bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2776: Inhibition of Nek2 by novel small molecules affects proteasome activity
Authors: Meng Lingyao , Warner Steven L. , Mollard Alexis , Vankayalapati Hariprasad , Sharma Sunil , Bearss David J.
Published: August 2012

Nek2 is a serine/threonine kinase that has been associated with centrosome function and cell cycle progression. Nek2 overexpression has been reported in several tumor types including breast and lung cancers. We have previously shown that elevated expression of Nek2 in clinical samples of multiple myeloma (MM) correlates with bortezomib resistance. Furthermore, the overexpression of Nek2 in MM cell lines decreases sensitivity to bortezomib and knockdown with RNAi sensitizes bortezomib resistant cells. Due to the proteaseome inhibitory activity of bortezomib, we hypothesized that Nek2 overexpression may increase proteasome activity. 26S proteasomes were isolated by ultracentrifugation from stably transfected Hela cells (+Nek2 or +GFP) and Nek2 was shown to be involved in the 26S proteasome complex by western blot. Proteasome activity assays were performed both biochemically and in cell culture demonstrating that proteasome activity in Hela+Nek2 is significantly higher than in Hela+GFP. Furthermore, the isolated 26S proteasomes were incubated with novel Nek2 inhibitors (HCI-2184 and HCI-2389) resulting in a significant reduction in proteasome function when used as single agents or in combination with bortezomib. HCI-2184, a reversible inhibitor of Nek2, demonstrated an IC50 value in the range of 13-38 nM, and HCI-2389, an irreversible inhibitor of Nek2, showed an IC50 value between 8-26 nM. Similarly, both compounds significantly increased the efficacy of bortezomib in inhibiting the proteasome activity. These results were confirmed in multiple cancer cell lines, including ARP1 (human MM cell line), H299 (human lung cancer cell line) and K28 (mouse Leydig tumor cell line). The effect of inhibiting Nek2 on cell cycle was also investigated. The expression levels of cyclinB1 and cdc2, proteins degraded by the proteasome for cells to exit mitosis, were increased in HCI-2389 treated Hela+Nek2 and to a lesser extent Hela+GFP treated cells. While Nek2 overexpression leads to the down-regulation of cyclinB1 and cdc2, HCI-2389 treatment successfully rescued this effect. Flow cytometry data showed that both treatments of HCI-2184 and HCI-2389 significantly arrested Hela+Nek2 and Hela+GFP in G2/M phase after 24 hours at concentration as low as 10nM. In conclusion, we have discovered a novel biological function of Nek2 related to elevated proteasome activity. We also demonstrated that our novel Nek2 inhibitors efficiently inhibit Nek2 function, resulting in reduced proteasome activity. These Nek2 inhibitors have the potential to be applied clinically for treatment of the MM patients resistant to bortezomib.

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 2776. doi:1538-7445.AM2012-2776
Abstract 2778: Development of non-covalent reversible proteasome inhibitors
Authors: Kazi Aslamuzzaman , Ozcan Sevil , Lawrence Harshani , Sebti Said M.
Published: August 2012

Numerous cell cycle, apoptosis and angiogenesis-regulatory proteins undergo ubiquitin proteasome-dependent proteolysis. A proteasome inhibitor Velcade was approved for the treatment of multiple myeloma. However, Velcade is a covalent and slowly reversible inhibitor and is associated with undesired side effects. Non-covalent inhibitors with potentially less toxicity are desirable. In this abstract we report on a family of non-covalent inhibitors that resulted from our HTS screen of a 50,000 compound library against the chymotrypsin-like (CT-L) activity of purified 20S proteasome. Our initial hit PI-1833 (IC50 value = 0.5 uM) was used as a starting point for structure activity relationship studies that led to highly potent (IC50 value = 20 nM) and cell permeable derivatives. In addition to inhibiting CT-L in human breast cancer cells, these derivatives also induced the accumulation of two proteasome target proteins Bax and p27KIP, indicating their ability to inhibit proteasome activity in intact cells. Furthermore, these proteasome inhibitors also induced apoptosis and inhibited tumor cell growth. Further mechanistic studies as well as in vivo anti-tumor efficacy studies are ongoing.

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 2778. doi:1538-7445.AM2012-2778
Abstract 2779: Withaferin A inhibits the malignant pleural mesothelioma growth in vitro and in vivo
Authors: Yang Huanjie , Wang Ying , Cheriyan Vino T. , Wu Wenjuan , Cui Cindy Qiuzhi , Polin Lisa A. , Pass Harvey I. , Dou Q. Ping , Rishi Arun K. , Wali Anil
Published: August 2012

The medicinal plant Withania somnifera has been used for over centuries in Indian Ayurvedic medicine to treat a wide spectrum of disorders. Withaferin A (WA), a bioactive compound that is isolated from this plant, has recently been found to have anti-inflammatory, immuno-modulatory, anti-angiogenic, and anti-cancer properties. Here we investigated malignant pleural mesothelioma (MPM) suppressive effects of WA and the molecular mechanisms involved. WA inhibited growth of the murine as well as patient-derived MPM cells in part by decreasing the chymotryptic activity of the proteasome that resulted in accumulation of ubiquitinated proteins. WA suppression of MPM growth also involved elevated apoptosis as evidenced by activation of caspase-3, elevated levels of pro-apoptotic Bax protein and cleavage of poly-(ADP-ribose)-polymerase. Our studies including gene-array based analyses further revealed that WA suppressed a number of cell growth and metastasis-promoting genes including c-myc. WA treatments also promoted cleavage of vimentin as well as stimulated expression of CARP-1/CCAR1, a novel transducer of cell growth signaling. Knock-down of CARP-1, on the other hand, interfered with vimentin cleavage and MPM growth inhibitory effects of WA. Administration of 5mg/kg WA by daily i.p injections inhibited growth of murine MPM cell-derived tumors in vivo in part by inhibiting proteasome activity and stimulating apoptosis. Together our in vitro and in vivo studies suggest that WA suppresses MPM growth by targeting multiple pathways that include blockage of proteasome activity and stimulation of apoptosis, and thus holds promise as an anti-MPM 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 2779. doi:1538-7445.AM2012-2779
Abstract 278: Post-transciptional activation of anti-oxidant enzymes by imatinib
Authors: Terry Emily N. , Diamond Alan M.
Published: August 2012

Patients with Chronic Myelogenous Leukemia (CML) can be treated with targeted bcr-abl kinase inhibitors, such as imatinib. Recently, we reported that mononuclear cells obtained from individuals treated with imatinib often exhibit an increase in the enzyme activity of the antioxidant selenoprotein, glutathione peroxidase (GPx). Treatment of the bcr-abl positive cell line KU812 resulted in increased GPx enzyme and GPx1 protein levels in a dose (100-150 nM) and time (0-7 days) dependent manner, with a maximal increase of 3-fold. Imatinib-induced changes in other anti-oxidant enzymes were also assessed. Of these, no change in the levels of either catalase or GPx-4 were detected, although the levels of manganese superoxide dismutase (MnSOD) increased by a maximal level of 4-fold in imatinib-treated cells. Similar results were obtained in another bcr-abl positive, CML cell line, MEG-01. The elevation in the levels of these proteins occurred via a post-transcriptional mechanism as no increase in the steady state levels of the corresponding mRNA was detected by RT-PCR. The effect of imatinib was likely to be dependent on the presence of the bcr-abl fusion product as no changes in either GPx-1 or MnSOD were detected in either the immortalized lymphocyte cell line GM10832 or the prostate cancer-derived LNCaP human cell lines, neither of which contain bcr-abl. Since the mammalian Target of Rapamycin (mTOR) is a known protein substrate for the abl kinase, and mTOR activity can regulate protein translation, it was investigated whether inhibiting mTOR with rapamycin (1ng/ml) affected GPx-1 and MnSOD levels. Exposure of the KU812 and MEG-01 cells, as well as LNCaP and GM10832 with rapamycin resulted in increased GPx-1 protein and enzyme levels (2- to 4-fold), but had no measurable effects on MnSOD. These results indicate that imatinib, likely by the inhibition of bcr-abl, can post-transcriptionally enhance both GPx-1 and MnSOD levels, although by different mechanisms. These data may have therapeutic significance as both of these anti-oxidant enzymes may influence several different clinically relevant biological processes, including DNA damage susceptability, cell signaling and apoptosis.

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 278. doi:1538-7445.AM2012-278
Abstract 2896: Nanoparticle co-delivery of RNAi and chemotherapy for the treatment of drug-resistant cancers
Authors: Shi Jinjun , Votruba Alexander R. , Xu Xiaoyang , Langer Robert , Zetter Bruce R. , Farokhzad Omid C.
Published: August 2012

The emergence of drug resistance has been a critical barrier for effective cancer chemotherapy. Recently, RNA interference (RNAi) has shown promise in suppressing the over-expression of drug resistance-associated proteins such as P-glycoprotein and Prohibitin1, and in treating multidrug resistant cancers through combination with chemotherapy. Nevertheless, the safe and effective delivery of RNAi therapeutics (e.g., siRNA and miRNA) to target cells remains a major hurdle for their clinical applications, and the co-delivery of RNAi and chemotherapeutic agents in a controlled fashion requires specific delivery vehicles. Herein, we present a novel nanoparticle (NP) platform to tackle the challenges associated with the delivery of siRNA and chemotherapeutic drugs, using biodegradable and biocompatible polymers and lipids. The hybrid lipid-polymer NP has a differentially charged hollow core/shell nanostructure which provides the delivery system with three distinct functional features: (i) a positively charged inner hollow core for dense loading of siRNA; (ii) a middle hydrophobic polymer layer for the encapsulation of anti-cancer drugs (e.g., taxanes) and for the controlled release of both siRNA and drug; and (iii) a relatively neutral lipid-polyethylene glycol (PEG) surface to keep the NP stable and prolong its systematic circulation. Moreover, such hybrid NPs can be formulated in a simple and robust way that could facilitate future scale-up. By screening various factors, such as inner cationic lipids, outer PEG chain length, and formulation parameters, the hybrid lipid-polymer NPs show excellent in vitro knockdown efficacy at low doses of siRNA, with greater than 80% luciferase silencing at an siRNA dose of 0.4 pmol (4.0 nM) and ∼ 95% silencing at a dose of 2.0 pmol (20 nM). These NPs can also efficiently reduce the expression of drug resistance-associated proteins (e.g., MDR1 and Prohibitin1), as evidenced by qPCR. Cellular cytotoxicity experiments further confirm that this NP co-delivery strategy can drastically improve the sensitivity of drug-resistant cells to chemotherapy. In addition, these hybrid NPs demonstrate promising in vivo results for co-delivering siRNA and chemotherapeutic drugs to treat drug-resistant 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 2896. doi:1538-7445.AM2012-2896
Abstract 291: Vitamin D induces KDM6B/JMJD3 histone demethylase gene in human colorectal cancer cells
Authors: Pereira Fábio , Barbáchano Antonio , Silva Javier , Bonilla Félix , Campbell Moray , Muñoz Alberto , Larriba María Jesús
Published: August 2012

Epidemiological and preclinical studies have shown that the active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) has protective effects against colorectal cancer (CRC). Histone tails are subjected to a variety of post-translational modifications, including methylation, which play an important gene regulatory role in development and physiology. Misregulation of histone modifications alters gene expression and leads to perturbations of the cellular identity, which may contribute to cancer initiation, progression and/or metastasis. We have found that 1,25(OH)2D3 regulates the expression of JMJD3 gene (also known as KDM6B) that encodes for a histone H3 lysine 27 demethylase with an important gene regulatory role in development and physiology. 1,25(OH)2D3 induces JMJD3 RNA expression in a time- and dose-dependent manner, activates the JMJD3 gene promoter and increases the level of JMJD3 RNA in human cancer cells of colon, breast and skin origin. JMJD3 upregulation is strictly dependent on vitamin D receptor (VDR) expression and is abolished by the protein synthesis inhibitor cycloheximide. We have also shown that JMJD3 modulates the gene regulatory action of 1,25(OH)2D3. In SW480-ADH CRC cells, JMJD3 knockdown by short-hairpin RNA or expression of an inactive mutant JMJD3 fragment decreases the induction by 1,25(OH)2D3 of CYP24A1, CDH1/E-cadherin and CST5/cystatin D genes and of an epithelial adhesive phenotype. Moreover, JMJD3 knockdown upregulates the epithelial-mesenchymal transition inducers SNAI1, ZEB1 and ZEB2 and the mesenchymal markers FN1/fibronectin 1 and LEF1, while it downregulates the epithelial genes CDH1/E-cadherin, CLDN1/claudin-1 and CLDN7/claudin-7. Additionally, JMJD3 knockdown blunts the nuclear export of β-catenin and abolishes the inhibition of β-catenin transcriptional activity caused by 1,25(OH)2D3. In line with data in cultured cells, the expression level of JMJD3 RNA is lower in tumour than in normal tissue in 56% of the CRC patients studied and correlates directly with VDR and inversely with SNAI1 RNA expression. Together, our results indicate that an epigenetic gene coding for a histone demethylase such as JMJD3 is a VDR target that partially mediate the effects of 1,25(OH)2D3 on human CRC 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 291. doi:1538-7445.AM2012-291
Abstract 2910: Selenium nanoparticles suppress prostate LNCaP cancer cells growth through induced endogenous apoptosis
Authors: Bi Xiaolin , Kong Ling , Yuan Qing , Zhu Huari , Gao Xueyun
Published: August 2012

Prostate cancer has the highest incidence in all kinds of cancers in males.Recent reports suggest androgen receptor (AR) plays a critical role in prostate cancer development.The trace element Selenium is suggested having cancer prevention activity and used as food supplement. Here, we investigated inherent anticancer mechanisms of Selenium nanoparticles. We found Selenium nanoparticles inhibit growth of prostate LNCaP cancer cells partially through caspases mediated apoptosis. Selenium nanoparticles suppress transcriptional activity of androgen receptor via down-regulating its mRNA and protein expression. Moreover, Selenium nanoparticles activate Akt kinase by increasing its phosphorylation, promote Akt-dependent androgen receptor phosphorylation and Mdm2 regulated degradation through proteasome pathway. Together, we suggest Selenium nanoparticles suppress prostate cancer cells growth by disrupting androgen receptor, implicating a potential application in cancer treatment.

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 2910. doi:1538-7445.AM2012-2910
Abstract 2911: SAR125844: a potent and selective ATP-competitive inhibitor of MET kinase
Authors: Schio Laurent , Nemecek Conception , Ugolini Antonio , Wentzler Sylvie , Grapinet Sandrine , Khider Jean , Albert Eva , Dischamps Nathalie , Gay Frédéric , Sonnefraud Véronique , Bacqué Eric , Kenigsberg Mireille , Goulaouic Hélène , Dagallier Anne , Vallée François , Bonche Fabrice , Lengauer Christoph
Published: August 2012

The tyrosine kinase MET is a membrane receptor that is essential for embryonic development and wound healing in normal cells. Stimulation of MET by its natural ligand, the hepatocyte growth factor (HGF), induces cell proliferation, migration, and invasion. Abnormal MET activation (over-expression of MET protein, amplification or mutations of the MET gene) has been observed in multiple human cancer types. We report herein the discovery of a potent and selective small molecule inhibitor (SAR125844) with potential therapeutic application in cancer patients with deregulated MET-dependent malignancies. Our initial hit identification approach was based on the biochemical screen of an in-house kinase inhibitor biased library where a series of benzimidazole sulfonate derivatives were identified with sub-micromolar MET inhibition. In particular, the initial hit exhibited an IC50 of 140 nM vs MET but it also had strong affinity for CDK9 (IC50= 6 nM), a CDK isoform involved in gene transcription. Chemical modifications of the series to dial out CDK9 affinity and remove potential normal cell cytotoxicity led to a more selective derivative with IC50's of 80nM and 1355nM vs MET and CDK9 respectively. Further sub-structural exploration allowed us to identify a heteorocyclic moiety which was shown by X-ray data to specifically interact with Tyr1230 in a non active conformation of the protein. The resulting highly favourable U-shape mode of binding in MET of representative examples from these series (e.g. IC50= 1nM) was not tolerated in CDK9 (IC50 > 10µM). Final multi-parametric medicinal chemistry optimisation led to SAR125844 with single digit nanomolar antiproliferative activity on MET-amplified cell lines. SAR125844 is highly selective for MET kinase in a panel of 275 kinases tested, with only 5 other protein kinases inhibited at IC50 values below 300 nM. This compound exhibits also satisfactory eADMET in vitro properties and has shown moderate total plasma clearance, large volume of distribution and moderate to long terminal elimination half-life in rats.

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 2911. doi:1538-7445.AM2012-2911
Abstract 2924: Constitutively active androgen receptor splice variants function independently of full-length androgen receptor in a cell-based model of castrate-resistant prostate cancer
Authors: Lonergan Peter E. , Nacusi Lucas P. , Schmidt Lucy J. , Regan Kevin M. , Ertem Furkan U. , Sun Zhifu , Boorjian Stephen A. , Tindall Donald J.
Published: August 2012

Recent studies suggest that progression to castrate-resistant prostate cancer (CRPC) is due to generation of androgen receptor (AR) splice variants, lacking the ligand binding domain. These AR variants originally identified in cell-based models of CRPC, have also been detected in benign and malignant human prostate tissue, with the highest levels observed in late state CRPC. The diversity and complexity of AR variants is well appreciated but not fully understood. Using RT-PCR we assessed the expression of a number of AR variants in cell-based models of both castrate-resistant and androgen-dependent prostate cancer. Several AR variants were expressed in both 22Rv1 and VCaP cells, but not in LNCaP cells. Coding sequences for AR variants were cloned into expression vectors and constitutive transcriptional activity was observed using a luciferase reporter assay. Augmented transcriptional activity of AR variants was observed in cells that express full-length AR, when full-length AR was silenced using shRNA targeting exons 7/8. This observation suggests the constitutive activity of AR variants may be independent of full-length AR. We profiled the AR variant transcriptome in 22Rv1 cells, using the Illumina HumanHT12 v4 whole-genome expression array platform. shRNA targeting exon 1 was used to silence all AR isoforms and shRNA targeting exons 7/8 was used to silence full-length AR. 5,256 genes were modulated by knocking down all AR isoforms and 5,086 genes were modulated by knocking down full-length AR. A total of 3,714 genes were common to both groups, leaving 1542 genes modulated by AR variants and 1372 modulated by full-length AR (p<0.05). The represents a subset of genes differentially regulated by AR variants independently of full length AR. Aggregate function of multiple AR variants may confer a CRPC phenotype independent of full-length AR. Pathway analysis of these genes differentially regulated by AR variants may reveal potential targets for therapeutic intervention in patients who progress despite androgen deprivation or anti-androgen therapy. (Supported by NIH grants CA125747, CA091956 and CA121277, T.J. Martell Foundation and the Mayo Clinic Prostate Cancer SPORE).

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 2924. doi:1538-7445.AM2012-2924
Abstract 2938: Polo-like kinase 1: a potential therapeutic target for the management of patients with triple negative breast cancer
Authors: Maire Virginie , Némati Fariba , Marty-Prouvost Bérengère , Dumont Aurélie , Richardson Marion , Vincent-Salomon Anne , Lang Guillaume , Tesson Bruno , Rigaill Guillem , Barillot Emmanuel , Marangoni Elisabetta , Decaudin Didier , Roman-Roman Sergio , Pierré Alain , Cruzalegui Francisco , Depil Stéphane , Tucker Gordon C. , Dubois Thierry
Published: August 2012

Breast cancers are composed of molecularly distinct subtypes with different clinical outcomes and responses to therapy. Our aim is to discover potential therapeutic targets for triple negative breast cancer (TNBC), a poor prognosis subgroup with no targeted therapy available yet. Gene expression profiling on a cohort of 160 breast cancers including 40 TNBC revealed that the polo-like kinase 1 (PLK1), a protein kinase, is specifically over-expressed in TNBC compared to the other breast cancer subtypes. High PLK1 expression was confirmed at a proteomic level by reverse phase protein array and tissue microarray. In TNBC cell lines grown as monolayers, RNAi-mediated PLK1 depletion or small compound (BI-2536)-mediated PLK1 inhibition induced an increase in phosphorylated H2AX, G2/M arrest and apoptosis, resulting in a decrease in cell viability. In addition, a soft-agar colony assay showed that PLK1 silencing impaired tumorigenicity of TNBC cell lines. With cells grown in extracellular matrix gels (Matrigel), BI-2536 induced apoptosis specifically in TNBC cancerous versus “normal” cell lines. The in vivo anti-tumor effect of BI-2536 was investigated in two TNBC xenografts derived from patient's biopsies. When administrated as a single agent, the PLK1 inhibitor significantly impaired the tumor growth. Most importantly, administration of BI-2536 in combination with adriamycin + cyclophosphamide chemotherapy led to 100% complete response (9/9 mice). Altogether, our observations point out that PLK1 may represent an attractive therapeutic target, in association with conventional chemotherapy, for the management of patients with TNBC.

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 2938. doi:1538-7445.AM2012-2938
Abstract 2953: Anticipating new biochemical mechanisms of resistance to Raf inhibition in Melanoma
Authors: Antony Rajee , Emery Caroline , Garraway Levi A.
Published: August 2012

More than 50% of melanoma's harbor the V600E activating mutation in BRAF, which constitutively activates the Mek/Erk signaling cascade leading to tumor cell proliferation. The BRAF(V600E) mutation predicts responsiveness to RAF inhibitors, which led to FDA approval of the RAF inhibitor vemurafenib (PLX4032) in 2011. Although several mechanisms of resistance to RAF inhibition have been described, no resistance-associated point mutations involving the RAF genes themselves have been detected in the clinical setting thus far. This is in contrast to well described resistance mechanisms to receptor tyrosine kinase inhibitors, which frequently involve secondary mutations within the target kinase. To gain additional insights in this regard, we created randomly mutagenized cDNA library of BRAF (V600E) and it's homolog C-RAF and expressed individually in A375(V600E B-RAF) cells in the presence of PLX4720, an analogue of PLX4032. The resistance clones emerged from the screen were characterized by massive parallel sequencing and the mutant alleles were further recreated via site directed mutageneis in the respective cDNA's. However, no candidate mutations occuring in BRAF (V600E) could be validated as bona fide resistance alleles. In contrast, we identified 11 alleles that engendered sustained Mek phosphorylation in the setting of PLX4720 exposure. Upon ectopic expression, three of these alleles conferred profound pharmacologic resistance to PLX4720. Two of these resistance alleles clustered within the 14-3-3 consensus binding site of C-RAF, and the third occurred in the kinase domain (ATP binding domain, activation segment). 14-3-3 proteins are known to stabilize and activate C-RAF through phosphorylation of key serine and tyrosine residues. Toward this end, some C-RAF resistance alleles promoted increased BRAF heterodimerization and increased kinase activity. Each of these alleles remained sensitive to the Mek inhibitor AZD6244. On the basis of these findings, we speculate that mutations in C-RAF that alter 14-3-3 binding, promoted dimerization, and/or enhance intrinsic kinase activity may conceivably mediate resistance to RAF inhibition - particularly as more potent inhibitors become utilized. Ongoing work involves investigating the clinical relevance of this phenomenon.

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 2953. doi:1538-7445.AM2012-2953
Abstract 2955: c-Myc suppresses DNA double-strand break repair
Authors: Li Zhaozhong , Owonikoko Taofeek K. , Sun Shi-Yong , Xiao Zhi-Qiang , Doetsch Paul W. , Khuri Fadlo R. , Curran Walter J. , Deng Xingming
Published: August 2012

c-Myc is a potent oncogenic molecule that promotes tumorigenesis through the induction of genomic instability. The exact mechanism(s) involved remains to be elucidated. Here we discovered that α-ray ionizing radiation (IR)-induced double-strand DNA breaks (DSBs) promote c-Myc to form foci and to co-localize with γ-H2AX. Conditional expression of c-Myc in HO15.19 c-Myc-null cells using the Tet-Off/Tet-On inducible expression system results in downregulation of Ku DNA binding and suppressed activities of DNA-PKcs and DNA end-joining, leading to inhibition of DSB repair and enhanced chromosomal and chromatid breaks. Expression of c-Myc reduces both signal and coding joins with decreased fidelity during V(D)J recombination. Mechanistically, c-Myc directly interacts with Ku 70 protein at the N-terminus via its MBII domain. Removal of the MBII domain from c-Myc abrogates its inhibitory effects on Ku DNA binding, DNA-PKcs and DNA end-joining activities, which results in loss of c-Myc's ability to block DSB repair and V(D)J recombination. Interestingly, c-Myc directly disrupts the Ku/DNA-PKcs complex in vitro and in vivo. Thus, c-Myc suppression of DSB repair and V(D)J recombination may occur through inhibition of the non-homologous end-joining pathway, which leads to increased DNA damage, genetic instability and carcinogenesis.

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 2955. doi:1538-7445.AM2012-2955
Abstract 2958: The novel protein kinase STK17A is a direct p53 target gene that mediates response to genotoxic and nutritional stress in a cancer cell context-dependent manner
Authors: Mao Pingping , Jardine Mary , Niemaszyk Lynn , Haghkerdar Jessica , Yang Eric , yanco Esty G. , Desai Damayanti , Beyrouthy Maroun , Kerley-Hamilton Joanna S. , Freemantle Sarah J. , Spinella Michael J.
Published: August 2012

STK17A (DRAK1) is a largely uncharacterized serine/threonine kinase that belongs to the death-associated protein kinase family. Prior microarray studies demonstrated that STK17A is induced with cisplatin in testicular cancer-derived human embryonal carcinoma (EC) cells. We now demonstrate the STK17A in a novel p53 target gene that is induced by a variety of DNA damaging agents in a p53-dependent manner in a number of different cell contexts. A consensus and functional p53 response element was found upstream of the STK17A promoter and endogenous p53 was shown to bind to this site in a cisplatin-dependent manner. Knockdown of STK17A conferred resistance to cisplatin-induced growth suppression and apoptotic cell death in EC cells. This was associated with up-regulation of detoxifying and antioxidant genes and reduction in reactive oxygen species (ROS), whereas overexpression of STK17A increased ROS levels in EC cells. Interestingly, we found that STK17A is highly overexpressed in clinical glioblastoma (GBM) and cell lines compared to its expression in normal brain tissue and other cancers. High STK17A expression in GBM patients correlated with poor clinical outcome and decreased survival. STK17A knockdown in GBM cells decreased clonagenic cell growth, soft agar tumorigenicity, and survival in response to genotoxic and nutritional stress. Preliminary data will be presented implicating STK17A in the regulation of autophagy and the proximal autophagy component, ULK1. These findings indicate that STK17A may be added to a growing list of direct p53 target genes involved in autophagy that includes TIGAR, Sestrins, DAPK1 and DRAM and that by impacting autophagy STK17A may modulate response to genotoxic and nutritional stress in a cell context-depend manner. This study also suggests that STK17A is a potential novel prognostic maker and therapeutic target for GBM.

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 2958. doi:1538-7445.AM2012-2958
Abstract 2967: CCL3 autocrine signaling regulate retention of metastasis associated macrophages and promote breast cancer metastasis
Authors: Qian Bin-Zhi , Kitamura Takanori , Pollard Jeffrey W.
Published: August 2012

Macrophages abundantly found in the tumor microenvironment enhance malignancy. At metastatic sites a distinct population of metastasis associated macrophages (MAMs) promotes tumor cell extravasation, seeding and persistent growth. These macrophages are derived from circulating inflammatory monocytes recruited by CCL2/CCR2 chemokine signaling. Our current study identified a distinct gene expression signature of the MAMs in comparison with resident macrophages in lung and spleen. Majority of these genes encodes cell surface proteins and secreted factors which mediate MAM's interaction with the metastatic microenvironment. Interestingly, we identified another chemokine, CCL3, is highly expressed by MAMs in a CCL2/CCR2 dependent manner. This indicates that CCL2 not only regulate MAM recruitment but also its function. RNA interference mediated CCL3 knockdown significantly reduced their potential in promoting tumor cell extravasation in vitro. Anti-CCL3 antibody and genetic ablation of CCL3 significantly reduced tumor cell metastatic potential in vivo. Importantly, MAM also express high level of CCR1 and CCR5, receptors for CCL3, upon differentiation. Anti-CCL3 inhibitory antibody significantly reduced MAM retention in pulmonary metastasis. Together, our data indicates an autocrine signaling via CCL3 regulated by CCL2/CCR2 controls MAM retention and promote breast tumor lung metastasis which identified a novel therapeutic target for treating metastatic breast disease.

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 2967. doi:1538-7445.AM2012-2967
Abstract 2985: Predicting the impact of mutations in cancer using an integrated pathway approach
Authors: Ng Sam , Thusberg Janita , Benz Stephen , Vaske Charles , Ellrott Kyle , Zhu Jing , Yau Christina , Collisson Eric , Mooney Sean , Benz Christopher , Haussler David , Stuart Joshua
Published: August 2012

The major mechanism by which cancer arises is through somatic mutations. These mutations can lead to alterations in gene regulation as well as changes in protein structure and function. Individual tumors can contain hundreds to thousands of mutations. It is critical to distinguish mutations that have an important role defining the cancer - driver mutations - from mutations that are unimportant to the tumor - passenger mutations. Differentiating driver and passenger events is essential for understanding cancer disease mechanisms, which can help guide treatment decisions as well as identify novel targets for treatment. Genomic probing with technologies such as expression arrays and high-throughput RNA sequencing provide insight into changes in gene regulation in cancer, but determining the tumorigenic role of a coding mutation is less clear. Genomic data coupled with pathway information may provide insight into the functional impact of a mutation to particular genes. We are developing a mutation prediction method based on integrated pathway analysis to discriminate loss-of-function, neutral, and gain-of-function mutations. Utilizing the set of regulatory interactions annotated for a given gene, we can detect a discrepancy in the downstream effects of an altered gene compared to what is expected from its upstream influences. We show that a score based on this discrepancy is highly predictive of the presence of a mutation and that the directionality of this discrepancy also reflects the gain- or loss-of-function in a gene. Application of our method to a set of known driver mutations reveals that there is a significantly strong signal for loss- and gain- of functional mutations in the surrounding network, demonstrating the sensitivity of this approach. In addition, when applied to the negative control of passenger mutations, the method predicts little pathway impact, indicating this approach also has high specificity. Application of this approach to all recurrent mutations in ovarian and glioblastoma multiforme cancers from the TCGA project identifies several important driver mutations across these cohorts. We also highlight the novel utility of this specific approach by comparison to earlier published approaches including MutSig, CHASM, and MutPred.

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 2985. doi:1538-7445.AM2012-2985
Abstract 2999: Gene expression signature of differential response to chemotherapy in sporadic pediatric osteosarcoma
Authors: Martin Jeff W. , Koti Madhuri , Chilton-MacNeill Susan , van Wijnen Andre J. , Squire Jeremy A. , Zielenska Maria
Published: August 2012

Osteosarcoma is the most common pediatric bone malignancy. Genomic instability, karyotypic heterogeneity and dysregulated gene expression are typical of osteosarcoma tumors, for which treatment is often multimodal and aggressive. Tumor necrosis in response to neoadjuvant chemotherapy is used to estimate outcome and survival, because reliable clinical diagnostic and prognostic biomarkers have not yet been described for this tumor. Our objective in the current study was to identify new molecular biomarkers for osteosarcoma based on differential mRNA expression levels of genes found to be consistently involved in the pathogenesis of osteosarcoma. The NanoString nCounter System utilizes color-coded probe pairs, which allow multiplexed expression analysis of specific genes in solution-phase hybridization with digital output. Advantages of the system are a higher sensitivity than microarrays and the utilization of small amounts of total RNA without requiring cDNA synthesis. We first determined the efficacy and consistency of the system using total RNA extracted from snap-frozen and formalin-fixed, paraffin-embedded osteosarcoma tissue specimens. We then verified that the NanoString assay was consistent with RT-PCR analysis of our samples, after which we subjected a cohort of osteosarcoma tumors to the expression assay. Seventeen genes, including many with well-described functions in osteosarcoma, were analyzed for expression in a cohort of tumors as well as normal human osteoblasts. Expression values of normal human osteoblasts were compared with those of tumors with more than 90% necrosis post-chemotherapy (an indicator of good prognosis) and of tumors with less than 90% necrosis post-chemotherapy (an indicator of poor prognosis). Of note, we found that tumors with poor response to chemotherapy had statistically significant up-regulation of CDKN1C, FOS, and RUNX2. The RUNX2 gene is located at chromosome 6p21.1, which frequently undergoes genetic amplification in osteosarcoma. Amplification-related overexpression of RUNX2 has been previously reported (Martin et al 2010, Sadikovic et al 2010) as a poor prognosticator for chemotherapy response, and the involvement of bone morphogenetic pathways downstream of RUNX2 are being addressed by multiplex analyses of differential gene expression.

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 2999. doi:1538-7445.AM2012-2999
Abstract 3009: Alternative pre-mRNA splicing in digestive tract malignancy
Authors: Miura Koh , Ohnuma Shinobu , Fujibuchi Wataru , Ishida Kazuyuki , Sasaki Iwao
Published: August 2012

Alternative pre-mRNA splicing plays an important role in the generation of functional diversity of genome. The process of pre-mRNA splicing is regulated by cis- and trans-elements, and their deregulations result in aberrantly-spliced individual variants and aberrant expression profiles. Accumulating evidence has revealed that aberrant splicing contributes to a number of diseases including human neoplasms. Germ line mutations in cis-element of tumor suppressor genes such as MMR genes, APC gene, and CDH1 gene are involved in Lynch syndrome, familial adenomatous polyposis, and hereditary diffuse gastric cancer, respectively. In addition, somatic mutations in cis-elements also play a role in tumorigenesis. These genetic alterations including nonsense, missense, or silent mutations in cis-elements, led to aberrant transcripts by exon skipping, retention of the intron, or introduction of new splice site (Miura et al., Cancer Science 2011 Feb;102(2):309-16). We present our results and current knowledge of the regulatory mechanism underlying alternative pre-mRNA splicing and aberrant splicing, particularly focusing on digestive tract malignancies.

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 3009. doi:1538-7445.AM2012-3009
Abstract 301: Ccl17 and Ccr4 co-localize in mouse lung adenomas in vivo and are associated with epithelial cell migration in vitro
Authors: Stein Lauren , Tichelaar Jay W.
Published: August 2012

Lung cancer is the leading cause of cancer deaths among men and women in the United States and improved treatments are urgently needed. Like many cancers, long-term inflammation is now recognized to be a contributing factor in the progression of lung cancer from a benign to a malignant state. Exactly how inflammation contributes to lung cancer is incompletely understood. Chemokines are a group of proteins that function by attracting immune cells to areas of higher chemokine concentrations. Specific chemokine-chemokine receptor pairs control the migratory response of many inflammatory cell types. It is known that chemokines and chemokine receptors can also have direct effects on cancer cells. Using in situ hybridization, we determined that the chemokine Ccl17 and its receptor Ccr4 are coordinately induced in tumor cells in transgenic and benzo(a)pyrene-induced mouse models of lung cancer. In addition, multiple subsets of T lymphocytes within mouse lung tissue express Ccr4. Immunohistochemistry localized CCR4 to cancer cells in 11 of 32 (34%) human non-small cell lung cancer biopsies while staining was not detected in 12 non-neoplastic lung biopsies. We identified human lung cancer cell lines expressing CCR4 using flow cytometry and immunofluorescence. Treatment of human NCI-H292 lung cancer cells with CCL17 peptide induced migration in a collagen-dependent manner in a transwell migration assay. The presence of Ccr4 on both lung cancer cells and lymphocytes suggests that this receptor has multiple roles in regulating lung tumor promotion and progression. Characterization of these roles may provide novel targets to inhibit lung tumor progression. Future studies will directly address the function of Ccr4 during lung tumorigenesis in vivo.

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 301. doi:1538-7445.AM2012-301
Abstract 3067: Regulation of PAX2 expression/activity by hypoxia and EGF in breast cancer cells
Authors: Lacombe Catherine , Beauchemin David , Annabi Borhane , Themsche Céline Van
Published: August 2012

Breast cancer is the most common cancer in women and is associated with the highest mortality rate after lung cancer. The leading cause of deaths associated with this cancer is metastasis, but cellular and molecular mechanisms controlling the metastatic ability of breast cancer cells are only partially elucidated. There is direct and indirect evidence suggesting that PAX2, a transcription factor preferentially activated in breast cancer cells of the luminal subtype, could reduce the invasive and metastatic ability of these cells, by downregulating the expression of ERBB2. It is thus primordial to understand the mechanisms regulating the expression and activity of PAX2 in breast cancer cells. Specifically, because hypoxic conditions prevailing in the center of solid primary tumours are known to regulate the expression of many genes controlling invasion and metastasis, we are investigating the impact of hypoxia-mimicking agent cobalt chloride (CoCl2) on PAX2 expression in breast cancer cells. Similarly, because EGF growth factor regulates the expression and/or activity of many genes controlling invasion and metastasis, we are determining the impact of EGF exposure on PAX2 activation and/or gene expression in ER+ MCF-7 cell line. The identification of factors and mechanisms regulating the expression and activity of PAX2 in breast cancer cells will help understand the biology of this emerging factor in breast cancer cells and may provide multiple strategies to control invasion and metastasis of these 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 3067. doi:1538-7445.AM2012-3067
Abstract 3071: Obesity-associated growth factor signaling upregulates aromatase expression and estrogen receptor activity in breast cancer cells
Authors: Bowers Laura W. , Angel Rebecca De , Tekmal Rajeshwar , Brenner Andrew , Hursting Stephen , deGraffenried Linda
Published: August 2012

Obesity increases the risk of breast cancer by approximately 50% in postmenopausal women and is also associated with a worse prognosis. Elevated estrogen synthesis by the local mammary epithelia and adipose tissue is thought to be the principal mediator of breast tumorigenesis in this population, which primarily develops estrogen receptor alpha (ERα) positive breast cancer. However, the elevated levels of free insulin-like growth factor 1 (IGF-1) that accompany obesity are also thought to play a role. IGF-1 has significant tumorigenic effects in the breast and also regulates aromatase, the key enzyme in the conversion of androgens to estrogen. Consequently, we hypothesized that obesity increases the risk of postmenopausal breast cancer via growth factor-induced aromatase expression and/or activity in the local mammary tissue. We have previously shown that MMTV-Wnt-1 mammary tumors from obese ovariectomized mice express higher levels of aromatase in comparison to tumors from lean ovariectomized mice. To examine the molecular pathways responsible for this effect, we have utilized an in vitro model of obesity in which ERα positive MCF-7 breast cancer cells were exposed to human sera obtained from postmenopausal women and pooled by BMI category. Our data indicates that exposure to obese (BMI ≤ 30 kg/m2) human serum stimulates greater aromatase expression in comparison to control (BMI: 18.5-24.9 kg/m2). This is coupled with enhanced ERα activity when exogenous androgen is present, indicating that the increased aromatase expression results in greater estrogen production. Obese human sera also activates MCF-7 cells’ Akt pathway to a greater degree than control, while MCF-7 cells expressing a constitutively active Akt demonstrate higher levels of aromatase expression in comparison to MCF-7 cells. This suggests that circulating growth factors in the obese sera, like IGF-1, may induce elevated aromatase expression via the downstream PI3K/Akt pathway. To expand on our findings, we plan to assess whether treatment with PI3K/Akt pathway inhibitors eliminates the difference in MCF-7 cells’ aromatase expression following exposure to obese versus control sera. We will also examine how inhibition of the PI3K/Akt pathway affects ERα activity in sera-exposed MCF-7 cells. Through elucidation of the signaling pathways responsible for obesity's upregulation of local aromatase expression, we ultimately hope to develop rational and effective chemopreventive and chemotherapeutic regimens for the high-risk obese postmenopausal population.

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 3071. doi:1538-7445.AM2012-3071