bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A9: Importance of C17orf37 prenyl modification in cancer cell migration and invasion
Authors: Dasgupta Subhamoy , Vishwanatha Jamboor K.
Published: May 2012

C17orf37 is a novel gene located in the hot spot locus of human chromosomal region 17q12-21. C17orf37 is frequently amplified in breast cancer patients along with ERBB2, and expression correlates with grade and stage of cancer. C17orf37 gene is also found to be activated in certain cancer cells and tissues irrespective of ERBB2 status, compared to minimal expression in normal cells. Recently, we have reported that C17orf37 is highly overexpressed in prostate cancer cells and tissues, and expression of the gene positively correlates with the migratory and invasive potential of cancer cells. Moreover C17orf37 acts as a signaling molecule via Akt/NF-κB pathway modulating the expression of key metastasis-related genes uPA, MMP-9 and VEGF. We identified a putative prenylation motif ‘CVIL’ at the C-terminal end of C17orf37 protein. Using confocal, total internal reflection fluorescence microscopy (TIRFM) and cellular fractionation studies we show C17orf37 is post-translationally modified by GGTase-I enzyme, which translocates the protein to the inner leaflet of plasma membrane. Mutation of the cys-112 or entire deletion of the ‘CVIL’ motif of C17orf37 protein blocks the protein localization to the membrane. Based on this, we hypothesize that prenylation at ‘CVIL’ motif is important for activation of C17orf37 protein and subsequent downstream signaling. To study the importance of prenylated C17orf37 in cancer cells, we generated stable cells expressing either wild type or mutant C17orf37 protein. Our studies show that prenylated C17orf37 protein (wild type) preferentially localize to the leading edge of migrating cancer cells (directional) thereby activating nucleation of F-actin, formation of lamellipodia and increased migration of cancer cells. Cells expressing mutant C17orf37 protein are mostly localized in the cytosol and fail to localize to the edge of the migrating cancer cells. In addition, we identified a canonical immunoreceptor tyrosine-based activation motif (ITAM) at the N-terminus of C17orf37 protein. Currently we are investigating the importance of ITAM domain for downstream signaling of C17orf37 through Akt/NF-κB pathway.

Citation Information: Cancer Res 2009;69(23 Suppl):A9.
Abstract B12: RB1-mediated regulation of osteoblast migration
Authors: Gunduz Volkan , Cardona Pedro Santiago , Hinds Philip W.
Published: May 2012

Alterations involving loss or gain of function of components of the retinoblastoma (RB1) tumor suppressor pathway are observed in a wide range of human cancers. They include loss of p16INK4a expression in melanomas and overexpression of cyclin D1 in breast tumors. RB1 itself, however, is inactivated in a reduced subset of tumors including retinoblastomas, osteosarcomas and small cell lung carcinomas. A common characteristic of these tumors is the lack of well-organized adherens junctions. Thus, in retinoblastoma, the cadherin/catenin complex fails to anchor to the actin cytoskeleton leading to loss of cell-cell contact inhibition. Likewise, osteosarcomas and small cell lung carcinomas show reduced expression of cadherins and beta-catenin. In addition, a strong correlation has been found between abnormal adherens junction structures and the invasive capacity of these tumors. In this study, we aim to investigate the role of RB1 on cell-cell adhesion and migration using calvarial osteoblasts that are deficient in RB1 expression. We observe that RB1-deficient immortal osteoblasts (MC3T3 cells) lack membrane localization of N-cadherin and Beta-catenin and display an increased migratory phenotype compared to the RB1 expressing counterparts. In addition, RB1-deficient MC3T3 osteoblasts express and secrete higher levels of IGF1 leading to the activation of the IGF1 receptor (IGF1R). Interestingly, the opposite pattern of expression/secretion was observed for IGFBP-2, with higher levels observed in RB1-expressing MC3T3 cells. Moreover, activation of the IGF signaling pathway correlated with higher levels of phosphorylated Akt in RB1-deficient MC3T3s and, accordingly, treatment of RB1-deficient MC3T3 osteoblasts with either a neutralizing antibody against IGF1 or an inhibitor of IGF1 receptor led to impaired migration. We have also observed reduced phospho-Akt levels in RB1-deficient MC3T3 osteoblasts that were exposed to the conditioned medium (CM) from RB1 wild type MC3T3 osteoblasts. This reduction in phospho-Akt levels correlated well with a reduced migratory phenotype of RB1-deficient MC3T3 osteoblasts under these conditions. Taken together our data suggest that IGF signaling is one of the mechanisms that might contribute to increased migration in RB1-deficient MC3T3 osteoblasts.

Citation Information: Cancer Res 2009;69(23 Suppl):B12.
Abstract B19: An integrative in vivo analysis of PIK3CA, PTEN and AKT alterations in head and neck squamous cell carcinoma
Authors: Bornstein Sophia , Owens Philips , Ruth White , Li Yuexin , Weber Stephen , Wang Xiao-Jing , Lu Shi-Long
Published: May 2012

In vitro studies indicate that deregulation of the PI3K/AKT pathway is important in head and neck squamous cell carcinoma (HNSCC) tumorigenesis, as has been observed in several other cancers. However, the in vivo role of PI3K/AKT pathway deregulation in HNSCC remains unknown. The objective of this study was to investigate the in vivo roles of PIK3CA, PTEN and AKT alterations in head and neck tumorigenesis. To this end, we developed inducible head and neck specific transgenic PIK3CA and AKT mice, and inducible head and neck specific knockout PTEN mice under the control of an RU486-responsive Keratin 5 promoter. Both PIK3CA or AKT transgene expression was induced in mouse head and neck epithelia, i.e. buccal mucosa, tongue, and esophagus, upon RU486 application in a dose- and time- dependent manner. PTEN deletion was also achieved in mouse head and neck epithelia after five consecutive RU486 applications. The PIK3CA and AKT inducible transgenic mice exhibited severe head and neck epithelial hyperplasia, dysplasia, and carcinoma in situ, but no visible tumor formation after one year of observation. Further, the inducible head and neck specific PTEN knockout mice developed oral, but not esophageal SCCs. To investigate whether the inducible PIK3CA or AKT mice harbored higher cancer susceptibility upon tobacco carcinogen exposure, we applied a subcarcinogenic dose of 4NQO, a surrogate of a tobacco carcinogen, to the PIK3CA and AKT mice. Both PIK3CA and AKT mice developed oral and/or esophageal SCCs as early as one month after 4NQO exposure, and by 4 months, all PIK3CA and AKT transgenic mice developed oral and/or esophageal SCCs. Interestingly, a higher incidence of esophageal in comparison with oral SCC (93% versus. 19% of mice) was observed in the AKT transgenic mice, while a higher incidence of oral versus esophageal SCC (74% vs. 44% of mice) was found in the PIK3CA mice, suggesting a tissue specificity of AKT and PIK3CA in head and neck tumorigenesis. Studies investigating the underlying molecular mechanism of each molecule in head and neck tumorigenesis are currently underway. In conclusion, our data demonstrate an oncogenic role of PIK3CA and AKT, and a tumor suppressive role of PTEN in head and neck tumorigenesis in vivo. Our inducible head and neck specific mouse models are powerful tools to evaluate the roles of PIK3CA, PTEN, and AKT during each stage and site involved in head and neck tumorigenesis, and to test novel therapeutics targeting this important signaling pathway.

Citation Information: Cancer Res 2009;69(23 Suppl):B19.
Abstract B2: Coup-tf1 is suppressed by androgen receptor in prostate cancer epithelial and normal stromal cells
Authors: Perets Ruth , Hidas Guy , Simon Itamar , Pikarsky Eli
Published: May 2012

Chicken ovalbumin upstream promoter-transcription factor 1 (Coup-tf1) is a transcription repressor previously known to play a role in neurogenesis and adipogenesis. It mainly acts through suppression of thyroid hormone receptor and vitamin D receptor. Coup-tf1 was implicated before in breast cancer as a negative regulator of the Aromatase gene, and in gastric cancer cell lines as a positive regulator of vegf-d.

We performed a genome wide location analysis of androgen receptor (AR) target genes in prostate cancer cells treated with androgen, an AR antagonist or androgen deprivation. This analysis revealed ligand dependent binding of the coup-tf1 promoter and enhancer by the AR which was confirmed by specific chromatin immunoprecipitation studies. AR activation by a ligand is correlated with decreasing mRNA levels implying that AR is a negative regulator of coup-tf1 in prostate cancer cells.

Using immunohistochemistry we have seen coup-tf1 staining in 21/28 primary human prostate cancer samples, regardless of Gleason score or prognosis. Coup-tf1 staining was in a nucleolar pattern and its levels in malignant cells were substantially higher than in the adjacent benign prostate glands. Stromal cells both in human and mouse prostate show nuclear coup-tf1 staining. There is an inverse correlation between Coup-tf1 staining and the rising levels of androgen with advancing puberty.

These results implicate coup-tf1 in prostate cancer for the first time, and show a novel AR repressed gene, that might play a role in prostate cancer.

Citation Information: Cancer Res 2009;69(23 Suppl):B2.
Abstract B22: Oncogene activation of K-ras in resident myofibroblasts leads to development of spontaneous bronchioalveolar carcinoma in mice
Authors: Vong Sylvia , Sugimoto Hikaru , Teng Yingqi , Xie Liang , Dewar Rajan , Kalluri Raghu
Published: May 2012

Stromal myofibroblasts, also known as carcinoma-associated fibroblasts, are recruited to neoplastic lesions and act as accomplices in promoting tumor progression. By secreting various growth factors, cytokines, extracellular matrix molecules, and matrix remodeling proteins, stromal myofibroblasts enhance tumor cell proliferation, angiogenesis, and tumor cell invasion. In contrast, the role of resident myofibroblasts in tumorigenesis is largely unknown. Our preliminary results strongly suggest that resident myofibroblasts may act as contributors to tumorigenesis. Selective expression of the oncogenic mutant of K-ras (K-rasG12D) in alpha smooth muscle actin-positive (αSMA+) stromal myofibroblasts is achieved by crossing αSMA-Cre+ transgenic mice to the LSL-KrasG12D f/+ mouse model (hereafter αSMAKrasG12D). While αSMAKrasG12D mice were phenotypically normal at birth, these mice developed spontaneous bronchioalveolar carcinoma by 5 weeks of age at 100% tumor penetrance. We speculate that genetic mutations in stromal myofibroblasts can disrupt stromal-epithelial homeostasis, driving hyperproliferation of epithelial cells. This fosters further accumulation of genetic mutations in the epithelial compartment, and thus, result in carcinogenesis.

Citation Information: Cancer Res 2009;69(23 Suppl):B22.
Abstract B23: Distinct roles for cancer-associated fibroblasts in breast cancer progression and metastasis
Authors: Tse Joyce C. , Sugimoto Hikaru , Cooke Vesselina G. , MacDonald Brian A. , Mehta Ankit I. , Neilson Eric G. , Zeisberg Michael , Kalluri Raghu
Published: May 2012

Cancer-associated fibroblasts (CAFs) are commonly identified and studied as an alpha-smooth muscle actin positive (αSMA+) population of myofibroblasts. However, recent work from our laboratory has shown that there are CAFs in the tumor microenvironment, which do not express αSMA. Most notably, fibroblast specific protein 1 (FSP1) marks a population of CAFs distinct from αSMA+ CAFs, as 90% of FSP1+ CAFs in the tumor microenvironment of the 4T1 murine breast cancer model are αSMA-negative. The functional heterogeneity of these subpopulations of CAFs remains unexplored. By using transgenic mice, which express viral thymidine kinase under control of the CAF promoters αSMA or FSP1 to selectively ablate either αSMA+ CAFs or FSP1+ CAFs in the context of breast cancer, we have identified distinct roles for the two CAF subpopulations in tumorigenesis and metastasis. While αSMA+ CAFs play a role in primary tumor growth and angiogenesis without affecting metastasis, FSP1+ CAFs do not contribute to primary tumor progression but facilitate the formation of macrometastases.

Citation Information: Cancer Res 2009;69(23 Suppl):B23.
Abstract B24: Thoc1 is required for mouse prostate tumorigenesis initiated by loss of Rb and p53
Authors: Chinnam Meenalakshmi , Wang Yanqing , Wang Xiaoling , Zhang Xiaojing , Gold David L. , Nikitin Alexander Yu , Goodrich David W.
Published: May 2012

A large percentage of prostate cancers show either mutational inactivation or deregulation of the Rb tumor suppressor gene. Rb mediates its tumor suppressor function through its association with other cellular proteins. Most of the Rb-binding proteins interact with the A/B pocket domain and the C terminal region. Less than 10 proteins are known to interact with Rb N terminal domain, a domain that appears to be required for normal Rb function in vivo. The Thoc1 gene encodes a protein that binds the Rb N terminal domain. We hypothesize that some of pRb functions in vivo could be mediated in part by interaction with pThoc1. Thoc1 protein has been found to be an essential component of the TREX (transcription/export) complex which is important for mRNP biogenesis and physically couples transcription elongation with RNA processing and export. We have previously reported that E1A/Ras transformed MEFs (mouse embryonic fibroblasts) but not normal MEFs were dependent on Thoc1 for their survival. These observations suggest that tumor cells specifically may be dependent on Thoc1 for survival and Thoc1 may play a role in tumorigenesis. To test our hypothesis, we used a mouse model of prostate cancer where prostatespecific deletion of Rb and p53 genes leads to development of metastatic adenocarcinoma. We find that compound loss of Thoc1, Rb and p53 increased the life-span of mice compared to mice with loss of Rb and p53 alone. Histopathological analyses of prostate tissue showed that initiation of tumorigenesis is delayed in the absence of Thoc1. Tumors that do arise in these mice retain expression of Thoc1. These findings indicate that Thoc1 is required for prostate tumorigenesis. Conditional deletion of Thoc1 alone in mouse prostate does not appear to affect normal prostate development. To test whether Thoc1 is relevant to human prostate cancer, we examined expression of Thoc1 protein in matched normal and prostate tumor tissue cores on tissue microarray. Analysis of nearly 600 patient samples reveals that Thoc1 is significantly overexpressed in tumor tissue compared to normal prostate tissue and pThoc1 levels positively correlated with tumor grade and negatively correlated with biochemical recurrence as indicated by elevated PSA (prostate-specific antigen) levels. Taken together the above findings suggest that Thoc1 is synthetic lethal with the genetic and epigenetic alterations in prostate tumor cells, and hence it may be a potential target for prostate cancer therapy.

Citation Information: Cancer Res 2009;69(23 Suppl):B24.
Abstract B25: DDB1 is a cell-nonautonomous tumor suppressor targeted by HBx in the liver
Authors: Yamaji Sachie , Zhang Mingjun , Zhang Jing , Bibikova Elena , Hodgson Amanda J. , Slagle Betty L. , Goff Stephen P. , Cang Yong
Published: May 2012

Hepatocellular carcinoma (HCC) is the fifth most frequent cancer and the third leading cause of cancer death worldwide. A major aetiologic risk factor for HCC is chronic infection of hepatitis B virus (HBV), which encodes a small regulatory x protein (HBx) critical for HBV-induced hepatocarcinogenesis. How HBx contributes to HCC is not clear, but is likely dependent on its association with host factors to activate transcription and regulate mitogenic pathways.

Here we demonstrate that deletion of one such HBx-interacting protein, the Damaged DNA Binding Protein 1 (DDB1), in liver parenchymal hepatocytes leads to development of HCC in mice, after prolonged inflammation resulting from continuous loss of DDB1-deficient hepatocytes and regeneration of DDB1-proficient hepatocytes. Expression of HBx in this regenerating liver impairs the proliferation of regenerated DDB1-proficient hepatocytes, and significantly accelerates the onset of tumorigenesis. Surprisingly, tumors arising from these animals are made up of cancer cells displaying no disruption of the DDB1 gene. This cell-nonautonomous function of DDB1 inactivation in HCC initiation can be further enhanced by deletion of the tumor suppressor p53 or administration of chemical carcinogen.

Our results indicate that cancer initiating mutations are not necessarily present in cancer initiating cells, but can be exploited by oncogenic signals such as HBx expression and p53 mutation to promote cancer progression.

Citation Information: Cancer Res 2009;69(23 Suppl):B25.
Abstract B26: Lack of cyclin D1 kinase activity leads to reduced levels of ErbB2-induced senescence and activation of the Wnt signaling pathway in the mammary gland
Authors: Brown Nelson E. , Kuperwasser Charlotte , Hinds Philip W.
Published: May 2012

Proper orchestration of the Oncogene-induced Senescence (OIS) checkpoint relies upon the activation of the ARF-p53 and/or the p16 INK4a-pRB tumor suppressor pathways. Overexpression of cyclin D1, as observed in a large proportion of human breast cancers, is believed to confer proliferative advantage through pRb inactivation. The aim of the current study is to assess the role of cyclin D1-associated kinase activity during ErbB2-induced senescence in the mammary epithelium using a kinase-deficient cyclin D1 knock-in mouse (cyclin D1KE/KE) as a model. The effect of cyclin D1WT/WT or cyclin D1KE/KE on MMTV-ErbB2-induced senescence was analyzed in mammary glands of double-transgenic animals. Surprisingly, we observed that cyclin D1KE/KE/MMTV-ErbB2 mammary glands fail to upregulate markers of cellular senescence when compared to preneoplastic cyclin D1WT/WT/ MMTV-ErbB2 mammary glands. These findings correlated with high levels of proliferation and increased cellularity per duct section despite the fact that the cyclin D1KE/KE/MMTV-ErbB2 mammary epithelial cells do not progress to tumor formation. Moreover, this ectopic proliferation was not accompanied by phosphorylation at Ser780 of pRb, suggesting that other D-cyclins were unable to compensate for the lack of cyclin D1-associated kinase activity. Interestingly, failure to implement the senescence program in mutant mammary epithelium was accompanied by an activation of the Wnt signaling pathway that led to stabilization of beta-catenin in mutant mammary epithelial cells, an observation that others have found to correlate with senescence bypass in MEFs. In addition, induction of senescence in cyclin D1WT/WT/ MMTV-ErbB2 mammary glands did not lead to the accumulation of the gene products encoded by the INK4a/ARF locus. Instead, p18 INK4c expression was increased in D1WT/WT/ MMTV-ErbB2 mammary glands above the basal levels observed in cyclin D1WT/WT mammary tissue that did not express MMTV-ErbB2. A comparable induction was not observed in cyclin D1KE/KE/ MMTV-ErbB2 mammary glands. Taken together, our results suggest a dual role for cyclin D1-associated kinase activity in ErbB2-induced senescence and tumor formation. Cyclin D1 kinase activity is necessary for the full transformation of ErbB2-expressing cells, but it may also play an active role in the cascade that leads ErbB2-overexpressing cells to senesce such that cells that are kinase-deficient can still proliferate due to Wnt signaling activation.

Citation Information: Cancer Res 2009;69(23 Suppl):B26.
Abstract B30: EBV LMP2A bypasses p53 inactivation in a MYC model of lymphomagenesis
Authors: Bieging Kathryn T. , Amick Alexandra C. , Longnecker Richard
Published: May 2012

Although Epstein-Barr virus (EBV) is linked to Burkitt's lymphoma (BL), the role of the virus in lymphomagenesis is unclear. LMP2A, encoded by EBV, is detected in BL biopsies and has pro-survival functions. We generated mice expressing MYC and LMP2A in B cells. Pretumor LMP2A/λ-MYC mice have significantly enlarged spleen size. In addition, LMP2A/λ-MYC mice show greatly accelerated tumor onset compared to λ-MYC mice. Inactivation of the p53 pathway and failure to induce the pro-apoptotic BH3-only protein, BIM are two major paths to tumor formation in MYC tumor models. Similar to previous work, we found p53 mutations in λ-MYC tumors, however we detected no p53 mutations in the rapidly arising LMP2A/λ-MYC tumors. We further demonstrate that the p53 pathway is functionally intact in LMP2A/λ-MYC tumors, which show increased levels of PUMA and sensitivity to p53 activation by Nutlin. We also show that BIM is induced in LMP2A/λ-MYC tumor cells. Despite the presence of p53 mutations in EBV+ human BL, our mouse model can be used to understand BL development in humans. We believe that LMP2A is important early in lymphomagenesis. The pro-survival function of LMP2A leads to an initial expansion of cells that have acquired a MYC translocation. This expansion is shown in our mouse model by enlarged spleens in pre-tumor mice. In humans, early expansion of cells increases the probability that a p53 mutation will occur in a cell with translocated MYC. Once the p53 mutation occurs, weak immune surveillance may select against high levels of LMP2A, consistent with low levels of LMP2A transcripts found in tumor biopsies. We have identified a novel mechanism that permits lymphomagenesis in the presence of an intact p53 pathway. This work also uncovers a contribution of Epstein-Barr virus (EBV) to molecular events that have documented importance in BL pathogenesis, and may underlie the poorly understood link between EBV and BL.

Citation Information: Cancer Res 2009;69(23 Suppl):B30.
Abstract B33: Dissecting the role of Nrf3 (NF-E2-related factor 3) in chemical carcinogenesis using knock-out mice
Authors: Chevillard Grégory , Nouhi Zaynab , Derjuga Anna , Paquet Marilene , Blank Volker
Published: May 2012

In this study, we investigated the role of the transcription factor Nrf3 in chemical carcinogenesis induced by benzo[a]pyrene, a common carcinogen found in cigarette smoke, car exhaust and charcoal-grilled meats. Wild type and Nrf3-deficient mice were treated weekly for 4 consecutive weeks with benzo[a]pyrene and then sacrificed 30 weeks after the first administration of carcinogen. Here, we report that mice exposed to benzo[a]pyrene rapidly exhibit labored breathing further accompanied with different signs of morbidity. Kaplan-Meyer survival rate analyses indicate that mice deficient for Nrf3 die earlier that wild type mice following benzo[a]pyrene exposure. Upon necropsy, gross morphology analyses showed the presence of an increased number of lung and thymus lesions in Nrf3-deficient mice compared to wild type mice whereas the number of stomach lesions is identical in both genotypes. Pathology analysis strongly suggests that observed lesions in lung and thymus are from lymphoma origin. Overall, our study reveals that mice deficient for Nrf3 are highly sensitive to chemicalinduced carcinogenesis suggesting a protective role of the transcription factor Nrf3 in this process. This provides new important insights into the molecular mechanisms involved in the development of lymphomas induced by chemical agents. We also propose the Nrf3-deficient mice as a new in vivo model to study tumor formation and response to chemoprotective agents.

Citation Information: Cancer Res 2009;69(23 Suppl):B33.
Abstract B34: Multigenic models of metastatic colon cancer in Drosophila
Authors: Bangi Erdem , Cagan Ross L.
Published: May 2012

Colon cancer is the third most common form of cancer and the second leading cause of cancer-related death in theWestern world. When it is detected at late stages with distant metastases already present, it is unlikely to be curable. This is largely due to the resistance of many late stage tumors to most targeted therapies and even chemotherapy. Development of new animal models of colon cancer that reflect the complex, multigenic and heterogeneous nature of human tumors will be essential in improving our ability to understand the biology of this disease and develop new targeted therapies. The Drosophila colon is remarkably similar to the human colon in terms of the signaling pathways that regulate its maintenance. We took advantage of this similarity to establish models of metastatic colon cancer in Drosophila that closely reflect features of human cancer. To this end, we utilized recent mutational profiling data from human colon tumors to generate flies carrying double, triple and quadruple combinations of mutations that are frequently found together in human tumors. We then investigated the tumorigenic and metastatic potential of these combinations by targeting them specifically to the adult Drosophila colon. We focused our initial analysis on three quadruple combinations: 1) rasG12V p53RNAi apcRNAi ptenRNAi, 2) rasG12V p53RNAi apcRNAi Smad4RNAi and 3) egfr p53RNAi apcRNAi Smad4RNAi. Colon epithelial cells carrying these combinations of mutations started expressing metastasis-related factors and established membrane processes; we are currently exploring the molecular nature of these tumor-like phenotypes. Eventually, these cells migrated through the basal membrane and the surrounding visceral muscle layer and colonized other sites within the body to form secondary tumors. Interestingly, these migratory cells were often found closely associated with trachea, a tubular network that provides oxygen to tissues in Drosophila. A more detailed characterization of the behavior of these cells as they leave the gut epithelium, as well as the secondary tumors are currently underway. We are also investigating emergent properties of these combinations by monitoring the behavior of cells carrying various double and triple combinations that make up the quadruple combinations we first analyzed. These models provide excellent paradigms to study tumorigenesis and metastasis in the context of a whole animal. Our findings will also shed light on the heterogeneity found in individual tumors, an important step towards personalized medicine.

Citation Information: Cancer Res 2009;69(23 Suppl):B34.
Abstract B36: The Ewing's sarcoma fusion oncogene EWS-FLI1 induces tumors and developmental phenotypes in zebrafish
Authors: Leacock Stefanie W. , Basse Audrey N. , Chandler Garvin L. , Kirk Anne , Amatruda James F.
Published: May 2012

Ewing's sarcoma, a member of the small round blue cell family of tumors, is an aggressive and malignant bone tumor of children and young adults. The cells are generally poorly differentiated, although can also have characteristics of neuroectodermal cells. Ewing's tumors are characterized by a t(11:22) that generates a fusion of the EWS protein and the FLI1 transcription factor. This fusion oncoprotein, EWS-FLI1, represents a potential therapeutic target for treatment of Ewing's sarcoma. However, the molecular mechanisms by which EWS-FLI1 mediates tumorigenesis remain unknown. Few animal models exist in which to study the function of EWS-FLI1. Here, we report the use of zebrafish as a new vertebrate model of EWS-FLI1 function and tumorigenesis.

Using the zebrafish Tol2 transposon system, we have developed several transgenic models to study the function of EWS-FLI1 in a vertebrate model organism. First, we have injected zebrafish with transposons containing human EWS-FLI1 driven by either the heat shock or the β-actin promoter. A subset of the injected fish developed tumors when raised to adulthood. These tumors strongly resemble Ewing's sarcoma. This finding demonstrates that the functional targets of EWS-FLI1 that mediate tumor development are conserved from zebrafish to human. We observe an increased incidence of tumors in a p53 mutant background, suggesting that the p53 pathway may suppress EWS-FLI1 driven tumorigenesis. We are performing microarray analysis to identify candidate downstream effector genes and to compare to previously identified EWS-FLI1 target genes.

In addition to these tumors, we identified transgenic lines that transmit the EWS-FLI1 transgene to their progeny. The embryos that express EWS-FLI1 exhibit abnormal embryonic development. This result suggests that EWS-FLI1 may regulate conserved developmental pathways. These transgenic lines represent a novel physiological context in which to study the cellular function of EWS-FLI1. From these two models of EWS-FLI1 expression in the zebrafish, we conclude that human EWS-FLI1 functions in zebrafish and that these models provide a novel context in which to study the function of this fusion oncogene. Additionally, the rapid development and small size of the zebrafish will allow us to utilize these models for genetic and chemical screens for specific EWS-FLI1 inhibitors for the treatment of Ewing's sarcoma.

Citation Information: Cancer Res 2009;69(23 Suppl):B36.
Abstract B39: Nkx2-1 down-regulation underlies lung adenocarcinoma progression towards malignancy
Authors: Winslow Monte M. , Dayton Talya L. , Snyder Eric L. , Chiang Derek Y. , Kim Caroline , Verhaak Roel , Crowley Denise , Bronson Roderick T. , Meyerson Matthew , Jacks Tyler E.
Published: May 2012

Metastasis leads to most cancer-related deaths, yet many of the molecular determinants and their mechanisms of action remain unknown. To uncover genetic programs that control the progression of lung adenocarcinoma towards malignancy, we developed and analyzed an autochthonous mouse model of this disease. Lentiviral-mediated somatic activation of oncogenic Kras and deletion of p53 in the lung epithelial cells of genetically-engineered mice induces the development of widely metastatic lung adenocarcinoma. Using cell lines derived from primary tumors and metastases, we determined the genome-wide expression changes that embody a metastatic profile. Here we show that the NK-2 related homeobox transcription factor, Nkx2-1 (Ttf-1/Titf1), controls cancer progression and malignant phenotypes. Lung adenocarcinoma progression in our model is associated with loss of Nkx2-1 expression, consistent with the poor prognosis for patients with NKX2-1 negative lung adenocarcinoma. In cells derived from non-metastatic tumors, shRNA-mediated reduction of Nkx2-1 results in increased metastatic potential in vivo and phenotypic alterations in vitro consistent with increased metastatic ability. Interrogation of Nkx2-1 regulated genes and analysis of tumors at defined stages of development indicate that Nkx2-1 may constrain tumors by repressing embryonically-restricted transcriptional regulators and components of the extracellular matrix.

Citation Information: Cancer Res 2009;69(23 Suppl):B39.
Abstract B40: Histone H3 methylation couples DNA damage detection to activation of the Tip60 tumor suppressor
Authors: Sun Yingli , Price Brendan
Published: May 2012

Cancer is a heterogeneous disease which arises primarily from the accumulation of mutations and deletions in the cells DNA. However, recent work has implicated both DNA methylation and histone modifications in the etiology and progression of cancer. For example, abnormal patterns of histone methylation have been identified in breast cancer, raising the possibility that altered patterns of histone methylation can be exploited both as a biomarker to monitor disease status and as a novel therapeutic target.

Here, we report on a novel role for histone methylation in regulating the ability of the Tip60 acetyltransferase to repair DNA double strand breaks (DSBs). Tip60 is a haplo-insufficient tumor suppressor, and loss of heterozygosity at the Tip60 locus and reduced levels of nuclear Tip60 have been detected in both breast and prostate cancer. Tip60 is an acetyltransferase, and can acetylate several DNA repair proteins implicated in breast cancer, including myc, p53 and ATM. Previously, we demonstrated that Tip60 acetylates and activates the ATM protein, a key component of the cells DSB repair mechanism. Here, we demonstrate that methylation of histones creates docking sites for the recruitment of the Tip60 DNA repair complex to sites of DNA damage. Further, interaction between Tip60 and methylated histones serves to specifically activate Tip60'fs acetyltransferase activity at DSBs. Reduction in histone methylation levels compromises Tip60 activation, leading to reduced DNA repair and increased genomic instability. These results indicate that the level and distribution of specific histone methylation marks across the chromatin is a critical determinant of cells ability to repair and survive DNA damage. Thus histone methylation may regulate the genomic stability of cells and their intrinsic sensitivity to agents, including radio- and chemo-therapy, which directly damage DNA. Altered histone methylation associated with breast cancer may therefore contribute to both genomic instability and to resistance to therapy. Finally, since histone methylation is reversible, the enzymes controlling histone methylation represent an exciting new target for therapy in breast cancer.

Citation Information: Cancer Res 2009;69(23 Suppl):B40.
Abstract B42: Methotrexate inhibits histone deacetylase activity and exhibits anti-tumor activity via acetylation and stabilization of p53
Authors: Chen Ching-Chow
Published: May 2012

Methotrexate (MTX) is a dihydrofolate reductase (DHFR) inhibit or that has been widely used as an anti-cancer drug in different kinds of human cancers. Histone deacetylase (HDAC) overexpression is usually found in tumors, which inhibits the expression of tumor suppressor genes. HDAC inhibitors (HDACi) reactivate these tumor suppressor genes and serve as potential anticancer drugs. In the present study, we found that MTX shared structural similarity with HDACi. Molecular modeling analysis provided an evidence for the interaction of MTX with HDAC. In vitro HDAC activity assay demonstrated its inhibition on HDAC activity in various human cancer cells. The global acetylation of histone-H3 was also induced by MTX. Post-translational modification of p53 plays a critical role in regulating its activity and stability. MTX promoted p53 acetylaion at Lys 373/382 and phosphorylation at Ser15 and Ser392, then increasing its stability and transcriptional activity. Stabilized p53 further induced the expression of its downstream gene, p21. MTX induced apoptosis and inhibited cell viability, which was dependent on p53 expression. This study provides the first time that MTX acts as a HDACi. In addition, MTX induces p53 acetylation and stabilization, and mediates p53-dependent apoptosis and growth inhibition in human cancer cells.

Citation Information: Cancer Res 2009;69(23 Suppl):B42.
Abstract B46: Essential roles of SIRT1 for BCR-ABL transformation and genetic mutations
Authors: Wang Zhiqiang , Yuan Hongfeng , Modi Hardik , Bhatia Ravi , Chen WenYong
Published: May 2012

Resistance of cancer stem cells and acquired genetic mutations are daunting challenges facing targeted cancer therapy, but the underlying mechanisms are not clear. This is best exampled by the tyrosine kinase inhibitor imatinib that effectively treats chronic myelogenous leukemia (CML) whereas resistance develops due to persistence of CML stem/progenitor cells and acquired mutations of BCR-ABL. SIRT1 is a mammalian stress response gene homologous to yeast silent information regulator 2 (Sir2), a nicotinamide adenine dinucleotide (NAD+) dependent protein deacetylase, and is involved in regulation of a variety of cellular functions including survival, glucose homeostasis and fat metabolism through deacetylating histones and non-histone target proteins. Here we demonstrate that BCRABL transformation activates SIRT1 in hematopoietic stem/progenitor cells in both clinical CML patients and a CML-like disease mouse model. Inhibition of SIRT1 by gene knockdown or pharmacological inhibitors suppresses CML cell proliferation and induces apoptosis in vitro. SIRT1 knockdown inhibits growth of CML tumor xenograft in immunodeficient mice. Homozygous knockout of SIRT1 gene inhibits BCR-ABL transformation of mouse bone marrow cells and the development of a CML-like myeloproliferative disease. We show that through deacetylation of Ku70, SIRT1 promotes BCR-ABL mutations upon imatinib treatment and de novo genetic mutations upon DNA damage. Our results suggest an essential role of SIRT1 for BCR-ABL oncogenic transformation and promoting cancer genome instability, and have implication for targeting SIRT1 to overcome resistance. These findings not only improve our understanding of SIRT1 functions in tumorigenesis, but also have translational implication for targeting SIRT1 for overcoming resistance in cancer treatment.

Citation Information: Cancer Res 2009;69(23 Suppl):B46.
Abstract B49: Smad4 suppresses Wnt signaling through downregulation of β-catenin
Authors: Smith J. Joshua , Freeman Tanner J. , Deane Natasha G. , Zi Jinghuan , Poon Sabrina , Chen Steven , Kis Christian , Lee Ethan , Dhawan Punita , Washington Mary Kay , Shyr Yu , Beauchamp R. Daniel
Published: May 2012

Introduction: Transforming Growth Factorβ (TGFβ) and Wnt signaling pathways modulate differential responses during embryonic development, and later in intestinal epithelial cell differentiation, proliferation and colorectal tumor promotion. β-catenin, the central mediator of canonical Wnt signaling, has been shown to pathologically promote intestinal epithelial cell de-differentiation and induce sustained proliferation in the absence of functional APC. Smad proteins are key developmental proteins involved in TGFβ/Bone Morphogenetic Protein (BMP) signaling involved in epithelial cell growth inhibition. Smad4, in particular, functions as a tumor suppressor and mutation of the Smad4 gene is a frequent and potentially late event in colon cancer. We recently found that expression of wild type Smad4 in Smad4-mutant colon cancer cells results in profound inhibition of β-catenin/TCF-driven transcriptional activation.

Purpose: In the current study, we set out to determine the mechanism of Smad4 inhibition of canonical Wnt signaling.

Experimental Procedures: We examined steady-state levels of β-catenin mRNA and protein in cells and human tissues, followed by analysis of components of β-catenin transcriptional and degradation machinery in the presence and absence of Smad4, and with and without inhibition of GSK3-β, a critical regulator of cytoplasmic β-catenin.

Results: We observed that wild type Smad4 expression resulted in significant repression of Wnt-specific transcriptional activity that could be restored with GSK-3β inhibition and overexpression of wild-type and mutant β-catenin in S W480 cells that have inactivating mutations in both APC and Smad4 genes. Transient overexpression of Smad4 also inhibited β-catenin-dependent transcriptional activity in HEK 293 cells that have wild-type APC and Smad4 genes. β-catenin phosphorylation is intact and unchanged in comparison of controls and Smad4-expressing S W480 cells, but the total level of β-catenin mRNA and protein is decreased in the Smad4-expressing cells. Assessment of human colorectal cancers using tissue microarray analysis revealed an inverse relationship between nuclear Smad4 and tumor cell β-catenin immunostaining. Furthermore, we observed that high Smad4 expression is associated with better disease-free survival and that high β-catenin expression is associated with worse disease-specific survival in analysis of outcomes from 250 patients.

Conclusions: Our data suggest that the tumor suppressor role of Smad4 involves reduction of β-catenin levels with resultant reduction of β-catenin/TCF-mediated transcriptional activity, and this regulatory mechanism has an important impact on clinical outcomes in colorectal cancer.

Citation Information: Cancer Res 2009;69(23 Suppl):B49.
Abstract B50: Smurf2-deficient mice exhibit increased tumorigenesis
Authors: Ramkumar Charusheila , Zhang Hong
Published: May 2012

Smurf2, a HECT family E3 ubiquitin ligase has been shown to ubiquitinate and target for degradation several proteins that promote tumorigenesis, including TGF-β receptors, Smad2/3, β-catenin, and Mad2. Furthermore, we have found previously that Smurf2 can induce senescence in early passage cells in culture. As senescence has been postulated to act as a tumor suppression mechanism, we hypothesized that Smurf2 functions as a tumor suppressor. To test this hypothesis, we generated a Smurf2-deficienct mouse strain using a gene trapping strategy. The homozygous Smurf2-deficient mice were obtained with the expected Mendelian ratio after intercrossing of heterozygous mice, and they exhibited no obvious developmental defects. Analysis of mouse embryonic fibroblasts from these Smurf2-deficient mice revealed that they exhibited delayed senescence entry and consequently had an increased tendency to immortalize in culture. Cohorts of Smurf2-deficient and wild-type mice were monitored for spontaneous tumor development. In this ongoing study, 20% (7 out of 35) of the Smurf2-deficient mice, but none of the wild-type mice showed signs of morbidity after 14-months of age. The moribund Smurf2-deficient mice were found to have enlarged spleen, pale kidneys and liver, and/or enlarged lymph nodes. Histopathology analysis indicated that most of the tumors are lymphomas. In addition, we found adenocarcinoma of the intestine in one Smurf2-deficient mouse. Collectively, our data suggest that Smurf2 is a tumor suppressor. The investigation of the mechanism by which Smurf2 deficiency leads to tumorigenesis is ongoing.

Citation Information: Cancer Res 2009;69(23 Suppl):B50.
Abstract B51: Dynamic remodeling of CEBPα complexes in myeloid differentiation
Authors: Koleva Rositsa I. , Ficarro Scott , Askenazi Manor , Parikh Jignesh , Marto Jarrod A.
Published: May 2012

Acute myeloid leukemia (AML) remains a highly lethal malignancy with limited therapeutic options. An integral component of the AML phenotype is an inability of hematopoietic progenitors to differentiate into mature myeloid cells. Mounting evidence indicates that the basic leucine zipper transcription factor C/EBPα plays a critical role in development of granulocytes, and that impaired function of C/EBPα may disrupt myeloid differentiation and ultimately lead to blast crisis in AML. Recent studies demonstrated that oncogenic Flt3 signaling leads to phosphorylation of C/EBPα on serine 21, with concomitant impairment of myeloid differentiation in cell line models and primary hematopoietic cells. While these data provide compelling evidence for the central role of C/EBPα in normal hematopoiesis and leukemia, the mechanism by which pS21 disrupts differentiation remains unresolved. Given the general thesis that cellular control of biological processes is orchestrated through a delicate balance of protein-protein interactions and post-translational modifications, we speculated that C/EBPα phosphorylation catalyzes the assembly of a transcriptionally inactive protein complex. To test this hypothesis we established affinity tagged C/EBPα under control of a tet-inducible promoter in myeloid progenitors with constitutive Flt3 activity. Treatment of these cells with Flt3 inhibitors modulated pS21 in a dose-dependent manner. Next advanced quantitative proteomics methodology, including true nanoflow LC coupled with multidimensional fractionation and on-column iTRAQ stable isotope labeling, was used to monitor remodeling of C/EBPα protein complexes as a function of pS21. Our data represent by far the largest catalog C/EBPα interactors involved in chromatin organization, transcriptional modulation, and cell cycle regulation. Furthermore, our quantitative proteomics data demonstrate that 1) C/EBPα interacts with proteins genetically linked to leukemia (DEK, SET and other); and 2) many of these interact with C/EBPα in a phosphorylation-dependent manner. Our functional validation studies show that DEK and SET bind to C/EBPα-target promoters in C/EBPα-dependent manner to enhance C/EBPα attachment to its DNA binding site. Knock-down of newly-identified, leukemia-associated interactors, DEK and SET, reduce the ability of C/EBPα to drive expression of granulocytic target genes. Furthermore the depletion of DEK and SET reduce capacity of myeloid cells to undergo terminal granulocytic differentiation. Our data demonstrate that phosphorylation on serine 21 modulates association of C/EBPα with protein partners that are functionally relevant for myeloid differentiation. Our ability to quantitatively monitor multiple leukemia-related gene products in the context of C/EBPα protein complexes provides valuable insight into the mechanisms by which oncogenic kinase activity disrupts transcription and leads to leukemogenesis.

Citation Information: Cancer Res 2009;69(23 Suppl):B51.
Abstract B55: The impact of inactivation of p53 and PTEN tumor suppressors on promotion and progression of human non-small cell lung carcinoma
Authors: Andjelkovic Tijana , Bankovic Jasna , Milinkovic Vedrana , Podolski Ana , Ruzdijic Sabera , Tanic Nikola
Published: May 2012

Lung cancer is the major cause of cancer-related death worldwide and the five-year survival rate is not improving significantly, remaining at about 15% for all stages. Inactivation of p53 and PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumor suppressors are molecular events highly related to the pathogenesis of lung cancer, particularly to non-small cell lung carcinoma (NSCLC). However, we still do not have clear image of the sequence of events in NSCLC promotion and progression, that is, whether inactivation of either of these tumor suppressors is early or late event and what the impact of these events is on NSCLC pathogenesis and patients survival. Therefore, we analyzed mutational inactivation of p53 and inactivation of PTEN by loss of heterozygosity (LOH) in 30 NSCLC patients and correlated obtained data with histopathological parameters that are indicators of initiation and progression of the disease: histological subtype, tumor grade, tumor stage, lymph node invasion and necrosis. PCR-single strand conformation polymorphism (PCR-SSCP) analyses revealed and sequencing procedures confirmed that 33.3% of patients had mutationaly inactivated p53 while fragment analysis of PTEN showed that 43.3% had inactivated PTEN by LOH. In total population of analyzed patients, 13.3% of them carried inactivated p53 only, 23.3% had inactivated PTEN only, while 20.0% of patients had inactivated both tumor suppressors. These data suggest that inactivation of PTEN, either alone or in combination with the inactivation of p53, prevalently occurs in NSCLC. Correlation analyses imply that inactivation of either p53 or PTEN is an early event in pathogenesis of NSCLC, while inactivation of both tumor-suppressors contributes to further tumor progression. In addition, correlation analysis of inactivated PTEN and p53 with the degree of genomic instability of each patient (obtained in previous study) disclosed that mutated p53 is associated with high genomic instability while inactivated PTEN was more frequent in patients with low genomic instability confirming important role of p53 in maintaining the stability of the genome. Finally, Kaplan & Meyer survival analyses revealed that patients with inactivated p53 have much worse prognoses. In order to conclude the study of p53 and PTEN inactivation, analyses of hypermetylation of PTEN promoter and LOH of p53 gene are underway.

Citation Information: Cancer Res 2009;69(23 Suppl):B55.