bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-317: Potent in vivo pharmacology of AZD9150, a next-generation, constrained ethyl-modified antisense oligonucleotide targeting STAT3 in multiple preclinical cancer models.
Authors: Kim Youngsoo , Hsu Jeff , Zhou Tianyuan , Zhang Nancy , Woessner Richard , Nadella Murali VP , Lawson Deborah , Reimer Corinne , He Guobin , Schmidt Joanna , Xiao Xiaokun , Greenlee Sarah , Bhattacharjee Gourab , Hung Gene , Monia Brett P. , MacLeod A. Robert
Published: August 2014

Next generation sequencing technologies have greatly expanded our understanding of cancer genomes, epigenomes and transcriptomes. This knowledge, however, has not yet been effectively translated into improved cancer therapeutics, partly due to the inability of available therapeutic modalities to target the most promising cancer driver pathways. In contrast to other therapeutic approaches, the druggable universe is not limited with antisense technology as inhibitors can be rationally designed based on sequence information alone. Recent human clinical data has demonstrated potent activity of systemically-administered, unformulated, antisense oligonucleotides (ASOs) when targeted to liver expressed genes. However, robust activity in extra-heptatic tissues and tumors has been limited with existing ASO chemistries. Here we evaluate the activity of high affinity next generation (constrained ethyl) ASOs in extrahepatic tissues and tumors of multiple preclinical cancer models including spontaneous tumors, human tumor xenografts and several primary patient-derived xenograft models. As a test case we employed next generation ASOs to inhibit the difficult to drug transcription factor STAT3. ASOs targeting mouse STAT3 sequences and the human-specific STAT3 ASO (AZD9150) demonstrate potent and selective inhibition of target RNA and protein levels in tumors and tumor-associated stromal cells of a broad range of cancer models, resulting in strong antitumor activity in several models. These findings suggest that next generation ASO technology is now poised to become a key therapeutic modality to bridge the pharmacogenomic divide in cancer drug discovery. The STAT3 ASO inhibitor, AZD9150 is currently in human clinical studies including patients with lymphomas.

Citation Format: Youngsoo Kim, Jeff Hsu, Tianyuan Zhou, Nancy Zhang, Richard Woessner, Murali VP Nadella, Deborah Lawson, Corinne Reimer, Guobin He, Joanna Schmidt, Xiaokun Xiao, Sarah Greenlee, Gourab Bhattacharjee, Gene Hung, Brett P. Monia, A. Robert MacLeod. Potent in vivo pharmacology of AZD9150, a next-generation, constrained ethyl-modified antisense oligonucleotide targeting STAT3 in multiple preclinical cancer 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 LB-317. doi:10.1158/1538-7445.AM2013-LB-317
Abstract LB-323: Deficiency of Kruppel-like factor KLF4 in mammary tumor cells resulted in inhibited tumor growth and pulmonary metastasis accompanied by compromised recruitment of myeloid-derived suppressor cells.
Authors: Yu Fang , Fan Daping , Ai Walden
Published: August 2014

Increasing evidence indicates that myeloid-derived suppressor cells (MDSCs) negatively regulate immune responses during tumor progression, inflammation and infection. However, the underlying molecular mechanisms of their development and mobilization remain to be fully delineated. Kruppel-like factor KLF4 is a transcription factor that has an oncogenic function in breast cancer development, but its function in tumor microenvironment, a critical component for tumorigenesis, has not been examined. By using a spontaneously metastatic 4T1 breast cancer mouse model and an immunodeficient NOD/SCID mouse model, we demonstrated that KLF4 knockdown delayed tumor development and inhibited pulmonary metastasis, which was accompanied by decreased accumulation of MDSCs in bone marrow, spleens and primary tumors. Mechanistically, we found that KLF4 knockdown resulted in a significant decrease of circulating GM-CSF, an important cytokine for MDSC biology. Consistently, recombinant GM-CSF restored the frequency of MDSCs in purified bone marrow cells incubated with conditioned medium from KLF4 deficient cells. In addition, we identified CXCL5 as a critical mediator to enhance the expression and function of GM-CSF. Reduced CXCL5 expression by KLF4 knockdown in primary tumors and breast cancer cells was correlated with a decreased GM-CSF expression in our mouse models. Finally, we found that CXCL5/CXCR2 axis facilitated MDSC migration and that anti-GM-CSF antibodies neutralized CXCL5-induced accumulation of MDSCs. Taken together, our data suggest that KLF4 modulates maintenance of MDSCs in bone marrow by inducing GM-CSF production via CXCL5 and regulates recruitment of MDSCs into the primary tumors through the CXCL5/CXCR2 axis, both of which contribute to KLF4-mediated mammary tumor development.

Citation Format: Fang Yu, Daping Fan, Walden Ai. Deficiency of Kruppel-like factor KLF4 in mammary tumor cells resulted in inhibited tumor growth and pulmonary metastasis accompanied by compromised recruitment of myeloid-derived suppressor 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 LB-323. doi:10.1158/1538-7445.AM2013-LB-323
Abstract LB-324: The response of infiltrating and resident macrophages to melanoma challenge in the mouse lung: Involvement of the CC-chemokine axis in the recruitment of pro-tumoral macrophages.
Authors: Tapmeier Thomas T. , Gal Annamaria , Muschel Ruth J.
Published: August 2014

In a pulmonary metastasis model using B16F10 melanoma, we found monocyte/macrophage infiltration gradually increasing in the course of metastatic progression. The gene expression pattern of the infiltrating macrophages revealed an early (d3) inflammatory response that shifted to a tumor-promoting late response (d21). The pro-tumoral expression pattern was characterized by Arg1 and elevated expression of Ccl2, Vcam1, Vegfa, Stab1, Lyve1 and Ptgs2. In contrast, while enhancing their anti-inflammatory phenotype, resident lung (alveolar) macrophages also expressed pro-inflammatory genes, notably Il12b.

We found at least three chemokine receptors involved in the recruitment of macrophages into the metastatic lung: CCR1, CCR2 and CCR5. The B16F10 cells secreted large quantities of RANTES which in turn activates CCR1 and CCR5, and thus contribute to macrophage chemotaxis. Infiltrating monocytes/macrophages, as well as alveolar macrophages, revealed an increased chemokine expression level in response to tumor challenge. These chemokines included the ligands for CCR1 and CCR5, Mip1α and Mip1β, thus providing a constant means of further macrophage recruitment. Interestingly, the expression of the CCR2 ligand, CCL2, was found strikingly increased by infiltrating monocytes/ macrophages, thus ensuring a positive feed-back loop for macrophage recruitment.

In order to explore the involvement of CC chemokine receptors in pulmonary metastasis, we treated the mice with antagonists against CCR1, CCR5 and CCR2 after i.v. B16F10 challenge. The CCR1 and CCR5 antagonist treatments resulted in a 20% decrease in the number of lung colonies each. FACS analysis of the B16F10 colony bearing lungs revealed a distribution of macrophage subsets quite different from untreated lungs, highlighting the importance of CC-chemokine receptors in the recruitment of pro-tumoral macrophages. The effect of a CCR2 antagonist on lung colony formation will be discussed, together with its effect on the phenotype of macrophage subpopulations.

Citation Format: Thomas T. Tapmeier, Annamaria Gal, Ruth J. Muschel. The response of infiltrating and resident macrophages to melanoma challenge in the mouse lung: Involvement of the CC-chemokine axis in the recruitment of pro-tumoral macrophages. [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 LB-324. doi:10.1158/1538-7445.AM2013-LB-324
Abstract LB-335: Autologous whole-tumor antigen vaccination in combination with adoptive T cell therapy for patients with recurrent ovarian cancer.
Authors: Kandalaft Lana E. , Tanyi Janos , Chiang Cheryl , Powell Daniel , coukos George
Published: August 2014

Novel therapeutic strategies are warranted in recurrent ovarian cancer. We report two independent consecutive studies of combinatorial immunotherapy comprising dendritic cell (DC)-based autologous whole tumor antigen vaccination in combination with antiangiogenesis therapy. 31 patients with recurrent progressive stage III and IV ovarian cancer with available tumor lysate from secondary debulking surgery enrolled in two different studies. First 6 underwent priming with intravenous bevacizumab and oral metronomic cyclophosphamide followed by vaccination with an autologous DC preparation pulsed with freeze-thaw autologous tumor lysate while the other 25 underwent vaccination with an enhanced vaccine where autologous DCs were loaded with HOCl-oxidized autologous tumor lysate administered intranodally every 2 weeks in combination with intravenous bevacizumab. Both studies were followed by lymphodepletion and transfer of autologous vaccine-primed, ex vivo CD3/CD28-costimulated peripheral blood T-cells, in combination with antiangiogenesis therapy and vaccination. Feasibility, safety, and biological and clinical efficacy were evaluated. Eleven subjects have completed vaccination and T cell transfer to date, while twenty-three additional subjects completed vaccination only. Vaccination was well tolerated and elicited tumor-specific T cell responses against various ovarian tumor antigen in both studies and clinical benefit of 65% correlated with the immune response with some experiencing prolonged progression free survival. Following lymphodepletion, adoptive transfer of vaccine-primed T-cells was well tolerated and resulted in durable reduction of T-regulatory cells and restoration of vaccine-induced antitumor immunity in patients who experienced clinical benefit. One patient exhibited no evidence of disease at end of study. Stable disease was observed in 8 subjects to date. Data on additional patients will be presented at the meeting. Our results suggest the use of combinatorial cellular immunotherapy comprising DC vaccination with whole tumor antigen and adoptive lymphocyte transfer using tumor antigen-specific T cells for the treatment of patients with recurrent ovarian cancer is promising yet warrants further investigation.

Citation Format: Lana E. Kandalaft, Janos Tanyi, Cheryl Chiang, Daniel Powell, George coukos. Autologous whole-tumor antigen vaccination in combination with adoptive T cell therapy for patients with recurrent 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 LB-335. doi:10.1158/1538-7445.AM2013-LB-335
Abstract LB-349: Tumor associated macrophages mediate gemcitabine resistance of pancreatic adenocarcinoma by upregulating cytidine deaminase.
Authors: Binenboum Yoav , Weissman Noam , Kerlin Yakov , Shabtai Ayelet , Amit Moran , Gil Ziv
Published: August 2014

Resistance to pharmacologic agents used in chemotherapy is a common phenomenon in most human carcinomas. Pancreatic ductal adenocarcinoma (PDA) is such a tumor, which is resistant to almost all drugs, including gemcitabine, a new nucleoside analog used as first-line treatment. For this reason the poor survival of patients with PDA have not change much during the last 4 decades. Recent data indicated that tumor-associated macrophages (TAMs) which are abundant in the microenvironment of several tumors, including PDA, secrete pro-tumorigenic factors which contribute to cancer progression and dissemination. In this study, we show for the first time that TAMs can also induce chemoresistance of PDA by reducing gemcitabine-induced apoptosis. Macrophages co-cultures with cancer cells or TAMs conditioned medium significantly reduced the levels of cleaved caspase-3 and caspase-3 during gemcitabine treatment. An in vivo PDA model of CCR2-deficient mice, which have reduced macrophage recruitment and activation, demonstrated improved response to gemcitabine compared to wild-types. Similarly, inhibition of monocytes/macrophages trafficking by a CSF1-receptors antagonist augmented the effect of gemcitabine in a transgenic mouse model of PDA that was resistant to gemcitabine alone. Analysis of multiple proteins involved in gemcitabine delivery and metabolism revealed that TAMs induced upregulation of cytidine deaminase (CDA), the enzyme that metabolizes the active form of the drug. Decreasing of CDA expression by PDA cells blocked the protective effect of TAMs against gemcitabine. Taken together, our results identify a paracrine effect of TAMs on PDA cells, which mediates acquire resistance of cancer cells to chemotherapy. Modulation of macrophage trafficking or inhibition of CDA may offer a new strategy for augmenting the response of PDA to chemotherapy.

Citation Format: Yoav Binenboum, Noam Weissman, Yakov Kerlin, Ayelet Shabtai, Moran Amit, Ziv Gil. Tumor associated macrophages mediate gemcitabine resistance of pancreatic adenocarcinoma by upregulating cytidine deaminase. [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 LB-349. doi:10.1158/1538-7445.AM2013-LB-349

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract LB-359: Inhibition of DNA repair by the dual PI3K/mTOR inhibitor NVP-BEZ235 as a novel radiosensitization strategy for glioblastoma.
Authors: Alcazar Carlos Gil Del , Gillam Molly , Tomimatsu Nozomi , Gao Xiaohuan , Mukherjee Bipasha , Burma Sandeep
Published: August 2014

Glioblastomas (GBM) are deadly brain tumors with extremely poor prognosis. The standard of care for GBM is tumor resection followed by treatment with ionizing radiation (IR) and the DNA alkylating agent temozolomide. As GBMs are highly radioresistant, it is crucial to identify effective radiosensitizers for improving therapeutic outcome. We find that NVP-BEZ235, a dual PI3K/mTOR inhibitor that is in clinical trials, potently inhibits both ATM and DNA-PKcs, the two major kinases that respond to IR-induced DNA double-strand breaks (DSBs). Consequently, this drug blocks non-homologous end joining (NHEJ) as well as homologous recombination (HR), the two major pathways of DSB repair. We find that low concentrations of NVP-BEZ235 (100 nM) can block DSB repair in a panel of human GBM lines resulting in significant radiosensitization. This radiosensitizing effect is more potent than that seen with much higher concentrations (10 μm) of current inhibitors of DNA-PKcs or ATM that are being optimized for clinical testing. In order to examine the effects of NVP-BEZ235 in vivo, we sub-cutaneously injected mice with radioresistant U87 glioma cells ectopically expressing EGFRvIII and treated with clinically-relevant doses of the drug. We find that NVP-BEZ235 blocks the activation of both DNA-PKcs and ATM in irradiated tumors resulting in significant attenuation of DSB repair. Tumors treated with a combination of IR and NVP-BEZ235 exhibit elevated DSBs and reduced Ki67 positivity compared to tumors treated with only IR or NVP-BEZ235. Consequently, a combination of IR and NVP-BEZ235 results in dramatic inhibition of tumor growth, in contrast to almost no inhibition with either treatment modality alone. Finally, we established that NVP-BEZ235 can cross the blood-brain barrier and therefore examined its effectiveness in an orthotopic brain tumor model. We find that NVP-BEZ235 given with IR can attenuate the growth of intracranial tumors generated from U87-EGFRvIII cells, thereby extending survival of tumor-bearing mice. Importantly, NVP-BEZ235 also sensitizes subcutaneous and intracranial tumors to temozolomide by attenuating the repair of secondary (replication-associated) DSBs that are generated upon temozolomide treatment. In sum, this study provides support for the potential use of NVP-BEZ235 as a radio- and chemo-sensitizer for improving the outcome of GBM therapy.

Citation Format: Carlos Gil Del Alcazar, Molly Gillam, Nozomi Tomimatsu, Xiaohuan Gao, Bipasha Mukherjee, Sandeep Burma. Inhibition of DNA repair by the dual PI3K/mTOR inhibitor NVP-BEZ235 as a novel radiosensitization strategy for glioblastoma. [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 LB-359. doi:10.1158/1538-7445.AM2013-LB-359
Abstract LB-38: VPS34 lipid kinase activity is positively regulated by Src phosphorylation and is required for Src-mediated cellular transformation.
Authors: Hirsch Dianne S. , Shen Yi , Dokmanovic Milos , Wu Wen Jin
Published: August 2014

VPS34 is a phosphatidylinositol 3-kinase (PI3K) that catalyzes phosphorylation of phosphatidylinositol. In response to nutrients, VPS34 contributes to the activation of mTOR/S6K1 signaling which regulates proliferation, protein synthesis, and cell survival. Intracellular mechanisms leading to VPS34 activation are just beginning to be elucidated. Src is a non-receptor tyrosine kinase that signals downstream of numerous pathways to regulate cell proliferation, survival, and cancer metastasis. Here, we report that Src directly phosphorylates VPS34 leading to activation of VPS34 lipid kinase activity, and that VPS34 lipid kinase activity is required for Src-mediated cellular transformation. Silencing VPS34 specifically inhibits Src-Y527F-induced colony formation in soft agar, but not Ras-G12V-induced colony formation. We have identified two novel VPS34 mutations, which either eliminate lipid kinase activity (KD mutant) or reduce tyrosine phosphorylation (Y231F mutant) by Src-Y527F. When VPS34-KD is stably expressed in Src-Y527F-transformed cells, proliferation and anchorage independent growth are significantly inhibited. Additionally, stable expression of VPS34-KD causes an increased number of binucleate and multinucleate cells, suggesting that VPS34 kinase activity is also required for cytokinesis. Stable expression of VPS34-Y231F in Src-Y527F transformed cells also reduces colony formation. We additionally found that VPS34 protein levels and tyrosine phosphorylation correlate with the tumorigenic activity of breast cancer cell lines. Together these data suggest that Src to VPS34 signaling may contribute to the development and progression of human cancers.

Citation Format: Dianne S. Hirsch, Yi Shen, Milos Dokmanovic, Wen Jin Wu. VPS34 lipid kinase activity is positively regulated by Src phosphorylation and is required for Src-mediated cellular transformation. [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 LB-38. doi:10.1158/1538-7445.AM2013-LB-38
Abstract LB-41: Rictor undergoes GSK3-dependent, FBXW7-mediated ubiquitination and proteasomal degradation.
Authors: Koo Junghui , Wu Xiayun , Yue Ping , Mao Zixu , Khuri Fadlo R. , Sun Shi-Yong
Published: August 2014

Rictor directly interacts with mTOR and other proteins to form an important complex (i.e., mTOR complex 2, mTORC2) that plays critical role in regulation of cellular homeostasis primarily by phosphorylating some AGC kinases such as Akt, PKC and SGK. Moreover, rictor also exerts biological functions such as regulation of protein degradation by interacting with other proteins independent of mTORC2. However the degradation of rictor itself and underlying mechanism are largely unknown and thus are the focuses of this study. We found that inhibition of proteasome with the proteasome inhibitor MG132 increased rictor levels accompanied with elevated rictor ubiquitination. Consistently inhibition of F-box and WD repeat domain-containing 7 (FBXW7; also known as FBW7 and CDC4) with various genetic means including knockdown, knockout and enforced expression of a dominant-negative mutant inhibited rictor ubiquitination and increased rictor levels, whereas enforced expression of FBXW7 decreased rictor stability and levels. Moreover, we detected an interaction between FBXW7 and rictor by immunoprecipitation. Hence, rictor is degraded through a FBXW7-mediated ubiquitination/proteasome mechanism. This process is dependent on GSK3 because GSK3 was associated with rictor and directly phosphorylated the Thr1695 site in a putative CPD motif of rictor; mutation of this site impaired the interaction between rictor and FBXW7, decreased rictor ubiquitination and increased rictor stability. Collectively we conclude that rictor undergoes GSK3-dependent, FBXW7-mediated ubiquitination and proteasomal degradation. (J Koo and X Wu contributed equally to this work. This study was supported by Georgia Cancer Coalition Distinguished Cancer Scholar award and NIH R01 CA118450, R01 CA160522 and P01 CA116676. FR Khuri and S-Y Sun are Georgia Cancer Coalition Distinguished Cancer Scholars.)

Citation Format: Junghui Koo, Xiayun Wu, Ping Yue, Zixu Mao, Fadlo R. Khuri, Shi-Yong Sun. Rictor undergoes GSK3-dependent, FBXW7-mediated ubiquitination and proteasomal degradation. [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 LB-41. doi:10.1158/1538-7445.AM2013-LB-41
Abstract LB-42: In vivo screen for novel breast cancer promoting genes.
Authors: Huo Yongliang , Macara Ian
Published: August 2014

Mis-regulation of oncogenes and tumor suppressor genes causes cancer. Although the evolution of cancer is not conclusive, most evidence supports that tumors arise from collaboration of multiple genes. To better understand the process of breast tumor initiation and cancer progression we carried out an in vivo screen, for genes that promote tumor formation. Our screen was performed using lentiviral transduction of primary murine mammary epithelial cells, and orthotopic implantation into syngeneic recipient mice. Wild type mammary stem cells can regenerate an entire mammary ductal tree in the recipients. We chose a well-characterized ErbB2/Her2 over-expression mouse model as a source of donor cells because HER2 over-expression occurs in >20% of human breast cancer patients. A cDNA library consisting of a “druggable” set of ~1000 genes was screened in 12 subsets segregated by ORF length. Ten months after transplantation the recipient mice were euthanized and DNA from mammary gland tumors was harvested. No tumors formed in mice transplanted with cells into which a control virus had been transduced. Genes from the library that were expressed in the tumors were identified by sequencing. The ability of these genes to drive tumor formation was confirmed by transduction and transplantation into a second cohort of mice. One of the genes identified in this screen is GTF2I repeat domain containing 1 (GTF2IRD1). Multi-focal atypical hyperplasia formed in mammary glands over-expressing GTF2IRD1 while no hyperplasia was seen in the control mice. This effect depends on the expression of ErbB2 because expression of GTF2IRD1 itself in wild type mammary glands did not cause abnormal growth before the end point. Analysis of the cancer genome atlas database demonstrates that GTF2IRD1 expression is significantly increased in invasive breast cancer compared to normal controls. Therefore, our screen has identified a novel tumor-promoting gene that is of potential clinical importance. A major advantage of our method is the use of normal cells of origin and immunocompetent mice. Moreover, it could be expanded to genome-wide searches for new tumor- and metastasis-promoting genes.

Citation Format: Yongliang Huo, Ian Macara. In vivo screen for novel breast cancer promoting genes. [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 LB-42. doi:10.1158/1538-7445.AM2013-LB-42
Abstract LB-57: Knockdown of RNF2 induces apoptosis by regulating MDM2 and p53 stability.
Authors: Wen Weihong , Peng Cong , Bode Ann M. , Dong Zigang
Published: August 2014

RNF2, also known as Ring1B/Ring2, is a component of the polycomb repression complex 1. RNF2 is highly expressed in many tumors, suggesting that it might have an oncogenic function, but the mechanism is unknown. Here, we show that knockdown of RNF2 significantly inhibits both cell proliferation and colony formation in soft agar, and induces apoptosis in cancer cells. Knockdown of RNF2 in HCT116 p53+/+ cells resulted in significantly more apoptosis than was observed in RNF2 knockdown HCT116 p53−/- cells, indicating that RNF2 knockdown-induced apoptosis is partially dependent on p53. Various p53-targeted genes were increased in RNF2 knockdown cells. Further studies revealed that in RNF2 knockdown cells, the p53 protein level was increased, the half-life of p53 was prolonged and p53 ubiquitination was decreased. In contrast, cells overexpressing RNF2 showed a decreased p53 protein level, a shorter p53 half-life and increased p53 ubiquitination. Importantly, we found that RNF2 directly binds with both p53 and MDM2 and promotes MDM2-mediated p53 ubiquitination. RNF2 overexpression could also increase the half-life of MDM2 and inhibit its ubiquitination. The regulation on p53 and MDM2 stability by RNF2 was also observed during the etoposide-induced DNA damage response. These results provide a possible mechanism explaining the oncogenic function of RNF2, and because RNF2 is important for cancer cell survival and proliferation, it might be an ideal target for cancer therapy or prevention.

Citation Format: Weihong Wen, Cong Peng, Ann M. Bode, Zigang Dong. Knockdown of RNF2 induces apoptosis by regulating MDM2 and p53 stability. [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 LB-57. doi:10.1158/1538-7445.AM2013-LB-57

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract LB-60: The BRAF:MEK complex reveals new therapeutic strategies for targeting KRASMT tumors.
Authors: Haling Jacob R. , Sudhamsu Jawahar , Hatzivassiliou Georgia , Song Kyung , Belvin Marcia , Hymowitz Sarah , Malek Shiva
Published: August 2014

Spatial activation and regulation of MAPK signaling is determined by genetic context. In BRAFV600E tumor models kinase activity of BRAF is insensitive to normal regulatory mechanisms, whereas in KRASMT tumor models CRAF is the primary driver of the pathway and its function is governed by RAF dimerization, translocation to the plasma membrane, and phosphorylation. Since MEK is a common downstream substrate, multiple allosteric MEK inhibitors are being tested in clinical trials in BRAFV600E and KRASMT cancer types. To date, the efficacy of MEK inhibitors has been substantially higher in BRAFV600E tumors than in KRASMT tumors suggesting that the mechanism regulating MEK activity is different in these settings. In this work, we have discovered that BRAF has a suppressive function in KRASMT tumor lines by forming a stable cytosolic complex with MEK which prevents MEK from being phosphorylated by CRAF. Enrichment of this complex in KRASMT cells results in dampened phospho-MEK levels compared to BRAFV600E cells where little cytosolic BRAF:MEK complexes are observed. Treatment of KRASMT tumor cells with particular classes of MEK inhibitors stabilize the BRAF:MEK complex and inhibit phosphorylation of MEK, while other MEK inhibitors dissociate the complex and enhance MEK phosphorylation. Further we find that the former class of MEK inhibitors selectively prefers binding to the BRAF:MEK complex compared to MEK alone based on the significantly slower off-rate for the BRAF:MEK complex. The crystal structure of a heterotetrameric BRAF:MEK complex in the presence of G-573, an allosteric MEK inhibitor, provides structural rationale for this observation and sheds light on the ability of G-573 and similar classes of MEK inhibitors to prevent RAF phosphorylation of MEK at Ser-218 and Ser-222. Although all MEK inhibitors function to inhibit ERK phosphorylation, our data explains the discrepancy of MEK activation between different classes of MEK inhibitors. Hence we conclude that more effective strategies to treat KRASMT tumors may require targeting signaling protein complexes such as BRAF:MEK as opposed to inhibiting individual components of the pathway. Importantly our work illustrates small molecule kinase inhibitors may not only affect enzymatic activity but also regulate protein-protein interactions thereby influencing pathway signaling.

Citation Format: Jacob R. Haling, Jawahar Sudhamsu, Georgia Hatzivassiliou, Kyung Song, Marcia Belvin, Sarah Hymowitz, Shiva Malek. The BRAF:MEK complex reveals new therapeutic strategies for targeting KRASMT tumors. [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 LB-60. doi:10.1158/1538-7445.AM2013-LB-60
Abstract LB-88: A new rapid diagnostic test to identify epidermal growth factor receptor mutations in non-small cell lung cancer.
Authors: Chikumi Hiroki , Takata Miyako , Matsunami Keiji , Matsumoto Shingo , Kotani Masahiro , Sakamoto Tomohiro , Touge Hirokazu , Yamaguchi Kosuke , Kurai Jun , Miyake Naomi , Nakamoto Masaki , Igishi Tadashi , Shimizu Eiji
Published: August 2014

An epidermal growth factor receptor (EGFR) mutation is the best marker of sensitivity to the EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Polymerase chain reaction (PCR)-based methods combined with the use of fluorescence probes, such as the Scorpion Amplification Refractory Mutation System (ARMS) or the PCR-Invader method, are frequently used to detect EGFR mutations before therapy. The sensitivities and specificities of these methods are satisfactory; however, there is potential to further improve the cost and detection time. We developed a new method for the rapid and reliable detection of EGFR mutations by using a combination of mutation-specific primers and the newly developed ultra-rapid real-time PCR (Hyper-PCR) amplification method that can be accomplished in <7 min. We designed specific ARMS primers for the common EGFR del E746-A750 mutations, and optimized the reaction conditions for the PCR machine that contained a thin disc-type reaction vessel, the temperature of which could be quickly altered by rotating it on thermal sources. We used Hyper-PCR to analyze the common EGFR mutations del E746-A750 and L858R in lung cancer samples from 143 non-small cell lung cancer (NSCLC) patients. The results were compared with those obtained by direct sequencing, conventional PCR, and the PCR-Invader method. Using the direct sequencing method, 13 (9.1%) and 15 (10.5%) NSCLC samples were positive for the del E746-A750 and L858R mutations, respectively, while the Hyper-PCR method revealed that 15 (10.5%) and 18 (12.6%) samples were positive for the del E746-A750 and L858R mutations, respectively. Five samples, whose results were discordant between the Hyper-PCR and direct sequencing methods, were analyzed by conventional PCR and the PCR-Invader method, and the results were found to be consistent with those obtained by Hyper-PCR. These results suggest that the novel Hyper-PCR method is an inexpensive, rapid, and reliable diagnostic test for EGFR mutations.

Citation Format: Hiroki Chikumi, Miyako Takata, Keiji Matsunami, Shingo Matsumoto, Masahiro Kotani, Tomohiro Sakamoto, Hirokazu Touge, Kosuke Yamaguchi, Jun Kurai, Naomi Miyake, Masaki Nakamoto, Tadashi Igishi, Eiji Shimizu. A new rapid diagnostic test to identify epidermal growth factor receptor mutations in non-small cell 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 LB-88. doi:10.1158/1538-7445.AM2013-LB-88
Abstract LB-99: The novel IGF-IR/Akt-dependent anticancer activities of glucosamine are affected by PIK3CA hot-spot mutations and PTEN deletion.
Authors: Kang Ju-Hee , Song Ki-Hoon , Nam Jeong-Seok , Min Hye-Young , Lee Ho-Young , Cho Sung-Dae , Kim Soo-Youl , Oh Seung Hyun
Published: August 2014

Background: Recent studies have shown that glucosamine inhibits the proliferation of various human cancer cell lines and downregulates the activity of COX-2, HIF-1α, p70S6K, and transglutaminase 2.

Objective: Because the IGF-1R/Akt pathway is a common upstream regulator of p70S6K, HIF-1α, and COX-2, we hypothesized that glucosamine inhibits cancer cell proliferation through this pathway.

Methods: Cell viability was assayed by MTT assay. Total RNA was isolated and reverse transcribed to cDNA for real-time PCR quantification of genes. Western blotting as performed for analyzing expression of proteins. Flow cytometry was performed for apoptosis and cell progression. Balbc/nu mice used in the study for validating results in vivo.

Results: We found that glucosamine inhibited the growth of human non-small cell lung cancer cells in vitro and in vivo and negatively regulated the expression of IGF-1R and phosphorylation of Akt. In other types of cancer cells, including head and neck, breast, prostate, and colon carcinoma cell lines, glucosamine-sensitive cell lines exhibited a more significant decrease in IGF-1R and pAkt levels than glucosamine-resistant cell lines. Interestingly, most of the glucosamine-resistant cell lines have “hot-spot” mutations in PIK3CA, the p110α subunit of PI3K, or loss of PTEN, a negative regulator of Akt activation. In contrast, most of the glucosamine-sensitive cell lines have normal PIK3CA and PTEN genes. Glucosamine decreased the pAkt level through activation of IGF-1R more efficiently in the glucosamine-sensitive than in the glucosamine-resistant cell lines. Furthermore, co-treatment of cells with glucosamine and LY294002, a specific inhibitor of PI3K, significantly enhanced the anticancer effect of glucosamine in the glucosamine-resistant cell lines, whereas transient inhibition of PTEN by siRNA partially decreased the glucosamine sensitivity in the resistant and sensitive cell lines.

Conclusions: Our results indicate that glucosamine is an effective inhibitor of the IGF-1R/Akt pathway and that glucosamine sensitivity of each cell line was affected by the mutation status of PIK3CA and PTEN.

Citation Format: Ju-Hee Kang, Ki-Hoon Song, Jeong-Seok Nam, Hye-Young Min, Ho-Young Lee, Sung-Dae Cho, Soo-Youl Kim, Seung Hyun Oh. The novel IGF-IR/Akt-dependent anticancer activities of glucosamine are affected by PIK3CA hot-spot mutations and PTEN deletion. [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 LB-99. doi:10.1158/1538-7445.AM2013-LB-99
Abstract PL02-01: Cancer genetic and epigenetic interactions.
Authors: Laird Peter W.
Published: August 2014

Cancer develops not only as a result of genetic mutations and genomic rearrangements, but also as a consequence of numerous epigenetic alterations, including extensive changes in the distribution of DNA methylation throughout the genome. DNA methylation changes contribute directly to cancer by transcriptional silencing of tumor-suppressor genes through promoter CpG island hypermethylation.

Cancer genome studies have uncovered somatic mutations in dozens of epigenetic regulators in almost all types of cancer, and integrative analyses have revealed associations between various somatic genetic alterations and broad epigenomic profiles. Some somatic mutations are very tightly associated with distinct epigenetic subtypes, such as CpG Island Methylator Phenotypes (CIMP). In some cases, the somatic mutation appears to contribute causally to the epigenomic profile, such as in the case of mutation of IDH1 in G-CIMP glioblastoma, while in other cases the epigenomic alterations appear to be a prerequisite for somatic mutation, such as colorectal CIMP-associated changes contributing to suppression of mutant BRAF-mediated senescence.

Other cancer epigenomic profiles are attributable to stochastic events or cell-of-origin chromatin states. For example, genomic loci targeted by Polycomb Group Repressors in embryonic stem cells, and involved in cellular differentiation are predisposed to aberrant DNA methylation in cancer cells, suggesting that an epigenetic block to cellular differentiation may sometimes be an initiating event in carcinogenesis. The analysis of whole methylomes at single-basepair resolution reveals that cancer-associated changes occur differentially across defined regions of the genome associated with the nuclear lamina.

It is apparent that epigenomic analysis is essential for a full understanding of the relationship between alterations in the cancer genome and the origin and clinical diversity of individual tumors.

Citation Format: Peter W. Laird. Cancer genetic and epigenetic interactions. [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 PL02-01. doi:10.1158/1538-7445.AM2013-PL02-01
Abstract SY01-01: Regulation of growth by the mTOR pathway.
Authors: Sabatini David M.
Published: August 2014

mTOR is the target of the immunosuppressive drug rapamycin and the central component of a nutrient-and hormone-sensitive signaling pathway that regulates cell growth and proliferation. We now appreciate that this pathway becomes deregulated in many human cancers and has an important role in the control of metabolism and aging. We have identified two distinct mTOR-containing proteins complexes, one of which regulates growth through S6K and another that regulates cell survival through Akt. These complexes, mTORC1 and mTORC2, define both rapamycin-sensitive and insensitive branches of the mTOR pathway. I will discuss new results from our lab on the regulation and functions of the mTORC1 and mTORC2 pathways.

Citation Format: David M. Sabatini. Regulation of growth by the mTOR pathway. [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 SY01-01. doi:10.1158/1538-7445.AM2013-SY01-01
Abstract SY05-02: Understanding colorectal cancer as a stem cell hierarchy.
Authors: Batlle Eduard
Published: August 2014

The inner layer of the intestinal tube, the intestinal epithelium, is in a constant process of renewal. Hundreds of millions of terminally differentiated intestinal cells are replaced by new cells every day during the life of an adult organism. This tremendous regenerative power is ultimately sustained by a small population of intestinal stem cells. It is believed that alterations in the biology of human colon stem cells (CoSCs) account for the pathophysiology of various large-bowel disorders, including colorectal cancer (CRC). Yet, the identification of human CoSCs remains elusive. We have recently achieved for the first time the isolation of stem cells of the human colonic epithelium. Differential cell surface abundance of the receptor EPHB2 allows the purification of different cell types from human normal colon mucosa biopsies. Colon epithelial cells with highest EPHB2 levels exhibit the longest telomeres and express markers characteristic of intestinal stem cells. Using culturing conditions that recreate the intestinal stem cell niche, a substantial proportion of EPHB2-high cells can be expanded in vitro as an undifferentiated and multipotent population. Furthermore, we have also discovered that most human CRCs are constituted by cell populations with phenotypes similar to either CoSCs or intestinal differentiated cells organized into well-defined compartments. CoSC-like cells purified from primary CRCs generate tumors in immunodeficient mice with high efficiency and display both self-renewal and differentiation capacity. We demonstrated that CRC shares a common hierarchy with the intestinal mucosa and that the acquisition of an intestinal stem cell gene program is a central process during disease relapse after therapeutic intervention. Here I will present our latest data on the mechanisms employed by CRC stem cells to regenerate a new tumor at the metastatic site. We have discovered that metastasis relies on a tumor cell non-autonomous program expressed in the microenvironment. This stromal program confers a survival advantage to the disseminated CRC stem cells during the initial phase of metastasis.

Citation Format: Eduard Batlle. Understanding colorectal cancer as a stem cell hierarchy. [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 SY05-02. doi:10.1158/1538-7445.AM2013-SY05-02
Abstract SY12-02: Computational cancer genomics: Biological discovery and translation.
Authors: Schultz Nikolaus , Sander Chris
Published: August 2014

The analysis of genes and cellular pathways that are altered in different cancers can lead to biological discoveries and potential patient-specific and subtype-specific therapeutic hypotheses. Several large-scale studies, including those from The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC), aim to survey the landscape of genomic and molecular profiles of thousands of tumor samples in tens of different tumor types from generous patients. We analyze these informative genetic and molecular profiles to better understand the processes of oncogenesis, vulnerabilities to therapeutic interventions and tumor-subtype specific differences. Hypotheses can then be tested in focussed cell line and mouse model experiments and, if validated, advanced to clinical trials. To assist the general community of cancer biologists, we make available a generally useful feature map of essential information in the Cancer Genome Atlas via the information service cbioportal.org. Analysis and information services reported are based on the work of Niki Schultz, Giovanni Ciriello, Nils Weinhold, William Lee, Jian Jiong Gao, Rileen Sinha, Anders Jacobsen, Erik Larsson, Arman Aksoy, Gideon Dresdner, Benjamin Gross, several clinical research collaborators and the TCGA network.

Citation Format: Nikolaus Schultz, Chris Sander. Computational cancer genomics: Biological discovery and translation. [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 SY12-02. doi:10.1158/1538-7445.AM2013-SY12-02
Abstract SY16-01: Transcriptional regulation and epigenetics in zebrafish cancer.
Authors: Zon Leonard I.
Published: August 2014

Melanoma is a devastating disease for which few therapies lead to long term remission after metastasis has occurred. We have been using the zebrafish to model melanoma. A transgenic fish carrying human V600E BRAF mutation in combination with p53 mutation leads to a rapid melanoma model in the zebrafish. Using a transposon based approach, we have been able to overexpress particular potential oncogenes to look for acceleration of the melanoma. One such oncogene (SETDB1) is amplified in 30% of human melanomas and accelerates tumor rate in the zebrafish. Using a library of chromatin factors that are full length, we have been able to overexpress over 80 factors and found several new factors that activate and induce more melanoma. This approach has led to a fundamentally new insight into melanoma and epigenetic regulation. We also have found a chemical (leflunomide) that lowers nucleotide levels and induces a transcriptional pause of neural crest genes. Leflunomide and a BRAF inhibitor is now in clinical trial for patients with metastatic melanoma. To understand the mechanism of action, we have focused on PTEFb that is a kinase that regulates the elongation step in transcription. We found that the HEXIM complex is able to sequester PTEFb into an inactive state. HEXIM is a new tumor suppressor gene for melanoma. Our studies have led to a better understanding of transcription in melanoma and have developed a novel therapeutic that is being tested in the clinic.

Citation Format: Leonard I. Zon. Transcriptional regulation and epigenetics in zebrafish 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 SY16-01. doi:10.1158/1538-7445.AM2013-SY16-01
Abstract SY21-03: Skin stem cells in morphogenesis and cancer.
Authors: Fuchs Elaine , Beronja Slobodan , Schober Markus
Published: August 2014

Stem cells have the ability to self-renew long term and differentiate into one or more tissues. Typically, stem cells are used sparingly to replenish cells during normal homeostasis. This is particularly true of adult tissues that undergo relatively infrequent or periodic turnover. However, even stem cells that are quiescent must be able to respond quickly to injury in order to fuel rapid tissue regeneration. How stem cells balance self-renewal and differentiation is of fundamental importance to our understanding of normal tissue maintenance and wound repair. Moreover, increasing evidence suggests that the regulatory circuitry governing this balancing act is at the root of some types of tumors both in mice and in humans.

We've been using skin as a paradigm to understand how stem cells function in normal tissue generation and how this process goes awry in cancer. We've elucidated many of the pathways involved in how extrinsic signaling mobilizes stem cells to make tissue. In so doing, we have revealed mechanisms which are also deregulated in a variety of different human cancers. This has led us to the realm of identifying cancer stem cells (tumor-initiating cells) of squamous cell carcinomas of the skin. The second most abundant form of cancer world-wide, skin squamous cell carcinomas also establish a paradigm for many life-threatening cancers of lung, esophagus, breast, cervix, prostate, throat and oral tissues. We've demonstrated that when purified and transplanted serially into the skins of host recipients, a single cancer stem cell from a skin squamous cell carcinoma can generate a new squamous cell carcinoma that is similar in properties to the parent tumor. We've also discovered that cancer stem cell numbers and aggressiveness are predicated on the tumor microenvironment and on the ability of cancer stem cells to respond to it. The striking differences between the microenvironments of cancer stem cells and their normal counterparts seems likely to contribute to why their expression profiles are also markedly distinct.

These findings underscore the importance of dissecting the molecular mechanisms of tumor progression and growth in vivo. To this end, we've developed a non-invasive method to knockdown genes rapidly, efficiently and selectively in the skin epithelium. We've exploited this technology to carry out a whole genome-wide screen in mice for genes that are selectively involved in regulating oncogenic growth in the skin. Our findings have major implications for our understanding of tumor progression and cancer.

[This work was supported by the HHMI and a grant from the National Institutes of Health]

Citation Format: Elaine Fuchs, Slobodan Beronja, Markus Schober. Skin stem cells in morphogenesis and 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 SY21-03. doi:10.1158/1538-7445.AM2013-SY21-03