bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3158: Evidence for a role of EWS in alternative splicing in Ewing's sarcoma
Authors: Riedmann Lucia T. , Kauer Maximilian O. , Jug Gunhild , Kovar Heinrich
Published: February 2011

Ewing's sarcoma family tumors (ESFT) are characterized in 85% of cases by the oncogenic fusion protein EWS-FLI1 resulting from a chromosomal translocation. While FLI1 is an ETS transcription factor, EWS belongs to the evolutionarily conserved FET family of RNA-binding proteins. So far, functional studies of oncogenic EWS fusion proteins have concentrated on their presumed role as transcriptional regulators. However, chimeric FET proteins are usually co-expressed with their intact counterparts and previous protein interaction studies have shown that EWS and EWS-FLI1 interact with each other. Therefore, it is possible that functional interference with normal EWS function accounts for part of the oncogenic activities of EWS-FLI1. However, the role of EWS in post-transcriptional and transcriptional gene regulation is poorly defined. Since FET proteins associate with a number of RNA processing factors, we investigated the influence of modulated EWS expression on genome wide splicing using Affymetrix HuEx-1.0stv2 arrays. For that purpose, we used two model systems: a unique ESFT cell line lacking endogenous EWS was studied upon stable restoration of EWS expression, and an ESFT cell line allowing for inducible RNAi-mediated EWS-FLI1 suppression was analyzed upon transient EWS knockdown. As a result, we found significant differences between cells with and without EWS expression in splicing patterns as well as alternative promoter usage for more than 50 genes. GO analysis of the top ranking genes is consistent with previously suggested functions of the FET protein family such as DNA repair, chromatin structure and RNA transcription. The alternative splicing patterns were verified by RT-qPCR of candidate genes which are currently tested for their functional relevance to ESFT. One of these candidate genes is structural maintainance of chromosome 5 (SMC5). As a core component of the SMC5-SMC6 complex it is involved in sister chromatid homologous recombination by recruiting the SMC1-SMC3 cohesin complex to DNA double-strand breaks during anaphase. Ongoing studies concentrate on the influence of EWS-FLI1 on EWS-dependent alternative RNA processing patterns in these model systems.

This study is supported by grant P20665-B12 of the Austrian Science Fund FWF

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 3158.
Abstract 3160: A redox-sensitive PEST sequence regulates Nrf2 degradation through proteasome pathway
Authors: Ma Quan , Yu Rong
Published: February 2011

Nrf2 transcription factor plays the pivotal role in protection against oxidative stress and chemical carcinogens by inducing a group of antioxidant/detoxification enzymes. Control of Nrf2 degradation is believed to be one of the major mechanisms in regulation of Nrf2-dependent gene expression. Here we report identification of a redox sensitive PEST sequence that targets Nrf2 protein to degradation through proteasome pathway. The PEST sequence locates between amino acids 332 and 359 in human Nrf2. Deletion of this sequence not only prolonged Nrf2 half-life, but also greatly enhanced Nrf2 transactivation function. Insertion of this sequence to the C-terminus of green fluorescence protein (GFP) caused rapid degradation of the fusion protein. Although deletion or insertion of the PEST sequence did not change the status of protein ubiquitination, its dependent protein degradation was sensitive to proteasome inhibition. Interestingly, PEST-mediated protein degradation was also sensitive to oxidant treatments. More over, delivery of a synthetic peptide that mimics PEST sequence caused accumulation of Nrf2 protein and activated downstream genes. Thus, we conclude that, in addition to a previously identified Keap1 pathway, Nrf2 is also actively degraded through a PEST sequence under homeostatic condition. Targeting this sequence would offer a novel approach for activation of Nrf2-dependent protective mechanisms.

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 3160.
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

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

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

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

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

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

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

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

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 > MCF-7 > 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

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.
Abstract 3183: Dysregulated expression and function of Plexin-B1 in ovarian cancers
Authors: Oscar Wong GW , Siu Michelle KY , Ngan Hextan YS , Cheung Annie NY
Published: February 2011

Background: Ovarian cancer has a tendency to spread within the peritoneal cavity early in the disease course. Understanding the metastatic pathways of ovarian cancer is thus of high importance for devising ovarian cancer treatment. Plexin-B1 is a cognate receptor of Semaphorin 4D (Sema4D). This study aims at investigating the role of Plexin-B1 in ovarian cancer.

Materials and Methods: Expression of Plexin-B1 and its ligand in ovarian epithelial cell lines was examined by western blot and qPCR. Their expression in benign, borderline, and malignant tumors was revealed by immunohistochemistrty of paraffin embedded tissues. High-resolution melting (HRM) curve analysis was used to screen for mutation in Rac binding region of PLXNB1. Recombinant Sema4D was purified and used to treat OVCA420 cells which express high level of Plexin-B1. siRNA was used to knock-down Plexin-B1 in OVCA420. Cell migration and invasion ability was assayed by transwell migration and invasion assay.

Results: Plexin-B1 was most highly overexpressed in serous ovarian cancer cell lines OVCA420, OVCA429, and SW626, whereas Sema4D was ovexpressed in OVCAR3, TOV21G, and PAI. In contrast, Plexin-B1 and Sema4D were not expressed in immortalized normal ovarian epithelial cell lines. Immunohistochemical studies also revealed high Plexin B1 expression in ovarian borderline tumors and invasive cancers while no Plexin B1 expression was detectable in benign tumor. On the other hand, downregulation of Plexin-B1 was observed in the metastatic foci compared with their corresponding primary tumors. There is also a tendency of higher Plexin-B1 expression in serous histotype than in other histotypes. A preliminary HRM screening of Plexin-B1 cDNA from 28 ovarian cancers detected no mutation in the Rac binding region. Knock-down of Plexin-B1 by siRNA caused increased migration and invasion of ovarian cancer cells. In contrast to Sema4D, treatment of ovarian cancer cells OVCA420 by follicle-stimulating hormone which enhances ovarian cancer growth, led to Plexin-B1 downregulation. Conclusion: Our study suggested that Plexin-B1 may contribute to early ovarian carcinogenesis. Its dyregulation in ovarian cancers is paradoxically associated with altered cancer cell invasion and migration. The role of Plexin B1 in ovarian cancer progression is complex.

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 3183.
Abstract 3186: The role of fibroblast growth factor 9 for androgen-independent growth of prostate cancer cells
Authors: Teishima Jun , Hayashi Tetsutaro , Shoji Koichi , Ohara Shinya , Matsubara Akio
Published: February 2011

Introduction & Objectives: In prostate tissue, impairment of communication between epithelial and stromal cells through fibroblast growth factor 9 (FGF9) signaling pathway has been thought to cause failure of tissue homeostasis and development of malignant disease. Previous studies have reported that FGF9 expression increased in the prostate cancer xenografts derived from bone metastasis. The aim of our study is to investigate the role of FGF9 for progression of prostate cancer.

Material & Methods: Immunohistochemical staining using anti-FGF9 antibody were performed for tissues derived from radical prostatectomy for 98 male patients. Cell viability and apoptosis of DU145, LNCaP and androgen-independent LNCaP subline (AI-LNCaP) were assessed with MTT assay and with Apopercentage apoptosis assay in the presence or absence of treatment with recombinant FGF9 or anti-FGF9 neutralizing antibody, respectively.

Results: In immunohistochemical staining, FGF9 positive cells were detected in 12 samples. In cases with Gleason score 8 or higher, rates of cases with FGF9 positive cells were 32.1%, significantly higher than those in case with Gleason score 6 (3.3%, p=0.016) and 7 (5.0%, p=0.0079), respectively. 5-year biochemical relapse-free survival rate in cases with FGF9 positive cells were 31.3%, significantly lower than those in cases that FGF9 positive cells were not detectable (77.6%, p=0.0008). In condition with androgen-deprivated medium, cell viability of LNCaP was significantly lower than that of AI-LNCaP, and enhanced to the same level by treatment with FGF9. Furthermore, cell viability of AI-LNCaP and DU145 were significantly suppressed by treatment with anti-FGF9 neutralizing antibody. Significantly more apoptotic cells were detected in LNCaP than in AI-LNCaP by androgen deprivation, and decreased to same level as in AI-LNCaP by treatment with FGF9.

Conclusions: These results indicate that FGF9 can contribute for progression of prostate cancer through stimulation for cell proliferation and anti-apoptotic effect. FGF9 may be candidates of novel therapeutic targets for hormone-refractory 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 3186.
Abstract 3187: Regulation of androgen receptor expression through angiotensin 2 receptor in prostate cancer cells
Authors: Hoshino Koji , Uemura Hiroji , Ishiguro Hitoshi , Ohta Junichi , Yoshida Shin-ichiro , Nagashima Yoji , Sasaki Takeshi , Umemura Satoshi , Kubota Yoshinobu
Published: February 2011

Background

We have investigated the involvement of renin-angiotensin system (RAS) in the development and progression of prostate cancer. Although the local RAS of prostate gland related to cell proliferation and angiogenesis has the potential in carcinogenesis of prostate, the detailed mechanism remains unclear. In the present study, we examined the effects of angiotensin 2 (Ang2) signal, especially through AT1 receptor (AT1R) in prostate cancer cells.

Method

Cell proliferations of prostate cancer, androgen dependent LNCaP and androgen independent DU145 cells, were examined after knocking down AT1R by AT1 small interfering RNA (siRNA) transfection. We confirmed the expression of AT1R, androgen receptor (AR) and its related proteins by Western blot analysis. Immunocytochemical staining of AR in LNCaP cells transfected with AT1R siRNA (LNCaP siRNA cells) was performed. We examined the promoter activities of AR and PSA in luciferase assay by transfection of AT1R siRNA.

Result

Cell proliferation of LNCaP siRNA and DU145 cells transfected with AT1R siRNA were decreased on 7 days by 75% and 52%, respectively. Western blot analysis showed the increase of cleaved PARP and survivin related to apoptosis. Interestingly, AR was dramatically suppressed in LNCaP siRNA cells. The flow cytometry of LNCaP siRNA cells showed G2 arrest or increase of apoptosis. In addition, PSA, COX2, NFκB, and c-myc expression were decreased in LNCaP siRNA cells. Luciferase assay showed that the activity of AR promoter or PSA promoter were inhibited by AT1R knocking down. In immunocytochemical staining, the suppression of AR translocation into nucleus by DHT was confirmed in LNCaP siRNA cells. Ang2 induced the cell proliferation associated with the enhancement of AR, PSA, and NFκB, and furthermore the activity of AR or PSA promoter.

Discussion

In this study, we focused on the involvement of RAS in the AR expression of prostate cancer cells. Knocking down AT1 receptor protein resulted in a significant inhibition of cell growth associated with a remarked decrease of AR protein. We also found that the expression level of AT1R could modulate the transcriptional level of AR by affecting c-myc and NFκB expression. Taken together, there seems to be a strong relationship between AR and Ang2-AT1R signals. These results indicate that the inhibition of AT1 receptor has a potential to influence the AR expression in prostate cells, presumably contributing to develop novel therapeutic agents for prostate cancer, especially hormone refractory 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 3187.
Abstract 3189: Aggressive renal cancer cell behavior is partly mediated by CD98hc
Authors: Poettler Marina , Kalinowska Wazlawa , Susani Martin , Haitel Andrea , Zielinski Christoph , Prager Gerald W.
Published: February 2011

CD98, a heteromeric transmembrane protein, consists of a heavy subunit (4F2hc, SLC3A2), extracellular linked to a light subunit. The light chains provide amino acid transport activity of CD98. Recently, we could show that CD98hc directly interacts with a conserved motif in integrin beta subunits, thereby mediating outside - in signaling. A genetic knockout of CD98hc in animals is embryonic lethal, whereas overexpression of CD98hc in somatic cells leads to malignant transformation.

Recently, we reported that high CD98hc expression is a novel and reliable diagnostic marker for the more malignant subtype II papillary renal cell cancer marker (pRCC). Thereby, CD98hc expression directly correlated with grade of differentiation.

In the present study, we identified the functional role of CD98hc in clear cell renal cell cancer (ccRCC) behavior. First, we generated stable low CD98hc ccRCCs by the use of a shRNA lentiviral construct and compared tumor cell behavior with high CD98hc expressing mock infected ccRCCs. We found that downregulation of CD98hc led to an altered tumor cell behavior including tumor cell survival, migration and proliferation. Expression of a silent mutation - lacking shRNA binding motif - reconstituted CD98hc expression in lowCD98hc ccRCC, thereby restoring aggressive tumor cell behavior. By introducing CD98hc mutations either lacking integrin binding capacities or light chain interactions, we could identify the functional role of leucine amino acid transport in tumor cell proliferation, as affected C14-leucine uptake was directly correlated with tumor cell proliferative activity. In contrast, mutants lacking interaction sites for integrins showed defects adhesion-induced FAK phosphorylation and c-src activation, leading to reduced tumor cell adhesion and spreading. A biological relevance of the in vitro finding was confirmed in in vivo tumor xenotransplant assays, whereby stable low CD98hc/ccRCCs either reconstituted with wild type CD98hc or with mutated CD98hc were used. Only wild type reconstitution was capable to restore full aggressiveness of tumor growth and metastasis formation.

From our data we conclude that CD98 partially mediates functional activity of renal cancer cell behavior. Thus, CD98hc-mediated tumor cell behavior may offer a potential novel therapeutic target for the treatment of cancer.

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 3189.
Abstract 3194: Epidermal growth factor receptor variant type III induces angiopoietin like 4 expression through mitogen activated protein kinase pathway and promotes tumor angiogenesis in malignant glioma
Authors: Katanasaka Yasufumi , Koizumi Fumiaki
Published: February 2011

Epidermal growth factor receptor variant III (EGFRvIII) is the most common mutation of EGFR in glioblastomas (GBMs) and is present in approximately 50% of GBMs with EGFR gene amplification and overexpression. This variant lacks the exon 2 to 7 (the extracellular ligand-binding domain) and results in a constitutively active form of the receptor. EGFRvIII has been shown to promote tumor progression in mouse models. Since it is assumed that EGFRvIII alters the tumor microenvironment, we examined the involvement of EGFRvIII in tumor angiogenesis in GBMs. For this purpose, we transfected cDNA for wild type EGFR or EGFRvIII into human glioma LN229 cells with retroviral vector (LN229-WT or LN229-vIII, respectively). The growth of LN229-vIII-tumors was significantly accelerated compared with that of LN229-WT-tumors. The microvessel density in LN229-vIII-tumors dramatically increased compared with that in LN229-WT-tumors. To study molecular mechanism of the EGFRvIII-induced angiogenesis, real time PCR analysis of angiogenesis-related molecules was performed. The analysis revealed that the expression level of angiopoietin like 4 (Angptl4) was significantly increased in LN229-vIII compared with LN229-WT in vitro. The mRNA expression of Angptl4 was remarkably increased in LN229-vIII-tumors. These results were confirmed at the protein level in vitro and in vivo. We then investigated which the signal pathway is associated with EGFRvIII-induced Angptl4 expression. MEK inhibitor U0126 remarkably inhibited Angptl4 mRNA expression in LN229-vIII, indicating that MAPK signal pathway could be an important pathway for the transcription. In summary, EGFRvIII may induce Angptl4 expression through MAPK signal pathway and strongly promote tumor angiogenesis in malignant glioma.

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 3194.
Abstract 3196: The role of Ga12 and Thromboxane receptor beta interaction in bladder cancer progression
Authors: Woolworth Julie A. , Watson Dennis , Halushka Perry V. , Moussa Omar
Published: February 2011

In the United States, it is predicted that there will be over 70,000 new cases of bladder cancer this year. Bladder cancer is the fourth most common cancer in men and eighth in women. Our lab has identified that Thromboxane receptor beta (TPβ) plays a role in bladder cancer progression. Our studies have previously shown that TPβ can induce malignant transformation in the immortalized non-transformed bladder cell line, SV-HUC. TPβ is a G-protein coupled receptor that is over-expressed in bladder cancer tumors and high levels of TPβ in bladder cancer patients correlate with decreased survival. G-proteins, such as Gα12, are involved in second messenger cascades that influence a variety of cellular functions, including transcription and motility. Constitutive activation of Gα12 has been shown to induce malignant transformation. Gα12 messenger RNA and protein are expressed in bladder cell lines and paraffin-embedded tissue obtained from bladder cancer patients. This study was designed to elucidate the TPβ and Gα12 dependent pathways that contribute to bladder cancer progression. We found that treatment with TP agonist, U46619, increases cell migration, while treatment with TP antagonist, GR32191, blocks agonist dependent increase in cell migration. We observe that knockdown of Gα12 with shRNA partially reduces U46619-induced cell migration and MAP kinase signaling. Together our results suggest that TPβ mediated malignant phenotypes are in part modulated by Gα12.

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 3196.
Abstract 3197: Differential senescent and apoptotic responses to HDM2 antagonism in p53 wild-type human solid cancer cells
Authors: Liu Suxing , Lin Yinghui , Mirza Asra , Seidel-Dugan Cynthia , Wang Yaolin , Hicklin Daniel J. , Bishop W. Robert
Published: February 2011

The p53 protein is a primary regulator of the cellular response to DNA damage and is a critical tumor suppressor. Restoration of p53 function in soft tissue sarcomas and hepatocellular carcinomas in mice was reported to cause tumor regressions associated with cellular senescence. These findings led us to investigate the ability of HDM2 antagonism to induce cellular senescence as well as apoptosis in a broad panel of wild-type p53 cell lines derived from human solid tumors. Here we report that the small molecule HDM2 antagonist Nutlin-3a activates p53 and induces cellular senescence in 10 out of 12 human tumor cell lines tested. Only two of these tumor cell lines also demonstrated an apoptotic response to Nutlin-3a. The kinetics of senescence induction by Nutlin-3a was slower than that of the apoptotic response, suggesting that apoptosis may be the first-line response, while senescence may be triggered as a back-up program when the apoptotic response fails to execute. A distinct expression profile of p53 target genes associated with apoptosis, cell cycle and senescence was observed and corresponded with the distinct phenotypic responses in this panel of cell lines. Depletion of p53 protein expression using shRNA blocked cellular senescence induced by HDM2 antagonism demonstrating the critical role for p53 in this process. The pronounced effects of Nutlin-3a on senescence in multiple wild-type p53 tumor cell lines underscores the unique anti-tumor mechanisms of HDM2 antagonism. This approach may provide a novel therapeutic strategy for future cancer treatment.

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 3197.
Abstract 3199: p53 represses TRF2 through E3 ubiquitin ligase Siah-1: Feedback regulation in telomere-initiated damage signaling
Authors: Horikawa Izumi , Fujita Kaori , Mondal Abdul M. , Vojtesek Borivoj , Bourdon Jean-Christophe , Lane David P. , Harris Curtis C.
Published: February 2011

p53 plays critical roles in tumor suppression, stem cell functions and aging in vivo. The telomere-capping protein complex (shelterin) prevents functional telomeres from undergoing erosion or end-to-end fusion and from initiating unwanted DNA damage response. Uncapped, dysfunctional telomeres at the end of cellular replicative lifespan lose this protective mechanism and trigger telomere-initiated DNA damage signaling to activate p53 and thereby induce cellular senescence. Here we report that p53 in turn controls a component of the shelterin complex, TRF2, through Siah-1, a p53-inducible E3 ubiquitin ligase. Endogenous TRF2 and Siah-1 were repressed and induced, respectively, in normal human fibroblasts at replicative senescence, when p53 was physiologically activated. Spontaneous allelic loss, shRNA-mediated knockdown, dominant-negative inhibition, nutlin-3a activation and overexpression of p53 all showed that p53 induced Siah-1 and repressed TRF2. TRF2 was subject to proteasomal degradation in a p53-dependent manner. Anti-TRF2 antibody-immunoprecipitated proteins were found to undergo p53- and Siah-1-mediated ubiquitination. In vitro and in vivo ubiquitination experiments showed that the E3 ligase activity of Siah-1 was responsible for TRF2 ubiquitination. Biologically, Siah-1 knockdown or TRF2 overexpression delayed the onset of replicative senescence, suggesting that the proteolytic control of TRF2 cooperates with the transcriptional regulation of p53 target genes (e.g., p21WAF1 and microRNA-34a) to regulate p53-mediated replicative senescence. This study reveals that p53, which is a downstream effector of the DNA damage signaling from uncapped telomeres, also functions upstream to regulate the telomere-capping complex, and suggests that the p53-Siah-1-TRF2 pathway takes an integral part in orchestrating the DNA damage response at telomeres. Given that TRF2 inhibits a p53-activating kinase ATM at telomeres, a positive feedback loop involving TRF2, ATM and p53 may function to amplify DNA damage-induced and p53-mediated cellular responses.

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 3199.