bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 460: The role of Cdc42 in cancer cell perineural invasion
Authors: Chernichenko Natalya , Omelchenko Tatiana , Deborde Sylvie , Bakst Richard , He Shuangba , Chen Chun-Hao , Hall Alan , Wong Richard J.
Published: August 2012

Perineural invasion (PNI) is the ability of cancer cells to invade in, around, and along nerves. PNI is widely recognized as an adverse pathologic feature of many malignancies. Glial-derived neurotrophic factor (GDNF) is secreted by nerve and glial cells, and is important in neural development. We previously showed that GDNF activation of the RET/GFR-alpha tyrosine kinase in cancer cells induces chemotaxis towards nerves and PNI. Cdc42 is a member of the Rho family of G proteins and a key regulator of cell polarity. We demonstrate here that GDNF-mediated activation of RET/GFR-alpha leads to activation of Cdc42 as a mediator of cancer cell chemotaxis and PNI. Exposure of MiaPaCa2 cells to GDNF or dorsal root ganglia (DRG) activates Cdc42. SiRNA inhibition of Cdc42 impairs MiaPaCa2 chemotaxis towards GDNF or DRG in Boyden chamber assays. SiRNA inhibition of RET decreases Cdc42 activity under GDNF stimulation and MiaPaCa2 chemotaxis. To study dynamic interactions between nerves and cancer cells in vitro, we used a DRG and cancer cell co-culture model of PNI in matrigel. Analysis of individual cell trajectories reveals that cell velocity remains unchanged, while directional migration is disrupted with shRNA inhibition of Cdc42. The disruption of directional response impairs the ability of shRNA Cdc42 MiaPaCa2 cells to migrate along neurites, as visualized by time lapse microscopy and measured by area of PNI. MRI imaging of live mice with sciatic nerves injected with shRNA Cdc42 MiaPaCa2 cells reveals significantly diminished length and volume of PNI as compared with injected shRNA control cancer cells when matched for equivalent overall tumor volume. Murine sciatic nerves injected with shRNA Cdc42 MiaPaCa2 cells exhibited preserved hindlimb and hind paw neurologic function, and diminished perineural invasion by histopathologic examination as compared with shRNA control MiaPaCa2. To identify potential activators of Cdc42 in this model, we performed a comprehensive siRNA screen of guanine nucleotide exchange factors (GEFs), identifying specific GEFs which are necessary for MiaPaCa2 migration towards DRG in Boyden chambers. We identified and validated six GEFs (FLJ10521, DOCK9, ARHGEF7, RASGRF1, ARHGEF5, PLEKHG1) that impair chemotaxis without affecting proliferation. These findings provide novel insights into the molecular mechanisms underlying cancer cell perineural invasion. The RET-GEF-Cdc42 axis may be an attractive target for novel therapies that may potentially interrupt perineural invasion.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 460. doi:1538-7445.AM2012-460
Abstract 4603: Optically-guided surgical approach using fluorescence visualization can significantly improve locoregional recurrence and overall survival for early stage oral cancer
Authors: Poh Catherine F.Y. , Anderson Donald W. , Durham J. Scott , Berean Kenneth W. , Williams P. Michele , MacAulay Calum E. , Rosin MIriam P.
Published: August 2012

Recurrence following excision of high-grade dysplasia (HGD, severe dysplasia and carcinoma in situ) or squamous cell carcinoma (SCC) implies that the presence of subclinical changes at the margins is not apparent at surgery, resulting in incomplete excision. Fluorescence visualization (FV) has shown its value in identifying clinically not apparent high-risk oral lesions. The objective of this study is to assess the effectiveness of FV-guided surgery in reducing the loco-regional recurrence and improving overall survival. Method: From September 1st 2004 to August 31, 2009, 264 consecutive patients diagnosed with T1/T2 SCC or HGD were registered at the BC Cancer Agency and treated with surgical excision with intent to cure. Among these, 246 patients (93%; SCC, 132; HGD, 114) had a follow-up period of at least 6 months. Among these patients, 149 had the surgery done under FV guidance (FV group) while the other 97 were treated with conventional surgery procedure (control group). The outcome measurements include pathology-proven local recurrence to severe dysplasia or higher requiring another surgical procedure, regional failure, and death at follow-up. Time to outcome curve was estimated by the Kaplan-Meier method. Results: There is no significant difference between FV and control groups in age, smoking habit, lesion anatomical site, diagnosis, tumor size, and previous cancer history. There were more females in the FV group (51% vs. 33%, P = 0.006). With an average of 40 months follow-up, the FV group shows significantly lower local recurrence (7% vs. 38%), regional failure (6% vs. 23%), and death (5% vs. 20%), and significant longer time to recurrence (P < 0.0001), regional failure (P = 0.0007), and death of disease (P = 0.004). Conclusion: Although they are retrospectively collected from a single centre, the data have strongly demonstrated that the use of FV for surgical margin decision can significantly improve outcomes of the early stage oral cancer. An on-going 5-year multi-centre phase III randomized surgical trial (the COOLS trial), funded by Terry Fox Research Institute, has started to recruit patients and the data will be used to validate the results of this study. (Supported by R01 DE17013 from the National Institute of Dental and Craniofacial Research, CCSRI-20336 from Canadian Cancer Society Research Institute, and TFRI-2009-24 from Terry Fox Research Institute. CFP is currently supported by a Scholar Award from the Michael Smith Foundation for Health Research).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4603. doi:1538-7445.AM2012-4603
Abstract 4613: A role of integrin-associated CD151 in human ovarian cancer
Authors: Jin Hongyan , Hoff John T. , Deng Xinyu , Drapkin Ronny I. , Yang Xiuwei H.
Published: August 2012

Despite being implicated in multiple types of human epithelia-origin cancer, laminin-binding (LB) integrins (e.g., α3α1 and α6β4) have rarely been investigated for their roles in human ovarian tumors. More recently, we and others have shown that CD151, a member of the tetraspanin family, not only forms tight surface complexes with these adhesion receptors, but also mediates the malignancy of human carcinomas largely in a LB integrin-dependent manner. Here we report our studies on the role of CD151 in human ovarian cancer. Initially, we stained human ovarian tumor tissue microarrays with CD151-specific antibody. Our data showed that the majority of ovarian tumors exhibited a reduced expression of CD151 protein, compared to the fallopian tube, implying a putative suppressing role of this tetraspanin in ovarian cancer. With this hint we next evaluated the impact of CD151 ablation on the behaviors and proliferation of cultured human ovarian cancer cells. While CD151 removal had a minimal impact on cell motility or invasion, it markedly enhanced cell proliferation. Also, CD151-deficeint cells appeared to have reduced cell-cell contacts, suggestive of epithelial-mesenchymal transition (EMT)-like changes. However, there was no dramatic difference in the surface expression of E-cadherin protein, according to our FACS analyses. To substantiate these observations, we also injected ovarian cancer cells with or without CD151 knockdown into immuno-deficient nude mice subcutaneously or orthotopically (intra-peritoneal). Results from these ex vivo analyses showed that disruption of CD151 accelerated ovarian tumor growth and production of ascites fluid in mice, further supporting a role of CD151 in ovarian tumor progression. While tumors in the control group had homogenous epithelial-like appearance, those derived from CD151-ablated cells exhibited mucin-related vacuoles and diverse morphologies. Our subsequent DNA array and protein analyses indicated that there was indeed a marked increase in the expression of a mucin family member in CD151-deficient tumor cells. Collectively, these results provide strong evidence that CD151 suppresses human ovarian cancer progression by regulating tumor cell proliferation and extracellular microenvironment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4613. doi:1538-7445.AM2012-4613
Abstract 462: Fer protein-tyrosine kinase promotes lung tumor progression and metastases
Authors: Ahn Joseph , Truesdell Peter , Boag Alexander H. , Craig Andrew W.B.
Published: August 2012

FER is a ubiquitously expressed non-receptor protein-tyrosine kinase implicated in regulating cell-cell and cell-matrix interactions, cell growth and migration. Although no activating mutations have been described for FER, recent studies implicate FER as a positive regulator of cancer progression in the prostate and liver. FER is composed of Fer/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR), extended F-BAR (FX), SH2, and kinase domains. FER oligomerization and membrane targeting is conferred by F-BAR/FX domains, while the SH2 domain allows FER recruitment to tyrosine phosphorylated ligands, including activated Epidermal growth factor (EGF) receptor (EGFR). Since gene amplification or gain-of-function mutations in EGFR are frequently observed in non-small cell lung carcinoma (NSCLC), we tested whether FER participates in EGFR-driven NSCLC migration, progression, and metastases. Here we show that FER is expressed in normal lung epithelial cells and at variable levels in NSCLC cell lines. In a panel of NSCLC homogenates we observed 5 out of 12 cases of increased FER expression over normal adjacent tissue. Furthermore, in a tissue microarray containing a range of lung cancer histological types we observed high FER expression in the NSCLC adenocarcinoma subtype and in SCLC. In human lung epithelial carcinoma H1299 cells that overexpress wild-type EGFR, we detected rapid EGF-induced activation of FER, and subsequent phosphorylation of several putative FER substrates including STAT3, Cortactin, and Vav2. To probe the function of FER, we used a lentiviral shRNA system to achieve stable knock-down (KD) of FER in H1299 cells. Compared to control cells, FER KD cells displayed a significant reduction in EGF-induced migration in scrape wound assays. FER KD cells were also defective in frequency and velocity of invasion under an agarose drop containing EGF. Of the putative FER substrates tested, we observed a significant decrease in Vav2 phosphorylation in Fer KD cells. Since Vav2 promotes Rac activation, we compared localization of active, GTP-bound Rac1 in control and FER KD cells treated with EGF. Interestingly, FER promotes localization of GTP-Rac1 to the leading edge of these cells. To test the role of FER in NSCLC progression and metastasis, H1299 vector control and FER KD cells were grown as subcutaneous tumors in immune compromised mice (Rag2-/-γc-/-) for four weeks. Although we observed no significant differences in tumor mass for FER KD cells compared to control, the potential role of FER in metastasis is currently under investigation. Together, this study may implicate FER as a viable new therapeutic target for treatment of EGFR-driven metastasis of lung cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 462. doi:1538-7445.AM2012-462
Abstract 4630: Integrin αVα6 is expressed on multiple solid tumors and is a potential therapeutic target for auristatin-based antibody-drug conjugates
Authors: Ryan Maureen C. , Kostner Heather , Meyer E. Bradley , Sussman Django , Torrey Lindsay , Anderson Martha , Lyon Robert , Meyer Dave , Jonas Mechthild , van Epps Heather , Benjamin Dennis
Published: August 2012

Antibody-drug conjugates (ADCs) are designed to deliver a targeted, potent payload directly to the tumor cell. This targeted cytoxicity can greatly augment the antitumor activity of a naked antibody and reduce the off target toxicity of free drug. Cancers of the head and neck, pancreas, bladder, lung, ovary and breast show frequent expression of integrin α6, suggesting they may be amenable to therapeutic targeting with an anti-integrin β6 ADC. We identified a therapeutic antibody that is optimal for drug delivery by screening a large panel of anti-integrin β6 ADCs on antigen-positive tumor lines. Monoclonal antibody 15H3 showed consistent potency as an ADC on solid tumor cell lines representative of the target indications. The ADC activity of 15H3 was detectable using either monomethyl auristatin E with a protease-cleavable linker (vcMMAE) or monomethyl auristatin F (mcMMAF), which requires antibody degradation. 15H3 was selected as the lead and humanized for in vivo testing. Humanized 15H3 (h15H3) retained all the properties of the mouse parental antibody, including the ability to cross-bind multiple species (human, monkey, rat, and mouse integrin α6). In vitro analysis showed that h15H3-vcMMAE and h15H3-mcMMAF internalize within a few hours and result in antigen-specific cytotoxicity of carcinoma lines. In vivo studies of integrin-α6 positive xenografts demonstrated antitumor activity. Our data suggest that integrin β6 is a promising potential ADC target for antigen-positive solid tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4630. doi:1538-7445.AM2012-4630
Abstract 4634: Engineered cysteine drug conjugates show potency and improved safety
Authors: Sussman Django , Torrey Lindsay , Westendorf Lori , Miyamoto Jaime B. , Meyer David W. , Lyon Robert , Duniho Steve , Hayes Dawn C. , Anderson Martha , Benjamin Dennis R.
Published: August 2012

Antibody-drug conjugates (ADCs) are emerging as an important therapeutic option for new oncology drugs. In this abstract we present data on engineered cysteine antibodies (EC-mAbs) with an additional unpaired cysteine residue introduced at position 239 on each heavy chain to facilitate more uniform drug loading while interfering with Fc gamma receptor function. With the EC-mAb, we have identified a site that blocks Fc gamma receptor III (CD16) binding and has a number of additional desirable biochemical and biophysical properties. We demonstrate that EC-mAb-drug conjugates, with two drugs per antibody, retain antitumor activity in mouse xenograft experiments and are well tolerated in non-human primates. These data suggest that engineered cysteine antibodies are a potential alternative to conjugation of native cysteines for future ADCs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4634. doi:1538-7445.AM2012-4634
Abstract 4635: Development of monoclonal antibodies against the GPCR CXCR4 and the ion channel Hv1 using virus-like particles
Authors: Rucker Joseph , Payne Riley , Mabila Manu , Patel Sonal , Hall Moniquetta , Willis Sharon , Doranz Benjamin J.
Published: August 2012

Antibodies against cancer targets are routinely used for facilitating early detection of disease, for diagnosing and predicting disease severity, and for guiding and monitoring therapeutic treatment. Ten of the 11 FDA-approved cancer therapeutic monoclonal antibodies (MAbs) target membrane proteins, and membrane proteins comprise >30% of existing cancer biomarkers. MAbs that recognize conformation-dependent epitopes on membrane proteins are usually the most valuable type of antibody because they can bind to critical structures of the receptor that can be exploited for its detection or inhibition. However, the development of conformational MAbs against membrane proteins is especially challenging because these proteins require a lipid environment to maintain their native structure, can be difficult to purify, and are usually poorly represented by peptide immunogens. To overcome these limitations, we have developed a novel technology, the ‘Lipoparticle’, which incorporates high concentrations (10-100 fold greater than cells or membrane preparations) of properly folded and oriented membrane proteins onto the surface of virus-like particles (VLPs). Here, we have used Lipoparticles as immunogens to generate monoclonal antibodies against two important membrane protein targets: the GPCR CXCR4, associated with tumor metastasis, and the ion channel Hv1, implicated in B cell activation. Lipoparticles containing each target were used to immunize mice, eliciting sera with strong and specific immunoreactivity. Screening of >900 hybridomas using Lipoparticles resulted in the identification of CXCR4-specific MAbs validated for reactivity against surface expressed antigen by immunofluorescence. Epitope mapping and functional studies demonstrate that each MAb binds a conformational-dependent epitope on the second extracellular loop capable of inhibiting ligand-dependent signaling in vitro. Similar studies are currently ongoing to isolate MAbs against the Hv1 ion channel, with strong sera responses against Hv1 detected in immunized mice. Our data highlight the utility of Lipoparticles for generating MAbs and screening hybridomas to isolate antibodies against challenging cancer membrane protein targets.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4635. doi:1538-7445.AM2012-4635
Abstract 4638: Suppression of tumorigenicity by targeting anterior gradient-2 (AGR2) through multiple epitopes binding of the monoclonal antibody 18A4
Authors: Li Dawei , Wu Zhenghua , Zhu Qi
Published: August 2012

Monoclonal antibody is playing an increasingly important role in the treatment of cancer. Specifically, secreted proteins and cell surface receptors that promote tumor growth and metastasis are emerging as important targets for antibody-based anti-cancer therapy. Recent discoveries reveal that Anterior Gradient-2(AGR2), a secreted signal molecule, is over-expressed in a series of adenocarcinoma cancer types and promotes cell growth and migration in vitro and in vivo that can be blocked by polyclonal antibodies. Thus, AGR2 might be a promising target for antibody-based anti-cancer therapy. Here, with a newly developed monoclonal antibody, we report evidence supporting the feasibility of antibody-based therapy against AGR2 as the target. Our study established a unique AGR2-blocking monoclonal antibody 18A4 which requires the co-existence of separate AGR2 epitopes and regions for efficient antibody-antigen binding. This monoclonal antibody against AGR2 can specifically inhibit the AGR2-induced growth and migration of several lines of tumor cells in vitro. Xenograft experiments showed marked repression of tumor growth by 18A4 treatment in nude mice. These results further validated the anti-AGR2 strategy in cancer therapy. In addition, our results suggest, at least in some cases, the necessity of using large regions or whole protein, not short peptides, in developing therapeutic antibodies against certain antigens.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4638. doi:1538-7445.AM2012-4638
Abstract 4646: Toward engineering of novel anticancer bio-agents
Authors: SHAH GOPITKUMAR R. , Wesener Shane , Cheng Yi-Qiang
Published: August 2012

Our research goal is to engineer the next generation of novel anticancer bio-agents that will target and eradicate a broad spectrum of solid tumors including breast cancer. Our research design was based on the hypotheses that (1) the next generation of bio-agents could be engineered to continuously produce doses of an anticancer drug (e.g. FK228 or tryprostatin) at the sites of bacterial spore germination and vegetative growth in the hypoxic/necrotic regions of solid tumors, and (2) the synergistic actions of bacterial consumption of tumor mass and anticancer drug activities could destroy many kinds of solid tumors regardless of cancer genotype, without the need for prodrug injections and could overcome the common side effects and less efficacy of current cancer therapies. Our research focuses on biosynthetic pathways of FK228 and tryprostatins. FK228 is an anticancer drug approved by FDA in 2009 in the name of Istodax® and acts as a potent histone deacetylase (HDAC) inhibitor. Tryprostatins are natural products of Aspergillus fumigatus BM939, with reported bioactivity as potent inhibitor of mammalian cell cycle and as an inhibitor of breast cancer resistance protein (BCRP). The specific aims of the project are (1) reconstitution of FK228 or tryprostatin biosynthetic pathways; (2) integration and expression of FK228 or tryprostatin biosynthetic genes in anaerobic/facultative anaerobic bacteria; (3) evaluation of antitumor efficacy of recombinant bacteria using animal model. In pilot experiments, we have succeeded in reconstituting the FK228 and tryprostatin biosynthetic gene cluster individually into E. coli using plasmid expression system and the engineered E. coli strains were able to express the genes and produced FK228 or tryprostatin under both aerobic and anaerobic fermentation conditions. Integration of the gene clusters into anaerobic/facultative anaerobic bacterial genome is in process. Successful integration and stable expression of anticancer compounds by bacteria would generate a new class of anticancer bio-agents that can target and eradicate solid tumors irrespective of genotypes. Project scheme:

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4646. doi:1538-7445.AM2012-4646
Abstract 465: NEDD9 adaptor protein in glioblastoma cell migration and invasion
Authors: Zhong Jessie , Turner Kylie , O'Neill Geraldine M. ,   Discipline of Paediatrics and Child Health of Sydney University
Published: August 2012

Gliomas are characterised by extensive infiltration of the surrounding normal brain parenchyma using neurite-like extensions. Significantly, glioma locomotion is highly similar to invading neural precursor cells. Earlier work has shown that the adhesion adaptor protein NEDD9 promotes active membrane extension in non-neuronal cells, is implicated in neuritogenesis and is enriched in neural precursor cells. Critically, NEDD9 depletion significantly inhibits neural precursor cell migration in vivo and reduces U87MG glioblastoma cell invasion through a brain homogenate. Therefore, we have sought to determine NEDD9 function in glioma cell morphology and 3D cell migration. We first categorized glioblastoma cell lines into mesenchymal, pro-neural and proliferative subtypes via expression of YKL-40 and DLL3. We then used representatives from each that expressed NEDD9 to investigate glioma cell morphology and behaviour. In two-dimensional (2D) culture, NEDD9 depletion caused the loss of microtubule-rich membrane protrusions that were observed in proliferative and mesenchymal cell types. Further, NEDD9 depletion caused increased cell size in 2D and reduced migration speed and persistence in 3D culture in all lines examined. Quantification of morphologies in 3D culture following either NEDD9 depletion or Rac GTPase inhibition revealed no detectable change in rounded versus elongated morphologies. To determine which partner molecules are required for NEDD9 maintenance of cell speed, we screened interacting partners in invading glioblastoma cells. We identified and confirmed the adaptor protein CIN85 as a novel interacting partner of the NEDD9 Src Homology 3 (SH3) domain. Immunofluorescence staining revealed that CIN85 localised to structures involved in cell migration. Our study has revealed that NEDD9 depletion can inhibit migration of lines derived from mesenchymal, pro-neural and proliferative glioblastoma cell types. These findings highlight NEDD9 signalling in glioma as potentially playing a role in the migration of different glioblastoma tumour subtypes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 465. doi:1538-7445.AM2012-465
Abstract 4650: Inhibiting oncogenic signaling by sorafenib activates PUMA via GSK3β and NF-κB to suppress tumor cell growth
Authors: Dudgeon Crissy , Buchanan Monica E. , Peng Rui , Wang Peng , Sebastiani Andrea , Yu Jian , Zhang Lin
Published: August 2012

Aberrant Ras/Raf/MEK/ERK signaling is one of the most prevalent oncogenic alterations and confers survival advantage to tumor cells. Inhibition of this pathway can effectively suppress tumor cell growth. For example, sorafenib, a multi-kinase inhibitor targeting B-Raf and other oncogenic kinases, has been used clinically for treating advanced liver and kidney tumors, and also has shown efficacy against other malignancies. However, how inhibition of oncogenic signaling by sorafenib and other drugs suppresses tumor cell growth remains unclear. In this study, we found that sorafenib kills cancer cells by activating PUMA, a p53 target and a BH3-only Bcl-2 family protein. Sorafenib treatment induces PUMA in a variety of cancer cells irrespective of their p53 status. Surprisingly, the induction of PUMA by sorafenib is mediated by IαB-independent activation of NF-κB, which directly binds to the PUMA promoter to activate its transcription. NF-κB activation by sorafenib requires GSK3β activation, subsequent to ERK inhibition. Deficiency in PUMA abrogates sorafenib-induced apoptosis and caspase activation, and renders sorafenib resistance in colony formation and xenograft tumor assays. Furthermore, the chemosensitization effect of sorafenib is dependent on PUMA, and involves concurrent PUMA induction through different pathways. Enhanced PUMA expression or BH3 mimetics potentiate the anticancer effects of sorafenib, and restore sorafenib sensitivity in resistant cells. We also developed a high-throughput screening assay for identifying anticancer compounds based on the activation of PUMA by sorafenib. Together, these results demonstrate a key role of PUMA-dependent apoptosis in therapeutic inhibition of Ras/Raf/MEK/ERK signaling. They provide a rationale for manipulating the apoptotic machinery to improve sensitivity and overcome resistance to the therapies that target oncogenic kinase signaling.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4650. doi:1538-7445.AM2012-4650
Abstract 4651: Sorafenib and its derivatives induce apoptosis and autophagy in canine mammary tumor cells
Authors: Lee Yi-Yen , Chen Kuen-Feng , Chou Chung-Hsi , Lin Chen-Si
Published: August 2012

Canine mammary tumors (CMTs) are the most common neoplasms in intact female dogs. Radiation treatment, chemotherapy and surgical removal are conventionally applied for treating CMTs; however, the efficacy is limited and a study showed around 48% of dogs died or were euthanized within one year due to recurrence or metastasis of the tumor after surgical removal. Recently, Sorafenib (Nexavar®), a multiple kinase inhibitor, has shown survival benefits in human patients with human hepatocellualr carcinoma (HCC) and is the first clinically approved drug for treating HCC. Except HCC, Sorafenib also showed certain effect on the treatment of human renal cell carcinoma, thyroid cancer, and, also, breast cancer. In this study, derivatives of sorafenib (SC-1 & CY-13) made by depleting the kinase inhibitory activities were investigated for their therapeutic efficacy in canine mammary tumors. Three CMT cell lines including CMT-1, CF41.mg and MPG were treated with sorafenib, SC-1 and CY-13. The results showed SC-1 owned stronger potency than other two compounds in suppressing tumor cell growth. Lower LD50 and earlier onset of cell death were found in SC-1-treated groups. The SC-1-mediated CMT cell death was through both apoptosis and autophagy since DNA fragmentation and autophagosome were observed in treated cells. These sorafenib derivatives were previously reported as novel inhibitors for STAT3. Here we also found that SC-1 down-regulated phospho-STAT3 (p-STAT3) at tyrosine 705 in all tested CMT cells. Expression of STAT3-driven genes, including cylcin D1 and survivin, was also repressed by SC-1. Although the in vivo anti-tumor efficacy of SC-1 will be soon evaluated, our preliminary results have provided a lead for drug development of canine mammary tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4651. doi:1538-7445.AM2012-4651
Abstract 4653: 5F203-induced reactive oxidative species, DNA damage and apoptosis involves aryl hydrocarbon activation in ovarian cancer cells
Authors: Callero Mariana A. , Luzzani Gabriela , Bradshaw Tracey D. , Pérez Andrea I. Loaiza
Published: August 2012

Ovarian cancer is the 2nd cause of death by gynecological malignancies after breast cancer. These tumors have poor prognoses because they are detected at advanced stages of disease and show acquired or intrinsic resistance to therapy.Benzothiazoles, agents with preclinical antitumor activity against ovarian and breast cancer models, belong to a novel mechanistic class which bind to the arylhydrocarbon receptor (AhR). Phortress the lysylamide prodrug of 2-(4-amino-3-methylphenyl)-5- fluorobenzothiazole (5F203) is currently under phase I clinical evaluation. We previously demonstrated that these compounds activate AhR signaling in sensitive breast cancer cells, the goal of this project was to evaluate the role of AhR in 5F203 activity in two human ovarian cell lines: IGROV-1 sensitive to 5F203 and SKOV-3 resistant to this agent. We observed by MTS assay IGROV-1 cell growth inhibition of 31.9±12.2% by 0.01 μM 5F203 and almost total growth inhibition (91.9±3.4%) after treatment of IGROV-1 cells with 1 μM 5F203. Pre-incubation with the AhR inhibitor ΔNF (1 μM), partially blocked the effect of 5F203 (inhibition of 21.6±4.7% and 27.7±5.3% respectively). In contrast, 5F203 failed to inhibit growth of SKOV-3 cells.5F203 (1 μM) induced AhR translocation from cytosolic to nuclear fractions evaluated by Western blot and confocal microscopy in IGROV-1 cells only. Moreover, activation of CYP1A1 related promoter sequences and EROD activity further indicated activation of AhR signaling. 5F203 treatment for 1h increased ROS and induced NFκB nuclear translocation only in IGROV-1 cells, effects which were attenuated by pre-treatment with ΔNF. Detection of γH2AX revealed DNA double strand breaks 2h post 5F203 treatment of IGROV-1 cells only. After 24h exposure, 5F203 resulted in increased IGROV-1 cell apoptosis, evaluated by DAPI staining of apoptotic bodies, increased pP53, caspase 3 and PARP cleavage. We also observed G1 cell cycle arrest, increased P21 expression and decreased cyclin D1 expression induced by 5F 203. We conclude that the antitumor/pro-apoptotic effects of 5F203 observed in IGROV-1 cells is mediated by AhR which may be a new molecular target in the treatment of ovarian tumors and that this agent may represent a potential novel treatment for ovarian cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4653. doi:1538-7445.AM2012-4653
Abstract 4663: The cyclooxygenase-2 inhibitor, celecoxib, suppresses proliferation and induces apoptosis in endometrial cancer cells
Authors: Schuler Kevin M. , Suri Anuj , Zhou Chunxiao , Bae-Jump Victoria A. , Gehrig Paola A.
Published: August 2012

INTRODUCTION: Obesity is associated with increased risk and worse outcomes for women with endometrial cancer. Inflammation may play a critical role in obesity-driven cancers; thus, the anti-inflammatory effects of COX-2 inhibitors may be a novel treatment strategy that is particularly beneficial in endometrial cancer patients. As a result, our objective was to evaluate the effect of COX-2 inhibitors on proliferation and apoptosis in endometrial cancer cell lines. METHODS: Two endometrial cancer cell lines (ECC-1 and Ishikawa) were used in these studies. Cell proliferation was assessed by MTT assay after exposure to the COX-2 inhibitor, celecoxib. Cell cycle progression was evaluated by flow cytometry. Apoptosis was assessed by ELISA for caspase-3. hTERT mRNA expression was assessed by real-time PCR. Western immunoblotting was performed to determine the effect of celecoxib on COX-2 expression in the endometrial cancer cell lines. RESULTS: Celecoxib significantly inhibited proliferation in a dose-dependent manner in both endometrial cancer cell lines (IC50 5-10 μM for ECC-1, 10-20 μM for Ishikawa; p<0.005 for each). Treatment with celecoxib resulted in G1 cell cycle arrest, induction of apoptosis and inhibition of hTERT mRNA expression. Western immunoblot analysis demonstrated that celecoxib treatment suppresses COX-2 expression in both endometrial cancer cell lines. CONCLUSION: Celecoxib potently inhibits cell growth via G1 arrest, decreases telomerase activity and increases apoptotic cell death in endometrial cancer cells. This work suggests that celecoxib may be a novel chemotherapeutic agent for endometrial cancer prevention and treatment, especially given the interplay between obesity, inflammation and cancer risk.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4663. doi:1538-7445.AM2012-4663
Abstract 4673A: DAPK-mediated phosphorylation of HSF1 enhances apoptosis level upon TNF in colorectal carcinoma cells
Authors: Benderska Natalya , Rau Tilman , Ivanovska Jelena , Chakilam Saritha , Gandesiri Muktheshwar , Ziesche Elisabeth , Agaimy Abbas , Schulze-Luehrmann Jan , Distel Luitpold , Fischer Thomas , Sticht Heinrich , Schneider-Stock Regine
Published: August 2012

Objectives: Tumor necrosis factor ≤ (TNF) is an inflammatory cytokine, which is released upon different stimuli, including irradiation. Recently it has been shown, that the Death-associated protein kinase (DAPK) mediates TNF-induced apoptosis in colon cancer cells [1]. Here, we aimed to identify new DAPK binding partners and to characterize the functional role of novel protein interaction complexes during TNF-induced apoptosis in colon cancer cells. Methods/Results: HCT116 colorectal cancer cells were cultured for 6 to 48 hours in either normal or TNF-conditioned medium. For phosphopeptide microarray (PPM) whole cell lysates were incubated on peptide platforms with radioactive-labeled P33. Apoptosis was detected by Annexin V staining and caspase 3 cleavage in Western Blotting. PPM analysis revealed heat shock transcription factor 1 (HSF1) as a new potential substrate of DAPK phosphorylation under TNF-stimulation. DAPK Co-IP, co-immunofluorescence (Co-IF), and mass spectrometry showed that DAPK interacts with HSF1 after TNF-treatment. Phosphorylation on serine residues of HSF1 is necessary for HSF1 nuclear translocation and the initiation of transcription of its target genes. Among them, Ser 230 is part of a consensus phosphorylation motif for DAPK. Maximal level of pHSF1Ser230 was observed at the time point where massive apoptosis was induced. Co-IF microscopy confirmed enrichment of pHSF1Ser230 in the nucleus already after 24 hours of TNF-stimulation. EMSA and Chromatin-IP revealed that pHSF1Ser230 binds to the heat shock response element in the DAPK promoter region and enhances its transcriptional activity. Exogenous over-expression of HSF1 protein led to a significant increase in mRNA DAPK levels and consequently to an enforcement of apoptosis. As expected, DAPK knockdown cells did not show any variation in pHSF1Ser230 level, supporting again that DAPK is an important mediator of the TNF-driven signaling pathway in colorectal cancer cells. The significance of the DAPK/pHSF1Ser230 interaction for response prediction was evaluated by immunohistochemical staining on tissue microarrays of colorectal cancer before and after radiotherapy. Conclusion: Our data show a novel functional interaction between HSF1 and DAPK under TNF-stress and highlight a positive feedback mechanism in DAPK-regulation. These results help to understand cell death pathways in response to radiotherapy. [1].Bajbouj K.et al., Am J Pathol. (2009)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4673A. doi:1538-7445.AM2012-4673A
Abstract 4677: Slug is a potential target to restore transforming growth factor-β mediated apoptosis in lung cancer cells
Authors: Reka Ajaya Kumar , Goswami Moloy , Kurapati Himabindu , Kaza Viritha , Chen Jun , Keshamouni Venkateshwar G
Published: August 2012

Transforming growth factor-β (TGFβ) inhibits carcinogenesis at early stages by inhibiting cell growth and inducing apoptosis. Cancer cells acquire resistance to TGFβ induced apoptosis and divert the TGF-β signaling to stimulate tumor promoting processes such as Epithelial-Mesenchymal-Transition (EMT), immunosuppression and angiogenesis. Hence, the identification of molecular targets/ pathways enabling cancer cells to overcome TGFβ-mediated apoptosis is critical for exploiting tumor suppressive functions of TGFβ for therapy. We have identified slug (snai2), a zinc-finger transcription factor, as a critical regulator of TGF-β mediated apoptosis in lung cancer cells. In EMT models of lung cancer slug is up-regulated by several fold upon TGFβ treatment. Knock down of Smad 3 or Smad 4, but not Smad 2, completely abrogated the TGF-β induced up-regulation of Slug. Chromatin immunoprecipitation analysis revealed increased occupancy of Smad 3 to the slug promoter on TGFβ treatment indicating slug might be a direct target gene of Smad 3. Slug is often described as a potent E-cadherin suppressor and an important regulator of EMT along with other E-box proteins. In contrast siRNA knock-down of slug (slug KD) in lung cancer cells did not prevent TGFβ induced E-cadherin suppression or acquisition of mesenchymal markers such as N-cadherin, vimentin and fibronectin; instead lead to increased cell death. Slug KD cells on TGFβ treatment underwent apoptosis through the typical apoptosis cascade characterized by increased annexin-v staining and caspase-3 activation. This apoptotic cell death is abrogated by inhibitors of caspase-3 and caspase-9. Inhibition of caspase-8, an initiator of extrinsic apoptotic pathway or siRNA knockdown of RIP1 that promotes necroptosis did not inhibit the death of TGFβ treated slug KD cells. Together these finding suggests slug KD results in cell death via intrinsic apoptosis pathway in response to TGFβ. This study indicates slug induction during EMT allows cancer cells to escape from apoptotic functions of TGFβ and might function as a critical regulatory switch to shift TGFβ role from tumor suppressor to tumor promoter. Thus slug inhibition might serve as a potential mechanism to reinstate the apoptotic functions of TGFβ in lung cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4677. doi:1538-7445.AM2012-4677
Abstract 4678: Silencing of TIMELESS induces growth suppression, apoptosis and enhanced cytotoxicity of cisplatin in H157 lung cancer cells
Authors: Yoshida Kenya , Sato Mitsuo , Hase Tetsunari , Oguri Tomoyo , Elshazley Momen , Yamashita Ryo , Kondo Masashi , Hasegawa Yoshinori
Published: August 2012

Background: TIMELSS (TIM) is a circadian clock gene in Drosophila, but its role in mammals in circadian clock systems is not well-understood. Mammalian TIM is involved in Chk1 activation and intra-S checkpoint through genotoxic stress (Unsal-Kaçmaz et.al, Molecular and Cellular Biology, 27(8), 3131-3142(2007)). TIM is also associated with doxorubicin-induced G2/M cell cycle arrest and TIM depletion sensitizes HCT116 colon cancer cells to doxorubicin-induced cytotoxicity (Yang et.al, Journal of Biological Chemistry, 285(5), 3030-3034(2010)). Mutations in TIM in breast cancer were reported (Sjöblom et.al, Science, 314(5797), 268-274(2006)). Methods and Findings: The mRNA expression levels of TIM in 17 non-small cell lung cancer (NSCLC) cell lines and 4 small cell lung cancer (SCLC) cell lines were analyzed by reverse transcriptase PCR. HCC44, H460, H157 (NSCLC), H146 and H740 (SCLC) expressed higher levels of TIM than cdk4/hTERT-immortalized normal human bronchial epithelial cell line HBEC4. Sequencing analysis of TIM revealed no mutation in 21 lung cancer cell lines. RNAi-mediated knockdown of TIM in H157 suppressed proliferation in WST-1 assay and clonogenic growth in liquid colony formation assay. Western blot analysis revealed increased level of cleaved caspase-3 after TIM-knockdown, suggesting that apoptosis was involved in growth inhibition. TIM-knockdown induced increased sensitivity to cisplatin in drug sensitivity assay using WST-1 (IC50 were 1.70μM and 0.91μM in TIM-knocked down H157 compared to 4.87μM in control). Conclusions: TIM inhibition causes growth suppression, apoptosis and enhanced cytotoxicity of cisplatin. Our results suggest that TIM inhibition has a potential utility in the treatment of lung cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4678. doi:1538-7445.AM2012-4678
Abstract 4679: Betulinic acid inhibits deubiquitinases to increase the degradation of pro-survival proteins and enhance prostate cancer-specific apoptosis
Authors: Reiner Teresita , Parrondo Ricardo , de las Pozas Alicia , Palenzuela Deanna , Perez-Stable Carlos
Published: August 2012

Inhibition of the ubiquitin proteasome (UPS) pathway is a valid anti-cancer target but the alternative strategy to increase activity in order to degrade pro-survival proteins is much less developed. Betulinic acid (BA) is a naturally occurring small molecule with multiple mechanisms for increasing apoptosis in cancer cells, making it an attractive anti-prostate cancer agent. There is evidence that BA can activate the UPS pathway to degrade multiple pro-survival proteins such as androgen receptor (AR), but the mechanism is not clear. Our western blot data in prostate cancer (PC) cells indicates that 10 μM BA decreases the levels of multiple pro-survival proteins including AR, cyclins, and cyclin-dependent kinases. Inhibition of the UPS pathway with 1 μM MG132 blocks the BA-mediated decrease of AR and AKT proteins and lowers cell death, suggesting a dependence on the UPS pathway. In TRAMP transgenic mice, BA treatment (10 mg/kg) inhibits tumor growth, increases apoptosis, decreases angiogenesis and proliferation, and lowers AR and cyclin D1 protein levels (immunohistochemistry) without toxic side effects to normal tissues. However, BA does not increase UPS activity (Proteasome-Glo Chymotrypsin-like Cell-Based Assay; Promega) in LNCaP PC cells suggesting an alternative mechanism for increasing protein degradation. BA-mediated inhibition of deubiquitinase (DUB) activity, as determined by DUB-Glo Protease Assay (Promega) and DUB labeling with HA-ubiquitin vinyl sulfone, results in the accumulation of poly-ubiquitinated (Ub) proteins that are recognized by the UPS pathway and degraded. The cyclin D1 T286A mutant that cannot be degraded by the UPS pathway is resistant to BA-mediated degradation. In non-cancer BJ fibroblast, however, BA does not inhibit DUB activity nor increases total poly-Ub proteins and this is associated with no effect on cell death. Our data suggests that BA-mediated inhibition of DUBs and induction of apoptotic cell death specifically in PC but not in non-cancer cells will provide an effective non-toxic and clinically selective agent.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4679. doi:1538-7445.AM2012-4679
Abstract 4798: Pilot study of plasma biomarker candidates for pancreatic cancer detection - a targeted proteomics approach
Authors: Pan Sheng , Chen Ru , Brand Randall , Hawley Sarah , Tamura Yasuko , Gafken Philip , Milless Brian , Goodlett David , Rush John , Brentnall Teresa
Published: August 2012

With the advances in genomics and proteomics, the number of biomarker candidates associated with pancreatic cancer has increased explosively in recent years. Many putative biomarkers have been discovered in pancreatic tumor tissue and pancreatic juice; such targets will need to be rigorously verified in plasma or serum for blood-based biomarker development. Currently, validation of putative biomarkers in blood largely relies on antibody creation for ELISA assays, and has been the bottleneck for biomarker development. Analytical techniques to robustly measure these proteins in plasma, which is arguably the most complex biological matrix with protein abundance exceeding 10 orders of magnitude, has been a challenge. In this study, we applied a mass spectrometry-based targeted proteomics platform to directly detect candidate biomarker proteins in plasma to evaluate their clinical utility for pancreatic cancer diagnosis. The characterization of these protein candidates used a clinically well-characterized cohort that included plasma samples from patients with pancreatic cancer, chronic pancreatitis and healthy age-matched controls. Based on the receiver operating characteristic (ROC) analysis, two of the five candidate proteins tested, demonstrated an Area Under Curve (AUC) value greater than 0.89 in the detection of pancreatic cancer from healthy controls and chronic pancreatitis controls. We further demonstrated that a composite biomarker by combining two complementary independent biomarkers improves the sensitivity for pancreatic cancer detection at 95% specificity. Based on this evidence, these biomarker candidates merit further validation in larger patient and control cohorts for their potential utility in clinical application. In addition, we provide an analysis of the reproducibility, accuracy, and robustness of the mass spectrometry-based targeted proteomics platform. This information addresses important technical issues that could aid in the adoption of the targeted proteomics platform for practical clinical utility.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4798. doi:1538-7445.AM2012-4798