bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3207: Monitoring changes in NF-kB pathway regulation using highly sensitive multiplex bioluminescent reporter assays
Authors: Dobbs Megan , Hughes Douglas , Narahari Janaki , Choi Jae , Deshpande Atul , Webb Brian
Published: August 2012

Monitoring gene activation and repression in cellular signaling pathways requires specific tools. Reporter gene assays use a technique of attaching a genetic regulatory element, or promoter, to a gene encoding for a protein with enzymatic, chromophoric or fluorescent properties. Cloned into a plasmid vector, these sequences can be transfected or transduced into animal, plant or tissue culture cells. Reporter gene expression can be measured via bioluminescent, chemiluminescent or fluorescent output to quantify the element or protein activity in the pathway affecting the regulatory element. Bioluminescent output is formed through the catalysis of a substrate and the natural enzyme luciferase. Present in many terrestrial and marine metazoans, bioluminescence is a powerful instrument for mutational analysis of gene promoters, transcription factor dynamics, and protein-protein interactions. Our study confirmed the beneficial use of sensitive bioluminescent enzymes in reporter gene assays with wide dynamic ranges and flexibility in signal identification. Mutation of several of the luciferase genes resulted in spectral shifting and narrowing of the photon reaction products relative to the wavelength of the natural state. The mutations provide the ability to spectrally resolve the modified luciferases in a single reaction. In addition, multiplex assays relying on sequential addition of substrates can be achieved through manipulation of the enzyme-substrate reaction conditions. Marine copepod and ostracod luciferase enzymes are naturally secreted, and reporter assays using these genes can capture real-time or time course data by sampling the media of transfected cells or blood from transgenic animals and assaying for luciferase. The distinct advantage of these assays is that they are not destructive to the cells or animals. Our study used spectral separation using Gaussia/firefly and multiplex assays relying on sequential addition of substrates and Gaussia/Cypridina to monitor changes in NFkB activity in response to small-molecule agonists. Our results demonstrate the usefulness of dual-secreted luciferase assays for sensitive real-time monitoring of NFkB activity in media. Furthermore, these assays enable simultaneous detection of spectrally resolvable luciferases using filter-based detection.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3207. doi:1538-7445.AM2012-3207
Abstract 321: IL-6 in the tissue microenvironment plays a direct role in normal prostatic neoplastic transformation
Authors: Wu Jennifer D. , Liu Gang
Published: August 2012

Increased elevated levels of serum IL-6, the major inflammatory mediator, has been shown to be associated with development of progressive diseases in many types of cancers. We have previously shown that chronic exposure of immortalized benign prostate epithelial cells to paracrine IL-6 can induce tumorigenic transformation and further EMT (epithelial-mesochymal-transition)-associated metastasis in xenografts through stimulating autocrine IL-6 signaling and transactivating type I Insulin-like Growth Factor Receptor (IGF-IR). Whether IL-6, as a result of inflammation, in the tissue microenvironment can solely initiate tissue-specific de novo tumorigenesis is unclear. To address this question, we have generated a novel line of prostate-specific IL-6 transgenic mouse, in which human IL-6 (hIL-6) was expressed under the prostate-specific probasin promoter. We found that enforced expression of hIL-6 transgene in normal mouse prostate epithelium is sufficient to induce neoplastic transformation of the prostate gland, demonstrated by disappearance of base cell layer of the prostate gland and loss of E-cadherin and increased proliferation of the epithelium. Expression of the hIL-6 transgene also induced autocrine mouse IL-6 secretion in the prostate gland and sustained activation of STAT3 pathway in the epithelium and the stroma. Strikingly, IL-6 transgenic mice have increased peri-prostatic adipose tissue in which STAT3 was highly phosphorylated in comparison to the wild-type littermate. There is no significant increase in infiltration of T cells or B cells in the prostate of IL-6 transgenic animals. However, significantly increased infiltration (P<0.01) of FoxP3+ lymphocytes was seen in the stroma of the prostate in the IL-6 transgenic animals in comparison to wild-type littermates. These data suggest that IL-6 as a pro-inflammatory cytokine alone can induce tissue-specific tumorigenesis through induction of autocrine IL-6 secretion and remodeling tissue microenvironment. Our finding suggests that inflammation may play a direct role beyond being the “stimuli” or “fuel” in tumorigenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 321. doi:1538-7445.AM2012-321
Abstract 3214: Resveratrol potentiates glycolytic inhibitor-induced neuroblastoma cell death independent of sirt activity
Authors: Graham Regina M. , Puerta Nataly , Webster Keith A. , Vanni Steven
Published: August 2012

Introduction: Low survival rates and severe side effects of current neuroblastoma treatments underline the need for more effective less toxic therapies. Compared to normal cells, cancer cells demonstrate high glucose consumption even under aerobic conditions, therefore; targeting the glycolytic pathway in cancer cells as a potential therapy is currently an area of intense investigation. The polyphenolic compound resveratrol which can be found in grapes, peanuts, red wine and purple grape juice, has demonstrated antitumor activities both in vitro and in vivo. Here we investigate the potential of inhibiting glycolysis in combination with resveratrol treatment. Methods: To inhibit glycolysis we used the glucose analog, 2-Deoxy-D-glucose (2-DG). N-MYC amplified neuroblastoma cells (NB1691) were exposed to low dose 2-DG (0.5 and 2mM) and resveratrol 10uM. Cell viability was determined at 72hr using MTS assay. Western blotting was used to determine cell signaling pathways and apoptotic protein levels. The role of the NAD+ dependent HDAC sirtuin 1 (sirt1) was determined using 50uM of the inhibitor sirtinol. Results: While 2-DG did reduce cell viability (2mM:82+4.4% of control), the combination of 2-DG and resveratrol significantly reduced cell viability (20+2.2%) compared to resveratrol alone (69+2.8%). Western blot analysis indicated a robust activation of AKT with 2-DG with treatment, however this activation was blocked with 10uM resveratrol. Furthermore, western blot analysis revealed that this was caspase dependent cell death. PI3K/Akt inhibitors wortmannin and LY-294002 (10uM each) also reduced cell viability in combination with 2-DG to approximately 40% compared to non-treated, confirming the protective role of AKT signaling in response to 2-DG treatment. To determine if resveratrol, a well-known activator of sirt1, inhibited AKT via sirt1, cells were pretreated with sirtinol. Inhibiting sirt1 did not prevent resveratrol's inhibition of AKT. Conclusions: The oncoprotein AKT mediates signaling processes involved in evading apoptosis and promoting cell proliferation and recently has been linked to glycolysis. Resveratrol significantly increased 2-DG-induced cell death by inhibiting the 2-DG induced increase in AKT activity independent of sirt1. Here we show that combining resveratrol with 2-DG, two relatively non-toxic agents can significantly reduce neuroblastoma cell viability.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3214. doi:1538-7445.AM2012-3214
Abstract 3221: Anticancer effect of PUFAs is mediated by inhibition of polyisoprenylated methylated protein methyl esterase
Authors: Amissah Felix , Ayuk-Takem Lambert T. , Lamango Nazarius S.
Published: August 2012

Purpose: Polyisoprenylation is a set of secondary modifications involving proteins whose aberrant activities are implicated in cancers and macular degeneration. This process is essential for the activities of oncogenic proteins such as Ras. The terminal reversible step of polyisoprenylation pathway is catalyzed by an ester forming polyisoprenylated protein methyl transferase and polyisoprenylated methylated protein methyl esterase (PMPMEase). PUFAs share structural similarities with the polyisoprenyl groups and may interfere with polyisoprenylated protein metabolism. Methods: The effect of PUFAs on purified PMPMEase activity was studied. The inhibitory PUFAs were further evaluated on the viability of various cancer cell lines. The PMPMEase activity in the lysates of treated cells was analyzed in the presence or absence of COX-2/celecoxib. The mechanism of PUFAs-induced cell death was determined using a modified ethidium bromide and acridine orange (EB/AO) staining assay. Results: PUFAs such as arachidonic (AA), eicosapentaenoic and docosahexaenoic acids inhibited purified PMPMEase activity with Ki values of 0.12 to 3.7 µM. Enzyme kinetics analysis with PMPMEase revealed a 2- fold decrease in KM from 16.2 ± 1.34 to 8.60 ± 1.20 and 7.84 ± 1.02 μM in the presence of 10 μM AA or EPA, respectively. AA also induced the degeneration of various cancer cells at physiologically relevant concentrations (EC50 from 1 to 24 μM). The cellular PMPMEase activity also followed similar profiles to those of the cell viabilities in concentration-dependent studies. Treated degenerating cells displayed diminished PMPMEase activities. The inhibited PMPMEase activities in PUFAs-treated cells were restored by the addition of COX-2 but not in the presence of celecoxib. Conclusions: These data suggest that AA and other PUFAs, functioning through PMPMEase inhibition, may be involved in the regulation of cell proliferation. NSAIDs may thus owe their anticancer effects by protecting the PMPMEase- and cell proliferation-inhibiting PUFAs from COX-2 metabolism.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3221. doi:1538-7445.AM2012-3221
Abstract 3231: A small molecule inhibitor of glucose transporter 1 (Glut1) down-regulates glycolysis, induces cell cycle arrest, and inhibits cancer cell growth in vitro and in vivo
Authors: Liu Yi , Cao Yanyan , Zhang Weihe , Bergmeier Stephen , Qian Yanrong , Akbar Huzoor , Colvin Robert , Ding Juan , Tong Lingying , Wu Shiyong , Hines Jennifer , Chen Xiaozhuo
Published: August 2012

Increased dependence on glycolysis and glucose metabolism, known as the Warburg effect, is a near-universal hallmark of cancer. The functional and therapeutic importance of the effect has been increasingly recognized and glycolysis has become a new target of anticancer strategies. We recently reported the identification of a group of novel small compounds that inhibit basal glucose transport and reduce cancer cell growth by using a glucose deprivation-like mechanism. Based on these and other results, we hypothesized that the compounds inhibit cancer growth by targeting Glut1 and they are efficacious in vivo as anticancer agents. Here we report that the representative and novel compound WZB117 not only inhibited cancer cell growth in multiple cancer cell lines but also inhibited cancer growth in a nude mouse model. Daily intraperitoneal (ip) injection of WZB117 at a dose of 10 mg/kg resulted in an over 70% reduction in the average size of xenografted tumor of human lung cancer A549 cell origin. Compound target study showed that WZB117 inhibited glucose transport in human red blood cells (RBC) and RBC-derived vesicles, both of which express Glut1 as their sole glucose transporter. Docking studies of WZB117-Glut1 interaction also demonstrate and support the binding of WZB117 to Glut1. Mechanism studies using multiple cancer cell lines revealed that treatment of WZB117 led to an induction of glycolytic enzymes, such as hexokinase, PKM2 and PGAM1; and then a decline of glycolysis as indicated by reduced lactate production with changes in Akt, AMPK and mTOR, protein factors in energy sensitive signaling pathways. These resulted in ER stress, followed by cell cycle arrest mediated through downregulation of cyclin E and Rb. Taken together, cell cycle arrest at G1/S phase plays major roles in the WZB117-induced inhibition of cancer cell growth while apoptosis is secondary. WZB117 can serve as a prototypic compound for further development of Glut1 inhibitors into novel anticancer therapeutics targeting glucose transport and glucose metabolism.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3231. doi:1538-7445.AM2012-3231
Abstract 3238: Frizzled-8 receptor is activated by the Wnt-2 ligant in non-small cell lung cancer
Authors: Bravo Dawn , Xu Zhidong , Kuchenbecker Kristopher , He Biao , Jablons David M. , You Liang
Published: August 2012

Introduction: Lung cancer is the leading cause of cancer-associated mortality throughout the world. We previously showed that the Wnt signaling pathway is aberrantly activated in lung cancer. In this report, we investigated the role of interactions between different Frizzled receptors and the Wnt-2 ligant in non-small cell lung cancer (NSCLC). Methods: Quantitative RT-PCR was used to measure expression of Frz-8 and Wnt-2 from fifty freshly resected lung tumors and matched normal lung tissue. A human dominant-negative Wnt-2 mutant (dnhWnt-2) was designed to target the interactions between Fzd-8 and Wnt-2 in vitro and in vivo. pTOPflash/pFOPflash luciferase activity assay was used to detect TCF/β-catenine transcription activity. Crystal violet staining assay was used to determine the colony formation. A mouse xenogrant model using A549 cells transfected with dnhWnt-2 vector or control vector was used to detect tumorigenic effects. Results: RT-PCR analysis showed that Wnt-2 overexpression is in correlation with overexpression of Fzd-8 in 45/50 (90%) tumor samples analyzed. Coexpression of the dnhWnt-2 construct together with Wnt-2 and Fzd8 expression vectors in 293T, 293 and A549 cells strongly reduced TCF-dependent transcriptional activity (TOPflash assay) as compared with cells transfected with control vectors. In lung cancer cell line A549, naturally overexpressing both Wnt-2 and Fzd8, the dnhWnt-2 construct induced cell death, inhibited cell proliferation. Results from colony formation assay showed 50% reduction in colonies formed from the dnhWnt-2 transfected cells as compared to control vector. A tumor xenograft model of A549 cells expressing the dnhWnt-2 mutant, demonstrated a marked reduction in tumor formation 43 days after cell inoculation (p<0.02) with reduced Wnt downstream target gene expressions (Survivin, c-Myc, Dvl-3 and Cyclin- D1) compared to control tumors. Conclusion: This report demonstrated a novel interaction between Frz-8 receptor and Wnt-2 ligant in non-small cell lung cancer. Inhibition of this interaction may provide a new therapeutic avenue in targeting the Wnt pathway in lung cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3238. doi:1538-7445.AM2012-3238
Abstract 3240: The Wnt secretion factor Evi/Wls/Gpr177 promotes glioma tumorigenesis
Authors: Augustin Iris , Goidts Violaine , Bongers Angelika , Kerr Grainne , Radlwimmer Bernhard , Hartmann Christian , Herold-Mende Christel , von Deimling Andreas , Reifenberg Guido , Boutros Michael
Published: August 2012

Malignant astrocytomas are highly aggressive brain tumors with poor prognosis. Despite multimodal therapy, including surgical resection followed by combined radio- and chemotherapy, the median survival of glioblastoma patients has remained as low as 12-14 months throughout the past decade. While a number of structural genomic changes and dysregulation of signaling pathways in gliomas have been described, the identification of biomarkers and druggable targets remains an important task for novel diagnostic and therapeutic approaches. Recent evidence supports the involvement of canonical and non-canonical Wnt signaling in glioma development and malignant progression. Here, we show that the Wnt-specific secretory protein Evi (also known as GPR177/Wntless/Sprinter) is overexpressed in astrocytic gliomas. Evi/Wls is a core Wnt signaling component and a specific regulator of pan-Wnt protein secretion, affecting both canonical and non-canonical signaling. We demonstrate that its depletion in glioma and glioma-derived stem-like cells led to decreased cell proliferation and apoptosis. Furthermore, Evi/Wls silencing in glioma cells reduced cell migration and the capacity to form tumors in vivo. We further show that Evi/Wls overexpression is sufficient to promote downstream Wnt signaling. Taken together, our study identifies Evi/Wls as an essential regulator of glioma tumorigenesis, identifying a pathway- specific protein trafficking factor as an oncogene and offering novel therapeutic options to interfere with the aberrant regulation of growth factors at the site of production.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3240. doi:1538-7445.AM2012-3240
Abstract 3244: Compensatory function of the homologous proteins MIF and D-DT in regulation of AMPK-dependent metabolic-stress pathways in non-small cell lung carcinoma
Authors: Brock Stephanie Erin , Rendon Beatriz , Mitchell Robert
Published: August 2012

Macrophage migration inhibitory factor (MIF) is over-expressed in most solid malignancies and is important for many pro-tumorigenic pathways. Several studies reveal that genetic deletion or inhibition of MIF results in anti-cancer phenotypes, but these phenotypes are not nearly as profound as one would anticipate given its reported activities. We recently discovered that MIF's only related family member, D-dopachrome tautomerase (D-DT), functionally compensates for MIF-dependent promotion of angiogenic potential in non-small cell lung carcinoma (NSCLC). This finding not only provides an explanation for why MIF individual targeting has not provided more efficacious anti-cancer activities, but also suggests that therapeutic targeting of MIF and D-DT simultaneously may provide a highly effective and novel new class of anti-lung cancer pharmacologic agents. Here we present further evidence of the important cooperative function of these two homologous proteins in NSCLC cells. We reveal a strong collaborative effect of MIF and D-DT on the negative regulation of AMP-activated protein kinase (AMPK) activation. AMPK is phosphorylated by the tumor suppressor, LKB1, in response to metabolic stress, resulting in AMPK-dependent p53 activation and mTOR inhibition. We show that while siRNA-mediated knockdown of endogenous MIF or D-DT individually has modest effects on AMPK activation in NSCLC cells, combined knockdown of both MIF and D-DT results in a significant activation of AMPK-dependent regulation of p53 and mTOR pathways. Importantly, this re-activation of AMPK by MIF and D-DT occurs in LKB1 mutant NSCLC cell lines. In addition, overexpression of MIF or D-DT alone by adenoviral delivery causes a substantial reduction in p53 expression, with a dramatic increase in mTOR pathway activation. Our findings suggest that MIF and D-DT promote a unique and well conserved adaptation pathway that is usurped by the malignant cells in order to counter metabolic stress-induced, AMPK-dependent, tumor suppression. Because the LKB1 tumor suppressor locus is mutated in over 30% of NSCLC lesions rendering these tumors insensitive to AMPK-dependent growth suppression, the identification of unique AMPK agonists would be expected to have a tremendous impact on NSCLC disease management for clinicians. Taken together, simultaneous therapeutic targeting of both MIF and D-DT represents an innovative and clinically efficacious approach to re-activating metabolic stress-induced tumor suppression in NSCLC malignant lesions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3244. doi:1538-7445.AM2012-3244
Abstract 325: Splice isoform switching: A new mechanism controlling EMT and breast cancer progression
Authors: Brown Rhonda L. , Reinke Lauren M. , Damerow Marin S. , Perez Denise , Chodosh Lewis A. , Yang Jing , Cheng Chonghui
Published: August 2012

Epithelial-mesenchymal transition (EMT) is a tightly regulated process that is critical for embryogenesis. When abnormally activated, EMT promotes cancer metastasis and recurrence. Here we show that splice isoform switching constitutes a novel mechanism that controls EMT. We identified a shift in CD44 expression from variant isoforms (CD44v) to the standard isoform (CD44s) during EMT. This isoform switch to CD44s, which is regulated at the level of alternative splicing, is essential for cells to undergo EMT and is required for the formation of breast tumors that display EMT characteristics in mice. We demonstrate that the splicing factor ESRP1 controls the CD44 isoform switch and is critical for regulating the EMT phenotype. Furthermore, the CD44s isoform activates Akt signaling and inhibits cell death, providing a mechanistic link to a key pathway that drives EMT. Finally, CD44s expression is upregulated in high-grade human breast tumors and correlates with the level of the mesenchymal marker N-cadherin in these tumors. Together, these data suggest that regulation of CD44 alternative splicing causally contributes to EMT and breast cancer progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 325. doi:1538-7445.AM2012-325
Abstract 3253: Bone morphogenetic proteins increase hepcidin in breast cancer cells
Authors: Clausen Kathryn A. , Di Xiumin , Torti Frank M. , Torti Suzy V.
Published: August 2012

Hepcidin is a systemic peptide hormone produced in the liver that regulates iron absorption and recirculation. Induction of hepcidin by inflammatory cytokines has been suggested to contribute to the anemia of inflammation and cancer. Our previous work has shown that synthesis of hepcidin is not confined to the liver, but is also observed in other tissues. In particular, we found that hepcidin is produced in mammary epithelial cells. Further, hepcidin levels increase in a subset of breast cancers, and increased synthesis of hepcidin is associated with poor prognoses in some breast cancer patients (Pinnix et al., 2010). In this work we sought to understand mechanisms regulating hepcidin synthesis in breast cancer, as well as the consequences of hepcidin upregulation. We demonstrate that treatment with bone morphogenetic proteins (BMPs) 2, 4, 6, or 7 results in hepcidin upregulation and concomitant Smad phosphorylation in breast cancer cells. The BMP7-induced hepcidin increase can be diminished by co-treatment of breast cancer cells with the BMP inhibitors, Wise or Noggin. We next assessed the consequence of hepcidin induction. Systemic hepcidin regulates iron by affecting the levels of the iron export protein, ferroportin. Extracellular hepcidin binds to ferroportin on the cell surface, initiating the internalization and degradation of ferroportin. This, in turn, decreases the ferroportin available to facilitate iron export, preventing iron efflux from the cell and increasing cellular iron retention. To test the functionality of this regulatory pathway in breast cancer, we used a hepcidin-specific antibody to deplete cultured breast cancer cells of extracellular hepcidin. We found that antibody-induced hepcidin depletion increased ferroportin protein levels, suggesting that hepcidin-mediated ferroportin regulation occurs in breast cancer cells. Consistent with this finding, we observed that breast cancer cells have increased hepcidin and decreased ferroportin relative to normal mammary epithelial cells. In all, these results raise the possibility that BMP signaling may control hepcidin expression in breast cells, decreasing ferroportin and increasing intracellular iron. The potential for hepcidin to alter iron availability to breast cancer cells on a paracrine level is a novel function for this key systemic iron regulator. Reference Z. K. Pinnix, L. D. Miller, W. Wang, R. D'Agostino Jr., T. Kute, M. C. Willingham, H. Hatcher, L. Tesfay, G. Sui, X. Di, S. V. Torti, F. M. Torti, Ferroportin and Iron Regulation in Breast Cancer Progression and Prognosis. Sci. Transl. Med. 2, 43ra56 (2010).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3253. doi:1538-7445.AM2012-3253
Abstract 3254: Cadherin-11 function is required for full neoplastic transformation by v-Src
Authors: Raptis Leda , Geletu Mulu , Arulanandam Rozanne
Published: August 2012

The signal transducer and activator of transcription-3 (Stat3) has emerged as an important signal transducer with a role in cancer etiology. Following activation of a number of growth factor or cytokine receptors or expression of oncogenes such as vSrc, Stat3 is phosphorylated at tyr-705, dimerizes and migrates to the nucleus where it activates transcription from a number of genes involved in cell division and survival. We recently discovered a novel pathway of activation of Stat3, triggered by cadherin engagement. Stat3 activation was caused by a striking upregulation of the Rac GTPase and IL6, and induced downregulation of the p53 antioncogene, upregulation of anti-apoptotic genes and a potent survival signal. A large number of oncogenes such as vSrc activate the E2F transcription factor, found to be hyperactive in many cancers. E2F induces the expression of genes involved in cell division, as well as a number of receptor tyrosine kinases, that can, in turn, activate Stat3. However, at the same time E2F is a potent apoptosis inducer, hence the high demand of transformed cells for survival signals. Since cadherin engagement activates Stat3, a potent survival signal, we examined the effect of downregulation of cadherin-11 upon transformation of mouse Balb/c3T3 fibroblasts by vSrc. Cadherin-11 knockdown through shRNA expression with a retroviral vector caused a dramatic reduction in Stat3 activity. At the same time, expression of vSrc in cadherin-11 knockdown cells failed to cause transformation; in the contrary, vSrc expression induced apoptosis. These data indicate that cadherin-11 is necessary for full neoplastic transformation of mouse fibroblasts by vSrc. This is especially remarkable because Src has been shown to have a negative effect upon, although not eliminate cadherin function, since Stat3 activity was, in fact increased in vSrc-expressing cells upon cadherin engagement. Therefore, it appears that the residual amounts of cadherin-11 present are sufficient to activate Stat3 and prevent apoptosis induced by the Src/E2F axis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3254. doi:1538-7445.AM2012-3254
Abstract 3385A: Synergistic effect of purified components from Antrodia Cinnamomea Mycelia on targeting oral cancer stem cell
Authors: Chang Ching-Wen , Lo Jeng-Fan
Published: August 2012

Background: Cancer stem cells (CSCs), also termed as Cancer initiating cells (CICs), represent a rare subpopulation of cells, which are responsible for tumor growth. CICs such as oral cancer -cancer initiating cells (OC-CICs), are often resistant to either radio- or chemotherapy. Therefore, development of drug candidates that specifically target OC-CICs will be effective for future oral cancer therapy. Methods: In this study, we used the in vitro cell-based ALDH activity assay system, which has been demonstrated as a CICs marker, to screen for the active purified components from Antrodia Cinnamomea Mycelia extract (ACME), which can target OC-CICs. In combination with the active components from ACME that targeting OC-CICs, we evaluated the synergistic effect of above active components from ACMS on target oral cancer stem cells. Results: We first found ACME purified compounds (CH-190-WS-A and CH-190-WS-D) can significantly down regulate the ALDH activity of oral cancer cells or OC-CICs, respectively. Treatment of CH-190-WS-A and CH-190-WS-D significantly induced cell death and enhanced the differentiation capability of OC-CICs. Moreover, we found that treatment of CH-190-WS-A and CH-190-WS-D reduced the self-renewal ability, tumorigenic properties and stemness properties of OC-CICs in vitro. Additionally, treatment with CH-190-WS-A or CH-190-WS-D suppressed tumor growth of nude mice bearing tumor xenografts, respectively. Furthermore, treatment of CH-190-WS-D results in down-regulation of mTOR/PI3K/pAKT molecular mechanisms which play an important role in CICs. Whereas treatment of CH-190-WS-A inactivated Notch pathway, which can restore AKT activity then overcome cell death. Finally, combined treatment of CH-190-WS-D and CH-190-WS-A also dramatically caused cell death of OC-CICs through dual-pathway blockage. Conclusion: Our studies reveal combination of ACME purified components can target OC-CICs. Eventually, we can develop therapeutic protocol for alternative oral cancer treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3385A. doi:1538-7445.AM2012-3385A
Abstract 3392: Molecular determinants for the bone-metastatic potential of prostate cancer cells
Authors: Liu Qingxin , Jernigan Danielle , Fatatis Alessandro
Published: August 2012

Skeletal metastasis is the major cause of morbidity and death in patients with advanced prostate cancer (PCa). The propensity of cancer cells to progress into macroscopic lesions at secondary sites relies on the successful adaptation of Disseminated Tumor Cells (DTCs) to the new microenvironment. This requirement explains the predilection of most tumors to produce clinical metastases in selected organs although they can seed malignant cells also in several other tissues. It seems plausible that the metastatic fate of DTCs is dictated by the pleiotropic effects of specific genes and their encoded products establishing functional interactions with the surrounding stroma. The main objective of this study was to identify these molecular mediators of cell survival and growth in the bone with the ultimate goal of identifying effective therapeutic targets to counteract PCa metastases. Using a pre-clinical animal model of experimental metastasis, we have recently shown that exogenous over-expression of PDGFRα in non-metastatic prostate phenotypes conferred bone-metastatic potential and a monoclonal antibody targeting the receptor reduced bone metastatic tumors by more than 70%. Here we report that comparative gene-expression analyses conducted with PCa cells showing different intrinsic metastatic potential, as well as prostate phenotypes that fail to increase their metastatic behavior upon PDGFRα expression, resulted in a bone-metastatic gene signature including three genes encoding for soluble, secreted proteins. This suggests that DTCs with aggressive metastatic behavior modify the bone microenvironment in their favor, not only coercing the stroma to support their own survival, but also allowing other malignant phenotypes, that would otherwise fail to thrive, to grow into macroscopic tumors. We are currently testing this cell-cooperation in metastasis as well as functionally validating our three-gene signature by stably silencing or over-expressing each of the three newly identified genes in bone-metastatic and non-metastatic PCa cells, respectively.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3392. doi:1538-7445.AM2012-3392
Abstract 3399: Detection of rare mutations in plasma by droplet digital PCR
Authors: So Austin , Hindson Benjamin , Koehler Ryan , Saxonov Serge , Karlin-Neumann George , Ericson Nolan , Bielas Jason
Published: August 2012

Detection and quantitation of specific mutations in circulating plasma holds promise for earlier and less invasive diagnosis of disease. This presents significant analytical challenges, particularly as the biomarker may differ from its highly abundant wildtype by only a single nucleotide. Conventional methods have poor selectivity and fail to detect mutant sequence below 1 in 100 wildtype sequences. Compounding this, the amount of circulating nucleic acid in plasma is low. Here we present a simple strategy using droplet digital™ PCR (ddPCR™) for the detection of somatic mutations with high selectivity and sensitivity. Based on the simple principle of sample partitioning into water-in-oil microdroplets, this ddPCR method increases the abundance of a mutant DNA sequence up to 20,000 times compared to an equivalent bulk PCR reaction. Using conventional TaqMan chemistries and workflow, selectivities of up to 1/100,000 can readily be achieved in any laboratory. We evaluated ddPCR for the detection and quantitation of several clinically important mutations in the EGFR and KRAS loci from clinical samples derived from normal and tumor plasma samples. We also demonstrate the feasibility of multiplexing of Kras and EGFR assays to improve sample processing efficiency.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3399. doi:1538-7445.AM2012-3399
Abstract 3401: Amplification of the STOML3, FREM2, and LHFP genes is associated with mesenchymal differentiation in gliosarcoma
Authors: Nagaishi Masaya , Kim Young-Ho , Mittelbronn Michel , Giangaspero Felice , Paulus Werner , Vital Anne , Nakazato Yoichi , Lachuer Joël , Ohgaki Hiroko
Published: August 2012

Gliosarcoma is a rare glioblastoma variant characterized by a biphasic tissue pattern with alternating areas displaying glial (GFAP-positive) or mesenchymal (reticulin-positive) differentiation. Previous analyses showed identical genetic alterations in both glial and mesenchymal tumor areas, suggesting that gliosarcomas are genetically monoclonal, and mesenchymal differentiation was considered to reflect the elevated genomic instability of glioblastomas. In the present study, we compared genome-wide chromosomal imbalances using array CGH (105K) in glial and mesenchymal tumor areas of 13 gliosarcomas. Patterns of gain and loss were similar, except for gain at 13q13.3-q14.1 (log2 ratio ≤ 3), containing the STOML3, FREM2, and LHFP genes, which was restricted to the mesenchymal tumor area of a gliosarcoma. Further analyses of 64 cases of gliosarcoma using quantitative PCR showed amplification of the STOML3, FREM2 and LHFP genes in 14 (21%), 10 (15%), and 7 (11%) of mesenchymal tumor areas but not in glial tumor areas. Immunohistochemistry confirmed that STOML3 and FREM2 overexpression was more extensive in mesenchymal than in glial tumor areas. These results suggest that mesenchymal components in a small fraction of gliosarcomas may be derived from glial cells with additional genetic alterations.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3401. doi:1538-7445.AM2012-3401
Abstract 3406: Impact of TJP-1 and TWIST expression on post-operative prognosis in hepatocellular carcinoma
Authors: Nagai Tomoyuki , Arao Tokuzo , Matsumoto Kazuko , Sakai Kazuko , Kudo Kanae , Kaneda Hiroyasu , Tamura Daisuke , Aomatsu Keiichi , Kimura Hideharu , Fujita Yoshihiko , Hagiwara Satoru , Sakurai Toshiharu , Ueshima Kazuomi , Haji Seiji , Kudo Masatoshi , Nishio Kazuto
Published: August 2012

Epithelial-Mesenchymal transition (EMT) plays a critical role in cancer progression and metastasis. However, whether the overexpression of EMT-related genes mediates the malignant phenotype in HCC remains largely unclear. In this study, we examined the relationship between mRNA expression levels of EMT-related genesand 12 clinical characteristics including recurrence-free survival and overall survival in HCC patients who had undergone curative hepatic resection. The mRNA expression levels of 15 genes (CDH1, CDH2, Snail, Slug, ZEB1, ZEB2, TWIST, Vimentin, Fibronectin, DDR2, S100A4, TJP-1/ZO-1, FOXC2, SIX1, GSC) was determined by quantitative real-time PCR in tissues from 72 HCC tumors. Twist expression was significantly upregulated in recurrence group (p=0.02). The upregulation of Twist and the downregulation of TJP1 were significantly related with poor prognosis in univariate and multivariate analysis. In conclusion, Twist and TJP1 may be predictive biomarkers in patients with HCC after hepatectomy. Our findings provide a novel insight into TJP1 and HCC, warranting further investigations.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3406. doi:1538-7445.AM2012-3406
Abstract 3407: Analysis of the MET dependent proteins expression in cervical carcinoma cell lines
Authors: Trzyna Elzbieta
Published: August 2012

MET receptor and its ligand, hepatocyte growth factor (HGF) are important players in mitogenesis, morphogenesis and acquisition of migratory phenotype. HGF/MET axis plays its role in neoplastic transformation through activation of key oncogenic pathways and influencing dissociation ability of cells leading to metastasis. Overexpression of MET receptor has been observed in a variety of tumors. Cervical carcinoma (CC) is the second most common malignancy in women worldwide, and it remains a leading cause of cancer-related death for women in developing countries. Clinical manifestation of CC is very diverse from in situ cancers to very invasive and strongly metastatic to neighbouring and distal organs (lungs, liver, bones). In this work we study the expression of MET dependent proteins in cervical carcinoma cell lines (HeLa, HTB34, HTB35) using mass spectrometry analysis. We prepared cervical carcinoma cell lines with silenced MET gene. Cells were transduced with lentiviral vector containing shRNA against MET. shRNA was designed based on siRNA sequence silencing this particular gene in 80%. Silenced and wild type cells were cultured and lysed. Proteins from various cell compartments were isolated. 2-dimentional electophoresis and silver staining of the gels were performed followed by software and mass spectrometry analysis. We detected differences in proteins expression among wild type samples and silenced HTB34 cell line in amount of proteins (examples: Heat shock protein 75 kDa, Leukocyte elastase inhibitor). Analysed proteins were also separated by 2D electrophoresis and we observed differences in pH of these proteins among wild type and silenced probes (examples: TAR DNA-binding protein 43, Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial, Leukotriene A-4 hydrolase). Other proteins were present either in wild type HTB34 (example: Importin subunit alpha-2, Putative elongation factor1-alpha-like3,) or silenced HTB34 (example: Serine/threonine-protein phosphatase PP1-alpha catalytic subunit, Serine/threonine-protein phosphatase PP1-beta catalytic subunit, LIM and SH3 domain protein 1). Analysis of these proteins gives as information about particular proteins involved in MET dependent regulation of cervical carcinoma cell lines. Characterization of HTB35 and HeLa proteins is pending. The results of this study can be used to better understand the biology of cervical carcinoma. They can be also used to develop new therapeutic strategies to treat cervical carcinoma patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3407. doi:1538-7445.AM2012-3407
Abstract 3428: Genome-stabilizing functions of NM23 in metastatic melanoma
Authors: Jarrett Stuart G. , Novak Marian , Merlino Glenn , Slominski Andrzej , Kaetzel David M.
Published: August 2012

The metastasis suppressor protein NM23-H1 exhibits two enzymatic activities in vitro with potential roles in the maintenance of genomic integrity: a nucleoside diphosphate kinase (NDPK) activity that maintains balance in nucleotide pools and a 3′-5′ exonuclease (3′-5′ EXO) activity that could provide proofreading during DNA repair and replication. Studies were conducted in cell culture and in vivo to determine the extent to which NM23-H1 expression contributes to maintenance of genomic integrity, as well as suppression of melanoma initiation and progression. Expression of NM23-H1, and possibly NM23-H2, was shown to be necessary for timely repair of UVR-induced (6-4) photoproducts (6-4PP) in melanoma cell lines, as well as in transgenic mouse-derived melanocytes and embryo fibroblasts, as determined by immunoslot-blot assay. NM23 protein expression also suppressed the incidence of both spontaneous and UVR-induced mutations as determined using the 6-thioguanine resistance colony-forming assay. Point mutations that either selectively or tandemly disrupted the NDPK and 3′-5′ EXO activities of NM23-H1 exhibited compromised DNA repair and mutation-suppressing activities, consistent with contributions of both enzymatic functions. UVR induced rapid (< 5 min) translocation of NM23-H1 to the nucleus, where it colocalized closely in concert with 6-4PPs, strongly suggesting direct participation in the repair of those lesions. Consistent with this proposed function, transgenic mice engineered to be hemizygous-null in expression of both NM23-M1 and NM23-M2 (the mouse homologs of NM23-H1 and NM23-H2, respectively) were rendered susceptible to UVR-induced melanoma and epithelioid follicular cyst formation. Moreover, this NM23-deficient genotype conferred an aggressively metastatic phenotype to the HGF-expressing transgenic mouse model of UVR-inducible melanoma. Metastasis of highly-pigmented cells to draining lymph nodes and the lungs was seen in most cases, with lesions also occasionally seen in liver, brain and bone. Taken together, these results demonstrate a critical role for NM23 proteins in DNA repair and suppressing mutations under basal and genotoxic conditions. Importantly, they highlight for the first time a tumor suppressor function in the context of UVR-induced melanoma in vivo, and suggest a novel metastasis suppressor activity through suppression of metastasis-driving mutations.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3428. doi:1538-7445.AM2012-3428
Abstract 3429: AKAP12/SSeCKS inhibits peritoneal metastasis by regulating chemokine production
Authors: Akakura Shin , Muramatsu Masashi , Ko Hyun Kyung , Gelman Irwin H.
Published: August 2012

AKAP12/SSeCKS/Gravin (AKAP12) is thought to act as a metastasis suppressor through its direct scaffolding of major signaling proteins such as PKC, PKA and Src, in a spatiotemporal manner (1). For example, AKAP12 inhibition of Src-mediated oncogenic signaling, including induction of VEGF secretion by tumor cells, correlates with inhibition of neovascularization at metastatic sites (2). We have established AKAP12-null (KO) mice and demonstrated that they are cancer-prone in regards to the formation of spontaneous prostatic dysplasia and to carcinogen-induced skin cancer formation (3, 4). However, the role of AKAP12 in host microenvironment in regards to tumor or metastasis control has yet to be elucidated. In this study, we utilized an orthotropic, subcutaneous injection model of B16F10 melanoma to address whether the loss of AKAP12 in the host facilitates metastasis. Although the size of the primary tumors was similar in WT vs. KO mice, KO mice exhibited increased rates of peritoneal metastasis. The peritoneal fluid (PF) of KO mice was more potent than WT-PF in inducing in vitro chemotaxis. Comparison of the cytokines/chemokines using antibody arrays showed a severe upregulation of multiple potential chemoattractants in the KO-PF, including CXCL9/10/11, known ligands for CXCR3, a chemokine receptor upregulated 12-fold in B16F10 versus the low-metastatic B16F0 variant. Pre-injection of KO-PF, but not WT-PF, into WT mice was sufficient to induce high levels of peritoneal metastasis from subcutaneous sites. Taken together, our study suggests that AKAP12 controls metastasis by regulating the production of chemoattractants in the peritoneum, produced either by local microenvironmental cells or by recruited inflammatory cells. References: (1) Gelman, I. H. (2002) Front Biosci 7, 1782-1797. (2) Su, B., Zheng, Q., Vaughan, M.M., Bu, Y., and Gelman, I.H. (2006) Cancer Res. 66, 5599-5607 (3) Akakura, S., Huang, C., Nelson, P. J., Foster, B., and Gelman, I. H. (2008) Cancer Res. 68, 5096-5103. (4) Akakura, S., Bouchard, R., Bshara, W., Morrison, C., and Gelman, I. H. (2011) Int. J. Can. 129, 2025-2031.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3429. doi:1538-7445.AM2012-3429