bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3833: Codon-optimized GM-CSF gene as an adjuvant to enhance the immunity of a human papillomavirus 16 E7 DNA vaccine in mice
Authors: Qiu Jian-Tai , Lin Cheng-Tao , Lai Chyong-Huey , Chang Kuo-Shiang , Wu Tzong-Shoon
Published: February 2011

Granulocyte macrophage-colony stimulating factor (GM-CSF) is a potent immunomodulatory cytokine. We have generated a novel codon optimized murine GM-CSF sequences as an adjuvant. The codon optimized murine GM-CSF plasmids significantly increased GM-CSF expression levels in all cells tested, including murine, monkey, and human cell lines. Here we show that coadministration of plasmids encoding the codon optimized GM-CSF sequence with the HPV-16 E7 DNA vaccine resulted in complete tumor regression in 100% of the C57BL/6 mice tested. Consistent with these findings, coadministarton of these plasmids elicited E7-specific CTL responses and a protective immune response against challenge of E7- expressing TC-1 tumors in mice. Histological analysis revealed that the tumor destruction occurred at the margins of the tumor, where there was a heavy lymphocytic infiltrate. We also created a TC-1 tumors expressing E7 and GM-CSF genes. Combining a GM-CSF secreting TC-1 vaccine with a systemic DNA vaccination successfully treated >90% of the mice bearing an established tumor but showed no resistance to an unrelated ovarian cancer. This novel codon optimized GM-CSF plasmid may prove a practical molecular strategy for overcoming this limitation of DNA vaccines and potentiating immune responses to vaccines as well as other immunotherapeutic strategies.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3833.
Abstract 3839: The role of RhoA in human normal mammary epithelial cell immortalization, stem/progenitor cell self-renew, and differentiation
Authors: Zhao Xiangshan , Band Hamid , Band Vimla
Published: February 2011

One of the earliest steps in oncogenic transformation of hMECs involves loss of senescence and continuous proliferation, a process referred to as immortalization. Delineation of biochemical pathways that mediate hMEC immortalization is therefore likely to provide answers to key questions about early breast cancer. RhoA GTPases are central players in many aspects of basic cell biology, There is little knowledge about the role of Rho in early breast cancer. Our recent findings have revealed the potential role of RhoA GTPase in early oncogenic transformation in breast cancer. We demonstrated that overexpression of constitutively-active RhoA, a Rho effector domain mutant, and the wild-type RhoA(WTRhoA), but not a dominant-negative Rho mutant, induces the immortalization of normal hMECs. This is highly significant as in breast and other cancers WTRhoA overexpression is seen but not mutation. Furthermore, we observed immortalization with a RhoA mutant T37A, that fails to interact with well known Rho effectors, Rho kinase, mDia1 and PKN, thus pointing towards a novel pathway that does not involve actin cytoskeleton organization, which may be a late event. In order to identify the molecular mechanism of RhoA-induced immortalization, we performed microarray expression profiling of RhoA-immortalized vs. parental cells and found alterations in expression of several genes (ELF3, ZNF217, S100P were overexpressed; DAB2, CLCA2, TFPI2, ALOX15B were downregulated) previously implicated in immortalization of mammary epithelial cells, and breast cancer progression. Our western blot and RT-PCR results showed that similar to immortal cells, the expression of these genes were altered in breast cancer cell lines. these results demonstrate that RhoA can induce the preneoplastic transformation of hMECs by altering multiple pathways linked to cellular transformation and breast cancer.

There is increasing evidence that breast cancer originates in and is maintained by cancer stem cell (CSC) Which has stem cell properties, including self-renewal, tumorigenicity and multi-lineage differentiation capacity. A major hurdle in addressing how normal stem cell become CSC is the lack of in vitro human stem/progenitor cells that are immortal and can be deliberately manipulated to ask questions related to origins of CSC.

Our current work showed that RhoA immortal cells express luminal, myoepithelial, basal, and stem cell markers (K8/18, E-cadherin, ESA, K5, K6, K14, p63, CD29, CD49f). Indicate that these cells may have stem/progenitor cell properties. It is important to further examine the role of RhoA in normal and cancer mammary stem/progenitor cell self-renewal and differentiation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3839.
Abstract 384: Effects of endostar on cell functions and biological characteristics in tumor-derived endothelial cells
Authors: He Yong , Jiang Li
Published: February 2011

Tumor microvessels are essential to tumor formation, growth, and metastasis. Interventions targeting the microvessels would lead to avascular necrosis of tumor cells. Therefore, specifically targeting endothelial cells in tumor vasculature had become one of the most important strategies for cancer therapy. Endostar, a novel recombinant human endostatin with an additional nine-amino acid sequence, is a novel anti-angiogenesis drug. This present study is to determine how endostar affects the behavior of endothelial cells and tumor-derived endothelial cells both in vitro and in vivo. Human umbilical vein endothelial cells (HUVEC) were cultured and differentiated to tumor-derived endothelial cells (Td-ECs) after co-culturing with conditioned medium from A549 cells, a lung cancer cell line. The anti-angiogenesis effects of endostar on Td-ECs were examined by MTT assay, cell migration assay, flow cytometry, and TUNEL assay. Endostar exhibited a significant inhibitory effect both time and dose dependently in Td-EC cells. In addition, Endostar significantly inhibited cell migration and induced G0/G1 and S phase cell cycle arrest and apoptosis. However, under the same concentrations, Endostar has little impact on HUVEC. To study the in vivo effects, a xenograft model in nude mice was established. Endostar was injected intratumorally as localized administration, and tumor growth was closely monitored during the following days. As a result, endostar showed significant anti-angiogenesis effect and tumor growth inhibition. The concentration of Ca2+ in the treatment groups measured by fluorescence spectrometry was elevated significantly compared to that in the control group. This study revealed a novel activity of endostar in inhibiting angiogenesis, more potently in tumor-derived endothelial cells than in normal endothelial cells. This finding suggests the potential use of endostar as cancer therapeutic to be tested in clinical trials.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 384.
Abstract 3846: Pleiotrophin in the tumor microenvironment induces expression of VEGFR2 on macrophages and promotes an anti-inflammatory phenotype
Authors: Lynn Kristi D. , Roland Christina L. , Dineen Sean P. , Sullivan Laura A. , Brekken Rolf A.
Published: February 2011

Macrophages are an abundant immune cell in the tumor microenvironment that contribute to tumor growth and metastasis. Macrophage recruitment into tumors is mediated by multiple cytokines, including vascular endothelial growth factor-A (VEGF). We are interested in investigating the effects of VEGF inhibition on macrophages and other stromal cells within the tumor. We demonstrated recently that macrophages (systemic and tumor associated) in tumor-bearing animals express VEGFR2 in contrast to macrophages from non tumor-bearing animals. Furthermore, we found that selective inhibition of VEGFR2 inhibits macrophage infiltration into tumors in vivo. These results demonstrate that when VEGFR2 is expressed on macrophages it is the dominant receptor driving VEGF-induced migration in vitro and in vivo. Additionally, we found that the cytokine pleiotrophin (PTN) is sufficient to induce VEGFR2 expression on macrophages. PTN is expressed by many human breast cancer cells (e.g., MDA-MB-231) and the majority of human primary breast tumors. However, the connection between PTN and macrophage phenotype is still unexplored. Macrophages can be divided into two functional sub-types: M1 or classically activated macrophages and M2 or alternatively activated macrophages. M1 macrophages are powerful effector cells that produce pro-inflammatory cytokines and are capable of destroying tumor cells. In contrast, M2 macrophages inhibit the inflammatory response and promote tumor growth and angiogenesis. We employed the MMTV-PyMT transgenic, spontaneous breast cancer model to characterize PTN expression, immune cell infiltration, and immune cell function during the course of tumor progression. We found that PTN levels correlate with VEGFR2+ macrophage and M2 macrophage levels, which all peak during the transition from hyperplasia to early malignancy. Whereas, MDSC and Treg levels as well as levels of the anti-inflammatory cytokines IL-10 and IL-1β peak during the early carcinoma stage. We identified that PTN not only induces VEGFR2 expression by macrophages but also promotes an M2 phenotype in vitro. In addition, we have evidence that VEGF can modulate the level of PTN expression through VEGFR1. These results suggest a mechanism through which anti-VEGF therapy modulates PTN and M2 macrophage levels in vivo. Based on these observations, we hypothesize that PTN is a critical cytokine involved in breast tumor progression through promotion of a VEGFR2+, M2 macrophage phenotype in the tumor microenvironment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3846.
Abstract 3849: Mast cell inhibition by Btk inhibitor PCI-32765 as a potential cancer therapy
Authors: Soucek Laura , Honigberg Lee , Kortlever Roderik , Buggy Joseph , Swigart Lamorna , Evan Gerard I.
Published: February 2011

An association between inflammation and cancer has long been recognized but the cause and effect relationship linking the two remains unclear. Myc is a pleiotropic transcription factor that is over-expressed in many human cancers and instructs multiple extracellular aspects of the tumor tissue phenotype, including remodeling of tumor stroma and angiogenesis. We have shown in a

β cell tumor model that Myc activation in vivo triggers rapid recruitment of mast cells to the tumor site, a recruitment that is absolutely required for macroscopic tumor expansion. Indeed, such tumors did not grow in mast cell deficient mice. Furthermore, treatment of established β cell tumors with cromolyn, a stabilizer that prevents mast cell degranulation, rapidly triggered hypoxia and cell death of tumor and endothelial cells. Inhibitors of mast cell function may therefore prove therapeutically useful in restraining expansion and survival of pancreatic cancer. However, such compounds have not yet been developed for systemic administration and applications in cancer therapy. In fact, there are no clinically effective mast-cell blockers. Therefore, we tested PCI-32765 (Pharmacyclics, Inc.), a Bruton's tyrosine kinase (BTK) inhibitor in clinical trials for B cell non-Hodgkin's lymphoma, for its ability to inhibit mast cell function and tumorigenesis in our mouse model of β cell neoplasia. Here we show that long term treatment with PCI-32765 prevents mast cell degranulation and causes tumor regression and vasculature collapse. Surprisingly, the compound also displays an unexpected anti-proliferative effect on tumor cells - but not surrounding normal tissue - which is still under investigation. Our results once more support the idea that mast cell inhibition could be beneficial in the treatment of insulinomas and possibly other pancreatic cancers and suggests that PCI-32765 could open the way to clinical studies assessing this potential.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3849.
Abstract 3858: Synergistic antitumor effects of combination therapy with S-1 and HER2 targeting agents in gastric cancer with HER2 amplification
Authors: Tanizaki Junko , Okamoto Isamu , Takezawa Ken , Tsukioka Sayaka , Uchida Junji , Kiniwa Mamoru , Fukuoka Masahiro , Nakagawa Kazuhiko
Published: February 2011

HER2 amplification is observed in 20-30% of gastric cancer and it has been correlated to poor outcomes and more aggressive disease. Combination therapies with HER2 targeting agents and cytotoxic agents are considered a therapeutic option for patients with HER2 amplified gastric cancer. We now investigated the effects of the combination treatment with S-1(or 5-fluorouracil (5FU)) and either lapatinib or trastuzumab on the growth of gastric cancer cells with or without HER2 amplification in vitro and in vivo. The combination of 5FU and either lapatinib or trastuzumab showed a synergistic antiproliferative effect in HER2 amplified gastric cancer cells. Combined treatment with 5FU and lapatinib or trastuzumab induced synergistic increases in the number of apoptotic cells and in the activity of caspase-3 in HER2 amplified cells, wheareas, such synergistic antiproliferative effect nor the enhancement of apoptosis was observed in cells without HER2 amplification. Thymidylate synthase (TS) is an important target enzyme for 5FU, with a reduced level of TS expression having been associated with a higher rate of response to 5FU-based chemotherapy. To elucidate the molecular mechanism of the synergistic effect by the combination of 5FU and lapatinib or trastuzumab, we examined the effects of lapatinib or trastuzumab on the expression and activity of TS in gastric cancer cells. Both lapatinib and trastuzumab induced down-regulation of TS expression and inhibition of TS activity in HER2 amplified cells, whereas neither lapatinib nor trastuzumab has such effects on TS expression and activity in cells without HER2 amplification. Similar to the effects of lapatinib or trastuzumab, TS depletion by siRNA resulted in the enhancement of 5FU-induced apoptosis, suggesting that TS down-regulation attributes, at least in part, to the synergistic proapoptotic interaction with 5FU. Furthermore, the combination of oral fluoropyrimidine S-1 and lapatinib or trastuzumab synergistically inhibited the growth of gastric cancer cell xenografts with HER2 amplification. Our results thus suggest that the combination of S-1 and lapatinib or trastuzumab has synergistic antitumor effects in HER2 amplified gastric cancer cells and that effect is likely attributable to the down-regulation of TS expression and inhibition of TS activity by lapatinib or trastuzumab. In conclusion, these observations provide a basis for clinical evaluation of combination chemotherapy with S-1 and HER2 targeting agents, lapatinib or trastuzumab, in gastric cancer patients with HER2 amplification.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3858.
Abstract 3861: A genome-wide association study of upper aerodigestive tract cancers identifies 4q21, 4q23, and 12q24 as susceptibility loci
Authors: McKay James D. , Truong Therese , Lathrop Mark , Brennan Paul ,   on behalf of the INHANCE consortium.
Published: February 2011

To identify risk variants for upper aero-digestive tract (UADT) cancers, we performed a genome-wide association study of 2,091 UADT cancer cases and 8,334 controls followed by replication of 20 variants in an additional 6,545 UADT cancer cases and 7,892 controls. Five common variants presented evidence for significant association in the replication series (one sided p ≤ 0.005 - preplication) and combined analysis (p ≤ 5×10−7 - pcombined). Three variants were in the alcohol dehydrogenase (ADH) gene cluster on 4q23 (rs1573496-ADH7, preplication=7×10−10 /pcombined=5×10−17 rs1229984-ADH1B, preplication=3×10−10 pcombined=5×10−21 and rs698-ADH1C, preplication=0.003 pcombined=3×10−7). The fourth variant (rs4767364, preplication=4×10−4/pcombined=7×10−8) was located in an extended linkage disequilibrium region at 12q24 that contains the aldehyde dehydrogenase 2 (ALDH2) gene and appears to have effects similar, albeit more modest, to that observed with heterozygote ALDH2 null allele (rs671) common in Asian populations. The fifth variant (rs1494961, preplication=9×10−6 pcombined=2×10−8) was located near DNA repair related genes HEL308 and FAM175A (or Abraxas) at 4q21. This 4q21 variant was also associated with lung cancer risk in a series of 5,652 lung cancer cases and 9,338 controls (rs1494961, p=3×10−4). These results further highlight the importance of the genes involved in the metabolism of alcohol in UADT cancer susceptibility, and also implicate 4q21 in susceptibility to both UADT and lung cancers.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3861.
Abstract 3862: Identification of a novel genetic variant in the KLK3 gene which predisposes to prostate cancer
Authors: Kote-Jarai Zsofia , Al Olama Ali Amin , Leongamornlert Daniel , Tymrakiewicz Malgorzata , Saunders Ed , Guy Michelle , Mahmud Nadiya , Giles Graham , Severi Gianluca , Hopper John , Hamdy Freddy , Neal David , Donovan Jenny , Muir Kenneth , Morrison Jonathan ,   The UK Genetic Prostate Cancer Study Collaborators/BAUS Section of Oncology, Easton Douglas , Eeles Rosalind
Published: February 2011

Prostate cancer (PrCa) is the commonest solid tumour in men in developed countries. To identify common PrCa susceptibilility alleles, we conducted a multistage genome-wide association study (GWAS). In the first stage (stage 1) 541,129 SNPs were genotyped using the Illumina Infinium platform in 1,854 PrCa cases with clinically detected PrCa diagnosed at <60 years or a family history of disease, and 1,894 population screened controls with low (< 0.5ng/ml) prostate specific antigen (PSA) level from the UK. We identified 7 novel PrCa susceptibility loci (P=2.2×10−8 to P=8.1×10−29) one of which contains a strong candidate susceptibility gene KLK3 which codes for PSA. PSA is widely used as a biomarker for PrCa detection and disease monitoring. The tag-SNP in this region, rs2735839, was associated with both PrCa disease (P=1.33×10−20) and with PSA level (P =6× 10−8). To refine the association of PrCa with variants in this region we imputed genotypes for SNPs that were not in our GWAS study, using the Mach program and the HapMap 2 CEU population as a reference panel. Among SNPs in the KLK3 region, the strongest evidence for association with PrCa cancer was observed for rs17632542, (P =5.9×10−26), a SNP not typed in the GWAS. We genotyped this novel SNP in our stage 1 (as above) and an additional stage2 sample set of 3650 PrCa cases and 3940 controls from the UK and Australia, not selected for PSA level, using Taqman assay and ABIPrism 7900HT sequence detection system. rs1763542 showed stronger evidence of association than the original tag-SNP in both stages (stage 2 P=2×10−5, overall P=3.2×10−31). Whereas rs2735839 is intergenic between the KLK3 and KLK2 genes, rs17632542 is a non-synonymous coding SNP in the KLK3 gene causing an Ile to Thr substitution at aa179. We conclude that this SNP more strongly associated with PrCa then other known SNPs in the KLK3 region, and is a strong candidate for a causative variant influencing PrCa risk.

Acknowledgements: This study was funded by Cancer Research UK, The Prostate Cancer Research Foundation, and NIHR support to the Biomedical Research Centre.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3862.
Abstract 3868: Long-term inhibition of cells at metaphase with intact mitotic spindles results in unscheduled chromatid separation and catastrophic arrest in mitosis
Authors: Daum John R. , Sivakumar Sushama , Daniel Jeremy J. , McAvoy Jennifer N. , Potapova Tamara A. , Gorbsky Gary J.
Published: February 2011

In the presence of unaligned chromosomes or other spindle abnormalities, the mitotic spindle checkpoint blocks both chromatid separation at anaphase and exit from mitosis. The spindle checkpoint is sustained by signaling from kinetochores that are not properly attached to microtubules of the mitotic spindle. Normally, when chromosomes align at the spindle equator at metaphase, microtubule-kinetochore interactions act to extinguish checkpoint signaling. The cessation of the checkpoint signal activates the Anaphase-Promoting Complex/Cyclosome (APC/C), an E3 ubiquitin ligase. The APC/C ubiquitylates the mitotic effectors, securin and cyclin B, which are then degraded by the proteasome. Securin and cyclin B degradation initiates chromatid separation at anaphase and triggers mitotic exit. We have found that long-term arrest of Hela cells with their chromosomes aligned at the metaphase plate results in chromatid separation without mitotic exit. Recently, we and others have characterized C13orf3/Ska3, a newly discovered component of the Ska (Spindle and kinetochore-associated) protein complex. We found that in mitosis Ska3 protein accumulates at kinetochores reaching maximal levels at metaphase. Upon anaphase onset, kinetochore-associated Ska3 levels decline and are eventually lost at telophase. We demonstrated that cells depleted of Ska3 by RNAi achieve metaphase alignment but fail to silence the spindle checkpoint. During this mitotic arrest with chromosomes aligned at the metaphase plate, kinetochores retain robust microtubule attachments. Kinetochores within these Ska3-depleted cells accumulate abnormally high levels of the mitotic spindle checkpoint protein, Bub1, presumably accounting for the inability to silence the checkpoint. Under extended metaphase arrest, the chromatids of individual chromosomes eventually separate, but cells remain arrested in mitosis. To test if other means of inducing metaphase arrest would also lead to chromatid separation, we treated cells in mitosis with the proteasome inhibitor, MG132. Again, after several hours at metaphase, chromatids separated. We speculate that for cells arrested at metaphase, continual pulling forces of the kinetochores on the mitotic spindle eventually overcomes the cohesin complex that holds chromatids together. This “cohesin slippage” results in abnormal chromatid separation for cells still in mid-mitosis. This response reflects a novel terminal phenotype for cells arrested in mitosis with an intact mitotic spindle. (Supported by the NIGMS and the McCasland Foundation.)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3868.
Abstract 3870: Distinct and cooperative functions of the RB and p53 tumor suppressors in genotoxic response and tumorigenesis
Authors: McClendon Kathleen , Dean Jeffry L. , Ertel Adam , Reed Christopher A. , Yang Xiaoping , Knudsen Erik S.
Published: February 2011

The compound inactivation of RB and p53 is a frequent occurrence in human cancers. At a molecular level, the RB and p53 pathways intersect at multiple points that have critical implications for cell cycle control and tumorigenesis. To examine the cooperation of these proteins in the response to genotoxic damage, we used a combination of genetic deletion and dominant negative strategies in vitro, and liver-specific genetic deletion in vivo. In vitro, RB proficiency was sufficient to maintain cell cycle checkpoint in response to genotoxic stress, independent of p53 status. In contrast, RB-deficient cells displayed deregulated cell growth, resulting in aggressive recovery from DNA damage. Strikingly, while disruption of the RB and p53 pathways alone does not result in transformation, the RB-deficient, recurred cell populations were tumorigenic. These in vitro findings indicate that RB is a critical suppressor of tumorigenesis, beyond that of p53, and a significant factor in the evolution of cells during genotoxic challenge. To further examine the cooperation of RB and p53 in vivo, a model of liver tumorigenesis was employed. Interestingly, these in vivo studies confirmed that while compound inactivation of the RB and p53 pathways in liver tissue promoted deregulated cell cycle control, this was not sufficient for transformation. However, loss of RB and p53 cooperated in promoting aberrant cell proliferation and mitosis in the presence of genotoxic challenge by DEN (diethyl-nitrosamine), ultimately resulting in enhanced tumorigenesis. Strikingly, loss of RB alone promoted S-phase entry as a result of genotoxic challenge, but there was an uncoupling of this event from productive mitosis. In contrast, the combined loss of RB/p53 resulted in further deregulation of DNA replication that was accompanied by a productive mitotic entry. These data indicate that, while both RB and p53 can contribute to appropriate replicative control, p53 plays a dominant role in mediating G2/M regulation. Furthermore, RB/p53-deficient livers displayed rapid progression to highly aggressive HCC within the span of six months, which was not observed in either RB-deficient or p53-deficient livers. Combined, these studies indicate that RB and p53 play distinct and cooperating roles in modulating response to genotoxic stress and tumorigenesis. Discovering the mechanism behind this cooperation could uncover a critical facet of tumor etiology and provide a means by which to treat this complex disease.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3870.
Abstract 3876: Cell cycle checkpoint in vivo in developing mouse embryo liver cells
Authors: Chan Kwok Kin , Wang XiaoQi
Published: February 2011

Embryonic stem (ES) cells have a distinct cell cycle distribution and cyclins/CDKs expression. ES cells are characterized by a short G1 phase and a high proportion of cell in S and G2 phase, maintain consistent level of cyclins expression seems not to be phase dependent. However, most cell cycle studies are on mouse or human ES cell line, whether that it is really reflective in vivo is not well defined. By extracting fresh liver cells from mouse embryo, we have investigated cell cycle distribution, correspondent cyclins and CDK expression in vivo in mouse embryo from embryonic days 10.5(E10.5, liver start to develop at E9.5) to post-natal 21 days(P21). We found that most of the cells (62%) were in S and G2 phase, 38% of cells in G1 at E11.5. Upon embryo development, the proportion of cells in G1 increased, although there were still large proportion of cells in S and G2 phase (55%) at P0. The short G1 phase in early embryonic day suggests that cell phase distribution may be related with embryo cells’ pluripotency in vivo. Distinct high levels of cyclin D1, E1, A, B1 and CDK1(cdc2) expressed at E10.5, which seemed to be pluripotency related but not cell phase dependent. The cyclins and CDK level started to drop from E11.5 to post-natal (P0) suggesting that cyclins/CDKs expression reduced and became regulated with differentiation. In ES cell line, where G1 population increased with differentiation, G1 DNA-damage checkpoint was still absent. However, in response to ionizing irradiation, P0 mouse liver cells showed a short G2 population increase followed by G1 cell phase arrest indicating that at P0 or even earlier stage, G1 DNA-damage checkpoint had been established in vivo. Thus, cell cycle and cell cycle checkpoint in vivo in mouse embryo liver cells displayed both similar and differential pattern compared to that in mES cell line in vitro. Underlying mechanism such as DNA-damage checkpoint patterns and its regulation will be further discussed.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3876.
Abstract 3879: YAP (Yes-associated protein), a proposed tumor suppressor in breast cancer, is regulated by cyclin D1
Authors: Lehn Sophie , Tobin Nicholas P. , Landberg Göran
Published: February 2011

In breast cancer, cyclin D1 over-expression and amplification has been linked to a worse prognosis. Conversely, lower levels of cyclin D1 have also been correlated to an unfavorable outcome. We have in our previous studies observed that lower levels of cyclin D1 correlate to a more migratory phenotype of breast cancer cells. In order to specifically delineate the possible effectors downstream of cyclin D1, we performed a whole-genome microarray experiment where cyclin D1 was downregulated in MDA-MB-231 cells. To avoid selecting for pure cell cycle-dependent changes, the cell cycle-related binding partners of cyclin D1; cyclin-dependent kinases 4 and 6 (Cdk4/6), were also downregulated and included in the microarray experiment.

One of the top downregulated genes upon cyclin D1 silencing was YAP, a recently proposed tumor suppressor gene implicated in regulation of breast cancer cell migration. We set out to clarify the link between cyclin D1 and YAP, both in an in vitro setting and by examining the expression of the two proteins in a clinical material of 179 primary invasive breast cancers.

To validate the microarray results, cyclin D1 and Cdk4/6 protein levels were transiently silenced up to 96 h in MDA-MB-231 and MCF-7 breast cancer cells using siRNA, and YAP protein levels were observed by western blot. The clinical material was stained using YAP and cyclin D1 antibodies, and intensity of the signal was scored. Also, chromogenic in situ hybridization was performed allowing for assessment of amplification of the CCND1 gene.

Transient downregulation of cyclin D1 in MDA-MB-231 and MCF-7 cells resulted in decreased YAP protein levels, as predicted by the microarray results. Downregulation of Cdk4/6 did not result in decreased YAP levels. In the clinical material, cyclin D1 intensity was inversely correlated to YAP intensity (Spearman's rho −0.220, P=0.031). Amplification of CCND1 was significantly correlated to none or low intensity of YAP expression.

We conclude that on protein level, the silencing of cyclin D1 decreases levels of YAP independently of cyclin D1's cell cycle-dependent association with Cdk4/6. On the chromosomal level, CCND1 gene amplification is correlated to loss of YAP expression which might explain the inverse correlation seen between cyclin D1 and YAP in the clinical material. Studies to further characterize the link between cyclin D1, YAP and migration are ongoing.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3879.
Abstract 3890: Induction of cell cycle arrest and apoptosis in mantle cell lymphoma by AZD5438, a novel cyclin-dependent kinase inhibitor
Authors: Lo Wann-Cheng , Growcott Jim , LeBrun David P. , Greer Peter A.
Published: February 2011

Mantle cell lymphoma (MCL) is recognized as a distinct subtype of B-cell lymphoma and it is characterized by cyclin D1 over-expression, which is caused by t(11;14)(q13;q32) translocation and unregulated cell cycle progression driven by increased cyclin-dependent kinase (CDK) activity. Accordingly, small inhibitors for CDKs represent a potential therapy for MCL. We are investigating a selective small molecule CDK inhibitor, AZD5438, which has nanomolar ranges of IC50 for cyclin E/CDK2, cyclin A/CDK2, cyclin B1/CDK1 and cyclin T/CDK9 and comparing its anti-tumor effects with a pan-CDK inhibitor, Flavopiridol. We hypothesize that inhibition of CDK1/2 by AZD5438 may lead to G1/S and G2 arrest, and therefore inhibit tumor cell proliferation and induce apoptosis in MCL. In Jeko-1 MCL cell lines, AZD5438 has shown both time- and dose-dependent inhibition of cell proliferation, inhibition of the phosphorylation of CDK substrates, and cell cycle arrest in G1 phase. The EC50 for AZD 5438 is 1μM as determined by inhibition of cell viability. We further developed an in vivo MCL xenograft assay: first, to guide the optimization of dosage and dosing schedule for clinical use; and second, to explore the selectivity and potency of this drug using phospho-histone H3 (Ser10) flow cytometry and phosopho-Rb (Ser249/Thr252) immunoblotting as our drug efficacy monitoring system. Recently we have established GFP transduced Jeko-1 cells and inoculated them into 8 to 12 week-old, immune-compromised NOD SCID mice. Investigations of AZD5438 drug dosage in this xenograft model are ongoing. Ultimately, this project is aimed at improving the treatment for MCL patients and reducing the toxic responses to the standard chemotherapeutic regimes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3890.
Abstract 3891: Reduced tumor formation by forced expression of cyclin-dependent kinase 6 in mouse epidermis
Authors: Wang Xian , Sistrunk Christopher , Rodriguez-Puebla Marcelo
Published: February 2011

The Cyclin-Dependent kinases (CDKs) are a family of essential serine-threonine kinases that are cell cycle regulated by association with cyclins at specific time-points during the cell cycle. Specifically, D-type cyclins form complexes and activate CDK4 and CDK6. We have previously determined that overexpression of CDK4 in mouse epidermis increases malignant progression in chemically induced skin tumors. In order to study the role of CDK6 in squamous epithelial tissues, we generated transgenic mice overexpressing CDK6, driven by the regulatory sequence of the keratin 5 promoter (K5CDK6). Keratinocytes from K5CDK6 mice have similar levels of proliferation compared to wild type mice, however, contrary to the role of CDK4 in mouse skin tumorigenesis, forced expression of CDK6 results in strong reduction of the multiplicity (papillomas per mouse) and incidence of skin tumors. Tumors derived from K5CDK6 or the wild type sibling show no histological differences nor changes in the rate of malignant progression, suggesting that CDK6 expression affects early stages of skin carcinogenesis. Our previous results have also shown that CDK6 preferentially binds to cyclin D3, which overexpression also decreases tumor development in mouse epidermis. Interestingly, lack of Cyclin D1 produces similar phenotype upon chemical carcinogenesis treatment. Thus, we hypothesize that overexpression of Cyclin D3 or CDK6 in a Cyclin D1-null background should synergize with the reduced tumor development previously observed in cyclin D1-null mice. As expected, a lower number of tumors were found in K5-cyclin D3/cyclin D1−/− compound mice compared to cyclin D1−/− siblings. Thus, we hypothesized that the reduced tumor development observed in these mice is a result of the CDK6/Cyclin D3 complex activity. Consequently, K5-CDK6/cyclin D3−/− compound mice are being generated to investigate whether CDK6-associated tumor inhibition depends on cyclin D3 expression.

Only in few cases, CDK6 has been associated with tumorigenesis as compared to CDK4 and CDK2, in particular, no association of CDK6 with epidermal tumors has been reported so far. We propose that CDK6/cyclin D3 complexes play an important role in maintaining normal epidermal homeostasis. Therefore we speculate that upregulation of one or both of its components will block tumor development which could be use as a therapeutic approach.

Supported by NIH grant CA116328.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3891.
Abstract 3920: EphA2 transcription is regulated by activation of Ras / MEK2 / cytoplasmic ERK pathway in pancreatic cancer
Authors: Levin Pavel A. , Arumugam Thiruvengadam , McConkey David J. , Reddy Shrikanth , Logsdon Craig D.
Published: February 2011

Background: We have previously shown that the EphA2 receptor is overexpressed in pancreatic cancer. Silencing of EphA2 receptor in combination with gemcitabine decreases tumor growth and metastases in a mouse model of gemcitabine resistant pancreatic cancer. However, regulation of EphA2 expression in pancreatic cancer is unknown. The objective of this study was to determine a possible relationship between Ras/MAPK pathway and EphA2. Methods: Pancreatic cancer cell lines, MPanc96 and MiaPaca-2, were used in this study. Ras was silenced with 2 different siRNA constructs; EphA2 protein and RNA expression levels were measured. ERK involvement in EphA2 regulation was analyzed by treating cells with 2 inhibitors for MEK (U0126 and PD 98059) and siRNA specific for either MEK1 or MEK2. Effect of MEK inhibition on EphA2 mRNA stability was examined by blocking mRNA synthesis with actinomycin D and measuring mRNA degradation by Q-PCR. In order to investigate the role of subcellular localization of ERK in regulation of EphA2 expression, we overexpressed cytoplasmic anchor protein PEA-15, which binds to ERK and sequesters it to the cytoplasm. Results: Silencing of mutant K-Ras decreased EphA2 mRNA and protein levels. EphA2 mRNA and protein levels decreased in a time- and dose-dependent manner after treatment with MEK chemical inhibitors. Inhibition of MEK activity did not affect EphA2 mRNA stability. Silencing of MEK2 but not MEK1 decreased EphA2 mRNA levels. Overexpression of PEA-15 depleted nuclear ERK, indicated by the absence of phosphorylated Fra-1. Depletion of the nuclear ERK did not affect EphA2 transcription, suggesting that EphA2 is regulated through the cytoplasmic ERK. Conclusions: These data suggest that EphA2 levels are regulated by the RAS / MEK2 / cytoplasmic ERK pathway via regulation of EphA2 mRNA transcription.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3920.
Abstract 3925: RNF138, a novel ubiquitin E3 ligase is overexpressed in colon cancer cell lines and interacts with RAD51D
Authors: Yard Brian D. , Bedenbaugh Michael K. , Pittman Douglas L.
Published: February 2011

The RAD51 protein family provides core components of the Homologous Recombination (HR) complex, which is critical for repairing DNA double-strand breaks and maintaining genome stability. The purpose of this study was to identify proteins that regulate HR processes through post-translational modification of the RAD51 paralogs. A yeast two-hybrid screen was performed using the RAD51 paralogs RAD51C, RAD51D, and XRCC2 as bait against a normalized universal mouse cDNA library. Fifty-eight primary candidates were isolated, and five are predicted to function in the ubiquitination pathway. Within this group, Ring Finger Protein 138 (RNF138) is of particular interest since RNF138 is a novel E3-ubiquitin ligase having both an N-terminal RING finger domain and a putative C-terminal ubiquitin interaction motif. Presence of both domains indicates RNF138 induces the transfer of ubiquitin to target substrates and also recognizes ubiquitin moieties on other proteins. Expression of RNF138 is increased by 12-fold in the HCC38 breast cancer cell line and 4-fold in SW-48, RKO, and Wi-DR colon cancer cell lines compared to non-tumorigenic controls. Yeast two-hybrid results indicate that RNF138 interaction is unique to RAD51D, as RNF138 failed to directly interact with RAD51C and XRCC2. Further analysis using N- and C-terminal truncated RAD51D constructs revealed that the RAD51D core domain rather than the N-terminal linker region that governs the interaction between RAD51D and XRCC2 mediates the association with RNF138. Moreover, K113A and K113R mutations of the highly conserved RAD51D Walker A ATP binding motif abolished RAD51D-RNF138 association, suggesting that ATP binding and hydrolysis may regulate the interaction. As further demonstration of this association, Myc-RNF138 co-immunoprecipitated with HA-RAD51D following transient transfections. Future studies will determine whether RNF138 is directly required for HR repair and if the mechanism specifically involves RAD51D ubiquitination. Understanding the regulation of HR proteins by ubiquitination processes will provide important insights into cancer progression and guide the identification of new therapeutic strategies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3925.
Abstract 3928: KU-0060019, a novel small molecule inhibitor of ATM
Authors: Shea Kerry F. , Albertella Mark , Bárdos Julia , Batey Michael A. , Cranston Aaron , Curtin Nicola , Griffin Roger , Lau Alan , Menear Keith , Newell David , O'Connor Mark , Richardson Caroline , Jones Louise , Smith Graeme , Thömmes Pia , Zhao Yan , Martin Niall
Published: February 2011

Ataxia telangiectasia mutated (ATM) is an important serine/threonine kinase involved in the DNA damage response and cell cycle checkpoint control. The 350kDa protein is a member of the PI3-kinase like kinase (PIKK) family and is mutated in the neurodegenerative disorder, Ataxia telangiectasia.

ATM exists in the inactive form as a dimer but following DNA damage is phosphorylated at a serine residue at position 1981 and dissociates to become an active monomer. It detects DNA double strand breaks (DSBs) through the Mre11/Rad50/NBS1 (MRN) complex and signals this information downstream to the cell cycle machinery, for example by the phosphorylation of p53 (Ser15) and Chk2 (Thr68).

ATM is a key signalling molecule for DNA double strand breaks that can regulate the homologous recombination repair pathway. Inhibition of ATM activity sensitises cells to the cytotoxic effects of DNA damaging agents such as some chemotherapies or ionising radiation that either directly or indirectly induce double stand breaks.

Here we present the biological profile of KU-0060019, a potent and specific novel small molecule inhibitor of ATM kinase activity. The compound has an IC50 value against the enzyme of 5 nM with above 1 μM IC50 value against DNA-PK and inhibits cellular phosphorylation of p53 at serine 15 with an EC50 value of 150 nM. It sensitises cells to chemotherapeutics, in particular topoisomerase II inhibitors, as well as radiotherapeutics in vitro in a variety of cancer cell lines. We also demonstrate potentiation of chemotherapeutics in in vivo mouse xenograft tumour models. Using the novel ATM inhibitor KU-0060019 these studies demonstrate the potential of targeting ATM as a sensitising mechanism by a small molecule in cancer therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3928.
Abstract 3940: Determining the mitochondrial DNA repair capacity in peripheral blood mononuclear cells using QPCR
Authors: Fonseca-Williams Sharon , Ayala-Torres Sylvette , El-Zein Randa , Torres-Ramos Carlos A.
Published: February 2011

Mutations in mitochondrial DNA (mtDNA) have been described in many cancer types. The functional significance of these mutations is not totally understood, but it has been suggested that mtDNA mutations may lead to increased ROS production, aerobic glycolysis and growth promotion in cancer cells. Increased mutagenesis in mtDNA may be due to decreased mtDNA repair capacity. However, there are a limited number of methods to specifically determine mtDNA repair capacity. The goal of this work is to apply a gene-specific PCR-based assay that can detect the presence of a variety of lesions in the mtDNA as a tool to determine the mtDNA repair capacity of peripheral blood mononuclear cells (PBMCs). The rationale of the QPCR assay is that lesions that stall the thermostable DNA polymerase on the DNA template during the PCR reaction will lead to a decrease in amplification of the fragment of interest. Because the DNA polymerase amplifies only undamaged templates, amplification is inversely proportional to the presence of damage. We performed dose-response and time-course experiments using the oxidizing agent H2O2 to define the optimal conditions of the QPCR assay to be used to determine mtDNA repair capacity in PBMCs from healthy human subjects. Our dose-response experiments show a dose-dependent reduction in the amplification of the mtDNA amplicon. In these experiments, exposure of PBMCs to 0.1 mM H2O2 for 1-hour resulted in a 50% decrease in the amplification of the mtDNA fragment. Time course experiments showed that 24 hours after treatment there was still a 50% reduction in the amplification of the mtDNA amplicon, suggesting that mtDNA lesions were still present. However, we observed that 48 hours after treatment, the amplification of the mtDNA amplicon reached levels similar to those from untreated control cells. These results indicate that human PBMCs take 48 hours to repair the mtDNA damage induced by 1-hour exposure to 0.1 mM H2O2. These results define the conditions to compare mtDNA repair capacity in PBMCs from cancer patients and healthy subjects. Sponsored by 5U54CA096297-07.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3940.