bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B59: Role of Nf1 signaling in regulating pigmentation in the mouse
Authors: Deo Mugdha A. , Fuchs Helmut , Hrabe Martin H. , Barsh Gregory S. , Van Raamsdonk Catherine D.
Published: May 2012

Introduction: Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominantly inherited disorders affecting 1 in 3500 individuals. The NF1 gene encodes a protein called neurofibromin that has a Ras-GAP activity and acts as a tumor suppressor by negatively regulating Ras. Loss of neurofibromin has a pleiotropic effect affecting a number of cell types. We are interested in using the elegance and sensitivity of the pigmentary system to understand the mechanisms by which Nf1 regulates cell survival and proliferation in vivo. We have found that an ENU-induced point mutation (Nf1Dsk9) or a targeted knockout of one Nf1 allele specifically and uniformly darken the mouse skin dermis. The Endothelin receptor B (Ednrb) is essential for melanocyte (pigment producing cell) survival in the dermis between E10–E12.5. Since Nf1Dsk9 causes uniform hyper pigmentation of the dermis, we wanted to know whether Nf1Dsk9 depends on Ednrb signaling to cause dermal hyper pigmentation. In an attempt to understand the effect of homozygous loss of Nf1 on melanocytes, we knocked Nf1 out specifically in melanocytes.

Methods: The Nf1Dsk9/+ mice were crossed to Ednrbs-l/s-l mice that have a complete deletion of the Ednrb gene. Pigmentation of the dermal tail skin and coat color spotting of 2–3 week old mice obtained from the above cross was quantified using ImageJ.

A melanocyte-specific knockout of Nf1 was made in vivo in the mouse by crossing existing Mitf-Cre line to the floxed-Nf1 line. Pigmentation of the dermal tail skin was quantified using ImageJ.

Results: A significant (p< 0.05, T-Test) hyper pigmentation of the dermis was observed in the Mitf-Cre/+; Nf1flox/flox homozygotes (now called Nf1mcko/mcko) with no change in epidermal pigmentation. In contrast to the Nf1Dsk9/+(germline heterozygote), the Nf1mcko/+ (melanocyte-specific knockout heterozygote) does not show dermal hyper pigmentation.

Comparison of the tail dermis of Nf1Dsk9/+;Ednrbs-l/s-l mice with +/+;Ednrbs-l/s-l mice showed that Nf1Dsk9 was unable to give a dark dermis in the absence of Ednrb. However, the Nf1Dsk9/+;Ednrbs-l/s-l mice showed a significant (p< 0.05, T-Test) increase in the percentage of coat that was pigmented.

Discussion: A significant darkening of the dermis in the Nf1mcko/mcko compared to wild type or Nf1mcko/+ littermates suggests a cell autonomous mechanism of dermal hyper pigmentation in the homozygote caused by the loss of Nf1 in melanocytes.

The Nf1mcko/+ heterozygote does not show dermal hyper pigmentation, unlike the Nf1Dsk9/+ heterozygote. This suggests that the effect of loss of Nf1 in melanocytes acts in a non-cell autonomous mechanism in the heterozygote.

The lack of dermal hyper pigmentation but an increased percentage of coat color spotting in the Nf1Dsk9/+;Ednrbs-l/s-l mice suggests that Nf1 regulates pigmentation by two independent mechanisms, one that depends on Ednrb and one that does not.

Citation Information: Cancer Res 2009;69(23 Suppl):B59.
Abstract B60: Suppressing malignant progression of prostate cancer by inhibiting expression of OPN and S100A4
Authors: Zhang Yu , Forootan Shiva S. , Foster Christopher , Ke Youqiang
Published: May 2012

The potential of using OPN and S100A4 as therapeutic targets has been tested in this study. Constructs expressing short silencing RNAs targeting OPN and S100A4 respectively were transfected into DU145 cells which express high levels of both OPN and S100A4 prior to the transfection. In the two established S100A4 transfectant lines, named SDRC2 and SDRC11 respectively, the level of S100A4 was reduced by 28- and 4- fold, compared with the control. In the two OPN-suppressed transfectants, named ODRC4 and ODRC10 respectively, the expression of OPN was either reduced by 8 times or almost completely suppressed. When tested in vitro, suppression of either OPN or S100A4 significantly inhibited the tumourigenicity and invasiveness, but not the motility and proliferation rate of the cancer cells. When inoculated in nude mice, all OPN-suppressed and S100A4-suppressed transfectants produced smaller tumors compared to the control group. Tumors produced by ODRC4 cells and ODRC10 cells showed significant reduction by 6- and 2.8- times in the average size compared to the control; and the difference between the average size of the tumors produced by OPN-moderately-suppressed ODRC4 cells and that produced by OPN-highly-suppressed ODRC10 cells was significant. The average size of tumors produced by S100A4-moderately-suppressed SDRC11 cells was reduced by 7.3 times while that produced by S100A4-highly-suppressed SDRC2 cells was only mild. Western blot and immunocytochemistry staining showed that the patterns of OPN and S100A4 expressions in tumor tissues were very similar to those correspondent cell lines inoculated. These results suggested that the inhibition of either OPN or S100A4 can suppress tumourigenicity of prostate cancer cells. While the tumor sizes appeared to be reduced as the reducing level of OPN, the tumor-suppressing and the S100A4-reduction did not exhibit a linear relationship. While the maximum tumor suppression was achieved when the level of S100A4 was reduced to the level similar to that expressed in benign cells, further S100A4 suppression did not appear to have any further effect on tumor size. Separate studies on the relationship between OPN and S100A4 indicated that S100A4 and OPN may promote the development and malignancy progression of prostate cancer in a coordinated manner. Our work suggested that both S100A4 and OPN may be potential targets for tumorgenecity-suppression.

Citation Information: Cancer Res 2009;69(23 Suppl):B60.
Abstract B61: Regulation of mammary stem/progenitor cells by p53
Authors: Tao Luwei , Roberts Amy , Dunphy Karen , Yan Haoheng , Jerry D. Joseph
Published: May 2012

Introduction: Breast cancer is the most frequent cancer among women in the United States. Understanding the biological behavior of mammary stem cells (MaSCs) and progenitor cells sheds light on the mechanism of mammary gland malignant transformation. The p53 tumor suppressor protein (TP53 in human, Trp53 in mouse) regulates cell growth and protects cells from malignant transformation and has been shown to also participate in regulation of tissue-specific stem cells.

Methods: Total mammary cells were isolated from 8–10 weeks old mice of different p53 genotype (Trp53+/+, Trp53+/−, Trp53−/−). Mammosphere assay were used to compare the self-renewal and ionizing radiation (IR) response of mammosphere-initiating cells. Limiting dilution and transplantation was done to compare the frequency of long-term regenerative mammary stem cells of Trp53+/+ and Trp53−/− epithelium. 3 weeks old BALB/c Trp53+/+ and Trp53−/− mice were injected with BrdU for 7 consecutive days and chased for 9 weeks. BrdU staining was done to compare the frequency of label-retaining cells in their mammary glands.

Results: Mammosphere assays showed that decreased p53 dosage in Trp53+/− and Trp53−/− mice led to increased numbers and size of mammospheres. Meanwhile, the number of secondary and tertiary mammospheres was not affected by (IR) regardless of their genotype. Serial dilution and transplantation experiments also showed that the Trp53−/− epithelium had significantly increased frequency of long-term regenerative MaSCs compared to Trp53+/+ epithelium. The BrdU labeling experiment showed that Trp53−/− mammary gland contains less label-retaining epithelial cells (LRECs) than Trp53+/+ epithelium.

Conclusions: Our data suggest that p53 negatively regulates the self-renewal of mammary stem cells. The MaSCs pool expands with decreased p53 dosage, which may results in a higher transformation risk. Meanwhile, the p53-mediated apoptosis pathway is compromised in the mammosphere-initiating cells, suggesting different functional status of p53 in cells of different differentiation stage.

Citation Information: Cancer Res 2009;69(23 Suppl):B61.
Abstract B62: WWOX expression suppresses tumorigenecity by inducing apoptosis and attenuating migration of metastatic cells
Authors: Abdin Suhaib , Salah Zaidoun , Aqeilan Rami
Published: May 2012

The WW domain-containing oxidoreductase (WWOX) spans one of the most active common fragile sites, FRA16D, involved in cancer. WWOX encodes a 46-kDa protein that contains two N-terminal WW domains and a central short-chain dehydrogenase/reductase (SDR) domain. Through its WW domain, Wwox interacts with its partners and modulates their functions; mainly by suppressing their transactivation functions through sequestering them in the cytoplasm. Wwox expression is absent or reduced in most cancer cells and its ectopic overexpression induces apoptosis in vitro and suppresses tumorigenecity in vivo suggesting tumor suppressor function of WWOX. Recently we demonstrated that targeted deletion of the Wwox gene in mice causes increased spontaneous tumor incidence confirming WWOX as a bona fide tumor suppressor. Furthermore, Wwox attenuates the migration and invasion ability of MDA-MB-231 breast carcinoma metastatic cells. In addition, its restoration results in reduced attachment and migration on fibronectin. By contrast, knocking down endogenous Wwox increases adhesion to fibronectin. Therefore, Wwox acts as a tumor suppressor not only by inducing apoptosis mediated by caspase activation but also through modulating the interaction between tumor cells and the extracellular matrix.

Citation Information: Cancer Res 2009;69(23 Suppl):B62.
Abstract B72: A computational approach integrating proteomic, transcriptional, and interactome data for discovery of cellular signaling networks
Authors: Huang Shao-shan Carol , Fraenkel Ernest
Published: May 2012

Cellular signaling and transcription are tightly integrated processes that have profound effects on every stage of tumor development and growth. Although there are now a variety of high-throughput methods for studying these processes, our knowledge of them remains fragmentary. Typically, the vast majority of hits identified by transcriptional, proteomic and genetic assays lie outside of the expected pathways. These unexpected components of the cellular response are often the most interesting, as they can provide new insights into biological processes and potentially reveal new therapeutic approaches. However, they are also the most difficult to interpret. We present a technique, based on the Steiner tree algorithm, that uses previously reported protein-protein interactions to determine how these hits are organized into functionally coherent pathways, revealing many components of the cellular response that are not readily apparent in the original data. Applied to simultaneously to phosphoproteomic and transcriptional profiling data for the yeast pheromone response, it identifies changes in diverse cellular processes that extend far beyond the expected pathways. We expect our current analysis of the experimental data from epidermal growth factor (EGF) signaling network in non-tumorigenic and tumorigenic cells will shed light on the regulation of cellular processes at the global level and the consequences of oncogenic mutations on signaling and transcription.

Citation Information: Cancer Res 2009;69(23 Suppl):B72.
Abstract C1: GLI1 induces CD133 expression in xenografts derived from transformed astrocytes but not in cultured cells: Extrinsic control of CD133 expression in gliomas
Authors: Sasai Ken , Nodagashira Miho , Tanaka Shinya
Published: May 2012

Overexpression of transcription factors is often observed in malignant gliomas and the roles of such factors in tumor malignancy have been widely investigated. However, the contribution of glioma-related transcription factors to tumor initiation are largely unclear, as the majority of brain tumor studies have employed glioma-derived cell lines, which recapitulate only a subset of late-stage human tumors. In contrast to tumor-derived cell lines, which harbor numerous undefined genetic elements, normal human cells allow us to generate experimental models of human cancers by direct manipulation with defined genetic elements. Here we demonstrate that the introduction of GLI1 is sufficient for immortalized human astrocytes to be transformed whereas FOXM1 fails to induce oncogenic transformation, suggesting that GLI1, but not FOXM1, directly contributes to gliomagenesis despite both transcription factors being overexpressed in malignant gliomas. While GLI1-transformed astrocytes have the ability to form tumors in nude mice, the oncogenic potential of GLI1 is subtle compared to the active form of H-RAS. To obtain insights into the mechanisms underlying less malignant features of GLI1-transformed astrocytes, expression of inhibitors of cyclin-dependent kinases in xenografts were analyzed by immunohistochemistry. We found that p27KIP1-positive cells were frequently observed in xenografts derived from GLI1-transformed astrocytes (GLI1-xenografts) compared to those from RAS-transformed cells (RAS-xenografts): more than 60% of cells were positive for p27KIP1-staining in GLI1-xenografts whereas only ∼20% in RAS-xenografts. We also made an interesting observation that CD133-positive cells were frequently detected in GLI1-xenografts whereas they were rarely seen in RAS-xenografts. The biological relevance and regulatory mechanisms of CD133 expression in human glioma are not completely understood although the use of CD133 as a marker to sort and enrich for brain tumor stem cells is common. Since GLI1 failed to induce CD133 expression in cultured astrocytes, it is possible that additional factors present in vivo and absent in culture might collaborate with GLI1 to modulate CD133 expression and that tumor microenvironment might contribute to maintenance of brain tumor stem cells. We propose that GLI1-tranformed astrocytes may prove to be useful tools as we strive to understand the molecular mechanisms underlying CD133 expression in gliomas as well as maintenance of brain tumor stem cells.

Citation Information: Cancer Res 2009;69(23 Suppl):C1.
Abstract C10: Neural stem cells and lineage-restricted progenitors can initiate malignant astrocytoma formation in somatic tumor suppressor mouse models
Authors: Alcantara Llaguno Sheila R. , Chen Jian , Kwon Chang-Hyuk , Jackson Erica , Li Yanjiao , Yan Jingsheng , Xie Yang , Burns Dennis K. , Johnson Jane E. , Alvarez-Buylla Arturo , Parada Luis F.
Published: May 2012

Malignant astrocytomas are infiltrative and incurable brain tumors. Despite profound therapeutic implications, the identity of the cell (or cells) of origin has not been rigorously determined. We previously reported mouse models based on conditional inactivation of the human astrocytoma-relevant tumor suppressors Nf1, p53, and Pten, wherein through somatic loss of heterozygosity, mutant mice develop tumors with 100% penetrance. In the present study, we show that tumor suppressor inactivation in neural stem cells induces malignant astrocytoma formation. Genetic targeting of lineage-restricted progenitors alone is also sufficient to initiate astrocytomas. We demonstrate in vivo that transformed cells and their progeny undergo infiltration and multi-lineage differentiation in situ during tumor development. Analysis of mutant mice several months prior to advanced disease shows defects in proliferation, differentiation, and migration, suggesting that these are requisite processes prior to tumor formation. Gene expression profiling in pre-symptomatic mutant mice provides insight into the molecular events involved during brain tumor development.

Citation Information: Cancer Res 2009;69(23 Suppl):C10.
Abstract C13: Targeting interleukin 6 signaling suppresses glioma stem cell survival and tumor growth
Authors: Wang Hui , Lathia Justin , Wu Qiulian , Wang Jialiang , Li Zhizhong , Heddleston John , Eyler Christine , Elderbroom Jennifer , McLendon Roger , Wang Xiao-Fan , Hjelmland Anita , Rich Jeremy
Published: May 2012

Glioblastomas are the most common and most lethal primary brain tumor. Recent studies implicate an important role for a restricted population of neoplastic cells (glioma stem cells (GSCs)) in glioma maintenance and recurrence. We now demonstrate that GSCs preferentially express two interleukin 6 (IL6) receptors: IL6 receptor alpha (IL6Ra) and glycoprotein 130 (gp130). Targeting IL6Ra or IL6 ligand expression in GSCs with the use of short hairpin RNAs (shRNAs) significantly reduces growth and neuro-sphere formation capacity while increasing apoptosis. Per-turbation of IL6 signaling in GSCs attenuates signal transducers and activators of transcription three (STAT3) activation, and small molecule inhibitors of STAT3 potently induce GSC apoptosis. These data indicate that STAT3 is a downstream mediator of prosurvival IL6 signals in GSCs. Targeting of IL6Ra or IL6 expression in GSCs increases the survival of mice bearing intracranial human glioma xenografts. IL6 is clinically significant because elevated IL6 ligand and receptor expression are associated with poor glioma patient survival. The potential utility of anti-IL6 therapies is demonstrated by decreased growth of subcutaneous human GSC-derived xenografts treated with IL6 antibody. Together, our data indicate that IL6 signaling contributes to glioma malignancy through the promotion of GSC growth and survival, and that targeting IL6 may offer benefit for glioma patients.

Citation Information: Cancer Res 2009;69(23 Suppl):C13.
Abstract C14: The thyroid hormone-sonic hedgehog cross-talk in the tumorigenesis of basal cell carcinoma cells
Authors: Dentice Monica , Luongo Cristina , Ambrosio Raffaele , Salvatore Domenico
Published: May 2012

The pro-hormone, thyroxine (T4), must be converted into T3 in order to be active. This metabolic process has been increasingly recognized as an essential mechanism that regulates thyroid hormone (TH) action at cellular level. TH action is regulated by the activity of the deiodinases, which can either activate (D1, D2) or inactivate (D3) the prohormone.

The Sonic hedgehog (Shh) pathway plays a critical role in tumor development, In skin, constitutive Shh signaling causes basal cell carcinoma (BCCs) the most common cancer in humans. We have recently demonstrated that Shh-Gli2 pathway induces D3 in human and mouse BCCs, however the role of D3 and T3-depletion in BCC tumor formation and maintenance is unknown. In the present study we tested the hypothesis that local control of TH action- through regulation of deiodinases- is a necessary step for the action of Shh pathway.

To this aim, BCC cells, G2N2c were grown in thyroid hormone deprived media (charcoal serum -CS), or CS plus exogenous T3 at different concentrations. We observed a reduction in the growing rate directly correlated to the concentrations of T3 in the media. Furthermore, in the same cells, T3 interferes specifically and directly with Gli2 expression, by reducing Gli2 protein level and activity. These data, together with the finding that Gli2 mRNA does not change in the same setting, indicate that T3 reduced Gli-2 protein at post-transcriptional level. Finally, to study the effect of thyroid hormone on the BCC tumor growth in vivo, we injected nude mice with BCC cells and treated the resulting tumors with a T3-based cream. Tumor weight, showed a significative reduction in tumors size when topically T3-treated.

In conclusion, we highlight a functional cross-talk between thyroid hormone and the Shh-signalling in BCC cells. This mechanism could have broad implications, not only to understand the general role of hormones, in particular T3, in the process of tumor formation and growth, but also may lead to the identification of novel therapeutic approaches aimed at interfering with these signals in a pathological context.

Citation Information: Cancer Res 2009;69(23 Suppl):C14.
Abstract C15: Combinatorial regulation of neuroblastoma tumor progression by HIF1α and N-myc transcriptional factors
Authors: Qing Guoliang , Mayes Patrick , Pawel Bruce , Martinez Daniel , Maris John M. , Simon M. Celeste
Published: May 2012

Deregulation of Myc and activation of hypoxia inducible factors (HIFs) frequently occur in solid tumors. In human neuroblastoma, amplification of the N-MYC gene predicts poor prognosis and resistance to therapy. Interestingly, we demonstrated here that HIF1α, but not HIF2α, is preferentially expressed in both N-MYC amplified neuroblastoma cells and primary tumors in comparison to non-amplified ones. Expression of HIF2α in N-MYC amplified cells appears to be silenced largely through DNA methylation and histone deacetylation. Interplay between N-Myc and HIF1α plays critical roles in progression of neuroblastomas. On one hand, N-Myc overrides the inhibitory function of HIF1α on cell cycle progression under hypoxia, which frequently occurs in solid tumors and is well known to inhibit cell growth. Of note, proliferation of N-MYC amplified cells is largely unaltered under hypoxia. On the other hand, HIF1α, together with N-Myc, cooperates to regulate aerobic glycolysis (the Warburg effect) of neuroblastoma. The near-universal glycolytic switch of N-MYC amplified neuroblastoma cells may be an essential tumor trait and a consequence of the cellular adaptation to intermittent hypoxia. Targeting either HIF1α or N-Myc significantly decreases expression of genes involved in glycolysis, such as lactate dehydrogenase A (LDHA), and inhibits cell proliferation in vitro under normoxia. Strikingly, N-MYC amplified cells are addicted to LDHA expression. Reduction in LDHA activity completely inhibits their proliferation under both normoxia and hypoxia. Taken together, our results demonstrate that neuroblastoma cells differentially respond to hypoxia with distinct HIFα induction patterns, and suggest the HIF1α/N-Myc axis may represent a pharmacologically tractable pathway for the potential treatment of N-MYC amplified neuroblastomas. In particular, we propose targeting LDHA may be a promising and nontoxic approach in treatment of N-MYC amplified neuroblastomas, given that depletion of LDHA expression completely inhibits growth of N-MYC amplified cells and individuals deficient in LDHA expression show no obvious symptoms under normal conditions.

Citation Information: Cancer Res 2009;69(23 Suppl):C15.
Abstract C16: Reactive oxygen species derivate of diethylnitrosamine metabolism participates in oxidation of thiol proteins
Authors: Aparicio-Bautista Diana Ivette , Gutiérrez-Nájera Nora Andrea , Arellanes-Robledo Jaime , Vásquez-Garzón Verónica Rocío , Jiménez-García Mónica Noemí , Pérez-Carreón Julio Isael , Villa-Treviño Saúl
Published: May 2012

Introduction: Conversion of Protein-SH into disulphides is an early event during the radical-mediated oxidation of proteins; this has prompted a great interest in the study of changes of these thiol proteins. The aim was to detect the most sensitive thiol proteins to reactive oxygen species generated by metabolism of Diethylnitrosamine (DEN) in the rat liver. DEN has been widely used to induce experimental hepatocellular carcinoma.

Methods: Twenty Male Fischer 344 rats were randomized into 4 groups: 3 groups were treated with DEN at 200 mg/kg weight. Each group was sacrificed at 6h (DEN 6), 12h (DEN 12) and 24h (DEN 24) after DEN administration. The last group was used as negative control (NC). To evaluate the formation of disulphide proteins in the liver, we compared the three DEN treated groups against the non treated rats. Total proteins were extracted and labeled with saturation dye according to method Redox-DIGE with some modifications, this method coupled to mass spectrometry was used to identify the differential oxidation protein profile.

Results: The analysis showed a tendency to increase the number of oxidized thiol proteins, through the time of DEN exposure together to a possible increase of proteins degradation. The differentially oxidized proteins at 6h, 12h and 24h were 407, 690, and 665 respectively; of those 6, 12 and 90 increased (up-oxidized) and 45, 12 and 51 decreased (down-oxidized), taking as reference two folds in their oxidation status respect to NC group. Some proteins were already identified from the different groups and according to functional classification, the identified proteins in our study are categorized as chaperone, oxidoreductase, activity isomerase, hidrolase, protein binding, cytoskeleton, activity transferase, among others.

Conclusions: DEN has an oxidative effect on thiol proteins; also, there is a tendency to increase the number of oxidized thiol proteins which depends of the time of DEN exposure and very likely related to the mechanism of carcinogenesis initiation. In addition to oxidation process, the proteins could be being degraded by DEN effect.

Citation Information: Cancer Res 2009;69(23 Suppl):C16.
Abstract C17: Diet-mediated enhancement of tumorigenesis in Drosophila
Authors: Hirabayashi Susumu , Baranski Thomas , Cagan Ross
Published: May 2012

Epidemiological studies have demonstrated that increased cancer risk is associated with metabolic disease including obesity and diabetes, but the underlying mechanism remains poorly understood. We have recently found that feeding Drosophila a high sugar-containing diet leads to a series of metabolic dysfunctions seen in human diabetes including hyperglycemia, increased insulin levels, and accumulation of fat. Here we demonstrate that a high sugar diet enhanced cell migration and invasion within Src-mediated tumor and massive overgrowth in multigenic Ras/Src-based tumor models. These diet-enhanced Ras/Src-based tumors produced aggressive metastasis-like behavior: transformed cells left the primary tumor to invade through the hemolymph to secondary sites that led to lethality of the animals. Genetic and inhibitor studies indicated that the insulin pathway plays a primary role in this diet-induced enhanced overgrowth through regulation of dTor. We further show that tumors with insulin pathway activation are resistant to dietary effects. Moreover, utilizing the diet-enhanced lethal phenotype, we developed a drug screening platform that relies on robotics as well as rescue-from-lethality. These studies should provide insights into mechanisms of the association between metabolic dysfunction and cancer progression and allow us to take the first steps towards identifying novel candidate therapeutic compounds.

Citation Information: Cancer Res 2009;69(23 Suppl):C17.
Abstract C18: A new IGF-I-regulated micronutrient carrier in cancer cells
Authors: O'Callaghan Katie , Ayllon Veronica , O'Keeffe Jean , Wang Yanru , Cox Orla , Loughran Gary , Forgac Michael , O'Connor Rosemary
Published: May 2012

Cancer cells are dependent on a continuous supply of nutrients to maintain cell proliferation and migration. Nutrients are acquired through transporters whose transcription, trafficking, and degradation are tightly regulated by growth factors, especially through activity of the Insulin/IGF-I-activated PI3K/mTOR signalling pathway. The intracellular trafficking of nutrient transporters requires an acidic endosomal pH that is maintained by the Vacuolar H+ ATPase (V-ATPase) proton pump. Recent studies have indicated a role for V-ATPase activity in cancer cells, but how this is regulated is not known. We recently isolated a series of new IGF-I-regulated genes whose expression is associated with cellular transformation (1, 2). Among these was a previously uncharacterized endosomal protein, which we determined associates with the V-ATPase, and which we called EVA. This protein was separately identified in C. elegans as a member of a family of heme transporters (HRG-1) that control heme homeostasis (3). We found that EVA is present throughout the endosome compartments; in early, recycling, and late endosomes, but not in lysosomes. Upon nutrient withdrawal EVA traffics to the plasma membrane. EVA interacts with the c subunit of the V-ATPase and enhances V-ATPase activity in isolated yeast vacuoles. Suppression of EVA expression increases endosomal pH and reduces V-ATPase holoenzyme assembly. This is accompanied by decreased migration, decreased transferrin receptor trafficking, decreased cellular heme uptake, and cell death. Over-expressed EVA enhances cellular heme uptake in a V-ATPase-dependent manner. We conclude that EVA/HRG-1 regulates V-ATPase-dependent acidification of endosomes necessary for trafficking of heme receptors as well as for heme transport within endosomes. Our data suggest that IGF-I induces expression of this micro-nutrient transporter to facilitate the enhanced metabolic requirements of cancer cells.

Citation Information: Cancer Res 2009;69(23 Suppl):C18.
Abstract C22: Telomerase activity and HR-HPV infection frequently are present before detection of high grade cervical lesions
Authors: Martín-Gámez Diana Carolina , Hernandez Gustavo Adolfo , Buitrago Oscar Leonardo , Huertas Antonio , Murillo Raúl , Bravo Maria Mercedes , Moreno Pablo , Muñoz Nubia , Molano-Luque Monica
Published: May 2012

Telomerase activation have been considered as a fundamental step in the immortalization process because majority nonmalignant somatic tissue shows undetectable or no telomerase activity. A few numbers of studies in cervical cancer have showed an increase in the telomerase activity with grade of lesion, and Human Papillomavirus (HPV) infection has been associated in some studies with telomerase activation. However until now there are not follow up studies that measure the telomerase activity along the time. We describe here results about telomerase activity and HPV infection during 7 years of follow up in women that belong to the Bogotá cohort.

Samples were obtained every six months during follow up and we had in average four samples by woman. Telomerase activity was analyzed in 58 samples of 17 women that during follow up developed High Grade Squamous Intraepithelial Lesions (HGSIL) and 149 samples of 34 women that during follow up had normal cytology using a TeloTAGGG Telomerase PCR ELISA kit (Roche Applied Science). The HPV infection was previously tested using a GP5+/GP6+mediated PCR-EIA and typing using a Reverse Line Blot assay (RLB).

Of the 17 women that developed HGSIL along follow up; 12 (70.5%) had HPV infection and telomerase activity at the same time of HGSIL diagnosis or in a previous visit, these women had infections with high risk types (HR) principally with those belong to the alpha 9 specie, 2 (17.7%) showed only transitory events of HPV infection and 2 (11.8%) had normal cytology without HPV infection or telomerase activity. Average time to develop HGSIL was 13.5 months. In normal women we observed diverse results. Of the 34 women; 21 (61.8%) had HPV transitory infections with high risk and/or low risk types sometime during follow up and only 12 (35.2%) of them were positive for telomerase activity sometime in follow up independently of HPV infection. Interestingly, 8 (23.5%) women had HPV-HR infection during follow up and they were never positive for telomerase activity and 5 (14.7%) women didn't have HPV infection and telomerase activity.

Detection of HR-HPV infection and telomerase activity at the same time likes to predict the risk of HGSIL. Our results support the idea that telomerase activity and HR-HPV infection can be used as progression markers for the development of HGSIL.

Citation Information: Cancer Res 2009;69(23 Suppl):C22.
Abstract C3: Gene expression profiling supports the hypothesis that human ovarian surface epithelia are pluripotent and capable of serving as ovarian cancer initiating cells
Authors: Bowen Nathan J. , Walker L. DeEtte , Matyunina Lilya V. , Logani Sanjay , Totten Kimberly A. , Benigno Benedict B. , McDonald John F.
Published: May 2012

Accumulating evidence suggests that somatic stem cells undergo mutagenic transformation into cancer initiating cells. The serous subtype of ovarian adenocarcinoma in humans has been hypothesized to arise from at least two possible classes of progenitor cells: the ovarian surface epithelia (OSE) and/or an as yet undefined class of progenitor cells residing in the distal end of the fallopian tube. Comparative gene expression profiling analyses were carried out on OSE removed from the surface of normal human ovaries and ovarian cancer epithelial cells (CEPI) isolated by laser capture microdissection (LCM) from human serous papillary ovarian adenocarcinomas. Differentially expressed genes were analyzed using gene ontology, molecular pathway, and gene set enrichment analysis algorithms. Consistent with pluripotent capacity, genes in pathways previously associated with adult stem cell maintenance are highly expressed in ovarian surface epithelia and are not expressed or expressed at very low levels in serous ovarian adenomcarcinoma. The results of the gene expression analyses were selectively confirmed using immunohistochemistry. Among the over 2000 genes that are significantly differentially expressed, a number of pathways and novel pathway interactions are identified that may contribute to ovarian adenocarcinoma development. Our results are consistent with the hypothesis that human ovarian surface epithelia are pluripotent and capable of serving as the origin of ovarian adenocarcinoma. While our findings do not rule out the possibility that ovarian cancers may also arise from other sources, they are inconsistent with claims that ovarian surface epithelia cannot serve as the origin of ovarian cancer initiating cells.

Citation Information: Cancer Res 2009;69(23 Suppl):C3.
Abstract C35: Rapid assessment of aneuploidy using 5 minute oligo-FISH probes
Authors: McGrath Patricia , Bradley Sean , Aurich-Costa Joan , Ng Carrie , Selvaggio Susan
Published: May 2012

More than 15 years ago, development of non-radioactive FISH probes revolutionized clinical cytogenetics and FISH is now routinely used for mapping genes, detecting pathogenic DNA, and diagnosing cancers and genetic diseases. However, most FISH applications rely on probes generated from large genomic fragments (BAC, YAC or plasmids), cDNA, or PCR generated fragments. The large probe size (200–600bp) results in poor cell penetration necessitating long, 8 to 16 hours hybridization. Furthermore, large BAC probes require high probe concentration and stringent wash conditions.

We recently developed short (30mer), synthetic Oligo-FISH probes targeting highly repetitive sequences which offer several advantages for assessing aneuploidy, including performance in < 10 min compared to 8–16 hours required for BAC probes. In addition, since Oligo-FISH probes hybridize at low stringency, it is possible to perform at least 6 successive 4 probe hybridizations without damaging cellular DNA, making it possible to determine aneuploidy in all 24 chromosomes on the same slide in < 24 hours.

Here we show results using Oligo-FISH probes specific for chromosomes: 7, 3, 9q12, and 17 designed to detect aneuploidy in urothelial cells. After optimizing probe design, we identified sequences available in the human genome database that contain chromosome specific alpha—satellite or satellite sequences. Next, we prepared urothelial slides and optimized denaturation and hybridization times. After establishing optimum Oligo-FISH conditions, we tested urine samples from 5 healthy volunteers. Our results, obtained in < 3 hours show Analytical Sensitivity > 98% and S/N> 2.

Urothelial carcinomas account for more than 90% of all bladder cancers and more than 54% of these newly diagnosed carcinomas are superficial, non-invasive transitional cell carcinomas, which has the highest rate of recurrence and requires life-long diagnostic surveillance. The current standard methods for bladder cancer include cystoscopy and cytology which are invasive, expensive and insensitive.

Citation Information: Cancer Res 2009;69(23 Suppl):C35.
Abstract C36: Using the Rb/E2f3 mutant mouse model to reveal markers for the progression to metastasis
Authors: Vormbrock Kirsten , Schreiber Caroline , Loddenkemper Christoph , Ziebold Ulrike
Published: May 2012

The retinoblastoma tumor suppressor protein (pRb) was the first tumor suppressor to be identified. The E2F family of transcription factors is considered the best-studied class of interaction partners of pRB. Specifically, E2F1-3 are all capable to control re-entry and progression through the S-phase of the cell cycle. The simplified view is that hypo-phosphorylated pRb is bound by E2F and recruits chromatin remodeling complexes to represses the transcription of E2F target genes. Upon cell cycle progression pRb is phosphorylated leading to a release of E2Fs and the subsequent progression through the G1/S-transition.

In order to understand the consequences of deregulation of the pRb/E2F-pathway, a number of groups have established mouse knock-out models of various members of this pathway. Using a combined knockout mouse model it was demonstrated that E2F3 contributes to both the inappropriate proliferation and p53-dependent apoptosis that arises in pRb-deficient embryos. Furthermore, it was shown that absence of E2F3 results on one hand in the attenuation of the growth of pituitary adenomas, on the other hand it promotes the development of medullary thyroid carcinomas (MTCs) yielding metastases at an increased frequency.

Since this is one of the few existing mouse models to study metastasis, we wished to use this model to identify novel markers and mechanisms that regulate this process. We established the whole genomic transcriptional profile of mouse MTCs harvested from Rb/E2f3 mutant mice. The comparison of metastatic MTCs with non-metastatic tumors defined a data set of 123 putative metastatic markers. Among this data set, we identified a cluster of 22 genes that are directly involved in the formation of the kinetochore, establishment of the centromere or play a distinct role in the spindle assembly checkpoint. We validated the expression of these novel metastatic markers using mouse RNA, as well as material from human metastatic MTC biopsies. Importantly, we were able to establish that the same genes were only upregulated in metastatic tumors utilizing immunhistochemistry on sections of human metastatic MTCs but not in non-metastatic counterparts. Moreover, chromatin immunoprecipitation assays support that these genes are direct targets of the pRB/E2F-pathway. In summary we identified novel markers of metastasis. Moreover, these markers are also putative E2F target genes, the fact of which emphasizes the important contribution of E2Fs to tumor progression.

Citation Information: Cancer Res 2009;69(23 Suppl):C36.
Abstract C37: Ensemble survival prediction for breast cancer using microarray data
Authors: Reddy Anupama , Ganesan Shridar , Bhanot Gyan
Published: May 2012

We present an algorithm, “Ensemble Logical Analysis of Survival Data”, for predicting survival of cancer patients based on patterns identified on gene-expression microarray data. A pattern is defined as a combination of expression levels of multiple genes, associated with risk of mortality or metastasis of the tumor. We illustrate our method on several breast cancer microarray datasets, including 286 samples from a study of node negative untreated samples from Wang et al 2005, 347 primary invasive samples from Ivshina et al. 2006, and 255 early-stage samples who received Tamoxifen as adjuvant treatment from Loi et al. 2008. We use the recent molecular stratification of the breast cancers into eight robust subtypes: Luminals: (LA, LB1, LB2, LB3), Basal-like: (BA1, BA2) and Her2+: (Her2+I, Her2+NI) from Alexe et al. 2007 and identify patterns of genes which drive the disease within each of these molecular types. This is followed by an analysis of the genes seen most frequently in patterns to determine the pathways responsible for progression/metastasis/death for each subtype.

At each event time point (event = death/recurrence/metastasis), we identify patterns that separate patients into high/low risk classes. Each pattern is then associated with a score defined as the integral of the Kaplan-Meier survival curve for the samples that satisfy the pattern. Pattern space is not disjoint and a sample can satisfy more than one pattern, even for a given time point. The average of the pattern scores for each patient across all time points defines a patient-specific survival score. All the above steps are computed using an ensemble method: two-thirds of the data is randomly selected, patterns are generated on this random subset, and patient scores are computed. This is repeated 100 times, and patient scores are aggregated over these 100 runs.

We find that these aggregated scores correlate very well with actual survival in the Wang dataset (c-statistic: 86% for LA, 95% for LB1, 88% for LB3, 77% for Basals, and 80% for Her2+). High/low risk class assignment based on the median of the predicted survival score is highly significant (log-rank p-value: 0.01 for LA, 0.0004 for LB1, 0.00006 for LB3, 3.3×10−8 for Basals and 0.0003 for Her2+). The method that we propose is general and can be used for predicting survival for any cancer type. It can also be applied to RT-PCR data, single nucleotide polymorphisms, microRNA data and data on copy number variations. Finally, we show that our risk score is significantly superior to an Oncotype DXTM score inferred for the samples from the gene expression data.

Citation Information: Cancer Res 2009;69(23 Suppl):C37.
Abstract C38: Metformin inhibits cancer cell growth in an AMP kinase-independent and -dependent manner
Authors: Rattan Ramandeep , Giri Shailendra , Hartmann Lynn C. , Shridhar Viji
Published: May 2012

Metformin is the most widely used drug for type 2 diabetes. Metformin activates AMP-activated protein kinase (AMPK), which regulates cellular energy metabolism via its upstream kinase LKB1. Activation of AMPK has been shown extensively to cause growth arrest in multiple tumor types by inhibiting the TSC-mTOR (mammalian target of rapamycin) pathway as well as by inducing cell cycle arrest. Our data indicate that metformin exerts an anti-proliferative effect on several chemo-responsive and -resistant ovarian cancer cell lines (A2780, CP70, C200, OV202, OVCAR3 and SKVO3ip, PE01, PE04), which is accompanied by activation of AMPK and cell cycle arrest. Interestingly, we have also determined that metformin is able to inhibit proliferation of ovarian cancer cells when AMPK is silenced by siRNA, where both the expression and activation of AMPK is significantly reduced. Also, metformin was able to attenuate proliferation of AMPK (−/−) mefs, although to a lesser degree in AMPK (+/+) mefs. Additional results indicate that metformin activates p21 and inhibits cyclinD1 activities partially in AMPK (−/) mefs. These data suggest a novel mechanism of metformin action, namely, an AMPK independent pathway which can modulate proliferation and possibly cell cycle regulated events in ovarian cancer. Whether the AMPK independent effects of metformin require LKB1 activity is currently under investigation. Collectively, our preliminary observations indicate that metformin can modulate proliferation and cell cycle arrest in an AMPK independent manner.

Citation Information: Cancer Res 2009;69(23 Suppl):C38.
Abstract C41: Heme oxygenase-1 and carbon monoxide modulate responses to DNA damage in prostate cancer
Authors: Wegiel Barbara , Bjartell Anders , Harris Clair , Belcher John , Vercellotti Gregory , Gallo Dave , Csizmadia Eva , Helczynski Leszek , Persson Jenny Liao , Otterbein Leo E.
Published: May 2012

Heme oxygenase (HO) and carbon monoxide (CO), a product of HO activity generated during heme catalysis, exert differential effects on the apoptotic response dependently on the environmental milieu and cell types involved. The role of HO-1 and CO in modulating chemotherapeutic treatment in prostate cancer has not been tested. Our focus in this work explored the role of HO-1 and CO in modulating PCa cell death via DNA damage mechanisms. We examined HO-1 expression in PCa samples from 482 patients and found strong nuclear staining in moderately differentiated tumors (Gleason grade 3). Treatment of PCa cells with the chemotherapeutic agents, camptothecin or doxorubicin increased HO-1 activity in the cytosol, which correlated with increased cell death. Nuclear translocation of HO-1 resulted in decreased enzymatic activity. Low activity of HO-1 in the nucleus may confer resistance to chemotherapy due to the antiapoptotic effects of HO-1. Overexpression of cytosolic and active HO-1 or exogenous delivery of CO at low non-toxic concentrations (250 parts per million) in combination with a chemotherapeutic led to enhanced tumor cell death via caspase-3 activation. CO exposure of mice with established PC3 xenografts delayed tumor growth via accelerated apoptosis (TUNEL staining) and mitotic catastrophe of cancer cells as well as strong inhibition of angiogenesis (CD31 staining). Further, CO sensitized tumors to doxorubicin treatment with enhanced apoptosis and blockage of mitosis. In contrast, CO blocked chemotherapeutic-induced cell death in normal cells supporting a new function for HO-1/CO as cellular sensors of DNA damage. Taken together, our data demonstrate the importance of cellular localization of HO-1 in PCa progression and importantly that CO, in part regulates cellular sensitivity to DNA damaging agents. We propose a novel strategy to overcome resistance to chemotherapy as well as reducing the chemotherapeutic burden by employing CO at safe, non-toxic concentrations that permits preservation of normal cells and tissue.

Citation Information: Cancer Res 2009;69(23 Suppl):C41.
Abstract C45: Somatostatin receptor activation by agonist-like antibodies can reverse neuroendocrine tumor BON cell dedifferentiation and increase cell death
Authors: Leu Frank , Nandi Minesh , Wong Chung
Published: May 2012

The somatostatin receptors (SSTR) are effective therapeutic targets for treating neuroendocrine tumors, primarily achieved through the inhibition of both cellular proliferation and hormone secretion. SSTR are comprised of five major subtypes, namely subtypes 1, 2 (A and B), 3, 4 and 5. The somatostatin signaling also works as a determinant in the TGF-β dependent neuroendocrine-mesenchymal transition (NMT) process. NMT is similar to the epithelial-mesenchymal transition (EMT), but has the opposite cellular differentiation response to TGF-β. For instance, TGF-β promotes cell dedifferentiation in EMT but promotes cell differentiation in NMT. The process of EMT is a critical morphogenic event during the progression of epithelial tumors. Recently, in epithelial tumors, the micro RNA 200c (miR-200c) has been found to negatively regulate E-cadherin's transcriptional repressor ZEB1 and capable of keeping the EMT invasiveness in check. In our study, the miR-200c repressor (Oligoengine Inc. designed by Todd Hauser) has been successful in up-regulating ZEB1 and ultimately driving the neuroendocrine tumor BON cells into the dedifferentiated mesenchymal state. The miR-200c repressor induces changes in the tumor metastasis markers, such as the ZEB1 increase, E-cadherin decrease, and ultimately with both Vimentin and Twist increases. Our anti-SSTR antibodies raised against receptor's ectodomain region (anti-SSTR ECL2 antibodies) were able to reverse the BON cell from its mesenchymal state back to the differentiated neuroendocrine state. Our results suggest that similar to EMT, both miR-200c and ZEB1 are also involved in the NMT dedifferentiation programming. In summary, we conclude that miR-200c/ZEB1 signaling can be regulated by the SSTR activation. This is evident because anti-SSTR agonist-like SSTR2, 3 and 5 antibodies are capable of overcoming neuroendocrine tumor's miR-200c commitment to metastasis. Furthermore, we also found that these anti-SSTR antibodies can sensitize tumor cells to death.

Citation Information: Cancer Res 2009;69(23 Suppl):C45.