bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1228: Identification of a novel p53 target regulating p53-induced apoptotic pathway
Authors: Mori Jinichi , Lo Paulysally , Tanikawa Chizu , Nakamura Yusuke , Matsuda Koichi
Published: August 2015

In response to various cellular stresses, p53 induces its target genes and suppresses tumor development. However, the anti-tumor function of p53 has not been fully elucidated. The purpose of our study is the identification and functional analysis of novel p53 downstream targets. We conducted cDNA microarray analysis by using Hct116p53+/+ and p53-/- cells treated with adriamycin and identified p53 inducible protein (p53IP) as a novel p53 downstream target. Induction of p53IP mRNA was confirmed by qRT-PCR. We also found that p53IP protein was increased only in Hct116p53+/+ cells by adriamycin treatment. The result of reporter assay and ChIP assay revealed the direct binding of p53 to p53-binding sequence within p53IP promoter. Colony formation assay by using HCT116 cells indicated that ectopic expression of p53IP significantly inhibited tumor cell growth. Furthermore, knockdown of p53IP with siRNA inhibited apoptosis of tumor cells induced by DNA damage. Our findings indicated that p53IP as a important mediator of p53-dependent apoptotic pathway.

Citation Format: Jinichi Mori, Paulysally Lo, Chizu Tanikawa, Yusuke Nakamura, Koichi Matsuda. Identification of a novel p53 target regulating p53-induced apoptotic pathway. [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 1228. doi:10.1158/1538-7445.AM2015-1228
Abstract 1245: Peroxiredoxin induction in breast cancer
Authors: Buurma Valerie , Freeman Brendan , Adzenyah Akosua , Pettit Gregory , Heinen Felicitas , Muhlbauer Jillian , Phelan Shelley A.
Published: August 2015

Cancer cells possess elevated levels of reactive oxygen species (ROS) that can enhance genomic instability and alter redox signaling. Aberrant antioxidant expression has also been recognized in many cancers, suggesting an adaptive response to the cancerous state. Breast cancers express significantly elevated levels of Peroxiredoxins (Prdxs), a family of thiol-specific antioxidant proteins involved in regulating cell growth and proliferation in many cell types. We previously reported overexpression of many Prdx proteins in the MCF-7 breast cancer line, as well as their importance in doxorubicin-resistance in this line. In the present study, we analyzed Prdx levels in several human breast tumor samples, and further investigated possible mechanisms of Prdx regulation in MCF-7 cells. Comparison of breast tumor and adjacent normal breast tissue from almost 20 patients showed that most patients exhibited elevated levels of several Prdx proteins in the tumor tissue. To begin to understand possible regulators of Prdxs in breast cancer, we focused on transcriptional regulators known to be aberrantly expressed in breast cancer, and also implicated in antioxidant regulation. Sequence analysis revealed potential binding sites for NF-κB, Nrf2, and AP-1 in several peroxiredoxin promoters. We found high levels of NF-κB in MCF-7 cells, as compared to normal breast epithelial cells. We further showed that a chemo-resistant MCF-7 culture generated from 14 days of doxorubicin treatment exhibited markedly decreased NF-κB expression. We then used both siRNA and chemical inhibition of NF-κB to examine the effect on Prdx regulation in these cells and found gene-specific effects. Similar studies are currently underway for Nrf2 and AP-1. This investigation will help to elucidate the role of peroxiredoxins in breast cancer cell survival, and the mechanism by which these cells alter Prdx levels to avoid death and achieve chemoresistance.

Citation Format: Valerie Buurma, Brendan Freeman, Akosua Adzenyah, Gregory Pettit, Felicitas Heinen, Jillian Muhlbauer, Shelley A. Phelan. Peroxiredoxin induction in breast 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 1245. doi:10.1158/1538-7445.AM2015-1245
Abstract 125: Protein kinase C loss-of-function mutations in cancer reveal role as tumor suppressor
Authors: Antal Corina E. , Hudson Andrew M. , Kang Emily , Zanca Ciro , Wirth Christopher , Stephenson Natalie L. , Trotter Eleanor W. , Gallegos Lisa L. , Miller Crispin , Furnari Frank , Hunter Tony , Brognard John , Newton Alexandra C.
Published: August 2015

Protein kinase C (PKC) remains an elusive chemotherapeutic target despite decades of research. To determine whether PKC isozymes function as oncogenes or tumor suppressors, we analyzed 8% of PKC mutations identified in human cancers. Surprisingly, the majority were loss-of-function and none were activating. Loss-of-function mutations were found in all PKC subgroups and acted by impeding 2nd messenger binding or preventing processing phosphorylations. Bioinformatic analysis revealed that PKC mutations might cooperate with co-occurring mutations in cancer drivers. Correction of a patient-identified, loss-of-function PKCβ mutation by CRISPR-mediated genome editing, in a colon cancer cell line, suppressed anchorage-independent growth and reduced tumor growth in xenograft models. Hemizygous deletion provided an anchorage-independent growth advantage, revealing PKC is haploinsufficient for tumor suppression. Several mutations were dominant-negative, suppressing global PKC signaling output. These data establish that PKC isozymes generally function as tumor suppressors, indicating that therapeutic strategies should focus on restoring PKC activity, not inhibiting it.

Citation Format: Corina E. Antal, Andrew M. Hudson, Emily Kang, Ciro Zanca, Christopher Wirth, Natalie L. Stephenson, Eleanor W. Trotter, Lisa L. Gallegos, Crispin Miller, Frank Furnari, Tony Hunter, John Brognard, Alexandra C. Newton. Protein kinase C loss-of-function mutations in cancer reveal role as tumor suppressor. [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 125. doi:10.1158/1538-7445.AM2015-125
Abstract 1252: Arginylation as a new link between oxidative stress response and prostate cancer progression
Authors: Zhang Fangliang , Birnbaum Michael D. , Kumar Akhilesh , Morgan William M.
Published: August 2015

In this study, we evaluate the role of post-translational arginylation in prostate cancer, and introduce its potential as a biomarker of prostate cancer progression. Arginylation has not yet been studied in cancer. Our lab has observed Aringyltransferase (Ate1) is required for oxidative stress-mediated apoptosis. Our preliminary data in multiple eukaryotic cell models demonstrate that a downregulation of Ate1 increases resistance to oxidative stress, and elevated Ate1 is sufficient to induce cell death. Preliminary data mining shows that a downregulation of Ate1 correlates with a poorer prognosis in prostate cancer. Because prostate cancer cells produce a high amount of oxidative stress, the decrease of Ate1 likely increases their survival. We hypothesize that Ate1 is essential for the normal cellular response to insurmountable oxidative stress leading to apoptosis, and that a downregulation of Ate1 will promote cancer cell progression. We found that a loss of Ate1 in a nontumorigenic fibroblast cell line promotes spontaneous tumorigenicity within immunocompromised mice, a disruption of normal cell-cell interactions, and loss of contact inhibition. Also consistent with our hypothesis, prostate cancer cell line PC-3 with stably knocked down Ate1 exhibit higher invasiveness in the Boyden Chamber invasion assay, and higher resistance to H2O2-induced cell death during a 12-hour variable dose treatment. Similarly, the highly metastatic subpopulation of PC-3, PC3-ML, has naturally reduced Ate1 and exhibits higher invasiveness and resistance to H2O2. Finally, histological analysis of primary patient prostate cancer samples show a dramatic reduction of Ate1 compared to normal prostate tissue. These findings warrant further study of Ate1 as a metastasis suppressor, as well as a potential biomarker for prostate cancer progression.

Citation Format: Fangliang Zhang, Michael D. Birnbaum, Akhilesh Kumar, William M. Morgan. Arginylation as a new link between oxidative stress response and prostate cancer progression. [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 1252. doi:10.1158/1538-7445.AM2015-1252
Abstract 126: PI3 kinase plays important role in c-Kit/V560D mediated oncogenic signaling
Authors: Sun Jianmin
Published: August 2015

Oncogenic mutations of c-Kit are often identified in mastocytosis, gastrointestinal stromal tumors (GISTs) and acute myeloid leukemia (AML). The activation mechanism of the most often occurring mutation, D816V in exon 17 of c-Kit, has been well studied while other mutations remain fairly uncharacterized in this respect. In this study, we show that the constitutive activity of the exon 11 mutant V560D is weaker than the D816V mutant except that the phosphorylation of the Src family kinase binding site, Tyr568, is similar in both c-Kit mutants. The ligand for c-Kit, stem cell factor (SCF), induced phosphorylation of downstream signaling proteins, such as Akt and Erk, in c-Kit/V560D expressing cells was stronger than that in c-Kit/D816V expressing cells. Although cells expressing c-Kit/V560D showed increased ligand-independent proliferation and survival compared to wild-type c-Kit expressing cells, these biological effects were less than c-Kit/D816V expressing cells. In contrast to cells expressing wild-type c-Kit, cells expressing c-Kit/V560D are independent of Src family kinases for downstream signaling. However, this independence of Src family kinases is not due to a Src-like kinase activity of the mutant receptor that c-Kit-D816V holds. Blockage of the association of PI3 kinase with c-Kit/V560D by point mutation, but not inhibition of PI3-kinase with pharmacological inhibitors, totally blocked ligand-independent activation of the receptor, suggesting a key role of the non-lipid kinase activity of PI3 kinase in c-Kit/V560D mediated oncogenic signal transduction. Thus, PI3 kinase is an attractive treatment target in c-Kit mutations induced malignancies independent of its lipid kinase activity.

Citation Format: Jianmin Sun. PI3 kinase plays important role in c-Kit/V560D mediated oncogenic signaling. [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 126. doi:10.1158/1538-7445.AM2015-126
Abstract 1270: Ribosome biogenesis is reduced by oncogenic stress in normal cells and is sufficient to trigger cellular senescence
Authors: Lessard Frédéric , Bourdeau Véronique , Igelmann Sebastian , Deschênes-Simard Xavier , Montero Marinieve , Ferbeyre Gerardo
Published: August 2015

Senescence is a stable arrest of cell proliferation in which the cells remain viable and metabolically active but display a constitutive activation of the DNA damage response and of the tumor suppressors p53 and RB. The senescent phenotype can be induced by multiple stresses including short telomeres and oncogenes. We have shown that senescence involves the ERK-dependent degradation of selective proteins involved in cell cycle progression and tumorigenesis. We called this process senescence associated protein degradation (SAPD) and it involves many nucleolar proteins that play a role in ribosome biogenesis. Using tritium pulse labelling we found a strong decrease of rRNA synthesis in senescent cells. In addition, qPCR with primers flanking known rRNA processing sites demonstrated defects in the processing of rRNA in senescent cells. Knocking down some of the nucleolar proteins degraded in senescence was sufficient to trigger the process indicating that a decrease in ribosome biogenesis is causal to cellular senescence. Senescence induced by depletion of ribosome biogenesis factors was characterized by accumulation of p53 and in some cases rescued by inhibition of p53 using shRNAs or the viral oncoprotein E6. We will discuss how reduced ribosome biogenesis impact protein biosynthesis in senescent cells.

Citation Format: Frédéric Lessard, Véronique Bourdeau, Sebastian Igelmann, Xavier Deschênes-Simard, Marinieve Montero, Gerardo Ferbeyre. Ribosome biogenesis is reduced by oncogenic stress in normal cells and is sufficient to trigger cellular senescence. [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 1270. doi:10.1158/1538-7445.AM2015-1270
Abstract 1274: Hallmark of immune responses to multiple tumor-associated antigens (TAAs) in immunodiagnosis of hepatocellular carcinoma (HCC) and other solid tumors
Authors: Dai Liping , Li Pei , Liu Weihong , Luo Chenglin , Tan Eng M. , Zhang Jianying
Published: August 2015

The immune system of the patient with cancer is a sensitive sensor of alterations in structure and/or function of participants in tumorigenesis pathways and is capable of making immune responses in the form of autoantibodies to tumor-associated antigens (TAAs). A hallmark of this immune response is the production of autoantibodies of multiple specificities indicating the activation of more than one tumorigenesis pathway. Novel autoantibodies appear prior to or coincident with clinical detection of transformation to cancer. Activation of more than one tumorigenesis pathway expressed as multiple autoantibodies to TAAs is a finding consistent with the synthetic lethality paradigm where mutation of more than one gene or in the case of cancer, more than one tumorigenesis pathway is required for malignant transformation. After analyzing 321 sera from patients with different type of cancer for autoantibodies to seven selected TAAs including p53, c-myc, cyclin B1, p62, KOC, IMP1 and Survivin, 34% of the sera had autoantibody to one TAA and 20% had two to six autoantibodies simultaneously. In a recent study, we have used a mini-array of 14 selected TAAs as an approach in the immunodiagnosis of HCC, and found that 68% of the sera contained one or more than one antibodies against these TAAs. In addition, this TAA array can identify 43.8% of HCC patients who had normal AFP levels in serum. Some autoantibodies appeared de-novo concurrent with conversion to malignancy. Many novel target antigens besides the above have been identified and shown to be participants in tumorigenesis. The immune system response to TAAs is a repository of information on partners in the synthetic lethality paradigm. Our study can also support the hypothesis that a customized TAA array used for detecting anti-TAA autoantibodies can constitute a promising and powerful tool for immunodiagnosis of HCC and other solid tumors.

Citation Format: Liping Dai, Pei Li, Weihong Liu, Chenglin Luo, Eng M. Tan, Jianying Zhang. Hallmark of immune responses to multiple tumor-associated antigens (TAAs) in immunodiagnosis of hepatocellular carcinoma (HCC) and other solid tumors. [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 1274. doi:10.1158/1538-7445.AM2015-1274
Abstract 1280: Clinical features of transcriptional factor Helios expression on regulatory T cells in patients with non-small cell lung cancer
Authors: Muto Satoshi , Owada Yuki , Inoue Takuya , Watanabe Yuzuru , Yamaura Takumi , Fukuhara Mitsuro , Okabe Naoyuki , Matsumura Yuki , Hasegawa Takeo , Osugi Jun , Hoshino Mika , Higuchi Mitsunori , Suzuki Hiroyuki , Gotoh Mitsukazu
Published: August 2015

Introduction

Foxp3+ regulatory T cells (Tregs) have been thought to have at least two subsets: natural Tregs (nTregs) and induced Tregs (iTregs). Recently, expression of the transcription factor Helios, a member of the Ikaros gene family, has been expected to be a molecular marker of nTregs, but this is still controversial. To date, several in vitro studies and animal model studies regarding Helios have been reported, but there has been no examination of the clinical impact of Helios expression on Tregs in patients with lung cancer to date.

Methods

A total of 45 non-small cell lung cancer (NSCLC) patients who underwent surgical resection and 19 advanced/recurrent NSCLC patients was evaluated. Peripheral blood mononuclear cells (PBMCs) taken from these patients were examined by flow-cytometry. Of these 45 patients, 18 who were evaluable for tumor tissue underwent immunohistochemical investigation for tumor-infiltrating lymphocytes.

Results

Helios-negative Tregs of CD4+ T cells were significantly higher not only in all cancer patients (2.4%), but also in stage IA patients (2.6%) as compared with normal controls (1.5%, p<0.001). Patients who had a lower level of Helios-negative Tregs in tumor-infiltrating lymphocytes had significantly longer survival (p = 0.038).

Discussion

Helios-negative Tregs may be involved in immune suppression, even in early-stage NSCLC. The frequency of Helios-negative Tregs could be a useful prognostic biomarker in patients with NSCLC. The biological functions of Helios-negative Tregs in immune suppression should be further examined.

Baseline demographicsCharacteristicN (%)Age (y)Mean±SD67±8SexFemale27 (42)Male37 (58)Pathological stageIA22 (34)IB16 (25)IIA4 (6)IIB3 (5)III0 (0)IV (Recurrence included)Chemotherapy1st-line6 (9)2nd-line6 (9)3rd or later-line7 (11)Pathological classificationAdenocarcinoma51 (80)Squamous cell carcinoma10 (16)Others3 (5)EGFR mutationPositive22 (34)Negative31 (48)Unknown11 (17)

Citation Format: Satoshi Muto, Yuki Owada, Takuya Inoue, Yuzuru Watanabe, Takumi Yamaura, Mitsuro Fukuhara, Naoyuki Okabe, Yuki Matsumura, Takeo Hasegawa, Jun Osugi, Mika Hoshino, Mitsunori Higuchi, Hiroyuki Suzuki, Mitsukazu Gotoh. Clinical features of transcriptional factor Helios expression on regulatory T cells in patients with non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1280. doi:10.1158/1538-7445.AM2015-1280
Abstract 1284: Analysis of the immune suppression mechanism in extra-hepatic cholangiocarcinoma
Authors: Wada Satoshi , Tsukagoshi Mariko , Watanabe Akira , Kubo Norio , Araki Kenichiro , Suzuki Hideki , Kuwano Hiroyuki
Published: August 2015

Introduction

Extra-hepatic cholangiocarcinoma (EHCC) has not been improved with the five-year survival rate for more than 10 years, which is a refractory cancer of poor prognosis. Development of novel therapeutic agents has been desired, because the standard chemotherapy has not been established yet. Anti-tumor immunotherapy is expected for less burden on the patient in recent years. However, it is limited by multiple tolerogenic mechanisms related to the tumor, the host immune system, and the tumor microenvironment. Therefore satisfactory results are not obtained in the clinical trials for several vaccinations. In this experiment, we focused on negative co-signaling molecules, especially immune checkpoints. The purpose of the present study was to investigate the relationship between the expression of immune checkpoints and clinico-pathological findings, including prognosis in EHCC patients.

Patients and specimens

We enrolled 81 patients (56 men and 25 women; age range: 36-84 years) with EHCC who underwent curative resection with lymphadenectomy at Gunma University Hospital and Saiseikai Maebashi Hospital (Maebashi, Japan) between 2000 and 2011. Patients who had undergone palliative resection, preoperative chemotherapy and radiation therapy were excluded from this study. Furthermore, none of the patients enrolled in this study had synchronous or metachronous cancer in other organs.

Results

Immunohistochemical staining of immune checkpoints in primary EHCC tissue was performed. Patients with high B7-H4 expression had a significantly poorer prognosis than those with low B7-H4 expression. However, it was not seen in B7-H3 expression in this experiment. Furthermore, in analysis for the expression of CD8, IFN-γ and FOXP3, patients with high FOXP3 expression had a significantly poorer prognosis than those with low FOXP3 expression.

Conclusion

The B7-H4 is a negative co-stimulatory molecule and a novel prognostic marker related to the T-cell mediated immune response. It's pathway may be a molecular target for controlling tumor progression in patients with EHCC.

Citation Format: Satoshi Wada, Mariko Tsukagoshi, Akira Watanabe, Norio Kubo, Kenichiro Araki, Hideki Suzuki, Hiroyuki Kuwano. Analysis of the immune suppression mechanism in extra-hepatic cholangiocarcinoma. [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 1284. doi:10.1158/1538-7445.AM2015-1284
Abstract 1299: Tumor-infiltrating B cells are predictive of human lung squamous cell survival
Authors: Centuori Sara , Kim Samuel , Gomes Cecil , Putnam Charles , Mount David , Garland Linda , Martinez Jesse
Published: August 2015

Recent studies in human lung squamous cell carcinoma (SqCC) have shown that upregulation of B cell-associated genes strongly correlate with patient survival. More than any genetic factor (immunologic or otherwise) CD79A, a pan-B cell marker, was found to have the strongest predictive value. Expression of this marker was capable of identifying short (< 2 years) and long (> 6 years) term lung SqCC survivors, with greater than 80% accuracy. These data suggest that the presence of CD79A-expressing tumor-infiltrating B (TIL-B) cells may be contributing to patient outcome. The aim of this study was to characterize CD79A+ TIL-B cells in SqCC patient samples and evaluate their role in lung SqCC survival. We evaluated human lung cancer specimens from long and short term survivors for the presence of CD79A+ TIL-B cells by immunofluorescence and found that their presence directly correlates with survival time. Further analysis showed that CD79A expression found in tumors of long term survivors (>6 years) co-localizes with CD20 staining but not CD138 staining, indicating that the cells responsible for producing a survival signature are not plasma cells. These data indicate that CD79A+ TIL-B cells are contributing to the generation of an efficient immune response in lung SqCC and may be doing so through non-humoral functions.

Citation Format: Sara Centuori, Samuel Kim, Cecil Gomes, Charles Putnam, David Mount, Linda Garland, Jesse Martinez. Tumor-infiltrating B cells are predictive of human lung squamous cell survival. [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 1299. doi:10.1158/1538-7445.AM2015-1299
Abstract 1303: Quantification of natural killer cell-mediated cytotoxicity using Celigo imaging cytometry
Authors: Chan Leo Li-Ying , Rosbach Kelsey , Somanchi Srinivas S. , Lee Dean
Published: August 2015

Cytotoxicity assays play a central role in studying the function of immune effector cells such as cytolytic T lymphocytes (CTL) and natural killer (NK) cells. Traditionally, cytotoxicity assays have been performed using 51Chromium (51Cr) and Calcein release assays. The assays involve labeling tumor cells (target) with radioisotope or fluorescent dyes, when the target cells are subjected to cytolysis by CTLs or NK cells (effector), they releases the entrapped labels into the media upon lysis. The amount of labels in the media is measured to determine the level of cytotoxicity the effectors have induced. These traditional methods may generate inconsistent results due to low sensitivity caused by poor loading efficiency and high spontaneous release of the reagents. In this work, we demonstrate a novel cytotoxicity assay using the Celigo imaging cytometry method. Utilizing imaging cytometry, direct cell counting of live fluorescent target cells can be performed, which is a direct method for assessment of cytotoxicity. Human NK cells from one healthy donor were used as effectors, and K562 (suspension) and IMR32 (adherent) were used as the target cells. Both target cells were first stained with Calcein AM, and seeded at 10,000 cells/well in a standard 96-well microplate. The donor NK cells were then added to each well at Effector-to-Target (E:T) ratios 10:1, 5:1, 2.5:1, 1.25:1, 0.625:1, and 0.3125:1. The 96 well plate was then scanned and analyzed using Celigo imaging cytometer at t = 1, 2, 3, and 4 h to measure the% lysis of target cells. The results showed increasing% lysis as incubation time and E:T ratio increased. The propose Celigo imaging cytometry is an accurate and simple method for direct quantification of cytotoxicity.

Citation Format: Leo Li-Ying Chan, Kelsey Rosbach, Srinivas S. Somanchi, Dean Lee. Quantification of natural killer cell-mediated cytotoxicity using Celigo imaging cytometry. [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 1303. doi:10.1158/1538-7445.AM2015-1303
Abstract 1304: Identification of responders for Anti-CTLA4 in refractory colorectal cancers using CANScript™ platform
Authors: Majumder Biswanath , Ulaganathan Baraneedharan , Thayakumar Allen , Thiyagarajan Saravanan , Brijwani Nilesh , Tewari Biplab , Santhappa Basavaraja U. , Radhakrishnan Padhma , Majumder Pradip K.
Published: August 2015

Predicting clinical response to anticancer drugs remains a major challenge in the treatment of cancer. Indeed, while biomarker-guided strategies for personalizing anticancer drugs have shown strong promise in certain cases, recent studies have shown that the tumor microenvironment and heterogeneity can limit the predictive power of biomarkers alone. Here we have engineered a personalized tumor ecosystems, termed CANScript™, that contextually conserve tumor heterogeneity and phenocopy the tumor ecosystem using thin tumor explants maintained in defined tumor grade-matched matrix support and autologous ligands from patients. We then demonstrated that the CANScript™ platform can be used to predict clinical response. Specifically, functional readouts obtained by exposing the CANScript™ ecosystems from more than 1100 patients to a panel of anticancer drugs, together with the corresponding clinical outcomes, were used to train a novel machine learning algorithm; the learned model was then applied to predict clinical response to anticancer drugs in a test group comprising of 900 new patients, where it achieved 100% sensitivity in its predictions while also keeping specificity in a desired high range. We have also observed that CANScript™ retains patient tumor immune environment which is important for clinical response of not only immunomodulators but other anti-cancer drugs. Here we report the effect of immunomodulators in refractory CRC tumors which remains difficult to treat. Data demonstrates that Anti-CTLA-4 has profound antitumor effect in refractory CRC tumors by increasing cytotoxic T-Cells. In this cohort of 16 patients’ tumors CANScript™ platform identifies 31% as responders (5/16) where conventional tumor sections culture model shows only 6% as (1/16) responders.

Citation Format: Biswanath Majumder, Baraneedharan Ulaganathan, Allen Thayakumar, Saravanan Thiyagarajan, Nilesh Brijwani, Biplab Tewari, Basavaraja U. Santhappa, Padhma Radhakrishnan, Pradip K. Majumder. Identification of responders for Anti-CTLA4 in refractory colorectal cancers using CANScript™ platform. [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 1304. doi:10.1158/1538-7445.AM2015-1304
Abstract 1313: Characterization and isolation of antigen-responding T cells in Sipuleucel-T treated patients
Authors: Pufnock Jeff , Vu Tuyen , Trager James , Sheikh Nadeem
Published: August 2015

Sipuleucel-T is an FDA-approved autologous cellular therapy for men with asymptomatic or minimally symptomatic metastatic castrate resistant prostate (mCRPC). Sipuleucel-T is manufactured from peripheral blood mononuclear cells (PBMCs) cultured with PA2024, a fusion antigen composed of prostatic acid phosphatase (PAP) fused to granulocyte macrophage-colony stimulating factor (GM-CSF). We have demonstrated that following three infusions this therapy results in T cell antigen specific responses directed against both PA2024 and PAP in treated patients. In this study we utilized the cellular dye Carboxyfluorescein succinimidyl ester (CSFE) and flow cytometric analysis to further characterize antigen responding T cells in Sipuleucel-T treated patients. We observed that CD4+ T cells predominate the antigen-responding T cell population following treatment, confirming our previous studies. Importantly, this approach has allowed us to also visualize a previously undetected population of antigen responding CD8+ T cells. The frequency of both of these populations is enhanced following treatment relative to baseline responses and the responding T cells display increased surface expression of the prototypical T cell activation markers CD134 and CD137 relative to non-proliferating T cells. Additionally, we are exploiting the increased surface expression of these activation markers on antigen-responding T cells to isolate these cells in an HLA and epitope-independent manner. Using this methodology we have demonstrated Sipuleucel-T is capable of inducing both CD4+ and CD8+ antigen-specific T cell responses in treated patients, and that we are capable of isolating these antigen-responding T cells specifically and with minimal manipulation.

Citation Format: Jeff Pufnock, Tuyen Vu, James Trager, Nadeem Sheikh. Characterization and isolation of antigen-responding T cells in Sipuleucel-T treated 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 1313. doi:10.1158/1538-7445.AM2015-1313
Abstract 1349: Tim-3/galectin-9 interaction might be associated with the immunosuppression of gastrointestinal stromal tumor (GIST)
Authors: KOMITA HIDEO , Koido Sigeo , Tajiri Hisao , Suzuki Masafumi , Homma Sadamu
Published: August 2015

Purpose: Although GIST shows high expression of Wilms’ Tumor 1 (WT1) and might be potentially immunogenic, there are no evidences that antitumor immunity suppresses GIST development. Elucidation of the mechanism of the immunosuppression of GIST might contribute to the establishment of the immunotherapy against GIST.

Methods: Nineteen GIST tissue samples were evaluated for the expression of various tumor-associated antigens, MHC class I, immune check point molecules and the infiltration of immune cells by immunohistochemistry. Tumor infiltrating lymphocytes (TILs) from freshly resected GIST tissues were analyzed for WT1 tetramer assay.

Results: All GIST tissues showed diffuse WT1 expression but not GP-100, NY-Eso1, Melan A and MAGEA4. They all expressed MHC class I and WT1 tetramer positive CD8+T cells were found in TILs from some GIST tissues. Seven GIST tissues showed marked CD8+ T cell infiltration and the infiltration of CD8+T cells and NK cells was strongly correlated. However, all the immune cells infiltrated into GIST tissues were CD69 negative inactivated cells. Although PD-1/PD-L1 expression was not seen in GIST tissues, galectin-9 was expressed in tumor cells and Tim-3 was expressed on NK cells but not CD8+T cells in all the GIST tissues.

Conclusions: Galectin-9 on tumor cells and Tim-3 on infiltrated NK cells might be associated with the immunosuppression of GIST.

Citation Format: HIDEO KOMITA, Sigeo Koido, Hisao Tajiri, Masafumi Suzuki, Sadamu Homma. Tim-3/galectin-9 interaction might be associated with the immunosuppression of gastrointestinal stromal tumor (GIST). [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 1349. doi:10.1158/1538-7445.AM2015-1349
Abstract 136: Evaluation of reversible BTK inhibitors in cell lines with clinically relevant Ibrutinib resistance mutations
Authors: Choy Regina Wai-Yan , Burton Luciana , Young Wendy , Crawford James J. , Penuel Elicia , Belmont Lisa D.
Published: August 2015

Background

Ibrutinib, a small molecule inhibitor that covalently binds Cys481 of Bruton's Tyrosine Kinase (BTK), has recently been approved in Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL). Although the majority of patients respond to this drug, eventually resistance develops. Known causes for resistance include the outgrowth of cancer cells with mutations in either the drug binding pocket of BTK or activating mutations in the downstream effector, PLCγ2.

Objective

To explore mechanisms of resistance to ibrutinib and evaluate the potential of next generation BTK inhibitors on ibrutinib resistant cancer, we selected for resistance in cultured lymphoma cells. We chose TMD8, a cell line model of diffuse large B-cell lymphoma (DLBCL) that has constitutive B-cell receptor signaling, and REC-1, an ibrutinib sensitive MCL line, to model resistance.

Methods

TMD8 and REC-1 were cultured in increasing concentrations of ibrutinib to generate resistant cell lines. Genetic changes that may account for resistance were evaluated using whole exome sequencing. Resistant cell lines were evaluated for sensitivity to a small panel of BTK inhibitors.

Results

We generated a TMD8 derived cell line that is 1000-fold resistant to ibrutinib. Whole exome sequencing revealed a C481Y mutation in BTK. Reversible inhibitors that are not reliant on this site may function as alternative inhibitors of C481 mutant BTK. Ibrutinib-resistant TMD8 cells were tested for response to novel, reversible BTK inhibitors. The potency was similar to or modestly reduced (3 to 10 fold) on the ibrutinib-resistant cells compared to the parental line. In addition, we generated ibrutinib-resistant cells derived from the REC-1 line. Unlike resistance generated in TMD8, REC-1 resistant cells harbor a L527W mutation in BTK and are resistant to all BTK inhibitors that we evaluated.

Conclusions

C481Y and L527W BTK mutations have been identified in CLL patients that have relapsed after response to ibrutinib, and C481S mutations have been identified in ibrutinib relapsed CLL and MCL patients. Our data suggest that reversible, next generation BTK inhibitors have the potential to be efficacious on ibrutinib relapsed patients that harbor mutations at C481 of BTK.

Citation Format: Regina Wai-Yan Choy, Luciana Burton, Wendy Young, James J. Crawford, Elicia Penuel, Lisa D. Belmont. Evaluation of reversible BTK inhibitors in cell lines with clinically relevant Ibrutinib resistance mutations. [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 136. doi:10.1158/1538-7445.AM2015-136
Abstract 1365: Visualizing the effect of BIBF1120 in lung cancer cells by imaging-massspectrometry (MS)
Authors: Arai Daisuke , Soejima Kenzo , Yasuda Hiroyuki , Ishioka Kota , Kagawa Shizuko , Hamamoto Junko , Naoki Katsuhiko , Emoto Katsura , Sugiura Yuki , Suematsu Makoto , Betsuyaku Tomoko
Published: August 2015

Background: Angiogenesis plays an important role for tumor maintenance, progression, and metastasis. BIBF1120 is a multiple tyrosine kinase inhibitor, targeting VEGFR, FGFR and PDGFR, and is known as an anti-angiogenic agent. The mechanism by which anti-angiogenic agents suppress the growth of tumors is supposed to be induction of under-nutrition. However, no study has demonstrated the regional differences in the nutrition status of tumors treated by anti-angiogenic agents. Imaging MS is a novel technology that quantitatively visualizes the distribution of hundreds of metabolites in the selected area of tissues.

Aim: The aim of this study is to evaluate the efficacy of BIBF1120 in lung cancer cells, and to examine whether BIBF1120 induces under-nutrition status in association with suppressed angiogenesis in xenograft models of lung cancer using Imaging MS.

Method: We used lung cancer cell lines driven by several types of histology, PC9, H1975, H3122, A549, H69, and H520. The efficacy of BIBF1120 was evaluated by MTS assay and mouse xenograft model. We treated the xenografted mice with BIBF1120 or vehicle for 4 weeks and harvested xenograft tumors. Micro vessel density and percentage of apoptosis cells in the tumors was evaluated by immunohistochemistry using anti-Ki-67 antibody and anti-cleaved caspase 3 antibody, respectively. The effect of BIBF1120 on metabolic status in xenograft tumors was evaluated by imaging MS.

Result: BIBF1120 inhibited the growth of all of the xenograft tumors tested, although it did not directly affect the proliferation rate of those cells in vitro. BIBF1120-treated tumors exhibited significantly lower micro vessel density compared to the vehicle-treated tumors (p<0.05), but BIBF1120 didn't increased the percentage of apoptosis cells. Imaging MS revealed that BIBF1120 treatment reduced 2-3 DPG (a maker of blood flow), glucose intake, and amount of ATP, whereas activated glycolysis via anaerobic metabolic pathway rather than oxidative phosphorylation in peripheral area of tumors.

Conclusion: To our knowledge, this is the first report to demonstrate the effect of BIBF1120 on the nutrition status of tumor cells in a site-specific manner.

Citation Format: Daisuke Arai, Kenzo Soejima, Hiroyuki Yasuda, Kota Ishioka, Shizuko Kagawa, Junko Hamamoto, Katsuhiko Naoki, Katsura Emoto, Yuki Sugiura, Makoto Suematsu, Tomoko Betsuyaku. Visualizing the effect of BIBF1120 in lung cancer cells by imaging-massspectrometry (MS). [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 1365. doi:10.1158/1538-7445.AM2015-1365
Abstract 1367: Anti-angiogenesis promotes venous thromboembolism through inducing PAI-1 in a mouse xenograft model of human lung carcinoma
Authors: Chen Ni , Ren Meiping , Li Rong , Deng Xin , Li Yongjie , Yan Kai , Xiao Lamei , Yang Yan , Wang Liqun , Luo Mao , Wu Jianbo
Published: August 2015

Thromboembolism is a major source of morbidity and mortality in patients with cancer. Increased incidence of venous thromboembolism (VTE) was associated with anti-vascular endothelial growth factor (VEGF) treatment in cancer. However, the mechanism of VTE initiation remains elusive. In this study, we addressed the effect of bevacizumab, a humanized monoclonal antibody against VEGF-A on either an inferior vena cava stenosis or a saphenous vein model using a xenograft mouse model. We found that treatment of bevacizumab accelerated thrombosis in a saphenous vein model with an increased thrombus weight in an inferior vena cava stenosis model. Administration of bevacizumab inhibited the growth of lung tumor by suppressing angiogenesis. Both intrathrombotic PAI-1 expression and plasma PAI-1 level was significantly increased by the treatment of bevacizumab. Furthermore, pharmacologic inhibition of PAI-1 with PAI-039 blocked the bevacizumab-induced venous thrombosis, but showed no change in tumor growth. Collectively, these findings indicate that PAI-1 plays a role in VET with anti-angiogenesis therapy, and have efficacy as a therapeutic strategy to prevent VET.

Citation Format: Ni Chen, Meiping Ren, Rong Li, Xin Deng, Yongjie Li, Kai Yan, Lamei Xiao, Yan Yang, Liqun Wang, Mao Luo, Jianbo Wu. Anti-angiogenesis promotes venous thromboembolism through inducing PAI-1 in a mouse xenograft model of human lung 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 1367. doi:10.1158/1538-7445.AM2015-1367
Abstract 1381: The vascular targeted and antitumor effects of gene electrotransfer of plasmid silencing endoglin with tissue specific and constitutive promoter
Authors: Stimac Monika , Dolinsek Tanja , Lampreht Ursa , Cemazar Maja , Sersa Gregor
Published: August 2015

Endoglin, the transforming growth factor-β (TGF-β) co-receptor, mediates proliferation, differentiation and migration of endothelial cells forming neovasculature. It is promising target for vascular targeted therapies for the treatment of cancer, although its specific, safe and long-lasting targeting remains the challenge. One of the options for targeted and prolonged silencing is gene electrotransfer (GET) of plasmid DNA encoding shRNA against endoglin. In our study we used two plasmid DNAs; one with tissue specific promoter for endothelial cells (TS plasmid) and the other with constitutive promoter (CON promoter). In vitro, we evaluated the transfection efficacy using flow cytometry measurement of transfected cells and determined the vascular targeted effects by tube formation assay. The therapeutic potential of the GET of plasmids and comparison of theirs efficacies in vitro was obtained by measuring tumor growth and by analyzing histological sections for determining area of necrosis and a number of activated blood vessels.

In vitro, we demonstrated the tissue specificity of TS plasmid on several cell lines, and the antiangiogenic efficacy of GET of either of both plasmids, as reduced tube formation of 2H11 endothelial cells. In vivo, on a murine mammary TS/A tumor model, we demonstrated good antitumor effect of GET of either of both plasmids in treatment of smaller tumors in avascular phase of growth, as well as on bigger tumors, already well vascularized, possibly due to both, the antiangiogenic and vascular disrupting effect. In order to support that, the histological analysis has demonstrated an increase in necrosis and a decrease in the number of blood vessels in therapeutic groups. Therefore, using GET of TS plasmid we were able to demonstrate that it is specific, safe and has quite the same efficacy as the GET of CON plasmid. Therefore, our approach can represent a novel alternative to other therapeutic approaches that aim at silencing endoglin, which represents also an alternative target in VEGF resistant (unresponsive) tumors.

Citation Format: Monika Stimac, Tanja Dolinsek, Ursa Lampreht, Maja Cemazar, Gregor Sersa. The vascular targeted and antitumor effects of gene electrotransfer of plasmid silencing endoglin with tissue specific and constitutive promoter. [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 1381. doi:10.1158/1538-7445.AM2015-1381
Abstract 1389: Study on cancer stem cell marker CD90 in liver cancer
Authors: Chen Wei-Ching , Lai Ming-Derg
Published: August 2015

CD90 is a glycophosphatidylinositol (GPI)-anchored glycoprotein, which is highly expressed in stem cells. CD90 involves in immunological function and nonimmunological functions, including cell adhesion, migration and cell death. The published data show that CD90 is a tumor suppressor gene in ovarian cancer and nasopharyngeal carcinoma. However, several studies indicate that CD90 is a potential marker for liver cancer stem cells (CSCs). It suggests that CD90 leads to different function in different tumors. In order to elucidate the role of CD90 in liver cancer progression, the HepG2 and Hep3B stable transfectants expressing exogenous CD90 were established and the tumorigenic ability was analyzed in vivo and in vitro. We further analyzed the stemness properties of the transfectants and found that CD90 increased the sphere formation ability and ALDH activity. We further analyzed the role of CD90 in ovarian cancer cell and found that there is a controversial phenotype in SKOV3 cell compared to liver cancer cell. Several cell surface markers have been used to identify liver cancer stem cells including CD90, EpCAM, CD133, CD44, and CD13. Among these markers, CD90 is particularly interesting since overexpression of CD90 inhibited the growth of another type of tumor. We analyzed the cross-talk interaction between CD90 and the other CSC markers and found that there was a cause-effect relationship. We further identified the signaling transduction pathway in mediating the processes of CD90-induced tumorigenic ability. Our findings provide a concept to target stemness-associated signaling axis as a strategy to treat cancer.

Citation Format: Wei-Ching Chen, Ming-Derg Lai. Study on cancer stem cell marker CD90 in liver 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 1389. doi:10.1158/1538-7445.AM2015-1389
Abstract 1398: Metastatic neuroblastoma cancer stem cells exhibit flexible plasticity and adaptive stemness signaling
Authors: Ramraj Satish Kumar , Khan Faizan H. , Aravindan Sheeja , Natarajan Mohan , Herman Terence S. , Aravindan Natarajan
Published: August 2015

High-risk neuroblastoma (HR-NB) presented with hematogenous metastasis is one of the most difficult cancers to cure with poor patient survival. Aggressive tumors contain populations of rapidly proliferating clonogens that exhibit stem cell properties, cancer stem cells (CSCs). Conceptually, CSCs that evade intensive multimodal therapy dictates tumor progression, relapse/recurrence and poor clinical outcomes. Herein, we investigated the plasticity and stem-cell related molecular response of aggressive metastatic neuroblastoma cells that fits CSC model. Well characterized clones of ‘metastatic site derive aggressive cells’ (MSDACs) from the manifold of metastatic tumors of clinically translatable HR-NB were characterized for their CSC fit. Alternated, yet stabilized (three generations) microenvironment simulation experiments with ‘serum-free stem cell medium’ to ‘growth medium with serum’ and vice versa identified flexible revocable plasticity. Signatures stem-cell related molecular responses consistent with phenotypic conversions are observed. Successive reintroduction to the favorable niche not only regained identical EMT, self-renewal capacity, pluripotency maintenance, and other stem-cell related signaling events, but also instigated additional events depicting the aggressive adaptive plasticity. Together, these results demonstrated the flexible plasticity of HR-NB MSDACs that typically fits in CSC model and further identified the intrinsic adaptivness of the successive phenotype switching that clarify the heterogeneity of HR-NB. Moreover, the continuous ongoing acquisition of stem-cell related molecular rearrangements may hold the key for the switch from favorable disease to HR-NB.

Citation Format: Satish Kumar Ramraj, Faizan H. Khan, Sheeja Aravindan, Mohan Natarajan, Terence S. Herman, Natarajan Aravindan. Metastatic neuroblastoma cancer stem cells exhibit flexible plasticity and adaptive stemness signaling. [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 1398. doi:10.1158/1538-7445.AM2015-1398