bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1724: Pancreatic cancer stem cell metastasis and mitochondrial respiration is dependent on the the ISG15-mediated ubiquitin-like modification process known as ISGylation
Authors: Sainz Bruno , Alcala Sanchez Sonia , Vallespinos Mireia
Published: July 2016

We recently discovered that CSCs of distinct solid tumors (e.g. pancreatic, colon, and liver) contain intracellular vesicles coated in the ATP-dependent transporter ABCG2, which act as a sink for the accumulation of riboflavin (vitamin B2). Using autofluorescence and other CSC markers, such as CD133, we have begun to dissect CSCs at the genetic and protein level. Using RNAseq to transcriptionally distinguish CSCs from non-CSCs, we observed that pancreatic CSCs of different primary PDAC tumors exhibit a transcriptional gene activation signature belonging to the Type I interferon (IFN) signaling pathway. In particular, CSCs up-regulate the interferon-stimulated gene 15, a 165 amino acid (17kDa) protein that is induced by Type I IFN treatment and functions to ISGylate proteins, a ubiquitin-like modification process whereby ISG15 can be covalently linked to cytoplasmic and nuclear proteins. We have discovered that CSCs not only upregulate the expression of monomeric ISG15, but numerous proteins within CSCs are ISGylated, the level of ISGylation correlates with their metastatic capacity and knockdown of ISG15 ablates pancreatic CSCs functions, such as self-renewal, migration and invasion in vivo and mitochondrial respiration. Thus, our data indicate that the ISGylayion pathway is active in CSCs and plays an important role in the “stem-like” properties of these cells. We have also elucidated the mechanism underlying the differential regulation of ISG15 in CSCs versus non-CSCs. Specifically, using over expression lentiviral vector systems and pharmacological inhibitors (i.e. BRD4 inhibitor JQ-1), we show that MYC controls the expression of miR22, a micro-RNA that targets, among others transcripts, a number of Interferon Regulatory Factors (IRFs) that are essential for intracellular IFN signaling, including ISG15 expression. CSCs express low levels of MYC and miR-22, relieving the inhibitory block on IRFs and subsequent ISG15 signaling. As expected, over expression of MYC increases miR-22 levels and blocks IRF-mediated ISG15 expression. While the intricacies of these complex and distinct pathways still need to be unraveled at the CSC level, our data suggest that MYC may be a key universal player in these pathways, and targeting ISG15 in CSCs may represent a unique approach to altering the biology of CSCs.

Citation Format: Bruno Sainz, Sonia Alcala Sanchez, Mireia Vallespinos. Pancreatic cancer stem cell metastasis and mitochondrial respiration is dependent on the the ISG15-mediated ubiquitin-like modification process known as ISGylation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1724.
Abstract 1789: Estrogen receptor alpha and beta gene expression in benign breast tissue of high risk premenopausal women treated with the SERM acolbifene
Authors: Kimler Bruce F. , Phillips Teresa A. , Fabian Carol J.
Published: July 2016

Background

We conducted a pilot study to assess the feasibility of a 4th generation selective estrogen receptor modulator (SERM) acolbifene as a breast cancer prevention agent in premenopausal women at high risk for development of breast cancer [Cancer Prev Res 8, December 2015]. Favorable modulation of biomarkers was observed in benign breast tissue acquired before and after intervention. This included reduction in proliferation (Ki-67 immunocytochemistry) and decreases in expression of genes that code for ERα, pS2, and PgR. While ERα is over-expressed and stimulatory in breast carcinogenesis, ERβ which would be inhibitory is down-regulated. Thus, a change in the ratio of gene expression might be most informative. We have now analyzed gene expression of ERβ so as to examine whether it, or the ratio of ERα to ERβ, may contribute to assessment of response.

Methods

Premenopausal women at elevated risk for breast cancer were screened by random periareolar fine needle aspiration (RPFNA) performed during the follicular phase of the menstrual cycle. Eligible women (n = 25) received 6-8 months of open-label acolbifene (20 mg/d), followed by repeat RPFNA. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) was performed on 16 paired specimens for several genes associated with estrogen activity: estrogen receptor 1 (ESR1) for ERα, trefoil factor 1 (TFF1) for pS2, GREB1 for growth regulation by estrogen in breast cancer 1, PGR for progesterone receptor, and estrogen receptor 2 (ESR2) for ERβ. The quantity of each biomarker transcript was expressed relative to the reference transcript HPRT1. Further normalization by epithelial cell markers (KRT19 for cytokeratin 19 or CDH1 for E-Cadherin) did not materially alter the results.

Results

Significant changes had been previously shown for transcripts that code for pS2 (15 decreases, 1 increases; p = 0.002), ERα (11↓, 5↑; p = 0.013), and PgR (15↓, 1↑; p = 0.001); plus a suggestion of effect of GREB-1α (10↓, 6↑; p = 0.074). ERβ exhibited a general increase, but this was not statistically significant (5↓, 11↑; p = 0.24). However, the reduction of the ratio of ERα to ERβ was statistically significant (15↓, 1↑; p = 0.002).

Conclusions

Assessment of the ratio of gene expression for ERα to ERβ, and possibly their protein products by immunocytochemistry, may provide a useful surrogate endpoint biomarker by which to monitor response to SERMs used for breast cancer chemoprevention in premenopausal women.

Supported by NO1-CN-35135.

Citation Format: Bruce F. Kimler, Teresa A. Phillips, Carol J. Fabian. Estrogen receptor alpha and beta gene expression in benign breast tissue of high risk premenopausal women treated with the SERM acolbifene. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1789.
Abstract 1818: Coregulation of progesterone receptor and O-GlcNAc in breast cancer
Authors: Trinca Gloria , Goodman Merit , Workman Lauren , Carroll Jason , D’Santos Clive , Slawson Chad , Hagan Christy R.
Published: July 2016

Progesterone receptor (PR) activity is highly regulated by post-translational modifications. Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) analysis identified O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) as a PR-interacting protein. OGT catalyzes the addition of N-acetylglucosamine sugar moieties to serine/threonine residues in order to coordinate several cellular processes—including transcription, metabolism, and cell fate. Further investigation revealed that PR is O-GlcNAcylated, and enhanced OGT activity subsequently decreases PR protein stability. Interestingly, PR-expressing breast cancer cells display lower OGT levels in comparison to PR-null counterparts, suggesting that PR may govern its own O-GlcNAcylation or that of other key regulatory proteins. Our lab previously identified a phosphorylation site at Ser-81 in PR that potentiates the expression of PR proliferative target genes. Phosphorylation and O-GlcNAcylation are known to compete for the same residue substrates; and our preliminary data suggests that PR O-GlcNAcylation may also alter its transcriptional activity for a subset of target genes. While the role of PR in breast cancer progression is well characterized, the overall effect of protein O-GlcNAcylation within these tumors remains highly controversial. Collectively, our data supports a potential regulatory mechanism wherein OGT serves to restrain PR function in early PR+ tumors; whereas those that have lost PR expression may exhibit the elevated pro-tumorigenic OGT activity previously associated with more advanced breast cancers. As such, the continued study of O-GlcNAcylation with respect to PR stability, phosphorylation, and transcriptional activity may lead to a clearer understanding of these effectors and their contribution to breast cancer progression.

Citation Format: Gloria Trinca, Merit Goodman, Lauren Workman, Jason Carroll, Clive D’Santos, Chad Slawson, Christy R. Hagan. Coregulation of progesterone receptor and O-GlcNAc in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1818.
Abstract 1891: PDGF signaling maintains survival of proneural glioma cells by regulating USP1 to stabilize ID2
Authors: Rahme Gilbert J. , Zhang Zhonghua , Young Alison L. , Cheng Chao , Bivona Eric J. , Fiering Steven N. , Hitoshi Yasuyuki , Israel Mark A.
Published: July 2016

Glioblastoma (GBM) is the most aggressive primary brain tumor and responds poorly to currently available therapies. Transcriptomic characterization of GBM has identified distinct molecular subtypes of GBM with the latest report identifying four molecular subtypes: proneural, neural, classical, and mesenchymal. Gain-of-function alterations leading to enhanced platelet-derived growth factor (PDGF) signaling are commonly observed in proneural GBM and can drive proneural gliomagenesis. However, little is known about the critical determinants of malignancy mediated by PDGF signaling in proneural glioma. Using a mouse model of proneural glioma and comparative transcriptomics, we determined that PDGF signaling upregulates ubiquitin specific peptidase 1 (Usp1) to promote survival of murine proneural glioma cells. Further experimentation revealed that PDGF-mediated expression of USP1 post-translationally stabilized Inhibitor of DNA-binding 2 (ID2), which we found to be required for glioma cell survival. Deletion of the Id2 gene delayed tumor-induced mortality. Pharmacological inhibition of USP1 decreased ID2 levels, and delayed tumorigenesis in mice. Importantly, decreased USP1 expression is associated with prolonged survival in patients with proneural GBM but not with other subtypes of GBM. Our data describe a signaling cascade downstream of PDGF that is required for the survival of proneural GBM cells and suggest that inhibition of the PDGF-USP1-ID2 axis could serve as a therapeutic strategy for the treatment of proneural GBM with increased PDGF signaling.

Citation Format: Gilbert J. Rahme, Zhonghua Zhang, Alison L. Young, Chao Cheng, Eric J. Bivona, Steven N. Fiering, Yasuyuki Hitoshi, Mark A. Israel. PDGF signaling maintains survival of proneural glioma cells by regulating USP1 to stabilize ID2. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1891.
Abstract 1936: Comprehensive long non-coding RNA expression profiling from the TCGA HNSCC RNA-sequencing data
Authors: Nohata Nijiro , Abba Martin , Amornphimoltham Panomwat , Gutkind J. Silvio
Published: July 2016

Objectives; Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer types in the world. Despite of considerable advances in multimodality therapy, including surgery, radiotherapy and chemotherapy, the overall 5-year survival rate for patients with HNSCC is only 40% - 50%. Although a growing body of evidence indicates that long non-coding RNAs (lncRNAs) contribute to the initiation and development of various types of cancers, the role of lncRNA expression in human HNSCC remains unknown. In this study, we aimed to establish the onco-lncRNAome profiling of HNSCC from The Cancer Genome Atlas (TCGA) project.

Materials and Methods; The Atlas of Noncoding RNAs in Cancer (TANRIC) database (http://ibl.mdanderson.org/tanric/_design/basic/index.html) was employed to retrieve the lncRNA expression information from the TCGA HNSCC RNA-sequencing data. Our recently generated RNA-sequencing data from 22 representative HNSCC cell lines (OPC-22 panel) were also included in this study. Bioinformatics approaches, such as differential gene expression analysis, survival analysis, principal component analysis, and Co-LncRNA enrichment analysis were performed.

Results; Using TCGA HNSCC RNA-sequencing data from 426 HNSCC and 42 adjacent normal tissues, we identified 728 lncRNA transcripts significantly and differentially expressed in HSNCC. Among the 728 lncRNAs, 55 lncRNAs were significantly associated with cancer prognosis (overall survival and/or recurrence free survival). We also identified 140 lncRNA transcripts significantly and differentially expressed between Human Papilloma Virus (HPV) positive tumors and HPV negative tumors. 30 lncRNA transcripts were distinctly expressed between TP53 mutated tumors and TP53 wild type tumors. Among them, there were 19 overlapping genes up-modulated in HPV positive and TP53 wild type tumors. Co-LncRNA enrichment analysis suggested that protein-coding genes that are co-expressed with cancer-associated lncRNAs might be involved in malignant progression. Of interest, many differentially expressed lncRNAs in HNSCC were reflected in the HNSCC cell line panel.

Conclusion; LncRNAs profiling could provide novel insights into the potential mechanisms of HNSCC oncogenesis. A unique set of lncRNAs may represent potential targets for diagnosis, therapy and prevention of HNSCC.

Citation Format: Nijiro Nohata, Martin Abba, Panomwat Amornphimoltham, J. Silvio Gutkind. Comprehensive long non-coding RNA expression profiling from the TCGA HNSCC RNA-sequencing data. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1936.
Abstract 1948: MiRNA profiling of dogs with transitional cell carcinoma of the bladder using blood and urine samples
Authors: Kent Michael , Zwingenberger Allison , Westropp Jodi , Barrett Laura , Ghosh Paramita , Vinall Ruth L.
Published: July 2016

Early symptoms of canine transitional cell carcinoma (TCC) are frequently assumed to be caused by other lower urinary tract diseases (LUTD) such as urinary tract infections, resulting in late diagnosis of TCC and decreased rates of survival. To determine whether miRNA can be used as non-invasive diagnostic biomarkers in this setting, we assessed miRNA expression in RNA extracted from urine and blood from dogs with clinically normal bladders (n = 28), LUTD (n = 25), and TCC (n = 18). Expression levels of 5 miRNA associated with TCC pathophysiology (miR-34a, let-7c, miR-16, miR-103, and miR-106b) were assessed by quantitative real-time PCR. Statistical analyses using ranked ANOVA identified significant differences in miR-103 and miR-16 levels between urine samples from LUTD and TCC patients (miR-103, fold change = 2.55, p = 0.015; and miR-16, fold change = 3.02, p = 0.002). However, logistic regression analyses determined miR-103, but not miR-16, can distinguish between canine LUTD and TCC patients (OR = 0.78 p = 0.031, AUC = 0.85). No statistically significant differences in miRNA levels were observed between blood samples from LUTD versus TCC patients. Expression levels of miR-34a trended with miR-16, let-7c, and miR-103 levels in normal urine samples, however, this coordination was completely lost in TCC urine samples. In contrast, cooordination of miR-34a, miR-16, let-7c, and miR-103 expression levels was maintained in blood samples from TCC patients. Our combined data indicate further investigation of miR-103 as a diagnostic urine biomarker for TCC, and investigation of the role of miR-103 as well as dysregulation of coordinated miRNA expression in bladder carcinogenesis, is warranted.

Citation Format: Michael Kent, Allison Zwingenberger, Jodi Westropp, Laura Barrett, Paramita Ghosh, Ruth L. Vinall. MiRNA profiling of dogs with transitional cell carcinoma of the bladder using blood and urine samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1948.
Abstract 1956: Mirnas hsa-miR-214-3p, -378a-3p e -34a-5p as biomarkers for uterine mesenchymal neoplasms
Authors: dos Anjos Laura G. , Maciel Gustavo A. , Cunha Isabela W. , Baracat Edmund C. , Carvalho Kátia C.
Published: July 2016

Background: Uterine mesenchymal tumors have specific mechanisms of development that are largely unknown. One of the molecular mechanisms which may be involved in the appearance and progression of these tumors is the regulation of gene expression by miRNAs. These molecules are small non-coding RNAs that play important roles in several biological pathways. The identification of miRNAs that show particular differences in expression profile can help broaden the diagnostic methods available for these tumors. Objectives: To evaluate the expression profile of microRNAs described as sequences related to tumors development in uterine leiomyosarcomas samples, comparing to leiomyomas and normal myometrium. Methods: We selected 37 samples of uterine leiomyosarcoma (LMS), 16 unconventional leiomyomas (LMA), 10 conventional leiomyomas (LM) and 2 myometrium (used as reference samples). Samples were obtained from the Discipline of Gynecology of the Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo and Department of Pathology of the A C Camargo Cancer Center, both in Sao Paulo, Brazil. The total RNA was obtained and used to perform the synthesis of cDNA.The real time PCR reactions was performed using the Kit miScript SYBR Green PCR (Qiagen) for analysis from the 84 miRNAs sequences already described in the literature as tumor development related sequences. Results: The data analysis from the 84 sequences of microRNA showed wide variation in the regulation of these molecules expression. After computational analysis, we selected only those miRNAs with different expression profile between LM, LMA and LMS. The goal was to obtain a molecular signature that differentiate and identify each tumor using normal tissue as a reference. We found that hsa-mir-214-3p and hsa-miR-378-3p (fold regulation of -2.32 and -2.97; respectively) presented down-regulation in samples from the uterine LMS. In LMA we found a down regulation of hsa-miR-34a-5p (-3.12 fold regulation). Conclusions: We identified three miRNAs (hsa-mir-214-3p, and -378-3p and -34a-5p) differentially expressed in LMA and LMS. Analyzes of target genes of these miRNAs may identify specific proteins for use in the differential diagnosis, prognosis and patients management in these tumors.

Citation Format: Laura G. dos Anjos, Gustavo A. Maciel, Isabela W. Cunha, Edmund C. Baracat, Kátia C. Carvalho. Mirnas hsa-miR-214-3p, -378a-3p e -34a-5p as biomarkers for uterine mesenchymal neoplasms. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1956.
Abstract 1957: MicroRNA analysis paired with a novel live cell viability assay: a complete epigenetic workflow in human cancer cell lines
Authors: Lewis Samantha , Hook Brad , Smith Don , Horejsh Douglas , Schagat Trista
Published: July 2016

The requirements for nucleic acid purification for use in RNA profiling have expanded with the growing interest in the role of microRNAs (miRNAs) and other small non-coding RNAs in cancer cell growth and metastasis. This evolution of expression analysis has highlighted the need for more sophisticated tools for total RNA isolation from cancer cells and tissues, beyond traditional translated messenger RNAs (mRNA). To address this need, we describe tools to isolate total RNA, for mRNA and miRNA analyses, in the context of an epigenetic workflow with cancer cell lines.

In these experiments, histone deacetylase (HDAC) inhibitor treatment of different cancer cell lines was performed and expression of miRNA and mRNAs measured in parallel with assays for cell viability, cytotoxicity, and HDAC activity. This workflow multiplexes RNA and miRNA expression profiling with assays for viability and cytotoxicity to efficiently capture a more complete understanding of expression results.

With this improved cell viability workflow, one experiment from the same well can yield information about cell health, cytotoxicity, HDAC activity, and mRNA plus miRNA expression profiling. Our workflow incorporates monitoring of cell health in real-time during experimental treatments, simple and effective total RNA purification, and robust quantitative PCR reagents for a comprehensive experimental design.

Citation Format: Samantha Lewis, Brad Hook, Don Smith, Douglas Horejsh, Trista Schagat. MicroRNA analysis paired with a novel live cell viability assay: a complete epigenetic workflow in human cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1957.
Abstract 1971: BRD4 amplification facilitates an oncogenic gene expression program in high-grade serous ovarian cancer
Authors: Rhyasen Garrett W. , Yao Yi , Dulak Austin , Castriotta Lillian , Jacques Kelly , Hattersley Maureen , Mills Gordon B. , Zinda Michael , Fawell Stephen , Lyne Paul , Clark Edwin , Chen Huawei
Published: July 2016

The bromodomain and extra-terminal (BET) protein, BRD4 functions as a transcriptional co-activator through recruitment of regulatory complexes to acetylated chromatin. Among all malignancies, the most focal, and recurrent BRD4 gene amplification occurs in high-grade serous ovarian cancer (HGSOC). Despite the elucidation of BRD4 dependencies in cancer, it is unclear whether BRD4 gene amplification can result in oncogenic activity, and hence serve as a patient selection strategy for BET bromodomain inhibitors. To assess the oncogenic potential of BRD4 amplification in HGSOC, we assayed enforced BRD4 expression in non-transformed immortalized surface ovarian cells (IOSE). Physiologically relevant expression of either long or short BRD4 spliced isoforms in IOSE (BRD4-IOSE) resulted in marked phenotypic changes indicative of cellular transformation, including altered growth kinetics, chromatin reorganization, and acquisition of colony formation potential. Transcriptional profiling of BRD4-IOSE and BRD4-amplified HGSOC patients revealed shared expression patterns, including enriched MYC, E2F1, and VEGF gene signatures. Interrogation of BRD4 inhibitor pharmacodynamic markers, and transcripts upregulated through enforced BRD4 expression uncovered Neuregulin-1 as a critical BRD4 transcriptional effector. RNAi-mediated Neuregulin-1 depletion was capable of abolishing the oncogenic activity of BRD4-IOSE, and exogenous Neuregulin-1 was sufficient to transform IOSE without BRD4. A novel BRD4-SWI/SNF interaction was required for driving the oncogenic activity of BRD4-IOSE through maintaining NRG1 expression. The sensitivity of patient-derived HGSOC xenografts to a novel clinical candidate BRD4 inhibitor, AZD5153, correlated with BRD4 amplification and a pharmacodynamic NRG1 response. This study demonstrates the oncogenic potential of BRD4 amplification, defines a relationship between BRD4 and NRG1, and further establishes HGSOC as an appropriate patient population in which to test the therapeutic potential of BET bromodomain inhibitors.

Citation Format: Garrett W. Rhyasen, Yi Yao, Austin Dulak, Lillian Castriotta, Kelly Jacques, Maureen Hattersley, Gordon B. Mills, Michael Zinda, Stephen Fawell, Paul Lyne, Edwin Clark, Huawei Chen. BRD4 amplification facilitates an oncogenic gene expression program in high-grade serous ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1971.
Abstract 1974: Consequence of the tumor-associated deletion of CHD1 on transcriptional output and tumor progression
Authors: Augello Michael A. , Blattner-Johnson Mirjam , Rubin Mark , Barbieri Christopher
Published: July 2016

CHD1 is a member of the super-family of chromatin modifier proteins, and is characterized by the presence of several chromo-domains as well as an ATP dependent helicase domain. A multitude of studies across several model systems have demonstrated that CHD1 functions as a transcriptional co-activator, responsible for efficient RNA polymerase II (RNA pol II) ejection from the transcriptional start site as well as maintenance of RNA pol II processivity across the gene body. This is accomplished largely though nucleosome remodeling ahead of RNA pol II, which efficiently reorganizes nucleosome position to allow for optimal RNA pol II function. Consequently, as CHD1 plays a critical role in transcriptional competency, tumor associated deletions and/or mutations of the CHD1 gene that compromise its function are rare. Interestingly, prostate cancer proves to be the exception, where the CHD1 locus is deleted with high frequency in primary disease. Tumors harboring homozygous deletion of CHD1 belong to a subclass of prostate cancer that are enriched for either SPOP or FOXA1 mutations, suggesting a prostate-specific, tumor suppressive role for CHD1 in this genomic context. Importantly, while the oncogenic functions of both FOXA1 and SPOP mutations have been largely defined, little is known as to how CHD1 loss contributes to tumor initiation or progression. Here, using 3D organoid cultures derived from GEM models of inducible CHD1 loss, prostate specific cells were found to have dramatically altered transcriptionally competency upon CHD1 depletion. These events were correlated with an aberrant, tumor-associated transcriptome, resulting in changes in morphology and growth. Furthermore, while genome wide assessment of CHD1 occupancy in human prostate cancer models uncovered an enrichment of CHD1 at sites of active transcription, loss of CHD1 did not dramatically affect expression of genes associated with tumor progression. Inversely, genomic loss of CHD1 was associated with changes in lineage dependent markers, suggesting that a major function of CHD1 is to regulate the differentiation state of prostate cells. Collectively, these data help to define the tumor suppressive role for CHD1 in prostate cancer tumorigenesis, and suggest that loss of CHD1 could alter the function of key transcriptional regulators associated with prostate cancer initiation and progression.

Citation Format: Michael A. Augello, Mirjam Blattner-Johnson, Mark Rubin, Christopher Barbieri. Consequence of the tumor-associated deletion of CHD1 on transcriptional output and tumor progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1974.
Abstract 1981: Histone demethyalase JMJD1A promotes the DNA damage response of prostate cancer cells
Authors: Fan Lingling , Peng Guihong , Sahgal Natasha , Fazli Ladan , Gleave Martin , Zhang Yuji , Hussain Arif , Rassool Feyruz , Qi Jianfei
Published: July 2016

DNA damage is a common feature of prostate cancer (PCa) therapies (e.g. radio-, chemo- and androgen deprivation therapies). Thus, the DNA damage response (DDR) is a key factor in determining the therapeutic response of PCa patients. JMJD1A regulates gene expression by demethylating H3K9. Here, we identified a new role for JMJD1A in the DDR of PCa cells. Inhibition of JMJD1A led to increased r-H2AX foci (marker for the double strand DNA breaks) in the non-irradiated cells, and the delayed clearance of r-H2AX foci in the irradiated cells. JMJD1A knockdown in PCa cells inhibited the DNA strand break repair in the reporter assays. We further found that phosphorylation and ubiquitination were key modifications that regulate the activity of JMJD1A in the DDR. Ectopic expression of the mutant JMJD1A at the phosphorylation or ubiquitination site attenuated the expression of DNA repair factors, DNA strand break repair, and PCa cell proliferation under irradiation and androgen deprivation conditions. Our results suggest that targeting JMJD1A may sensitize the response of PCa to the major anti-PCa therapies.

Citation Format: Lingling Fan, Guihong Peng, Natasha Sahgal, Ladan Fazli, Martin Gleave, Yuji Zhang, Arif Hussain, Feyruz Rassool, Jianfei Qi. Histone demethyalase JMJD1A promotes the DNA damage response of prostate cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1981.
Abstract 201: Tribble 3 as integrator of metabolic stresses during lung cancer progression
Authors: Almiman Abeer , Adom Jamila , Guo Yande , Wang Jiuhui , Nie Daotai
Published: July 2016

Introduction: Tribbles pseudokinase 3 (TRIB3) has been identified as a stress related gene. Recent studies reported the increased expression of TRIB3 in cancers and its correlation with the poor prognosis in cancer patients. However, other studies suggest TRIB3 as a tumor suppressor due to its inhibitory activities toward PI3K/Akt and Mek-Erk signaling. In the present study, we aim to investigate the expression of TRIB3 in lung cancer cells and whether it plays a role in lung cancer progression and responses toward chemotherapy.

Methods: TRIB3 expression was determined by RT-PCR and Western blot. A lentiviral vector was used to overexpress TRIB3 in lung cancer cells. Small hairpin RNA constructs targeting TRIB3 were used to deplete TRIB3 expression. A CRISPR-CAS9 construct with guiding sequence matching to TRIB3 gene was used to knock out its expression. Cell proliferation, survival, and migration were determined using standard techniques.

Results: Through in silico analyses, we found that high level of TRIB3 RNA was associated with poor survival of lung cancer patients. In lung cancer cells (NCI-H358), TRIB3 expression was markedly stimulated during metabolic stresses or ER stresses. Overexpression of TRIB3 in cells led to decreased cell proliferation and increased arrest at G0/G1 phase, even when the cells were not under overt metabolic stresses. RT-qPCR analyses of cell cycle proteins revealed an upregulation of p27, a CDK inhibitor, in cells overexpressing TRIB3. Overexpression of TRIB3 reduced Akt phosphorylation at threonine 308. Depletion or knockout of TRIB3 led to increased cell growth but cells became more sensitive to stressors, leading to increased apoptotic cell death.

Conclusion: Our study reveals complex roles of TRIB3 in lung cancer progression. Induced by metabolic stresses, TRIB3 can block cell cycle progression through p27 in adaptive responses. Further studies are needed to determine whether and how TRIB3 functions as a metabolic gatekeeper during lung cancer progression.

Citation Format: Abeer Almiman, Jamila Adom, Yande Guo, Jiuhui Wang, Daotai Nie. Tribble 3 as integrator of metabolic stresses during lung cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 201.
Abstract 207: Regulation of mitochondrial biogenesis in primary human clear cell renal cell carcinoma cells
Authors: Nilsson Helén Å. , Johansson Martin E.
Published: July 2016

Clear cell renal cell carcinoma, ccRCC, is the most prevalent type of kidney cancer. In over 90% of ccRCCs the tumor suppressor protein von Hippel Lindau is inactivated, leading to a pseudohypoxic phenotype with vast effects on angiogenesis as well as energy metabolism. In accordance with the Warburg effect, aerobic glycolysis increases, while mitochondrial oxidative phosphorylation is decreased.

We have previously published data showing that also the number and activity of mitochondria is strikingly reduced in primary human ccRCC cells compared to normal kidney epithelial cells. This was not seen in any of the established RCC cell lines included in the study. We are now continuing these studies by investigating the regulation of mitochondrial biogenesis in primary human ccRCC cells, and how the number and activity of mitochondria influences the tumorigenic properties of these cells.

RNA has been isolated from ccRCC tissue areas identified as mitochondria high or low. The expression profile in these samples will be analyzed by RNA sequencing.

Human primary ccRCC cells obtained from patient nephrectomies are kept in culture and the regulation of mitochondrial biogenesis in these cells is investigated. How the mitochondrial content affects tumorigenic properties such as proliferation, migration and resistance to apoptosis is also investigated.

Preliminary data indicate that primary ccRCC cells have an absent or severly limited capability to increase the number of mitochondria on metabolic demand, and that the regulation of mitogenesis is defective.

These results point towards a malfunctioning energy sensing and mitochondrial biogenesis in ccRCC cells that might be part of the explanation to the very low mitochondrial content in these cells.

Citation Format: Helén Å. Nilsson, Martin E. Johansson. Regulation of mitochondrial biogenesis in primary human clear cell renal cell carcinoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 207.
Abstract 2084: Pharmacokinetic and pharmacodynamic assessment of autophagy inhibition following hydroxychloroquine in mice
Authors: Jackson Kristen M. , Hansen Ryan J. , Gustafson Daniel L.
Published: July 2016

Hydroxychloroquine (HCQ) is being tested in a number of human clinical trials to determine the role of autophagy in response to standard anticancer therapies. However, preclinical studies in mouse models are equivocal with regard to HCQ exposure (pharmacokinetics) and inhibition of autophagy (pharmacodynamics) in various tissues. Understanding HCQ pharmacokinetics (PK) and pharmacodynamics (PD) in mice allows for a comparison to human PK as reported in recent trials and as such the subsequent PD associated with autophagy inhibition in tissues. In this study, female BALB/c mice were treated with a single intraperitoneal dose of 20, 40, or 80 mg/kg HCQ and tissues and whole blood collected at 3, 6, 12, 24, 48 and 72 hours. Levels of HCQ and desethylhydroxychloroquine (dHCQ), an active metabolite, in whole blood and tissues were determined via a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. Non-compartmental analysis (NCA) measured exposure, as determined by area under the drug concentration versus time curve (AUC0-inf), to both HCQ and dHCQ was dose proportional in whole blood, liver and brain. The maximum concentration (Cmax) of HCQ was 1.3, 3.1 and 4.7 μg/mL and 20.6, 41.7 and 114.7 μg/mL in whole blood and liver, respectively, following 20, 40 and 80 mg/kg. Brain exposure was far less with Cmax achieving 272, 717 and 2220 ng/mL. In general, the time of maximal concentration (Tmax) of HCQ and dHCQ was observed at 3 hr or 6 hr, regardless of dose and location measured. PD assessment for autophagy inhibition was determined by western blotting. The LC3 protein, which is involved in membrane dynamics during autophagy, was visualized and the ratio of LC3-II to tubulin was measured. In the liver, autophagy was inhibited approximately two-fold following all doses at 24 and 48 hr compared to controls. Autophagy was inhibited in both the gut and kidney at 24 and 48 hr at all doses. In the brain, autophagy inhibition was not dose related and may be a result of low HCQ levels. These results demonstrate that a dose of 20 mg/kg is sufficient to inhibit autophagy in most tissues investigated; however, while PK parameters appear to be dose proportional, increased tissue drug levels does not necessarily equate to increased PD effects/autophagy inhibition. In addition, the data establishes a dose of 40 mg/kg in mice results in Cmax and AUC0-inf of HCQ equivalent to that observed in human clinical trials targeting autophagy modulation following a dose of 600 mg daily. Studies are ongoing to assess this relationship in solid tumors.

Citation Format: Kristen M. Jackson, Ryan J. Hansen, Daniel L. Gustafson. Pharmacokinetic and pharmacodynamic assessment of autophagy inhibition following hydroxychloroquine in mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2084.
Abstract 21: The mTORC2 target Akt is regulated in response to glutamine metabolite levels
Authors: Jacinto Estela , Moloughney Joseph , Kim Peter K. , Vega-Cotto Nicole M. , Wu Chang-Chih , Lynch Thomas , Zhang Sisi , Adlam Matthew , Guntaka Sai , Chou Po-Chien , Rabinowitz Joshua D. , Werlen Guy
Published: July 2016

Highly proliferating cells are particularly dependent on glucose and glutamine for bioenergetics and to fuel biosynthesis of macromolecules. The signals that respond to the fluctuations of these nutrients and how they control metabolic pathways remain poorly understood. mTOR, as part of mTOR complex 1 (mTORC1), responds to amino acids and plays a central role in metabolism. On the other hand, little is known on how mTORC2, consisting of the core components mTOR, rictor, SIN1 and mLST8 is regulated and its metabolic functions. The phosphorylation of the mTORC2 substrate, Akt, is enhanced in cancer cells, suggesting that mTORC2 becomes deregulated during tumorigenesis. Here we found that the activity of mTORC2 is enhanced by diminishing glutamine-derived metabolites. mTORC2 activity is required by glutamine-requiring biosynthetic pathways such as the hexosamine biosynthetic pathway (HBP). Acute nutrient withdrawal augments Akt phosphorylation but does not affect GFAT1 expression. However, extreme starvation that eventually depletes intracellular glutamine metabolites inactivates mTORC2 and downregulates GFAT1 expression. Thus, while mTORC1 senses glutamine abundance to promote anabolism, mTORC2 responds to declining glutamine catabolites in order to restore metabolic homeostasis. Our findings uncover the role of mTORC2 in metabolic reprogramming and provide insights on more effective therapeutic strategies for glutamine-dependent tumors.

Citation Format: Estela Jacinto, Joseph Moloughney, Peter K. Kim, Nicole M. Vega-Cotto, Chang-Chih Wu, Thomas Lynch, Sisi Zhang, Matthew Adlam, Sai Guntaka, Po-Chien Chou, Joshua D. Rabinowitz, Guy Werlen. The mTORC2 target Akt is regulated in response to glutamine metabolite levels. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 21.
Abstract 2102: Targeting mTOR downstream of LIN28 in atypical teratoid rhabdoid tumors promotes apoptosis and suppresses tumorigenicity
Authors: Rubens Jeffrey , Price Antoinette , Orr Brent , Eberhart Charles , Raabe Eric
Published: July 2016

LIN28 is a somatic cell reprogramming and stem cell factor that binds to and regulates RNA involved in growth, invasion and metabolic genes. We have previously shown that LIN28 is upregulated in atypical teratoid rhabdoid tumors (AT/RT) and contributes to the aggressive nature of these tumors. One of the canonical downstream targets of LIN28 is the mTOR pathway. We hypothesized that AT/RT tumorgenicity is dependent on mTOR signaling downstream of LIN28 and inhibition of this pathway would disrupt tumor growth. We found that primary human AT/RT samples have high expression of the mTOR pathway as determined by immunohistochemistry staining for P-S6 and P-AKT Ser473 (21% of tumors with 2+ P-S6 staining; 87% with 2+ p-AKT staining). The dual TORC1/2 inhibitor MLN0128 has good brain penetration and is currently being tested in phase I clinical trials. Treatment of AT/RT cell lines with MLN0128 inhibits TORC1/2 targets in vitro and suppresses cell proliferation at 100nM concentration in multiple AT/RT cell lines (MTS assay for BT12 p<0.005 vs DMSO control; BT37 p = 0.006 vs DMSO control; and CHLA-06 p<0.005 vs DMSO control by t-test). MLN0128 also slows cell proliferation via BrdU assay (BT12 p<0.005 vs DMSO control; BT37 p<0.005 100nM vs DMSO control; CHLA-06 p<0.005 100nM vs DMSO control by t-test) and induces apoptosis measured by Western Blot for cleaved PARP and cleaved caspase 3 assay (BT12 p<0.005 100nM vs DMSO; BT37 p<0.005 100nM vs DMSO; CHLA-06 p = 0.007 100nM vs DMSO by t-test). MLN0128 induces apoptosis synergistically with cisplatin, which is the backbone of conventional AT/RT therapy (CC3 assay BT37 p<0.005 vs DMSO by t-test) and slows cell growth (MTS assay BT37 p<0.005 vs DMSO control by t-test). MLN0128 treatment of AT/RT xenograft mouse models extends overall survival from a median of 23 days to 39 days (Log-rank test p = 0.002). Targeting the mTOR pathway with MLN0128 leads to potent in vitro and in vivo activity against AT/RT and can be combined with conventional chemotherapy to provide an important survival benefit. MLN0128 may be a candidate for future clinical trials to treat this deadly tumor.

Citation Format: Jeffrey Rubens, Antoinette Price, Brent Orr, Charles Eberhart, Eric Raabe. Targeting mTOR downstream of LIN28 in atypical teratoid rhabdoid tumors promotes apoptosis and suppresses tumorigenicity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2102.
Abstract 2105: c-Met hyperactivation is an universal resistance mechanism to both first and third generation EGFR inhibitors
Authors: Shi Puyu , Oh You-Take , Zhang Guojing , Yao Weilong , Yue Ping , Kanteti Rajani , Riehm Jacob , Salgia Ravi , Owonikoko Taofeek , Ramalingam Suresh S. , Chen Mingwei , Sun Shi-Yong
Published: July 2016

c-Met amplification and acquisition of a second T790M mutation are key mechanisms accounting for majority of resistant cases to first generation EGFR-tyrosine kinase inhibitors (EGFR-TKIs; i.e., erlotinib). The third generation EGFR-TKIs (e.g., AZD9291), which selectively and irreversibly inhibit EGFR activating and T790M mutants while sparing wild-type EGFR, represent very promising therapeutic options for NSCLC patients who have become resistant to 1st generation EGFR-TKIs due to T790M mutation. However, eventual resistance to the 3rd generation EGFR-TKIs has already been described in the clinic, resulting in disease progression. Therefore, there is a great challenge and urgent need to understand how this resistance occurs and to develop effective strategies to delay or overcome the resistance. We show that c-Met amplification and hyperactivation is an universal mechanism to both 1st and 3rd generation EGFR-TKIs since both erlotinib- and AZD9291-resistant HCC827 cell lines possessed elevated levels of c-Met (due to gene amplification) and p-c-Met and were cross-resistant to AZD9291 or erlotinib. Both chemical and genetic inhibition of c-Met overcame the resistance of these cell lines to AZD9291 including enhancement of apoptosis or G1 cell cycle arrest. Consistently the combination of AZD9291 and c-Met inhibition effectively inhibited the growth of both erlotinib- and AZD9291-resistant HCC827 xenografts in nude mice. Hence, we suggest that inhibition of c-Met is also an effective strategy to overcome resistance of EGFR-mutated NSCLCs with c-Met amplification or hyperactivation to AZD9291, providing the rationale for clinical development of this novel combination strategy. (SSR, TKO and SYS are Georgia Research Alliance Distinguished Cancer Scientists)

Citation Format: Puyu Shi, You-Take Oh, Guojing Zhang, Weilong Yao, Ping Yue, Rajani Kanteti, Jacob Riehm, Ravi Salgia, Taofeek Owonikoko, Suresh S. Ramalingam, Mingwei Chen, Shi-Yong Sun. c-Met hyperactivation is an universal resistance mechanism to both first and third generation EGFR inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2105.
Abstract 2128: Influence of estrogen receptor status on acquisition of doxorubicin resistance in breast cancer cells
Authors: Ponnusamy Logeswari , Mahalingaiah Prathap Kumar Salakatte , Singh Kamaleshwar P.
Published: July 2016

Breast cancer is the most commonly diagnosed invasive cancer in women. Doxorubicin is being the mainstay treatment for solid cancers including breast cancer. However, chemotherapy resistance, either innate or acquired, is a major limitation in breast cancer treatment. The mechanism of resistance as well as the role of estrogen receptor (ER) status in doxorubicin resistance in breast cancer is not clear. Therefore, objective of this study was to determine whether ER status influence the doxorubicin resistance, and to further identify the molecular mechanism in this process. To address this question, ER-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cell lines were given continuous treatment of clinically relevant concentration of doxorubicin and the pattern of resistance was monitored. Resistance was evaluated using various parameters such as cell count, cell growth and cytotoxicity by MTT assay, cell cycle analysis using flow cytometer. Expression of genes and protein related to cell cycle and cell survival, drug transport, DNA damage repair, metastasis/invasion and epigenetic regulatory complex were measured by qRT- PCR and western blot respectively. Soft agar assay and wound healing assay were performed to determine the anchorage-independent growth and migration potential of resistance cells. Results of MTT assay, and cell cycle revealed that ER-positive MCF-7 cells developed relatively earlier and high level of resistance when compared to MDA-MB-231 cells. This was further confirmed by additional features such as, cancer stem cell markers, epithelial to mesenchymal transition, and increased tumorigenicity in MCF-7 cells than in MDA-MB-231 cells during resistance development. These changes were associated with alteration in drug transporters, cell cycle genes and epigenetic regulatory proteins such as HDAC1 and DNMT1. Pretreatment with demethylating agent 5-aza-deoxycytidine and HDAC inhibitor Trichostatin A significantly resensitized resistance MCF-7 and MDA-MB-231 cells to doxorubicin in comparable to sensitive parental cells. However, this resenstitivty was higher in MCF-7 cells when compared to MDA-MB-231 cells. In summary, result of this study suggests that acquisition of doxorubicin resistance depends on status of estrogen receptor in breast cancer cell lines. Additionally, this differential resistance pattern could be due to differences in epigenetic changes in these two types of breast cancers, thus the acquired resistance could be potentially resensitized using epigenetic therapy

Citation Format: Logeswari Ponnusamy, Prathap Kumar Salakatte Mahalingaiah, Kamaleshwar P. Singh. Influence of estrogen receptor status on acquisition of doxorubicin resistance in breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2128.