bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 747: p53 activation through stress induction mediates differential regulation of target genes.
Authors: Mittal Shilpi , Kadamb Rama , Bansal Nidhi , Saluja Daman
Published: August 2014

p53 is a sequence specific transcription factor that gets up regulated in response to stress such as DNA damage, microtubule disruption or oxidative stress. p53 protein gets stabilized upon stress induction and functions by activating and repressing large number of downstream genes involved in varying functions like cell cycle, DNA repair, apoptosis or senescence depending upon type of stress. Gene activation and repression is crucial for p53 to perform its functions in the cell. p53 mediates its activation functions through its ability to bind to cis-acting DNA elements within regulatory regions. However, p53 regulates negative expression of genes either through direct recruitment on the promoter or through indirect mechanisms. Studies show that genotoxic as well as nongenotoxic stress leads to accumulation of p53 protein in cell, though the downstream effects vary under both conditions. The various cellular mechanisms that take place in the cell following DNA damage are well studied; however less is characterized about agents that disrupt microtubule assembly.

To put an insight into such differential regulation mechanism, we focused to study the difference in cellular fate upon genotoxic and non genotoxic stress. In the present study, we observed that two varying stresses that include DNA damage caused by bleomycin and microtubule disruption by colchicine mediate cell cycle arrest and increased levels of p53. At the gene level, it was observed that upon bleomycin treatment, Sin3/HDAC corepressor complex is docked onto the promoters of p53 target genes whereas colchicine treatment results in dissociation of Sin3/HDAC complex from the same promoters. Moreover, recruitment of Sin3/HDAC complex is reflective of downregulation of p53 target genes and dissociation of this complex from the promoter brings about transactivation of the same genes.

Therefore, our studies highlight that mere activation of p53 in varying kind of stress is not a decisive factor in gene regulation, rather interaction of p53 with corepressor or coactivator directs succeeding processes depending upon the type of stress.

Citation Format: Shilpi Mittal, Rama Kadamb, Nidhi Bansal, Daman Saluja. p53 activation through stress induction mediates differential regulation of target 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 747. doi:10.1158/1538-7445.AM2013-747
Abstract 75: CpG methylation signature to predict outcome in preoperatively treated rectal cancer.
Authors: Gaedcke Jochen , Claus Rainer , Leha Andreas , Grade Marian , Beißbart Tim , Ghadimi Michael , Weichenhahn Dieter , Plass Christoph
Published: August 2014

Introduction: Introducing a preoperative therapy in locally advanced rectal cancer significantly decreased the local recurrence rate whereas outcome was not improved. To identify patient subgroups that could benefit from an adjusted therapy a prediction prior to the preoperative therapy is necessary.

Material and methods: Whole genome promoter methylation analyses were applied to a pilot group of patients (n=11) to identify targets that separate a good and a bad prognosis group based on their methylation level. Using a mass spectrometry based approach (MassArray) candidate CpG probes were assessed in set of 63 patients (training set). Relevant probes were then validated independently in an additional patient set (n=93). Data were then compared to the previously identified biomarkers (CpG island methylatior phenotype (CIPM)) and clinical data.

Results: Relevant DNA regions from whole genome screen were filtered and the number was reduced down to 20 DNA regions located within 17 different genes (244 different CpGs). In the training set thirteen regions could be confirmed and a clear separation into a good and a bad prognosis group was possible (p=0.0394). Validation in an independent cohort revealed a highly significant result (p=0.000341). Comparing the results the methylation panel outperformed CIMP or clinical data.

Conclusion: Using the retrieved methylation panel of 13 different DNA regions allows a highly significant assignment to a good and a bad prognosis group. These data can now be used to identify patients that are well treated by standard therapy and others that benefit from alternative treatment modalities.

Citation Format: Jochen Gaedcke, Rainer Claus, Andreas Leha, Marian Grade, Tim Beißbart, Michael Ghadimi, Dieter Weichenhahn, Christoph Plass. CpG methylation signature to predict outcome in preoperatively treated rectal 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 75. doi:10.1158/1538-7445.AM2013-75
Abstract 753: Reciprocal regulation of HOXB13 and androgen receptor in prostate cancer.
Authors: Chen Shuangling , Johng Dorhyun , Isaacs William B.
Published: August 2014

The androgen receptor (AR), a member of the nuclear receptor superfamily of ligand-regulated transcription factors, plays a pivotal role in the initiation and progression of prostate cancer. HOXB13, a prostate-specific homeodomain-containing transcription factor, interacts with AR to regulate the cellular response to androgens (Norris et al. Mol Cell 2009). Our group recently identified a rare but recurrent germline mutation (G84E) in HOXB13 as a prostate cancer susceptibility allele by sequencing the 17q21-22 region of the genomic DNA from families with hereditary prostate cancer. (Ewing et al. NEJM 2012). Our research goal is to study how HOXB13 regulates AR function and how this mechanism might be affected by mutant HOXB13. The overexpression of wild type HOXB13 in LNCaP cells led to an increased expression of AR at the mRNA and protein levels; this induction of AR was attenuated with HOXB13 G84E over-expression. Upon the reduction of endogenous HOXB13 by siRNA in LNCaP cells, the amount of AR decreased at the mRNA and protein levels, indicating that HOXB13 promotes the expression of AR. Conversely, when AR was knocked down with siRNA, the level of HOXB13 transcripts and proteins increased, indicating that AR suppresses HOXB13 expression. Furthermore, co-immunoprecipitation experiments demonstrate that FOXA1, the pioneer factor implicated to control prostate-specific transcription of HOXB13 (McMullin et al. 2009), interacts with AR and HOXB13. These analyses implicate at least a three factor mechanism whereby the levels of critical components of prostate-specific androgen signaling network might recruit and/or titrate one another, and suggest this network may be perturbed by mutations in HOXB13. Since LNCaP cells contain a mutated copy of HOXB13 (L144P), these analyses need to be performed in additional prostate cancer cells before general conclusions can be reached.

Citation Format: Shuangling Chen, Dorhyun Johng, William B. Isaacs. Reciprocal regulation of HOXB13 and androgen receptor in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 753. doi:10.1158/1538-7445.AM2013-753
Abstract 763: Hyperthermia promotes c-myc turnover and reduces ODC expression in colorectal cancer cells.
Authors: Wales Christina T.K. , Jacobs Aaron T.
Published: August 2014

Hyperthermia therapy is an FDA-approved procedure that is used to improve the clinical response to radiation and chemotherapy in a variety of solid malignancies. This method involves raising the temperature of tumor-bearing tissues to 41-43°C, which is commonly achieved by using microwave or radiofrequency (RF)-emitting devices. Despite a multitude of treatment centers that are now providing this type of therapy, the cellular and molecular processes are not fully understood. Here, we report that that the oncogenic transcription factor, c-myc has a potentially important role in the anti-cancer mechanism of hyperthermia. By treating colorectal cancer cells (RKO cell line) with heat (incubation at 42°C), we observe a rapid decrease in c-myc levels that is not seen with other transcription factors. We have also observed a similar effect in other cell types, including lung (A549) and neuroblastoma (SH-SY5Y). It is already known that the turnover of c-myc protein is enhanced by phosphorylation at Thr58. Our Western blot data reveal that hyperthermia causes a significant increase in Thr58 phosphorylation, which is accompanied by a reduction in total c-myc levels. c-Myc is frequently deregulated in human cancers, where it promotes the expression of genes that drive tumor cell proliferation. To assess the role of c-myc target genes in the response to hyperthermia, we looked at one such target gene, ornithine decarboxylase (ODC), which catalyzes the rate-limiting step in polyamine biosynthesis. We see that the hyperthermia-induced degradation of c-myc is accompanied by a drastic reduction in ODC mRNA and protein levels. Using actinomycin D, we observe that heat has no effect on ODC mRNA stability, suggesting that the decrease in ODC is due to a reduction in gene transcription. In support of this, we find that silencing c-myc expression with siRNA inhibits ODC mRNA and protein levels by a similar amount as hyperthermia. We propose that the down-regulation of ODC caused by c-myc degradation may be an important part of the clinical response to hyperthermia, and future work will evaluate the effect of heat on cellular polyamine levels.

Citation Format: Christina T.K. Wales, Aaron T. Jacobs. Hyperthermia promotes c-myc turnover and reduces ODC expression in colorectal cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 763. doi:10.1158/1538-7445.AM2013-763
Abstract 766: Phenformin is a MYCN/MYC destabilizer that can specifically target neural crest derived tumors.
Authors: Ikegaki Naohiko , Lomahan Srah , Tang Xao X.
Published: August 2014

We have been interested in identifying MYCN/MYC destabilizers that can specifically target neural crest derived tumors (neuroblastoma, melanoma, and malignant pheochromocytoma). MYCN amplification and its over-expression are associated with the worst neuroblastoma disease outcome. Non-MYCN amplified neuroblastomas express high-levels of MYC, and prognosis of these neuroblastomas is very poor. Melanoma and malignant pheochromocytoma are also known for high MYC expression. Interestingly, these neural crest derived tumors characteristically express the norepinephrine transporter (NET). This feature can be used to specifically target the tumor cells with agonists of NET, such as Meta-Iodo-BenzylGuanidine (MIBG). In the previous AACR meeting, we presented our finding that non-radio-labeled MIBG can suppress growth of neuroblastoma cells in vitro, and this effect is in part related to its ability to destabilize MYC and MYCN. We have also shown that FCCP, a well-known mitochondrial respiration inhibitor, destabilized MYCN and MYC in neuroblastoma cells and caused growth suppression. Recently, it has been reported that Phenformin, a Type II diabetic drug, binds NET, suggesting that NET positive cells preferentially uptake Phenformin. It is also known that Phenformin inhibits the mitochondrial electron transfer system. In this study, we have investigated a possible effect of Phenformin on growth and MYC/MYCN stability in neuroblastoma cells in vitro. SKNBE(2)C and CHP134 neuroblastoma cell lines were treated with various doses (0-2.5mM) of Phenformin for two days. MTS assay was used to assess the effect of Phenformin on growth of the cells. There was a dose-dependent growth suppression of the NB cell lines tested. Phenformin was more effective against growth of SKNBE(2)C than CHP134 cells. The effect of Phenformin on MYC/MYCN expression in four neuroblastoma cell lines [SKNBE(2)C, CHP134, SKNAS, and SY5Y] was also examined by Western blot assay. SKNBE(2)C and CHP134 express MYCN, whereas SKNAS and SY5Y express MYC at high levels. Treatment of these cells with Phenformin at the dose of 1mM for 24 hours resulted in destabilization of MYCN/MYC, suggesting that the growth suppressive effect of Phenformin on neuroblastoma cells was in part due to reduction in MYC/MYCN expression. We are currently examining whether the above observations made in neuroblastoma are also applicable to other neural crest derived tumors. In addition, we are investigating how Phenformin-related mitochondrial dysfunction causes destabilization of MYC/MYCN in neuroblastoma cells.

Citation Format: Naohiko Ikegaki, Srah Lomahan, Xao X. Tang. Phenformin is a MYCN/MYC destabilizer that can specifically target neural crest derived 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 766. doi:10.1158/1538-7445.AM2013-766
Abstract 775: Mutant Wilms' tumor gene 1 devoid of zinc-fingers promotes proliferation of human hematopoietic progenitor cells in vitro.
Authors: Vidovic Karina
Published: August 2014

The transcription factor Wilms’ tumour gene 1 (WT1) is present in some primitive hematopoietic cells and is highly expressed in the majority of lymphoid and myeloid leukemias, suggesting a role for WT1 in leukemogenesis. Mutations within the WT1 gene are present in 10-15 % of patients with cytogenetically normal acute myeloid leukemias and may be an independent predictor of poor clinical outcome. The mutations are most often heterozygous frameshift mutations leading to premature termination of the reading frame and therefore deletion of the entire DNA-binding zinc-finger domain. We have investigated the cellular effects of mutant WT1 protein lacking all four zinc-fingers, WT1(delZ). We report that expression of WT1(delZ) prolongs the proliferation of human hematopoietic CD34+ progenitor cells in vitro. Moreover, WT1(delZ) transduced cells express erythroid markers, including hemoglobin and elevated STAT5 protein levels, in part independently of addition of erythropoietin. STAT5 was increased by a posttranscriptional mechanism. The erythroid phenotype was also observed at global gene expression level, where WT1(delZ) caused coordinated up-regulation of genes associated with erythroid maturation, as well as genes related to cell division, while monocytic and neutrophil genes were down modulated. Our results indicate that WT1(delZ) promotes cell proliferation and erythroid differentiation at the expense of monocytes and granulocytes.

Citation Format: Karina Vidovic. Mutant Wilms' tumor gene 1 devoid of zinc-fingers promotes proliferation of human hematopoietic progenitor cells in vitro. [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 775. doi:10.1158/1538-7445.AM2013-775
Abstract 78: Fractionated whole brain irradiation modulates Homer1a expression in a brain region specific manner.
Authors: Moore Elizabeth , Kooshki Mitra , Miller Lance , Robbins Mike
Published: August 2014

About 500,000 new cancer patients will develop brain metastases in 2012. The primary treatment modality for ∼250,000 of these patients is partial or whole brain irradiation. While effective for controlling metastatic brain tumors, there are significant late effects (> 6 months) following radiotherapy which include a progressive, irreversible cognitive impairment. Although the exact mechanism(s) behind this radiation-induced brain injury are unknown, preclinical studies suggest that radiation alters neuronal plasticity. Indeed irradiating the rodent brain decreases long term potentiation, alters N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid (GABA) receptor expression and ablates neurogenesis. In this study, we used microarray analysis to show that Homer1a, a transcriptionally-regulated early response gene, was transiently upregulated in the hippocampus and downregulated in the cortex at 48 hours after fractionated (total dose of 40 Gy delivered as 2 Gy/fraction, 2X/week) whole brain irradiation (fWBI) in young adult male Fischer 344 X Brown Norway rats. Two months after fWBI Homer1a gene expression was downregulated in both the hippocampus and cortex. Homer1a binds the consensus sequence on the C-terminus of the group I metabotropic glutamate receptors (mGluR); thereby, disrupting the cross-linking of constitutively expressed forms of Homer1 and activating mGluR in the absence of glutamate. Radiation did not alter phosphorylation of mGluR1, total GluR2 or phosphorylation of GluR2/3 in the hippocampus or cortex at 2 months after fWBI. However, the radiation-induced changes in Homer1a expression appeared to produce a significant downregulation in mGluR1 in the hippocampus, and a significant upregulation in the cortex. Memory/learning paradigms suggest that trafficking of 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA) receptors containing mGluR1 from intracellular compartments to the plasma membrane is an “early” event during neural plasticity; thus decreased mGluR1 may result in alterations in hippocampal neural plasticity following fWBI. These data suggest a novel mechanism for the development of radiation-induced cognitive impairment. (Supported by CA122318)

Citation Format: Elizabeth Moore, Mitra Kooshki, Lance Miller, Mike Robbins. Fractionated whole brain irradiation modulates Homer1a expression in a brain region specific manner. [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 78. doi:10.1158/1538-7445.AM2013-78
Abstract 788: Effects of ATF3 on the gain-of-function of mutant p53.
Authors: Wei Saisai , Malmut Sarah , Wang Hongbo , Yan Chunhong
Published: August 2014

Activating Transcription Factor 3 (ATF3) is a member of the ATF/CREB family of transcription factors and can be rapidly induced by DNA damage and other cellular stresses. We previously found that ATF3 activates p53 by binding to the C-terminus of the tumor suppressor and blocking its ubiquitination, and consequently inhibits cellular transformation. Since TP53 is mutated in half of human cancers and mutant p53 proteins (mutp53) acquire oncogenic activities including promoting drug resistance and driving cancer cell invasion and metastasis, we were interested to explore effects of ATF3 on the mutp53 gain-of-functions (GOF). One major mechanism by which mutp53 promotes tumorigenesis is through binding another p53 family member p63 and inhibiting its tumor suppressor functions. We found that ATF3, but not a mutated ATF3 protein (▵102-139) devoid of its p53-binding region, bind the p53R175H mutant and disrupted the interactions between mutp53 and p63. As a consequence, while mutp53 inhibited the p63 transcriptional activity, ATF3 but not the ▵102-139 protein counteracted mutp53 and resumed p63 activity. Consistent with these results, ATF3 expression inhibited mutp53-promoted cell migration and invasion. In addition to effects on the mutp53-p63 interaction, we also found that ATF3 repressed mutp53-induced NFκB activity, thereby sensitizing mutp53-expressing cells to treatments with etoposide and cisplatin. We conclude that ATF3 suppresses the GOF of mutant p53 and thus may also contribute to tumor suppression in p53-mutated cancer cells.

Citation Format: Saisai Wei, Sarah Malmut, Hongbo Wang, Chunhong Yan. Effects of ATF3 on the gain-of-function of mutant p53. [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 788. doi:10.1158/1538-7445.AM2013-788
Abstract 796: Utilizing TCRβ and whole exome sequencing to determine the clonality and mutational spectrum of p53-null thymic lymphomas.
Authors: Dudgeon Crissy , Chan Chang , Sun Yvonne , Levine Arnold J.
Published: August 2014

The tumor suppressor p53 is nonfunctional in over 50% of cancers. The ability for p53 to protect the cell from tumorigenesis is rooted in its ability to induce cell cycle arrest, apoptosis, and senescence following genotoxic stress. Furthermore, loss of p53 in mice gives rise to spontaneous thymic lymphomas and sarcomas. In this study, we used the p53-KO mouse as a model for progression of tumorigenesis. We first analyzed genomic DNA of p53-KO and WT normal thymus at various ages using TCRβ sequencing. This procedure allowed us to determine the clonality of the T cell repertoire. We then analyzed the clonality of p53-KO thymic lymphomas. Our results indicated that while the highest T cell clone was 0.08% for normal thymus, the highest T cell clone for the p53-KO tumor was 70%, indicating that the p53-KO thymic lymphomas were monoclonal. Building on this data, we analyzed the mutations occurring in the thymic lymphomas by mouse exome sequencing. We believe that combining TCRβ sequencing with mouse exome sequencing will allow for a better understanding of the clonality and genetic mutations required for cellular transformation.

Citation Format: Crissy Dudgeon, Chang Chan, Yvonne Sun, Arnold J. Levine. Utilizing TCRβ and whole exome sequencing to determine the clonality and mutational spectrum of p53-null thymic lymphomas. [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 796. doi:10.1158/1538-7445.AM2013-796
Abstract 820: Genome-wide aberration analysis of rare cancer cell populations derived from formalin fixed paraffin embedded tissue.
Authors: Juskevicius Darius , Ruiz Christian , Dietsche Tanja , Rufle Alex , Barrett Michael T. , Bubendorf Lukas , Dirnhofer Stephan , Tzankov Alexander
Published: August 2014

Introduction. The majority of clinically described patient material worldwide is available as formalin fixed parafin embedded (FFPE) tissue blocks. Despite being a cheap and convenient way of archiving tissue suitable for review under the microscope, FFPE blocks are difficult to interrogate with modern molecular biology methods due to changes occurring during the process of fixation, embedding and aging. Genetic analysis of tumours with limited malignant cell count, like classical Hodgkin lymphoma (cHL) or T-cell/histiocyte-rich B cell lymphoma (TCHRBCL) is impeded by a bulk mass of non-malignant tumour infiltrating cells. Laser capture microdissection is currently a method of choise for rare tumor cell isolation, however it induce serious damage for gDNA, therefore additional techniques for enrichment are needed to enable meaningful genome-wide aberration analysis of tumors with low cancer cell content.

Methods. For extraction of nuclei from FFPE blocks of cHL and TCHRBCL cases enzymatic digestion and mechanical force were utilized. Pure populations of cancer cell nuclei were isolated by fluorescence-activated cell sorting (FACS) based on cancer type-specific nuclear markers Pax-5, Mum-1, Bcl-6, Ki-67. Array comparative genomic hybridization (aCGH) was utilized for genome-wide aberration analysis.

Results. We have optimized a set of methods to successfully isolate intact, high quality nuclei from FFPE tissue blocks. Nuclei retain their antigenicity and, therefore, are suitable for labelling with frourophore-conjugarted antibodies, which enables flow-sorting based not only on physical parameters and DNA content, but also on positivity for highly specific nuclear markers. Tumour nuclei populations from cHL and TCHRBCL of 70-90% in purity were isolated. DNA extracted from as little as 500 flow-sorted nuclei was sufficient to generate high-resolution and reproducible aCGH data.

Conclusions. Our proposed methodology allows the use of tissues in FFPE blocks more widely. Efficient isolation of malignant cells from the tumour mass was achieved. In the cases where starting material is scarce and very low number of target nuclei can be isolated, DNA extraction with improved recovery together with commercially available whole genome amplification methods enable genome-wide aberration analysis.

Citation Format: Darius Juskevicius, Christian Ruiz, Tanja Dietsche, Alex Rufle, Michael T. Barrett, Lukas Bubendorf, Stephan Dirnhofer, Alexander Tzankov. Genome-wide aberration analysis of rare cancer cell populations derived from formalin fixed paraffin embedded tissue. [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 820. doi:10.1158/1538-7445.AM2013-820
Abstract 832: MicroRNA-30c inhibits human breast tumor chemo-resistance by regulating twinfinlin-1 (TWF1) and IL-11.
Authors: Liu Huiping , Bockhorn Jessica , Dalton Rachel , Nwachukwu Chika , Huang Simo , Prat Aleix , Yee Kathy , Chang Ya-Fang , Huo Dezheng , Lu Jun , Dolan Eileen , Perou Charles M. , Olopade Olufunmilayo I. , Clarke Michael F. , Greene Geoffrey
Published: August 2014

Chemotherapy resistance remains a challenging problem in the clinic and the underlying molecular mechanisms are poorly characterized. We hypothesize that epithelial-to-mesenchymal transition (EMT) is involved in therapy resistance and cancer progression, but the functional link and signalling pathways need to be elucidated. Our work discovered that miR-30c, a human breast tumour prognostic marker, plays a pivotal role in chemo-resistance and apoptosis by a direct targeting of TWF1, which encodes an actin-binding protein and promotes EMT. We also identified IL-11 as a secondary target of TWF1 in the miR-30c signalling pathway. Expression of miR-30c inversely correlated with TWF1 and IL-11 levels in primary breast tumours and low IL-11 associated with relapse-free survival in breast cancer patients. Furthermore, our study demonstrates that miR-30c is transcriptionally regulated by GATA3 in breast tumours. Identification of a novel miRNA-mediated pathway that regulates chemo-resistance and apoptosis in breast cancer will facilitate the development of novel therapeutic strategies.

This study was supported in part by The University of Chicago Women's Board (J.B.) and Chicago Fellows Program (H.L.), DOD W81XWH-09-1-0331, NIH K12 CA139160-02, NCI K99 CA160638-01A1, CTSA UL1 RR024999 (H.L.), Segal Fund and Ludwig Fund (G.L.G.).

Citation Format: Huiping Liu, Jessica Bockhorn, Rachel Dalton, Chika Nwachukwu, Simo Huang, Aleix Prat, Kathy Yee, Ya-Fang Chang, Dezheng Huo, Jun Lu, Eileen Dolan, Charles M. Perou, Olufunmilayo I. Olopade, Michael F. Clarke, Geoffrey Greene. MicroRNA-30c inhibits human breast tumor chemo-resistance by regulating twinfinlin-1 (TWF1) and IL-11. [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 832. doi:10.1158/1538-7445.AM2013-832
Abstract 835: hRrp15, a ribosome RNA processing protein, has profound function on nucleoli construction, ribosome biogenesis and cell proliferation.
Authors: Dong Zhixiong , Zhu Changjun , Jiang Wei
Published: August 2014

Nucleolus is the sub-organelle that controls ribosome biogenesis in cells, and ribosome biogenesis is a highly regulated process that ensures cell growth (increase in biomass) coordinated with cell proliferation. In the nucleolus, ribosomal RNA precursor (47S rRNA) is initially transcribed from rDNA and then subsequently processes into 5.8S, 18S and 28S rRNAs, together with numerous ribosome proteins and nonribosomal factors, to form 60S and 40S pre-ribosomal particles before they are exported to the cytoplasm. A large body of evidence demonstrates that perturbation of rRNA synthesis, process and/or pre-ribosomal factor function impairs ribosome biogenesis and induces cell cycle arrest. This is attributed to nucleolar disruption and diffusion of ribosomal proteins, such as Rpl11 and Rpl5, into the nucleoplasm in which these ribosomal proteins bind to and inhibit MDM2 function, resulting in p53 induction and activation. In this study, we described the identification and characterization of a human protein, hRrp15, that has sequence homology with yeast pre-ribosomal factor, Rrp15p. Previous studies showed that Rrp15p is a ribosome RNA processing protein required for 60S pre-ribosomal particle formation and/or maturation in yeast. Using immunofluorescence and sucrose gradient centrifugation analyses, we demonstrated that hRrp15 was mainly localized at nucleoli in human cells. Depletion of hRrp15 expression disrupted nucleolar structure, inducing diffusion of nucleolar protein, nucleolin, in the nucleus. In contrast to Rrp15p, hRrp15 mainly associated with 60S pre-ribosomal particle but it also presented in 40S pre-ribosomal particle. Consistently, inhibition of hRrp15 expression perturbed nucleolar formation and impaired ribosomal biogenesis in both 60S and 40S pre-ribosomal subunits. In addition, depletion of hRrp15 in human non-transformed cells, RPE1, resulted in the induction of p53 and p21 proteins and cell cycle arrest at G1-G1/S whereas depletion of hRrp15 in human tumor cells, HeLa and MCF7, caused DNA damage response with detectable γ-H2AX and apoptosis. These results indicated that hRrp15 plays a critical role in regulating formation of both 60S and 40S pre-ribosomal particles, ribosome biogenesis, construction of nucleoli, cell growth and cell proliferation in human cells. Hence, hRrp15 may serve as a potential target for cancer therapy.

Citation Format: Zhixiong Dong, Changjun Zhu, Wei Jiang. hRrp15, a ribosome RNA processing protein, has profound function on nucleoli construction, ribosome biogenesis and cell proliferation. [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 835. doi:10.1158/1538-7445.AM2013-835
Abstract 838: Distinct effects of S6K homologues on breast cancer cell survival.
Authors: Sridharan Savitha , Basu Alakananda
Published: August 2014

The 40S ribosomal protein S6 kinase (S6K) acts downstream of the mammalian target of rapamycin (mTOR), which is an important target for cancer therapy. mTOR inhibitors are, however, of limited success, partly due to the activation of Akt caused by persistent inhibition of mTOR/p70 S6 kinase or S6K1 via a negative feedback loop. S6K exists as two homologs, S6K1 and S6K2. While both homologs are overexpressed in breast cancer, little is known about the function of S6K2. The objective of the present study is to determine whether S6K homologs perform redundant or distinct functions with respect to Akt activation and breast cancer cell survival. Silencing of S6K1 inhibited whereas knockdown of S6K2 potentiated cell death by various apoptotic stimuli, including tumor necrosis factor-alpha (TNF) and TNF-Related Apoptosis Inducing Ligand (TRAIL). In contrast to S6K1, depletion of S6K2 decreased Akt phosphorylation in MCF-7 and T47D cells. Ectopic expression of constitutively-active Akt in MCF-7 cells restored cell survival in S6K2-depleted cells. We have previously shown that activation of Akt induces downregulation of Bid via p53. Knockdown of S6K2 caused an increase in p53 and silencing of Bid blunted the ability of S6K2 deficiency to enhance TNF-induced apoptosis. Thus, our study demonstrates for the first time that the two homologs of S6K have distinct effects on Akt activation and cell survival and that S6K2 depletion enhanced cell death via the proapoptotic Bcl-2 family member Bid. Thus, targeting S6K2 may be an effective therapeutic strategy to treat cancers. (This work was supported by the grant CA71727 from the NCI).

Citation Format: Savitha Sridharan, Alakananda Basu. Distinct effects of S6K homologues on breast cancer cell survival. [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 838. doi:10.1158/1538-7445.AM2013-838
Abstract 839: An uncharacterized mechanism of cell death independent of p53 mutation status.
Authors: Morrison Bethanie L. , Russell Elisabeth , Mitala Joseph , Bernal Federico
Published: August 2014

Resistance to chemotherapeutic agents is often a rate-limiting event in the treatment of metastatic cancers. Mutations of TP53 or dysfunctions in the p53 pathway are known to play primary roles in the resistance to apoptosis-inducing agents. A stapled alpha-helical p53 peptide (SAH-p53) capable of disrupting the p53-HDM2 and p53-HDMX complexes and restoring p53 function has been found to induce a death response in breast, melanoma and choriocarcinoma cancer cells independent of p53 mutational status. We have recently discovered that the mechanism of death induced by SAH-p53 does not exhibit all of the characteristics of classical apoptosis, necrosis or autophagy independently. The binding of SAH-p53 to HDM2, HDMX, or a yet unidentified target results in a mechanism that leads to the intracellular aggregation of proteins and lipids that are extruded from the cell, resulting in catastrophic damage to the integrity of the cell membrane and culminating in cell death. This type of cell death involves the loss of cytoplasm, generation of phase-clear vacuole-like structures, apparent degradation of cellular membrane components, and the significant release of uniform non-vesicular cellular components. The cellular debris, while yet undefined, displays orange staining with acridine orange as well as accumulation of MitoTracker, suggesting that the debris contains hydrophobic regions. Thus far it has been established that there is a lack of any membrane blebbing, no chromatin condensation, no apoptosis signaling events or typical necrotic bubble formation after treatment with SAH-p53 in cells harboring wild-type p53, mutant p53, or null p53, suggesting an alternative mechanism of cell death. Data suggest the manifestation of early autophagy within 6 hours of SAH-p53 treatment as evidenced by LC3 accumulation, yet this phenotype is lost within 24 hours post-treatment. In addition, the pretreatment of the cells with chloroquine does not inhibit the release of debris and changes in morphology characteristic of SAH-p53 treatment, suggesting that the mechanism of death is independent of classical autophagy. We have begun to uncover the precise mechanism of cell death through the identification and validation of the transcriptional program regulated by SAH-p53 in both wild-type and mutant p53-containing cells via microarray analysis. The complete understanding and characterization of this novel mechanism may be exploited to overcome resistance to apoptosis and thus lead to a more effective way of treating chemotherapy-resistant metastatic cancers.

Citation Format: Bethanie L. Morrison, Elisabeth Russell, Joseph Mitala, Federico Bernal. An uncharacterized mechanism of cell death independent of p53 mutation status. [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 839. doi:10.1158/1538-7445.AM2013-839
Abstract 840: Caspase 2 short isoform interacts with membrane-associated cytoskeleton proteins to inhibit apoptosis.
Authors: Han Chunhua , Zhao Ran , Wani Altaf A. , Wang Qi-En
Published: August 2014

Caspase-2 (casp-2) is the most conserved caspase across species, and is one of the initiator caspases activated by various stimuli. The casp-2 gene produces several alternative splicing isoforms. The exclusion of exon 9 results in the dominant casp-2 long isoform (casp-2L) mRNA, while the inclusion of exon 9 leads to the inclusion of an in-frame stop codon in casp-2, resulting in a short isoform (casp-2S) mRNA. It is believed that casp-2L promotes apoptosis, whereas casp-2S inhibits apoptosis. However, the effect of casp-2S on apoptosis is still controversial, and the underlying mechanism for casp-2S-mediated apoptosis inhibition is unclear. Here, we analyzed the effects of casp-2S on DNA damage induced apoptosis by “gain-of-function” and “loss-of-function” effects in ovarian cancer cell lines. We have clearly demonstrated that over-expression of casp-2S inhibited, while knockdown of casp-2S promoted, the cisplatin-induced apoptosis in ovarian cancer cells, as revealed by the detection of PARP cleavage and Annexin V staining. To explore the mechanism for casp-2S-mediated apoptosis inhibition, we analyzed proteins interacting with casp-2S in cells by using mass spectrometry (MS) and immunoprecipitation (IP). We have identified two cytoskeleton proteins, Fodrin and α-Actinin 4, associated with casp-2S in HeLa cells and confirmed this interaction by reciprocal IP. Furthermore, we have demonstrated that casp-2S primarily resides in the cytoplasm, and inhibits DNA damage-induced cytoplasmic Fodrin cleavage independently of cellular p53 status. Given that Fodrin cleavage is critical to the apoptotic membrane blebbing, we hypothesized that casp-2S may inhibit the blebbing during apoptosis. Interestingly, immunofluorescence studies did show that over-expression of casp-2S inhibited the cisplatin-induced membrane blebbing. Taken together, our data suggest that casp-2S interacts with the membrane-associated cytoskeleton protein Fodrin, protecting it from cleavage, to affect the formation of membrane blebbing and apoptosis. (Supported by NIH grant CA151248 to QEW, and CA93413 to AAW)

Citation Format: Chunhua Han, Ran Zhao, Altaf A. Wani, Qi-En Wang. Caspase 2 short isoform interacts with membrane-associated cytoskeleton proteins to inhibit apoptosis. [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 840. doi:10.1158/1538-7445.AM2013-840
Abstract 868: HER2-targeted engineered toxin body demonstrates selective binding and cell kill of HER2-overexpressing breast cancer.
Authors: Rajagopalan Sangeetha , Willert Erin , Wirth Rachel , Null William , Liu Jen-Sing , Higgins Jack P.
Published: August 2014

The HER2 receptor is overexpressed in 20-30% of breast cancers. HER2 overexpression in breast cancer has been successfully targeted by both antibody and antibody-drug conjugate approaches. Trastuzumab, a humanized monoclonal antibody is approved for the treatment of HER2 positive metastatic breast cancer. More recently, trastuzumab-DM1, an antibody drug conjugate (ADC), has shown promising clinical results in patients refractory to trastuzumab. Because DM1 is a chemotherapeutic and refractory breast cancer patients have typically progressed through several rounds of chemotherapy, we were interested in developing a new ADC approach with trastuzumab that utilized a novel mechanism of action. 5G2 is an engineered toxin body comprised of the trastuzumab scFv and a modified ribosome-inhibiting protein derived from Shiga-like toxin 1 A. 5G2 has exhibited high binding affinity to HER2 protein and HER2-expressing cells. 5G2 exerts a potent HER2-specific cytotoxic effect on HER2-expressing cells, but little activity on various HER2 negative cells. These results demonstrate that 5G2 is a promising HER2-targeted therapeutic agent against breast carcinomas and is currently under further development.

Citation Format: Sangeetha Rajagopalan, Erin Willert, Rachel Wirth, William Null, Jen-Sing Liu, Jack P. Higgins. HER2-targeted engineered toxin body demonstrates selective binding and cell kill of HER2-overexpressing breast 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 868. doi:10.1158/1538-7445.AM2013-868
Abstract 900: Chemoresistance is induced by butyrate in colon cancer cells.
Authors: Lim Soo-jeong , Choi Hyeon Gyeom , Jeon Chae Kyung , Kim So Hee
Published: August 2014

Purpose.

Butyric acid is a short chain fatty acid produced naturally by fermentation of dietary fiber in the colonic mucosa. It is known to play an important role in preventing the initiation and the progression of colon cancer by suppressing the proliferation of cancer cells. However, some colon cancer still develops and progresses to a more malignant and chemoresistant one despite of the presence of butyric acid. The present study was undertaken to investigate a possibility of butyrate-involved mechanism by which the colon cancer cells acquire resistance to chemotherapeutic drugs.

Methods.

Human HCT116 colon cancer cells were exposed to progressively increasing concentrations of butyrate for three months to establish a colon cancer cell line with acquired resistance to butyrate. After obtaining butyrate-resistant cells, the sensitivity against multiple chemotherapeutic drugs were compared between parental and butyrate-resistant cells. The expression of proteins related to chemotherapeutic response was also compared by immunoblotting analysis.

Results.

Butyrate-resistant HCT116 cancer cell lines could be established by culturing cells in the presence of butyrate up to 0.8 mM concentration. The concentrations inhibiting the cell growth by 50% (IC50) of butyrate was 0.62 and 4.56 mM in parental and butyrate-resistant HCT116 cells. Butyrate-resistant HCT116 cells were also resistant to widely used chemotherapeutic drugs: the IC50 after paclitaxel, doxorubicin or 5-FU treatment were 2.10-, 3.98- and 1.6-fold higher in butyrateresistant cells compared with parental cells. The expression of multidrug resistance-related proteins that function as drug efflux pumps such as P-glycoprotein and the multidrug resistance-associated protein 1 were not changed at protein level. Among the apoptosis-involved proteins, the expression of anti-apoptotic BcL-2 protein was increased while that of proapoptotic BAD protein was reduced in butyrate-resistant cells, suggesting that the above chemoresistance was associated with cellular resistance to apoptosis.

Conclusion.Our data herein suggests that the colon cancer cell grown in the presence of butyric acid may acquire resistance to apoptotic cell death and thus lead to resistance to chemotherapeutic drugs.

Citation Format: Soo-jeong Lim, Hyeon Gyeom Choi, Chae Kyung Jeon, So Hee Kim. Chemoresistance is induced by butyrate in colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 900. doi:10.1158/1538-7445.AM2013-900
Abstract 901: Overexpression of inhibitors of apoptosis (IAP) family members in human breast and ovarian cancer models of non-MDR1 taxane resistance.
Authors: Duran George E. , Brozovic Anamaria , Martinez Francisco J. , Francisco E Brian , Wang Yan C. , Sikic Branimir I.
Published: August 2014

Four breast cancer cell lines (MCF-7, BT-549, MDA-MB-231 and T-47D) and four ovarian cancer cell lines (1A9/A2780, ES-2, MES-OV and OVCAR-3) were selected for taxane resistance by exposing cells to either docetaxel or paclitaxel in the presence of the P-glycoprotein inhibitor, PSC-833 (valspodar, 2 μM). All of these co-selected variants are MDR1/ABCB1(-), and the resistance to taxanes is not transporter-mediated. We have previously reported elevated class III β-tubulin (TUBB3), reduced BRCA1, elevated CDKN1A (p21), and altered epithelial-mesenchymal transition (EMT) genes in the majority of the non-MDR1 taxane variants. Inhibitors of apoptosis (IAP) proteins directly bind and inhibit several caspases, and may play a critical role in determining cell fate after exposure to chemotherapeutic agents. In this study, we profiled the expression of IAP proteins and found elevated content in this panel of taxane-resistant human breast and ovarian cancer cell lines. In both the paclitaxel- (TP) and docetaxel-selected (TxTP) ovarian cancer cell lines we observed significantly overexpressed cIAP1 (BIRC2), cIAP2 (BIRC3), XIAP (BIRC4), and Livin (BIRC7) relative to parental cell lines. Expression of cIAP2 was undetectable in OVCAR-3 and its taxane resistant cell lines, OVCAR-3/TP20 and OVCAR-3/TxTP5. Elevated cIAP1 and XIAP levels were detected in the human breast cancer variant BT-549/TxTP50, and Livin was overexpressed in all taxane-resistant breast cancer cell models. Survivin (BIRC5) is highly expressed in cancers and has been associated with taxane resistance via its effects on apoptosis and the cell cycle. We observed reduced Survivin content in the majority of the variants established in our laboratory except for the ovarian ES-2/TP80 cell line where we found slightly elevated expression resulting from paclitaxel selection relative to its parental control. In addition to these alterations in IAP content, we observed elevated Bcl-2 in ovarian (MES-OV/TP40 and MES-OV/TxTP50) and breast (MCF-7/TxTP50, BT-549/TxTP50, and MDA231/TxTP50) cancer cell lines at the transcript level by rt-PCR and confirmed by immunoblotting. Specific gene silencing by RNAi and treatment with small molecule inhibitors will test the functional significance of IAP alterations in these models of taxane resistance.

Citation Format: George E. Duran, Anamaria Brozovic, Francisco J. Martinez, E Brian Francisco, Yan C. Wang, Branimir I. Sikic. Overexpression of inhibitors of apoptosis (IAP) family members in human breast and ovarian cancer models of non-MDR1 taxane resistance. [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 901. doi:10.1158/1538-7445.AM2013-901
Abstract 904: TP-0413 is a dual-specific inhibitor against Jak2 and Alk2 with therapeutic potential for treating anemia of chronic disease.
Authors: Stephens Bret J. , Mollard Alexis , Meng Lingyao , Bearss David J. , Warner Steven L.
Published: August 2014

Anemia of chronic disease is a debilitating condition commonly found in cancer patients. Studies suggest that this syndrome is largely the result of the body's production of hepcidin, a master regulator of human iron metabolism. TP-0413 is a small molecule that targets two important components of the hepcidin pathway, Janus Kinase 2 (Jak2) and Activin A receptor, type I (Alk-2). Jak2 facilitates hepcidin function by phosphorylating ferroportin upon hepcidin binding, inducing its degradation and thus preventing iron entry into plasma. Alk2 on the other hand, is a component of the signal transduction pathway that leads to hepcidin expression. Here we report on recent efforts aimed at further characterization of TP-0413. In biochemical assays TP-0413 inhibits Jak2 and Alk2 at 3.11 nM and 70 nM, respectively (IC50). In cell culture, HepG2 cells treated with TP-0413 exhibit reduced hepcidin levels, and TP-0413 is able to inhibit BMP-2 induction of hepcidin. Furthermore, TP-0413 ameliorates hypoferremia induced by LPS treatment in mice, and modulates cytokine levels in these animals. TP-0413 shows favorable drug-like properties in pharmacokinetic models, and further studies are being undertaken to advance TP-0413 towards clinical trials.

Citation Format: Bret J. Stephens, Alexis Mollard, Lingyao Meng, David J. Bearss, Steven L. Warner. TP-0413 is a dual-specific inhibitor against Jak2 and Alk2 with therapeutic potential for treating anemia of chronic disease. [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 904. doi:10.1158/1538-7445.AM2013-904