bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 3162: MDM2 regulates MYCN mRNA stabilization and translation in human neuroblastoma cells
Authors: Zhang Hailong , Gu Lubing , Li Jiansha , He Jing , Zhou Muxiang 
Published: February 2011

Abstract
The MYCN gene plays a critical role in determining the clinical behavior of neuroblastoma. Genomic amplification occurs in high-risk subsets, but it remains unclear how MYCN expression is regulated in human neuroblastomas. Here, we report that the expression of MYCN is regulated by the oncoprotein MDM2 at the post-transcriptional level. Ectopic overexpression of MDM2 or an increase in the cytoplasmic levels of endogenous MDM2 by inhibition of PI3K/Akt activity enhanced both MYCN mRNA and protein expression. UV cross-linking and RNA-protein binding assays demonstrated that the C-terminal RING domain of MDM2 bound to AU-rich elements within the 3’-untranslated region (3’-UTR) of MYCN mRNA. Furthermore, gene transfection and reporter assay confirmed that MDM2 increased MYCN 3’-UTR-mediated mRNA stability and induced translation of reporter luciferase. Conversely, MDM2 silencing by specific siRNA rendered the MYCN mRNA unstable, reducing the abundant MYCN protein found in MYCN-overexpressing neuroblastoma cell lines, which are a consequence of gene amplification. Importantly, silencing of MDM2 in MYCN-amplified neuroblastoma cell lines resulted in a remarkable inhibition of cell growth and induction of cell death, through a p53-independent pathway. These results indicate that MDM2 plays a p53-independent role in regulating MYCN mRNA stabilization and protein translation, and suggest that MDM2-mediated MYCN expression is an important mechanism for tumor growth and for disease progression, specifically in MYCN-associated neuroblastoma.
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 3162.
Abstract 317: p38γ MAPK regulates topoisomerase IIα activity by phosphorylation
Authors: Qi Xiaomei , Hou Songwang , Lou Zhenkun , Chen Guan 
Published: February 2011

Abstract
DNA topoisomerase IIα (Topo IIα) is a critical target for cancer therapy and its protein expression, however, is frequently depleted by its inhibitors and strategies to maintain Topo IIα expression and thereby to sustain the Topo II drug sensitivity are still lacking. Here we show that p38γ MAPK is specifically activated by Topo II inhibitors and activated p38γ in turn phosphorylates Topo IIα, leading to increased Topo IIα protein expression through attenuating its proteasome-dependent degradation. The sensitivity of a panel of human breast cancer cell lines to Topo II inhibitors was found not to correlate with the intrinsic but with inducible Topo IIα protein expression and with endogenous protein expression of p38γ MAPK that is specifically phosphorylated in response to Topo II toxins. p38γ activation and inhibition alone were shown to increase and decrease Topo IIα expression and Topo drug toxicities. Mechanistically, p38γ MAPK phosphorylates Topo IIα at Ser 1524, leading to a decreased proteasome-dependent Topo IIα degradation. These results reveal a novel mechanism by which p38γ MAPK specifically confers Topo II drug toxicity in breast 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 317.
Abstract 3171: Expression of PKC iota in breast cancer
Authors: Paget Judith , Daneshmand Manijeh , Amin Md. Shahrier , Islam Shahidul , Lorimer Ian 
Published: February 2011

Abstract
One in nine (11%) women is expected to develop breast cancer during their lifetime. Abnormal activation of the PI3K signaling cascade is common in breast cancer. The p110α catalytic subunit of PI3K is mutated in 20-40% of breast cancers. The two most common mutations are E545K in the helical domain and H1047R in the kinase domain; these mutations render PI3K constitutively active. PKC iota is a member of atypical class of PKC family serine/threonine kinases and a downstream effector in the PI3K signaling pathway. PKC iota has been implicated in carcinogenesis and oncogenic signaling in lung, colon and ovarian carcinomas but its role in breast cancer progression is unknown. Using immunohistochemistry (IHC), we have evaluated PKCι expression and localization in breast cancer tissue microarrays (TMA). Weak PKCι staining was detected in normal breast tissue. PKCι was over-expressed in subset of breast cancers with no staining in the surrounding stroma. Positive tumor staining was mainly cytoplasmic with nuclear staining in some cases. There was no significant correlation of positive PKCι staining with tumour type. PKCι overexpression was also seen in a subset of ductal carcinoma in situ samples. In vitro, we have also shown that PKCι is over-expressed and has higher levels of phosphorylation in a subset of breast cancer cell lines when compared to mammary epithelial cell lines. Stable cell lines expressing E545K or H1047R mutations were generated in the mammary myoepithelial cell line MCF-10A by retroviral transduction. Using Western blotting, we have shown that these mutations are sufficient to increase PKCι expression and activation. These results suggest a possible mechanism for the PKCι overexpression previously demonstrated by IHC. Two structurally independent PI3K inhibitors, Wortmannin and LY294-002, inhibited PKCι phosphorylation and activation in mutant PI3K expressing cells, confirming that the PKCι activation is PI3K-dependent. These inhibitors did not inhibit the low level of PKCι activation seen in MCF10A cells expressing wild-type PI3K control, suggesting that this activation occurs by a PI3K-independent mechanism. These results demonstrate that PKCι is overexpressed in some breast cancers, and that PI3KCA mutations may drive this overexpression. These results also indicate a potential role for PKCι in breast 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 3171.
Abstract 3172: ZEB1 regulation in breast epithelial cells: Role of IGF-1 in the CCN6 modulation pathway
Authors: Lorenzatti Guadalupe , Pal Anupama , Huang Wei , Cabanillas Ana M. , Kleer Celina G. 
Published: February 2011

Abstract
Background: During cancer progression, the transcription factor ZEB1 has been implicated in the regulation of key genes triggering the EMT (Epithelial to Mesenchymal Transition) program. We previously reported that the matricellular protein CCN6 is lost or reduced in highly aggressive breast carcinomas. Furthermore, we have identified CCN6 as a tumor suppressor in breast cancer whose down-regulation enhances IGF-1 signaling on breast epithelial cells. Recently, our lab observed that stable inhibition of CCN6 in benign mammary epithelial cells causes EMT and invasion with marked up-regulation of ZEB1. Since IGF-1 is shown to up-regulate ZEB1 expression in epithelial prostate cancer cells we hypothesize that CCN6 may regulate EMT in breast cancers by modulating IGF-1 signaling and thereby regulating ZEB1 expression. Methods: Stable CCN6-deficient HME (Human Mammary Epithelial cells)(HME-CCN6 KD) were incubated with 500 ng/mL rhCCN6 protein. Western blots (WB) for ZEB1 and vimentin, inmunofluorescence, invasion and proliferation assays were performed. ZEB1 mRNA levels were evaluated by RT-qPCR (Taqman). IGF-1 expression was analyzed by RT-qPCR and WB on MDA-MB-231 and HME-CCN6 KD (pre- and post-rhCCN6 treatment) while IGF-1R, phospho-IGF-1R, IRS-1 and phospho-IRS-1 expression were investigated by WB. Lentivirus-mediated (pLK.O1) short hairpin RNA targeting IGF1-R was performed in HME control and HME-CCN6 KD cells. WBs were done to assess expression of proteins of interest. mRNA content was evaluated by RT-qPCR. Results: CCN6 incubation of HME-CCN6 KD cells induced down-regulation of ZEB1 mRNA and protein levels as well as subcellular localization changes of the mesenchymal marker vimentin (“cap” pattern) evidenced as a decrease in vimentin expression by WB. hrCCN6 protein treatment also reduced proliferation and invasion abilities of HME-CCN6 KD cells compared to controls. In Addition, cell lines expressing low levels of CCN6 such us HME-CCN6 KD and MDA-MB-231 secreted IGF-1 in the conditioned media and showed activation of the IGF-1 signaling pathway. This phenomenon was reverted by CCN6 treatment, with reduction of both, IGF-1 protein and mRNA levels. IGF-1R down-regulation in HME-CCN6 KD cells decreased ZEB1 expression and induced the re-expression of cytokeratin-18 (epithelial marker). Conclusions: Our results suggest that CCN6 regulates ZEB1 expression and EMT in breast epithelial cells by modulating the IGF-1 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 3172.
Abstract 3173: The role of the Src/Stat3 axis in autocrine HGF signalling in invasive human breast cancer
Authors: Carefoot Esther I. , Raptis Leda , SenGupta Sandip , Elliott Bruce E. 
Published: February 2011

Abstract
Recent studies have shown that the proto-oncogene Met is frequently over-expressed in breast cancers, particularly the highly aggressive basal-like subtype. The Elliott lab has previously shown co-expression of HGF and Met in invasive human breast cancers, but not in normal breast epithelium (CJPP 80:91-102, 2002), and a novel activating role of Src and Stat3 on HGF transcription and transformation of breast epithelial cells (Oncogene 25:2773-84, 2006). Together, these findings suggest that the Src/Stat3 axis promotes autocrine HGF/Met signalling, and thereby contributes to invasion and metastasis in breast cancer. To assess the role of Src/Stat3 activation and autocrine HGF/Met signaling in breast metastasis, we have used the human breast carcinoma cell line MDA-MB-231, which exhibits basal-like characteristics. To directly assess the role of Src, we have expressed an activated Src mutant in MDA-MB-231 cells (MDA-Src). Using western blotting analysis, we observed increased levels of active Stat3 (pY705) and Met (pY1234/1235), as well as increased total Stat3, HGF and Met proteins in MDA-Src cells, compared to MDA-MB-231 cells. This finding supports a role of Src in activation of Stat3 and HGF/Met signalling in MDA-MB-231 cells. Treatment of the above cell lines with Dasatinib, an ATP-competitive inhibitor of Src family kinases, resulted in a decrease in the activity of Src and Met, while the levels of activated Stat3 (pY705) and HGF protein were unchanged. In contrast, treatment with CPA7, an inhibitor of Stat3-mediated gene transcription, produced a marked decrease in total HGF protein in addition to decreased Src and Met activity. These results suggest that Src inhibition alone is not sufficient to inhibit the Stat3-dependent expression of HGF in MDA-MB-231 cells, and that inhibition of Stat3 gene transcription rather than its activation is key to the inhibition of the HGF/Met autocrine loop. We are currently performing immunohistochemical analyses of Src/Stat3 and HGF/Met expression on a cohort of human breast cancer tissues (n=59). Preliminary results have shown increased expression and nuclear localization of active Stat3 in breast tumours compared to normal mammoplasty tissues, which is concurrent with HGF expression. These findings implicate a role of the Src/Stat3 axis in regulating autocrine HGF/Met signaling in a human breast cancer cell line model, and may lead to a predictive marker for targeting of breast cancer subtypes which may exploit this pathway. (Supported by CBCRA and CIHR).
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 3173.
Abstract 3174: Visualization of the activation/deactivation cycle of RhoC in inflammatory breast cancer
Authors: Kennedy Elizabeth J. , Ventura Alejandra C. , Wu ZhiFen , Garcia Hector , Maddur Shwetha , Merajver Sofia D. 
Published: February 2011

Abstract
A key event in the development of Inflammatory Breast Cancer, a particularly aggressive, metastatic form of breast cancer, is the over-expression of the GTPase protein RhoC. RhoC plays a role in regulating cell shape, attachment and motility and over-expression is likely associated with tumor proliferation. RhoC is activated into a GTP-bound state by the regulatory proteins GEFs (guanine nucleotide exchange factor proteins) and is deactivated to a GDP-bound state by GAPs (GTPase activating proteins). Stimulation of IBC cells with LPA (lysophophatidic acid), an activator of the cycle, showed a transient RhoC-GTP increase, peaking at 2 minutes and then diminishing until 20 minutes. It has been suggested that LPA can activate both GEF and GAP proteins, and that a delay in the GAP protein activation could explain this RhoC-GTP behavior. Because RhoC activity occurs at the plasma membrane, the translocation of GEF and GAP proteins was studied as an indication of RhoC interaction. Immunofluorescent stains for the three key proteins_RhoC, GAP, and GEF_were performed on an IBC cell line before and after stimulation with LPA and observed using confocal microscopy. Preliminary images revealed a co-localization of three proteins RhoC, p190B (a GAP protein), and PDZ (a GEF protein) in the cytosol, forming a gradient of high to low protein concentration from the nuclear membrane to the plasma membrane. Further analysis with ImageJ software showed slight differences in protein concentration at the two membranes upon stimulation with LPA. RhoC protein levels increased at both membranes, PDZ quantities increased slightly at the plasma membrane, and the p190B levels decreased at both membranes as a result of stimulation. This data suggests that after 5 minutes, LPA has a positive regulatory effect on the GEF protein and a negative one on the GAP protein at the plasma membrane. In addition, co-localization of the proteins RhoC and p190B was analyzed at different time points. The data indicates a decrease in co-localization after LPA stimulation that is slightly recovered 20 minutes after treatment. A possible explanation for these results is that 5 minutes after stimulation, RhoC undergoes translocation to the nuclear and plasma membranes, and is not followed by the GAP protein until later times. As of now, these conclusions are preliminary. These results will be verified using a new RhoC antibody, and additional co-localization analysis.
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 3174.
Abstract 3175: Importance of CXCL12, CXCR4 and CXC7 in breast cancer cells
Authors: Hawsawi Nahed M. , May Felicity E.B. 
Published: February 2011

Abstract
The purpose of this study is to understand the relative importance of the two chemokine receptors CXCR4 and CXCR7 in the response of estrogen-responsive and estrogen-nonresponsive breast cancer cells to the chemokine CXCL12. CXCR4 has long been accepted, whereas CXCR7 was identified more recently as a CXCL12 receptor. CXCL12 is expressed by stromal and epithelial cells in breast tumors and has been shown to be regulated by estrogen in estrogen-responsive breast cancer epithelial cells. CXCL12 activates the two seven transmembrane G protein-coupled receptors, CXCR4 and CXCR7, that transduce signals via heterotrimeric G-proteins.
Methods
The expression of CXCL12, CXCR4 and CXCR7 was measured in five estrogen-responsive and five estrogen-nonresponsive breast cancer cell lines by qRT-PCR and western transfer analysis. The ability of CXCL12 to activate the CXCR4 receptors was measured by western transfer analysis with a phospho-specific antibody. The motogenic responses of different breast cancer cell lines to CXCL12 were measured in modified Boyden chamber and in vitro wounding assays. The relative importance of CXCR4 in the CXCL12 response was probed with a specific inhibitor of CXCR4 receptor.
Results
The expression of CXCL12, CXCR4 and CXCR7 varied greatly between the ten breast cancer cell lines analyzed. CXCL12 expression was highest in estrogen-responsive T-47D cells whereas CXCR4 and CXCR7 expression was highest in estrogen-nonresponsive MDA-MB-231 cells. CXCL12 protein expression was not detected in protein extracts from breast cancer cells. CXCL12 was detected however after secretion into medium conditioned by T-47D and MCF-7 breast cancer cells. Chemotaxis assays showed that CXCL12 stimulated breast cancer cell migration in a concentration-dependent manner and cells that expressed higher concentrations of the receptors were more responsive to CXCL12. CXCR4 phosphorylation was stimulated by CXCL12 in some but not all breast cancer cells. AMD3100, a competitive inhibitor of CXCL12 binding to CXCR4, inhibited CXCL12-induced migration. Maximum inhibition of migration stimulated by 12.5 nM CXCL12 was achieved with 5 µM AMD3100.
Conclusion
This study shows that CXCL12 is an autocrine motogen in estrogen-responsive breast cancer cells. CXCL12 stimulates migration of both estrogen-responsive and estrogen-nonresponsive breast cancer cells. The sensitivity of the migratory response of breast cancer cells to CXCL12 depends on the relative concentrations of CXCR4 and CXCR7.
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 3175.
Abstract 3176: Connexin-43 facilitates breast cancer cell proliferation through upregulation of ERK-dependent beta-catenin, c-Myc and cyclin D1 expression
Authors: Shih-Feng Yu , Tze-Sing Huang 
Published: February 2011

Abstract
Connexin-43 (Cx43) is one of the major components of gap junction which functions in maintaining cellular homeostasis. Despite Cx43 was reportedly deficient in several cancers such as glioma and colorectal cancer, its abnormally elevated expression was noted in the breast cancer tissues. In this study, we analyzed and detected differential Cx43 expression levels in three breast cancer cell lines: hs578T &gt; MCF-7 &gt; MDA-mB-231 cells. The same order was also observed with their growth rates, i.e., greatest in hs578T cells, followed by MCF-7 cells and then MDA-mB-231 cells. To explore if Cx43 was associated with breast cancer cell proliferation, we established Cx43-overexpressing clones from MCF-7 and MDA-mB-231 cells and found these cells obviously exhibited increased DNA synthesis and cell proliferation rates. We also cloned Cx43-knockdown hs578T cells and found their DNA synthesis and cell proliferation rates were significantly reduced. In these breast cancer cells, ectopic Cx43 overexpression was accompanied with up-regulation of β-catenin, phospho-ERK, c-Myc and cyclin D1 proteins, and vice versa. Furthermore, these Cx43-related increases of β-catenin, c-Myc, cyclin D1, and cell proliferation were drastically prevented by the inhibitor PD98059, suggesting that Cx43 overexpression induced β-catenin through ERK pathway, which in turn led to the increase of c-Myc and cyclin D1 to promote breast cancer cell proliferation.
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 3176.
Abstract 3179: Functional profiling of multiple signal pathway proteins in breast cancer patients
Authors: Liu Xinjun , Kim Phillip , Kirkland Richard , Lee Tani , Magonova Katya , Liu Limin , Ohrmund Linda , Lin Frederick , Kuller Anne , Leesman Glen , Barham Robert , Ybarrondo Belen , Singh Sharat 
Published: February 2011

Abstract
The COllaborative Proximity ImmunoAssay (COPIA) is a multiplexed protein microarray platform that utilizes the formation of a unique immuno-complex requiring co-localization of two detector-antibodies. The detector-antibodies are conjugated with corresponding channeling-enzymes, glucose oxidase (GO) and horseradish peroxidase (HRP). Once target proteins are bound by the capture antibodies, the channeling events between GO and HRP in proximity enables the profiling of the target proteins with extreme sensitivity. COPIA delivers extremely high analytical specificity as it requires multiple entities within target specific proximity for the signal generation/amplification. COPIA can also be configured for each specific target protein to allow differential detection of truncated targets (i.e., p95HER2) from their normal counter parts (i.e., full length-HER2). We applied COPIA to investigate the levels of expression and activation of HER1, HER2, p95HER2, HER3, IGF1-R, c-MET, PI3K, Shc, VEGFR, panCK, and other targets in signal transduction pathways.
Here, we report the functional pathway signatures for multiple proteins in 250 frozen tissues obtained from BCA patients with various primary histology and from 50 fine needle aspirate (FNA) samples collected from metastatic sites (mFNA) in advanced BCA patients with various ER/PR/HER2 status. There was a high concordance between primary HER2-IHC status and COPIA-HER2 expression analysis. Significant levels of p95HER2 were observed in over 40% of HER2-positive (HER2: 3+ and 2+ with FISH+) patients, and low but detectable levels in some sample tissues with IHC-HER2 negative (2+ with FISH - / 1+ /0) were also observed. Over 50% of p95HER2-expressors had activated p95HER2, and over 25% of HER2-positive samples also had HER1, HER3, IGF1-R and other RTKs and transduction protein expression and/or activation. As the disease profile often shifts in recurrent breast cancer, our unique assay format can be utilized to provide valuable clinical information on limited samples obtained from evolving disease to help oncologists adjust their disease treatment options for each patient according to their ‘personal’ cancer profile-shift. Having the ability to profile tumors at different metastatic sites with an expanded pathway panel could provide information on their differential metastatic potentials; hence minimally invasive single-passage-mFNA samples may be utilized to tailor therapy options accordingly.
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 3179.