bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3941: Altered helical structures: Repair and induction of genomic instability
Authors: Rogers Faye A.
Published: February 2011

Naturally occurring DNA sequences capable of forming H-DNA (triplexes) are found in the human genome as frequently as 1 in every 50,000 base pairs. These sequences, typically located in promoters and exons have been found to be intrinsically mutagenic and are believed to be involved in the regulation of expression of several cancer-linked genes. To counteract the potentially devastating effects of altered helical structures on genomic integrity, an intricate balance between DNA repair and apoptosis is critical. In the present work, we have investigated the potential for non-canonical structures to trigger pro-apoptotic pathways. In conjunction with the use of a fibroblast line containing multiple chromosomal target sites, sequence-specific triplex-forming oligonucleotides (TFOs) were used to synthetically create altered helical structures. Our studies demonstrate that DNA damage induced by these structures can trigger the switch from DNA repair to apoptosis. Additional studies were initiated to determine whether key proteins that contributed to cellular survival by participating in the repair of helical distortions also played a role in triggering the cell to activate a pro-apoptotic pathway. The TFIIH factor, XPD has been implicated in our studies to occupy a central role in triggering apoptosis in response to excessive H-DNA induced damage. Knock-down of XPD resulted in a significant decrease in H-DNA induced apoptosis, thus suggesting that key proteins that contribute to cellular survival by acting in DNA repair become executioners in the face of excess DNA damage. The maintenance of this mechanism may be of central importance for avoiding progression to cancer, because the default mechanism of apoptosis prevents expansion of cells in which unrepaired damage would lead to mutation and carcinogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3941.
Abstract 3942: Novel mechanism for inactivation of the DNA damage response during apoptosis
Authors: Solier Stéphanie , Pommier Yves
Published: February 2011

Apoptosis, programmed cell death, is required in multicellular organisms for various physiological processes. Disruption of the equilibrium between cell death and proliferation is one of the mechanisms of tumor formation. Therefore, one of the challenges and one of the ongoing approaches for cancer therapy is to develop pro-apoptotic agents. One such agent is TRAIL (Tumor necrosis factor-Related Apoptosis-Inducing Ligand), a promising therapy in development because it induces apoptosis preferentially in cancer cells compared to normal cells. We recently identified the “apoptotic ring”, a previously unnoticed process of cellular apoptosis by the nuclear peripheral staining of phosphorylated DNA damage response (DDR) proteins including γ-H2AX, ATM, DNA-PK and Chk2. The γ-H2AX apoptotic ring forms in the early phase of the apoptotic response to a broad range of stimuli including TRAIL in different cell types. It differs from the focal pattern response (DDR nuclear foci) to DNA damaging agents. Because γ-H2AX is a fundamental chromatin modification in response to DNA double-strand breaks and serves as a molecular platform for the docking of DDR protein complexes and for amplifying the DDR, we were particularly interested to determine whether the apoptotic γ-H2AX response led to the full recruitment of the DDR factors. Here we looked at MDC1 (mediator of DNA damage checkpoint 1) which normally binds to γ-H2AX in response to DNA damage and amplifies the DDR. We will report that during TRAIL-induced apoptosis, MDC1 does not exhibit an apoptotic ring. Lately a new phosphorylation of histone H2AX on tyrosine 142 (P-Y142-H2AX) has been described, thereby preventing the binding of MDC1 to γ-H2AX. However, we find that Y142 phosphorylation is reduced in response to TRAIL and uncovered a novel mechanism to explain the inactivation of MDC1-mediated transduction of the DDR during apoptosis. Indeed, we demonstrate for the first time that MDC1 is cleaved by caspase-3 during apoptosis, separating the FHA (forkhead-associated) and the BRCT (breast cancer C-terminal) domains, and consequently disrupting the linkage to γ-H2AX and other DNA damage responsive proteins.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3942.
Abstract 3947: PARP-1 as a therapeutic target in gliomas
Authors: Seoane Marcos , Iglesias Pablo , Castro Lydia , Arce Victror M. , Costoya Jose A.
Published: February 2011

Gliomas are the most common primary tumors in the brain. We use this tumor type as a paradigm of oncogenic transformation. Deregulation of signal transduction pathways are commonly found both in low- and high-grade gliomas. Moreover, all WHO grade III and IV (GBM) show a disruption of cell cycle due to alterations in at least one out of three families of proteins involved in G1/S transition, such as: CDKs, INK4a-ARF or Rb.

Oncogenic HRas (HRasV12) expression recapitulates gain-of-function mutations in several growth factor receptors in glial cells, resulting in the activation of different downstream effectors such as Raf/MEK/ERK and PI3K/AKT signaling pathways. The hyperactivation of growth signaling pathways, phenocopied by HRasV12, increases cell proliferation rate in the absence of senescence, facilitating their transformation into tumoral cells. Furthermore, our data indicate that HRasV12 is able to cooperate with Rb loss in the tumor malignization.

Poly(ADP-ribose) polymerase-1 (PARP-1) facilitates DNA repair by binding to DNA breaks and attracting DNA repair proteins to the site of DNA damage. Despite its important role in the cellular response to genotoxic stress, PARP-1 is not required for survival and Parp-1−/− mice are viable and fertile. These mice do not develop early onset tumors and tumor latency is increased in PARP-1 knockout mice that are deficient for p53. Accordingly, here we observed that Parp-1−/− astrocytes display a marked proliferative decrease when compared with Parp-1+/+ astrocytes which express HRasV12, either in a Rb+/+ or in a Rb−/− context.

Given that loss of PARP-1 are less susceptible to HRasV12 induced transformation, the inactivation of PARP-1 with chemical inhibitors is a potential therapeutic approach to restrain the tumor progression. Interestingly, in our model of gliomagenesis PARP-1 chemical inhibition (PJ34) leads to a significant fall in the proliferative rate in HRasV12 astrocytes comparable to Parp-1−/− astrocytes, and is also able to reduce tumor progression even when HRasV12 cooperates with Rb loss. These results indicate that treatment with PARP-1 inhibitors might be a potential therapy for this type of tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3947.
Abstract 395: Rapid engraftment of human primary acute leukemic cells in NOD/SCID mice by intrasplenic inoculation: a robust human primary leukemia (Hukemia) model for preclinical evaluation of new therapies
Authors: Liang Huang , Zhang Amy , Wang Na , Ma Shuyan , Liu Jingping , Qi Xiaoxu , Song Xiaoming , Chen Taiping , Xia Michelle , Wery Jean-Pierre , Chen Xingjun , Chen Yiyou , Zhou Jianfeng , Zhou Pengfei
Published: February 2011

The ability to engraft human leukemia cells directly from patient samples into immunodeficient rodents such as NOD/SCID mice would be uniquely valuable in preclinical prioritization of novel therapies for future clinical trial. The aim of this study was to develop an animal model for human acute leukemia in which the kinetics, characteristics and chemosensitivity of leukemia can be sequentially monitored in individual immunodeficient mouse model. Continuous xenografts from 28 acute myeloid leukemia (AML) patients and 18 acute lymphoblastic leukemia (ALL) patients with AML-ETO, CBFβ/MYH11, TAL/SIL1, MLL/AF4 fusion genes were established in NOD/SCID mice by intrasplenic inoculation of human leukemic cells from born marrow. Progression of leukemia was monitored throughout the development of disease by determination of immunophenotyping and karyotyping of the engraftment tumor cells. Chemosensitivity and in vivo efficacy to standard of care therapies were screened in xenografts from both AML and ALL patients. Genetic lesions and special surface markers in different passages of cells in recipient mice were also examined by gene array and FACS analysis. Intrasplenic inoculation of leukemia cells significantly enhanced the engraftment rate and expedited disease development when compared with I.V. route. Relative to engrafted human primary cells, negligible changes in growth rates, chromosomal aberrations and immunophenotype were observed at second, third and fourth passages. The in vivo sensitivity of xenografts to standard of care correlated significantly with clinical course and patient outcome. We have also uncovered key molecular events and serum immunoassay biomarkers which correlate with the efficacy and potency of the drugs for AML and ALL models. The biologic and genetic characteristics and patterns of chemosensitivity of xenografts from acute leukemia patients accurately reflect the clinical disease. The efficient engraftment and rapid expansion of the leukemia within NOD/SCID mice affords a robust model for the preclinical evaluation of new therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 395.
Abstract 3954: A role for the cholecystokinin 2 receptor (CCK-2R) in promoting cancer progression
Authors: Mayne Cerys M. , Grabowska Anna M. , Watson Susan A.
Published: February 2011

Background and aims: The gastrointestinal (GI) hormone, gastrin, promotes cancer progression and its down-regulation has been linked to reduced cancer stem cell numbers. Gastrin acts through the cholecystokinin 2 receptor (CCK-2R) and its biological effects are blocked by CCK-2R inhibitors. We investigated a potential role for CCK-2R in promoting survival of cancer stem cells using RNAi combined with a sensitive method to detect CCK-2R mRNA.

Materials and methods: A panel of cancer cell-lines was used, including GI, glioblastoma and small cell lung cancer (SCLC), with CCK-2R-transfected cells as a positive control. Linear-after-the-Exponential (LATE)-PCR was used to quantify CCK-2R gene expression and its sensitivity compared with a Taqman-based assay. Flow cytometry was used to investigate receptor protein expression. The activity of CCK-2R promoter reporters constructed in pGL4, using between 250 and 2000bp of DNA upstream of the CCK-2R start codon, was quantified using luciferase assays.

Results: LATE-PCR for CCK-2R gene expression is 1000-fold more sensitive than the Taqman-based assay. Cell-lines from the panel, including HCT116 (colorectal) and H209 (SCLC), in which CCK-2R mRNA was not detectable by the Taqman assay, were positive using the LATE-PCR. CCK-2R siRNAs resulted in up to 86% knockdown of the receptor in CCK-2R-transfected AGS cells, confirming the specificity of the LATE-PCR. FACS analysis for the CCK-2R protein suggests the presence of a small population of cells within HCT116 (colorectal) and AGS (gastric) cell-lines that express CCK-2R very highly. CCK-2R expression was enriched when cells were grown as colospheres and RNAi demonstrated a role for the CCK2R in promoting cell survival. The CCK-2R promoter constructs were active in cancer cell-lines, indicating that the 250bp proximal to the CCK-2R start codon are most crucial for transcription. However, transcriptional activity did not always correlate with gene expression.

Conclusions: LATE-PCR provides a highly sensitive method for detection of genes such as CCK-2R which have important biological functions but low expression. An element just upstream of the CCK-2R transcriptional start site appears to be controlling transcription in cell-lines showing low levels of endogenous expression of the CCK-2R genes, but lack of correlation between the RNA levels and transcriptional activity suggests possible post-transcriptional regulation of this gene. CCK-2R protein expression is elevated in a subset of cells, and may play a role in promoting survival of cancer stem cells.

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

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3954.
Abstract 3957: Identification of activated receptor tyrosine kinases in gastric cancer cells
Authors: Yoon Sang Hyun , Lee Myung Eun , Jeung Hei-Cheul , Jung Jae-Joon , Kim Tae Soo , Chung Hyun Cheol , Shin Sang Joon , Rha Sun Young
Published: February 2011

To improve gastric cancer treatment, relevant molecular targets need to be identified. Receptor tyrosine kinases (RTKs) are multifunctional transmembrane proteins and important mediators of the signaling cascade, determining key roles in diverse biological processes like proliferation, growth, differentiation, and apoptosis of cancer cells. To determine if any of these RTKs are activated in gastric cancer, we performed phospho-receptor tyrosine kinase array with 25 gastric cancer cell lines using Human Phospho-RTK Antibody Proteome Profiler Array (R&D Systems) which includes 42 different tyrosine kinases. Relative levels of tyrosine phosphorylation of RTKs among these cell lines were determined as a ratio relative to four positive phosphorylated tyrosine kinase control's intensity in each membrane.

We found that various growth factor receptor tyrosine kinases were differentially activated and 15 of 42 RTKs were highly activated with mean intensity >5 among 25 gastric cancer cell lines. The highly activated RTKs were: EGFR (93.2), ErbB2 (33.0), HGFR (27.6), ErbB3 (26.2), IGF-IR (17.2), InsulinR (13.9), Mer (12.9), FGFR2α (11.4), Dtk (10.1), EphA7 (9.5), Tie-2 (8.9), MSPR (6.5), c-Ret (6.4), FGFR3 (5.4), and ErbB4 (5.1). These receptors basically belong to the EGF, HGF, FGF, Insulin, Tie, and Ephrin growth factor gene families_all of which are involved in cell proliferation, differentiation or signal transduction. We confirmed that increased RTKs phosphorylation observed by RTK array analysis was correlated with the expression level of each protein by immunoblotting. Additionally, we showed that Met gene amplification by quantitative real-time PCR is associated with Met activation in gastric cancer cell lines. In conclusion, these activated RTKs may work as potential therapeutic targets in gastric cancer and helpful to understand the molecular mechanisms underlying tumor development.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3957.
Abstract 3958: IL-6 stimulates cell motility in squamous cell carcinoma of head and neck (SCCHN) cells through CK2 phosphorylation and stabilization of TWIST
Authors: Su Yingwen , Xie Tongxin , Fredrick Mitchell J. , Daisuke Sano , Myers Jeffrey N.
Published: February 2011

Squamous cell carcinoma of head and neck (SCCHN) is the tenth most common cancer in the world. Unfortunately, the survival of patients with SCCHN has not improved in the last 40 years. Therefore new targets for therapy are needed, and to this end we are studying signaling pathways activated by IL-6 which we have found stimulates cell migration and soft agar growth in SCCHN. Our data show that IL-6 increases TWIST expression in a transcription-independent mechanism in many SCCHN cell lines. Further investigation reveals TWIST can be phosphorylated upon IL-6 treatment. By computation prediction (http://hits.isb-sib.ch/cgi-bin/PFSCAN), we found that TWIST has a putative phosphorylation site for casein kinase 2 (CK2) suggesting that this kinase could serve as a link between IL-6 stimulation and Twist stability. To test this hypothesis, we used a CK2 inhibitor and shRNA to CK2 and found that these interventions inhibited IL-6 stimulation of TWIST stability. In addtion, mutation of the putative CK2 phosphorylation site (S18/S20) in TWIST decreased the amount of phospho- ATP incorporated by TWIST in an in vitro kinase assay and altered TWIST stability. In soft agar colony forming assays, the CK2 inhibitor, DMAT, was found to decrease the number of IL-6 stimulated colonies formed by SCCHN cells. To our knowledge, this is the first report to identify the importance of IL-6 stimulated CK2 phosphorylation of TWIST in SCCHN and supports the previous findings from other researchers. As CK2 inhibitors are currently under phase I clinical trials, our findings indicate that CK2 may be a viable therapeutic target in SCCHN. Therefore, further pre-clinical studies of this inhibitor are underway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3958.
Abstract 3962: Wnt signaling modulates the Hedgehog pathway by stabilizing GLI1 mRNA
Authors: Noubissi Felicite , Goswami Srikanta , Sanek Nicholas A. , Kawakami Kazuyuki , Minamoto Toshinari , Moser Amy , Grinblat Yevgenya , Spiegelman Vladimir S.
Published: February 2011

Wnt and Hedgehog signaling pathways play central roles in embryogenesis, stem cell maintenance, and tumorigenesis. These two pathways being fundamental in the coordination of developmental transitions, have been postulated to interact, or cross-regulate at multiple levels yet the mechanisms of these interactions are not well-understood.

Here, we show that Wnt signaling pathway stimulates the transcriptional output of Hedgehog signaling. We demonstrate that Wnt/β-catenin signaling induces the expression of an RNA-binding protein, CRD-BP, which, in turn, binds and stabilizes GLI1 mRNA, causing an elevation of GLI1 expression and transcriptional activity.

The newly described mode of regulation of GLI1 appears to be important to several functions of Wnt, including survival and proliferation of colorectal cancer cells.

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

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3962.
Abstract 3964: Transforming growth factor-beta dependent regulation in breast cancer progression
Authors: Kim Hye Ok , Song Junkyu , Cho Chang Hoon , Yoon Kyung-sik ,   Department of Biochemistry and Molecular Biology (BK21 project), MRC for Bioreaction to ROS and Bi
Published: February 2011

Transforming growth factor-ß (TGF-ß) has a wide range of biologic functions such as the regulation of cell growth, differentiation, and immunologic response in various types of cells. The involvement of TGF-ß in the pathogenesis of breast cancer has been proposed based on its capacity to stimulate the production of extracellular matrix (ECM) proteins and to regulate their metabolism. Induction of plasminogen activator inhibitor-1 (PAI-1) by TGF-ß may contribute to the degradation of extracellular matrix during cancer invasion by decreasing the adhesive strength of cells. Thus, it is important to clarify the mechanism of TGF-ß-induced PAI-1 production. Several polymorphisms have been described in the PAI-1 gene. We examined the influence of the G/A (−844) and 4G/5G (−675) polymorphisms in the promoter region on TGFß-induced PAI-1 expression by breast cancer cells (MDA-MB231, MCF7). The genotype of breast cancer cells was identified by sequencing. TGF-ß-induced PAI-1 mRNA expression was determined by real time polymerase chain reaction (PCR). Activation of MAPKs and Smad proteins was evaluated by an immunoblot analysis. The activities of PAI-1 promoter were measured by a luciferase reporter assay. TGF-ß increased PAI-1 expression in a genotype-dependent manner, higher values observed for MDA-MB231 (4G/4G, A/A) compared with MCF7 (5G/5G, G/G). These results suggest that regulation by TGF-ß influences the PAI-1 polymorphism-dependent expression in breast cancer cells. Analysis of PAI-1 genotype may help identifying subgroups of breast cancer risk.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3964.
Abstract 3967: Dihydrotestosterone downregulates expression of smad3 in prostate epithelial cells through a transcriptional mechanism, promoting androgenic protection against TGF-beta-induced apoptosis
Authors: Song Kyung , Wang Hui , Krebs Tracy L. , Wang Bingcheng , Kelley Thomas J. , Danielpour David
Published: February 2011

Androgens suppress TGF-beta responses in the prostate through mechanisms that are not fully explored. We have recently reported that 5a-dihydrotestosterone (DHT) suppresses the ability of TGF-beta to inhibit proliferation and to induce apoptosis of prostatic epithelial cells, and provided evidence that such suppression was fueled by the transcriptional downregulation of TGF-beta receptor II (TbRII). We now show that AR activated by DHT suppresses the TGF-beta-induced phosphorylation of Smad3 in LNCaP cells overexpressing TbRII under the control of a CMV promoter, suggesting that transcriptional repression of TbRII alone does not fully account for the impact of DHT on TGF-beta responses. Instead, we demonstrate that such suppression occurs through loss of total Smad3, resulting from transcriptional suppression of Smad3. Consistent with this mechanism, we report that loss of Smad3 mRNA inversely correlates with elevated AR mRNA in a cohort of human prostate tissues (p < 0.0001). We provide evidence that DHT downregulates the promoter activity of Smad3 in various prostate cancer cell lines, including NRP-154+AR, DU145+AR, LNCaP, and VCaP, at least partly through androgen-dependent inactivation of Sp1. Moreover, we show that overexpression of Smad3 reverses the ability of DHT to protect against TGF-beta-induced apoptosis in NRP-154, supporting our model that loss of Smad3 by DHT is involved in the protection against TGF-beta-induced apoptosis. Together, these findings suggest that deregulated/enhanced expression and activation of AR in prostate carcinomas may intercept the tumor suppressor function of TGF-beta through transcriptional suppression of Smad3, thereby providing new mechanistic insight on the development of castration-resistant prostate cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3967.
Abstract 3969: Stromal depletion of transforming growth factor receptor 2 promotes the development of forestomach squamous cell carcinoma
Authors: Achyut B R. , Bader David A. , Yan Hannah H. , Li Chaoyang , Pang Yanli , Yang Li
Published: February 2011

Deletion or attenuation of transforming growth factor beta (TGFβ) signaling pathway has significant impact on human tumor development and progression. Specific deletion of transforming growth factor receptor 2 gene (Tgfbr2) in a subset of fibroblast (Tgfbr2fspKO, cre-mediated DNA recombination driven by fibroblast specific protein, FSP) specific promoter, leads to the development of squamous cell carcinoma of the forestomach in 100% of Tgfbr2fspKO mice by 6 Wks of Age. Here we dissect the underlying mechanisms. We first confirmed that deletion of Tgfbr2 is primarly restricted to the stromal components but not epithelial cells using TBR2 (TGFβR2) immunofluorescence and immunohistochemistry, which was done in both tumor tissue sections and cancer epithelial cells derived from tgfbr2fspKO mice. The functionality of TGFβ signaling was further examined in vivo by p-smad2 expression and nuclear localization which was clearly observed in the epithelial but not the stromal components. There was also a clear nuclear localization of p-smad2 protein in cultured cancer epithelial cells when treated with TGFβ1 (10 ng/ml). We further observed that stromal deletion of Tgfbr2 resulted in elevated proliferation of fibroblasts. In addition, there was a significantly increased inflammation in forestomach lesion which was featured by increased CD45+ leukocyte infiltration and elevated expression of inflammatory mediators COX2, NFkB, Akt. Interestingly, the alterations in epithelial cell compartment in Tgfbr2fspKO mice is very evident compared with floxed control littermates Tgfbr2fspKO mice, showing a significant enrichment of epithelial cancer stem cells. Our data demonstrate the importance of stromal TGFβ signaling mediated inflammation and carcinogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3969.
Abstract 3970: DRAK2 inhibits the TGF-B signaling pathwsy through interaction with TGF-B receptors
Authors: Yang Kyung-Min , Kim Seong-Jin
Published: February 2011

Transforming growth factor β (TGF-β) controls a diverse set of cellular responses, including proliferation, differentiation, migration, apoptosis and immune modulation. In recent years, research has begun to unravel the mechanisms that tune the TGF-β signaling pathway. These mechanisms include interactions and modifications that regulate the stability of TGF-β's signaling machinery, receptors and Smads. In addition, they are known to be of major importance for maintaining proper physiological TGF-β signaling. In this study, we have identified a novel TGF-β receptor binding protein called DRAK2 (DAP kinase-related apoptosis-inducing kinase 2). Here, we show that DRAK2 inhibits TGF-β-induced Smad2/3 phosphorylation. To identify the interaction between TGF-β receptors and DRAK2, we performed immunoprecipitation, immunofluorescence staining and in situ proximity ligation assay. DRAK2 specifically bound to the TGF-β receptors. Although ectopic expression of DRAK2 did not inhibit the interaction between TGF-β receptor type I and type II, transient transfection of DRAK2 reduced TGF-β-dependent reporter activity. In stable cell lines that overexpress DRAK2, DRAK2 suppressed the phosphorylation of Smad2 and Smad3 and activity of Smad2-specific (ARE) and Smad3-specific (CAGA) reporters. Furthermore, the expression of DRAK2 attenuated TGF-β-induced translocation of Smad2 and Smad3 from the cytoplasm to nucleus. Conversely, the knockdown of DRAK2 enhanced the TGF-β signaling pathway including TGF-β-dependent promoter activity and phosphorylation of Smad2 and Smad3. Thus, these results suggest that DRAK2 may be a negative regulator in the TGF-β signaling pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3970.
Abstract 3971: The type III tgf-β receptor mediates bmp signaling in normal and cancerous mammary epithelial cells
Authors: Gatza Catherine E. , Blobe Gerard C.
Published: February 2011

BMP signaling has been shown to enhance breast cancer invasion and bone metastasis, while inhibition of BMP signaling abrogates bone metastasis in an in vivo model. The type III TGF-β receptor (TβRIII) is known to mediate BMP signaling and can inhibit migration, invasion, and metastasis in breast cancer, however the role of TβRIII in regulating BMP signaling in breast cancer has not been examined. The MDA-MB-231, MCF7, and mouse 4T1 breast cancer cell lines, which express low levels of TβRIII, are responsive to BMP2 and 4 treatment as demonstrated by increased Smad1/5/8 phosphorylation, cell migration, and invasion. Restoring TβRIII expression in these cell lines or treatment of MDA-MB-231-Neo cells with recombinant soluble TβRIII (sTβRIII) or with conditioned media from MDA-MB-231-RIII cells inhibited BMP2 and BMP4 induced Smad1/5/8 phosphorylation (pSmad1/5/8) and BMP mediated cell migration. MDA-MB-231-RIII cells also exhibited enhanced growth arrest in response to BMP2 compared to MDA-MB-231-Neo cells, mediated through the up-regulation of p21. In reciprocal studies, shRNA-mediated knockdown of TβRIII expression in the normal mammary epithelial cell lines HMEC and MCF10A, abrogated BMP mediated induction of pSmad1/5/8, demonstrating that TβRIII mediates BMP signaling in normal mammary epithelial cells. Additionally, treatment with both recombinant sTβRIII and conditioned media from MDA-MB-231-RIII cells inhibits BMP mediated induction of pSmad1/5/8, suggesting that sTβRIII is able to inhibit BMP signaling in normal mammary epithelial cells by sequestration of ligand. BMP2 treatment induces cell migration in HMEC cells, which was attenuated by knockdown of TβRIII or treatment with sTβRIII. These data demonstrate that TβRIII mediates BMP signaling in normal mammary epithelial cells and breast cancer cell lines, regulating cell migration, invasion, and proliferation. The effects of sTβRIII suggest that the ratio of membrane bound versus soluble TβRIII plays an important role in mediating BMP signaling and cellular effects in normal and cancer mammary epithelial cells. Further work is underway to elucidate the mechanism by which TβRIII regulates BMP signaling in breast cancer in vitro and in vivo.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3971.
Abstract 3975: CLIC4 induces a Smad7 variant to enhance TGF-β signaling in keratinocytes
Authors: Shukla Anjali , Yang Yihan , Madanikia Sara , Ho Yan , Yuspa Stuart H.
Published: February 2011

CLIC4 (Chloride intracellular channel 4) is a member of a family of intracellular chloride channel proteins that are ubiquitously expressed in multiple tissue types. It is a dimorphic protein that exists in both soluble and membrane-bound forms. CLIC4 is multifunctional, traffics between the cytoplasm and nucleus, and participates in cell cycle control and differentiation in keratinocytes and other cell types. CLIC4 expression is lost in multiple human epithelial cancers. We have recently shown that nuclear CLIC4 enhances transforming growth factor beta (TGF-β) signaling through stabilization of phospho-Smad2/3. The TGF-β signaling pathway is a major growth regulator of multiple cell types, and its regulation is altered in many human cancers suggesting that CLIC4 could participate in altering TGF-β regulation in tumor cells. Here we show that in addition to stabilizing phospho-Smads 2/3, CLIC4 alters Smad7, a feedback inhibitor of the TGF-β pathway. Elevated levels of CLIC4 in primary keratinocytes lead to the expression of a truncated form of Smad7 (Smad7Δ), which is detectable as a PCR product. The Smad7Δ variant contains a missing region of 94bp in exon 4 of Smad7 and appears to be an alternately spliced form. Absence of these 94bp leads to a frameshift mutation and an early protein truncation causing the Smad7Δ variant to lack the MH2 domain of Smad7, the domain responsible for inhibition of TGF-β signaling. While wild-type Smad7 expression inhibits TGF-β signaling, exogenously expressed Smad7Δ variant does not inhibit TGF-β signaling, based upon analysis of TGF-β dependent proliferation, reporter assays and phosphorylation of Smad proteins. Together, these data suggest that CLIC4 regulates TGF-β signaling by multiple mechanisms. In light of this, CLIC4 may serve as a target for modifying TGF-β function in cancer and other conditions where this pathway is involved in pathological changes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3975.
Abstract 3976: A novel violet-laser excitable ratiometric probe for the detection of membrane asymmetry breakdown during apoptosis
Authors: Hicks Justin , Bradford Jolene
Published: February 2011

Apoptosis is carefully regulated and essential part of normal tissue development and homeostasis. Regulatory changes in the apoptotic pathway have been implicated in many diseases; most notable are many types of cancers and autoimmune disorders. Normal cells exhibit a remarkable asymmetry in lipid distribution between the outer and inner cell membranes characterized by phosphatidyl-serine (PS) and phosphatidylethanolamine (PE) normally located on the inner leaflet of the cell membrane. During apoptosis translocation of PE and PS to the external cellular environment facilitates recognition and elimination of these cells by macrophages. The Violet Ratiometric Membrane Asymmetry Probe, 4′-N, N-diethylamino-6-(N, N, N-dodecyl-methylamino-sulfopropyl)-methyl-3-hydroxyflavone (F2N12S), is a novel violet excitable dye for the detection of membrane asymmetry changes during apoptosis. The dye exhibits an excited-state intramolecular proton transfer (ESIPT) reaction resulting in a dual fluorescence with two emission bands corresponding to 530 nm and 585 nm, producing a two-color ratiometric response to variations in surface charge. This ratiometric probe is a self-calibrating absolute parameter of apoptotic transformation, which is independent of probe concentration, cell size, and instrument variation. The two-color ratiometric response of the F2N12S reagent will detect apoptotic cells in samples that vary in cell concentration by as much as 100 fold. The Violet Ratiometric Membrane Asymmetry Probe rapidly targets the plasma membrane requiring only a 5 minute room temperature incubation without additional buffers or wash steps reducing the chance of cell death that can occur from prolonged sample processing. The Violet Ratiometric Membrane Asymmetry Probe will work in both suspension and adherent cell lines and its efficacy is confirmed by caspase and mitochondrial apoptosis markers. Annexin V staining produced a larger population of apoptotic cells during early time points in both suspension and adherent cell lines that do not correlate with caspase and mitochondrial apoptosis markers. The violet 405 nm excitation of F2N12S expands the capability of multi-parameter apoptosis assays to study the apoptotic process while reducing or eliminating the need to create complex compensation controls by taking advantage of the time and spectral separation a violet-excitable reagent provides.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3976.
Abstract 3985: New quantification tools for perfusion and VEGFR2 Expression in tumors
Authors: Foster F. Stuart , Coulthard Tonya , Needles Andrew , Theodoropoulos Catherine
Published: February 2011

Microbubble contrast agents are used to enhance the visualization of the microcirculation in human and mouse tumors. Specifically, contrast agents allow for the relative quantification of tissue perfusion and vascular volume. The in vivo expression of endothelial cell surface markers can also be studied using targeted microbubbles. In this study a nonlinear contrast agent imaging technique, employing amplitude modulation on a linear array-based micro-ultrasound system was used to visualize and quantify time to peak enhancement (TP) and vascular volume (VV) in subcutaneous tumors, as well as to assess expression levels of VEGFR2.

A Vevo 2100 high resolution ultrasound imaging system (VisualSonics Inc, Toronto, Canada) was used and all images were acquired with the MS-250 (center operating frequency of 20MHz, axial resolution 75um) probe. Tumor flow was assessed using MicroMarker contrast agents. A bolus delivery was employed to generate time-intensity curves of contrast wash-in; prototype analytical software was used to perform curve fit analysis and to generate parametric images visually depicting VV and TP as an overlay on the anatomical images. Additionally, Target-Ready MicroMarker contrast agents were conjugated to VEGFR2 antibodies to assess the expression level of this endothelial cell surface receptor. Again, prototype analytical software was used to quantify the relative expression of this target and to generate parametric images of its expression pattern within the tumor. All images were acquired from athymic nude mice implanted with hepatocellular carcinoma cells (Hep3B-Luc-C4) subdermally in the hind limb 4 to 5 weeks before imaging.

Results showed heterogeneity in TP and VV in all tumors. TP ranged from 3.5 seconds in well perfused regions to 12 seconds in poorly perfused tumor regions. The graphical appearance of the associated parametric images readily revealed these differences. Assessment of VEGFR2 expression completed in 2D and 3D showed 2-3 times enhancement in VEGFR2 binding over that observed with the isotype control antibody. These results are consistent with previous methods using linear contrast processing. Overall this study shows the utility of nonlinear processing of contrast signals in assessment of various flow related parameters of the tumor microcirculation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3985.
Abstract 3989: Ovarian cancer protein biomarker identification from the tumor tissue microenvironment
Authors: Hoskins Ebony R. , Hood Brian , Conrads Tom , Edwards Robert P.
Published: February 2011

Objective: In order to circumvent the challenges associated with serum-based biomarker discovery for ovarian cancer screening development, we utilized a novel sampling strategy to harvest shed and secreted proteins from tissue interstitial fluid (TIF) from the ovarian tumor tissue microenvironment. The motivating hypothesis is TIF, the proximal fluid that bathes ovarian cancer cells and stroma, is rich in cancer-specific proteins that are readily identifiable by mass spectrometry (MS)-based proteomics.

Materials: Ovarian tumor tissue and ascites were collected within 30 minutes of removal from the patient, at the time of debulking surgery. Samples used in the analysis were from four chemotherapy naïve patients with pathology-proven papillary serous ovarian cancer. We implemented a standardized sampling strategy, involving incubation of tumor tissue in saline for 1 hour immediately following resection resulting in protein rich TIF. The TIF and ascites samples were analyzed by MS for protein sequence identification and relative quantitation.

Results: Over 2700 total proteins were identified in TIF across all patient samples, while ascitic fluid contained over 2000 proteins across all patient samples. 13 proteins were identified in all TIF samples that were 3-fold or greater in quantity compared to ascitic fluid samples. For comparison, 14 proteins that were 3-fold greater in ascitic fluid compared to TIF, was also identified. Interestingly, these select proteins from two microenvironments had contrasting function and location. TIF samples contained largely intracellular proteins, which are secreted and/or shed while ascitic fluid sample contained extracellular proteins, similar to serum, thus confirming the utility of TIF representing the tumor microenvironment, see figure. The results demonstrate the ability to readily identify hundreds of proteins in TIF, which are at higher abundance compared to those, identified in ascitic fluid from the same patients.

Conclusions: We developed a standard workflow for the discovery of candidate biomarkers from the ovarian cancer tissue microenvironment. The TIF proteome contains more lower abundant and cancer specific proteins compared to ascitic fluid. We are prospectively validating candidate biomarkers from this microenvironment in patient serum by western blot analysis and immunohistochemistry. Ultimately we will expand our standard protocol to a cohort of patients with likely early stage cancers in order to translate to a serum-based screening test.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3989.
Abstract 399: A special childhood T-ALL model of NOD/SCID mice can recapitulate the clinical patient disease
Authors: Zhang Amy , Huang Liang , Ma Shuyan , Wang Na , Liu Jingping , Song Xiaoming , Chen Taiping , Wery Jean-Pierre , Chen Yiyou , Zhou Jianfeng , Zhou Pengfei
Published: February 2011

Despite recent advances in the management of childhood acute lymphoblastic leukemia (ALL), the risk of relapse remains about 30 percent. Here we report an interesting childhood T-acute lymphoblastic leukemia (T-ALL) patient case, engrafting into NOD/SCID mice successfully, recaptured the clinical course accurately including the rapid remission induction, minimal residual disease and relapsing. Immunophenotypes of primary, secondary and third xenografts indicated the cells expressing CD3/CD5/CD7/CD4, as well as the genetic lesions, were unaltered compared with the original patient sample. Immunohistochemistry (IHC) studies showed more than 90% infiltration of leukemic cells in spleen, liver, lung and variable level in other organs include brain within 4 weeks in primary xenografts and 2 weeks in secondary xenografts. The response to Vinscristine and Dexamesathone of primary engrafts was similar to what the original patient did in the clinic. A minimal residual disease phase and relapsing model had also been established in this model after complete remission induced by Vincristine plus Dexamesathone. In addition, Dexamesathone treatment alone was able to prolong the survival of the T-ALL hosts for more than two weeks in the presence of over 90% cancer cell infiltration in the peripheral blood. This study supports previous finding that the NOD/SCID model of childhood T-ALL is an accurate representation of the patient disease and can be used in the screening of anticancer drugs for childhood ALL. This model also warrants further investigation in T-ALL at relapse and potentially in the study of minimal residual disease for chemotherapy-resistant T-ALL subgroups.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 399.
Abstract 3990: Methods for Fine Needle Aspiration (FNA) biopsy sampling and “ex vivo” signal transduction analysis of murine xenografted human tumors
Authors: Aguero Martin F. , Jeffries Emiko , Schayowitz Adam , Clark Douglas P.
Published: February 2011

Fine Needle Aspiration (FNA) biopsy is a common procedure for sampling human tumors to enable diagnosis and, increasingly, molecular diagnostics on fixed samples. However, FNA is rarely used to sample murine xenografted human tumors. FNA sampling of xenografted tumors offers several advantages over surgical excision: 1) efficiency; 2) enables serial sampling of individual tumors over time; 3) provides live cells to perform “ex vivo” analysis of signal transduction pathways. Objectives: To define a protocol for FNA sampling of murine xenografted tumors using an HCT-116 human colon carcinoma model; to characterize the cellular and molecular composition of such samples and to present proof-of-concept studies for “ex vivo” analysis from FNA samples. Methods: FNA biopsies were performed on HCT-116 xenografts using a standardized protocol that will be defined. Samples were assessed by microscopic analysis of direct smears stained with the Diff-Quik or Papanicolaou stains. Tumor cell number was determined by hemocytometer count and cell viability by trypan blue exclusion. Samples were lysed and total protein and RNA were extracted and quantitated by spectrophotometry. RNA quality was determined by RIN calculation. “Ex vivo” stimulation was performed by treating tumor cells obtained from FNA with TPA and EGF and subsequently analyzing phosphoprotein levels relative to baseline using Western blots. Results: FNA sample: Cellular composition: 25 % tumor cells, 50% macrophages, 20% lymphocytes, and 5% degenerate cells (n=5). Tumor cell viability median: after 1 hour post-FNA: 42% (range 22-88%) n=15. Cell viability decreases by 50% after 2 h post-FNA, (n=5). Total Protein median/FNA: 555 ug/ml (range 36-4222 ug/FNA) n=33; Total RNA median 14.80 ug/FNA (range 3.24-26.37 ug) n=18; RIN number median/FNA: 8.40 (range 3.10-9.50) n=15. “Ex vivo” analysis of signal transduction: a 60%, transient increase in phospho-ERK was identified within the first 5 min. post-FNA procedure, which returned to baseline. No increase in p-AKT or p-JNK was noted during this time period. Following “ex vivo” EGF or PMA stimulation, FNA samples showed a 1-3 fold increase -respectively- in p-ERK relative to baseline. Conclusions: Murine xenografts can be efficiently sampled by FNA biopsy. Such samples yield sufficient high-quality biomolecules for molecular analysis. FNA samples also enable live cell analysis, such as “ex vivo” stimulation of signal transduction pathways.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3990.