bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A13: Decitabine treatment of c-Myc transformed cancer cells is augmented by the removal of the checkpoint kinase Chk1
Authors: Höglund Andreas , Nilsson Jonas A.
Published: May 2012

Decitabine, or DacogenTM (also known as 5-Aza-2′-deoxycytidine), is a FDA approved drug currently in clinical use against myelodysplastic syndrome, a pre-stage of acute myeloid leukemia (AML). Decitabine is a DNA methyltransferase inhibitor which causes rapid genome-wide demethylation, reactivating genes silenced during tumorigenesis. We have recently shown that Decitabine is a valid therapeutic agent in treatment of c-Myc transformed tumor cells and that overexpression of c-Myc sensitized mouse embryonic fibroblasts to Decitabine treatment. In vivo modeling using cells overexpressing c-Myc showed a delayed tumor development in animals treated with a single dose of Decitabine. Futhermore, Decitabine treatment was shown to cause DNA damage that was further relayed in a p53-independent manner through the checkpoint kinase Chk1. In the light of this, we decided to combine Decitabine treatment with a commercially available Chk1 inhibitor (SB218078). In this study we show that combinatorial treatment with Chk1 inhibitor and Decitabine augments the separate treatment regimes. Furthermore, we also show that RNAi against Chk1 sensitizes tumor cells from the λ-Myc transgenic mice to Decitabine treatment. This demonstrates the therapeutic possibilities of using Chk1 inhibitors in combination with DNA damage agents in treatment of c-Myc transformed cells.

Citation Information: Cancer Res 2009;69(23 Suppl):A13.
Abstract A16: Requirement of functionally active BRCA2 protein for the expression of IRF9-regulated genes in human breast cells
Authors: Mittal Mukul K. , Chaudhuri Gautam
Published: May 2012

Interferons are cytokines that induces antiviral, immunomodulatory and antiproliferative effects to cells by inducing the expressions of many effector proteins. Type I interferons are often used to treat advanced stages of breast cancer. Unfortunately, in many cases interferon treatment does not produce any significant effect. We found that transient ablation of the breast cancer susceptibility gene BRCA2 in the human breast cells impairs the expressions of many type I interferon regulated genes. Thus, it appears that type I interferons need functional BRCA2 for their actions. We tested the promoter activity of several of the interferon target genes, such as GBP1, GBP2, ISG15, MX1, MX2, PKR etc. in the BRCA2 knocked-down human breast cells with or without added INF-alpha2a. The interferon-stimulated activity of the promoter decreased significantly in the BRCA2 knocked-down cells, further suggesting a possible role of BRCA2 in the interferon signal transduction pathway. Over expression of BRCA2 in the BRCA2-deficient pancreatic cancer cell Capan1 significantly increased the interferon-stimulated activity of the IFN-alpha-regulated gene promoters in these cells. We tested whether BRCA2 helps mediating the interferon pathway by interacting with the components of this pathway. The classical signal transduction pathway for the type I interferons involve the formation of the ternary complex ISGF3 among phosphorylated STAT1 and STAT2 with IRF9. Immuno-pull-down experiments revealed the binding of STAT1, STAT2 and IRF9 with BRCA2 in the human breast cells. Our study thus proposes a not-known-before role of the breast cancer susceptibility protein BRCA2 in the type I interferon pathway. Supported by the generous funding from US Department of Defense IDEA grant #W81XWH-08-1-0446 to GC.

Citation Information: Cancer Res 2009;69(23 Suppl):A16.
Abstract A17: Transforming growth factor beta stimulates STAT3 tyrosine phosphorylation and cellular invasion through interleukin-6 in human breast cancer cell lines
Authors: Parker Nicole NT , Law Mary E. , Müller-Newen Gerhard , Corsino Pat , Davis Brad , Jahn Stephen , Law Brian K.
Published: May 2012

The signaling mechanisms involving Transforming Growth Factor β (TGFβ) and Signal Transducer and Activator of Transcription 3 (STAT3) are characterized in breast cancer, but the mechanism of crosstalk between these two pathways is poorly understood. In this study we addressed the question of TGFβ-STAT3 cross-signaling in nontransformed epithelial cells and in human breast cancer cell lines.

Breast cancer continues to be a serious disease in the United States. A large component of novel chemotherapeutics specifically target overexpressed or overactivated proteins in cancers. Two examples of these oncogenic molecules are the cytokine TGFβ and the transcription factor STAT3. Although these proteins have each been implicated in early and late stages of cancer and invasion, the mechanism of cross-talk between the TGFβ and STAT3 pathways has not been well characterized.

We have observed that TGFβ treatment is sufficient to induce phosphorylation of STAT3 on its activating Tyr (705) site in nontransformed mouse mammary NMuMG epithelial cells. The time dependence of this relationship implies de novo expression of an intermediate factor that data suggest is Interleukin-6 (IL-6). TGFβ treatment induces gene upregulation of IL-in a time-dependent manner similar to that of TGFβ stimulated STAT3 tyrosine phosphorylation. Inhibition of IL-6 with the mIL-6 Receptor Fusion Protein Inhibitor (mIL-6.RFPI) abrogated TGFβ stimulation of STAT3 tyrosine phosphorylation. Further, TGFβ treatment is sufficient to induce cellular invasion in NMuMG cells. This effect was recapitulated by IL-6 treatment, while IL-6 inhibition markedly reduced TGFβ stimulated invasion.

The human breast cancer cell lines MDA-MB-231 and MDA-MB-361, which are basal-like and luminal breast cancer models, respectively, display varying levels of constitutive STAT3 tyrosine phosphorylation. The MDA-MB-361 cell line exhibits no endogenous STAT3 phosphorylation but responds to TGFβ treatment in the same manner as nontransformed NMuMG mammary epithelial cells. Conversely, the MDA-MB-231 cell line displays high levels of constitutive STAT3 tyrosine phosphorylation, which data suggest is dependent in part on TGFβ signaling and is completely dependent on IL-6 signaling. Taken together, interruption of the TGFβ-IL-6-STAT3 signaling loop in MDA-MB-231 cells is sufficient to abrogate this signaling and dramatically reduce endogenous cellular invasiveness, while induction of the TGFβ-IL-6-STAT3 pathway in MDA-MB-361 cells confers an invasive phenotype on this otherwise noninvasive cell line.

Citation Information: Cancer Res 2009;69(23 Suppl):A17.
Abstract A2: Oncogenic and drug-sensitive ERBB2 mutations in glioblastoma
Authors: Greulich Heidi , Chheda Milan , Chen Tzu-Hsiu , Tanaka Kumiko , Dutt Amit , Wong Kwok-Kin , Hahn William , Meyerson Matthew
Published: May 2012

The Cancer Genome Atlas (TCGA) Consortium recently completed a multidimensional analysis of glioblastoma, characterizing somatic alterations across a large sample set, and identifying genetic lesions that likely contribute to tumor fitness (TCGA Network, Nature, 2008). However, functional experiments are required to determine whether significantly altered genes are in fact oncogenes or tumor suppressor genes in glioblastoma. We experimentally analyzed mutations in one of the significantly mutated genes, ERBB2. Unlike ERBB2 mutations in lung adenocarcinoma, ERBB2 mutations in glioblastoma are primarily located in the extracellular region, clustering around the domain that mediates receptor dimerization. We have determined that a subset of the reported mutations are gain-of-function and oncogenic, require kinase activity for oncogenic transformation, and are sensitive to specific small molecule kinase inhibitors. This discovery has therapeutic implications for glioblastoma patients who harbor somatic ERBB2 mutations.

Citation Information: Cancer Res 2009;69(23 Suppl):A2.
Abstract A38: Human kinase screen for breast cancer oncogenes
Authors: Shrestha Yashaswi , Schafer Eric J. , Weir Barbara , Boehm Jesse , Thomas Sapana , Hahn William C.
Published: May 2012

The heterogeneity of breast cancer requires identification of novel genes that promote breast tumor development. Targeting such oncogenes therapeutically promises to improve clinical outcomes. We conducted an in vitro screen to identify human kinases that promote anchorage-independent growth of immortalized human mammary epithelial cells. The screen is based on a transformation model that requires cooperative activation of the PI3K/AKT and MEK/ERK pathways. The pooled screen identified three transforming kinases - PTK6, PAK1 and CAMK4. The functions of these candidates in the PI3K/AKT and/or MEK/ERK pathways are being currently studied to determine their role in breast tumorigenesis.

Citation Information: Cancer Res 2009;69(23 Suppl):A38.
Abstract A4: Guanylate binding protein-1 promotes glioma cell invasion through induction of MMP1 expression
Authors: Li Ming , Furnari Frank B. , Cavenee Webster K.
Published: May 2012

Glioma is the most common and deadly human brain tumor. Epidermal growth factor receptor (EGFR) amplification or constitutively active mutation (EGFRVIII) is one of the most frequent genetic lesions in gliomas. To identify target genes regulated by EGFR signaling, we performed expression array analysis and found that human guanylate binding protein-1 (GBP-1), a large GTPase that is induced by inflammatory cytokines, is one of the predominant genes induced by EGFR/EGF or EGFR VIII kinase activity. Our results demonstrate that EGF/EGFR signaling could dramatically induce Gbp-1 expression at both the mRNA and protein level through the p38 kinase signaling cascade that is distinct from Stat1 signaling used by interferon gamma. Interestingly, we show that Gbp-1 is required for EGFR-mediated matrix metallopeptidase-1 (MMP1) expression, and plays a role in glioma cell invasion in vitro. Overexpression of Gbp-1 induced MMP1 expression in glioma cells, which was independent of Gbp-1 GTPase activity. In contrast, siRNA-mediated knockdown of Gbp-1 inhibited MMP1 expression and in turn blocked cell invasion as well. Consistently, we found that GBP-1 is overexpressed and positively correlated with EGFR/EGFRVIII and MMP1 expression in human glioma clinical samples and glioma cell lines. Therefore, this study suggests that Gbp-1 may be a novel therapeutic target in glioma.

Citation Information: Cancer Res 2009;69(23 Suppl):A4.
Abstract A47: Fucoidan from Laminaria cichorioides inhibits AP-1 transactivation and cell transformation in mouse epidermal JB6 cells
Authors: Namgoog Gwang Mo , Cho Hae-Gook , Choi Hong Seok
Published: May 2012

Fucoidan, a sulfated polysaccaride extracted from brown seaweeds, has anticoagulant and antithrombotic activities. Unlike heparin, fucoidan is known to exhibit anticarcinogenic activities. However, the underlying molecular mechanisms of the chemopreventive activities of fucoidan are not understood. Here we report that fucoidan from L. cichorioides inhibited the epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic cell transformation, but had less cytotoxic effects on JB6 mouse epidermal cells. The EGF-induced phosphorylation of extracellular signal-regulated kinases 1/2 and c-Jun N-terminal kinases, and c-Jun was inhibited by fucoidan, resulting from the inhibition of phosphorylation of epidermal growth factor receptor (EGFR). Fucoidan dose-dependently attenuated the c-fos or c-jun transcriptional activity, and thereby inhibited the associated AP-1 transactivation activity. In vitro binding assay revealed that fucoidan directly interacted with EGF, suggested that anti-tumor promoting effect of fucoidan might be due to preventing the binding of EGF to its cell surface receptor (EGFR). These findings are the first to reveal a molecular basis for the anticarcinogenic action of fucoidan and may partially account for the reported chemopreventive effects of brown seaweeds.

Citation Information: Cancer Res 2009;69(23 Suppl):A47.
Abstract A52: Mechanisms of 1,25-dihdyroxyvitamin D-mediated growth arrest and apoptosis in transformed mammary cells
Authors: Keith Meggan E. , LaPorta Erika , Welsh JoEllen
Published: May 2012

1,25-Dihdyroxyvitamin D (1,25D), the biologically active form of vitamin D, exerts pro-differentiating and anti-proliferative actions on epithelial cells, but the exact role of the vitamin D receptor (VDR) in mediating these effects remains to be defined. The goal of this project is to link specific functional domains of the VDR with regulation of cancer cell proliferation, invasion, and apoptosis. Epithelial cell lines derived from DMBA induced mammary tumors generated in VDR knock-out (KO) mice and their wild-type (WT) littermates were characterized for growth arrest and apoptosis following 1,25D treatment. Cells derived from WT tumors expressed VDR and exhibited growth arrest and apoptosis in response to 1,25D. In VDR positive cells, 1,25D mediated apoptosis was associated with activation of multiple caspases and calpain, resulting in PARP cleavage. In contrast, cells derived from KO tumors lacked VDR and failed to undergo growth arrest and apoptosis upon 1,25D treatment. To recreate VDR signaling in KO cells, human VDR was stably expressed in the KO240 cell line, generating the KOhVDR cell line. KOhVDR cells expressed transcriptionally active VDR protein at levels comparable to that of WT cells. Re-introduction of VDR did not affect doubling time of KOhVDR cells in the absence of ligand, but did sensitize cells to growth arrest and apoptosis in response to VDR agonists. PCR screening arrays and independent qPCR validation were used to identify VDR target genes in this model system. In WT cells, 6-24h treatment with 100nM 1,25D induced PDGF-B (up to 10 fold) and VEGF-A (up to 5 fold). No changes in PDGF-B or VEGF-A expression were observed in KO cells treated with 1,25D, but both genes were inducible by 1,25D in KOhVDR cells stably expressing hVDR. Thus, 1,25D can activate either human or murine VDR to induce endogenous PDGF-B and VEGF-A gene expression in these murine cells. In summary, these data indicate that VDR does not exert ligand-independent effects on mammary cell growth, but is essential to trigger gene expression, growth arrest and apoptosis in response to 1,25D. Supported by NIH CA69700.

Citation Information: Cancer Res 2009;69(23 Suppl):A52.
Abstract A57: Nonsense-mediated decay escaping mutations in microsatellite-unstable colorectal cancer
Authors: Gylfe Alexandra , Niittymäki Iina , Ahonen Leena , Lehtonen Heli , Laakso Marko , Sirkiä Johanna , Nousiainen Kari , Pouwels Jeroen , Järvinen Heikki , Nuorva Kyösti , Mecklin Jukka-Pekka , Ristimäki Ari , Ørntoft Torben , Hautaniemi Sampsa , Karhu Auli , Kallio Marko , Aaltonen Lauri
Published: May 2012

Genomic instability drives tumorigenesis by allowing the accumulation of genetic alterations that provide cells with growth advantage. Microsatellite-instability (MSI) and the underlying mutator phenotype caused by a defect in mismatch repair (MMR) functions is the hallmark of Lynch syndrome, and is also observed in a subset of all colorectal cancers (CRC). In cells with a defective MMR system, spontaneous length changes of repetitive microsatellite sequences accumulate all over the genome at highly increased rates. At coding regions instability may lead to frameshift mutations and altered protein products. Genes that mutate this way under MMR deficiency giving selective advantage to cells in tumorigenesis are called MSI target genes. It is generally anticipated that the frameshift mutation-containing transcripts that lead to prematurely terminated proteins undergo nonsense-mediated decay (NMD), followed by a reduction in gene expression levels. However, when a premature stop occurs in the carboxyl-terminal end of the gene it might escape decay mechanisms, which may lead to either dominant-negative or oncogenic effects. Aim of this study was a genome-wide unbiased identification of new MSI CRC target genes that escape NMD. By combining bioinformatic search to expression profiling, we created a list of 330 genes that contained mononucleotide repeats from 6 to 10 base pairs and were likely to be translated despite potential mutations. A novel frameshift predictor software was developed to search all repeat-containing transcripts in the human genome that would escape NMD after one nucleotide deletion. To enhance the odds of identifying oncogenic mutants, the analysis was restricted to genes that were overexpressed in MSI CRC versus normal colonic mucosa. All of these genes were screened initially by sequencing the given repeat in a panel of 30 MSI CRCs.Whenever the mutation frequency exceeded 20% in the tumor set, which was considered evidence for possible selection in MSI tumorigenesis, an additional set of 70 MSI CRCs was sequenced. The great majority of the successfully sequenced genes had no mutations. Altogether four genes were mutated in over 20% of the samples in the extended 100 MSI tumor panel. These candidate driver target genes are being evaluated further by various methods, including sequencing of MSI CRC cell lines and microsatellite-stable (MSS) CRCs, statistical analyses, and functional in vitro experiments.

Citation Information: Cancer Res 2009;69(23 Suppl):A57.
Abstract A62: Phosphorylation of Ser205 by the protein kinase CK2 persists on Pax3-FOXO1, but not Pax3, throughout myogenic differentiation
Authors: Dietz Kevin N. , Miller Patrick J. , Hollenbach Andrew D.
Published: May 2012

The myogenic transcription factor Pax3 plays an essential role in early skeletal muscle development and is a key component in Alveolar rhabdomyosarcoma (ARMS), a childhood solid muscle tumor. ARMS, which is associated with a four year survival rate of only 17%, is most commonly characterized by a t(2;13) chromosomal translocation resulting in the fusion of the 5′ Pax3 sequences to the 3′ FOXO1 sequences to encode the oncogenic fusion protein, Pax3-FOXO1. Posttranslational modifications such as phosphorylation are common mechanisms by which transcription factors are regulated. Consistent with this fact, we demonstrated in a previous report that Pax3 is phosphorylated on Ser205 in proliferating, but not differentiated, primary myoblasts. However, the kinase that mediates this phosphorylation event has yet to be identified. More recently it was demonstrated that Pax3-FOXO1 is also phosphorylated at unidentified sites in non-physiologically relevant cells. Despite this information, it is not known whether Pax3-FOXO1 is phosphorylated at Ser205 or how the phosphorylation of the fusion protein changes during myogenic differentiation. In this report, we use standard in vitro kinase assays to identify CK2 (formerly termed “casein kinase II”) as the kinase responsible for phosphorylating Pax3 and Pax3-FOXO1 at Ser205 in proliferating mouse primary myoblasts. Furthermore, we use standard Western blot analysis to demonstrate that in contrast to wild-type Pax3, phosphorylation at Ser205 persists on Pax3-throughout early myogenic differentiation. Finally, we use standard Western blot analysis to show that Pax3-FOXO1 is phosphorylated at Ser205 in a variety of translocation-containing ARMS cell lines. The results presented in this report not only suggest a possible mechanism by which the disregulation of Pax3-FOXO1 may contribute to tumorigenesis, but also identifies a novel target for the development of therapies to be used for the treatment of ARMS.

Citation Information: Cancer Res 2009;69(23 Suppl):A62.
Abstract A71: Distinct functions of epithelial growth factor receptor (EGFR) pathways in pancreatic acinar cell differentiation and initiation of pancreatic ductal adenocarcinoma
Authors: Shi Guanglu , DiRenzo Daniel , Konieczny Stephen F.
Published: May 2012

Significance: Pancreatic ductal adenocarcinoma (PDA) is one of the most fatal malignancies, partially due to the lack of early detection methods.

Background: Efforts to understand the early stages of PDA progression have led to identification of Kras mutations as the initiating event and pancreatic intraepithelial neoplasias (PanINs) as one of the cellular precursors of PDA. Recently, several labs have demonstrated that PanINs can be generated from pancreatic acinar cells upon expression of Kras mutants in mouse models. This dramatic shift from differentiated acinar cells to duct-like PanIN cells, generally termed acinar-ductal metaplasia (ADM), is accelerated by loss of Mist1, an acinar-restricted basic helix-loop-helix transcription factor. Despite this recent progress, the transcription and signaling networks that regulate ADM remain largely unknown.

Results: To study these networks, we employed a collagen-based 3-dimensional (3D) culture model of ADM, where primary acinar cells can be transformed into duct-like cysts when treated with transforming growth factor-alpha (TGF-α). A similar acinar-to-ductal conversion was observed with primary acinar cells that express the KrasG12D mutant. Initial characterization of the ADM process revealed that the MAPK pathway is required in both settings. However, EGFR activity is required for TGF-α-induced conversion but not for KrasG12D-induced conversion. Consistent with previous data generated from mouse models, loss of Mist1 accelerates KrasG12D-induced conversion to ductal cysts in vitro. This accelerated ADM formation can be reversed by inhibition of EGFR, without affecting the basal level conversion induced by KrasG12D, suggesting that loss of Mist1 promotes transformation through an EGFR-dependent pathway. Further investigation has revealed that Mist1 directly activates transcription of the epidermal growth factor (EGF) gene, which encodes a classic ligand of EGFR.

Conclusion: These data suggest that EGFR has a complex role in this ADM model. EGFR activity either promotes maintenance of acinar differentiation, or cooperates with KrasG12D-induced transformation, possibly depending on the functional specificity of different ligands.

Citation Information: Cancer Res 2009;69(23 Suppl):A71.
Abstract A74: Estrogen receptor α-regulated growth and transcription is dependent on X-box binding protein-1 in breast cancer cells
Authors: Kiewlich David , Rosser Mary , Kretschmer Peter , Zajchowski Deborah
Published: May 2012

This study was designed to investigate the impact of X-Box Binding protein 1 (Xbp1) mRNA knockdown on the estrogen-responsive growth and gene expression in an estrogen receptor α (ERα) positive breast cancer cell line model. Xbp1 is a basic leucine zipper-containing transcription factor that plays a key role in the unfolded protein response (UPR), a cell program that is activated by unfolded or misfolded proteins in the endoplasmic reticulum. MCF7 cells, a model of ERα positive breast cancer, express high levels of Xbp1 and are dependent on estrogen for growth and ERα regulated transcription. Previous studies have revealed that further overexpression of Xbp1 in ERα positive breast cancer cells leads to estrogen independent ERα regulated transcription and anti-estrogen resistance, suggesting that Xbp1 expression influences ERα regulated transcription. Unknown is whether ERα requires Xbp1 as a cofactor for transcription. Here we show that Xbp1 mRNA knockdown by specific siRNAs prevents estrogen responsive growth of MCF7 cells as well as ERα regulated gene expression, while non-specific control siRNAs had little effect. These results, together with the high expression of Xbp1 seen in ERα positive breast cancers, but not in normal mammary ductal cells, suggests that high levels of Xbp1 enable ERα tumor cells to use estrogen as a growth factor. Thus, high levels of Xbp1 expression could represent an early transformation step leading to ERα positive breast cancer, and could explain why normal ERα positive breast ductal cells have not been established as cell lines.

Citation Information: Cancer Res 2009;69(23 Suppl):A74.
Abstract A79: Genome-wide RNAi screen for tetraploid-specific lethality in cancer cells
Authors: Kuroda Taruho S. , Dagher Regina K. , Pellman David
Published: May 2012

Cancer cells are often genetically unstable, resulting in aneuploid genomes. The consequences of aneuploidy are poorly understood, but have been suggested to play roles in tumor aggressiveness, chemotherapy resistance, and metastasis.

One important route to an aneuploid genome may be through doubling of the genome, leading to unstable tetraploid cells. Many cell division errors are ultimately manifest as a failure of cytokinesis. Especially in cells defective in p53 function, tetraploid cells proliferate, but generate both high rates of whole chromosome aneuploidy and defects in the maintenance of chromosome structural integrity. Our laboratory has demonstrated that, in p53-primary cells, tetraploidy promotes tumor development and the resulting tumors display markedly abnormal genomes (Fujiwara T. et al. Nature 2005, 437: 1043). It is now known that several human cancer-mutations — e.g. Rb loss or loss of the adenomatous polyposis coli tumor suppressor — also predispose cytokinesis failure and tetraploidy. These findings highlight a need to understand in detail the physiological changes that accompany tetraploidy.

To gain a comprehensive understanding of physiological alterations associated with tetraploidy in cancer cells, we have established tetraploid cell lines derived from a near diploid human colorectal cancer cell line HCT116. Using these paired tetraploid and diploid cell lines, we have performed a genome-wide RNAi screen to identify genes whose knock down selectively affect the viability of either diploids or tetraploid cells. We are currently validating the result of our ploidy-specific lethality screen and we would like to update our progress at this conference.

Citation Information: Cancer Res 2009;69(23 Suppl):A79.
Abstract A80: Somatic cancer mutations in human mtDNA show neutral and positive selection
Authors: Stafford Peter , Chen-Quin Estella B.
Published: May 2012

The majority of cancers exhibit somatic mutations in the mitochondrial chromosome (mtDNA). Whether these mutations play a causal role or are merely an effect is an issue of on-going uncertainty. To study the role of somatic mtDNA mutations in cancer, we present a meta-analysis of 356 tumor sequences of multiple cancer types from the literature. As tumor formation is a type of somatic evolution, we examine the mutation pattern for whether the cancer DNA variation is consistent with drift (random forces) or selection. We also compare the cancer mutation pattern to that observed at the organismal or population level in order to gain insight on the mutation process in cancer cells. Among germline sequences, all mtDNA genes show a strong deficiency of amino acid changes, reflecting negative selection during human history and consistent with functional constraints at the organismal level. In somatic cancer sequences, mtDNA genes show either relaxed negative selection relative to germline or a mutation pattern consistent with neutrality. ND3, CO3, and ND4L in particular show cancer missense mutation rates nine - 18 times that of their counterpart genes at the germline level. Bootstrap analysis of the cancer-specific missense mutations show Cytochrome B and ND6 to have changes occurring in positions of unusually high conservation, suggesting that tumors select for mutations in residues of high functionality. These results support relaxed negative selection or neutrality for somatic mtDNA mutations in cancer, with the later potential for positive selection of alterations that impair mitochondrial function.

Citation Information: Cancer Res 2009;69(23 Suppl):A80.
Abstract B1: Chemotherapy induces NEDP1-mediated destabilization of MDM2
Authors: Watson Ian R. , Li Bryan K. , Roche Olga , Blanch Alvaro , Ohh Michael , Irwin Meredith S.
Published: May 2012

MDM2 is an E3 ligase that promotes ubiquitin-mediated destruction of p53. Cellular stresses such as DNA damage can lead to p53 activation due in part to MDM2 destabilization. Here, we show that the stability of MDM2 is regulated by an ubiquitin-like NEDD8 pathway and identify NEDP1 as a chemotherapy-induced isopeptidase that deneddylates MDM2, resulting in MDM2 destabilization concomitant with p53 activation. Concordantly, RNA-imediated knockdown of endogenous NEDP1 blocked diminution of MDM2 levels and increased chemoresistance of tumor cells. These findings unveil the regulation of MDM2 stability via NEDP1 as a common molecular determinant governing chemotherapy-induced p53-dependent cell death.

Citation Information: Cancer Res 2009;69(23 Suppl):B1.
Abstract B17: Stepwise prostate carcinogenesis in vitro culture
Authors: Ke Xisong , Li Wencheng , Qu Yi , Hovland Randi , Rotter Varda , Akslen Lars A. , Øyan Anne Margrete , Kalland Karl-Henning
Published: May 2012

The loss of epithelial characteristics and the acquisition of a migratory phenotype, referred to as epithelial to mesenchymal transition (EMT), is a crucial event in tumor metastasis. Our previous work reported an EMT model based on primary prostate epithelial cells (EP156T) which gave rise to mesenchymal cells (EPT1) without malignant transformation. To achieve transformed prostate cells, EPT1 cells were kept growing in extended saturation density culture to select for cells overriding quiescence. Many foci formed in EPT1 monolayers after 4 weeks at confluence, but foci never appeared in EP156T monolayers in parallel controls. Cells from the foci formed robust colonies in soft agar suggesting full malignant transformation. The transformed cells were named EPT2. In this stepwise transformation model, EPT2 cells showed much higher abilities to proliferation at confluence, migrated better and were less dependent on exogenous growth factors than EP156T and EPT1 cells. Foci were more easily derived from EPT1 cells at later passages following EMT. Gene expression profiling showed progressive downregulation of CDH1 from EP156T, through EPT1 to EPT2 together with a number of additional changes. Our findings provide a novel stepwise transformation model according to which EMT can occur independently of transformation, but can promote subsequent malignant transformation in prostate carcinogenesis.

Citation Information: Cancer Res 2009;69(23 Suppl):B17.
Abstract B18: A role for E2f3 in DNA damage-induced apoptosis
Authors: Martinez Luis A.
Published: May 2012

The E2F transcription factors have emerged as critical apoptotic effectors. Herein we report that the E2F family member, E2f3a, can be induced by DNA damage through transcriptional and post-translational mechanisms. We demonstrate that the post-translational induction of human E2f3a is dependent on the checkpoint kinases. Moreover, we show that human E2f3a is a substrate for the chk kinases and that mutation of the chk phosphorylation site eliminates the DNA damage inducibility of the protein. Furthermore, we demonstrate that all three activating E2Fs (E2f1, E2f2 and E2f3a) are transcriptionally induced by DNA damage. siRNA knockdown of E2f3 in human cells or E2f3 genetic deficiency in mouse embryonic fibroblasts eliminates the DNA damage induction of the transactivating E2fs. Finally, using both in vitro and in vivo approaches, we establish that E2f3 is required for DNA damage induced apoptosis, regardless of p53 status. Thus, our data reveal the novel ability of E2f3 to function as a master regulator of the DNA damage response.

Citation Information: Cancer Res 2009;69(23 Suppl):B18.
Abstract B20: Acetaminophen enhances the cytotoxicity of cisplatin in vitro and in a subcutaneous tumor xenograft model of human ovarian carcinoma
Authors: Wu Yingjen Jeffrey , Neuwelt Alexander J. , Muldoon Leslie L. , Neuwelt Edward A.
Published: May 2012

Background: High dose acetaminophen causes hepatotoxicity by depleting glutathione and increasing production of toxic metabolites. The objective of this study was to examine if these mechanisms of acetaminophen can enhance the cytotoxicity of cisplatin in human ovarian cancer cells both in vitro and in vivo.

Methods: In vitro studies (glutathione concentration, cell viability and immunoblotting assays) were performed using SKOV3 human ovarian cancer cells cultured in combinations of acetaminophen and cisplatin. For the tumor model, female nude rats were pre-treated with cyclophosphamide (i.p. 100 mg/kg) one day prior to subcutaneous inoculation of SKOV3 cells with matrigel on the left flank. At 7 days after implantation, rats were randomly assigned into vehicle, cisplatin, and acetaminophen plus cisplatin treatment groups (n=3–5 per group). Subcutaneous tumor volume was measured by dial caliper. Acetaminophen pharmacology and the toxicity of treatments were monitored.

Results: In vitro, acetaminophen decreased SKOV3 cellular glutathione level by approximately 30% over 2 hr time frame. Acetaminophen combined with cisplatin decreased cell growth and viability and enhanced apoptosis compared to cisplatin alone in vitro. In vivo, high dose acetaminophen showed uniform tissue distribution, but only decreased glutathione in the liver but not in the serum, brain, or subcutaneous tumor of nude rats. Acetaminophen pretreatment 2–4 hr prior to cisplatin enhanced cisplatin chemotherapeutic effect as determined by a reduction in subcutaneous tumor volume by 15–25% (55–85% vs 40–60%). Serum BUN and creatinine levels were elevated by the combination of acetaminophen with cisplatin treatment when compared to cisplatin alone (48.2±10.9 vs 24.4±4.1 mg/dL; 0.65±0.06 vs 0.30±0.04 mg/dL; p<0.01).

Conclusions: Our results suggested that a chemotherapeutic regimen containing both high dose acetaminophen and cisplatin could be applied not only in hepatic tumors but also in ovarian or other tumor malignancies as a potentially promising approach for pre-clinical trial.

Citation Information: Cancer Res 2009;69(23 Suppl):B20.
Abstract B21: Defining the DNA damage response-mediated barrier to ErbB2-induced transformation of somatic mammary cells in vivo
Authors: Reddy Jay P. , Peddibhotla Sirisha , Bu Wen , Zhao Jing , Haricharan Svasti , Du Yi-Chieh Nancy , Podyspanina Katrina , Rosen Jeffrey M. , Donehower Larry A. , Li Yi
Published: May 2012

The DNA damage response (DDR) represents a signaling cascade, which is comprised of DNA damage sensors (such as γH2AX and 53BP1), ATM, Chk2, p53, among others, that becomes activated when confronted with DNA double strand breaks and other forms of DNA damage. The activation of the DDR, in addition to the ARF pathway, has recently been found to elicit apoptosis and senescence in response to oncogene activation. However, the significance of a DDR in blocking cancer progression has not been tested in breast carcinogenesis. Preneoplastic lesions in four germline transgenic models of breast cancer that express polyoma middle T antigen (PyMT), ErbB2, c-Myc, or H-Ras do not exhibit a DDR. We have previously reported a novel mouse model of mammary cancer, where oncogenes can be introduced into somatic mammary epithelial cells via retroviral infection of RCAS vectors into MMTV-tva mice (termed RCAS-TVA). Hyperplastic lesions in the mammary gland induced by somatic introduction of PyMT using the RCAS-TVA system display a robust DDR accompanied by increased p53 and apoptosis. This observation represents the first study, to our knowledge, to fully recapitulate DDR signaling in response to acute oncogenic stress in the in vivo mammary gland.

20–30% of all breast cancers display amplification of ErbB2, a cellular oncogene which, when activated in somatic murine mammary cells, forms sporadic tumors with a long latency. Like RCAS-PyMT, we found that p53 stabilization and apoptosis, as well as senescence, are induced by somatic activation of RCAS-ErbB2. In advanced RCAS-ErbB2-induced tumors, DDR signaling and ARF induction persist, yet p53 stabilization and apoptosis are lost. Surprisingly, attributes of senescence remain in some of the ErbB2 tumor cells. To demonstrate that the DDR is critical for oncogene-induced apoptosis and senescence, we generated RCAS-ErbB2 lesions in ATM-deficient mice. Strikingly, p53 stabilization, apoptosis, and senescence were no longer activated following ErbB2 activation in the absence of ATM signaling. These data suggest that a DDR plays a critical role in inhibiting breast carcinogenesis by inducing senescence and apoptosis, the latter, but not the former, of which is lost in progression to tumors. Furthermore, these findings indicate that therapies designed to bolster the activity of DDR signaling kinases and effectors may have a broad role in breast cancer prevention.

Citation Information: Cancer Res 2009;69(23 Suppl):B21.
Abstract B29: Inactivation of suppressor of fused alone is not sufficient to cause medulloblastoma
Authors: Satkunendran Thevagi , Ward Ryan J. , Dirks Peter B. , Hui Chi-chung
Published: May 2012

Hedgehog (Hh) signaling is essential for embryonic development and adult homeostasis. Aberrant pathway activity can result in various developmental disorders and tumors. The Hh receptor Patched (PTCH1) is a negative regulator of the pathway and acts as a tumor suppressor. We and others have shown that suppressor of fused (SU(FU)) and KIF7 are two major negative regulators of the pathway downstream of PTCH1.

Medulloblastoma (MB), the most common and aggressive pediatric brain tumor arising in the cerebellum, involves deregulation of Hh signaling. Similar to those observed in patients with PTCH1 mutations, Ptc1+/− mice spontaneously develop MB. In Ptc1+/− mice, the incidence of MB increases upon exposure to DNA damaging agents or a compounded loss of other tumor suppressor genes, such as p53. SU(FU) mutations have been found in MB, suggesting that SU(FU) is also a tumor suppressor in the cerebellum. However, we found that Su(fu)+/− mice do not develop any spontaneous MB, and that upon neonatal exposure to X-ray irradiation as well as a chemical carcinogen, these mice do not form any tumors. To examine the potential tumor suppressor function of Su(fu) in the brain, we performed conditional deletion of Su(fu) in neural progenitors. In mice, deletion of Su(fu) in neural progenitors using the Nestin-Cre line resulted in a disorganized cerebellum and motor defects. Unexpectedly, unlike Nestin-Cre;Ptc1f/- mice which developed MB, MB formation was not observed in Nestin-Cre;Su(fu)f/- mice. In contrast, similar to those reported previously, 100% of Su(fu)+/−;p53−/− mice developed MB between 2.5–4 months of age. Molecular analysis of primary tumor cell lines from these MBs revealed a loss of the wild type Su(fu) allele in the tumors. Together, these observations indicate that loss of Su(fu) alone is not sufficient for transformation into MB and suggest that other molecules, such as Kif7, may possess compensatory functions in the suppression of the tumor phenotype. Currently, we are examining the effects of Kif7 inactivation alone and in conjunction with Su(fu) inactivation on MB formation.

Citation Information: Cancer Res 2009;69(23 Suppl):B29.
Abstract B37: PSCA haploinsufficiency enhances prostate tumor growth in prostate-specific Pten knockout mice
Authors: Moore Miranda L. , Tang Shuai , Willingham Mark C. , Dubey Purnima
Published: May 2012

Prostate cancer is a heterogeneous disease, with multiple stages of cancer evident within an individual organ. Although some of the genetic changes necessary to initiate or advance prostate cancer have been identified, in order to develop effective therapies, it is critical to understand the different means by which this disease develops and progresses. Prostate Stem Cell Antigen (PSCA) is a GPI-anchored cell surface protein that is expressed in the normal epithelial cells of a restricted set of organs, including prostate, bladder, stomach, kidney and pancreas. Over-expression of PSCA is found in all stages of prostate cancer, from PIN to metastasis.

We are investigating the biological function of PSCA in prostate tumorigenesis. We recently reported that deletion of PSCA increases metastasis of TRAMP-induced prostate tumors in mice, suggesting that PSCA may function as a tumor suppressor. To determine the effect of modulating PSCA dosage in the context of another oncogenic signal implicated in human prostate cancer development, we crossed PSCA knockout mice to prostate-specific Pten knockout mice, and analyzed the animals at 30 weeks of age. Pten −/−;PSCA +/− mice developed significantly larger prostate tumors than Pten −/− mice that are wild-type for PSCA. These results also suggest that PSCA may function as a tumor suppressor. In contrast, mice with deletion of both copies of the PSCA gene (Pten −/−; PSCA −/− mice) developed tumors similar in size to Pten −/−;PSCA +/+ animals, consistent with the possibility that PSCA function may contribute to the early stages of cancer development.

Together, our results suggest that modulation of PSCA gene dosage affects cancer initiation and progression, indicating a putative dual role for PSCA in epithelial cancer. Our current experiments are designed to determine the molecular mechanism of PSCA function.

Citation Information: Cancer Res 2009;69(23 Suppl):B37.
Abstract B53: Post-translational modification of p53 by HDAC inhibitors leads to selective assembly of transcriptional complexes at the p21 locus
Authors: Keith James , Roy Somdutta , Tenniswood Martin
Published: May 2012

CG-1521 is a novel histone deacetylase (HDAC) inhibitor that stabilizes the acetylation of p53 at lysine 373, triggering G2/M phase arrest and apoptosis in LNCaP cells. This is in contrast to the well known HDAC inhibitor trichostatin A (TSA) which stabilizes the acetylation of lysine 382 on p53 and triggers G1/S phase arrest and senesence. The known p53 target gene, p21, contains two p53 response elements (RE) and 6 Sp1 binding sites in its promoter. Previous work in LNCaP cells indicates acetylated p53 isoform-modulated assembly of an active or inactive transcriptional complex at these p53REs in response to CG-1521 or TSA, respectively. Consistent with this finding, p21 mRNA levels were increased only upon treatment with CG-1521. Treatment with either HDAC inhibitor led to the upregulation of p21 protein levels, with a difference in the speed and duration of this increase. A second protein, p21B, coded within the exons and introns of the classical p21 protein, has been implicated in the apoptotic response, should the cellular DNA repair machinery fail to resolve a genomic insult. p21B appears to be controlled by a p53RE within the first intron of p21. This RE exhibits the same qualitative loading characteristics of the classical p21 p53RE in response to cellular stress and the acetylation state of p53, yet is temporally expressed later than p21. Using chromatin immunoprecipitation assays at narrow timepoints, we have determined the temporal loading characteristics of this region upon treatment with the HDAC inhibitors CG-1521 and TSA. Our results demonstrate a clear difference in the components of the basal transcriptional complex depending on the HDAC-stabilized acetylation state of p53. This indicates the acetylation state of the p53 protein is an important regulator of the cellular response to stress and suggests HDAC inhibitors may have an important role as cancer therapeutics beyond their traditional role in histone modification.

Citation Information: Cancer Res 2009;69(23 Suppl):B53.
Abstract B58: Neurofibromin: A required GAP in lens development
Authors: Carbe Christian J. , Zhang Xin
Published: May 2012

Human neurofibromatosis 1 (NF1) is an autosomal dominant genetic disease characterized by multiple discrete dermal neurofibromas as well as optic and other central nervous system gliomas. NF1 patients have a high incidence of additional clinical symptoms including cafe-au-lait spots, hamartomas in the iris (Lisch nodules), macrocephaly, short stature, seizures, and learning disabilities. Neurofibromin, the protein product of the Nf1 gene has been shown to function as a Ras-GTPase activating protein (GAP), thus making it a tumor suppressor protein. Neurofibromin may also regulate adenyl cyclase activity and intracellular cyclic-AMP generation. Here, we show that neurofibromin, as a functional GAP protein, is required for normal murine lens induction. Utilizing a previously developed strain of Nf1-deficient mice, we demonstrate that Nf1−/− embryos exhibit severe lens hypoplasia, and in some cases, anophthalmia. By analyzing RNA (Ras target gene expression) as well as target protein expression patterns (Phopho-ERK), we demonstrate that MAPK signaling is indeed upregulated in the lens region of the developing Nf1−/− embryo at E9, but down regulated after E10. Interestingly we observe a simultaneous down-regulation of transcription factors Pax6, and Sox2, both necessary for normal lens development after E10, which, is the same time we begin to observe poor or absent lens induction. However, the smaller deformed lenses that do form in the Nf1 mutants still express key lens marker proteins (alpha-A crystalline, Prox1), indicating that lens cell differentiation appears to occur normally after lens induction. Cell proliferation studies (BrdU immunohistochemistry) at both E9 and E10 revealed E9 Nf1−/− lens cell proliferation to be normal, however, significantly decreased at E10. In addition, TUNNEL staining performed at both E9 and E10 indicate normal apoptosis in the developing Nf1−/− lens. To test our hypothesis that increased MAPK signaling is the mechanistic cause of the hypoplastic lens, we initiated rescue experiments by injecting E10-pregnant females with MEK inhibitor UO126 and then analyzed the lenses of E12-harvested embryos. Our results do show that attenuation of the MAPK pathway does indeed rescue the mutant lens phenotype, thus confirming our assertion that neurofibromin is an essential GAP protein required for normal lens development.

Citation Information: Cancer Res 2009;69(23 Suppl):B58.
Abstract B64: Telomeric DNA sequence determines the mechanism of dysfunctional telomere fusion in human cancer cells
Authors: Stohr Bradley A. , Xu Lifeng , Blackburn Elizabeth H.
Published: May 2012

Cancer cells rely on the ribonucleoprotein enzyme telomerase to maintain their telomeres, which consist of tandem DNA repeats bound to protective protein factors collectively termed shelterin. Experimentally-induced telomere dysfunction in cancer cells, either through critical telomere shortening or disruption of shelterin binding, typically leads to telomere fusions, ultimately resulting in genome instability and cell death. For reasons that are not well understood, the mechanism of telomere fusion varies depending on the cause of telomere dysfunction. We have investigated telomere fusions caused by overexpression of mutant telomerases that add mutated telomeric repeats, thereby disrupting shelterin binding and inhibiting cancer cell proliferation. We previously showed that the effect of one such telomerase in human cancer cells is ATM dependent (Stohr BA and Blackburn EH, Cancer Research 68:5309–5317, 2008). Here, we have tested additional mutant telomerases that add either palindromic or non-palindromic telomeric repeats. We show that while all mutant telomeric sequences tested induce heterodicentric chromosome fusions, only palindromic telomeric repeats induce ATM-independent sister chromatid and isodicentric chromosome fusions in human cancer cells. Thus, the telomeric sequence itself influences the fate of telomeres that have become dysfunctional. These results implicate DNA strand annealing in an alternative telomere fusion pathway in human cells. The need to avoid genome instability caused by such fusions is one explanation for the characteristic lack of self-complementarity in naturally-occurring telomeric sequences. Finally, our results have significant implications for the translational application of mutant telomerases as a cancer therapeutic strategy.

Citation Information: Cancer Res 2009;69(23 Suppl):B64.