bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1037: Histone lysine methyltransferase EHMT2 is involved in proliferation, apoptosis, and DNA methylation of human neuroblastoma cells
Authors: Lu Ziyan , Salwen Helen R. , Raj J Usha , Yang Qiwei
Published: August 2012

Background: Neuroblastoma (NB), a childhood neoplasm arising from neural crest cells, is characterized by a diversity of clinical behaviors ranging from spontaneous remission to rapid tumor progression and death. In addition to genetic abnormalities, recent studies have indicated that epigenetic aberrations also contribute to NB pathogenesis. However, the epigenetic mechanisms underlying the pathogenesis of NB are largely unknown. Methods: Cell proliferation was measured by cell counting and BrdU incorporation assay in a human NB cell line (LA1-55n). Expression of MYCN was measured by real-time PCR. Caspase 3/7 activity was measured by Caspase-Glo 3/7 assay kit. The level of global DNA methylation was measured by liquid chromatography-mass spectroscopy. Results: BIX-01294, a specific inhibitor of EHMT2 (a key enzyme for histone H3 dimethylation at lysine-9), decreased proliferation of NB LA1-55n cells and induced apoptosis via increasing caspase 3 activity. BIX-01294 at a concentration of 1µg/ml inhibited NB cell proliferation, decreased expression of the MYCN oncogene, and increased global DNA methylation levels. At a concentration of 5 µg/ml and 10 µg/ml, BIX-01294 significantly increased caspase 3/7 activity. Conclusion: Our results demonstrate that histone lysine methylation is involved in cell proliferation, apoptosis, and global DNA methylation in human NB cells. Further understanding of this mechanism may provide insight into the pathogenesis of NB progression and lead to novel treatment strategies.

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 1037. doi:1538-7445.AM2012-1037
Abstract 1048: DNMT1: An emerging target in the treatment of invasive urinary bladder cancer
Authors: Dhawan Deepika , Ramos-Vara José A. , Hahn Noah M. , Zheng Rong , Stewart Jane C. , Knapp Deborah W.
Published: August 2012

Introduction: More than 14,000 people die from invasive transitional cell carcinoma (InvTCC) of the urinary bladder each year in the US, and more effective therapies are needed. Naturally-occurring canine InvTCC very closely resembles the disease in humans and serves as a highly relevant translational model for novel therapy of human InvTCC. Work was undertaken to identify new entry points for anticancer therapy in dogs with the goal of translating successful therapeutic strategies into humans with InvTCC. Methods: Microarray expression analyses were conducted on canine normal bladder from dogs with no urinary tract disease (n=4) and InvTCC tissues (n=4) using Genome Array 1.0 and analyzed by GeneSpring GX 11, with the stringency of p < 0.02 and a ≥2-fold change. The genes thus identified were further analyzed for functional and pathway analysis using PANTHER (Protein ANalysis THrough Evolutionary Relationships) Classification System. In selecting genes for further study, consideration was also given for evidence of a role of the gene in human InvTCC. From these analyses, DNA methyltransferase 1 (DNMT1) was selected for further study. Immunohistochemistry (IHC) of canine normal bladder and InvTCC tissues was performed to confirm the microarray expression analyses. The effects of targeting DNMT1 in vitro was assessed through MTT assay and Western blot of canine InvTCC cells treated with 5-azacitidine (5-azaC) and trichostatin A (TSA). Results: DNMT1 was expressed in 0 of 6 normal canine bladder samples and in 10 of 22 (45%) canine InvTCC samples. The proliferation of canine InvTCC cells was inhibited by 5-azaC (at concentrations > 5µM) and by TSA (at concentrations > 0.1µM). Western blot results were supportive of DNMT1-related effects having a role in the antiproliferative activity. Discussion and Conclusions: Microarray expression analyses on canine tissues identified DNMT1 as a potentially “targetable” gene. Expression of DNMT1 in canine InvTCC was confirmed by IHC, and in vitro studies confirmed that drugs that inhibit DNMT1 have antiproliferative effects. These findings are similar to those recently reported by Wu et al. (Cancer, 2011) in human InvTCC. These findings are also in line with results of a pre-clinical (pre-human) trial of 5-azaC in dogs with naturally-occurring InvTCC, in which remission or cancer control occurred in 72% of dogs. In conclusion, DNMT1 has excellent potential as a target for InvTCC therapy in humans.

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 1048. doi:1538-7445.AM2012-1048
Abstract 1059: SUV39H1 promotes HCC tumorigenesis and is targeted by tumor suppressive miRNA-125b
Authors: Fan Dorothy Ngo Yin , Tsang Felice Ho Ching , Au Sandy Leung Kuen , Wei Larry Lai , Tam Aegean Hoi Kam , Wong Chun Ming
Published: August 2012

Epigenetic alternation is a common dysregulated event in hepatocellular carcinoma (HCC) development. Through interrogating the expression of 90 major epigenetic regulating genes, we identified SUV39H1, the prototype histone methyltransferase as one of the most frequently up-regulated epigenetic regulators in human HCC (61%, 23/38). SUV39H1 is responsible for H3K9 trimethylation establishment and essentially involves in heterochromatin formation and transcriptional repression. We found that SUV39H1 up-regulation in human HCC was significantly associated with increased Ki67 expression (P < 0.001) and the presence of venous invasion (P = 0.020), suggesting the importance of SUV39H1 in HCC tumor growth and metastasis. To investigate the functions of SUV39H1 in human HCC, both gain- and loss-of-function models were established. Ectopic expression of SUV39H1 remarkably enhanced cell clonogenicity and migratory ability of immortalized hepatocytes. Conversely, stable knockdown of SUV39H1 by shRNA substantially reduced cell proliferation, colony formation and migration of HCC cells. In addition, an elevated lysosomal β-galactosidase activity in SUV39H1 knockdown cells was observed and suggested the senescence-suppressive role of SUV39H1 in cancer progression. Consistent with previous observations, treatment of SUV39H1 inhibitor (Chaetocin) drastically suppressed cell proliferation and induced apoptosis in HCC cells. The oncogenic function of SUV39H1 was further demonstrated by in vivo experiments, knockdown of SUV39H1 significantly inhibited tumorigenicity of HCC cells in both subcutaneous and orthotopic mouse model. Most importantly, SUV39H1 knockdown abolished pulmonary metastasis of HCC cells in orthotopic tumor implantation model. To further investigate the underlying mechanism of SUV39H1 up-regulation in HCC, we explored the potential regulation of SUV39H1 by microRNA. In silico analysis suggested SUV39H1 as a putative target of miR-125b. Ectopic expression of miR-125b precursor in HCC cells significantly inhibited SUV39H1 3′UTR luciferase reporter activity and suppressed endogenous SUV39H1 expression at both mRNA and protein levels. Interestingly, down-regulation of miR-125b was frequently observed in human HCC (70%, 38/54) and was inversely correlated with SUV39H1 expression (P = 0.001). Taken together, we demonstrated that SUV39H1 was frequently up-regulated in human HCC and functioned as an oncogene in promoting HCC progression and metastasis. miR-125b negatively regulated SUV39H1 expression and downregulation of miR-125b may account for SUV39H1 up-regulation in human HCC.

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 1059. doi:1538-7445.AM2012-1059
Abstract 1065: Growth factor-mediated resistance to BRAF/MEK inhibitors in BRAF mutant melanoma
Authors: Lutterbach Bart , Ware Chris , Davis Lenora , Zhang Qing , Zawel Leigh , Reilly John , Pan Bo-Sheng
Published: August 2012

BRAF and MEK inhibitors induce striking tumor regressions in BRAF V600E melanoma patients. However, relapse occurs in the majority of patients within several months. Reported resistance mechanisms include acquisition of NRAS mutations, PDGFR or IGF1R activation, Cot amplification, or truncated BRAF upregulation. We report here that hepatocyte growth factor (HGF), neuregulin-1 (NRG1), and fibroblast growth factor-2 (FGF2) cause robust resistance in vitro to BRAF or MEK inhibitors in BRAF mutant melanoma cell lines. NRAS mutant lines were similarly resistant to MEK inhibition in the presence of these growth factors. By contrast other mitogenic factors such as PDGF, EGF, NGF, BDNF, MSP, MDK, GRO, and IGF1 did not maintain cell viability or growth. HGF, NRG1 and FGF2 prevented growth arrest and cell death and in some cell lines maintained cells in progressive growth. In addition, HGF and NRG1 could prevent growth arrest after dual treatment with BRAF and MEK inhibitors. Western analysis revealed that HGF and NRG1 reactivated Erk signaling and activated S6RP and AKT phosphorylation. Combined pharmacological inhibition mTor signaling and Akt signaling was required to fully block growth factor-mediated proliferation. In addition, small molecule Met inhibitors and an anti-HGF antibody blocked HGF-mediated resistance, while lapatinib and an anti-NRG1 antibody blocked NRG1-mediated resistance. While combined Akt and mTor inhibition potently inhibited cell line growth independent of BRAF or MEK inhibition, lapatinib and Met inhibitors had minimal effect on growth, suggesting potential for less adverse events with receptor inhibitors. In vitro co-culture experiments revealed that primary cells present in normal skin can cause resistance, and we have identified growth factors produced by these cells that contribute to resistance. We are analyzing receptor activation (Met and Erbb3) and FGF2 expression in wildtype, NRAS, and BRAF mutant primary melanoma samples. In future experiments we plan to test clinical samples from patients treated with BRAF/MEK inhibitors, and we hypothesize that HGF, NRG1, and FGF2 signaling is elevated in patients with poor initial responses or relapse to BRAF or MEK inhibitors.

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 1065. doi:1538-7445.AM2012-1065
Abstract 1067: Targeting the fibroblast growth factor receptor axis in malignant pleural mesothelioma
Authors: Hoda Mir Alireza , Schelch Karin , Pirker Christine , Ghanim Bahil , Klikovits Thomas , Laszlo Viktoria , Setinek Ulrike , Filipits Martin , Dome Balazs , Hegedues Balazs , Marian Brigitte , Grasl-Kraupp Bettina , Klepetko Walter , Berger Walter , Grusch Michael
Published: August 2012

Background: Malignant pleural mesothelioma (MPM) is an aggressive malignancy characterized by frequent resistance to chemo- and radiotherapy. Fibroblast growth factors (FGF) and their high-affinity receptors (FGFR) contribute to malignant growth in several tumor types including thoracic malignancies while a role in MPM remains widely undefined. Therefore, aim of the present study was to investigate the expression and impact of FGFs and FGFRs in MPM and to evaluate their potential suitability as new therapeutic targets. Material und Methods: Expression of all known FGF and FGFR genes was assessed by expression array analysis and confirmed by qRT PCR in MPM cell lines (n=10) and normal mesothelial cells. Selected FGFs were also investigated by immunohistochemistry on tissue samples. FGFR-specific tyrosine kinase inhibitors and an adenovirus expressing dominant-negative FGFR1 were used to block FGF signal transduction in MPM cell models. MTT and clonogenic assays as well as spheroid formation assays and videomicroscopy were performed to analyze cell growth, survival and migration. The effect on downstream signal transduction was assessed by immunoblotting with phosphorylation site-specific antibodies. Results: FGFR1 as well as the ligands FGF2 and FGF18 were highly expressed in almost all MPM cell lines investigated. Moreover, immunohistochemistry demonstrated high FGF expression also in tissue sections. Stimulation with exogenous FGF2 in contrast, led to a remarkable increase in cell migration and clonogenic growth. In contrast, inhibition of FGFR1 by the specific small molecule kinase inhibitor PD166866 lead to decreased proliferation, survival, migration and spheroid formation in the majority of cell lines tested. Adenoviral expression of dominant-negative FGFR1 further confirmed these results. In addition, concomitant FGFR inhibition increased the efficacy of currently used chemotherapeutic agents such as cisplatin. Also combination of FGFR inhibition and radiation lead to enhanced cytotoxicity. Conclusion: Taken together, these data suggest that FGFR signals contribute to proliferation, survival, migration and chemotherapy resistance of MPM cells and their inhibition should be further evaluated as a potential new treatment strategy in MPM.

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 1067. doi:1538-7445.AM2012-1067
Abstract 1187: Inactivation of p53 in HPV-infected lung cancer is correlated with p53 codon 72 genotypes and HPV 16/18 E6 protein expression but not with HPV 16/18 DNA
Authors: Cheng Ya-Wen , Hsu Nan-Yung
Published: August 2012

Background. A polymorphism at codon 72 of the p53 tumor suppressor gene is a possible determinant for cancer risk, particularly for carcinomas associated with HPV infection. We recently reported that high-risk HPV 16/18 E6 protein was associated with p53 protein degradation in lung cancer. The present study addressed the relationship between the different p53 genotypes and HPV oncoprotein expression with respect to p53 protein degradation in 319 primary lung cancer patients. Methods. The p53 codon 72 polymorphisms, HPV 16/18 infection, HPV 16/18 E6 and p53 protein expression were determined for all patients by PCR-RFLP, nested-PCR and immunohistochemical analysis. Results. The presence of HPV 16/18 DNA and E6 protein was inversely correlated with p53 expression. The frequency of p53 protein degradation in HPV 16/18 E6-positive/Arg/Arg lung cancer tumours was also much higher than in the HPV 16/18 E6-negative/Arg/Arg, HPV 16/18 E6-positive/Arg/pro+pro/pro, and HPV 16/18 E6-negative/Arg/pro+pro/pro groups. After adjusting for polymorphism, HPV 16/18 E6 protein, HPV 16/18 DNA, gender, smoking habit and tumor type, the major contributors to p53 degradation in lung cancer patients were p53 codon72 polymorphism and HPV 16/18 E6 oncoprotein expression. This correlation was not found for HPV 16/18 DNA infection. Conclusion. These results suggested that the involvement of HPV 16/18 E6 protein in the p53 inactivation that contributes to HPV-infected lung tumourigenesis is correlated with the p53 codon 72 genotype.

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 1187. doi:1538-7445.AM2012-1187
Abstract 1188: Reprimo, a p53-dependent G2 arrest mediator, reduces tumorigenic capacity in human gastric cancer cell lines
Authors: Olivares Wilda , Torres Veronica , Maturana María José , Montecinos Viviana , Aguayo Francisco , Corvalan Alejandro H.
Published: August 2012

Introduction: Gastric cancer (GC) is one of the leading cause of cancer-related deaths worldwide. The progression of GC is related to genetic as well as epigenetic changes, such as DNA methylation, that involves the downregulation of tumor suppressor genes. Recently we have identified Reprimo (RPRM), a p53-dependent G2 arrest mediator candidate as a putative biomarker for early diagnosis of GC (Clin Cancer Res 2008;14:6264-9). However, the role of RPRM in GC is unknown. Methods: The expression and sequence of the promoter and the coding region of RPRM were performed in five GC cell lines (AGS, NCI-N87, KATOIII, MKN28, MKN45). Two expression negative RPRM cell lines were stable transfected with a pCMV6 vector containing the full-length of the coding region of RPRM (pCMV6-RPRM). Cell growth and proliferation were analyzed by MTS, colony formation and soft agar assays. In addition, we analyzed the ability of migration through wound/healing assay. Results: Only two cell lines were negative for RPRM expression (AGS & MKN45). Bisulfite sequence analysis of the promoter region showed a dense CG-rich CpG island suggesting a role for methylation in regulation of gene expression. No mutations in the coding region were found. A significant decreased of cellular proliferation and anchorage independent growth were observed after the transfection with pCMV6-RPRM. The migration ability was also decreased in both cell lines. Conclusion: Our results suggest that RPRM reduces tumorigenic capacity in GC cell lines. Taken together we suggest that downregulation of RPRM is not only a putative biomarker for early diagnosis but also might play a role in the pathogenesis of GC. Grants-in-Aid for Scientific &Technological Research (Fondecyt #1111014 & Fondef #D09I1137) to AH Corvalán from the Government of Chile.

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 1188. doi:1538-7445.AM2012-1188
Abstract 1192: ZMIZ1 is elevated in epithelial ovarian cancer due to an upregulated alternative promoter and is associated with p53 mutations
Authors: Nadkarni Niyati J. , Rogers Laura , Samuelson Megan , Neff Traci , Bender David P. , Ahmed Amina , Geest Koen De , Button Anna , Dupuy Adam , Goodheart Michael J.
Published: August 2012

Objectives: The ZMIZ1 gene has recently been found to be a possible, candidate oncogene through a transposon mutagenesis screen. It is widely known that one of the most common genetic alterations in epithelial ovarian cancer involves mutations of the p53 tumor suppressor gene. Previous studies have shown that ZMIZ1 appears to transcriptionally regulate the p53 gene. We hypothesize that ZMIZ1 expression is caused by the presence of an alternative promoter and that the interaction between ZMIZ1 and p53 may impart worse survival. Methods: A retrospective chart review was performed to identify 138 patients with epithelial ovarian cancer. Data collected included clinico-pathologic factors, patient follow-up, and mortality. ZMIZ1 staining and p53 sequencing were performed on all patients. For the determination of ZMIZ1 alternative promoter relative gene expression, RT-PCR was performed on 33 tumors after cDNA had been isolated. Statistical analyses included chi-square analysis and the binomial test. The log rank test was used to evaluate survival analysis. Results: Immunostaining for the ZMIZ1 protein was performed with the following results: 55 (40%) tumors stained positive and 83 (60%) were negative. Of those patients that were found to be ZMIZ1 positive, over half were noted to have a p53 gene mutation. The median survival of the ZMIZ1 positive/p53 mutated cohort was approximately 36 months while the median survival for the ZMIZ1 negative/p53 wild type cohort was 84 months (p = 0.012). ZMIZ1 alternative promoter relative gene expression was noted to be elevated in patients with ZMIZ1 positive immunostaining compared to patients with negative staining. Conclusions: ZMIZ1 staining appears to be present in 40% of patients with epithelial ovarian cancer. In tumors that are ZMIZ1 positive, half contain p53 gene mutations; this combination imparts worse survival. ZMIZ1 upregulation may be due to the presence of a newly identified alternative promoter. Additional studies are ongoing to better define the role of this alternative promoter.

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 1192. doi:1538-7445.AM2012-1192
Abstract 1195: Erk activity promotes Notch-dependent HES1 expression in pancreatic cancer cells
Authors: Tremblay Isabelle , Bintz Jennifer , Boucher Marie-Josee
Published: August 2012

The oncogene Ras is well-recognized for its involvement in tumorigenesis in part through activation of downstream pathways including the PI3K/Akt and Mek/Erk pathways. Recent studies have suggested that the ability of Ras to transform cells also depends on cooperation with the Notch signalling pathway. However, the molecular and cellular mechanisms involved in this cooperation remain largely unknown. The aim of this study was to evaluate whether specific activation of the Ras/Raf/Mek/Erk pathway impacts Notch-dependent signalling. METHODS. Experiments were done using the pancreatic cancer cells MIA PaCa-2 and BxPC-3. Both cell lines express endogenous levels of the activated Notch1 (NIC). To inhibit Mek/Erk activity, cells were treated with the Mek inhibitor U0126 (10uM). Activation of the Mek/Erk pathway was achieved by treatment with phorbol 12-myristate 13-acetate (PMA, 100nM). The hes1-luciferase reporter gene was used to evaluate Notch-dependent transcriptional activity. The gamma-secretase inhibitor DAPT (25µM) was used to inhibit Notch activation. RESULTS. 1- Treatment of pancreatic cancer cells with U0126 reduced the activity of the hes1-luciferase reporter gene to the same extent than treatment with the gamma-secretase inhibitor DAPT. 2- Combined treatment (U0126 + DAPT) did not result in an additive inhibition of hes1-luciferase activity suggesting a Notch-dependent impact of the Mek inhibitor on hes1 transcription. 3- Increased HES1 mRNA expression levels were observed following treatment with PMA, an effect prevented by addition of U0126. 4- Massive activation of the Notch receptors by treatment with EGTA promoted HES1 protein expression. 5- Interestingly, strong stimulation of Erk activity by PMA promoted the EGTA-induced HES1 protein expression. 6- Conversely, inhibition of Erk activity completely prevented both the EGTA-induced and EGTA+PMA-induced HES1 protein expression. CONCLUSION. Taken together, our results suggest that Erk activity promotes Notch-dependent transcriptional activity and consequently promotes the expression of Notch target genes such as HES1.

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 1195. doi:1538-7445.AM2012-1195
Abstract 1197: Soluble uPAR plays a critical role in the progression of meningioma by autocrine signaling
Authors: Gogineni Venkateswara Rao , Nalla Arun Kumar , Velpula Kiran Kumar , Rao Jasti S.
Published: August 2012

In previous studies, we have reported on the vital role of the uPA-uPAR system in the progression of meningioma. Recent studies on the molecular structure of uPAR confirm that its shedding characterizes a further level of regulation in cellular processes. Here, we show that malignant meningioma cells release higher levels of soluble uPAR (SuPAR). Further, SuPAR concentration increased with radiation treatment (7 Gy) while uPAR knockdown diminished it. Deglycosylation analysis illustrated the existence of both SuPAR and cleaved SuPAR in vitro and in vivo. The cells shed SuPAR and phospholipase C mediates radiation-induced shedding of uPAR. SuPAR had a physical interaction with uPA and influences its activity in a concentration-dependent manner. Nonetheless, SuPAR did not adversely influence cell proliferation or cell cycle characteristics, but instead activated the MEK- ERK pathway. Receptor tyrosine kinase arrays and function blocking assays revealed the role of FPRL 1 receptors in SuPAR-mediated signaling. Further, SuPAR acted as a chemo-attractant for the invasion and migration of meningioma cells. Tumor bearing mice showed a significant rise in serum SuPAR concentrations, which also correlated with radiation treatment and morbidity. In conclusion, our results demonstrate the pro-invasive role and prognostic significance of SuPAR in the progression of meningioma.

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 1197. doi:1538-7445.AM2012-1197
Abstract 1198: Conversion of triple negative breast cancer cells into HER2 positive cells - a novel therapeutic approach
Authors: Bernemann Christof , Hülsewig Carolin , Kiesel Ludwig , Liedtke Cornelia
Published: August 2012

Introduction: Triple negative breast cancer (TNBC) is defined by the lack of estrogen-receptor alpha (ERα) and progesterone receptor (PGR) expression as well as absence of human epidermal growth factor receptor2 (HER2/neu) overexpression. This type of breast cancer is characterized by a poor prognosis and significantly reduced survival rate compared to other breast cancer subtypes. This is mostly due to the lack of targeted agents like endocrine or anti HER2 targets. Breast cancers which express HER2 are usually treated by chemicals targeting the human epidermal growth factor receptor; either by blocking the extracellular domain of HER2 through trastuzumab or the intracellular domain of HER2 through the small molecule lapatinib. Both therapeutics lead to inhibition of downstream signaling pathways like MAPK and PI3K signaling, resulting in increased apoptosis as well as reduced proliferation. Methods: We hypothesize that downstream effects e.g. reduced proliferation or apoptosis might be induced in triple negative breast cancer cells when HER2 is artificially overexpressed by transfection and cells get treated as HER2 positive cells. Therefore, triple negative breast cancer cell lines were stably transfected with HER2. These cells will be treated with anti-HER2 agents. Molecular analyses will demonstrate whether transfection with HER2 will yield a HER2 positive breast cancer phenotype in that all downstream signaling mechanisms act similarly to a priori HER2 positive cells. Results: Preliminary experiments suggest that proliferation of breast cancer cells transfected with HER2 does not change significantly. Treatment with HER2-specifically blocking antibody trastuzumab leads to significant decrease of proliferation in HER2 transfected, initially triple negative MDA-MB-231 breast cancer cells. Only a moderate decrease in proliferation was observed when lapatinib, a small molecule directed against both EGFR and HER2, was used in both MDA-MB-231 wildtype and HER2-transfected cell lines. Additional results will be presented. Conclusion: We surmise that firstly, transdifferentiation of cancer might become a clinical tool to treat cancer of poor prognosis and secondly that our results might shed light on future therapeutic approaches e.g. small molecule compound screening for endogenous HER2 reactivation / overexpression and subsequent targeted treatment of cancers.

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 1198. doi:1538-7445.AM2012-1198
Abstract 1214: Effects of dovitinib on fibroblast growth factor receptor-2 (FGFR2) expressing pancreatic cancer
Authors: Zhang Hao , Hylander Bonnie , LeVea Charles , Repasky Elizabeth , Straubinger Robert , Adjei Alex , Ma Wen Wee
Published: August 2012

Introduction. FGF signaling has been implicated in pancreatic cancer (PC) tumorigenesis and tumor-stromal interactions. Dovitinib is a potent inhibitor of the FGF receptors. We examined the effect of dovitinib in pancreatic cancer in relation to tumor FGFR2 expression using cell lines and patient-derived primary PC models. Methods. FGFR2 expression in 6 PC cell lines (L3.6PL, Panc4.30, AsPC1, Panc2.13, SU86.86, Panc02.03) and 13 patient-derived primary PC tumors was assessed using immunoblotting and RT-PCR. Dovitinib efficacy was assessed in vitro following FGF2 stimulation (IC50 < 10 μM). Contribution by FGF signaling inhibition was evaluated by knockdown and constitutive activation of FRS2, Mcl-1 and Akt. Effects on downstream signaling pathways were evaluated by immunoblotting. Tumor-bearing mice were treated with dovitinib 40mg/kg (daily oral gavage) and tumor growth was measured. Effects of dovitinib in tumors were evaluated by immunohistochemistry (H&E, ki67, TUNEL, CD34, aSMA, collagen IV). Results. Dovitinib and FRS2 shRNA induced significant in vitro cell kill in bFGF-stimulated PC cell lines that had elevated FGFR2 mRNA expression. This was associated with inhibition of p-Akt and decreased Mcl-1 level in sensitive PC cells. Ectopic Mcl-1 and constitutively active Akt1 overexpression reversed the sensitivity whereas Mcl-1 knockdown sensitized resistant PC cells to dovitinib-induced apoptosis. In in vivo xenograft studies, compared to controls, dovitinib caused significant growth inhibition in high FGFR2 mRNA expressing PC (L3.6PL; T/C=0 after 10 days) but not low expressing PC (SU86.86; T/C=0.8 after 28 days). We then selected a primary tumor with high FGFR2 mRNA expression (#12424), which dovitinib exerted anti-proliferative and pro-apoptotic effects, causing significant growth inhibition (T/C=0.1 after 28 days), with reduced tumor-associated stroma and microvessel density at ≤ 20 mg/kg in a dose-dependent manner. Dovitinib caused only moderate tumor growth inhibition in #10978 (T/C= 0.78), a low FGFR2 mRNA expressing primary tumor. Conclusions. Dovitinib demonstrated significant anti-tumor efficacy that was mediated via the Akt/Mcl-1 pathway, and this effect was dependent on FGFR2 activity. We propose that FGFR2 mRNA level may be a predictive marker for efficacy of dovitinib in PC, and will be evaluated in an on-going clinical trial using a dovitinib-containing regimen.

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 1214. doi:1538-7445.AM2012-1214
Abstract 1231: Differential roles of trans-phophorylated EGFR, HER2, HER3 and RET as heterodimerization partenrs of MET in lung cancer with MET amplification
Authors: Tanizaki Junko , Okamoto Isamu , Sakai Kazuko , Nakagawa Kazuhiko
Published: August 2012

Background: MET is a receptor tyrosine kinase (RTK) whose gene is amplified in various tumor types. We investigated the roles and mechanisms of RTK heterodimerization in lung cancer with MET amplification. Methods: With the use of an RTK array, we identified several phosphorylated RTKs in lung cancer cells with MET amplification. We examined the roles and mechanisms of action of these RTKs with immunoprecipitation, annexin V binding, and cell migration assays. Results: We identified EGFR, HER2, HER3, and RET in addition to MET as highly phosphorylated RTKs in lung cancer cells with MET amplification. Immunoprecipitation revealed that EGFR, HER2, HER3, and RET each formed a heterodimer exclusively with MET and that these associations were markedly reduced in extent by treatment with a MET kinase inhibitor, but not by treatment with their spcific inhibitors. RNA interference-mediated depletion of EGFR, HER2, or HER3 induced apoptosis in association with inhibition of AKT and ERK signaling pathways, whereas depletion of HER2 or RET inhibited both cell migration and STAT3 signaling. Conclusion: Our data suggest that heterodimers of MET with EGFR, HER2, HER3, or RET play differential roles in tumor development, and they provide new insight into the function of trans-phosphorylated RTKs as heterodimerization partners of MET in lung cancer with MET amplification.

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 1231. doi:1538-7445.AM2012-1231
Abstract 1234: GRK2 negatively regulates IGF-1R signaling pathway and cyclins in HepG2 cells
Authors: Wei Zhengyu , Hurtt Reginald , Bodzin Adam S. , Gu Tina , Doria Cataldo
Published: August 2012

G protein-coupled receptor kinase 2 (GRK2) plays a central role in the regulation of a variety of signaling pathways associated with many important cellular processes. Alternation of GRK2 expression has profound effects on cell physiological functions and induces diseases such as heart failure, rheumatoid arthritis, and cancer. In this study, we investigated the role of GRK2 in human hepatocellular carcinoma (HCC) HepG2 cells. We created a GRK2 knockdown cell line using a lentivirus containing GRK2 specific shRNA. Under IGF-1 (50 ng/ml) stimulation, HepG2 cells with reduced GRK2 expression showed increased tyrosine phosphorylation of IRS1 at the residue 612 and increased phosphorylation of Akt. We then treated the cells with gefitinib, an EGFR inhibitor. We found HepG2 cells displayed gefitinib sensitiveness with the increase of drug concentration. Moreover, gefitinib treated cells with reduced expression of GRK2 showed decreased phosphorylation of Akt in a dose dependent manner, but had relatively more IGF-1R expression. However, HepG2 cells with reduced expression of GRK2 did not display any growth difference in culture as compared with the scramble control cells. We further detected that reduced expression of GRK2 induced a small G2/M phase arrest by overexpressing cyclin A, B1, and E. DNA microarray analysis showed decreased transcription levels of IGF binding protein 1 and 4, which were confirmed by quantitative PCR analysis. Our results demonstrated that GRK2 has a role in the regulation of IGF-1R signaling pathway and involves in cell cycle progression in HCC HepG2 cells.

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 1234. doi:1538-7445.AM2012-1234
Abstract 127: miR-141 confers anoikis resistance of ovarian cancer cells by targeting KLF12
Authors: Mak Sze Ling
Published: August 2012

Anoikis resistance is one of the fundamental capacities of metastatic cancer cells to survive during invasion and dissemination. Emerging evidences have documented that deregulation of miRNAs have been associated with different aspects of tumorigenesis. In this study, we aimed to identify putative miRNA candidates that govern this process during metastasis of ovarian cancers. Using miRCURY™ LNA Array profiling (1264 miRNAs) analyze on two categories of ovarian cancer cell lines: high and low anchorage-independent growth ability. We firstly sorted out a list of 9 miRNAs which were upregulated and associated with high anchorage-independent growth ability in ovarian cancer cells. Amongst these candidates, miR-141 retained the strongest anchorage-independent growth ability examined by soft agar assay. Further real-time Q-PCR analysis found that miR-141 was frequently upregulated in ovarian cancer cell lines (n = 6) and clinical samples (n = 49) as compared with normal ovaries (n = 12). Functional studies showed that enforced expression of miR-141 increased both number and size of colonies formed in soft agar assay, and cell growth in focus formation assay. Moreover, miR-141 could enhance cell survival and proliferation in ovarian cancer cells in low serum media. Conversely, inhibition of miR-141 expression by miR-141 inhibitor could abrogate the above tumorigenic functions in ovarian cancer cells. On the other hand, by computational miRNA target prediction and pMIR-REPORT miRNA luciferase assay, we identified a tumor suppressor, Krüppel-related zinc finger protein AP-2rep (KLF12), was post-transcriptionally downregulated by miR-141. Previous studies have documented that KLF12 is a tumor suppressor in inhibiting AP2-induced tumorigenicity including anoikis resistance. Indeed, our biochemical analyses using luciferase reporter assays in combination with mutational analyses and Western blotting confirmed that miR-141 specifically targeting KLF 3′UTR and its expression. Taken together, our findings suggest that miR-141 is an oncogene that can enhance anoikis resistance in ovarian cancer cells through downregulation of KLF12. However, further investigation in delineating molecular mechanism of miR-141 and KLF12 in mediating ovarian cancer oncogenesis is warranted.

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 127. doi:1538-7445.AM2012-127
Abstract 1273: Identification of differentially expressed proteins in human papillary thyroid carcinoma using proteomic analysis
Authors: Sugimoto Chizuru , Susuki Dai , Kimura Yuichi , Narita Norihiko , Fujieda Shigeharu
Published: August 2012

Papillary thyroid carcinoma (PTC) is one of the most common malignant tumors of the thyroid glands. It tends to grow slowly but is associated with a poor prognosis when metastasis happens. To better understand basic mechanisms of tumor development and identify potential new biomarkers of PTC, we examined protein expression profiling in clinical PTC tissue and matched normal thyroid tissue using fluorescence two-dimensional difference gel electrophoresis (2D-DIGE). To identify the proteins, peptide mass fingerprinting via MALDI-TOF mass spectrometry was carried out. Using these strategies, 85 up-regulated or down-regulated proteins were found in PTC. Imaging software determined 26 proteins to be differentially expressed at the two-fold (or greater) level. Among them, Cathepsin B and Lamin A/C were selected for further analyses. Western blotting and immunohistochemical staining showed a higher expression of these proteins in clinical PTC tissue compared to normal thyroid tissue. We are presently investigating those roles in PTC. Proteomic analysis of PTC using 2D-DIGE and mass spectrometry could provide novel potential biomarkers and insights into global pathophysiologic changes in PTC.

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 1273. doi:1538-7445.AM2012-1273
Abstract 1276: Application of high-throughput proteomics Micro-Western array in studying molecular mechanism of anticancer effect of liver X receptor agonists against human colon cancer cells
Authors: Lin Ching-Yu , Huo Chiech , Lin Hui-Ping , Jiang Shih Sheng , Chuu Chih-Pin
Published: August 2012

We recently developed a high-throughput proteomics technology, the Micro-Western arrays (Ciaccio MF, Nature Methods 2010). Micro-Western array is a nano-litter scale array-based Western blotting assay. This method allowed us to measure 96 to 384 different phosphosites on multiple proteins of a set of 6 to 15 different samples simultaneously. We applied this technology in investigating molecular mechanism of anticancer effect of small molecular compound. Colon cancer is the third leading cause of cancer-related death in the world. Previously, we reported that agonists for liver X receptors (LXRs) suppressed the proliferation of several human cancer cell lines. LXRβ and LXRβ are nuclear receptors and are important regulators of cholesterol, fatty acid, and glucose homeostasis. 96-well proliferation assay and colony formation assay showed that three commonly used LXR agonists 22(R)-hydroxycholesterol, T0901317, and GW3965 suppressed proliferation of HT-29 and HCT-116 human colon cancer cells dose-dependently. Flow cytometry analysis indicated that LXR agonists induced G1 cell cycle arrest but not apoptosis in colon cancer cells. We then used gene overexpression and shRNA/siRNA knockdown to verify that LXRβ and LXRβ were directly involved in the growth inhibition caused by LXR agonists. To understand the molecular mechanism, we used gene array and Micro-Western Array with more than 240 phosphorylated antibodies targeting signaling network regulating receptor tyrosine kinases (RTKs), cell cycle, cell stress, and PI3K/Akt to systematically investigate signaling proteins being affected by LXR agonists in multiple time points. With help of bioinformatics modeling, our result revealed a potential mechanism how nuclear receptor LXR regulating cell cycle in human colon cancer cells via EGFR/PI3K/Akt and ABCA1/SREBP-1/LDLR signaling. Effect of LXR agonists treatment with or without chemotherapy agents on HT-29 xenograft model and validation of target proteins for LXR agonists in patients’ tissues are currently ongoing. Our observations suggested that LXR agonists treatment may be a potential adjuvant therapy for colon cancers.

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 1276. doi:1538-7445.AM2012-1276
Abstract 1286: Elucidating the role of E2F2 in mediating human breast cancer metastasis
Authors: Yuwanita Inez , Barnes Danielle , Andrechek Eran
Published: August 2012

In human breast cancer it is not the primary tumor that is the cause of mortality but tumor metastasis to distant sites. Therefore, it is important to elucidate the biological mechanisms that underlie the metastatic processes. To address this, we utilized bioinformatic methods in combination with biochemical assays and genetic studies. Specifically, we crossed MMTV-Myc transgenic mice with various activator E2Fs mutants. We found that the tumor incidence decreased in MMTV-Myc mice interbred with E2F2 and E2F3 mutant backgrounds. Surprisingly, we also found that the loss of E2F2 sharply increased the percentage of lung metastasis in MMTV-Myc mice. Here, we test whether these observations in mice translate to human breast cancer and identify the genes under the control of E2F2 transcription factor that are responsible for mediating breast cancer metastasis. To achieve these aims, we examined the probability of activation of the E2F2 transcription factors in human breast cancer. We calculated the probability of E2F2 activation and used this data to stratify the human breast cancer samples and their associated metastatic clinical data. Examining this data we found that a low probability of E2F2 pathway activation led to decreased time to distant metastasis. To account for this discrepancy, we performed hierarchical clustering of human and mouse gene expression datasets. We found two clusters of human breast tumors in which E2F2 played distinctly different roles in mediating metastasis. One particular cluster clustered with our mouse gene expression data and exhibited the same role for E2F2 where it increased time to distant metastasis. In addition, we examined the ontology of the genes in this cluster and found that the majority of these genes had putative E2F binding sites. To further identify genes that are involved in metastasis, we stratified human breast cancer metastasis clinical annotation using gene expression values from these genes. We found that there were seven genes that are uniquely differentially regulated in this cluster. Interestingly, we observed that this group of seven genes included genes that were implicated in the metastatic process such as MMP16. Taken together, our results showed that there is an important role for E2F2 in mediating breast cancer metastasis and using our bioinformatic methods, we have begun to elucidate this role of E2F2 in mediating breast cancer metastasis. Our future directions include further validation of these target genes in vitro and in vivo to establish the mechanism by which E2F2 mediates breast cancer metastasis.

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 1286. doi:1538-7445.AM2012-1286
Abstract 1296: Identification of human nuclear factor 1 (NF-1) as a potential transcription factor which regulates the gene expression of Sulfotransferase isoform 1A1 (SULT1A1) in human breast cancer
Authors: Yao-Borengasser Aiwei , Yu Xinfeng , Dhakal Ishwori B. , Edavana Vineetha K. , Williams Suzanne , Kadlubar Susan A.
Published: August 2012

Sulfotransferase isoform 1A1 (SULT1A1) plays a central role in drug metabolism. SULT1A1 expression is tissue specific, with little to no expression in normal breast epithelia but plentiful protein expression in most breast tumors. Revelation of the regulators of SULT1A1 expression in breast tissues is important in predicting breast tissue response to therapeutic agents that are substrates of SULT1A1, particularly tamoxifen which can be used for both breast cancer treatment and chemoprevention. In order to identify the appropriate transcription factors that regulate the gene expression of SULT1A1, we performed transcription factor (TF) Activation Profiling Plate Array assay (Signosis, Sunnyvale, CA) in normal breast MCF10A cells and breast cancer ZR-75 cells. TF activation profile differences in MCF10A cells and ZR-75 cells were quantitatively analyzed and compared. Out of 48 TFs DNA-binding activities allowed per plate, 18 TFs’ activities were at least 1.5 fold higher in ZR75 cells compared to that of in MCF10A cells. Matching of the 18 TFs to the possible TF binding consensus sequences in SULT1A1, which were predicted by software Alibaba 2.0, was performed and we found that nuclear factor 1 (NF-1) and GATA transcription factor 1 (GATA-1) were putative transcription factors involved in the regulation of SULT1A1 expression. To further study the relationship between NF-1 and GATA-1 and SULT1A1, we measured mRNA levels of NF-1, GATA-1, and SULT1A1 using total RNA isolated from 97 human liver samples. The liver samples were obtained from 43 female and 54 male health subjects (age range 26-102 year, mean 59.0 years). The real-time RT-PCR results showed that only NF-1, not GATA-1, mRNA level showed significant correlation with SULT1A1 mRNA level (r=0.64, p<0.0001). Therefore, we have identified that NF-1 as a new strong candidate for SULT1A1 gene expression regulation. The finding has laid foundation for the study on the effect of NF-1 on SULT1A1 gene expression, which could provide a mechanistic explanation for the differential expression of SULT1A1 between normal breast tissue and breast tumors.

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 1296. doi:1538-7445.AM2012-1296