bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 703: Caffeine modulates the cell cycle in cancer cells to enhance cisplatin efficacy.
Authors: Miwa Shinji , Yano Shuya , Hiroshima Yukihiko , Tome Yasunori , Uehara Fuminori , Mii Sumiyuki , Kimura Hiroaki , Hayashi Katsuro , Tsuchiya Hiroyuki , Digman Michelle , Gratton Enrico , Hoffman Robert M.
Published: August 2014

Caffeine has been shown to enhance the efficacy of chemotherapy, but the mechanism is not well understood. In this study, we investigated cell-cycle modulation induced by caffeine in combination with cisplatinum treatment of human cancer cells. Proliferation inhibition was determined with a clonogenic assay. Mitotic and apoptotic changes were observed by imaging of 143B dual-color cells, in which GFP is expressed in the nucleus and RFP in the cytoplasm. Modulation of the cell-cycle in combination with cisplatinum was observed using time-lapse imaging analyses of HeLa cells expressing a fluorescent cell-cycle indicator, FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator). The clonogenic assay showed that caffeine increased the efficacy of cisplatin on inhibition of cell proliferation. Dual-color imaging demonstrated that cisplatin decreased mitosis and induced apoptosis in 143B cells. The combination of cisplatin and caffeine reduced the decrease of mitosis and increased apoptosis. Time-lapse FUCCI imaging showed that cisplatin strongly induced cell-cycle arrest at the S/G2/M phases in HeLa cells. Caffeine released the cell-cycle arrest induced by cisplatin, resulting in increased cell-killing efficacy. These data indicates that caffeine can effectively modulates the cell-cycle in cancer cells treated with cisplatin and possibly other drugs.

Citation Format: Shinji Miwa, Shuya Yano, Yukihiko Hiroshima, Yasunori Tome, Fuminori Uehara, Sumiyuki Mii, Hiroaki Kimura, Katsuro Hayashi, Hiroyuki Tsuchiya, Michelle Digman, Enrico Gratton, Robert M. Hoffman. Caffeine modulates the cell cycle in cancer cells to enhance cisplatin efficacy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 703. doi:10.1158/1538-7445.AM2013-703
Abstract 711: Small molecule compounds that target cell division cycle 7 (Cdc7) kinase inhibit cell proliferation and tumor growth.
Authors: Bailis Julie , Fang Li , Orf Jessica , Heller Scott , Bush Tammy , Bourbeau Matthew , Escobar Sonia , Frohn Michael , Harrington Paul , Hsieh Faye , Pickrell Alexander , Sham Kelvin , Siegmund Aaron , Tan Helming , Zalameda Leeanne , Allen John , Wickramasinghe Dineli
Published: August 2014

Cdc7 is an essential, serine/threonine protein kinase that activates the initiation of DNA synthesis at replication origins. Cdc7 also promotes cell cycle checkpoint activation in response to replication stress. As a key regulator of S phase entry and progression, Cdc7 kinase is a potential target for cancer therapy, with a distinct mechanism of action from known drugs that inhibit DNA replication. Following a high throughput screen for inhibitors of Cdc7 kinase activity, we investigated structure-activity relationships of azole-based compounds and optimized the compounds for potency and pharmacokinetic properties. Here we present the characterization of one of these compounds as a potent, selective, bioavailable Cdc7 kinase inhibitor. In cells, Cdc7 inhibition decreases MCM2 phosphorylation and DNA synthesis, causes DNA damage, and slows S phase progression. Cdc7 inhibition also induces chromosome missegregation leading to cell lethality in vitro and tumor growth inhibition in vivo. Cdc7 inhibition provides a new approach to target cancers, either as a single agent or in combination with chemotherapy.

Citation Format: Julie Bailis, Li Fang, Jessica Orf, Scott Heller, Tammy Bush, Matthew Bourbeau, Sonia Escobar, Michael Frohn, Paul Harrington, Faye Hsieh, Alexander Pickrell, Kelvin Sham, Aaron Siegmund, Helming Tan, Leeanne Zalameda, John Allen, Dineli Wickramasinghe. Small molecule compounds that target cell division cycle 7 (Cdc7) kinase inhibit cell proliferation and tumor growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 711. doi:10.1158/1538-7445.AM2013-711
Abstract 721: Multiplexed systems pathology for in-depth analysis of the tumor microenvironment: a strong correlation between pAkt and androgen receptor in the epithelial component of prostate cancer.
Authors: Blom Sami , Mäki-Teeri Petra , Mirtti Tuomas , Rannikko Antti , Säilä Jenni , Pellinen Teijo , Kallioniemi Olli
Published: August 2014

It is important to understand the heterogeneity across the cancer tissue as well as the spatial interactions and signaling between the different components that constitute the tumor microenvironment; epithelium, stroma, inflammatory cells, etc. The state of the tumor microenvironment and its variability within and in between tumors may give important insights on cancer initiation and progression, diagnostics, prognostics and therapeutic assessment. Thus, there is a major need to assess multiple molecular targets simultaneously, quantitatively, and within the specific tumor sub-compartments. Here, we demonstrate a systems pathology approach for quantitative detection of multiple proteins in formalin-fixed paraffin-embedded patient tissue samples using a combination of immunohistochemistry and high-throughput fluorescence imaging.

The study cohort consisted of 50 prostate cancer tissue samples from 25 patients exhibiting different disease stages in a tissue microarray (TMA) format. We developed a multiplexed assay for detecting pAkt, AR, and cytokeratin 8 (CK8) simultaneously using fluorescent nanoparticle labeling and high-throughput quantitative imaging. Fluorescent signals of pAkt and AR were automatically quantified within the segmented CK8-positive prostate epithelium. The stromal compartment and the non-malignant prostate tissue were analysed separately.

Our results showed that the expression of pAkt and AR correlated strongly in the prostate cancer epithelium (Pearson correlation coefficient 0.89, p < 0.001). This suggests that the androgen-dependent signaling is strongly associated with the pAkt signaling in cancer, with potential ramifications for administrating and monitoring anti-androgens and/or Akt-inhibitor therapy. The systems pathology approach enables quantitative in situ multianalyte detection for any key cancer signaling proteins. The analyses can be readily expanded to TMAs of large prospective or clinical trial cohorts with automated imaging. The ability to quantitatively study the spatial arrangement of the tumor microenvironment could have decisive advantages and improve the diagnostic and therapeutic predictions.

Citation Format: Sami Blom, Petra Mäki-Teeri, Tuomas Mirtti, Antti Rannikko, Jenni Säilä, Teijo Pellinen, Olli Kallioniemi. Multiplexed systems pathology for in-depth analysis of the tumor microenvironment: a strong correlation between pAkt and androgen receptor in the epithelial component of prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 721. doi:10.1158/1538-7445.AM2013-721
Abstract 726: Fluorophore-conjugated antibodies improve surgical resection of pancreatic cancer leading to prolonged disease-free survival and overall survival in orthotopic mouse models.
Authors: Metildi Cristina A. , Kaushal Sharmeela , Talamini Mark A. , Luiken George A. , Hoffman Robert M. , Bouvet Michael
Published: August 2014

We established orthotopic mouse models of human pancreatic cancer with fragments of BxPC-3-RFP tumor. Two weeks after implantation, mice were randomized to FGS or bright-field surgery (BS). FGS was performed 24 hrs after tail vein injection of anti-CEA-Alexa 488. Completeness of resection was assessed from pre- and postoperative images obtained with the OV-100 Small Animal Imaging System. Whole body images of mice were obtained postoperatively to assess for recurrence and mice were followed until premorbid to measure survival. We achieved a complete resection of pancreatic cancers in 92% of mice in the FGS group as compared to 58% in the BS group (p=0.002). Preoperative tumor burden was not significantly different between groups (p=0.424). However, FGS afforded a significantly smaller mean postoperative tumor burden compared to BS: 0.0004 ± SE 0.0003 mm2 vs. 0.0841 ± SE 0.027 mm2, p=0.004. On average, mice in the FGS experienced a greater reduction in tumor burden: 99.99% vs 97.72%, p=0.01. FGS reduced recurrence rates from 95% to 50% (p=0.002) and lengthened mean disease-free survival from 7 weeks to 30.5 weeks (p<0.001). The FGS group experienced longer overall survival compared to the BS group: 16.5 weeks vs. 37 weeks (p=0.001). Cure rate was improved with FGS from 5% to 50% (p=0.003). Furthermore, FGS more than doubled the one-year postoperative survival rate from 3 mice in the BLS group to 8 mice in the FGS group (p=0.005). This novel approach has potential to improve outcomes in the surgical treatment of pancreatic cancer.

Survival and recurrence according to surgical mode. Surgical Mode Disease-Free Survival Overall Survival Recurrence Rate Bright-Field Surgery 7 weeks 16.5 weeks 95% Fluorescence-Guided Surgery 30.5 weeks 37 weeks 50%

Citation Format: Cristina A. Metildi, Sharmeela Kaushal, Mark A. Talamini, George A. Luiken, Robert M. Hoffman, Michael Bouvet. Fluorophore-conjugated antibodies improve surgical resection of pancreatic cancer leading to prolonged disease-free survival and overall survival in orthotopic mouse models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 726. doi:10.1158/1538-7445.AM2013-726
Abstract 738: Neutrophil tracking study on mammary tumors using bioluminescence imaging in vivo.
Authors: Alshetaiwi Hamad , Balivada Sivasai , Pyle Marla , Shrestha Tej B. , Bossmann Stefan H. , Troyer Deryl L.
Published: August 2014

Polymorphonuclear neutrophils (PMNs) are the most abundant circulating blood leukocytes. They are part of the innate immune system and provide a first line of defense by migrating toward areas of inflammation in response to chemical signals that have been released from the site. Like inflammation, cancer also causes activation of PMNs and release of myeloperoxidase. In this study, we demonstrate that giving a small amount of luminol to mice that have been transplanted with 4T1 mammary tumor cells allows detection of myeloperoxidase activity. Luminol allowed detection of activation of PMNs only two days after cancer cell transplantation, even though tumors were not yet palpable. In conclusion, luminol-bioluminescence imaging (BLI) can provide a way to image certain tumors at an early stage.

Citation Format: Hamad Alshetaiwi, Sivasai Balivada, Marla Pyle, Tej B. Shrestha, Stefan H. Bossmann, Deryl L. Troyer. Neutrophil tracking study on mammary tumors using bioluminescence imaging in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 738. doi:10.1158/1538-7445.AM2013-738
Abstract 740: Investigating the role of epigenetic mechanisms upon aberrant signaling network activation.
Authors: Nabet Behnam , Braun Rosemary , Popovic Relja , Licht Jonathan D.
Published: August 2014

Growth factor (GF) stimulation of quiescent cells activates critical receptor tyrosine kinase (RTK) signaling pathways as well as waves of downstream gene expression to drive cell proliferation, survival, and migration. Aberrant activation of RTK signaling networks is a root cause of the uncontrolled cell growth and cell survival seen in cancer. In this study, we aim to uncover the role of epigenetic mechanisms in controlling the dynamic gene expression changes that occur as a cell is activated from a quiescent state, in normal and oncogenic cellular contexts. In order to compare controlled RTK signaling with hyperactive RTK signaling cellular states, we created a cell culture system to modulate the duration and intensity of RTK activation. We genetically deleted the Sprouty (Spry) genes, a family of negative feedback inhibitors of RTK signaling that function as putative tumor suppressors. The Spry deficient cells are hypersensitive to several GFs and show significantly enhanced and prolonged activation of several critical RTK signaling pathways, including the Ras-MAPK pathway. Additionally, the Spry deficient cells possess accelerated cell growth and increased tumorigenic potential compared to their wild-type counterparts. To determine whether this deregulated signaling results in aberrant gene expression, quiescent Spry wild-type and deficient cells were stimulated with GFs, and microarrays were used to profile changes in gene expression over time. Deregulated RTK signaling due to the absence of Spry leads to abnormal expression of a large subset of genes. Specifically, many genes display display not only changes in the level of expression, but aberrant patterns of response to GF stimulation. We determined that genes with deregulated activation in Spry deficient cells display significant differences in histone tail modifications at promoter regions and distal regulatory elements. Interestingly, these changes are evident in the quiescent cellular state as well as upon GF-mediated RTK network activation. Our work suggests that dynamic changes in a cell's epigenetic landscape contribute to the deregulated activation of genes upon hyperactive RTK signaling. Our study will provide a comprehensive view of how epigenetic mechanisms contribute to aberrant gene expression in oncogenic cellular contexts.

Citation Format: Behnam Nabet, Rosemary Braun, Relja Popovic, Jonathan D. Licht. Investigating the role of epigenetic mechanisms upon aberrant signaling network activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 740. doi:10.1158/1538-7445.AM2013-740
Abstract 741: CpG methylation abrogates KLF2-mediated transcriptional repression of telomerase in human T cells.
Authors: Hara Toshifumi , Yoshita Manami , Nakamura Masataka
Published: August 2014

Many genes in tumor cells are DNA-methylated and transcriptionally repressed. Constitutive expression of the limiting catalytic subunit of telomerase, hTERT, is one of the common signatures of tumor cells, even though its promoter is DNA-methylated. On the other hand, in normal somatic cells the promoter is unmethylated and transcription of hTERT is strictly regulated. In this study, we examined transcriptional regulation of the hTERT gene in normal and tumor cells. We identified a novel promoter element, which bound the zinc-finger transcription factor Krüpple-like factor 2 (KLF2) in normal human T Lymphocytes. The binding of KLF2 to the hTERT promoter element paralleled repression of hTERT transcription in resting normal T cells. T cell growth induced KLF2 release from the element and hTERT transcription. Forced reduction of KLF2 expression by siRNA in normal resting T cells increased the level of endogenous hTERT mRNA. The hTERT promoter is methylated in the KLF2-positive human T leukemic cell line, Kit 225, and KLF2-negative human T leukemic cell line, Jurkat. Treatment with the DNA methyltransferase inhibitor, Zebularine, reduced levels of CpG methylation in the hTERT promoter, resulting in downregulation of hTERT mRNA expression in KLF2-positive Kit 225 cells, while the Zebularine treatment did not cause any appreciable effects on hTERT expression in KLF2-negative Jurkat cells. Methylation of CpG sequences in endogenous and exogenous hTERT promoter elements inhibited the KLF2 binding to the element. Furthermore, KLF2 binding to the promoter is associated with histone H3 repressive marks, indicating KLF2-mediated epigenetic silencing of the hTERT promoter. Our results suggest that KLF2 partly controls strict transcriptional regulation of the hTERT gene through association and dissociation with the promoter in normal T cells, but in tumor cells these important interactions are prevented by DNA methylation.

Citation Format: Toshifumi Hara, Manami Yoshita, Masataka Nakamura. CpG methylation abrogates KLF2-mediated transcriptional repression of telomerase in human T cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 741. doi:10.1158/1538-7445.AM2013-741
Abstract 750: Negative regulation of bcl-2 transcription by DNA damage-binding protein complex in human ovarian cancer cells.
Authors: Zhao Ran , Han Chunhua , Eisenhauer Eric , Kroger John , Gong Li , Zhu Qianzheng , Zhao Weiqiang , Yu Jianhua , Karuppaiyah Selvendiran , Liu Xingluo , Wani Altaf Ahmad , Wang Qi-En
Published: August 2014

Elevated expression of the anti-apoptotic factor Bcl-2 is believed to be one of the contributing factors to an increased relapse rate associated with multiple cisplatin-resistant cancers. DNA damage-binding protein 2 (DDB2) has recently been revealed to play an important role in sensitizing human ovarian cancer cells to cisplatin-induced apoptosis through the down-regulation of Bcl-2, but the underlying molecular mechanism remains poorly defined. Here, we report that DDB2 functions as a transcriptional repressor for Bcl-2. DDB2 binds to a specific response element at the 5’-end of bcl-2 P1 promoter, concomitant with marked losses of histone H3 acetylation along the bcl-2 promoter and enhancer regions. Quantitative chromatin immunoprecipitation analysis revealed that HDAC1 was locally enriched in the bcl-2 P1 core promoter following DDB2 over-expression. Lysine 244 in DDB2 is essential for the transcriptional regulation of bcl-2, evidenced by the fact that the K244E mutant failed to inhibit bcl-2 promoter activity. Interestingly, the simultaneous DDB1 knock-down and DDB2 over-expression are required to potentiate bcl-2 promoter activity in the human ovarian cancer cell line CP70, while the knockdown of DDB1 alone failed to produce any significant effect. Furthermore, quantitative analysis of DDB2 and Bcl-2 expression in ovarian cancers provides clinical evidence that DDB2 and Bcl-2 are inversely correlated. Collectively, our data supports a model in which DDB2 and DDB1 function cooperatively in repressing bcl-2 transcription by recruiting HDAC1 to deacetylate histone H3 across bcl-2 regulatory regions, with DDB2 being responsible for binding to a specific cis-acting element in the bcl-2 P1 promoter. These findings have immediate implications for clinical trial designs of DDB2-based therapies for diverse cisplatin-resistant malignancies.(Supported by NIH grant CA151248 to QEW, and CA93413 to AAW)

Citation Format: Ran Zhao, Chunhua Han, Eric Eisenhauer, John Kroger, Li Gong, Qianzheng Zhu, Weiqiang Zhao, Jianhua Yu, Selvendiran Karuppaiyah, Xingluo Liu, Altaf Ahmad Wani, Qi-En Wang. Negative regulation of bcl-2 transcription by DNA damage-binding protein complex in human ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 750. doi:10.1158/1538-7445.AM2013-750
Abstract 759: Regulation of androgenic signaling by yes-associated protein, YAP, in prostate cancer cells.
Authors: Alptekin Ahmet , Abali Gamze Kuser , Wang Qiang , Cinar Bekir
Published: August 2014

Androgenic signaling plays a central role in prostate tumor development, progression, and metastasis. The molecular circuitry contributing to these pathological events is still a mystery. Previously, we reported that the hippo-like protein kinase Mst1/2 antagonized androgen receptor (AR) and prostate tumor cell growth (Cinar et al., Cancer Res., 2011). Here, we studied the role of yes-associated protein (YAP) oncogene, a prominent nuclear effector of the Mst/Hippo tumor suppressor network, in regulating AR and in prostate tumor cell growth in vitro. As revealed by co-immunoprecipitation (IP) and western blot experiments, we first showed that endogenous YAP formed protein complexes with endogenous AR. The complex formation between the two proteins mainly occurred in cell nuclei was further enhanced by androgen stimulation. Our luciferase-promoter reporter analysis demonstrated that enforced YAP expression increased the androgen-mediated and AR-responsive promoter reporter gene activation, and the carboxyl-terminal site of YAP, which consists of PDZ and coiled-coiled protein-protein interaction domains, appeared to be responsible for AR transactivation. Chromatin-IP and PCR analysis of the bound and uncross-linked genomic DNA revealed that YAP interacted with the DNA regions where AR also binds, and androgen enhanced the interaction between YAP and chromatin DNA. We also provided evidence that, compared to vector control, enforced YAP expression promoted androgen-dependent and independent cell growth, and silencing YAP by RNAi reduced the growth of castration-resistant prostate tumor cells in vitro. Our findings indicate that YAP is a physiologic binding partner and positive regulator of androgenic signaling, and suggest that the Hippo-YAP pathway plays a critical role in human prostate tumor progression.

Citation Format: Ahmet Alptekin, Gamze Kuser Abali, Qiang Wang, Bekir Cinar. Regulation of androgenic signaling by yes-associated protein, YAP, in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 759. doi:10.1158/1538-7445.AM2013-759
Abstract 761: Importance of protein interactions in mediating AF9/MLLT3 function.
Authors: Malik Bhavna , Hemenway Charles S.
Published: August 2014

AF9/MLLT3, located on chromosome band 9p22, was first identified as a fusion partner of the Mixed Lineage Leukemia (MLL) gene in acute myeloid leukemia. The AF9 protein has been shown to play an important role in erythrocyte/ megakaryocyte development by regulating the expression of genes encoding hematopoietic transcription factors. Moreover, in hematopoietic precursor cells, expression of a chimeric protein comprised of the C-terminus of AF9 fused to the N-terminus of MLL leads to leukemic transformation. Thus AF9 contributes both to normal as well as neoplastic blood cell development. Our lab and others have shown the direct or indirect association of the C-terminus of AF9 with several distinct proteins including activators and repressors of transcription (AF4, pTEFb, DOT1L, CBX8, RING1b, and BCoR), thus suggesting that the function of AF9 can be substantially changed depending upon the nature of molecules that specifically associate with it. We hypothesize that AF9 is an important component of several distinct macromolecular complexes, each of which has a unique function. Specifically, DOT1L is a chromatin modifying enzyme that methylates histone H3 at lysine 79. CBX8 is component of the repressive Polycomb PRC1 complexes. Here, we report that the association of AF9 with CBX8 competes with DOT1L for AF9 binding and alters expression of genes regulated by DOT1L. In this context, CBX8 enhances rather than represses gene expression.

Citation Format: Bhavna Malik, Charles S. Hemenway. Importance of protein interactions in mediating AF9/MLLT3 function. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 761. doi:10.1158/1538-7445.AM2013-761

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 763A: SUMOylation of ATF3 alters MC2R, NR5A1, STAR, and TP53 gene activities.
Authors: Wang Chiung-Min , Brennan Victoria , Gutierrez Ninoska , Wang Xirui , Wang Lizhong , Yang Wei-Hsiung
Published: August 2014

Cyclic AMP-dependent transcription factor-3 (ATF3), a stress sensor/mediator, plays an essential role in cells to maintain homeostasis and has diverse functions in cellular death and survival signal pathways. ATF3 binds to the consensus ATF/cAMP response element (CRE) to regulate its downstream target genes, such as CCNE2 (Cyclin E2), CD82 (KAI1), DDIT3 (GADD153), LDLR (LDL receptor), SNAI1, TP53, and TWIST1. A previous report has shown that the activities of ATF3 are modulated by ubiquitination; however, whether it is modified by small ubiquitin-related modifier (SUMO), an important modification in regulating a wide range of cellular processes, remains unknown. The aim of this study was to investigate whether ATF3 is post-translationally modified by SUMO proteins and also to determine the role of SUMOylation of ATF3 on MC2R, NR5A1, STAR, and TP53 gene activities. Here we demonstrate that ATF3 is clearly defined as a SUMO target protein both in vitro SUMOylation assay using recombinant proteins and at the cellular levels. Furthermore, ATF3 interacted with UBE2I (Ubc9), the only SUMO E2 enzyme found so far. In addition, PIAS3β (a SUMO E3 ligase) enhanced and SENP2 and SENP7 (two SUMOylation proteases) decreased SUMOylation of ATF3, respectively. In addition, we found that ATF3 is selectively SUMOylated at lysine residue 42 but the SUMOylation/deSUMOylation does not alter subcellular localization of ATF3. We next characterized the functional role of ATF3 SUMOylation on MC2R, NR5A1, STAR, and TP53 gene expression. We found that SUMOylation of ATF3 is required for full repression of NR5A1 and TP53 genes. While SUMOylation of ATF3 is required for full activation of MC2R gene activity, SUMOylation of ATF3 further enhances repression of STAR gene activity. Overall, we provide the first evidence that ATF3 is post-translationally modified by SUMO to regulate MC2R, NR5A1, STAR, and TP53 gene activities.

Citation Format: Chiung-Min Wang, Victoria Brennan, Ninoska Gutierrez, Xirui Wang, Lizhong Wang, Wei-Hsiung Yang. SUMOylation of ATF3 alters MC2R, NR5A1, STAR, and TP53 gene activities. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 763A. doi:10.1158/1538-7445.AM2013-763A
Abstract 769: Integrin-Linked Kinase (ILK) breaks Merlin's spell to inhibit the Hippo tumor suppressor pathway and activate the YAP oncogene.
Authors: Serrano Isabel , Muller William J. , Dedhar Shoukat
Published: August 2014

The Hippo signalling pathway is gaining recognition as an important player in tumor progression. Components of the pathway, which include transcriptional co-activators YAP/TAZ, are dysregulated in a significant fraction of human cancers, and YAP activation has recently been shown to promote Epithelial to Mesenchymal Transition (EMT) and metastasis. However, how Hippo signalling is dysregulated in tumors remains unclear. Here, we demonstrate that Integrin-Linked Kinase (ILK), an actin-interacting, focal adhesion adaptor and serine/threonine protein kinase whose expression is also significantly upregulated in many types of cancers, inhibits the Hippo tumor suppressor pathway by stimulating the phosphorylation, and inhibiting the activity of Merlin, via the protein phosphatase inhibitor, CPI-17, an established target of ILK, and an inhibitor of Merlin (Deng JT et al. Biochem J 2002; Huang J et al. Biochem J 2006). We have assessed the pathway in human breast and prostate cancer cell lines, in which the Hippo pathway is switched OFF, as determined by the phosphorylation status of Merlin, MST1/2, LATS1/2, and YAP, and the nuclear YAP localization. We demonstrate that inhibition of ILK expression, or inhibition of its activity in these cells turns the Hippo pathway ON, resulting in the stimulation of YAP phosphorylation, sequestration in the cytoplasm, and inhibition of TEAD transcriptional activity. In addition, we have determined that YAP/TAZ protein expression is significantly upregulated in vivo in ErbB2 induced mammary tumors from transgenic mice, and that deletion of ILK from these tumors, which significantly delays tumor growth and inhibits metastasis, dramatically suppresses the expression of these transcription co-activators. Finally, we demonstrate that ILK plays a critical role in the suppression of the Hippo pathway by TGF-b, EGF and LPA. Treatment of human breast and prostate epithelial cells with these growth factors results in dephosphorylation of LATS 1/2, and YAP, and nuclear localization of YAP. However, inhibiting ILK activity under these conditions results in the stimulation of phosphorylation of LATS1/2 and YAP, as well as in the sequestration of YAP/TAZ in the cytoplasm. These novel data demonstrate that ILK inhibits the Hippo tumor suppressor pathway leading to nuclear activation of YAP/TAZ, thereby contributing to tumor progression and metastasis.

Citation Format: Isabel Serrano, William J. Muller, Shoukat Dedhar. Integrin-Linked Kinase (ILK) breaks Merlin's spell to inhibit the Hippo tumor suppressor pathway and activate the YAP oncogene. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 769. doi:10.1158/1538-7445.AM2013-769
Abstract 77: Genetic variations in the stem cell pathway predict for toxicity following radiotherapy in lung cancer.
Authors: Skinner Heath D. , Pu Xia , Chang Joe , Lu Charles , Komaki Ritsuko , Wu Xifeng
Published: August 2014

Lung cancer is a devastating malignancy leading to over 150,000 deaths in the US annually. One primary modality in the treatment of locally advanced lung cancer is radiotherapy in combination with chemotherapy. However, this treatment can lead to significant local toxicity in as many as 50% of all patients and, in some cases, is fatal. Though post-radiation toxicity significantly affects adherence to treatment, quality of life and clinical outcome, it is difficult to predict. Further, no genetic markers have yet been found to be useful in a clinical setting. Because stem cell recruitment is part of the post-radiation tissue repair response, we hypothesize that alterations in stem cell signaling may predict for toxicity following radiotherapy. To investigate this question we examined 441 SNPs related to stem cell signaling in the peripheral blood lymphocytes in 159 patients with stage III-IV lung cancer treated with chemoradiotherapy. After adjusting for treatment-related and demographic factors, we found 47 SNPs significantly associated with symptomatic radiation pneumonitis (p<0.05) and 51 SNPs associated with symptomatic esophagitis (p<0.05). For example, patients with at least one risk allele for WNT9Brs:2165846 had a 46% risk of symptomatic pneumonitis, compared to 31% in those who did not, for a 2.23 fold increase in risk (CI: 1.05-4.07, p=0.04). On analysis of a 141 patient validation set with similar characteristics, variant WNT9Brs:2165846 was confirmed to be associated with symptomatic radiation pneumonitis (OR 2.6, CI: 1.1-6.16, p=0.03). Analysis on this confirmation set is ongoing; however, the preliminary results are indicative of the importance of stem cell signaling in modulating repair following radiotherapy and, in combination with clinical factors, may allow for better management of these patients.

Citation Format: Heath D. Skinner, Xia Pu, Joe Chang, Charles Lu, Ritsuko Komaki, Xifeng Wu. Genetic variations in the stem cell pathway predict for toxicity following radiotherapy in lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 77. doi:10.1158/1538-7445.AM2013-77
Abstract 771: Pin1 regulates the dynamics of c-Myc DNA binding to facilitate target gene regulation and oncogenesis.
Authors: Farrell Amy , Pelz Carl , Wang Xiaoyan , Daniel Colin , Su Yulong , Janghorban Mahnaz , Impey Soren , Sears Rosalie C.
Published: August 2014

The c-Myc oncoprotein is generally considered to be a master regulator of gene transcription by virtue of its ability to activate or repress the expression of a large percentage of all genes. However, mechanisms that direct c-Myc's recruitment to DNA and target gene selection to elicit specific cellular functions have not been well elucidated. Here, we report that the Pin1 prolyl-isomerase regulates c-Myc transcriptional activity by altering c-Myc's association with target gene promoters. Specifically, we show that Pin1 enhances the recruitment of Serine62-phosphorylated c-Myc along with its co-activators to selected target gene promoters during gene activation, followed by promoting c-Myc's release associated with its degradation, resulting in cyclic c-Myc DNA binding. This facilitates c-Myc's activation of target genes associated with an increase in elongating RNA Polymerase II in the gene body. Genes impacted by this mechanism are involved in cell growth and metabolism, resulting in enhanced pro-proliferative activity, even while controlling c-Myc expression. In cancer cells with impaired c-Myc degradation, Pin1 still regulates cyclic c-Myc DNA binding, even though it no longer enhances c-Myc degradation. This allows for simultaneous high expression of Pin1 and c-Myc in cancer, which can drive a gene expression pattern that we show is enriched in poor outcome subtypes of breast cancer. This study provides new insight into mechanisms regulating c-Myc DNA binding and oncogenic activity, it reveals a novel role for Pin1 in transcription factor regulation, and it elucidates a mechanism that can contribute to oncogenic cooperation between Pin1 and c-Myc.

Citation Format: Amy Farrell, Carl Pelz, Xiaoyan Wang, Colin Daniel, Yulong Su, Mahnaz Janghorban, Soren Impey, Rosalie C. Sears. Pin1 regulates the dynamics of c-Myc DNA binding to facilitate target gene regulation and oncogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 771. doi:10.1158/1538-7445.AM2013-771
Abstract 772: Rap2b, a novel p53 downstream target, promotes cell survival after DNA damage.
Authors: He Yunlong , Li Mangmang , Dubois Wendy , Kovalchuk Alexander , Wu Xiaolin , Huang Jing
Published: August 2014

The tumor suppressor p53 is a critical regulator of apoptosis and cell survival. It evokes both pro-survival and pro-apoptosis programs upon DNA damage. However, the mechanisms underlying this cell fate decision are largely unclear. To identify p53 targets in a global and unbiased manner, we performed gene expression microarray and ChIP-chip assays using mouse embryonic fibroblasts (MEFs) and mouse embryonic stem (mES) cells. I treated MEFs and mES cells with adriamycin, a DNA damage agent, for 8 hours. Comparing to the untreated cells (negative controls), I obtained common genes that were dramatically induced both in MEFs and mES cells. Subsequently, we further analyzed these genes and obtained a list of 90 common genes. Then, I compared these 90 genes with published p53 targets in human cell lines U2OS and HCT116, and found 10 genes (Alox5, Eda2r, Btg2, Mdm2, Adrb2, Cdkn1a, Tnfrsf10b, Rap2b, Ddit4, Bbc3) that are conserved p53 targets between mouse and human. Most of these genes have been well studied except for Rap2b. Both mouse and human Rap2b share the same amino acid residue sequence, which indicating Rap2b might have important conserved functions both in human and in mouse. Using conventional chromatin immunoprecipitation (ChIP) assay and luciferase reporter assay, I identified one p53 binding motif in Rap2b promoter region. After DNA damage, p53 binds to the promoter of Rap2b and activates its transcription. The reduction of Rap2b levels by small interference RNA increases the apoptosis of cells under the damaged condition, suggesting that Rap2b helps cell survive upon DNA damage. This pro-survival role of Rap2b is very similar to the function of another well-known p53 target, Mdm2. Bioinformatic analysis revealed that Rap2b is over-expressed in many types of tumors, consistent with its pro-survival function. Anchorage independent growth assay showed that Rap2b only has weak transformation activity, suggesting that it is not a typical oncogene. Importantly, Rap2b activates RalGDS-Ral survival pathway after DNA damage. Therefore, our results identified a novel player, Rap2b, in the pro-survival program conducted by p53 and revealed a connection between the p53 signaling and the RalGDS-Ral cell survival pathway. Future studies will focus on investigating whether the inhibition of Rap2b may increase the apoptosis of tumor cells.

Citation Format: Yunlong He, Mangmang Li, Wendy Dubois, Alexander Kovalchuk, Xiaolin Wu, Jing Huang. Rap2b, a novel p53 downstream target, promotes cell survival after DNA damage. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 772. doi:10.1158/1538-7445.AM2013-772
Abstract 777: TAp73 depletion unleashes p53-mediated apoptotic response to lethal DNA damage.
Authors: Chen Dongshi , Ming Lihua , Zou Fangdong , Yu Jian , Zhang Lin
Published: August 2014

DNA damage response is essential for maintenance of genomic integrity and functions as a guardian against oncogenic transformation. Ionizing radiation and chemotherapeutic drugs used in anticancer therapies often incur DNA damage in killing of tumor cells. The central regulator of DNA damage response is the tumor suppressor p53, which inhibits cell cycle progression by activating p21 and other cell cycle regulators, or induces apoptosis through proapoptotic targets such as PUMA and Bax. In addition to p53, other p53 family members, such as p73 and p63, also play a significant role in DNA damage response. However, it has remained unclear how coordination of different p53 family members modulates DNA damage response. In this study, we found that sub-lethal levels of DNA damage up-regulated TAp73, the transcriptionally active form of p73, whereas lethal doses of DNA damage triggered TAp73 depletion. In response to mild DNA damage, TAp73 occupied p53 responsive elements in the promoters of p53 target genes to keep the transcriptional activity of p53 in check, which resulted in cell cycle arrest. Following excessive DNA damage, the depletion of TAp73 resulted in enhanced induction of p53 target genes such as PUMA, Noxa and Bax, leading to apoptosis initiation. Furthermore, our results demonstrated that TAp73 depletion after excessive DNA damage was caused by ubiquitin/proteasome-mediated degradation of E2F1, which activates TAp73 expression to maintain cell survival. Collectively, our results suggest that modulation of p53 activity by TAp73 mediates differential responses to genotoxic stress.

Citation Format: Dongshi Chen, Lihua Ming, Fangdong Zou, Jian Yu, Lin Zhang. TAp73 depletion unleashes p53-mediated apoptotic response to lethal DNA damage. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 777. doi:10.1158/1538-7445.AM2013-777
Abstract 787: Tumor suppressor p53 inhibits expression of the pro-metastasis protein Toca-1.
Authors: Chander Harish , Brien Colin D. , Truesdell Peter , Germain Doris , Craig Andrew W. B.
Published: August 2014

Transducer of Cdc42-dependent actin assembly-1 (Toca-1) is an adaptor protein that recruits key actin regulatory proteins to invadopodia in metastatic breast cancer cells. We recently showed that stable silencing of Toca-1 led to reduced invadopodia formation, cell invasion and tumor metastasis in an orthotopic xenograft model in mice. These results raise the possibility that Toca-1 upregulation may occur during tumor progression to metastatic disease. Here we show that Toca-1 expression is higher in triple negative breast cancers (HER2/ER/PR) compared to other subtypes. Since triple negative breast cancers frequently harbor mutations in the tumor suppressor p53, we tested whether p53 regulates Toca-1 expression. In wild-type (WT) p53-expressing normal breast epithelial and breast cancer cells, stabilization of WT p53 by inducing DNA damage, or treating cells with Mdm2 inhibitor Nutlin-3, led to dose-dependent reductions in Toca-1 mRNA and protein expression. Likewise, ectopic expression of WT p53 in p53-null HCC1806 cells also led to repression of Toca-1 levels. To test if this was a direct effect of p53 on the Toca-1 gene, chromatin immunoprecipitation (ChIP) studies were performed. Indeed, the proximal promoter region and intron 2 of the Toca-1 gene (which contained a predicted p53 binding site) were detected in p53 ChIP assays in WT p53-expressing cells, but not mutant p53 expressing breast cancer cells. Together, these results identify a novel pathway to inhibit Toca-1 expression in cells with WT p53, and imply that the loss of p53 in highly metastatic breast cancer subtypes may lead to elevated Toca-1 expression and progression to metastatic disease.

Citation Format: Harish Chander, Colin D. Brien, Peter Truesdell, Doris Germain, Andrew W. B. Craig. Tumor suppressor p53 inhibits expression of the pro-metastasis protein Toca-1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 787. doi:10.1158/1538-7445.AM2013-787
Abstract 79: Immunomodulation in response to radiation-induced lung injury.
Authors: Cappuccini Federica , Wirsdörfer Florian , Drabczyk Malagorzata , Eldh Therese , Sak Ali , Jendrossek Verena
Published: August 2014

Introduction.

Pneumonitis and fibrosis constitute dose-limiting side effects of thorax and whole body irradiation. Although several novel disease biomarkers have been described during recent years, the cellular and molecular factors responsible for disease initiation and progression are still not sufficiently understood. Here, we characterized local and systemic changes in the immune cell compartment in response to radiation-induced lung injury and defined the role of lymphocytes for fibrosis development in a murine model.

Material and methods

A single dose of hemithorax or whole thorax irradiation (15 Gy) was applied on C57BL/6 wild type mice (WT) or immunodeficient RAG-2-/- mice. Mice were sacrificed at defined time points and lungs were isolated for histological and immunohistochemical analysis as well as immune cell phenotyping. Moreover, immune cells were isolated from spleen, blood and cervical lymph nodes and also characterized by flow cytometry. Bronchioalveolar lavage fluid was collected for determination of cytokine levels.

Results and discussion.

Whole thorax irradiation triggered time-dependent changes in the composition and activation state of immune cells isolated from the lung tissue. These changes were paralleled by specific alterations in the cytokines present in the bronchoalveolar lavage fluid. In WT mice whole thorax irradiation with 15 Gy led to a time-dependent increase in collagen deposition and lung fibrosis. Irradiated RAG-2-/- mice showed an earlier onset of collagen deposition and an increased number of fibrotic foci in the lung when compared to WT mice.

Conclusion.

Thorax irradiation triggers local and systemic changes in the composition and the activation state of immune cells. The increased sensitivity of RAG-2-/- mice to radiation-induced fibrosis suggests a role of mature lymphocytes in the suppression of fibrotic changes. An improved understanding of the contribution of specific immune cells to the pathogenic process in the lung may open novel routes to prevent or treat radiation-induced pneumopathy.

Citation Format: Federica Cappuccini, Florian Wirsdörfer, Malagorzata Drabczyk, Therese Eldh, Ali Sak, Verena Jendrossek. Immunomodulation in response to radiation-induced lung injury. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 79. doi:10.1158/1538-7445.AM2013-79
Abstract 799: Global genomic analysis identified PRKCI as a potential therapeutic target gene for human clear cell ovarian cancer.
Authors: Tsang Tsun Yee , Mohapatra Gayatry , Itamochi Hiroaki , Mok Samuel C. , Birrer Michael J.
Published: August 2014

Clear cell ovarian cancer (CCOC), a rare epithelial ovarian cancer histotype, is more resistant to standard chemotherapy and has a worse prognosis than serous and endometrioid histotypes. We attempted to identify genes that are responsible for the aggressive behavior of CCOC and could be potential therapeutic targets using genomic and expression profilings. To identify gene amplification, genome-wide DNA copy number alterations were measured in 13 CCOC cell lines using high-resolution oligonucleotide array CGH (Agilent 105k Human Genome CGH Microarray). GISTIC analysis identified 16 amplified regions containing 391 genes. After comparison to our database of differentially expressed genes between human CCOC specimens and normal ovarian surface epithelium specimen (Affymetrix U133 Plus 2.0 microarrays), 45 of the amplified genes showed mRNA overexpression in CCOC specimens. Among the 45 genes, protein kinase C iota (PRKCI) was amplified in 9/13 CCOC cell lines and was overexpressed (6.7 fold) in CCOC specimens. Analysis using PathwayStudio software identified PRKCI mediated pathways for cell migration and invasion. Knock-down of PRKCI by transfection with PRKCI specific siRNAs suppressed cell migration and invasion in KOC-7c and OVISE cells. On the other hand, ectopic expression of PRKCI by transfection with PRKCI expressing vector enhanced cell migration and invasion in ES2 and TOV21G cells. The results from the present study therefore suggested that PRKCI may promote metastasis and serve as a potential target gene for treatment of human clear cell ovarian cancer.

Citation Format: Tsun Yee Tsang, Gayatry Mohapatra, Hiroaki Itamochi, Samuel C. Mok, Michael J. Birrer. Global genomic analysis identified PRKCI as a potential therapeutic target gene for human clear cell ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 799. doi:10.1158/1538-7445.AM2013-799