bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1006: Soluble epoxide hydrolase gene deficiency attenuates colitis-induced carcinogenesis in IL-10 knockout mice
Authors: Zhang Wanying , Liao Jie , Li Haonan , Dong Hua , Bai Han , Yang Allison Liao , Hammack Bruce D. , Yang Guang-Yu
Published: August 2012

Soluble epoxide hydrolase (sEH) can quickly inactivate epoxyeicosatrienoic acids (EETs) anti-inflammatory function via converting it to dihydroxyeicosatrienoic acid (DHETs). Inhibition of sEH has shown effects against inflammation. But little is studied about the effect of sEH on colitis-induced carcinogenesis. In this study, we determined the role of sEH gene deficiency in suppressing the development of colitis-induced tumor using chronic colitis mouse model in IL-10 knockout mice combined with sEH gene deficiency. Tumor formation in the bowel was examined at age of 25 weeks for male mice and 35 weeks for female mice. Our results showed that sEH (-/-)/IL-10 (-/-) mice exhibited a significant decrease of tumor multiplicity (2 tumor/mouse vs. 1 tumor/mouse) and tumor size (344.55±71.73 vs. 126.94±23.18 mm3, p=0.028) as compared to IL-10(-/-) mice. But no difference was observed for tumor incidence. Colitis-induced dysplasia was further identified by Ki67-labeled luminal surface epithelial cell proliferation combined with morphologic features and revealed that the number of dysplasia was significantly decreased in sEH (-/-)/IL-10 (-/-) mice. Histopathological analysis of inflammatory activity in the bowel showed the significantly lower inflammation scores, including inflammation-involved area (1.4±0.18 vs. 0.70±0.16, P=0.007), ulcer (2.15±0.18 vs. 1.55±0.35, P=0.032), and myeloperoxidase-labeled inflammatory cell infiltrates in sEH (-/-)/ IL-10(-/-) mice compared to IL-10(-/-) mice. qPCR and western blotting assays showed a downregulation of inflammatory cytokines and NF-kB signals. Analysis of eicsanoid acid metabolic profile confirmed sEH functional deficiency and showed a significant increase in the ratios of EETs/DHET and EpOME/DiOME in sEH (-/-)/IL-10 (-/-) mice compared to IL-10(-/-) mice. The results indicate that sEH gene plays an important role in the development of chronic colitis-induced carcinoma and could serve as a crucial target of cancer chemoprevention. (Supported by NIH R01 grant CA137467)

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 1006. doi:1538-7445.AM2012-1006
Abstract 1008: Retinoids and TRAIL target early stage intestinal polyps in ApcMin/+ mice
Authors: Davis Jennifer S. , Huang Shaoyi , Ren Xiaoyang , Wu Xiangwei
Published: August 2012

The purpose of this study is to determine the optimal timing of treatment for a novel chemoprevention approach in a mouse model of Familial Adenomatous Polyposis (FAP). FAP is an autosomal dominantly inherited disorder caused by mutations in the tumor suppressor Apc (adenomatous polyposis coli) gene, which results in the formation of hundreds to thousands of polyps, predominantly in the colon and rectum. Left untreated, these polyps will form colorectal adenocarcinomas. Although surgical removal of the colon eliminates the colon cancer risk, these individuals are also at increased risk for other primary cancers, including tumors of the small intestine and stomach. Systemic chemopreventive interventions to specifically remove the pre-cancerous cells in these individuals could greatly improve their outcomes and quality of life. Recently, we have developed a new method based on the ability of combined retinoid and TRAIL (tumor necrosis factor related apoptosis inducing ligand) treatment to induce apoptosis and regression of intestinal polyps in ApcMin/+ mice, a mouse model of FAP. Treatment of these mice with retinoid causes an increase in TRAIL death receptors DR4 and DR5 as well as a concomitant decrease in the decoy receptors. APC deficiency leads to a decreased level of the anti-apoptotic protein FLIP (flice-like inhibitory protein). Thus, the combination of TRAIL and retinoid specifically targets APC deficient cells for apoptosis. Following up on these findings, we have tested the hypothesis that early stage adenomas of the intestine will respond more readily to TRAIL and retinoid treatment than those at a later stage. We used ApcMin/+ mice at either two months of age or five-six months of age. Each group of mice was given three treatments of retinyl acetate and TRAIL within one week. Following the last treatment, the intestine was harvested from each mouse and the polyps were counted. The number of polyps in each group of mice was compared with the polyp number in age matched non-treated mice. Results: When treated at two months of age, ApcMin/+ exhibit a nearly 50% decrease in polyp number following only one week of therapy. Conversely, ApcMin/+ treated at five-six months of age did not show any significant difference in polyp number compared to non-treated mice. Conclusion: Earlier stage adenomas respond more readily to the combined treatment of retinoid and TRAIL. This is likely due to additional mutations acquired by the later stage polyps. The cause of this resistance is a current area of study.

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 1008. doi:1538-7445.AM2012-1008
Abstract 1021: HDM2 inhibition augments the antitumor and antiangiogenic effects of sunitinib by suppressing sunitinib-induced SDF-1 expression and tumor recruitment of CD11b/Gr1+ MDSC in RCC xenografts
Authors: Panka David J. , Liu Qinjun , Signoretti Sabina , Mier James W.
Published: August 2012

The VEGFR2 inhibitor sunitinib induces transient growth arrest of 786-0 RCC xenografts during which p53 as well as several p53-dependent genes (e.g. p21, HDM2, and collagen prolyl hydroxylase) are induced. Within a short period of time tumor growth resumes despite continued administration of the sunitinib. However, the development of resistance to sunitinib is associated with continued expression of p53 but the loss of p53 transcriptional activity as determined by the suppressed expression of the above target genes and the increased expression of the p53 transcriptional inhibitor hdmx. Recently, several small molecule antagonists of the p53 negative regulator hdm2 have been developed which stabilize p53 expression. One of these, MI-319 (Sanofi-Aventis), in addition to inhibiting hdm2 also inhibits hdmx. We have combined MI-319 with sunitinib in two separate xenograft model of RCC resistance. The addition of MI-319 prevents the development of resistance to sunitinib. The enhanced antitumor activity of the drug combination is associated with enhanced suppression of the vasculature and increased evidence of apoptosis relative to sunitinib alone. The addition of MI-319 to sunitinib reestablishes p53 activity in 786-0 and A498 xenografts as determined by p21 and hdm2 expression. Furthermore, whereas sunitinib alone results in increased expression of SDF-1 and the recruitment of CD11b, GR-1 myeloid suppressor cells, the addition of MI-319 to the sunitinib decreases expression of SDF-1 and suppresses the recruitment of the myeloid suppressor 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 1021. doi:1538-7445.AM2012-1021
Abstract 1047: Suppression of the chromatin remodeling protein CHD3 and platinum resistance in ovarian cancer
Authors: Cutter Noelle , Vigliotti Michele , Lum Elena , Wrzeszczynski Kazimierz O. , Varadan Vinay , Dimitrova Nevenka , Levine Douglas A. , Lucito Robert
Published: August 2012

Epithelial ovarian cancer is the leading cause of death from gynecological malignancies. Currently platinum-based chemotherapy, coupled with a taxane based drug is the primary treatment for ovarian cancer. Approximately 25% of patients either present with or rapidly develop resistance to platinum based chemotherapy and all recurrent tumors ultimately become resistant. Epigenetic modifications have been associated with tumor formation and progression and may contribute to therapy response. We hypothesize that Epigenetic changes such as DNA CpG methylation is in part responsible for the onset of chemoresistance of EOC. To identify epigenetically regulated genes associated with ovarian cancer chemotherapy resistance, a genome wide approach was used. For the most significant genes an in vitro culture system was developed to study platinum resistance. Candidate genes were screened by addition of shRNAs to model the transcriptional suppression caused by DNA methylation and genes that scored positive for increasing resistance were identified, one of them being the CHD3 gene. Here we show that loss of expression of CHD3, a member of the Mi-2/NuRD complex, causes increased resistance to platinum chemotherapy drugs. Additionally, ovarian cell lines transcriptionally silenced for CHD3 are more invasive, and have increased migratory ability. Recent evidence suggests molecular and phenotypic associations between chemo resistance and the acquisition of epithelial-mesenchymal transition of cancer cells. The transition of epithelial cell to a mesenchymal cell requires an alteration in morphology, cellular architecture, adhesion, and migration capacity. Cancer cells undergoing EMT can acquire invasive properties and enter the surrounding stroma, resulting in the creation of a favorable microenvironment for cancer progression and metastasis. Our results indicate that when CHD3 is silenced, cells undergo an EMT-like transition thereby allowing them to bypass apoptosis and resist platinum based therapy. Taken together, we provide the first evidence of a role for CHD3 as an important epigenetic regulator of chemoresistance in ovarian cancer and hypothesize EMT as one of the underlying mechanisms. Furthermore, CHD3 expression might represent a therapy response predictor and could be a future therapeutic target 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 1047. doi:1538-7445.AM2012-1047
Abstract 1053: DNMT3B7 alters E-cadherin expression and cell proliferation in breast cancer cells
Authors: Brambert Patrick R. , Rehbein Jennifer , Slezak Izabela , Kelpsch Daniel J. , Ostler Kelly R. , Godley Lucy A. , Raimondi Stacey L.
Published: August 2012

Breast cancer is the second-leading cause of cancer death among women in the United States and the progression from non-invasive to invasive/metastatic disease correlates with an increased risk of death. Changes in DNA methylation have been shown to be important in breast cancers. However, the mechanism by which cancer cells acquire and maintain abnormal methylation is not well understood. DNA methyltransferases (DNMTs) are responsible for monitoring and altering DNA methylation patterns in cells. Interestingly, we have identified many aberrant DNMT3B transcripts in cancer cells compared to normal cells. Therefore, we hypothesize that aberrant DNMT3B isoforms alter gene and protein expression to promote tumor progression in breast cancer cells. Our studies indicate that the breast cancer cell lines MCF-7 and MDA-MB-231 express many aberrant DNMT3B isoforms including DNMT3B7 and ΔDNMT3B6. Interestingly, the invasive MDA-MB-231 cells express increased levels of DNMT3B7 and ΔDNMT3B6 compared to the poorly invasive MCF-7 cells. To further our studies, poorly invasive MCF-7 cells were stably transfected with a DNMT3B7 expression construct to determine the role of this aberrant transcript in tumor progression. Preliminary data indicate that the increased expression of DNMT3B7 in these cells correlates with a significant increase in growth. Specifically, cells expressing more DNMT3B7 grow faster than those with lower levels of the aberrant protein. Furthermore, E-cadherin expression is inversely correlated with DNMT3B7 expression indicating that the expression of this aberrant protein may lead to a more aggressive phenotype. Finally, soft agar assays have been performed to determine the functional significance of these findings. The novel results presented in this study suggest the need for additional studies to determine the role of aberrant DNMT3B transcripts in cancer. Ultimately, this work may lead to a better understanding of breast cancer progression and the development of novel therapeutics.

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 1053. doi:1538-7445.AM2012-1053
Abstract 1054: The role of DNMT1 in the DNA damage response
Authors: Lee Gun Eui , Robertson Keith D.
Published: August 2012

DNA methyltransferases (DNMTs) are proteins reponsible for establishing and maintaining genome-wide patterns of DNA methylation, an epigenetic signature regulating gene expression and genome integrity. DNMT1 is regarded as the maintenance enzyme while DNMT3A/3B are involved in the de novo synthesis of DNA methylation pattern. Besides its known role in copying methylation during replication, DNMT1 has been reported to be involved in the DNA damage response by recruitment to damaged sites on DNA. However, the mechanism of how DNMT1 takes part in DNA damage repair (DDR) remains to be elucidated. Our previous study showed that DNMT1 is an early responder to DNA double-strand breaks and interacts with components of the DDR machinery (Ha, KS et al, 2011, Hum. Mol. Genet., 20 (1): 126-140). In order to further characterize the role of DNMT1 in DDR, we analyzed DNMT1 in cis and trans. In our previous study, we observed that DNMT1 is recruited to DNA damage sites rapidly and transiently. Since DNMT1 is present throughout the cell cycle, this rapid and transient localization suggests that, upon DNA damage, in order to localize on DNA damage sites, a fraction of the DNMT1 protein pool might be modified and/or its complement of binding partners transiently altered. To this end, we purified DNMT1 from mammalian cells before and after ionizing radiation and analyzed its post-translational modifications (PTMs) by mass spectrometry. DNMT1 shows multiple unique patterns of PTMs including acetylation and methylation before and after ionizing radiation. In order to characterize trans-acting partners of DNMT1 after DNA damage, we purified DNMT1 complexes after protein cross-linking and used mass spectrometry to determine the identity of interacting factors. Consistent with changes in the localization and PTM patterns on DNMT1 in the presence and absence of DNA damage, several known and novel DNMT1-protein interactions are also altered in cells with damaged DNA. Finally, in order to understand the role of DNMT1 in the DDR mechanism in a physiological context, we used siRNA to acutely reduce levels of DNMT1 in the presence and absence of DNA damage. Knock-down of DNMT1 reduced the activation of DDR signaling molecules such as ATM, CHK1, and γ-H2AX in a colon cancer cell line. Collectively, our results demonstrate that DNMT1 is a critical molecule involved in coordinating DNA damage signaling with DNA repair mechanisms.

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 1054. doi:1538-7445.AM2012-1054
Abstract 1077: Bufalin inhibited cell proliferation and expression of insulin-like growth factor 2 and type 1 receptor in human colorectal cancer cells
Authors: Zhang Yong , Feng Dian-Xu , Zhao Ronghua , Chen Chao , Wang Jie , Zuo Qing-Song , Chen Ya-Feng , Wang Xu , Chen Teng
Published: August 2012

Objective: To investigate the inhibitory effect of bufalin on the proliferation of human colorectal cancer cell, HCT116, and its relationship with expression of insulin-like growth factor 2 (IGF-2) and type 1 receptor (IGF1R). Methods: HCT116 cells were cultured with bufalin and cell proliferation was measured by MTT assay. The inhibitory rate of cell proliferation and the half maximal inhibitory concentration (IC50) were calculated. Cell cycle was determined by use of flow cytometry. mRNA and protein expressions of IGF-2 and IGF1R were analyzed by real time polymerase chain reaction (PCR) and Western Blot, respectively. The distribution of IGF-2 and IGF1R in cells was also observed by immunocytochemical staining. Results: The IC50 of bufalin at 24, 36, 48, 60, and 72 hours were 0.79±0.10, 0.37±0.10, 0.25±0.03, 0.17±0.02, and 0.10±0.03 uM, respectively. We selected 0.3 uM and 48 hours as the concentration of bufalin and culture time in following experiments. When cultured with bufalin, the ratio of cells in G2 phase to cells in M phase was increased in a dose dependent manner, indicating a delay from G2 to M caused by bufalin. For IGF-2, the mRNA and protein expressions were increased when cells were cultured with bufalin; For IGF1R, the mRNA increased but protein decreased. Both IGF-2 and IGF1R were seen in cytoplasm. Conclusion: Bufalin can significantly inhibit the proliferation of HCT116 cells, its underlying mechanism may relate with the up-regulation of IGF-2 expression and down-regulation of IGF1R protein 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 1077. doi:1538-7445.AM2012-1077
Abstract 1078: ErbB/Pi3K signaling contributes to EGFR ligand expression in colorectal cancer cells
Authors: Jacobs Bart , Tejpar Sabine
Published: August 2012

Background: Cellular expression of EGFR ligands is tightly regulated and is thought to be maintained by an autocrine signaling component in colorectal cancer cells. More importantly, the expression levels of individual ligands have gained recent interest since their expression of their mRNA was found to be linked to anti-EGFR therapy response. Since the expression of these ligands can be regulated by EGFR signaling elicited by EGF, we decided to investigate the contribution of MAPK as well as the PI3K axis, to EREG expression, in K-Ras wild-type colon cancer cells. Methods Colon cancer cells were used to interrogate EGFR downstream signaling. Several inhibitors targeting the ErbBs and the MAPK and PI3K/Akt pathways were used to dissect the pathways downstream of EGFR maintaining ligand mRNA levels in the lim1215 colon cancer cell line. Additionally, we used ER and AR reporter assays to selectively evaluate involvement of the EGFR pathway in transcriptional regulation of the pathway. Consequently, blockade and activation of EGFR pathway members was assessed by Western Blot. Results We show that Ereg and Areg mRNA levels are specifically maintained by erbb1 signaling. Treatment of lim1215 cells with inhibitors blocking Erbb1 (cetuximab) or ErbB1/Erbb2 (lapatinib) did results in near complete inhibition of Ereg and Areg expression in Kras/pi3K wild-type cells, while blockade of ErbB2 alone (which is activated in these cells) with Trastuzumab did not decrease Ereg nor Areg mRNA levels. Downstream of EGFR, we show that MEK signaling is not required for maintenance of EREG and AREG levels in lim1215 cells. Conversely, blockade of PI3K signaling by Akt inhibitors or PI3K inhibitors abolished EREG mRNA levels in these cells. This was reflected by the ability of overexpressed Akt to induce EREG and AREG reporter activity in 293T cells. In contrast, this activation could be rescued by the addition of Akt and PI3K inhibitors, but not MEK inhibitors in cellular media. Conclusions In this work we show that PI3K signaling is required for the transcription of EGFR ligands in colon cancer cells. In this regard, this provides additional insights to the recent findings that link PI3K/Akt signaling to Receptor Tyrosine Kinase expression and activation in cancer cells. Therefore, a global understanding of the signaling networks that control ligand availability and receptor abundance will be crucial in combining ErbB pathway inhibitors in the treatment of kras wild-type 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 1078. doi:1538-7445.AM2012-1078
Abstract 1189: Discovery of RPS25-MDM2/MDMX-p53 regulatory feedback loop
Authors: Zhang Xu , Wang Wei , Xu Wenrong , Wang Ming-hai , Wang Hui , Zhang Ruiwen
Published: August 2012

There is an increasing interest in elucidation of ribosomal proteins (RPs) in cancer development and progression. Ribosomal proteins have extraribosomal functions and participate in a surveillance network for cell growth, division, and apoptosis. Accumulating evidence suggests that the p53 protein, together with ribosomal proteins, takes part in a surveillance mechanism for genome integrity and protein translation, and is activated by nucleolar stress. We herein identified a regulatory role of RPS25 in p53-mediated cellular response to ribosomal stress. We demonstrated that RPS25 bound to MDM2 by using immunoprecipitation and GST pull-down assays. RPS25 inhibited the E3 ligase activity of MDM2, resulting in the stabilization and activation of p53. Overexpression of RPS25 induced cell cycle arrest and apoptosis through p53. The MDM2-binding and nucleolar location domains of RPS25 were critical for regulating p53 stability and activity. Knockdown of RPS25 by siRNA attenuated the activation of p53 induced by ribosomal stress. Furthermore, we found that MDMX was involved in the regulation of the E3 ligase activity of MDM2 by RPS25, suggesting that RPS25 modulates p53 in an MDM2 and MDMX-dependent manner. Finally, RPS25 was identified to be a transcriptional target of p53. Ribosomal stress-induced p53 activation inhibited RPS25 protein level, while knockdown of p53 restored RPS25 expression. In conclusion, these results demonstrate that RPS25 is a novel regulator of p53 in ribosomal stress and define a unique S25-MDM2/MDMX-p53 regulatory feedback loop in ribosomal stress. (This work was supported by NIH/NCI grants R01 CA112029 and R01 CA121211 and a Susan G Komen Foundation grant BCTR0707731.)

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 1189. doi:1538-7445.AM2012-1189
Abstract 1194: p53 negatively regulates the hepatoma growth factor HDGF
Authors: Tokino Takashi , Idogawa Masashi , Sasaki Yasushi
Published: August 2012

Tumor suppressor genes may exert a significant part of their activity by regulating autocrine and paracrine growth factor pathways. This study reveals how the transcriptional repression function of p53 mediates cancer cell growth and migration. Hepatoma-derived growth factor (HDGF) is a secreted heparin-binding growth factor that has been implicated in cancer development and progression. Here we report that HDGF is a critical target for transcriptional repression by the tumor suppressor p53. Endogenous HDGF expression was decreased in cancer cells with introduction of wild-type p53, which also downregulated HDGF expression after DNA damage. In support of the likelihood that HDGF is a critical driver of cancer cell growth, addition of neutralizing HDGF antibodies to culture media was sufficient to block cell growth, migration and invasion. Similarly, these effects were elicited by conditioned culture medium from p53-expressing cells and they could be reversed by the addition of recombinant human HDGF. Interestingly, we found that HDGF was overexpressed also in primary gastric, breast and lung cancer tissues harboring mutant p53 genes. Mechanistic investigations revealed that p53 repressed HDGF transcription by altering HDAC-dependent chromatin remodeling. Taken together, our results reveal a new pathway in which loss of p53 function contributes to the aggressive pathobiological potential of human cancers by elevating HDGF 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 1194. doi:1538-7445.AM2012-1194
Abstract 1203: Targeting FGFR4 in colorectal cancer cells
Authors: Heinzle Christine , Erdem Zeynep , Sonvilla Gudrun , Stättner Stefan , Karner Josef , Klimpfinger Martin , Grasl-Kraupp Bettina , Holzmann Klaus , Grusch Michael , Berger Walter , Marian Brigitte
Published: August 2012

Fibroblast growth factors (FGFs) and their receptors (FGFRs) are involved in the regulation of cell proliferation, differentiation and survival as well as tumor development and therefore constitute prominent targets for cancer therapy. Over-expression of FGFR4 has been described in various cancer types and was also related to tumor aggressiveness. Reports concerning the role of FGFR4 in colorectal cancer (CRC) are still under dispute. In our study we found FGFR4 expression to be significantly up-regulated in human CRC tissue compared to normal mucosa. CRC cell line models over-expressing FGFR4 were created by transfecting SW480, HCT116 and HT29 colon carcinoma cells with a FGFR4 expression vector. This resulted in an increase of cell proliferation and colony formation under standard tissue culture conditions and colony outgrowth in soft agar was stimulated. In xenografted SCID mice tumor growth was significantly increased by FGFR4 over-expression. siRNA mediated knock down of FGFR4 in the high FGFR4 expressing cell lines HCT116 and HT29 resulted in decreased cell proliferation, viability and colony outgrowth in 2-dimensional growth assays. Additionally a dominant negative FGFR4 adenoviral construct inhibited 3-dimensional anchorage independent growth and suppressed tumor growth in vivo almost completely. Ongoing experiments analyze the effects of a soluble FGFR4 variant on 2-dimensional and 3-dimensional growth. FGFR4 up-regulation resulted in activation of survival pathways via FRS2 and PI3K leading to phosphorylation of GSK3β and S6. Signaling effects by blockage of FGFR4 dependent signaling via siRNA and the dominant negative adenoviral construct are currently being investigated. Based on our results we identified the FGFR4 as an important oncogene in the carcinogenesis which is involved in the tumor growth regulation of CRC in vitro and in vivo. Consequently FGFR4 should be regarded as a new therapeutic target in CRC.

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 1203. doi:1538-7445.AM2012-1203
Abstract 1217: Detection of HER2 expression and phosphorylation in breast cancer CTC samples by CEER™
Authors: Hazra Saswati , Liu Limin , Webster Edvelyn , Fithian Andrew , Lee Tani , Magonova Katya , Barham Robert , Kim Phillip , Singh Sharat
Published: August 2012

Survival rates of metastatic breast cancers (mBCAs) are considerably low. Often, tumor cells at the primary site may not reflect the profile of the tumor cell population in recurrent disease. Circulating tumor cells (CTCs) isolated from peripheral blood offer a non-invasive disease monitoring modality. Identification of reliable molecular markers within (CTCs) from patients with recurrent disease may further improve breast cancer survival. Hence, we have developed a multiplexed immunoassay for monitoring activated HER2 protein and their expression levels using a multiplexed immuno-microarray platform. Collaborative Enzyme Enhanced Reactive-immunoassay (CEER™) was utilized to analyze HER2 profiles in CTCs isolated from 76 BCA patients (stages III to IV) with HER2 negative primary disease. The CEER technology utilizes the formation of a unique immuno-complex requiring co-localization of two detector antibodies around capture antibodies immobilized on immuno-array to profile HER2 protein expression and activation. The collaboration between two channeling-enzymes conjugated on two detection antibodies in proximity, enables the profiling of the target proteins with extreme sensitivity and specificity. Approximately 25% of HER2 negative BCA patients in this cohort showed varying levels of HER2 activation in CTCs isolated from the recurrent disease. About 8% of patients with HER2 activation in CTCs also showed significant HER2 over-expression. This suggests mechanisms of HER2 activation either through formation of receptor heterodimer (in samples without HER2 over-expression) or homodimer (in samples with HER2 overexpression) formation. The distinct HER2 discordance between primary tumor and recurrent disease demonstrates an urgent need for routine monitoring of HER2 status in CTCs found in mBCA patients. Incidence of HER2 alterations in CTCs should be considered in selecting effective treatment regimens for BCA patients with relapsed disease. Furthermore, CEER can be used for profiling other druggable targets for guiding effective clinical strategies including rational targeted agents.

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 1217. doi:1538-7445.AM2012-1217
Abstract 1223: Relief of feedback inhibition of RTK and RAS signaling in V600E BRAF melanomas exposed to RAF inhibitors buffers their effects on signaling
Authors: Pratilas Christine A. , Joseph Eric W. , Tadi Madhavi , Poulikakos Poulikos I. , Gabay Maytal , Lito Piro , Wong Wai , DeStanchina Elisa , Chandarlapaty Sarat , Rosen Neal
Published: August 2012

Activation of ERK signaling causes feedback inhibition of receptor tyrosine kinase and RAS signaling by multiple mechanisms and limits the steady-state output of the pathway. The V600E BRAF mutant does not depend on RAS-dependent dimerization and is not susceptible to feedback inhibition of RAS. In V600E BRAF melanomas, ERK pathway output and the expression of ERK genes such as DUSP6 and members of the SPROUTY family are markedly elevated. In these tumors, RTK signaling and RAS activation are actively suppressed. In the context of elevated DUSP6, RAF inhibitors rapidly inhibit ERK phosphorylation and output. This relieves the feedback inhibition of various RTKs and induces RAS activity and the formation of activated wild type RAF dimers that are insensitive to RAF-inhibitors. This results in a rebound in ERK phosphorylation and output that is sensitive to MEK, but not RAF inhibitors and suggests that the latter cannot maximally inhibit ERK signaling in these tumors. In support of this idea, combined treatment with MEK and RAF inhibitors reduces ERK output and the growth of V600E BRAF melanoma xenografts to a greater degree than either alone.

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 1223. doi:1538-7445.AM2012-1223
Abstract 1227: Modulation of EGF-receptors signaling by small peptidomimetic molecules in breast cancer cells
Authors: Banappagari Sashikanth , Satyanarayanajois Seetharama , Corti Miriam , Pincus Seth
Published: August 2012

Protein-protein interactions play a crucial role in regulating many biological processes as well as in disease pathology. Understanding the structural basis and modulating protein-protein interactions has a tremendous impact in developing new therapeutic strategies for many human diseases. Human epidermal growth factor receptor family of proteins (EGFR, HER2, 3 and 4) plays an important role in cell growth and proliferation. Aberrant interactions of EGFR family of proteins lead to tumor growth and metastasis. HER2, a member of EGFR family of proteins is overexpressed in approximately 25% of breast cancers. Deregulation of HER2 signaling pathways and overexpression of HER2 is known to occur in many cancers. HER2 is known to form heterodimers and is a major therapeutic target in breast cancer treatment. Formation of HER2 receptor heterodimerization with other EGFR family of proteins such as EGFR, HER3 is essential for intracellular kinase activation and is the basis for exploring direct inhibition of EGFR activation by blocking dimerization for therapeutic purposes. We have designed several small peptidomimetics to inhibit HER2-mediated signaling for cell growth. One of such peptidomimetics, Compound 5 (Arg-[3-amino-naphthyl-propionic acid]-Phe) designed, exhibited antiproliferative activity with IC50 values in the nM range against HER2 overexpressing breast cancer cell lines, SKBR-3 and BT-474. Binding studies using Florescence Microscopy, ELISA, Circular Dichroism (CD) and Surface Plasmon Resonance (SPR) confirmed that compound 5 binds specifically to HER2-extracellular domain. SPR studies revealed that compound 5 specifically binds to domain IV of HER2 extracellular region. Also, Compound 5 inhibited HER2-HER3 as well as HER2-EGFR heterodimerization evaluated by SPR. Pathhunter and proximity ligation assays confirmed the inhibition of HER2 heterodimerization with EGFR and HER3 by compound 5. Western blots revealed that compound 5 blocks the transphosphorylation of HER2 protein. Based on these observations small peptidomimetic inhibitor molecules can be potential therapeutic agents in disabling HER2 mediated heterodimerization.

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 1227. doi:1538-7445.AM2012-1227
Abstract 1235: p120ctn is a key effector of Ras-PKC∈-mediated oncogenic signaling
Authors: Dann Stephen G. , Golas Jonathon , Miranda Miriam , Shi Celine , Upeslacis Eric , Rosfjord Edward , Jin Guixian , Wu Jiang , Klippel Anke
Published: August 2012

Within the family of protein kinase C (PKC) molecules only the novel isoform member PRKCE (PKC∈) acts as a bona fide oncogene in in-vitro and in-vivo models of tumorigenesis. Previous studies have reported cancer-specific misexpression of PKC∈ at levels well above that of normal adjacent tissue in breast, prostate and lung tumors. We find that oncogenic Ras signaling promotes PKC∈ expression and results in hyperphosphorylation of CTNND1/p120-catenin (p120ctn). In this context, loss of PKC∈ by genetic or pharmacological means results in normalization of morphology and signaling responses. In a KRasD13-dependent breast cancer model loss of PKC∈ function results in growth inhibition in 2-dimensional (2D) and 3-dimensional (3D) culture systems as well as in orthotopic xenografts concomitant with the normalization of a subset of Ras-induced signaling responses. Using phospho-proteomic profiling analysis we observe that CTNND1 (p120ctn) phosphorylation at serine 268 (S268) occurs in a strictly PKC∈ dependent manner. Treatment with a specific PKC∈ inhibitor, PF-5263555, recapitulates the genetic loss of function phenotype and interferes with breast cancer cell growth in-vitro and in-vivo. We also show that PKC∈-mediated phosphorylation at S268 further stabilizes additional p120ctn phosphorylation sites and total protein levels of β-catenin. We demonstrate that p120ctn phosphorylation at S268 represents a specific readout for PKC∈ activity and as such can serve as a suitable biomarker for PKC∈ dysregulation in human cancer and for monitoring therapeutic response.

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 1235. doi:1538-7445.AM2012-1235
Abstract 1237: TGF-β signaling pathway analysis using MILLIPLEX MAP TGF-β multiplex panel
Authors: Johnston Randy , Warmke Tim , Wiese Rick , Hayes David , Hwang Joseph
Published: August 2012

Transforming growth factor beta (TGF-β) is a secreted protein that controls cellular proliferation, differentiation, apoptosis, migration, adhesion, immune response, and other functions in most cells. The binding of TGF-β to its receptor, TGFβRII, can lead to activation of both the SMAD-dependent canonical pathway as well as the SMAD-independent non-canonical pathways (such as PI3K/Akt, ERK, JNK, and p38). The TGF-β signaling pathway is coordinately regulated by phosphorylation and understanding the role of this pathway in normal physiological processes and in diseases such as cancer and diabetes requires the ability to simultaneously measure phosphorylation status of multiple protein targets. In recent years, bead-based assays, such as those using Luminex xMAP technology, have enabled the measurement of phosphorylation levels of up to 12 proteins simultaneously. Using the TGF-β Signaling Pathway multiplex panel, we observed increased levels of phosphorylated Smad2 and Smad3, but not phosphorylated ERK or Akt in TGF-β treated cells when compared to their unstimulated counterparts. Modifications in phosphorylation levels were confirmed by the treatment of cells with specific inhibitors against TGFβRI, which blocked phosphorylation of Smad2 and Smad3 in TGF-β treated HepG2 cells. Furthermore, significant increase in phosphorylated Smad3 was observed in human breast cancer versus matched breast normal tissues. The TGF-β Signaling Pathway multiplex panel allowed for simultaneous detection of multiple analytes in both the SMAD-dependent canonical pathway as well as the SMAD-independent non-canonical pathways in a specific, sensitive, and reproducible manner.

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 1237. doi:1538-7445.AM2012-1237
Abstract 124: Hypoxia-inducible miR-210 is involved in hypoxia-induced cell migration and invasion in human hepatocellular carcinoma
Authors: Kai Alan KL , Lo Regina CL , Lee Joyce MF , Wong Jack CM , Ng Irene OL
Published: August 2012

MicroRNA dysregulation is implicated in many human malignancies, yet little is known regarding the regulatory mechanisms of these small noncoding RNAs. Hypoxia is a prevalent tumor microenvironment in hepatocellular carcinoma (HCC) and plays a key role in modulating tumor aggressiveness. In the present study, we investigated the effects of hypoxia on microRNA expression in human HCC. First, we examined the miRNA expression profiles with TaqMan human microRNA Low-Density Array on HCC cell lines put under normoxic and hypoxic conditions to identify expression changes of microRNA. From the 664 microRNAs examined, miR-210 was overexpressed in SMMC-7721, Huh-7 and MHCC-97L HCC cell lines under 0.1% O2 hypoxic condition. The hypoxic induction of miR-210 was further validated in another two HCC cell lines (Hep3B and PLC/PRF/5) by individual real-time quantitative PCR (qPCR) and the same expression pattern was observed, suggesting that miR-210 is consistently induced in HCC cells under hypoxic conditions. The abundance of mature human miR-210 was also examined by qPCR in a human HCC cohort consisting of 48 pairs of primary HCC tissues and their corresponding non-tumorous livers. We found that miR-210 expression was significantly higher in the HCC tissues as compared with the corresponding non-tumorous livers (P=0.0003). Furthermore, we used the hypoxia marker carbonic anhydrase IX (CAIX) coupled with microdissection technique to isolate tissue samples in the peri-necrotic areas, which are considered pathologically hypoxic, in clinical formalin-fixed paraffin-embedded (FFPE) HCC samples. We extracted the microRNA from the tissues and compared the miR-210 expression with those from histologically preserved HCC areas away from necrotic areas. In line with the in vitro observations, with qPCR, miR-210 was remarkably overexpressed in hypoxic HCC regions. Furthermore, miR-210 overexpression significantly and inversely correlated with the overall survival and disease-free survival rates (P= 0.029 and 0.043, respectively) of HCC patients, indicating that overexpression of miR-210 in HCC was associated with a poorer prognosis. The biological function of the miR-210 induction in HCC was also investigated. Notably, with transwell and cell invasion assays, we demonstrated that the cell migratory and invasive abilities of SMMC-7721 were dramatically enhanced during hypoxia, and this enhancement was abolished upon miR-210 suppression using locked nucleic acid (LNA) inhibitor. Taken together, our present data indicate that miR-210 is a hypoxia-inducible microRNA in HCC, which may enhance HCC aggressiveness under hypoxic 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 124. doi:1538-7445.AM2012-124
Abstract 1241: In vivo analysis of the role of Lyn tyrosine kinase in castrate-resistant LNCaP xenograft tumor growth
Authors: Zardan Anousheh , Cox Michael , Gleave Martin , Zoubeidi Amina
Published: August 2012

Introduction: It is proposed that advanced Prostate Cancer (PCa) tumors develop mechanisms, which re-activate the androgen receptor (AR) axis via oncogenic pathways, in which tyrosine kinases play a crucial role. Genetic manipulation of several tyrosine kinases in vivo revealed that only a knockout of Lyn tyrosine Kinase, compromised prostate gland development suggesting that Lyn plays a critical role in the regulation of AR in normal prostate development. Our results demonstrated that Lyn kinase enhances AR transcriptional activity and promotes prostate cancer cell survival. Thus, we hypothesized that Lyn kinase overexpression accelerates castrate-resistant LNCaP tumor growth and serum PSA relapse to pre-castration levels. Method: LNCaP cells stably overexpressing Lyn kinase (LNCaPLyn) or Empty vector (LNCapEmpty) were generated using clonal selection. 1 × 106 LNCaPLyn and LNCaPEmpty were injected into the male nude mice. Serum PSA level and tumor volumes were followed weekly and mice were castrated when serum PSA values reached 50ng/ml. Results: Interestingly, a higher rate of tumor growth and PSA relapse was observed after casteration in mice bearing the LNCaPLyn compare to the LNCapEmpty-bearing mice. Total protein was extracted from the xenograft tumors and levels of AR, PSA, FKBP52, AKT, pAKT were analyzed. Results of Protein analysis further confirmed the role of Lyn kinase in prostate cancer cell survival and progression to castrate resistant stage. Conclusion: Together, these results suggest that Lyn plays a key role in CRPC development and that inhibiting Lyn expression could be considered as a potential therapeutic target for CRPC.

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 1241. doi:1538-7445.AM2012-1241
Abstract 1262: Simple western and NanoPro analysis of key EGF signaling cascade proteins
Authors: Nguyen Uyen , Dermody Jessica , Ramirez Francisco , Kazakova Irina , Yang Tom , Gavin Robert , Boge Annegret
Published: August 2012

Aberrant expression and signaling in the EGF signaling pathway is a common occurrence in a variety of cancers including breast cancer. Understanding how EGF signaling impacts disease progression is key to the development of novel therapeutics. Detection of important protein targets in cancer samples frequently employs Western blot analysis. In-depth phosphorylation analysis often requires 2D gels which are extremely variable and labor intensive, followed by MS analysis. Each of these methods have their advantages and challenges. This study employs the use of two novel nano-scale capillary-based electrophoresis and immunodetection technologies to characterize and validate subtle changes in key protein targets involved in the regulation of disease progression. The Simon system performs size-based separation of proteins, known as Simple Western assays. Alternatively, NanoPro systems utilize charge-based separation via isoelectric focusing (IEF). Both methods utilize capillary electrophoresis (CE) to perform the separation events. Both Simon and NanoPro instruments provide automated workflow eliminating the need for manual processing of multiple steps traditionally needed to carry out either of these separation and detection processes. Proteins from a single sample preparation were analyzed on two platforms: either in a native conformation using charge-based separation (NanoPro assays) or in a denatured state followed by size-based separation (Simple Western assays). Both methods incorporate immunodetection to monitor both subtle and specific changes to key signaling protein targets. Utilizing both platforms allowed for detailed analysis of post-translational modifications (charge-based) and protein abundance (size-based) within a prepared sample. As a validation of synergy between the platforms, we investigated key members of the EGF signaling cascade in different phosphorylation states. We showed that the same cellular lysate preparation can be used for charge- or size-based separation. Advantages of Simple Western and NanoPro assays over gels (1D and 2D) and traditional Westerns include ease of use, minimal user intervention, automatic analysis and excellent reproducibility which ultimate results in better data quality to characterize key regulatory events involved with cancer progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1262. doi:1538-7445.AM2012-1262