bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4186: miR-218 suppresses invasion and migration via inhibition of ZEB2 and N-Cadherin in colon cancer.
Authors: Batliner Jasmin , Abba Mohammed , Hauser Christine , Allgayer Heike
Published: August 2014

The great majority of cancer deaths are due to metastasis. A hallmark of cancer metastasis is the process of epithelial to mesenchymal transition (EMT), in which cells alter their gene expression program resulting in loss of their epithelial characteristics and gain of typical mesenchymal properties. Recently, microRNAs have been shown to play key roles in the dynamic process of EMT, orchestrating a coordinated multi-target repression of genes and, together with master transcription factors, act in regulatory networks to alter cell function. We used a miRNA-microarray platform to identify differentially expressed microRNA genes in primary tumor, distant metastatic and corresponding normal samples in a cohort of resected patient tissues. From our list of differentially regulated microRNAs we focused on miR-218 as so far very little information has been accumulated regarding its function in cancer. In our microarray analysis, miR-218 was significantly downregulated in tumor/metastatic tissue compared to normal colon tissue. We validated miR-218 expression in an additional series of 42 colorectal cancer patient tissues, where we found a significant downregulation of miR-218 in tumor as compared to the corresponding normal tissues in over 80% of cases. Furthermore, ectopic expression of miR-218 significantly reduced in vitro invasion and migration of invasive colorectal cancer cell lines and upregulated the key epithelial marker E-cadherin, while silencing of miR-218 lead to enhanced migration and invasion. In addition, we identified two novel miR-218 target genes that are associated with EMT including ZEB2, a zinc-finger transcription factor, and N-Cadherin, using reporter gene, RT-PCR and Western blot analysis. In our present study, we show that miR-218 is a direct suppressor of EMT by its ability to downregulate ZEB2 and N-Cadherin expression and represents an additional molecule contributing to the complexity of the EMT regulatory network.

Citation Format: Jasmin Batliner, Mohammed Abba, Christine Hauser, Heike Allgayer. miR-218 suppresses invasion and migration via inhibition of ZEB2 and N-Cadherin in colon 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 4186. doi:10.1158/1538-7445.AM2013-4186
Abstract 4279: Isolation of a high affinity, specific human monoclonal antibody that binds to FGFR-2 IIIc.
Authors: Schwertschlag Ullrich S. , Turner Katherine J. ,   Central Lab Naval Hospital Bejing.
Published: August 2014

Fibroblast Growth Factor Receptor-2 (FGFR-2) expression and activation have been increasingly implicated in a number of human cancers, including prostate, ovarian, breast, gastric, bladder, pancreatic, endometrial and lung malignancies. Mechanisms include stimulation of angiogenesis, direct stimulation of tumor growth and promotion of tumor metastasis. Similar to other FGFRs, FGFR-2 binds a subset of FGF ligands with high affinity and consists of an extracellular region comprised of two or three immunoglobulin-like (Ig) domains, a single transmembrane region and an intracellular kinase domain. Variable splicing in the second half of the third Ig-like domain generates two isoforms, IIIb or IIIc, with different ligand binding specificity and expression patterns. An isoform switch from epithelial cell-expressed FGFR-2IIIb to mesenchymal cell-expressed FGFR2-IIIc has been shown to associate with an epithelial to mesenchymal transition (EMT), which increases cell migration and invasion and facilitates metastasis of epithelial carcinomas, such as prostate and bladder cancer. Several groups are developing antibodies to block FGFR-2 as therapeutics. Small molecule drugs that block FGFR-2 signaling are currently being evaluated in clinical trials. Recently, there have been reports of FGFR-2 IIIb-specific antibodies. In the same studies, monoclonal antibodies that bind both isoforms IIIb and IIIc were described, but, to date, no detailed studies describing the isolation of an FGFR-2 IIIc-specific antibody have been reported. The high degree of sequence conservation between the IIIb and IIIc exons is presumably the major reason why this has been technically challenging. In this study, we report the isolation of a high affinity, human monoclonal antibody, ATTO-1, and a murine mAb, 5H11, that bind to human and mouse FGFR-2 IIIc, but not FGFR2-IIIb, respectively. ATTO-1 binds to established mesenchymal tumor cell lines, such as AT3 and BT459 (FGFR-2 IIIc+), but not to MCF-7 or DT (FGFR-2 IIIb+). These antibodies may be valuable research tools for studying and developing novel therapies for blocking tumor invasion and metastasis.

Citation Format: Ullrich S. Schwertschlag, Katherine J. Turner, Central Lab Naval Hospital Bejing. Isolation of a high affinity, specific human monoclonal antibody that binds to FGFR-2 IIIc. [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 4279. doi:10.1158/1538-7445.AM2013-4279
Abstract 4305: FBXO31 functions as an oncoprotein by suppressing apoptosis via regulation of MAPK pathway in esophageal squamous cell carcinoma cell lines.
Authors: Liu Jia , Li Bin , Tsao George SW , Cheung Annie ML
Published: August 2014

FBXO31 is a DNA-damage responsive F-box protein, found to be down-regulated in breast and hepatocellular carcinoma but up-regulated in esophageal squamous cell carcinoma. The role of FBXO31 in carcinogenesis is not fully determined. Mitogen activated protein kinases (MAPKs) play pivotal role in cellular response to diverse stresses, and are involved in regulating cell proliferation, differentiation and apoptosis. In this study, data from gain-of-function and loss-of-function experiments revealed that FBXO31 regulated cell apoptosis upon UV stress, as evidenced by changes in expressions of cleaved caspase 3 and PARP in FBXO31 modified cell lines compared to control cell lines, as well as regulation of sub-G1 population measured by flow cytometry analysis. The effects of FBXO31 on apoptosis were associated with changes in MAPK p38 and JNK phosphorylation. In order to verify the role of FBXO31 in cell apoptosis and MAPK regulation, shRNA rescue experiments were performed. The results showed that ectopic overexpression of wild type FBXO31 in stable cell line which expressed shRNA targeting the 3’ UTR of FBXO31 could reverse the effects of FBXO31-knockdown on apoptosis, and on MAPK p38 and JNK phosphorylation. Furthermore, we found that JNK was a dominant mediator of FBXO31 in regulating cell apoptosis upon UV stress since treatment with JNK inhibitor, but not p38 inhibitor, reversed apoptosis induced by FBXO31-knockdown. Consistent with this finding, soft agar assay and in vivo xenograft experiments showed that knockdown of FBXO31 reduced the tumorigenic potential of ESCC cells. In conclusion, our data suggest that FBXO31 functions as an oncogenic protein in ESCC by suppressing cell apoptosis via MAPK pathway. This mechanism may favor the survival of tumor cells in stringent environment and contribute to ESCC development.

Citation Format: Jia Liu, Bin Li, George SW Tsao, Annie ML Cheung. FBXO31 functions as an oncoprotein by suppressing apoptosis via regulation of MAPK pathway in esophageal squamous cell carcinoma 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 4305. doi:10.1158/1538-7445.AM2013-4305
Abstract 4354: Dihydroartemisinin inhibits mTORC1 signaling in tumor cells.
Authors: Odaka Yoshinobu , Xu Baoshan , Huang Shile
Published: August 2014

Dihydroartemisinin (DHA) is a derivative of artemisinin, a natural product isolated from the plant, Artemisia annua. DHA is also the active metabolite of first-generation artemisinin derivatives (artemisinin, artesunate, artemether, etc.). Artemisinins have been clinically used to treat malaria for decades. Recent studies have demonstrated that artemisinin and its derivatives possess anticancer activity. This study was set to investigate the anticancer mechanism of DHA. We found that DHA inhibited proliferation in a spectrum of tumor cells, by arresting cells in G1/G0 and G2/M phases, which was linked to downregulation of cyclin D1, CDK4 and CDC25A, as well as CDK1 and CDC25C expression, respectively. We also observed that DHA induced caspase-dependent apoptosis, which was related to downregulation of survivin, Mcl-1 and Bcl2. As the mammalian target of rapamycin (mTOR) is a master kinase that regulates cell proliferation and survival, we further tested whether DHA inhibits mTOR signaling. Interestingly, we observed that DHA inhibited phosphoryaltion of mTOR and its downstream effector molecules, p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), in a concentration- and time-dependent manner, but did not obviously affect mTORC2-mediated phosphorylation of Akt, which is in contrast to the conventional mTORC1 inhibitor, rapamycin. The results suggest that DHA may represent a novel class of mTORC1 inhibitor, and may execute its anticancer activity primarily by blocking mTOR-mediated signaling pathways in the tumor cells.

Citation Format: Yoshinobu Odaka, Baoshan Xu, Shile Huang. Dihydroartemisinin inhibits mTORC1 signaling in tumor 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 4354. doi:10.1158/1538-7445.AM2013-4354
Abstract 4391: MET is a potential therapeutic target and a candidate for combination therapy with EGFR inhibition in triple-negative/basal-like breast cancer.
Authors: Kim Yu Jin , Choi Jong-Sun , Song Ji-Young , Jung Kyungsoo , Oh Ensel , Shin Young Kee , Choi Yoon-La
Published: August 2014

Triple-negative breast cancer (TNBC)/basal-like breast cancer (BLBC) are known as poor outcome subtypes with deficiency of targeted molecular therapy. Previous studies showed that MET may play a critical role in the development of TNBC and may be a rational therapeutic target. In order to evaluate MET as a potential therapeutic target for TNBC, we investigated genomic aberration and expression status of MET and evaluated efficacy of MET inhibitor, PHA-665752 in TNBC cell lines. MET protein expression was relatively high in TNBC cell lines. The depletion of MET using small interfering RNA (siRNA) led decreased cell proliferation and migration. PHA-665752 treatment resulted in decreased cell viability, and most of these cell lines were found to be moderately sensitive in their response to PHA-665752. The most resistant cell line, MDA-MB-468, showed exclusively high expression of EGFR, and then EGFR depletion using siRNA induced increased sensitivity to PHA-665752. In our cohort, BLBC was the subtype showing the highest relationship with MET expression among 5 subtypes (77/134, 57.5%, P<0.001). MET expression had a poorer OS (P=0.001) and DFS (P=0.010). Interestingly, TNBC patients with co-expression of MET and EGFR worsen the DFS significantly compared with EGFR expression only. Our findings highly suggest that MET can be a potential therapeutic target in TNBC, and the combined inhibition of MET and EGFR may benefit for the treatment of BLBC patients.

Citation Format: Yu Jin Kim, Jong-Sun Choi, Ji-Young Song, Kyungsoo Jung, Ensel Oh, Young Kee Shin, Yoon-La Choi. MET is a potential therapeutic target and a candidate for combination therapy with EGFR inhibition in triple-negative/basal-like breast 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 4391. doi:10.1158/1538-7445.AM2013-4391
Abstract 4420: Generating preclinical models to assess bone marrow toxicity induced by the PARP inhibitor olaparib in combination with chemotherapy.
Authors: O'Connor Mark J. , Mason Helen , Horner Steve , Slater Ian , Lau Alan , Cadogan Elaine , Oplustilova Lenka
Published: August 2014

Inhibitors of the DNA damage response (DDR) provide an exciting opportunity as new anti-cancer therapies. A prominent example is olaparib, an inhibitor of poly (ADP-ribose) polymerase-1 (PARP-1), that has shown potential as a monotherapy for tumours bearing BRCA1/2 defects in clinical trials. Apart from monotherapy based on the concept of synthetic lethality, PARP inhibitors also have the potential to provide additional benefit when used in combination with DNA damage-inducing chemotherapies. In the clinic, however, this approach has been challenging due to enhanced bone marrow toxicity.

Our aim is to generate preclinical models to provide a better understanding of the mechanisms for this limiting bone marrow toxicity. We characterized in vitro responses to DNA damage induced by IR and chemotherapy in human hematopoietic multipotent progenitors and mesenchymal stem cells, both components of bone marrow. In parallel, we modelled the effects of a platinum chemotherapy (carboplatin) in combination with olaparib in wild type rats with the aim of reproducing the clinical combination-induced bone marrow toxicity.

We will present data that provide initial insights into the suitability of the rat pre-clinical model to guide combination dose and schedules with olaparib and an assessment of the ability of different bone marrow and peripheral blood cell populations to act as useful biomarkers to guide better tolerated combination schedules.

Citation Format: Mark J. O'Connor, Helen Mason, Steve Horner, Ian Slater, Alan Lau, Elaine Cadogan, Lenka Oplustilova. Generating preclinical models to assess bone marrow toxicity induced by the PARP inhibitor olaparib in combination with chemotherapy. [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 4420. doi:10.1158/1538-7445.AM2013-4420
Abstract 447: Hypoxia induces tumor cell escape from T cell-mediated immunity via up-regulation of B7-H1.
Authors: Barsoum Ivraym B. , Smallwood Chelsea A. , Siemens D. Robert , Graham Charles H.
Published: August 2014

In the current research we describe a novel mechanism by which hypoxia contributes to tumour cell escape from lysis mediated by cytotoxic T lymphocytes (CTLs). Specifically, we show that when exposed to hypoxia for 24 h, human DU145 prostate and MDA-MB-231 breast cancer cells, as well as mouse B16-OVA melanoma cells, increased their expression of the immune inhibitory ligand B7-H1 at both the mRNA and protein levels in a manner dependent on hypoxia-inducible factor-1α (HIF-1α). Moreover, our findings revealed that the hypoxia-induced up-regulation of B7-H1 expression in tumour cells resulted in increased tumour cell resistance to lysis mediated by primed CTLs. We previously demonstrated that activation of nitric oxide (NO) signalling blocks HIF-1α accumulation in tumour cells exposed to hypoxia. Here we show that the NO mimetic glyceryl trinitrate (GTN; 10 nM) prevented the hypoxia-induced up-regulation of tumour cell B7-H1 expression as well as the increased resistance to CTL-mediated lysis. Furthermore, co-culture studies revealed that the increased expression of B7-H1 following exposure of tumour cells to hypoxia led to the apoptotic death of CTLs as well as Jurkat leukemia cells in a manner that could be prevented by blocking B7-H1, programmed cell death-1 receptor (B7-H1 receptor expressed by T cells) or by incubation with low concentrations of GTN. These results indicate that a potential immunotherapy of cancer could involve interference with B7-H1 function in hypoxic tumour cells via administration of NO mimetics.

Citation Format: Ivraym B. Barsoum, Chelsea A. Smallwood, D. Robert Siemens, Charles H. Graham. Hypoxia induces tumor cell escape from T cell-mediated immunity via up-regulation of B7-H1. [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 447. doi:10.1158/1538-7445.AM2013-447
Abstract 4470: Mechanisms of acquired resistance to the PI3K inhibitors in colorectal cancer cell lines.
Authors: Edgar Kyle A. , Wagle Marie-Claire , Yan Yibing , Belvin Marcia , Friedman Lori , Wallin Jeffrey
Published: August 2014

Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutation of the PIK3CA gene or loss of the tumor suppressor PTEN. The dysregulation of this pathway has been implicated in tumor initiation, cell growth and survival, invasion and angiogenesis, thus, PI3K is a promising therapeutic target for cancer. There are multiple PI3K inhibitors in clinical trials and the current study investigates preclinical mechanisms of resistance to two clinical PI3K inhibitors, GDC-0941 and GDC 0032. Acquired resistance to PI3K inhibitors was generated in vitro, in SW48 parental and SW48 knock-in clones harboring hotspot PIK3CA mutations. We found that GDC-0941 resistant clones and GDC-0032 resistant clones demonstrated a marked signaling increase in the PI3K/Akt/mTOR pathway when released from drug inhibition, and different nodes in the pathway were activated. GDC-0941 resistant clones have elevated pAKT levels due to loss of PTEN, and the pAKT is further increased when drug is removed. GDC-0032 resistant clones retained functional PTEN protein, but had increased pS6 levels in the absence of GDC-0032. The result of increased PI3K pathway signaling in cells with acquired resistance to PI3K inhibitors resulted in a decreased sensitivity to inhibitors of the MAPK pathway upon removal of drug. Resistant cells maintained on PI3K inhibitors gained sensitivity to MAPK pathway inhibitors. These preclinical data may provide rationale for combination therapy in the clinic.

Citation Format: Kyle A. Edgar, Marie-Claire Wagle, Yibing Yan, Marcia Belvin, Lori Friedman, Jeffrey Wallin. Mechanisms of acquired resistance to the PI3K inhibitors in colorectal 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 4470. doi:10.1158/1538-7445.AM2013-4470
Abstract 4534: Mutant KRAS2 mRNA PET imaging in spontaneous transgenic lung cancers.
Authors: Wickstrom Eric , Chen Chang-Po , Paudyal Bishnuhari , Wampole Matthew E. , Zhang Kaijun , Wang Antican , Lu Bo , Thakur Mathew L.
Published: August 2014

Treatment of lung cancer with anti-EGFR antibodies or tyrosine kinase inhibitors (TKIs) fails when KRAS2 cancer gene is activated by mutation. Mutation of the KRAS2 12th codon from the wild type GGU that encodes glycine (G) to a mutant GAU that encodes aspartate (D) occurs in some lung cancers. For this reason, we hypothesize that determining KRAS2 mutation status in vivo by external mRNA PET imaging will enable physicians to decide on alternatives to EGFR-directed therapy. To test the hypothesis, we designed wild type and mutant KRAS2 hybridization agents based on peptide nucleic acid (PNA), due to its strong, specific basepairing, and resistance to nucleases and proteases. At the C-terminus, we included a cyclized tetrapeptide based on insulin-like growth factor 1 (IGF1) to enable IGF1R-mediated cellular uptake. We included an N-terminal N2S2 chelator in order to bind 64Cu. We then administered the 64Cu-labeled agents by tail vein into mutant KRAS2 transgenic mice that develop spontaneous lung tumors. We demonstrated sequence-specific positron emission tomography (PET) imaging and quantitation of mutant KRAS2 G12D mRNA in the mice. The wild type control agent, differing by one base in the middle of the 12th codon, showed neglible images in the same mice. Wobble base agents with a hypoxanthine to detect either G12D or G12V KRAS2 mutants are under investigation. Supported by NIH CA148565; IP owned by EW/MLT, licensed to MTTI.

Citation Format: Eric Wickstrom, Chang-Po Chen, Bishnuhari Paudyal, Matthew E. Wampole, Kaijun Zhang, Antican Wang, Bo Lu, Mathew L. Thakur. Mutant KRAS2 mRNA PET imaging in spontaneous transgenic lung cancers. [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 4534. doi:10.1158/1538-7445.AM2013-4534
Abstract 4570: Development of a proteolytically activatable EGFR Probody for cancer therapy.
Authors: Desnoyers Luc R. , Yang Annie , Liang Tony W. , Moore Stephen , Sagert Jason , Hostetter Daniel R. , Menendez Elizabeth , Han Fei , Krimm Michael , Wong Ken , Richardson Jennifer , West Jim W. , Liu Shouchun , Vasiljeva Olga , Lowman Henry B.
Published: August 2014

Antibodies directed to specific disease-related antigens have proven to be very successful therapeutics for a variety of disease indications. In spite of their high affinity and specificity for target antigen, target-mediated toxicity constitutes a major limitation for the development of antibodies to certain targets. We have addressed this type of on-target toxicity by developing a new class of targeting antibodies (Probody™ therapeutics) that remain in an inert, masked form until proteolytically activated at the site of disease. As a proof-of-concept for the construction of a Probody, we used cetuximab as a starting point. Cetuximab is an EGFR-targeted antibody approved for the treatment of colorectal and head-and-neck cancers that produces an on-target toxicity in the form of a skin rash that afflicts 88% of patients treated with the antibody. We engineered an EGFR Probody by incorporating an inhibitory masking peptide fused to the antibody light chain. Masking of the Probody is achieved through a linker that also incorporates a substrate that is cleaved by one or more proteases up-regulated in cancer. In vitro, EGFR binding and cell-based activities of the masked Probody were diminished compared to those of cetuximab, but treatment with exogenous target proteases activated the Probody and restored activity comparable to cetuximab. Using tumor xenograft models in mice, we demonstrated that the Probody remained masked in systemic circulation but was activated and accumulated in the tumor microenvironment. Tumor activation of the Probody translated to efficacy similar to that seen with cetuximab. Consistent with our results in mice, the Probody remained efficiently masked in non-human primates and did not cause skin toxicity such as that observed in animals treated with cetuximab. Together, these results demonstrate that antibody activity can be specifically targeted to diseased tissue by utilizing locally overexpressed proteases as activating agents, suggesting that a variety of antigens not previously amenable to an antibody therapeutic approach may be successfully addressed with Probodies.

Citation Format: Luc R. Desnoyers, Annie Yang, Tony W. Liang, Stephen Moore, Jason Sagert, Daniel R. Hostetter, Elizabeth Menendez, Fei Han, Michael Krimm, Ken Wong, Jennifer Richardson, Jim W. West, Shouchun Liu, Olga Vasiljeva, Henry B. Lowman. Development of a proteolytically activatable EGFR Probody for 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 4570. doi:10.1158/1538-7445.AM2013-4570
Abstract 4611: Multimodal activation of apoptosis by a hydrocarbon-stapled PUMA BH3 helix.
Authors: Edwards Amanda L. , Gavathiotis Evripidis , LaBelle James L. , Braun Craig R. , Opoku-Nsiah Kwadwo , Bird Gregory H. , Walensky Loren D.
Published: August 2014

The BH3 domain of BCL-2 family proteins is a critical interaction motif that mediates the crosstalk among anti- and pro-death members. We have applied hydrocarbon stapling to restore alpha-helical structure to synthetic BH3 domain peptides, recapitulating the architecture observed in the native context and/or when bound to their physiologic targets. By generating Stabilized Alpha-Helices of BCL-2 domains (SAHBs) modeled after the BID and BIM BH3 domains, we previously detected direct, quantifiable, and functional binding interactions with both anti- and pro-apoptotic BCL-2 family proteins. Like BIM BH3, PUMA BH3 is well known to broadly engage the variety of anti-apoptotic targets. However, the ability of PUMA BH3 to directly bind and activate BAX remains unresolved. Here, we report the synthesis of PUMA SAHB, a structurally-stabilized PUMA BH3 helix that, in addition to targeting the complete spectrum of anti-apoptotic proteins, directly binds to BAX. Structural analysis revealed that PUMA SAHB engages the α1/α6 trigger site of BAX in a manner analogous to the BIM BH3 helix. PUMA SAHB binding triggers functional BAX activation as reflected by dose-responsive and sequence-dependent BAX-mediated liposomal and mitochondrial release. Importantly, biotin-PUMA SAHB and Flag-PUMA protein pull-downs from cellular lysates confirm binding to the corresponding native multidomain anti- and pro-apoptotic targets. As PUMA has been implicated in driving apoptosis of a variety of neuronal cell types, we explored the therapeutic potential of PUMA SAHB activity in the context of neuroblastoma. Treatment of a variety of neuroblastoma cell lines, including SH SY5Y, Kelly and IMR5, induced dose-responsive caspase 3/7 activation and cell death. To confirm the on-target effects of PUMA SAHB, genetically modified cells lacking BAX and BAX were similarly treated, but no caspase 3/7 activation or viability effects were observed. Thus, we find that a stapled PUMA BH3 helix is a dual anti-apoptotic inhibitor and pro-apoptotic direct activator that may serve as an effective pharmacologic trigger of apoptosis in cancers of neural origin.

This work was supported by NIH grant R01 CA050239 to L.D.W. and an NSF Graduate Research Fellowship to A.L.E.

Citation Format: Amanda L. Edwards, Evripidis Gavathiotis, James L. LaBelle, Craig R. Braun, Kwadwo Opoku-Nsiah, Gregory H. Bird, Loren D. Walensky. Multimodal activation of apoptosis by a hydrocarbon-stapled PUMA BH3 helix. [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 4611. doi:10.1158/1538-7445.AM2013-4611
Abstract 4674: Pilot study of intrahepatic artery chemotherapy in combination with sorafenib in hepatocellular carcinoma patients.
Authors: Feun Lynn G. , Narayanan Govindarajan , Yrizarry Jose , Dinh Vy , Savaraj Niramol , O'Brien Christopher , Martin Paul
Published: August 2014

Background: Sorafenib and chemoembolization of the liver (TACE) have both produced increased survival in patients (pts) with hepatocellular carcinoma (HCC). However, not all HCC pts can tolerate or accept TACE due to portal vein thrombosis or risk of liver failure.Chemotherapy infused via intrahepatic artery infusion (IA) may potentially increase drug delivery to the tumor while reducing systemic toxicity. In this pilot trial, we combined IA cisplatin or IA carboplatin with oral sorafenib for pts with unresectable HCC and report our results herein.

Methods: Major inclusion criteria into this study include:unresectable HCC, Child's A or early Child's B (maximum 7 points on Child-Pugh scale), serum bilirubin <3 mg/dl, ECOG performance status 0-1.

Treatment plan:IA cisplatin 60 mg/m2 or carboplatin AUC =6 was infused percutaneously IA on day 1 and then every 6 weeks. Sorafenib 400 mg po bid was given on day 1 and continuously. MRI or CT abdomen was done every 6 weeks. Recist criteria was used to measure response.

Results: 11 pts were accrued onto the study.Median age was 65 (range 48-84), median number of cycles 2 (range 1-7). Of 10 evaluable pts, 6 had clinical benefit (4 partial responses for 2+, 3+, 8+ and 18 months, 2 minor responses). Two of the responding pts withdrew to have ablation therapy or liver transplant after their HCC was able to be downstaged and remain free of disease for 32+ and 36+ months. Toxicity was generally tolerable and no pt withdrew due to drug toxicity.

Conclusion: Preliminary results with IA cisplatin or IA carboplatin with sorafenib are encouraging and this combination may downstage some pts with unresectable HCC. Further studies with this regimen are indicated.

Supported by Bayer HealthCare and Onyx Pharmaceuticals, Inc.

Citation Format: Lynn G. Feun, Govindarajan Narayanan, Jose Yrizarry, Vy Dinh, Niramol Savaraj, Christopher O'Brien, Paul Martin. Pilot study of intrahepatic artery chemotherapy in combination with sorafenib in hepatocellular carcinoma patients. [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 4674. doi:10.1158/1538-7445.AM2013-4674
Abstract 4677: Non-prostate cancer death within 5 years after radical therapy in men with localized prostate cancer.
Authors: Kobayashi Takashi , Kamba Tomomi , Terada Naoki , Yamasaki Toshinari , Ikeda Itaru , Inokuchi Haruo , Norihisa Yoshiki , Mizowaki Takashi , Inoue Takahiro , Okubo Kazutoshi , Yoshimura Koji , Hiraoka Masahiro , Ogawa Osamu
Published: August 2014

Background: As AUA guideline for the Management of Clinically Localized Prostate Cancer: 2007 Update states “An assessment of the patient's life expectancy, overall health status, and tumor characteristics should be undertaken before any treatment decisions can be made," definitive therapy for localized prostate cancer is usually recommended for patients with 5-year life expectancy or more. In some cases, however, unexpected lethal disease is diagnosed and causes death within 5 years after prostate cancer treatment. It seems important to elucidate the characteristics of other causes of death during follow up period after definitive therapy for localized prostate cancer (PCa). Patients and Methods: We reviewed 664 consecutive patients who had received radical prostatectomy (RP, n = 107) or external beam radiotherapy (EBRT, n = 557) and been followed up over 5 years at our institute. Survival status was evaluated and cause of death was identified in dead cases. All cases underwent chest and abdominal computed tomography (CT) and bone scan that were negative for metastasis. Results: At latest follow up, 103 (15.5%) patients were dead of PCa (n = 48) and other causes (n = 55), respectively. Of the 55 patients who died of other causes, 26 did not survive 5 years after radical treatment for PCa. 54% of them died of other malignancies including lung (n = 7) and gastrointestinal (GI) tract (n = 3), lymphoma (n = 2), liver (n = 1), and pancreatic (n = 1) cancers. Non-cancer causes included cerebrovascular disease (n = 3), pulmonary disease (n = 3), ischemic heart disease (n = 2), and unknown (n = 3). Conclusions: Since all patients had negative chest CT for primary lung cancer, some patients with localized PCa seemed to have developed more lethal and rapid malignancy after treatment for PCa. Urologists may need to pay their attention that their patients undergo regular mass screening for common diseases even if they were under urological follow up. Pretreatment test for fecal occult blood may be useful to screen GI tract cancer before treatment initiation.

Citation Format: Takashi Kobayashi, Tomomi Kamba, Naoki Terada, Toshinari Yamasaki, Itaru Ikeda, Haruo Inokuchi, Yoshiki Norihisa, Takashi Mizowaki, Takahiro Inoue, Kazutoshi Okubo, Koji Yoshimura, Masahiro Hiraoka, Osamu Ogawa. Non-prostate cancer death within 5 years after radical therapy in men with localized prostate 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 4677. doi:10.1158/1538-7445.AM2013-4677
Abstract 4682: Phase II study of first line chemotherapy with tegafur/uracil and oral leucovorin in elderly patients with advanced or metastatic colorectal cancer.
Authors: Murata Kohei , Yamamoto Hirofumi , Fukunaga Mutsumi , Kato Takeshi , Ohnishi Tadashi , Uemura Yoshio , Ohta Hirofumi , Kimura Fumihiko , Ohue Masayuki , Nezu Riichiro , Sekimoto Mitsugu , Ikeda Masataka , Mizushima Tsunekazu , Doki Yuichiro , Mori Masaki ,   Clinical Study Group of Osaka University (CSGO), Colorectal Group
Published: August 2014

Objective: This phase II trial was performed to evaluate the efficacy and tolerability of tegafur/uracil (UFT) and oral leucovorin (LV) in elderly patients with advanced or metastatic colorectal cancer who had not received prior chemotherapy. Methods: Patients aged ≥70 years were eligible. UFT and LV were taken orally on days 1-28 of the cycle at doses of 300 mg/m2/day and 75 mg/body/day, respectively. Treatment was administered on an outpatient basis every 35 days and consisted of at least two cycles until disease progression.

Results: A total of 30 patients were enrolled in this study. The median age of the patients was 81.5 years (range: 74-88 years). The observed overall response rate was 17.9%. The estimated median overall survival time was 23.5 months. Two patients (7%) experienced toxicities with a worst grade of 3, and one patient (4%) experienced toxicities with a worst grade of 4. There were no treatment-related deaths. No patients experienced grade 3 or 4 hematological adverse events.

Conclusions: Although the response rate to UFT/LV was moderate, a favorable survival time and good tolerability were observed. UFT/LV can be one of the optional treatments for elderly colorectal cancer patients.

Citation Format: Kohei Murata, Hirofumi Yamamoto, Mutsumi Fukunaga, Takeshi Kato, Tadashi Ohnishi, Yoshio Uemura, Hirofumi Ohta, Fumihiko Kimura, Masayuki Ohue, Riichiro Nezu, Mitsugu Sekimoto, Masataka Ikeda, Tsunekazu Mizushima, Yuichiro Doki, Masaki Mori, Clinical Study Group of Osaka University (CSGO), Colorectal Group. Phase II study of first line chemotherapy with tegafur/uracil and oral leucovorin in elderly patients with advanced or metastatic colorectal 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 4682. doi:10.1158/1538-7445.AM2013-4682
Abstract 4711: Targeted inhibition of STAT3 dimerization by garcinol suppresses the growth of human hepatocellular carcinoma in vitro and in vivo.
Authors: Shanmugam Muthu K. , Chatterjee Snehajyoti , Peramaiyan Rajendran , Li Feng , Senapati Parijat , Wong Kwong Fai , Wong Kwong Fai , Kumar Alan Prem , Kumar Alan Prem , Kumar Alan Prem , Luk John M. , Luk John M. , Luk John M. , Hui Kam M. , Hui Kam M. , Sethi Gautam , Sethi Gautam , Kundu Tapas K.
Published: August 2014

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and the third cause of global cancer mortality. Activation of signal transducer and activator of transcription 3 (STAT3) is commonly encountered in tumor cells and is a critical mediator of oncogenic signaling in HCC and controls the expression of several genes involved in proliferation, survival, metastasis and angiogenesis. Current drug-targeted therapies, besides being expensive, are associated with serious side effects and morbidity. Thus, novel agents that can suppress STAT3 activation have potential for both prevention and treatment of HCC. In the present report, we investigated whether the potent HAT/KAT inhibitor, garcinol, (a polyisoprenylated benzophenone), could suppress STAT3 activation in HCC cells and nude mice model. We found that garcinol could inhibit constitutive STAT3 activation in a dose- and time-dependent manner both by inhibiting STAT3 phosphorylation and acetylation in HCC cells. When investigated for molecular mechanism(s), we found that garcinol interferes with the dimer formation of STAT3 thereby inhibits its nuclear localization. Computational modeling showed that garcinol could bind to the SH2 domain of STAT3 and suppresses its dimerization in vitro. To understand the cellular mechanism(s) of inhibition of STAT3 function by garcinol, we observed that upon inhibition of STAT3 dimerization by garcinol, STAT3 DNA binding ability gets repressed. The inhibition of STAT3 activation by garcinol led to the suppression of various gene products involved in proliferation, survival, and angiogenesis. Finally, when administered i.p., garcinol inhibited the growth of human HCC xenograft tumors in male athymic nu/nu mice. In conclusion, results from in vitro and in vivo studies suggest that garcinol exerts its anti-proliferative and pro-apoptotic effects through suppression of STAT3 signaling cascade in HCC by inhibiting its phosphorylation, acetylation and ultimately dimerization.

Citation Format: Muthu K. Shanmugam, Snehajyoti Chatterjee, Rajendran Peramaiyan, Feng Li, Parijat Senapati, Kwong Fai Wong, Kwong Fai Wong, Alan Prem Kumar, Alan Prem Kumar, Alan Prem Kumar, John M. Luk, John M. Luk, John M. Luk, Kam M. Hui, Kam M. Hui, Gautam Sethi, Gautam Sethi, Tapas K. Kundu. Targeted inhibition of STAT3 dimerization by garcinol suppresses the growth of human hepatocellular carcinoma 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 4711. doi:10.1158/1538-7445.AM2013-4711
Abstract 4718: Withaferin A, a potent and abundant component of Withania somnifera root extract, reduces myeloid-derived suppressor cell function.
Authors: Sinha Pratima , Ostrand-Rosenberg Suzanne
Published: August 2014

Myeloid cells play a crucial role in growth and metastasis of malignant tumors. Tumor infiltrating myeloid cells includes myeloid-derived suppressor cells (MDSC), tumor-associated macrophages (TAMS) and dendritic cells (DC). These cells infiltrate into tumor and suppress tumor immunity by their inherent immune suppressive activity which is enhanced by interactions with each other (cross-talk). Therapeutic strategies targeting manipulation of these cells are being developed. The root extract of the plant Withania somnifera (Ashwagandha) (WRE) has been reported to reduce tumor cell proliferation and angiogenesis. Whether the anti-tumor effects of WRE are due to direct effects on malignant cells, or whether WRE also impacts immune suppressive myeloid cells is unknown. We hypothesize that WRE or its constituents impact tumor infiltrating myeloid cells and thereby boost anti-tumor immunity. HPLC and mass spectrometry analysis revealed that Withaferin A (WA) is the most abundant constituent of Withania somnifera root powder (ChromaDex, Inc. Irvine, CA). In comparison with the other constituents, WA has the most potent effect on reducing suppressive functions of myeloid cells. A prominent effect of MDSC is their production of IL-10 which increases upon cross-talk with macrophages, thus polarizing immunity to a tumor promoting type-2 phenotype. WA reduces inherent and cross-talk induced IL-10 secretion from MDSC in a dose dependent manner. Macrophage secretion of IL-6 and TNFα cytokines that are characteristic of M1-type macrophages and that also increase MDSC accumulation and function, are also reduced by WA. Much of the T cell suppressive activity of MDSC is due to MDSC production of reactive oxygen species (ROS), and WA significantly reduces MDSC production of ROS. Thus adjunctive treatment with WA has the potential to concomitantly reduce myeloid cell mediated immune suppression, to polarize immunity towards a tumor-rejecting type I phenotype, and to facilitate the development of anti-tumor immunity.

Supported by NIH RO1CA115880, RO1CA84232, and American Cancer Society IRG-97-153-07

Citation Format: Pratima Sinha, Suzanne Ostrand-Rosenberg. Withaferin A, a potent and abundant component of Withania somnifera root extract, reduces myeloid-derived suppressor cell function. [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 4718. doi:10.1158/1538-7445.AM2013-4718
Abstract 4732: Depletion of Tregs from PBMCs by treatment with defucosylated anti-CCR4 mAb.
Authors: Kurose Koji , Eikawa Shingo , Mizote Yu , Ohue Yoshihiro , Isobe Midori , Uenaka Akiko , Oka Mikio , Nakayama Eiichi
Published: August 2014

Objectives: Tregs down-regulate immune responses against various antigens including tumor cells. Impairment of Tregs cause autoimmune responses. CC chemokine receptor 4 (CCR4) is expressed selectively on Foxp3+CD4 T-cells, so that the treatment of PBMCs with KM2760, anti-human CCR4 mAb with a defucosylated Fc region, could deplete CCR4+Tregs by ADCC. In this study, we analyzed depletion of Tregs phenotypically and functionally after treatment of PBMCs with KM2760.

Methods and main results: CCR4 expression was analyzed on CD4 T-cells purified from healthy donor PBMCs by FACS. CCR4 positivecells were 21% in CD4 T-cells, and 71% in Foxp3+cells. Depletion of CCR4 positive cells by treatment with KM2760 was examined. PBMCs were incubated with KM2760 (0, 0.01, 0.1, 1, 10 μg/ml) for 4 or 20 hours, and analyzed by FACS. Efficient depletion was observed with higher antibody concentration and longer incubation time. Then we examined inhibition of CFSE dilution of labeled CD4 or CD8 T cells stimulated with anti-CD3 mAb after culture with an equal number of CD4+25+127low Tregs and CD4-8- cells pretreated with or without KM2760. Proliferating responder cells were 68% in culture with CD4+25+127low Tregs pretreated with KM2760, whereas those cells with CD4+25+127low Tregs without pretreatment was 38% in CD4 T-cells, 77%and 40% in CD8 T-cells,respectively.

Conclusions: Efficient Treg depletion was observed in vitro by treatment of PBMCs with KM2760. In vivo treatment with the same mAb may induce tumor immune responses by Treg depletion.

Citation Format: Koji Kurose, Shingo Eikawa, Yu Mizote, Yoshihiro Ohue, Midori Isobe, Akiko Uenaka, Mikio Oka, Eiichi Nakayama. Depletion of Tregs from PBMCs by treatment with defucosylated anti-CCR4 mAb. [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 4732. doi:10.1158/1538-7445.AM2013-4732
Abstract 4745: Quantitative single-cell functional characterization of CD19-specific CAR+ T Cells for immunotherapy.
Authors: Varadarajan Navin , Liadi Ivan , Romain Gabrielle , Singh Harjeet , Cooper Laurence J.N.
Published: August 2014

Adoptive cell therapy (ACT) utilizing chimeric antigen receptor (CAR) T cells rendered specific for CD19 have demonstrated significant anti-tumor effects in patients with CD19+ chronic lymphocytic leukemia (CLL) that were considered untreatable, including the use of stem-cell transplantation. In spite of the clinical promise of ACT in achieving complete responses, their efficacy remains unpredictable and new approaches are needed to address a priori define the therapeutic potential of T-cell based therapies. In our current work, we characterize the in vitro functionality of CD19-specific (CD19RCD28) CAR+ T cells propagated using artificial antigen presenting cells expressing membrane bound IL-21, by employing a novel methodology single-cell nanowell screening that determines their cytotoxic ability and cytokine secretion capability at single-cell resolution.

We show that CAR+ T cells exert specific cytotoxicity against NALM6 cells (31 ± 8 %) when co-incubated at a 1:1 ratio in nanowell containers. Furthermore, single CAR+ T cells were capable of engaging and killing multiple targets; 17 ± 8% of T cells killed two target cells and 9 ± 3% killed three target cells within the 6 hour window of observation. In parallel, microengraving was used to determine the cytokine secretion profile of these same cells. Hierarchical clustering of the two functions indicated that interferon-gamma (IFNγ) secretion is not correlated to cytotoxicity or the ability of T cells to kill multiple target cells. Simultaneously, monitoring apoptosis on CAR+ T cells allowed us to quantify their activation-induced cell death (AICD). CAR+ T cells that secreted IFNγ upon target ligation did not undergo AICD whereas T cells that engaged in repeated killing showed an increased propensity to undergo AICD (p = 0.04).

Finally, automated time-lapse microscopy of hundreds of these effector target interactions allowed us to quantify the kinetics of effector mediated lysis and effector cell velocity profiles before and after lysis. These analyses demonstrate that individual CAR+ T cells demonstrate vastly different individual killing profiles, at least in vitro.

In summary, our SNS based methodology allows the deep functional characterization of clinical grade CAR+ T cells and can be used to determine in vitro functions of CAR+ T cells that correlate with clinical efficacy.

Citation Format: Navin Varadarajan, Ivan Liadi, Gabrielle Romain, Harjeet Singh, Laurence J.N. Cooper. Quantitative single-cell functional characterization of CD19-specific CAR+ T Cells for immunotherapy. [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 4745. doi:10.1158/1538-7445.AM2013-4745