bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1076: Ids act as downstream effectors in the transforming growth factor-β pathway in prostate cancer cells
Authors: Strong Nicole , Millena Cecille , Walker Lindsey , Chaudhary Jaideep , Khan Shafiq
Published: August 2012

Transforming growth factor-β (TGFβ) regulates proliferation, differentiation and differentiated function in a wide variety in cells. In prostate cancer cells, TGFβ inhibits proliferation in the earlier stages; however the cancer cells become refractory to growth inhibitory effects in advanced stages where TGFβ promotes cancer progression and metastasis. Intracellular mechanisms involved in these varied effects of TGFβ on prostate cancer cells are poorly understood. Inhibitor of Differentiation (Id) family of closely related proteins (Id1, Id2, Id3, Id4) inhibit binding of bHLH transcription factors to DNA and inhibit their effects on cell proliferation and differentiation. In the present study, we have investigated the involvement of Id1 and Id3 proteins in the effects of TGFβ on prostate cancer cells. Id1 and Id3 isoforms of protein are expressed in DU145 cells, which have previously been shown to be responsive to growth inhibitory effects of TGFβ. TGFβ caused a significant increase of protein levels of Id1 and Id3 as determined by Western blot analyses. This early induction was followed by a late downregulation of Id1, while Id3 induction remained stable. However in PC3 cells, this early induction of Id1 and Id3 proteins remained stable. Proliferation marker p21 was also induced in DU145 cells, while p21 protein was undetected in PC3 cells upon TGFβ1 treatment. Since previous studies have revealed that the loss of Id1 or Id3 is indicative of loss of proliferation in prostate cancer, Id1 effects were further studied between DU145 and PC3 cell lines. Id1 knockdown in DU145 cells resulted in enhanced inhibitory effects of TGFβ on cell proliferation. However, Id1 knockdown in PC3 did not influence TGFβ effects on proliferation. These results indicate that Id1 downregulation by TGFβ may be required for its effects on proliferation in DU145 cancer cells. We next investigated TGFβ effects on migration in PC3 cells, which TGFβ stimulation has been shown to increase its migration properties. Both Id1 and Id3 knockdown in PC3 cells decreased migratory response to TGFβ. This indicated that both Id1 and Id3 are necessary for TGFβ migratory properties of PC3 cells. Taken together, these results show that Id1 and Id3 act as necessary proteins in the TGFβ pathway on both proliferation and migration. Grant support: NIH 1P20MD002285-02 and RCMI G12RR03602

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 1076. doi:1538-7445.AM2012-1076
Abstract 1191: Managing the cross talk: BRCA1 and its regulation of the p53 family, E2f1/Rb and apoptosis
Authors: Scata Kimberly A. , El-Deiry Wafik S.
Published: August 2012

Under constant assault from internal and external stresses, the cell constantly monitors cellular damage, repairing damage when it is able. If the cell deems the damage too great to repair, it activates the pathways that induce programmed cell death. This life or death decision is controlled, in part, by the activation of the tumor suppressor, p53. Upon stabilization and activation, p53 activates a variety of genes including those genes capable of inducing apoptosis. Our lab has previously demonstrated that the tumor suppressor, BRCA1, is capable of stabilizing p53 and directing it to cell cycle arrest genes at the expense of apoptotic genes. In our efforts to understand the mechanism by which BRCA1 is able to prevent expression of apoptotic genes, we have discovered that BRCA1 also stabilizes E2F1, an oncogene that is able to induce p53-dependent and p53-independent apoptosis. Interestingly, we have found that BRCA1 also induces DNp73, a pro-survival member of the p53 family. We hypothesize that BRCA1 may be interfering with the interaction between E2F1 and Rb that has been shown to be important for the apoptotic response to DNA damage. Furthermore, we are currently using siRNA to inhibit expression of these genes to determine the relative contribution of DNp63, DNp73 and E2F1/Rb signaling to control the cell's decision to undergo apoptosis in response to BRCA1 expression.

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 1191. doi:1538-7445.AM2012-1191
Abstract 1196: Identification of lung cancer associated oncoantigens as targets for active immunotherapy
Authors: Riccardo Federica , Quaglino Elena , Arigoni Maddalena , Ercole Elisabetta , Iezzi Mannuela , Longo Dario L. , Forni Guido , Cavallo Federica , Calogero Raffaele A.
Published: August 2012

Non small cell lung cancer (NSCLC) is the leading cause of cancer death in men and women. Therefore, the development of new therapeutic strategies is essential for improving the prognosis and treatment of patients. An interesting experimental model to study human lung adenocarcinoma is represented by p53R172HΔg/K-rasG12D mice. They develop aggressive NSCLC that metastasize to multiple sites and display a stepwise, directly age-related progression, mimicking several features observed in lung cancer patients. Lung cancer progression in p53R172HΔg/K-rasG12D mice was characterized by non-invasive magnetic resonance imaging (MRI), histopathological and immunohistochemical analysis. p53R172HΔg/K-rasG12D mice develop lung adenocarcinomas already evident in 10 week-old mice, with a variable degree of differentiation. A significant gender difference in tumor progression was observed, with females developing more tumors than males. Moreover, by using qRT-PCR, some of the antigens deregulated in human lung cancer turned out to be up-regulated also during cancer progression in our mouse model, suggesting that p53R172HΔg/K-rasG12D mice recapitulate the metastatic nature of human NSCLC. Identification of oncoantigens expressed during tumor development could provide an unprecedented opportunity to address the immune system against these molecules. RNA was extracted from lungs of 10, 20 and 30 week-old wild type (wt) and p53R172HΔg/K-rasG12D mice. Transcription profiling was done using Mouse Exon 1.0 ST arrays: we detected 282 genes significantly differentially expressed during different stages of cancer progression. Interestingly, some of the genes that were already deregulated in the early phases of the tumor development remained deregulated also in advanced stages, suggesting their possible application as early biomarkers or as vaccination targets. These genes were ranked to full-fill the minimal requirement for an oncoantigen; within the plasma membrane proteins we identified potential candidates: CXCR1, SLC16A6, SLC26A9 and ROS1. On the basis of the literature data and mouse data we consider ROS1 an interesting putative oncoantigen to be further investigated.

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 1196. doi:1538-7445.AM2012-1196
Abstract 1211: Antitumor effect of prostaglandin E2 receptor inhibitors in gastric carcinoma
Authors: Fukuoka Tatsunari , Yashiro Masakazu , Takeda Hiroshi , Maruyama Takayuki , Morisaki Tamami , Hasegawa Tsuyoshi , Hirakawa Toshiki , Aomatshu Naoki , Matsuoka Junko , Sakurai Katsunobu , Kimura Kenjiro , Nagahara Hisashi , Amano Ryosuke , Noda Eiji , Kubo Naoshi , Tanaka Hiroaki , Muguruma Kazuya , Yamada Nobuya , Maeda Kiyoshi , Ohira Masaichi , Ishikawa Tetsuro , Hirakawa Kosei
Published: August 2012

Introduction: Prostaglandins (PGs) have been suggested to play an important role for the proliferation and migration of cancer. Cyclooxygenase2(COX-2) stimulates PG synthesis. The selective COX-2 inhibitors have reported to show antitumor effects on various types of cancers, including colorectal cancer and gastric cancer. Gastric cancer remains to be poor prognosis because few anti-cancer drugs are available for advanced gastric cancer. PGE2 binds to PGE2 receptor, EP1, EP2, EP3 and EP4 and activates MAPKinase. The aim of this study is to investigate the effects of the EP2 inhibitors on the proliferation and motility of gastric cancer cells. Methods: Human gastric cancer cell lines (OCUM-2M and OCUM-12) were used in this study. Cell proliferation was studied by 3-(4,5-Dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Cell migration was studied by a wound healing assay. Results: PGE2 significantly stimulated proliferation and migration of gastric cancer cells, but not PGE1. EP2 and EP4 inhibitors significantly decreased the proliferation and migration ability of gastric cancer cells in a dose dependent manner, but not EP1, EP3. Conclusion: PGE2 is associated with progression of gastric cancer cells.PGE2 receptor inhibitors might be promising anti-cancer drug for gastric carcinoma.

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 1211. doi:1538-7445.AM2012-1211
Abstract 1212: The dual action antibody MEHD7945A targeting EGFR and HER3 enhances chemotherapy induced cytotoxicity in vitro and in vivo
Authors: Crocker Lisa M. , Fields Carter , Shao Lily , Sliwkowski Mark X. , Phillips Gail D. Lewis , Schaefer Gabriele
Published: August 2012

Dysregulation of the epidermal growth factor receptor family (EGFR, HER2, HER3, HER4) by mutation and/or overexpression plays an important role in tumorigenesis, and targeted agents directed against two members of the HER/ErbB family, epidermal growth factor receptor (EGFR/HER1), and HER2/ErbB2, are used in the treatment of cancer. Extensive crosstalk seen among these receptors implies that blocking signaling of more than one receptor may be more effective in inhibiting tumor growth and circumventing resistance mechanisms than targeting individual receptors. In particular, HER3 is considered a key mediator of resistance to many targeted agents. We generated a “two-in-one” antibody, MEHD7945A, that binds to EGFR and HER3 with high affinity, inhibits receptor function and is more broadly efficacious in various tumor types when compared to monospecific anti-EGFR or anti-HER3 antibodies. Given the ability of HER3 to potently activate the PI3K survival pathway, we investigated if antagonizing ligand-dependent HER signaling with MEHD7945A in the presence of chemotherapy augments cytotoxicity. We calculated combination index values the effects of combining MEHD7945A with commonly used chemotherapeutic agents in NSCLC and colorectal cell lines in vitro and in vivo. The NSCLC lines NCI-H292, NCI-H1666, NCI-H358 and HCC827 were treated with MEHD7945A plus gemcitabine over a wide range of drug concentrations. Cell proliferation data were analyzed using CalcuSyn software and all combination index values were <1, demonstrating that the combination of MEHD7945A and gemcitabine inhibited proliferation synergistically in these cells. To further explore the enhanced cytotoxic effect we evaluated the combination of MEHD7945A and gemcitabine, versus each single agent, in the NCI-H1975 NSCLC xenograft model, and observed enhanced tumor regression. Similar results were observed when MEHD7945A was combined with a range of chemotherapeutic agents, including pemetrexed, docetaxel and irinotecan in H1975, H441 or SW948 xenograft models. In summary, these in vitro and in vivo results demonstrate that MEHD7945A potentiates various cytotoxic agents in a variety of tumor types.

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 1212. doi:1538-7445.AM2012-1212
Abstract 1218: PTPRO as a candidate phosphatase in regulating EGFR signaling in colorectal cancer
Authors: Asbagh Layka Abbasi , Vecchione Loredana , Sablina Anna , Tejpar Sabine
Published: August 2012

Background Protein tyrosine phosphatases (PTPs) are known to be responsible for the negative regulation of many receptor tyrosine kinases by serving as antagonists to tyrosine kinase signaling. Several PTPs have been identified as candidate regulators of EGFR phosphorylation such as PTP1B, RPTP-sigma, RPTP-kappa, and PTPRJ (DEP-1). Here, we focused on PTPRO, a member of R3 family. The Drosophila ortholog of this family, Ptp4E/Ptp10D, negatively regulates EGFR signalling. Gene expression analysis of 688 primary tumours led us to identify PTPRO as a gene with strongly reduced expression in colorectal cancers with poor prognosis in particular the ones belonging to the Braf mutant subgroup. This suggests that PTPRO could act as a tumour suppressor by negative regulation of EGFR in colorectal cancer. Material and methods HEK293 cells were transiently transfected with wild-type (WT) PTPRO and with its catalytic mutant (C/S) and stimulated with EGF. After cell lysis, extracts were analyzed by SDS-PAGE and probed by western blotting with an anti-phosphotyrosine antibody (pY99, 4G10). The total level of EGFR is detected with anti-EGFR antibody. EGFR phosphorylation antibody arrays are used to identify the specific tyrosine phosphorylation sites. Results and discussion Upon expression of the PTPRO (C/S) mutant, we observe a delay in EGF induced EGFR degradation. Our preliminary data suggests that PTPRO might play a role in EGFR trafficking through the endocytic pathway and its catalytic activity is required for efficient endocytic progression of EGFR. Ongoing experiments will answer to this question. WT-PTPRO over-expression specifically decreases the Tyr 845 phosphorylation of EGFR, a known Src family tyrosine kinase (SFK) phosphorylation site. SFKs also regulate receptor turnover both at the level of endocytosis and cbl-mediated ubiquitination by phosphorylation. However, underlying mechanism how PTPRO can modulate EGFR signalling through the regulation of endocytic machinery is not known yet.

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 1218. doi:1538-7445.AM2012-1218
Abstract 122: MiR-9-3 and miR-193a are targets for DNA methylation in non-small cell lung cancers
Authors: Heller Gerwin , Weinzierl Marlene , Noll Christian , Babinsky Valerie , Ziegler Barbara , Altenberger Corinna , Minichsdorfer Christoph , Lang György , Döme Balazs , End-Pfützenreuter Adelheid , Arns Britt-Madeleine , Grin Yuliya , Klepetko Walter , Zielinski Christoph C. , Zöchbauer-Müller Sabine
Published: August 2012

DNA methylation (referred to as methylation) is part of the epigenetic gene regulation complex which is relevant for the pathogenesis of different malignant diseases including non-small cell lung cancers (NSCLC). Recently, it has been reported that besides protein encoding genes also microRNA (miRNA) encoding genes may be targets for methylation in NSCLCs, however, the number of known methylated miRNA genes is still small. To investigate the role of methylation on miRNA gene silencing in NSCLCs, we performed microarray expression analyses of 856 miRNA genes in NSCLC A549 cells before and after treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (Aza-dC) and with a combination of Aza-dC and the histone deacetylase inhibitor trichostatin A. Comparing microarray data of untreated and drug treated A549 cells, we identified 33 miRNA genes whose expression was upregulated after drug treatment and which are associated with a CpG island. Methylation of these miRNA genes was determined in 11 NSCLC cell lines by methylation-sensitive high resolution melting (MS-HRM) analysis and 30 of 33 (91%) miRNA genes were found to be methylated in at least 1 NSCLC cell line. In addition, methylation of the miRNA genes miR-9-3 and miR-193a was also analysed in primary tumors and corresponding non-malignant lung tissue samples of 101 stage I-III NSCLC patients and was found to be tumor-specific. Moreover, we compared miR-9-3 and miR-193a methylation results and clinico-pathological characteristics of NSCLC patients. We found a shorter disease-free survival of miR-9-3 methylated lung squamous cell carcinoma (LSCC) patients compared to miR-9-3 unmethylated LSCC patients by multivariate analysis (HR = 3.8, 95% CI = 1.3 to 11.2, p = 0.017) and a shorter overall survival of miR-9-3 methylated LSCC patients compared to miR-9-3 unmethylated LSCC patients by univariate analysis (p = 0.013). In conclusion, our results suggest that methylation is an important mechanism for silencing certain miRNA genes in NSCLCs and that miR-9-3 methylation may serve as a prognostic parameter in LSCC patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 122. doi:1538-7445.AM2012-122
Abstract 1226: Lapatinib could not prevent heregulin-induced HER2-HER3 signalling, and addition of pertuzumab to lapatinib increases drug sensitivity and inhibition of HER signalling
Authors: Leung Wing Yin , Kong Anthony H.
Published: August 2012

Lapatinib is an EGFR/HER2 tyrosine kinase inhibitor (TKI) that has been approved for clinical use in HER2 positive breast cancer patients who have progressed on previous Trastuzumab containing regimens. However, lapatinib monotherapy only delays tumour growth in HER2 positive xenograft models and has only modest clinical activity in breast cancer patients. There is a further need to understand its mechanisms of action and resistance. We aimed to assess the acute effect of lapatinib on HER2 phosphorylation in HER2 positive breast cancer cells since previous work from our lab showed that inadequate suppression of HER2 phosphorylation by gefitinib and trastuzumab may lead to acquired drug resistance. Traditionally, the phosphorylation status of HER proteins is determined by phosphor-tyrosine western blotting followed by immunoprecipitation. The limitation of this technique includes false positive due to signals from co-precipitated proteins of the same size as target, which is even more prominent with the HER family of protein. With the use of phosphorylated protein-specific antibody, we found that, contrary to general belief, lapatinib could not abolish HER2 phosphorylation in HER2 over-expressed breast cancer cell lines under serum-supplemented condition. It was thought that the growth factors in the serum activate other HER receptors to maintain HER2 phosphorylation. Indeed, Lapatinib could reduce phospho-HER2 level in serum-starved breast cancer cell lines; however, it failed to reverse heregulin-induced HER signalling activation and downstream MAPK and AKT signalling. The combination of lapatinib and pertuzumab drastically inhibited ligand-induced HER family phosphorylation and downstream signalling. HER3 was reported as an important biomarker for resistance in HER family targeted therapy. This novel combination could inhibit HER3 phosphorylation and its downstream AKT pathway more than lapatinib or pertuzumab alone; this finding also correlated with cancer cell viability, as the combination of lapatinib and pertuzumab reduced cell viability more than either drug alone. These results might indicate a new strategy of drug combination to tackle resistance in HER2 targeted therapy.

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 1226. doi:1538-7445.AM2012-1226
Abstract 123: DNA methylation of miRNA promoters in breast cancer
Authors: Vrba Lukas , Muñoz-Rodríguez José L. , Stampfer Martha R. , Futscher Bernard W.
Published: August 2012

MicroRNAs (miRNA) are short single-stranded RNA molecules that regulate gene expression and deregulation of their expression is linked to human diseases including cancer. Epigenetic mechanisms including DNA methylation play an important role in the regulation of miRNA expression, and a number of reports describe silencing of miRNA expression linked to the aberrant DNA methylation of individual miRNA genes in cancer. In this study, we have used a comprehensive miRNA custom tiling microarray to evaluate aberrant DNA methylation of miRNA gene promoters in human breast cancer, with selected hypermethylated promoters being validated by Sequenom MassARRAY technology. We found more than 25% of miRNA gene promoters analyzed have significant hypermethylated regions in breast cancer cell lines (CCL) relative to logarithmically growing normal pre-stasis finite lifespan human mammary epithelial cells isolated from healthy individuals. In addition to in vitro grown breast cancer cell lines, we analyzed DNA methylation of miRNA promoters in a dozen breast tumor tissue specimens and five normal breast specimens. Aberrant DNA hypermethylation of miRNA promoters in breast cancer specimens displayed a perfect overlap with those hypermethylated in breast cancer cell lines, albeit the number of miRNAs promoters was smaller in the heterogeneous tissue samples. These aberrantly methylated miRNA promoters readily clustered breast cancer samples and cell lines together, and separate from normal beast tissue and mammary cell strains. Integration of our data with publically-available H3K27me3 ChIP-Seq data from stem cells reveals that miRNA promoters targeted by aberrant DNA methylation in cancer cells significantly overlap with promoters targeted by polycomb repression complexes in stem cells, similar to the results other have seen with protein coding genes promoters in cancer. We have found previously that the proportion of miRNA genes targeted by polycomb repression in normal mammary cell types is about 27% - three times higher than seen in protein coding genes, perhaps explaining why the proportion of miRNA genes with aberrant DNA methylation in our analysis of breast cancer is so high. In summary, our data show that a large fraction of miRNA gene promoters are targets of aberrant DNA methylation in human breast cancer, and that the miRNA promoters aberrantly methylated in breast cancer cell lines are also targeted in tumor tissue samples, indicating that these changes in DNA methylation are not artifacts of cell culture and might thus have clinical relevance. Similar to protein coding genes, miRNA gene promoters targeted by polycomb complexes in stem cells are more prone to hypermethylation in cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 123. doi:1538-7445.AM2012-123
Abstract 1240: The role of hematopoietic protein kinases in GBM
Authors: Moncayo Gerald E. , Grzmil Michal , Wang Yuhua , Frank Stephan , Merlo Adrian , Brian Hemmings A.
Published: August 2012

Glioblastoma multiforme (GBM) is the most common and deadly form of brain cancer. Despite intensive research the median survival of GBM remains at 1 year, with less than 10 % of patients surviving over 5 years. Our analysis of fresh GBM, secondary GBM, high grade astrocytoma, oligodendroglioma, normal brain and normal human astrocytes showed increased expression of 19 novel protein kinases in GBM and targets previously associated with gliomagenesis (e.g. EGFR or PDGF). Two of these kinases which are mainly expressed in hematopoietic cells were the cytoplasmic kinases SYK and LYN. We confirmed the overexpression in GBM tumours and cell lines through western blot and immunohistochemistry analysis. More importantly, SYK expression was derived from the GBM tumours and not from infiltrating leukocytes as shown through colocalization with GFAP, and not with CD45. SYK was found strongly activated at the membrane after EGF and treatment with three specific small molecule inhibitors and siRNA targeting SYK strongly blocked proliferation and migration of GBM cells. This was confirmed through wtSYK and kinase dead SYK overexpression. In addition, SYK phosphorylation was found to be cell cycle regulated after synchronization. Due to our preliminary results we will test the therapeutic potential of SYK inhibitors and epistatic kinase interactions on our xenograft model measuring the efficacy of therapeutics crossing the blood brain barrier with the ultimate goal of discovering alternative treatments for GBM.

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 1240. doi:1538-7445.AM2012-1240
Abstract 1242: A broad activity screen in support of a chemogenomic map for kinase signaling research and drug discovery
Authors: Gao Yinghong , Davies Stephen , Augustin Martin , Harvey Kevin J.
Published: August 2012

Since protein kinases play a fundamental role in cell signaling, especially as components of critical growth and differentiation pathways linked to cancer, kinase inhibitors have been commercialized as both therapeutics and research tools. This has been enabled to some degree by the chemical tractability of protein kinase ATP binding pockets, the automation of activity measurements, and the correlation of in vitro, cellular, and in vivo activities. Despite the development of a number of efficacious kinase inhibitors, the strategies for rational design of these compounds have been limited by target promiscuity. In an effort to better understand the nature of structure and activity across the kinome, especially as it relates to off-target effects, we screened a well-defined collection of kinase inhibitors using “gold standard” radiometric assays for inhibitory activity toward 234 kinases, which represent all branches of the kinome tree. We screened 158 small molecules initially identified in the literature as potent and specific inhibitors of kinases important from a therapeutic target as well as a signal transduction biology perspective. We performed hierarchical clustering of these benchmark kinase inhibitors based upon their kinome activity profiles and illustrate how they relate to chemical structure similarities; this provides new insights into inhibitor specificity and potential applications for probing new targets. Additionally, we developed a selectivity score for each kinase which we believe reflects general binding site accessibility to small molecules. Knowledge of such scoring indices can play an important role in therapeutic target selection and drug discovery strategies. Using this broad set of data, we provide a framework for assessing polypharmacology. Examples will be shown to demonstrate that we not only identify likely off-target inhibitor activities but also potential new uses for known small molecules. Moreover we demonstrate how activity data can be used to help explain the effects of small molecules observed in phenotypic screening.

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 1242. doi:1538-7445.AM2012-1242
Abstract 1245: Hepatitis B virus X protein induces Src homology 2-containing protein tyrosine phosphatase, SHP2, expression through the NF-kB pathway in HBV-infected hepatocellular carcinomas
Authors: Kim Hyun Mi , Kim Ga-hye , Yu Eunsil , Choi Jene
Published: August 2012

Hepatitis B virus X protein (HBx) has a crucial role in the development of hepatocellular carcinoma and activates many transcription factors, including AP-1, NF-kB and CREB. In the present study, we showed that increased SHP2 expression was observed in 16 (53%) of the 30 cases of HBV-positive hepatocellular carcinomas tissues. We investigated the relationship of SHP2 and HBx in stably HBx-overexpressing Huh7-X and transiently HBx expressing HEK293 cells. The level of SHP2 proteins was concomitantly increased with HBX in a dose-dependent manner in HEK293 cells. We identified a putative NF-kB binding site within 500 bp of the SHP2 promoter and also observed the decrease of SHP2 proteins after the treatment of the NF-kB/p65 inhibitor, parthenolide in HBx expressing cell. In addition, we confirmed that the complex of NF-kB and HBx proteins direct bound to the SHP2 promoter by chromatin immunoprecipitation (ChIP)-real time PCR assays. Taken together, we conclude that HBV infection induces SHP2 overexpression through the HBx-NF-kB pathway. Currently, we are investigating the effects of SHP2 overexpression in the proliferation of hepatocellular carcinoma 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 1245. doi:1538-7445.AM2012-1245
Abstract 1247: ARHI downregulates PI3K and Ras-MAP signaling and induces autophagy by promoting integrin and EGFR membrane protein degradation
Authors: Lu Zhen , Liao Warren S.L. , Yang Maojie , Gao Fengqin , Bast Robert C.
Published: August 2012

Autophagy selectively degrades cellular proteins, protein aggregates and organelles, providing energy in a nutrient-deprived environment. We have reported that autophagy can be induced by re-expression of ARHI (DIRAS3), a maternally imprinted Ras-related tumor suppressor gene that is down regulated in more than 60% of ovarian cancers. Whereas prolonged expression of ARHI in cultured cells induces autophagic cell death, ARHI expression in xenografts sustains dormant cancer cell survival for weeks. When ARHI levels were reduced in dormant cells, xenografts grew rapidly. Inhibition of ARHI-induced autophagy with chloroquine dramatically reduces regrowth of xenografts, suggesting that autophagy contributes to the survival of dormant cells. Re-expression of ARHI inhibits both PI3K and Ras-MAPK signaling. Growth factors (VEGF, IL-8, IGF) and ECM components (fibronectin) found in the cancer microenvironment can rescue ovarian cancer cells from ARHI-induced autophagic cell death in culture, associated with partial reversal of ARHI-mediated inhibition of PI3K signaling. Consequently, we have hypothesized that autophagy together with growth factors and ECM in the microenvironment are all required to maintain dormancy, where autophagy provides energy for xenografts and survival factors prevent autophagic death by partially reversing inhibition of PI3K-AKT signaling. The mechanism by which ARHI regulates PI3K and Ras-MAPK signaling has not been fully elucidated. Here, we show that re-expression of ARHI in SKOv3 ovarian cancer cells inhibits expression of integrin β1 and EGFR. ARHI expression enhanced EGF-mediated receptor endocytosis and colocalization of EGFR with Rab11 and Rab7, markers for multivesicular endosomes and lysosomes, respectively. Our results suggest that ARHI enhances membrane receptor turnover, in part, by facilitating the internalization and association of membrane receptors with vesicular trafficking proteins targeted for degradation in the lysosome. ARHI-mediated downregulation of membrane receptors reduces both PI3K-AKT and Ras-MAPK signaling, ultimately contributing to the inhibition of tumor growth.

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 1247. doi:1538-7445.AM2012-1247
Abstract 1254: FAK activity regulates αvβ5 integrin and osteopontin expression to control breast and ovarian cancer anchorage-independent growth
Authors: Tancioni Isabelle , Lawson Christine , Miller Nichol L.G. , Uryu Sean , Jean Christine , Chen Xiao L. , Lim Ssang-Taek , Ward Kristy , Schlaepfer David D.
Published: August 2012

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that controls cell motility and survival downstream of integrin receptors. FAK expression and activity are elevated during breast and ovarian cancer progression and this is associated with a poor clinical prognosis. Small molecule FAK inhibitors (FAK-I) block breast and ovarian tumor progression through a novel mechanism that is associated with the apoptosis under anchorage-deprived conditions. Metastatic cells can grow as anchorage-independent spheroids and we hypothesize that a spheroid micro-environment remains dependent on integrin- and FAK-associated signaling for survival. Here we show that intraperitoneal (ip) injection of murine ID8 ovarian carcinoma cells into mice, recovery of ascites-associated tumor cells (ID8ip) and growth ex vivo, yielded tumor cells with elevated levels of phosphorylated FAK (Y397 and Y576 phosphorylation), αvβ5 integrin, and osteopontin (OPN) matrix protein expression compared to parental ID8 cells. Analyses of paraffin-embedded breast and ovarian tumor sections revealed significantly higher pY397 FAK (p<0.05), β5 integrin (p<0.0001), and OPN expression (p<0.005) in stage II-III compared to stage I tumors. Treatment of ovarian ID8ip, human ovarian HEY carcinoma, or human MDA-MB-231 breast carcinoma cells with FAK-I inhibited cell growth, prevented FAK Y397 phosphorylation, reduced β5 integrin and OPN levels independent of effects on β1 or β3 integrins or changes in matrix proteins such as fibronectin. FAK-I impairs anchorage independent cell growth and orthotopic tumor growth but not growth of cells in plastic-adherent cell culture. Stable knockdown of either FAK or β5 integrin in HEY cells prevented anchorage-independent growth. Re-expression of wildtype or kinase-dead FAK within HEY cells verified the importance of FAK activity in promoting β5 integrin and OPN expression associated with enhanced tumor growth. Ongoing studies are focused on determining the molecular connections of FAK to the regulation of αvβ5 and OPN expression. Together, our studies support a novel role for a signaling loop involving OPN and αvβ5 integrin leading to FAK activation and reinforcement of OPN and β5 integrin expression within the spheroid and tumor micro-environment enhancing breast and ovarian cancer cell survival and tumor progression. Supported by Susan G. Komen for the Cure (KG111237) and NIH grant CA102310

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 1254. doi:1538-7445.AM2012-1254
Abstract 1257: Microarray analysis of BRONJ-like oral mucosal lesions in mice
Authors: Bae Susan , Williams Drake , Oh Ju Eun , Shi Songtao , Kang Mo , Shin Ki-Hyuk , Park No-Hee , Kim Reuben H.
Published: August 2012

Bisphosphonates (BPs) are potent anti-resorptive agents that are widely used to treat bone-associated diseases including metastatic bone cancer. Many reports have indicated that long-term use of BPs can develop BP-related osteonecrosis of the jaw (BRONJ) that occurs largely at the site of tooth extraction; however, the pathophysiology of BRONJ is still poorly understood. Clinically, BRONJ is defined as exposed necrotic bone with unhealed and open oral mucosa, implying that the lack of wound closure may be caused either directly by BPs or indirectly via biological signals (i.e., cytokines) from the BP-affected surrounding structures. We hypothesize that oral mucosal tissues lose their capacity to close the wound properly at the affected sites and that the genes involved in wound healing processes are differentially expressed between BRONJ lesions and the normal counterparts. To test our hypothesis, we utilized the established BRONJ mice model. Zometa was administered via tail vein 1 week prior to tooth extraction. After extracting the upper first molar, the extracted socket was allowed to heal, and the overlaying oral mucosal tissues were harvested after 2 weeks from control and BRONJ groups (n=3). The fresh tissue samples were subjected to RNA isolation, cDNA sysnthesis, and cDNA hybridization onto microarray chip (Affymetrix MG 430 2.0). The data analysis by functions using IPA software revealed that the genes involved in skeletal and muscular disorders, dermatological diseases, cellular movement, cellular assembly and organization, and inflammatory disease were significantly associated with BRONJ. In particular, we found that growth factors (i.e., Igf2, Fgf7, Pdf2bp2), collagens (i.e., Col1a1, Col1a2, Col2a1, Col2a2, Col5a1, Col5a2, Col11a1, and Col11a2), and non-collagenous proteins (i.e., DMP-1, ITBP, and SPP1) were significantly down-regulated in BRONJ groups. Taken together, our study suggests that the diminution of growth factors as well as collagenous and non-collagenous structural proteins may be associated with the development of BRONJ. This study was supported by NIDCR/NIH (R03DE021114 and K08DE017121).

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 1257. doi:1538-7445.AM2012-1257
Abstract 1270: Identification and functional analysis of Pim-1 kinase substrates in prostate cancer
Authors: Barkley Nicole , Li Xiang , Bieberich Charles
Published: August 2012

Protein kinases constitute one of the largest and most pleiotropic families of proteins. Mutations leading to activation of protein kinases and loss of function of phosphatases can have deleterious consequences that lead to disease. Pim-1, a serine/threonine kinase, is one of many kinases that may play a role in the etiology of malignant diseases including lymphoma and prostate cancer. Recent studies indicate Pim-1 is overexpressed in prostate cancer and is a highly plausible therapeutic target. Paradoxically, a decrease in Pim-1 expression in later stages of prostate cancer correlates with poor prognosis. Pim-1 has been known to be an oncogene for more than two decades, however, our understanding of how it functions is incomplete. A key underlying factor in this lack of knowledge is that, to date, fewer than fifteen Pim-1 substrates have been identified. The identification and validation of novel Pim-1 substrates could elucidate biomarkers of Pim-1 activity and provide insight into the molecular basis of Pim-1 function. A recent, innovative kinase-substrate profiling method termed the Reverse In-gel Kinase Assay (RIKA) reverses the role of kinase and substrate in a traditional in-gel kinase assay. The assay involves the partial immobilization of an active kinase in a denaturing polyacrylamide gel to determine which substrates from cell lysates can be phosphorylated by the kinase within the gel. The potential substrates are excised from the gel and identified using mass spectrometry. Using a Pim-1 RIKA approach, 40S Ribosomal Protein 7 (RPS7) was identified as a potential novel Pim-1 substrate. RPS7 was expressed in E.coli to conduct preliminary in vitro studies. These in vitro studies demonstrated that Pim-1 robustly phosphorylates RPS7. Further studies were performed to identify sites of phosphorylation on RPS7, and alanine-substitution mutants were generated that completely abolished phosphorylation of RPS7 by Pim-1. These data indicated that RPS7 may be a novel Pim-1 substrate. Studies to validate RPS7 as a direct target of Pim-1 in vivo, and to determine the functional consequences of Pim-1 phosphorylation of RPS7 are in progress.

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 1270. doi:1538-7445.AM2012-1270
Abstract 1271: Aberrant activation of Spleen Tyrosine Kinase in ovarian cancer identified through a global phosphorylation profiling of protein tyrosine kinases
Authors: Shoni Melina , Du Jinyan , Yang Junzheng , Ng Shu-Kay , Muto Michael George , Welch William , Crum Christopher , Berkowitz Ross , Golub Todd , Ng Shu-Wing
Published: August 2012

Aberrations in protein kinase activity and the consequent deregulation of downstream signaling pathways are considered a hallmark of oncogenesis. The purpose of this study was to investigate the phosphorylation state, the ultimate readout of dysregulated activity, of protein tyrosine kinases in ovarian tumors. An innovative Luminex multiplex platform was employed to systematically determine phosphorylation status of protein tyrosine kinases in a total of 69 protein lysates derived from normal human ovarian surface epithelial (HOSE) cells, ovarian tumors, and cancer cell lines. Significance Analysis of Microarrays (SAM) was performed to identify the signature of phosphorylated kinases with statistical significance. Profiling results were confirmed by using phosphor-specific kinase antibodies in immunoblot analysis of the profiled lysates and immunohistochemistry on formalin-fixed paraffin-embedded archived ovarian and fallopian tissues, as well as immunofluorescence of ovarian cancer cells growing as monolayer or 3D Matrigel cell cultures. A signature of twenty-two phosphorylated protein tyrosine kinases was identified by SAM analysis of the profiling data. Spleen Tyrosine Kinase (SYK), a novel tyrosine kinase included in the signature, was found significantly phosphorylated in lysates from ovarian cancer tissues compared to normal tissues by both Western blot analysis and immunohistochemistry on 94 clinical ovarian tissues (P=0.024). SYK phosphorylation was also identified in tubal intraepithelial carcinomas, the hypothesized precursors of high-grade serous ovarian carcinomas. However, SYK activation was not observed in ovarian cancer cell lines growing as attached monolayer cells, but was present when they grew as mouse xenografts or in 3D Matrigel in vitro cultures. We are the first to report that SYK, an extensively investigated tyrosine kinase in hematopoietic cells, is aberrantly phosphorylated and activated in ovarian cancer. SYK phosphorylation in ovarian cancer might be induced by extracellular stimuli that are exclusively present in the tumor microenvironment. These findings provide additional options in targeted therapy for ovarian cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1271. doi:1538-7445.AM2012-1271
Abstract 1284: Estrogen receptor alpha transcriptionally regulates p53 in MCF7 human breast cancer cells
Authors: Berger Crystal , Qian Yingjuan , Chen Xinbin
Published: August 2012

The p53 tumor suppressor plays a critical role in maintaining genome stability. In response to genotoxic stress, p53 induces target genes for cell cycle arrest, apoptosis, and DNA repair. While p53 is the most commonly mutated gene in all cancers, it is only mutated in 20% of breast cancers. Interestingly, 70% of all breast cancer cases are estrogen receptor (ER) positive and are associated with wild type p53 expression. An ER-positive breast cancer diagnosis generally indicates good patient prognosis and treatment responsiveness with anti-estrogens such as tamoxifen. The relationship between ERα and p53 remains poorly defined. Here, we found that ERα positively regulates p53 expression. Specifically, knockdown of ERα decreases the amount of p53 along with its downstream target p21, a regulator of cell cycle arrest, in MCF7 breast cancer cells. Furthermore, we found that ERα transcriptionally regulates p53 expression via binding to the p53 promoter. Luciferase promoter reporter experiments showed that two estrogen receptor element half sites located at nt −1828 and −1611 from the p53 transcriptional start site responded to addition of ERα with increased reporter activity. Mutation of those two sites resulted in no reporter activity. To determine the biological significance of ERα regulation of p53, we found that knockdown of ERα desensitized cells to growth arrest and cell death upon treatment with DNA damage-inducing drugs in a p53-dependent manner. Conversely, cells overexpressing ERα were sensitized to growth arrest and cell death upon DNA damage-inducing drugs via the p53 pathway. Together, these results provide an insight into the reciprocal regulation of p53 and ERα at the transcript level and how their pathways interact in ER-positive breast cancers under basal and stress conditions.

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 1284. doi:1538-7445.AM2012-1284
Abstract 1295: Regulation of PAX2 activity by estradiol in breast cancer cells
Authors: Beauchemin David , Lacombe Catherine , Themsche Céline Van
Published: August 2012

Most breast cancer deaths arise from tumour cells ability to produce distant metastases. Among breast tumour subtypes, luminal A tumours, which express the highest levels of estrogen receptor alpha, are associated with the poorest metastatic phenotype and with longest relapse-free survival. Usually active during embryogenesis, PAX2, a homeobox transcription factor, is of a particular interest in this context. It is preferentially activated in luminal A tumours in vivo and it was shown to downregulate the expression of ERBB2 (erythroblastic leukemia viral oncogene homolog 2, HER-2/neu), a well-documented proinvasive and pro-metastastic gene, in these cells in response to estradiol in vitro. However, the ability of estradiol to regulate PAX2 activity and ERBB2 expression in non-luminal breast cancer cells, and the intracellular mechanisms involved in estradiol-induced PAX2 activity in breast cancer cells, have not been investigated. Objective: The objective of our work was to compare the impact of estradiol exposure on PAX2 activation in luminal and non-luminal breast cancer cells, and to determine the molecular mechanisms mediating estradiol-induced activation in breast cancer cells. Hypothesis: Estradiol would induce PAX2 activity in breast cancer cells of the luminal subtype only, in an estradiol receptor alpha-dependent manner. Results: Basal PAX2 activity was higher in cell lines from luminal compared to non-luminal subtype. Activation of PAX2 by estradiol, as assessed by measuring PAX2 phosphorylation on serine 393 residue and ERBB2 expression, was selectively achieved in breast cancer cell lines of the luminal subtype. This process was blocked by estrogen receptor alpha antagonist ICI 182780. Conclusion: PAX2 activation by estradiol is selectively achieved in breast cancer cells of the luminal subtype, via ERα. We are currently investigating intracellular signaling pathways downstream of estrogen receptor alpha which could mediate estradiol-induced activation of PAX2 in luminal breast cancer 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 1295. doi:1538-7445.AM2012-1295