bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4525: Distinctive chromatin organization of p53 binding sites in normal and cancer cells
Authors: Cui Feng , LoVerso Peter , Bao Feifei
Published: July 2016

The tumor suppressor p53 is arguably one of best known proteins in humans, which induces or represses hundreds of genes to prevent tumor development. Now, it becomes clear where p53 binds across the human genome and how it regulates gene expression. Recent studies have established that p53 can access its target sites on the nucleosomal DNA and the rotational positioning of a nucleosome is critical to determine the accessibility of p53 sites. We have found that p53 response elements (REs) associate with cell cycle arrest such as p21 REs are likely to be exposed on the nuclesomal surface. However, it remains unclear if other chromatin elements such as histone modifications and DNA methylation affect p53 in vivo binding and if such chromatin organization differs in normal cells versus cancer cells.

We analyzed the pre-existing chromatin landscape of thousands of p53 binding sites identified so far in normal (GM12878) and cancer (K562) cells. The p53 sites derived from the normal cells are often found in the vicinity of the transcription start sites (TSSs) of nearby genes, characterized with relative low nucleosome occupancy, high levels of transcriptionally active histone marks such as H3K4me3 and low levels of DNA methylation. By contrast, the p53 sites found in the cancer cells are located remotely from TSSs, with relatively high nucleosome occupancy, low levels of H3K4me3 and DNA methylation. The shift in p53 genomic binding patterns in the normal and cancer cells reflects the cancer-associated epigenetic dysregulation. Overall, in contrast to most of DNA-binding transcriptional factors, p53 binding sites occur in nucleosome-enriched regions. These findings indicate that p53 belong to a set of transcription factors including the pluripotency factors Oct4, Sox2 and Klf4 that can directly interact with nucleosomes in the context of chromatin.

Citation Format: Feng Cui, Peter LoVerso, Feifei Bao. Distinctive chromatin organization of p53 binding sites in normal and cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4525.
Abstract 4532: Deregulation of retinoic acid (RA) epigenetic control of sphingolipid signaling
Authors: Rossetti Stefano , Gagliostro Vincenzo , Sacchi Nicoletta
Published: July 2016

All-trans retinoic acid (RA), the bioactive derivative of vitamin A, can paradoxically exert both anti-cancer and cancer-promoting actions. According to our studies, RA promotes breast cancer cell growth and invasion whenever it fails to exert its genome-wide epigenetic control of transcription via the RA receptor alpha (RARA). Factors that negatively affect RARA genomic function lead to genome-wide deregulation of the transcriptome epigenetically regulated by RA, including RARA-target genes controlling critical sphingolipid functions. We show here that functional inhibition of genomic RARA concomitantly leads to epigenetic repression of neutral sphingomyelinase 2 (nSMase2/SMDP3), a critical enzyme involved in the synthesis of pro-apoptotic ceramide, and upregulation of S1PR1, a key receptor of sphingosine-1-phosphate (S1P), a sphingolipid with pro-proliferative and pro-invasive activity. Selective activation of S1PR1 recapitulates RA pro-proliferative/pro-invasive action, while selective inhibition of S1PR1 counteracts it. Apparently, activation of the S1P-S1PR1 axis, combined with lack of RARA-mediated epigenetic control of SMPD3-ceramide axis, contributes to determine RA tumorigenic action.

This study was supported by the NCI R01 CA127614 grant (NS).

Citation Format: Stefano Rossetti, Vincenzo Gagliostro, Nicoletta Sacchi. Deregulation of retinoic acid (RA) epigenetic control of sphingolipid signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4532.
Abstract 4533: Disruption of intrachromosomal interactions by deficient cohesin initiate epithelial-mesenchymal transition and stemness in cancer
Authors: Yun Jiyeon
Published: July 2016

PURPOSE

Although EMT-MET process is a crucial reversible event in development of human tumorigenesis controlled by complex networks, very little is known about how EMT-MET occurs reversibly and timely in cancer. Recently, different levels of Rad21 protein, a subunit of cohesin high-order-chromatin-architectural complex, between mesenchymal and epithelial cancers have been described.

METHODS

To elucidate how Rad21 determines EMT states, we silenced Rad21 in epithelial cancers using lentiviral shRNAs. We performed microarray to compare transcriptional changes by Rad21-KD. Additionally, we measured cell cycles and mitotic chromosome segregations to rule out the role of Rad21 as a sister chromatid segregation molecule. We observed cell morphology through confocal microscopy and checked high-order-chromatin structures using Chromosome-Conformation-Capture (3C) and Chromatin immunoprecipitation (ChIP) assay.

RESULTS

We identified high-order-chromatin structures mediated by Rad21 plays critical regulatory roles in transcription of EMT related genes for the first time. Rad21-KD in epithelial cancers caused reduction of Rad21 enrichment on TGFB1 and ITGA5 genes, leading to disruption of the-gene-specific-chromatin-looping structures. This dramatically recruited PolII and AcH3, active transcription marks, causing rapid induction of the gene transcriptions. Indeed, induced EMT-related genes directly led to morphological changes with EMT traits. We ultimately found that cancer stem cell-like cells showed same results with mesenchymal cancer and Rad21-KD epithelial cells.

These findings indicate that the initiation of EMT in cancer might crucially require changes in high-order-chromatin structures of EMT-related genes mediated by cohesin, contributing to rapid and precise reversible changes in EMT or MET state to adapt to phenotypes of human cancer development.

Citation Format: Jiyeon Yun. Disruption of intrachromosomal interactions by deficient cohesin initiate epithelial-mesenchymal transition and stemness in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4533.
Abstract 4540: Use of SIAH E3 ubiquitin ligase as a biomarker in pancreatic adenocarcinoma
Authors: Svyatova Elizaevta
Published: July 2016

The dismal prognosis of patients diagnosed with pancreatic cancer points to our limited arsenal of effective anticancer therapies. Oncogenic K-RAS pathway activation is commonly detected in pancreatic cancer. These activations confer drug resistance, drive aggressive tumor growth and metastasis, and ultimately contribute to poor clinical outcome. Currently, there is no curative treatments for pancreatic cancers. Thus, early detection, biomarker discovery, and identifying novel means of countervailing hyperactive K-RAS signals are urgently needed.

Seven-In-Absentia Homolog (SIAH), E3 ubiquitin ligase, is an essential downstream component of the RAS signal transduction pathway. SIAH is a member of a highly evolutionarily conserved family of RING finger E3 ubiquitin ligases.

Our previous immunohistochemistry (IHC) studies have shown SIAH is a tumor-specific biomarker and SIAH expression is progressively increased as pancreatic tumor progresses from pancreatic intraepithelial neoplasia (PanIN) to pancreatic adenocarcinoma (PDCA) at late stages. In this study, we expand our observations by comparing the expression patterns of SIAH and several newly identified SIAH-interacting proteins using Mayo clinic pancreatic cancer SPORE tissue microarrays (TMA) in a hope of identifying new vulnerability and novel prognostic biomarkers in pancreatic cancer. We hypothesized that SIAH and SIAH-interacting proteins may serve as useful biomarkers and will help to assess effectiveness of anticancer treatment, disease progress, predict clinical outcome and patient survival in the clinical settings. Moreover, we are evaluating SIAH's role in the regulation of the SIAH-interacting proteins and validating the potential mechanisms of these dynamic interactions identified in vitro using pancreatic cancer patients TMA. Ultimately, we aim to explore the new cancer vulnerability, develop new therapeutic strategies, and validate novel prognostic biomarkers for pancreatic cancer patients.

This study is performed in a double blind control settings and we are currently awaiting the disclosure of the clinical information from the pathologist.

Citation Format: Elizaevta Svyatova. Use of SIAH E3 ubiquitin ligase as a biomarker in pancreatic adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4540.
Abstract 4543: Substrate trapping proteomics reveals novel mechanism for regulation of mTORC1 signaling by βTrCP-FNIP1/2-FLCN axis
Authors: Kim Tai Young , Chang Jee Yun , Ku Jin Mo , Hong Se Hyang , Kim ji Hye , Choi Hyeong Sim , Lee Kangwook , Kim Myeong-Sun , Woo Sang Mi , Major Michael B. , Ko Seong-Gyu
Published: July 2016

Defining the full complement of substrates for each ubiquitin ligase remains an important challenge. Improvements in mass spectrometry instrumentation and computation and in protein biochemistry methods have resulted in several new methods for ubiquitin ligase substrate identification. Here we used the parallel adapter capture (PAC) proteomics approach to study βTrCP2, a substrate adaptor for the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex. The processivity of the ubiquitylation reaction necessitates transient physical interactions between βTrCP2 and its substrates, thus making biochemical purification of FBXW11-bound substrates difficult. Using the PAC-based approach, we inhibited the proteasome to “trap” ubiquitylated substrates on the SCF (βTrCP2) E3 complex. Comparative mass spectrometry analysis of immunopurified βTrCP2 protein complexes before and after proteasome inhibition revealed 21 known and 23 putatively novel substrates. Interestingly, many novel substrates for βTrCP2, including TBC1D4, HCFC1, DENND4C, FNIP1, and FLCN are related to cell metabolism. TBC1D4 encodes a GTPase activating protein for the small GTPase Rab that controls insulin-dependent trafficking of the GLUT4 glucose transporter in adipocytes. DENND4C acts as a guanine nucleotide exchange factor for Rab10 and its activity is required for insulin-stimulated GLUT4 translocation to plasma membrane in adipocytes. HCFC1 is a member of the host cell factor family, affecting gluconeogenesis by modulating PGC-1α stability. These suggest βTrCP2 might play important roles in glucose homeostasis by regulating stability of several different target proteins. Here, in focused study, we found that βTrCP1/2 bound, polyubiquitylated, and destabilized FNIP1, FLCN interacting protein 1. We further demonstrated that FNIP1 degradation was promoted by AMPK activation after glucose depletion and expression of a degradation-resistant FNIP1 mutant results in sustained activation of mTORC1 signaling. Hence, our findings reveal that βTrCP1/2 is involved in nutrient sensing through the AMPK-FLCN-FNIP1 and mTORC1 signaling pathways.

Citation Format: Tai Young Kim, Jee Yun Chang, Jin Mo Ku, Se Hyang Hong, ji Hye Kim, Hyeong Sim Choi, Kangwook Lee, Myeong-Sun Kim, Sang Mi Woo, Michael B. Major, Seong-Gyu Ko. Substrate trapping proteomics reveals novel mechanism for regulation of mTORC1 signaling by βTrCP-FNIP1/2-FLCN axis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4543.
Abstract 4546: Ubc9 sumoylation is required for its interaction with CDK6 through SUMO-interacting motif (SIM) and regulates CDK6 sumoylation in glioblastoma
Authors: Bellail Anita , Hao Chunhai
Published: July 2016

Small ubiquitin-like modifier-1 (SUMO1) is a conserved member of the ubiquitin-related protein family. Recently we have shown that the cell cycle G1 phase cyclin-dependent kinase-6 (CDK6) is modified by SUMO1 in glioblastoma (Bellail et al., Nat Commun. 2014). CDK6 sumoylation stabilizes CDK6 protein and its kinase activity and drives cell cycle progression for the cancer development and progression. SUMO1 is covalently attached to the Lysine 216 of the CDK6 protein through catalytic reactions by an E1 (SUMO-activating enzyme 1/2) and an E2-conjugating enzyme (UBC9). In this study, we further showed that UBC9 is phosphorylated by CDK1 kinase during mitosis and UBC9 phosphorylation increases the interaction of UBC9 with CDK6 and enhances SUMO1-CDK6 conjugation. Knockdown of SUMO1 by small hairpin RNA (shRNA) abolishes the interaction of UBC9 and CDK6 and SUMO1-CDK6 conjugation. UBC9 is conjugated by SUMO1 and recognizes its substrate protein through interaction of SUMO1 and SUMO-Interacting motif (SIMs) on the substrate protein. Indeed, the survey of CDK6 amino acid sequence and structure identified four SIMs on CDK6 protein. Each SIM was mutated to abrogate its binding property. Of the 4 SIMs, the CDK6-SIM4 is responsible for UBC9-CDK6 interaction as demonstrated by the finding that mutation of the first two amino-acids of the SIM sequence abolishes UBC9-CDK6 interaction and SUMO1-CDK6 conjugation. Our data show that UBC9 sumoylation is required for its interaction with CDK6 protein through SIM and thus involved in glioblastoma progression.

Citation Format: Anita Bellail, Chunhai Hao. Ubc9 sumoylation is required for its interaction with CDK6 through SUMO-interacting motif (SIM) and regulates CDK6 sumoylation in glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4546.
Abstract 4550: High-level nuclear GSK3β promotes glioma tumorigenesis by phosphorylating KDM1A and inducing its deubiquitination by USP22
Authors: Zhou Aidong , Lin Kangyu , Zhang Sicong , Huang Suyun
Published: July 2016

Glioblastoma multiforme (GBM) is the most lethal brain tumors and is resistant to most therapeutic endeavors. Glioma stem cells (GSCs) in GBM are responsible for glioma propagation and resistance to conventional therapies. GSK3β is a multifunctional serine/threonine kinase. Growing evidences have supported the oncogenic roles of GSK3β in diverse cancers, including in GBM; however, the functional mechanism of GSK3β in tumor initiation and propagation remains elusive. Our study showed that nuclear GSK3β is responsible for overexpression of the histone demethylase KDM1A and critically regulates histone H3K4 methylation during glioma tumorigenesis. GSK3β phosphorylates KDM1A, which induces its binding and deubiquitination by USP22, leading to KDM1A stabilization. GSK3β and USP22-dependent KDM1A stabilization is required for the demethylation of histone H3K4, thereby repression of BMP2, CDKN1A, and GATA6 transcription, and result in GSCs self-renewal and brain tumorigenesis. Moreover, introduction of a GSK3β-phosphorylation-mimic mutant KDM1A rescues the effect of GSK3β depletion on the inhibition of glioma tumorigenesis. In human glioblastoma specimens, KDM1A levels are correlated with nuclear GSK3β and USP22 levels. Furthermore, a GSK3 inhibitor tideglusib in a GBM mouse model inhibits the tumor initiating ability of GSCs, sensitizes tumors to temozolomide,

and prolongs mice survivals via KDM1A down-regulation. Our findings demonstrate that nuclear GSK3β and USP22-mediated KDM1A stabilization is essential for glioma tumorigenesis and highlights that targeting GSK3β as a therapeutic target for GBM.

Citation Format: Aidong Zhou, Kangyu Lin, Sicong Zhang, Suyun Huang. High-level nuclear GSK3β promotes glioma tumorigenesis by phosphorylating KDM1A and inducing its deubiquitination by USP22. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4550.
Abstract 4556: Activation of sonic hedgehog signaling is essential for non-alcoholic steatohepatitis induced by liver-specific disruption of Nrf1
Authors: Cao Mingnan , Guo Limei , Li Juan , Liu Chun , Zhang Yiguo , Yu Siwang , Kong Tony A-N.
Published: July 2016

Liver-specific inactivation of NF-E2 p45-related factor 1 (Nrf1) results in sequential development of non-alcoholic steatohepatitis (NASH), fibrosis, and finally hepatocellular carcinoma (HCC). However, the detailed mechanisms responsible for this phenotype remain uncertain. In the present study, a β-naphthoflavone (β-NF)-inducible liver-specific Nrf1 knockout mouse line which harbors an Nrf1flox/flox allele and a rat CYP1A1-Cre transgene was employed to investigate potential mechanism underlying Nrf1 deficiency-induced NASH.

β-NF-induced liver-specific knockout Nrf1 resulted in a reversible typical NASH and mild fibrosis phenotype. β-NF treatment almost abolished Nrf1 expression after 2 weeks, and induced dramatic elevation of serum transaminase, inflammation, necrosis, ballooning degeneration and HSC activation, but these phenotype diminished after 16 weeks, accompanied by restoration of Nrf1 expression. These observations clearly demonstrated the dependency of NASH and fibrosis on loss of Nrf1, and provide a novel model of reversible non-alcoholic liver diseases.

Notably, disruption of Nrf1 caused a sustained activation of sonic hedgehog (shh) signaling pathway accompanied by pronounced endoplasmic reticulum (ER) stress. More importantly, vismodegib, a hedgehog signaling inhibitor, dramatically reversed the deleterious effects caused by loss of Nrf1, such as elevation of serum transaminase activities, inflammatory cells infiltration and hepatocyte necrosis. It has been reported that hepatocytes undergoing endoplasmic reticulum stress and showed elevated expression of hedgehog ligands. However, the mRNA level of hedgehog ligand was not changed in Nrf1 deficiency mice, while the protein levels of both shh and Gli2 were significantly increased. Furthermore, chemical chaperone 4-PBA which can promote protein degradation partially reversed the effects of Nrf1 deficiency on shh signaling. Collectively, these data demonstrate that activation of sonic hedgehog signaling is essential for non-alcoholic steatohepatitis induced by liver-specific disruption of Nrf1, possibly through post-translational modification/procession of shh, and hedgehog signaling could be targeted to treat non-alcoholic liver diseases.

The present works were supported by the National Natural Science Foundation of China (No. 91429305, 81272468, and 81372266)

Citation Format: Mingnan Cao, Limei Guo, Juan Li, Chun Liu, Yiguo Zhang, Siwang Yu, Tony A-N. Kong. Activation of sonic hedgehog signaling is essential for non-alcoholic steatohepatitis induced by liver-specific disruption of Nrf1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4556.
Abstract 4557: Tip110-mediated regulation of NF-κB activity is key to tumorigenesis
Authors: Timani Khalid Amine , Whitmill Amanda , Liu Ying , He Johnny J.
Published: July 2016

HIV-1 Tat-interacting protein (Tip110), also known as squamous cell carcinoma antigen recognized by T cells 3 (SART3), is a multifaceted nuclear protein and has been shown to function in tumor antigenicity. Tip110 protein expression is very low in normal tissues and non-proliferating cells but becomes highly elevated in a number of malignant tumor cell lines and cancerous tissues, pointing to its possible role in tumorigenesis. Our previous studies have shown that Tip110 specifically interacted with oncogenic deubiquitinating enzyme, USP15, and that ectopic expression of the Tip110 protein led to re-distribution of USP15 from the cytoplasm to the nucleus. USP15 is known to inhibit TNF-α-induced NF-κB activity by modulation of IκB-α ubiquitination. In the current study, we demonstrated that Tip110 augmented TNF-α-induced transcription activity of NF-κB, and that deletion of the nuclear localization domain in Tip110 obliterated the observed enhancement of TNF-α-induced NF-κB activity, providing the very first evidence to support the specific role of Tip110 in NF-κB activation. In addition, we found that Tip110 expression modulated IκB-α phosphorylation and that activation of NF-κB by Tip110 was mediated through the p38/MAPK pathway. Moreover, we showed that ectopic expression of Tip110 induced translocation of NF-κB p65 from the cytoplasm to the nucleus, reminiscent of the aforementioned translocation of USP15, in the absence of direct interaction of Tip110 with either p65 or IκB-α. Taken together, these results indicate that opposing regulation of TNFα-induced NF-κB activity by Tip110 and USP15 could be pivotal for modulation of tumorigenesis. Further delineation of the underlying signaling cascades will likely provide new insights into development of anti-tumor agents.

Citation Format: Khalid Amine Timani, Amanda Whitmill, Ying Liu, Johnny J. He. Tip110-mediated regulation of NF-κB activity is key to tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4557.
Abstract 4558: PIN1 expression level is maintained by a negative feedback loop through regulation of Myc and miRNAs
Authors: Leong Ka Wai , Cheng Chi Wai , Kwong Yok Lam , Tse. Wai Choi Eric
Published: July 2016

Introduction: PIN1 is a peptidyl-prolyl-isomerase (PPIase) that binds specific motifs (serine/threonine preceding a proline) in proteins, thereby catalyzing cis/trans isomerization of the peptide bond between the phosphorylated serine/threonine and proline. As a result of such conformational changes, stability, sub-cellular localization, functional activity as well as protein-protein interactions of the PIN1-bound proteins may be altered. PIN1 has been demonstrated to play an important role in controlling various cellular processes such as cell proliferation, apoptosis and migration. Consequently, its expression has to be tightly controlled. Previously, we found that two miRNAs, miR-296-5p and miR-874-3p, served as negative regulators of PIN1. However, the mechanism regulating these two miRNAs remains to be defined.

Methods: The online database-UCSC Genome Bioinformatics (NCBI/hg18) was used to search for the upstream regulator of miR-296-5p and miR-874-3p. Luciferase reporter assay was conducted to examine the effect of the identified regulator on the promoter regions of miR-296 and miR-874-3p. Quantitative polymerase chain reaction (Q-PCR) was used to detect the expression levels of miR-296-5p and miR-874-3p. Protein expression levels of the identified regulator and PIN1 were examined by western immunoblotting.

Results: By screening the online database-UCSC, Myc was identified as a potential transcription factor for the miRNAs, miR-296-5p and miR-874-3p. As demonstrated by luciferase reporter assay, expression of Myc activated the promoters of miR-296-5p and miR-874-3p. Myc over-expression also increased the expression levels of these two miRNAs, which in turn led to down-regulation of PIN1. Interestingly, we found that over-expression of PIN1 increased the expression of Myc, miR-296-5p and miR-874-3p. We therefore hypothesized that PIN1 expression is maintained via a negative feedback loop of Myc-miRNAs-PIN1. Consistent with this hypothesis, we demonstrated that over-expression of exogenous EGFP-tagged PIN1 led to the enhancement of Myc, miR-296-5p and miR-874-3p expression, and thereby subsequently decrease the endogenous PIN1 level.

Conclusion: Taken together, our results suggested that the expression level of PIN1 is maintained by a negative feedback loop through the regulation of Myc, miR-296-5p and miR-874-3p.

Citation Format: Ka Wai Leong, Chi Wai Cheng, Yok Lam Kwong, Wai Choi Eric Tse. PIN1 expression level is maintained by a negative feedback loop through regulation of Myc and miRNAs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4558.
Abstract 4562: Inhibition of STAT3 signaling in human liver cancer cells using Evista
Authors: Wang Yina , Zhao Chongqiang , Ma Haiyan , Li Huameng , Lu Jiagao , Li Chenglong , Lin Jiayuh , Lin Li
Published: July 2016

Liver cancer is one of the major causes of morbidity and mortality in China and also around the World. Constitutive activation of Signal Transducers and Activators of Transcription 3 (STAT3) signaling is frequently detected in human cancer including liver cancer and has emerged as a viable molecular target for liver cancer treatment. Novel therapeutic approaches of more effective treatments are much needed. To date, few STAT3 inhibitors are available for the therapy of liver cancer yet. Evista (Raloxifene HCI) is used for the prevention and treatment of osteoporosis and was approved for reducing the risk of invasive breast cancer in postmenopausal women with osteoporosis and in postmenopausal women at high risk for invasive breast cancer. We reported the discovery of Raloxifene as novel inhibitors of IL-6/GP130 protein­protein interactions (PPIs) using multiple ligand simultaneous docking (MLSD) and drug repositioning. The possible effect of Evista in STAT3 signaling or liver cancer cells is still unclear. The ability of Evista to inhibit STAT3 was tested. Evista inhibited the P-STAT3 stimulated by IL-6 but not the induction of STAT1, STAT2, and STAT4 phosphorylation by IFN-α and IL-4. Evista inhibited STAT3 phosphorylation and resulted in the induction apoptosis on human liver cancer cell lines HEPG2, 7721, and HUH-7 as evidenced by caspase-3 cleavage. Evista also inhibited the cell viability, cell migration and colony formation in liver cancer cells. Furthermore, administration daily of Evista suppressed the HEPG2 tumor growth in mice in vivo. In summary, our study is the first report indicating that Evista also exhibits biology activity against persistent STAT3 signaling in human liver cancer cells. These results suggest that Evista may also be a chemoprevention agent for liver cancer by targeting persistent STAT3 signaling.

Citation Format: Yina Wang, Chongqiang Zhao, Haiyan Ma, Huameng Li, Jiagao Lu, Chenglong Li, Jiayuh Lin, Li Lin. Inhibition of STAT3 signaling in human liver cancer cells using Evista. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4562.
Abstract 4564: Elucidating the mechanism of the STAT3/NF-κB/EP4 signaling axis in pancreatic cancer
Authors: Munoz Amanda R. , Freeman James W. , Kumar Addanki P.
Published: July 2016

Despite recent advances to pancreatic cancer (PanCA) management, the 5 year survival rate remains only 7%. This poor survival rate highlights the importance of increasing our molecular understanding of PanCA pathogenesis to develop effective therapies to improve patient outcomes. We previously demonstrated that targeting the STAT3/NF-κB signaling axis with Nexrutine® (a Phellodendron amurense bark extract) was able to inhibit autophagy, ROS production, and proliferation in multiple PanCA cell lines. Our data also indicated the potential involvement of prostaglandin receptor EP4 in mediating these effects. To our surprise, we observed decreased levels of EP4 in human pancreatic tumors implicating a possible protective role. However, the mechanism behind STAT3/NF-κB signaling and its link to EP4 are still unknown. Using genetic and chemical inhibition of STAT3 and EP4, we have sought to define the STAT3/NF-κB/EP4 signaling axis. Our results indicate that inhibition of STAT3 via knockdown (STAT3 KD) using shRNA does not affect the proliferative ability or viability of PanCA cell lines when compared to non-transfected control (NTC) cells. Even though proliferation and viability were un-affected, STAT3 KD increased Cox-2 levels in a KRAS mutant cell line (Capan-2) and inhibited Cox-2 in a KRAS WT cell line (BxPC-3). Inhibition of EP4 increased STAT3 and NF-κB activity in KRAS mutant cell lines while decreasing activity in the KRAS WT cell line. Furthermore, EP4 inhibition increased protein levels of IL-6 and dual inhibition of STAT3 and EP4 yielded increased NF-κB activity and decreased Cox-2 levels. Converse experiments in cells treated with IL-6 revealed inhibited EP4 expression and decreased NF-κB activity. This may be the reason behind the increased STAT3 activity noted in EP4 inhibited KRAS mutant cells and supports the notion that EP4 may be protective against PanCA development. Taken together, these data suggests a feedback loop in regulating the STAT3/NF-κB/EP4 signaling cascade and that STAT3/NF-κB signaling axis members are differentially regulated upon STAT3 or EP4 inhibition possibly depending on KRAS status. Supported by Initiative for Maximizing Student Development (IMSD) Training Grant (R25 GM095480; NMJB), NCCIH (R01 AT007448; APK), and VA-MERIT Award (I01 BX 000766; APK)

Citation Format: Amanda R. Munoz, James W. Freeman, Addanki P. Kumar. Elucidating the mechanism of the STAT3/NF-κB/EP4 signaling axis in pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4564.
Abstract 4571: Semaphorin/plexin signalling promotes nucleocytoplasmic trafficking of nuclear receptors
Authors: Williamson Magali , Masters John R.
Published: July 2016

Androgen receptor (AR) signalling is critical to prostate cancer progression and androgen deprivation therapy (ADT) is the first line of treatment for metastatic prostate cancer. Acquired resistance to ADT results from androgen-independent reactivation of the AR pathway primarily. Prostate tumours also bypass androgen blockade by the upregulation of the glucocorticoid receptor (GR). Both AR and GR are nuclear receptors that bind their respective ligands in the cytoplasm and then translocate to the nucleus where they act as transcription factors. Plexins are cell surface receptors for semaphorins. PlexinB1 is overexpressed and mutated in several cancers including prostate cancer and deletion of the plexinB1 gene reduces metastasis in mouse models, indicating an oncogenic role for plexinB1 in prostate cancer.

We have found that activation of plexinB1 by semaphorin4D (Sema4D) results in AR phosphorylation at serine 81, activation of AR-transcriptional activity and increased expression of androgen-responsive genes, effects reversed by plexinB1 knockdown. PlexinB1 activation increases the translocation of both AR-GFP constructs and endogenous AR to the nucleus in prostate cancer cells. Furthermore knockdown of AR in the prostate cancer cell line LNCaP reduces Sema4D-induced anchorage-independent growth of these cells.

To determine if the Sema4D/plexinB1 signalling pathway also affects translocation of other nuclear receptors to the nucleus, we have also studied the effect of Sema4D treatment on nuclear trafficking of the glucocorticoid receptor (GR). Prostate cancer cells were transfected with a GR-GFP construct and the subcellular localisation of GR-GFP was monitored upon Sema4D treatment by immunocytochemistry and confocal microscopy. The nuclear translocation of endogenous GR was also assessed following Sema4D stimulation by immunocytochemistry and subcellular fractionation studies.

We found that activation of plexinB1 by Sema4D results in a significant increase in the percentage of cells with nuclear GR-GFP and in the nuclear localisation of endogenous GR in prostate cancer cells.

These findings show that plexinB1 activation has a role in the trafficking and activation of nuclear receptors and so may have a role in resistance to ADT in late stage prostate cancer.

Citation Format: Magali Williamson, John R. Masters. Semaphorin/plexin signalling promotes nucleocytoplasmic trafficking of nuclear receptors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4571.
Abstract 4573: Complex interactions among different types of androgen receptor splice variants and the full-length androgen receptor in mediating castration resistance
Authors: Zhan Yang , Zhang Guanyi , Wang Xiaojie , Qi Yanfeng , Zhang Haitao , Dong Yan
Published: July 2016

Androgen receptor splice variants (AR-Vs) have been implicated in the pathogenesis and progression of castration-resistant prostate cancer after androgen-directed therapies. Approximately 20 AR-Vs have been identified to date. When expressed alone in cells, some AR-Vs (e.g., AR-V7 and ARv567es) localize primarily to the nucleus, whereas some others (e.g., AR-V4 and AR-V6) distribute equally between the nucleus and the cytoplasm. Significantly, the latter is often co-expressed with the nucleus-localized AR-Vs and the full-length AR (AR-FL). Thus, an important question that needs to be addressed is whether these different types of AR-Vs interact with each other as well as with AR-FL and the consequence of the interactions. In the present study, we used the bioluminescence resonance energy transfer (BRET) assay to show that both AR-V6 and AR-V4 can heterodimerize with AR-V7 and AR-FL. Consequently, AR-V7 and androgen-bound AR-FL induced nuclear localization of AR-V6 and AR-V4, leading to an increased ability of AR-V6 and AR-V4 to transactivate target genes and to confer castration-resistant cell growth. On the other hand, neither AR-V6 nor AR-V4 affected the subcellular localization of AR-V7 or AR-FL. These findings support the ability of AR-V7 and androgen-bound AR-FL to activate AR-V6 and AR-V4. Since AR-FL and nucleus-localized AR-Vs are the predominant forms of AR expressed in clinical specimens, it is likely that most of the AR-Vs in these cells are in the nucleus and are transcriptionally active. Our data therefore highlight the importance of the complex interactions among different AR-Vs and AR-FL in mediating castration resistance.

Citation Format: Yang Zhan, Guanyi Zhang, Xiaojie Wang, Yanfeng Qi, Haitao Zhang, Yan Dong. Complex interactions among different types of androgen receptor splice variants and the full-length androgen receptor in mediating castration resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4573.
Abstract 4575: Altered catalytic properties of a subset of Met cytoplasmic domain variants occurring in renal cell carcinoma
Authors: De Silva Dinuka , Lee Young , Roy Arpita , Bottaro Donald P.
Published: July 2016

TCGA provisional data of papillary renal cell carcinoma (PRCC) kidney cancer, a malignant hereditary cancer syndrome, reveals that 23% of patients (n = 161) have MET gene amplification and/or mRNA up-regulation (2-fold or greater; RNA-Seq V2) and 10% of patients possess MET mutations. The Met tyrosine kinase is the cell surface receptor for hepatocyte growth factor (HGF). HGF stimulation induces Met protein autophosphorylation and consequently enhances epithelial cell motility, morphogenesis, and proliferation. Chronic and/or overstimulation of Met contributes to tumor growth and metastasis. We determined Km and Vmax for ATP binding to full-length Met protein isolated from the PRCC derived cell line ACHN, which possesses the MET mutation T992I (numbered per SWISSPROT accession P08581) in the juxtamembrane domain, and the clear cell RCC derived cell line Caki-1, which possesses the MET V1220I mutation in the kinase domain. Catalytic constants were determined using two-site immunoassays for total Met and phospho-Met. Both variants were associated with increased Vmax relative to wild type Met protein isolated from B5/589 breast epithelial cells. The observed increased catalytic activity of receptor variants may confer a higher overall rate of signaling at lower ATP concentrations, potentially contributing to enhanced tumor cell motility and proliferation in both papillary and clear cell RCC.

Citation Format: Dinuka De Silva, Young Lee, Arpita Roy, Donald P. Bottaro. Altered catalytic properties of a subset of Met cytoplasmic domain variants occurring in renal cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4575.
Abstract 4579: Simultaneous activation of C-X-C chemokine receptor 4 and cannabinoid receptor 2 results in decreased cell signaling and tumor cell intravasation
Authors: Fennell Imani N. , Scarlett Kisha A. , Coke Christopher J.
Published: July 2016

G-protein-coupled receptor (GPCR) heterodimerization has emerged as a means by which new signaling entities can be created with respect to agonist binding. Preliminary data from our lab indicates that agonist-bound GPCR C-X-C Chemokine Receptor 4 (CXCR4) and agonist-bound cannabinoid receptor 2 (CB2R) interact at the cell membrane and that receptor interaction mitigates the effects of CXCL12-mediated CXCR4 signaling. RhoA, Rac1 and Cdc42 are small GTP-binding proteins of the Rho family that are activated downstream of CXCR4 signaling. The effects of RhoA, Rac1 and Cdc42 on the actin cytoskeleton have led to established roles for Rho proteins in cancer cell motility, metastasis and intravasation. Here we report the novel finding that CXCR4/CB2R heterodimerization decreases Rho signaling, resulting in decreased intravasation. To test the effect of receptor heterodimerization on RhoA, Rac1, and Cdc42 activation, PC3 cells were treated with the CXCR4 agonist SDF1α, the CB2R agonist AM1241 or with a combination of SDF1α and AM1241 for 1 minute. Cell lysates were collected and immunoprecipitation experiments were performed to detect RhoA, Rac1 and Cdc42. To test the effects of CXCR4/CB2R heterodimerization on intravsation, we performed a zymography assay for the detection of metalloproteinase-2 (MMP2) and metalloproteinase-9 (MMP9), which are commonly secreted by CXCR4 signaling. Immunopreciptation results indicated an increase in RhoA and Rac1 protein levels after 1 minute in PC3 cells compared to the untreated control. However, when treated with SDF1α and AM1241 for 1 minute, RhoA and Rac1 levels decreased compared to SDF1α independent treatment; there was no observable change in Cdc42 protein levels. The results from the zymography experiments demonstrated that co-treatment with SDF1α and AM1241, which induces CXCR4/CB2R heterodimerization, led to decreased MMP-2 and MMP-9 activity, as compared to the SDF1α-independent treatment. Preliminary results provide novel evidence that CXCR4 and CB2R heterodimerize at the cell membrane, which may result in decreased signaling through RhoA and Rac1, but not through Cdc42. Furthermore, CXCR4/CB2R heterodimerization results in decreased MMP activation, thus abrogating intravsation.

Citation Format: Imani N. Fennell, Kisha A. Scarlett, Christopher J. Coke. Simultaneous activation of C-X-C chemokine receptor 4 and cannabinoid receptor 2 results in decreased cell signaling and tumor cell intravasation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4579.
Abstract 4584: Apoptosis-mediated AXL activation: Contribution to metastatic cancers
Authors: Zweemer Annelien , Meyer Aaron , Lauffenburger Douglas
Published: July 2016

AXL is a receptor tyrosine kinase that is widely expressed in a variety of cancers and is predictive of poor patient outcome. This receptor is an essential epithelial-to-mesenchymal transition-induced regulator of cancer metastasis and is related to the occurrence of treatment resistance. AXL activation requires both the lipid moiety phosphatidylserine (PtdSer) and the bridging protein ligand Gas6. PtdSer is only available to mediate AXL activation when it is externalized on cell membranes, an event that occurs specifically during apoptosis. For that reason, we aimed to identify if apoptotic cell debris is capable of activating AXL on cancer cells and how this would affect the metastatic capacity of these cells.

In a non-AXL expressing tumor cell line (HCC827) apoptosis was induced by means of UV-irradiation. We detected AXL activation of AXL-expressing MDA-MB-231 cells that were treated with HCC827 cell debris, suggesting that apoptotic debris of dying tumor cells is capable of activating AXL in surviving surrounding tumor cells. Next we determined the contribution of PtdSer-mediated AXL activation to the migration of MDA-MB-231 cells in a wound scratch assay. The addition of warfarin, which prevents autocrine Gas6 binding to PtdSer, decreased migration to a similar extent as the AXL inhibitor R428.

This study revealed that PtdSer-mediated AXL activation contributes to the migration properties of MDA-MB-231 cells. As we detected AXL activation upon addition of PtdSer-containing apoptotic cells debris, this suggests that the induction of apoptosis by cancer therapeutics might enhance the metastatic capacities of neighboring surviving tumor cells. This finding may broadly impact the oncology field given AXL's widespread expression among different types of cancers, and provides a rationale for the design of novel combination therapies for decreasing tumor metastasis.

Citation Format: Annelien Zweemer, Aaron Meyer, Douglas Lauffenburger. Apoptosis-mediated AXL activation: Contribution to metastatic cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4584.
Abstract 4585: ROR1 sustains caveolae and RTK-mediated survival signaling as a scaffold of cavin-1 and CAV1 in lung cancer
Authors: Yamaguchi Tomoya , Lu Can , Ida Lisa , Yanagisawa Kiyoshi , Usukura Jiro , Cheng Jinglei , Hotta Naoe , Shimada Yukako , Isomura Hisanori , Suzuki Motoshi , Fujimoto Toyoshi , Takahashi Takashi
Published: July 2016

TTF-1/NKX2-1, a homeobox-containing transcription factor indispensable for lung morphogenesis, was previously identified by four independent groups including us, as a “lineage-survival” oncogene involved in the pathogenesis of lung adenocarcinoma. We have further shown that TTF-1/NKX2-1 induces expression of the receptor tyrosine kinase-like orphan receptor 1 (ROR1), which in turn plays a “sustainer” role for EGFR-mediated signaling. Interestingly, ROR1 knockdown effectively overcame EGFR-TKI resistance-conferring, HGF-mediated bypass signaling through MET, suggesting that ROR1 sustains not only EGFR but also additional RTK signaling. However, the underlying mechanism remained rather elusive.

We report here an unanticipated function of this receptor tyrosine kinase (RTK) as a scaffold of cavin-1 and caveolin-1 (CAV1), two essential structural components of caveolae. Caveolae are 50-100 nm invaginations of the plasma membrane that play various physiological roles, including function as a platform for insulin-induced signaling in adipose tissue. We found that ROR1 facilitates the interactions of cavin-1 and CAV1 at the plasma membrane in a kinase-independent manner, thereby preventing the lysosomal degradation of CAV1, as well as consequential caveolae formation. Caveolae structures and prosurvival signaling towards AKT through multiple RTKs such as MET and IGF-IR are consequently sustained. The present findings thus provide mechanistic insight into how ROR1 inhibition can overcome EGFR-TKI resistance due to bypass signaling via diverse RTKs, which is currently a major clinical obstacle.

Inhibition of prosurvival signaling and proliferation of lung cancer cells could be recapitulated by knocking down cavin-1 or CAV1, but they are unlikely to be suitable molecular targets for NSCLC treatment due to their crucial physiological functions in various organs even at low expression levels. In this regard, it should be noted that ROR1 is considered to be an onco-embryonic antigen with tumor-specific expression in adults. Taken together, development of novel therapeutic means to inhibit the scaffold function of ROR1 and thereby attack the cancer's “Achilles heel” is thus anticipated to be an attractive approach for improved treatment of this devastating cancer.

Citation Format: Tomoya Yamaguchi, Can Lu, Lisa Ida, Kiyoshi Yanagisawa, Jiro Usukura, Jinglei Cheng, Naoe Hotta, Yukako Shimada, Hisanori Isomura, Motoshi Suzuki, Toyoshi Fujimoto, Takashi Takahashi. ROR1 sustains caveolae and RTK-mediated survival signaling as a scaffold of cavin-1 and CAV1 in lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4585.
Abstract 4590: Mechanism of PI3K activation by the HER3/ErbB3 receptor
Authors: Michael Nicole , Hopkins Michael , Jura Natalia
Published: July 2016

Human epidermal growth factor receptor 3 (HER3, also known as ErbB3) is a receptor tyrosine kinase that lacks catalytic activity but is essential for cellular homeostasis due to its ability to allosterically activate its heterodimeric partners, EGFR and HER2 (also known as HER1/ErbB1 and ErbB2, respectively). HER3 is an important therapeutic cancer target because the signaling emanating from HER3 heterodimers is critical for the progression and maintenance of several human epithelial malignancies. This is primarily due to the potent ability of HER3 to recruit and activate the phosphoinositide 3-kinase (PI3K). This ability has been attributed to the presence of multiple direct binding sites for PI3K located in the intracellular tail of HER3. The advantage of having multiple recruitment sites is unclear and our recent data show that they provide little advantage to the ability of HER3 to activate PI3K. In addition, we have identified an alternative mode by which HER3 can activate PI3K, demonstrating a complex mechanism by which HER3 phosphorylation is coupled to activation of PI3K/Akt signaling pathway. Our results have important implications for therapeutic targeting of a number of human cancers in which HER3/PI3K signaling axis is abnormally activated and contributes to disease progression.

Citation Format: Nicole Michael, Michael Hopkins, Natalia Jura. Mechanism of PI3K activation by the HER3/ErbB3 receptor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4590.