrupted TP73 gene region.

Citation Format: Hesham M. Hassan, Michelle Varney, Shantaram S. Joshi, Rakesh K. Singh, Bhavana J. Dave. Cycloxygenase-2 inhibitor regulates p73 isoform expression pattern, inhibit growth, and induce apoptosis in NHL cell line model with 1p36 chromosomal disruption. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4353. doi:10.1158/1538-7445.AM2013-4353