bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2113: Elucidating mechanisms of regulation of homologous recombination utilizing a PALB2 fusion protein that contains the BRCT repeats of BRCA1
Authors: Zhang Fan , Bick Gregory , Andreassen Paul R.
Published: August 2012

The products of the breast cancer susceptibility genes, BRCA1 and PALB2, directly interact through coiled-coil domains present in each protein. How PALB2 is recruited to sites of DNA damage and how the BRCA1-PALB2 interaction regulates the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) have remained unclear, however. Here, the BRCT repeats of BRCA1, which are required for its localization, were fused to a PALB2 mutant incapable of binding BRCA1. We introduced this fusion protein into HCC1937 breast cancer cells that contain a truncated BRCA1 or into cells in which full-length BRCA1 was depleted using a siRNA. The fusion protein corrected the deficiency for PALB2 foci in both systems, either with or without exposure to ionizing radiation. Importantly, expression of the fusion protein also corrected a defect in PALB2-dependent assembly of foci by the RAD51 recombinase. Further, we also introduced the fusion protein into PALB2/FANCN-deficient cells from a Fanconi anemia patient. The fusion protein restored both PALB2 and RAD51 foci in these cells without binding to BRCA1. Strikingly, the fusion protein also conferred resistance to mitomycin C (MMC) and corrected a defect in DSB-initiated HR in PALB2-deficient cells. Our data convincingly demonstrate that BRCA1 localizes PALB2 to sites of DNA damage, and that this is necessary for recruitment of RAD51 and for HR. Contrary to a published report, since the fusion protein is incapable of binding to PALB2, our results suggest that PALB2 functions, such as HR, are mediated by the BRCA1-PALB2 interaction rather than the PALB2-PALB2 dimer. Given that BRCA1 and PALB2 participate in a pathway of DNA repair by HR, our results suggest that mutations which disrupt the BRCA1-PALB2 interaction may lead to the genetic instability that underlies the development of cancer. Additionally, the BRCA1-PALB2 interaction may be a potential therapeutic target for sensitization of cancer cells to DNA damaging agents.

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 2113. doi:1538-7445.AM2012-2113
Abstract 2114: Trypanosoma brucei: A model to evaluate joint contribution of BRCA2 and PARP in DNA damage repair
Authors: Cooper Rachel , Misra Smita , Chaudhuri Minu , Chaudhuri Gautam
Published: August 2012

The DNA repair protein BRCA2, initially discovered as a tumor suppressor for familial breast cancer cases, is now found to play critical role in the biology of many eukaryotes. The extracellular protozoan parasite Trypanosoma brucei significantly relies on its BRCA2 protein for stress management as well as surviving from host humoral immunity during its mammalian stage of its complex digenetic life cycle. An interesting aspect of BRCA2 deficiency in cancer cells is that these cells are ultra-sensitive to the inhibitors of the chromatin modifying enzyme poly(ADP-ribose) polymerase (PARP). In the absence of PARP, spontaneous single-strand breaks collapse replication forks and trigger homologous recombination for repair. Thus, PARP activity is essential in homologous recombination-deficient BRCA2-mutant cancer cells. BRCA2 and PARP are not only expressed in the mammalian cells but also present in high levels in many other eukaryotic cells including the parasitic protozoan T. brucei. To evaluate the universality of the importance of these two proteins in the management of DNA damage, we determined the effect of PARP inhibitors such as 3-AB, Veliparib, PF-01367338, Olaparib and Iniparib in BRCA2 knocked down T. brucei cells treated with the DNA damaging agent cisplatin. Our in vitro data strongly suggest that a combination of a BRCA2 inhibitor (e.g. BRCA2 BRC peptide), a PARP inhibitor and a DNA damaging agent (e.g. cisplatin) will be a chemical regimen for the management of trypanosomiasis and similar protozoan infections. Supported by NIH grants 2SC1GM081146-05 to MC, 1U54RR026140-01 to SM and NIH grants R01AI042327, R21AI076757 to GC.

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 2114. doi:1538-7445.AM2012-2114
Abstract 2120: Hepatitis B virus X protein interacts with PARP1 that inhibits DNA repair
Authors: Na Tae Young , Lee Mi Ock
Published: August 2012

Hepatitis B virus (HBV) is strongly associated with the development of hepatocellular carcinoma (HCC). Among the HBV viral proteins, X protein (HBx) is regarded as a major risk factor of HCC. HBx contributes to HCC development through regulating expression of many host or viral genes and interacting with other cellular proteins. In this study, proteins that interact with HBx were analyzed by immunoprecipitation and subsequent LC/MS/MS. We identified 60 HBx interacting proteins that function in a wide spectrum of biology. Among these proteins, we were interested in PARP1, Poly (ADP-ribose) polymerase, a crucial factor in base excision repair (BER) pathway, since HBx was known to inhibit DNA repair capacity in cancer cells. Therefore, we characterized the cross-talk between HBx and PARP-1. HBx and PARP-1 colocalized in the nucleus and they are physically associated. Coimmunoprecipitation experiments revealed that the catalytic domain of PARP-1 bound to HBx. Chromatin immunoprecipitation showed that HBx inhibited the recruitment of DNA damage induced by paraquat treatment. HBx overexpression significantly inhibited the protein expression of the DNA damage response factors that respond to the paraquat-induced oxidative stress. Finally, HBx further enhanced the level of 8-OHdG, the marker of DNA damage, under oxidative stress. Together, these data suggest that the interaction between HBx and PARP1 inhibits DNA repair, which could result in onset of hepatocarcinogenesis.

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 2120. doi:1538-7445.AM2012-2120
Abstract 2129: Regulation of DNA repair by Kruppel-like factor 8
Authors: Lu Heng , Hu Liu , Li Tianshu , Mukherjee Debarati , Lahiri Satadru , Wason Melissa , Yu Lin , Zhao and Jihe
Published: August 2012

Krüppel-like factor 8 (KLF8) regulates critical gene transcription and cellular events associated with cancer. Recently we have identified poly(ADP-ribose) polymerase-1(PARP-1) as a novel KLF8-interacting and regulating protein. However, whether KLF8 is involved in PARP-1 relulated DNA repair is unknown. Here, we report that KLF8 plays a role in the PARP-1-dependent DNA repair. Western blotting and immunofluorescence staining indicated that overexpression of KLF8 attenuated the αH2A.X levels in doxorubicin-treated PARP-1+/+ MEF cells but not in the PARP-1−/− MEF cells, whereas the PARP-1-interaction-defective KLF8 mutant did not affect the αH2A.X levels. Clonogenic assay and tetrazolium salt (WST-1) assay showed that KLF8 promoted cell survival in human breast cancer cells. Comet assay revealed that KLF8 expression could decrease DNA damage levels. Interestingly, we found that in response to DNA damage KLF8 was phosphorylated by DNA-PKcs and subsequently sumoylated, which depended on the interaction between KLF8 and PARP-1. These results suggest a novel role and mechanism for KLF8 in the regulation of DNA repair in 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 2129. doi:1538-7445.AM2012-2129
Abstract 2133: MCPH1 interacts with the anaphase-promoting complex subunit Cdc27
Authors: Wiltshire Timothy D. , Singh Namit , Mer Georges , Couch Fergus
Published: August 2012

Microcephalin (MCPH1), the first gene identified as causative for primary recessive autosomal microcephaly, is aberrantly expressed in autism-like disorders and human malignancy of breast and ovarian origin. MCPH1, the encoded protein product, has been implicated in various cellular processes including the DNA damage checkpoint, DNA repair and transcription. While our understanding of the cellular context in which MCPH1 operates continues to develop, a structural understanding of the C-terminal tandem BRCT domains of MCPH1 remains unexplored. Here, we identify Cdc27, a component of the anaphase-promoting complex (APC/C), as a novel interacting partner of MCPH1. We provide in vitro and in vivo evidence that the C-terminal tandem BRCT domains of MCPH1 (C-BRCTs) bind Cdc27 in a phosphorylation-dependent manner. To further characterize this interaction, we determined the structure of MCPH1 C-BRCTs in complex with a phosphorylated Cdc27 peptide (pCdc27) using X-ray crystallography. Based on this structure, we identified single amino acid mutations targeted at the binding interface that disrupted the MCPH1-pCdc27 interaction. Collectively, our data defines the biochemical, structural and cellular determinants of the novel interaction between MCPH1 and Cdc27 and suggests that this interaction may occur within the larger context of MCPH1-APC/C.

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 2133. doi:1538-7445.AM2012-2133
Abstract 2136: Nuclear trafficking of Tie2 is associated with radioresistance of gliomas
Authors: Hossain Mohammad B. , Gururaj Anupama , Cortes-Santiago Nahir , Gabrusiewicz Konrad , Fueyo Juan , Gomez-Manzano Candelaria
Published: August 2012

Resistance and relapse are still primary causes that result in poor effectiveness of chemo- and radiotherapy in glioblastoma multiforme (GBMs), the most frequent subtype of primary brain tumors in adults. Patients suffering from this disease exhibit a median survival that ranges from 9 to 15 month, and the disease invariably recur after therapy due to the presence of cells exhibiting a multidrug-resistance phenotype. Aberrant DNA repair pathways can enable tumor cells to survive DNA damage that is induced after chemotherapy and radiotherapy. Therefore, development of new therapeutic strategies requires the identification of key molecular pathways regulating the resistant phenotype of these tumors. Previous work from our laboratory showed that Tie2, a previously considered specific vascular tyrosine kinase receptor, was expressed in glioma tumors and in brain tumor stem cells. The degree of expression of Tie2 was related to the malignancy of these tumors, and played an essential role in the multidrug resistance phenotype of gliomas. Our current research is focused on deciphering the mechanisms underlying this critical function. We found that density of Tie2 receptors in the membrane decreased upon exposure to both ligand (angiopoietin 1, Ang1) and ionizing irradiation (IR). Immunoflurorescence studies using confocal microscope and subcellular fractionation followed by Western blot showed that Tie2 traffics from the cellular membrane to the nucleus upon both stimuli. Western blot analysis using antibodies against N- and C-terminus, and against phosphorylated-Tie2, indicated that the nuclear Tie2 is a full-length protein, and importantly that is phosphorylated at Tyr992. Of biological importance, to find the role of Tie2 in the nucleus, we irradiated the glioma cells, U251.Tie2, and found that ionizing radiation also helps Tie2 to move to the nucleus and importantly it binds with γH2AX one of the key DNA repair protein complex. By using confocal microscopy we found that Tie2 makes foci with γH2AX after DNA damage. We also found that after genotoxic stress like ionizing radiation or DNA-damaging drugs, Tie2-expressing cells were more resistant to DNA damaging effect, as assessed by cell viability assay and comet assay. Collectively, the nuclear Tie2 plays important role in the DNA repair which will result in the design of Tie2-targeting combinational therapies for patients with glioblastomas.

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 2136. doi:1538-7445.AM2012-2136
Abstract 214: High expression of CKS1B antagonizes MLN4924, a nedd8-activating enzyme inhibitor, that induces myeloma cell growth inhibition through decreased neddylation of SCF complex
Authors: Zhan Fenghuang , Wang He , Zangari Maurizio , Tricot Guido
Published: August 2012

Background: MLN4924 binds to the NEDD8-activating enzyme (NAE), and promotes neddylation of SCF (Skp1-Cul1 / Cdc53-F-box protein) complex resulting in inhibition of proteasomal degradation of several substrates. CKS1B acts as an adaptor of SCF complexes involved in cell cycle progression and DNA damage repair in many cancers, including multiple myeloma (MM). In this study, we investigate the functional role of CKS1B expression on MLN4924 in myeloma and determine whether CKS1B can be used as a potential biomarker for sensitivity to MLN4924 treatment in MM. Methods: Three myeloma cell lines, KMS12, KMS28PE, and OCI-MY5 in which CKS1B was artificially over-expressed (CKS1B-OE) as well as their empty vector (EV) controls were treated with MLN4924 at different concentrations. The IC50 and growth inhibition of cells were analyzed via a fluorencent screeing assay. APC-labeled annexinV and PI were assessed by flow cytometry to detect apoptosis and cell cycle changes. The expression of Cullin-NEDD8 and a series of ubiquitined SCF substrates were analyzed by non-reducing western blot. Results: MLN4924 effectively inhibited cell growth and induced cell apoptosis in both CKS1B-OE and EV MM cells. However, MM cells transfected with EV were more sensitive than those transfected with CKS1B-OE as evidenced by the IC50 values (KMS28PE OE vs. EV 99: 65 nM; KMS12 OE vs. EV 125: 72 nM; and OCI-MY5 OE vs. EV 106:89 nM). The cell viabilities at 100 nM were 49.6% vs. 26.7%, 54.5% vs. 34.2%, and 53.6% vs. 36.8% in CKS1B-OE versus EV KMS28PE, KMS12, and OCI-MY5 cells (P<0.05). Western blots showed that over-expression of CKS1B significantly decreased expression of cullin1, nedd8, and the conjugated complex of cullin-nedd8 in MLN4924 treated cells (100 nM, 24h) compared with EV in all tested three cell lines. We also found that high expression of CKS1B was negatively correlated with SCF substrates including p21, CDT-1, p27 and CDT-1. MLN4924 induced a dose-dependent accumulation of these substrates in all three MM cell lines. Conclusion: High expression of CKS1B induces SCF substrate ubiquitination and degradation in MM cells. The NAE inhibitor MLN4924 induces cell apoptosis through decreased neddylation of SCF complex resulting in increase of p27, p21, CDT-1 and p130. CKS1B can be used as a marker of drug resistance to MLN4924, suggesting that a combination of MLN4924 with other drugs will be necessary to treat myeloma patients with high endogenous expression of CKS1B.

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 214. doi:1538-7445.AM2012-214
Abstract 2145: Coupling S100A4 to Rhotekin alters Rho signaling output in breast cancer cells
Authors: Chen Min , Bresnick Anne R. , O'Connor Kathleen L.
Published: August 2012

S100A4 is a calcium binding protein and tumor metastasis associated factor that has been suggested to promote motility and invasiveness of different types of cancer. This migratory promoting effect, in part, is due to the interaction of S100A4 with actin and actin binding proteins, such as myosin IIA. We found that S100A4 interacts with Rho binding domain of Rhotekin (TRBD), one of the Rho effectors, thus suggesting a connection between the Rho and S100A4 pathways. To test whether this interaction is specific, we performed GST pull-down assays using Rho binding domain from different Rho effectors such as Rhotekin, Rhophilin, PKN, ROCK II, and Citron. These results showed that S100A4 specifically interacts with Rho binding domain of Rhotekin but not of other Rho effectors. We further determined that this interaction is direct and calcium-dependant by performing GST-TRBD pull-down assays with purified S100A4 in the presence and absence of calcium. The in vivo interaction of these two proteins was further confirmed by immunoprecipitation of exogenously expressed mutated forms of Rhotekin with endogenous S100A4 from MDA-MB-231 cells. Confocal microscopy showed that Rhotekin and S100A4 co-localize at the edge of membrane ruffles in response to EGF. We demonstrated that S100A4 did not bind TRBD using the same residues as Rho, as determined by using a triple-A mutant of Rhotekin. Interestingly, we found that active Rho, S100A4 and Rhotekin are in a complex by using constitutively active GST-RhoQ63L pull-down assay. To examine the function of this interaction, RNAi was used to suppress Rhotekin and/or S100A4 in MDA-MB-231. F-actin staining showed that suppression of both RTKN and S100A4 leads to the loss of Rho-dependent membrane ruffling in response to EGF stimulation, an increase in contractile F-actin “stress” fibers in the cell body and a reduction in invasive growth in three-dimensional culture. Taken together, we showed that S100A4 specifically and directly interacts with Rhotekin in a calcium-dependent manner. This coupling permits S100A4 to complex with RhoA and switch RhoA function from stress fiber formation to membrane ruffling. Based on the function of this interaction, we propose that interaction of S100A4 and Rhotekin alters the functional output of Rho signaling to confer an invasive phenotype in breast 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 2145. doi:1538-7445.AM2012-2145
Abstract 2149: Cooperation of Notch and RAS/MAPK pathway leads to poor outcome in breast cancer patients
Authors: Mittal Suruchi , Kumar Rekha V. , Rangarajan Annapoorni
Published: August 2012

Breast cancer is a leading cause of cancer-related deaths in women the world over, particularly in the Western population. However, emerging trends indicate an alarming rise in breast cancer incidences in other parts of the world including India. A major impediment in breast cancer treatment is occurrence of metastasis leading to poor prognosis and survival in certain patients. Therefore understanding the molecular pathways responsible for such phenomenon is critical. Recent studies have implicated aberrant Notch signaling in breast cancers. We undertook an extensive study analysing the expression of the Notch pathway members in breast biopsies of Indian origin. We observed increased expression of several Notch receptors including Notch 1, 2 3, and 4, Notch-ligands including Jagged1, Jagged2, and Delta-like 4, and downstream targets of the Notch pathway Hes1/Hes5 in breast cancer tissues compared to the normal counterpart. Additionally, we detected cleaved (active) Notch1 and Hes1/5 in early precursors of breast cancers - hyperplasia and ductal carcinoma in situ - suggesting that aberrant Notch activation may be an early event in breast cancer progression. In in vitro transformation experiments, we observed that while constitutively active Notch1 alone failed to transform HMLE immortalized breast cells, it synergized with the Ras/MAPK pathway to mediate transformation in vitro. Interestingly, compared to only cleaved Notch positive cases, those positive for both pERK1/2 and Notch showed poor survival, and majority of such cases were triple negative breast cancers. This suggests that in highly aggressive triple negative breast cancers that exhibit poor survival, the Notch and Ras/MAPK pathway cooperation might play a crucial role and thus targeting these two pathways together might offer a novel therapeutic strategy for this aggressive form of 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 2149. doi:1538-7445.AM2012-2149
Abstract 2150: The role of SHP2 in HER2+ breast cancer
Authors: Wang Xiaonan
Published: August 2012

Breast cancer is the most common malignancy and the second most common cause of cancer related death in women. HER2+ over-expressing (HER2+) tumors comprise 20% to 25% of all cases and are associated with a poor prognosis. The discovery of HER2 monoclonal antibody-based therapeutics has greatly improved patient prognosis. However, patients suffer relapse and de novo resistance. Thus it is important to determine additional therapeutic targets in HER2+ breast cancer that might be used in combination with current therapies to improve patient survival. SHP2 (PTPN11) is a non-receptor protein-tyrosine phosphatase (PTP) that contains two SH2 domains at its N-terminus, a central PTP domain and a C-terminal proline-rich tail. Previous studies suggest that Shp2 is required for Ras/Erk signaling downstream of most growth factor receptors and integrins. Heterozygous germ-line PTPN11 mutations cause Noonan Syndrome and LEOPARD syndrome, which are associated with leukemia and cancer predisposition. Somatic PTPN11 mutations cause juvenile myelomonocytic leukemia (JMML) and several other neoplasms. We have discovered that Shp2 is required in Neu-mediated tumorigenesis in the MMTV-NIC mouse model; furthermore, SHP2-depleted human HER2+ BT-474 has defective tumorigenesis in nude mice. SHP2 is required in anchorage-independent growth in 4 HER2+ breast cancer cell lines, though its effect on growth on plastics appears minimal. I hypothesize that SHP2 is required for HER2+ breast tumor initiation and maintenance. I further hypothesize that SHP2 functions through specific substrates/interacting proteins in HER2+ breast cancer cells, possibly molecules that are involved in anchorage-independent proliferation.

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 2150. doi:1538-7445.AM2012-2150
Abstract 2151: Activation of NF-kB and MAP kinases by TNF Family Receptors is regulated by cellular IAPs
Authors: Varfolomeev Evgeny , Goncharov Tatiana , Maecker Heather , Zobel Kerry , Komuves Laszlo , Deshayes Kurt , Vucic Domagoj
Published: August 2012

Activation of NF-kB, JNK and p38 MAPK signaling pathways is innermost activity of TNF receptor protein signaling complexes that is important for proper function of the immune system. However, excessive activation of these signaling pathways may result in tumor development and progression. Therefore, elucidation of the detailed molecular mechanisms of TNF receptor signaling should gain advantage in our understanding of the tumor biology and invention of the novel anticancer therapies. The formation of TNF receptor complexes, as well structural and functional associations between different signaling molecules is regulated by ubiquitination in TRAF-dependent fashion. TNF receptors employ several proteins possessing ubiquitin E3 ligase activities including cellular Inhibitor of Apoptosis, c-IAP1 and c-IAP2 proteins. We demonstrate that c-IAPs are required for canonical NF-kB and MAPK activation by TNF receptors. By regulating the recruitment of Nemo, IKK2 and HOIP to TNFR signaling complexes c-IAPs promote induction of gene expression by TNF ligands. We also found that TNF receptors that stimulate the noncanonical NF-kB pathway trigger c-IAPs, TRAF2 and TRAF3 intracellular translocation followed by their proteasomal and lysosomal degradation. Finally, we establish that BR3-induced cytosolic depletion of TRAF3 results in noncanonical NF-κB activation.

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 2151. doi:1538-7445.AM2012-2151
Abstract 2156: Preclinical combinations of vemurafenib, a selective BRAF inhibitor, with other targeted therapies in BRAFV600E colorectal cancer models
Authors: Yang Hong , Higgins Brian , Kolinsky Kenneth , Li Jiakui , Margolis Richard , Go Zenaida , Railkar Aruna , Packman Kathryn , Bollag Gideon , Su Fei
Published: August 2012

Mutations in BRAF at codon 600 promote catalytic activity and are associated with 8% of all human (solid) tumors, including about 10% of colorectal cancers (CRCs). Vemurafenib (RG7204, PLX4032) is a first-in-class, BRAF-specific small molecule inhibitor that dose-dependently inhibits tumor growth in BRAFV600E CRC xenografts. Despite these encouraging preclinical findings, unlike the remarkable responses observed in melanoma, single agent vemurafenib in a Phase I extension trial of 21 patients with previously treated metastatic CRC resulted in a modest 5% response rate. We therefore explored a range of combinations of vemurafenib with different novel targeted therapies including a MEK inhibitor, an AKT inhibitor, a PI3K inhibitor, an mTOR/PI3K dual inhibitor and an EGFR inhibitor in BRAFV600E CRC cell lines. The consequences of these combinations on proliferation were analyzed by MTT assay and the combined effect was determined by combination index (CI) calculated using CalcuSyn software. Western analysis and Annexin V staining were utilized to evaluate combination effects on downstream signaling events and apoptosis induction. Optimized doses of both vemurafenib and a MEK inhibitor were tested as single agents and in combination in CRC xenograft models in nude mice. Synergistic anti-proliferative effects were observed with combinations of vemurafenib and other targeted therapies in the BRAFV600E positive CRC cell lines tested. More effective signaling inhibition and apoptosis induction were observed with the combinations than either agent alone. Combining vemurafenib with a MEKi delivered greater anti-tumor activity and increased life span of animals in the LS411N CRC xenograft model. In a BRAF-mutant CRC xenograft model with inherent resistance to vemurafenib (RKO), tumor growth inhibition by vemurafenib was enhanced by combining with an AKT inhibitor. These in vitro and in vivo data suggest that the administration of vemurafenib in combination with novel targeted therapies may be effective in delivering enhanced and sustained clinical antitumor efficacy in colorectal cancers harboring the BRAFV600E mutation.

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 2156. doi:1538-7445.AM2012-2156
Abstract 2158: RAF signaling and SHOC2 mediate mutant NRAS acquired resistance to RAF inhibitors in melanoma
Authors: Kugel Curtis H.
Published: August 2012

RAS signaling through extracellular signal-regulated kinase 1/2 (ERK1/2)/mitogen-activated kinase (MAPK) pathway is critical for normal cellular homeostasis and is frequently aberrantly regulated in cancerous cells. Although often depicted as a linear sequence of RAS-RAF-MEK-ERK1/2, this pathway is also regulated by expression of multiple isoforms of proteins at each level, scaffold proteins that enhance pathway activation, and negative feedback mechanisms. Some of these factors may also impact on the response of the pathway to pharmacological inhibitors. To test this notion, we analyzed resistance to the RAF inhibitor, PLX4720, using mutant BRAF melanoma cell models. In a screen to isolate cells displaying resistance to the inhibitory effects of PLX4720, we observed a subset of cells harboring an acquired mutation in NRAS. In parental cells, expression of mutant NRAS was sufficient to prevent PLX4720-mediated inhibition of ERK1/2 signaling, entry into S phase and susceptibility to apoptosis in mutant BRAF cells. Effects of mutant NRAS were associated with the maintenance of RAF binding. Analysis of isolated clones of RAF inhibitor-resistant mutant BRAF cells demonstrated that re-activation of the ERK1/2 pathway in the presence of PLX4720 was dependent on NRAS, BRAF, CRAF, and the RAS-RAF scaffold protein, SHOC2/Sur-8. Depletion of SHOC2 also rendered resistant cells susceptible to PLX4720-mediated apoptosis in 3D collagen. Together, these data demonstrate the role of RAF isoforms and a RAS-RAF scaffold protein in the acquired resistance to RAF inhibitors induced by secondary mutations in NRAS.

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 2158. doi:1538-7445.AM2012-2158
Abstract 2160: Regulation of DHX33 during oncogenic stress and its crucial role in Ras-initiated tumor formation
Authors: Zhang Yandong , Weber Jason D.
Published: August 2012

Previously we found that DHX33 is pivotal for RNA polymerase I transcription, changes in DHX33 levels have a dramatic impact on RNA polymerase I activity. In this report, we explore the regulation of DHX33 protein levels by ARF/p53 pathway and Ras signaling. In wild type primary fibroblasts, Ras V12 infection induced a transient increase in DHX33 protein level as well as rRNA transcriptional rate which was eventually overcome by suppression from ARF/p53 pathway. ARF/p53 dependent reduction of DHX33 occurred at the translational level, while Ras activation induced DHX33 levels both at the level of transcription and translation. The induction of DHX33 by Ras activation is sensitive to inhibitors of PI3K, mTOR as well as MAPK. Induction of DHX33 by Ras V12 is pivotal for enhanced rDNA transcription, enhanced protein translation, and thus, the oncogenecity of Ras V12, as DHX33 knockdown abolished the increase in rRNA transcription and protein translation, and completely inhibited the tumorigenecity of Ras V12 both in semisolid agar and xenografts. Our results implicate that DHX33 level could be an important factor to adjust rDNA transcriptional rate in response to ARF induction and Ras activation. Most importantly, we identified DHX33 as a novel downstream target of Ras signaling to regulate rRNA transcription and pointed toward its pivotal role in Ras-initiated tumor formation and therapeutic value to treat Ras mutated cancer patients.

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 2160. doi:1538-7445.AM2012-2160
Abstract 2166: In silico identification and biological evaluation of potent small molecule inhibitors targeting GAB1 pleckstrin homology domain
Authors: Dumas Sabrina , Du-Cuny Lei , Moses Sylvestor , Song Zuohe , Zhang Shuxing , Meuillet Emmanuelle J
Published: August 2012

The Grb2-associated binder-1 (GAB1) over-expressed in many cancers represents a novel therapeutic target that integrates signals from different systems such as MET and PI3K/Akt pathways. It mediates processes involved in tumor progression and metastasis. In this study we have identified novel inhibitors targeting the pleckstrin homology (PH) domain of GAB1 for cancer treatment. To this end, a homology model of GAB1 PH domain was constructed, followed by structural refinement using molecular dynamics. The resulting structure was applied to high-throughput virtual screening of 5 million compounds and 19 hits were selected for experimental evaluation. We found that 6 compounds bound to the PH domain of GAB1 as measured by surface plasmon resonance spectroscopy, with a KD. Out of these 6 hits, all of them inhibited the HGF-I induced phosphorylation of GAB1 Tyr627 in T47D and inhibited T47D and MDA-MB-231 breast cancer cell lines proliferation. Three of them exhibited selective binding to GAB1 PH domain as compared to the PH domain of IRS-1 and AKT. This demonstrates for the first time the identification of novel GAB1 PH domain inhibitors which may be used for targeted breast cancer therapy.

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 2166. doi:1538-7445.AM2012-2166
Abstract 2169: FHL1 on chromosome X is a single-hit gastrointestinal tumor-suppressor gene and contributes to the formation of an epigenetic field defect
Authors: Asada Kiyoshi , Ando Takayuki , Ushijima Toshikazu
Published: August 2012

Tumor-suppressor genes on chromosome X can be inactivated by a single hit, any of point mutations, chromosomal loss, and aberrant DNA methylation. Among these mechanisms, aberrant DNA methylation can be present not only in tumor tissue but also in normal-appearing tissues. Levels of aberrant DNA methylation in non-cancerous tissues are known to correlate with cancer risk for gastric cancers and other cancers (epigenetic field defect). However, only a limited number of genes that functionally contribute to the field defect have been identified. To identify a tumor-suppressor gene on chromosome X that contributes to the formation of an epigenetic field defect, we here searched for genes on chromosome X whose expression was up-regulated by treatment of AGS gastric cancer cell line with a DNA demethylating agent, 5-aza-2′-deoxycytidine (5-aza-dC). Among the 495 genes up-regulated at 4-fold or more by treatment with 5-aza-dC, 69 genes were located on chromosome X. Among the 69 genes, 11 genes had low expression (signal intensity < 200) in non-treated AGS cells and high expression (signal intensity > 500) in a pool of gastric mucosae of three healthy volunteers. Among the 11 genes, FHL1 was frequently methylation-silenced in gastric and colon cancer cell lines, and in primary gastric (11/58) and colon (5/50) cancers. In HCT116 cells, knock-down of FHL1 accelerated cell growth (shRNA-1, 243 % of control cells at 120 hours, P < 0.001, and shRNA-2, 191%, P < 0.001) and sizes of xenografts in nude mice (shRNA-1, 2.7-fold larger tumors than that of control cells, P < 0.001). Also in HSC39 cells, knock-down of FHL1 significantly increased cell growth. Expression of exogenous FHL1 in a non-expressing AGS cell line significantly reduced its growth (72.2% of control cells, P < 0.05). A somatic mutation (G642T; Lys214Asn) was identified in one of 144 colon cancer specimens, and the mutant FHL1 was shown to lack a growth-suppressive effect. Among the healthy volunteers, FHL1 methylation was elevated only in H. pylori-positive individuals (0/16 vs. 10/16, P < 0.001). Since potent methylation induction by H. pylori can mask a difference in H. pylori-positive individuals, FHL1 methylation levels were compared between healthy volunteers and gastric cancer patients among the H. pylori-negative individuals, and FHL1 methylation levels were shown to be elevated only in gastric cancer patients (0/16 vs. 5/26, P = 0.06). These data showed that FHL1 is a methylation-silenced tumor-suppressor gene on chromosome X in gastrointestinal cancers, and that its silencing contributes to the formation of an epigenetic field for cancerization.

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 2169. doi:1538-7445.AM2012-2169
Abstract 217: Modulation of Hsp90 chaperone activity by hypoxic condition: Hypoxia stimulates ARD1-mediated Hsp90 post-translational modification
Authors: Woo Jong Kyu , Lee Ho-Young
Published: August 2012

Arrest defective-1 protein (ARD1, also known Naa10p) is an N-acetyltransferase known to be involved in cell cycle control. Several reports have implicated ARD1 in regulation of cell proliferation and apoptosis in mammalian cells. Although a potential role for ARD1 in controlling cell proliferation and apoptosis has been identified, little is known about the relevance of ARD1 to cancer development. Heat Shock Protein 90 (Hsp90), an essential molecular chaperone in eukaryotic cells, maintains the functional conformation of a subset of proteins that are typically key components of multiple regulatory and signaling networks mediating cancer cell proliferation, survival and metastasis. Reversible acetylation has been implicated as a regulatory post-translational modification of Hsp90. Although an acetyltransferase responsible for acetylating Hsp90 has yet to be identified, several groups recently identified Histone deacetylase 6 (HDAC6) as an one of the Hsp90 deacetylase. This study, we show that hypoxia leads to increased Hsp90 acetylation via ARD1 binding to Hsp90 and disrupted interaction between Hsp90 and client proteins and co-chaperone proteins. We found that treatment with the HDAC inhibitor trichostatin A (TSA) or sodium butyrate (NaB) enhanced Hsp90 acetylation and led to the dissociation of Hsp90 and client protein Hif-1a. To gain further insight into protein acetylation in the cytoplasm, we searched cellular targets for Hypoxia. By using an immunoprecipitation approach, we identified ARD1 as a prominent Hsp90 interacting partner. Transfection of ARD1 leads to Hsp90 hyperacetylation, its dissociation from an essential co-chaperone proteins and a loss of chaperone activity. Transfected ARD1 and Hsp90 can be abundantly and specifically co-immunoprecipitated under hypoxia condition. Those interactions prevented the formation of the Hsp90 chaperon complex, resulting in reduced chaperone function of Hsp90. We suggest that ARD1 functions as a tumor suppressor by disrupting the function of Hsp90, and thus inactivating of PI3K/Akt pathway and Hif-1a in cancer cells under hypoxic condition. We conclude that a coordinated interaction between Hsp90 and ARD1 under hypoxic condition may be an important mechanism for hypoxic cell signal to homeostasis.

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 217. doi:1538-7445.AM2012-217
Abstract 2170: The SWI/SNF nucleosome-remodeling gene PBRM1 - Another tumor suppressor gene in renal cell carcinomas
Authors: Krause Hans , Ikromov Odiljon , Kamal Eymad All , Miller Kurt , Schostak Martin , Jandrig Burkhard
Published: August 2012

The polybromodomain containing gene PBRM1 was recently discovered to harbor 41% truncating somatic mutations in clear cell renal cell carinomas (ccRCC). Next to the VHL tumor suppressor gene this gene is thought to be the second major cancer gene in ccRCCs. PBRM1 (also called BAF180) belongs to the SWI/SNF-B or PBAF complex and provides functional specificity to this class of chromatin remodelers by binding to acetylated histones via its six bromodomains, two BAH domains, and one HMG domain (the latter binding to nucleosomal DNA). PBRM1 is located on chromosome 3p21 in close vicinity to VHL and SETD2 genes arguing for a putative gatekeeper function in cases where VHL loss alone is insufficient for ccRCCs tumorigenesis. PBRM1 codes for at least three major transcripts of about 7500nt in length whose expression was measured by Q-PCR in 49 cases of paired normal and tumor tissue samples of RCC patients who underwent radical tumor nephrectomy at our clinic between 06/2009 and 10/2010. 20/49 cases showed a marked upregulation of PBRM1 expression whereas in 29/49 cases PBRM1 was clearly downregulated. To assess the contribution of somatic mutations for PBRM1 downregulation we resequenced 29 PBRM1 exons of 48 tumor tissues. Specific truncating mutations were detected in 6 (15.4%) of 39 ccRCCs. These mutations include 5 deletions in exons 1, 10, 16, 23, and 24, as well as one insertion in exon 16. All these heterozygous mutations lead to a decrease of PBRM1 mRNA expression, that was verified by Q-PCR measurements. In addition, 6 coding SNPs (cSNPs) were revealed as well as 5 SNPs in intronic regions, one of the latter is in close vicinity to the splice acceptor site of exon 9. Concurrent expression of three major transcripts (var. 1,2, and 4) is seen in all 46 matched normal and tumor samples analyzed so far. In 22 (47.8%) samples we detected a dominance of var.1 (NM_018165) in normal tissue when compared to the corresponding tumor sample, that preferentially expresses var.4 (NM_181042). So far our data set of 15.4% truncating mutations in 39 cases of ccRCC could not match the exceptional high number of 41% reported in the initial report by the Sanger Institute. Truncating mutations of PBRM1 have been described in breast cancers but were absent in lung carcinomas, despite the fact that LOH at 3p21 is frequently seen in these cancers. Understanding the contribution of PBRM1 expression changes, be it on the genomic level (mutation) and/or mRNA (differential splicing), to clinical disease progression and outcome in kidney cancer needs further investigation.

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 2170. doi:1538-7445.AM2012-2170
Abstract 2171: Human FLCN delays cell cycle progression through late S and G2/M-phases: Effect of phosphorylation and tumor-associated mutations
Authors: Laviolette Laura A. , Wilson Jonas , Koller Julia , Zimmer Michael , Iliopoulos Othon
Published: August 2012

The Birt-Hogg-Dube (BHD) autosomal dominantly inherited syndrome has clinical features that include the development of skin fibrofolliculomas, lung cysts, and renal neoplasia. Renal tumors occur in approximately one third of individuals with BHD. The gene responsible for BHD syndrome is Folliculin (FLCN), which encodes a 68kD phosphoprotein (FLCN). One allele of the FLCN gene is mutated in the germline of >90% of BHD patients and somatic mutations or loss of the wild type FLCN allele are observed in the renal tumors of patients with BHD, suggesting that FLCN acts as a tumor suppressor protein. The human renal cell carcinoma cell line UOK257, derived from a patient with a germline mutation in the FLCN gene, harbors a truncated version of the FLCN protein. Reconstitution of the wild type FLCN protein into the FLCN-deficient UOK257 cells decreased their proliferation rate. Here we tested whether this difference in cell proliferation rate may be partly attributed to the effect of FLCN on cell cycle progression. We synchronized FLCN-deficient UOK257 cells and their isogenic, wild type or mutant FLCN counterparts, by double thymidine block and followed cell cycle progression after release. UOK257 cells expressing wild type FLCN exhibited a slower progression through the late S and G2/M-phases of the cell cycle compared to the vector only controls (FLCN-deficient). The reintroduction of tumor-associated FLCN mutants failed to delay cell cycle progression in UOK257 cells, indicating that the effect of wild type FLCN on cell cycle progression may contribute to its tumor suppressor function. FLCN phosphorylation on serines S62 and S73 fluctuates throughout the cell cycle and peaks in cells arrested at the G2/M boundary following treatment with nocodazole. In keeping with this observation, the reintroduction of a FLCN S62/73 phosphomimetic mutant into the UOK257 cell line resulted in faster progression through the cell cycle compared to those expressing the wild type FLCN protein. These findings suggest that orderly changes in FLCN phosphorylation during the cell cycle are linked to and may be required for its effect on cell cycle progression and possibly its activity as a tumor suppressor protein. We expect that these studies will provide insight into the role of FLCN in kidney tumorigenesis and will identify putative new targets for therapeutic intervention.

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 2171. doi:1538-7445.AM2012-2171
Abstract 2173: Heterozygosity for Mtap accelerates lymphomagenesis in Emu-Myc and Pten +/− mice
Authors: Kruger Warren D. , Kadariya Yuwaraj , Tang Baiqing , Al-Saleem Tahseen , Haykawa Kyoko
Published: August 2012

The gene encoding the methionine salvage pathway methylthioadenosine phosphorylase (MTAP) is a tumor suppressor gene that is frequently inactivated in a wide variety of human cancers. Loss of MTAP is associated with elevated expression of ornithine decarboxylase (ODC), a key enzyme in the synthesis of polyamines and a known oncogene. Here, we have examined if heterozygosity for a null mutation in Mtap could accelerate tumor development in two different mouse cancer models, Eα-myc transgenic and Pten+/−. We found that the median age of tumor formation in both models was significantly accelerated in Mtap+/− compared to Mtap+/+ mice. In Eα-myc mice, Mtap mutations accelerated the formation of lymphomas from cells in the early pre-B stage and these tumors tended to be of higher grade and have higher expression levels of ODC protein compared to those observed in control Emu-myc Mtap+/+ mice. Supprisingly, examination of Mtap mRNA and protein in highly lymphoma infiltrated tissue in heterozygous animals indicates that >80% of the tumors maintain expression of the Mtap allele. Our findings show that germline inactivation of a single Mtap allele enhances lymphomagenesis in both Emu-myc and Pten+/− mice and is associated with elevated levels of ODC.

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 2173. doi:1538-7445.AM2012-2173