bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A01: ER stress disrupts circadian rhythms via miR-211
Authors: Bu Yiwen , Diehl John Alan
Published: May 2016

The Endoplasmic Reticulum, ER, senses both intracellular and extracellular stimuli that perturb the folding and maturation of secretory proteins that transit the ER. In the context of neoplastic growth, oncogenes such as c-Myc, H-Ras and BRAF trigger ER stress and activate the Unfolded Protein Response (UPR), a cell adaptive signaling pathway composed of three primary signal transducers, PERK, IRE-1 and ATF6.

Bmal1 and Clock form a core heterodimeric transcription complex regulates both central and peripheral circadian rhythms. There are multiple lines of evidence suggesting that deregulation of circadian rhythms contribute to neoplastic cell growth, altered cellular metabolism and tumorigenesis. Analogously, UPR signaling contributes to tumor cell survival and tumor growth under conditions of micro-environmental and metabolic stress. However, whether UPR interactively regulates stress with the circadian clock is largely unknown.

Herein, we describe the molecular mechanisms whereby UPR signaling integrates with the circadian clock. We provide evidence that ER stress-dependent activation of PERK alters circadian oscillations via micro-RNA 211-dependent suppression of BMAL1 and Clock. We provide evidence that the mechanism of regulation of BMAL1 versus clock is distinct, reflecting novel ability of miR-211 to function in both the nucleus and cytoplasm. We provide additional support for this model through using a mouse model to interrogate the role of PERK signaling in a liver specific response to ER stress. With regard to the impact of PERK signaling and circadian clock disruption in cancer, we assessed PERK-miR-211 signaling in c-Myc and MLL-AF9 driven malignancy. Here, expression of either c-Myc or MLL-AF9 expression was directly associated with activation of PERK-miR-211 and loss of both BMAL1 and Clock expression. We also found that Myc driven human Burkitt's lymphoma has PERK hyperactivation, higher miR-211 expression, and loss of circadian oscillations. As the Bmal1 promoter is hypermethylated in multiple lymphoma cancer cell lines, we hypothesize that this is likely to reflect the action of nuclear miR-211. Consistently, anti-miR211 partially released Bmal1 repression and Inhibition PERK or suppression miR-211 rescued circadian rhythms in lymphoma cells thereby affecting their survival and proliferation. Our data suggested a model where ER stress disrupts peripheral circadian rhythms via PERK-miR211-Bmal1/Clock pathway. Targeting activated ER stress or abnormal miR-211 expression to rescue circadian rhythms could be a new potential therapy for Myc- (perhaps also other oncogenes) driven hematological malignancy.

Citation Format: Yiwen Bu, John Alan Diehl. ER stress disrupts circadian rhythms via miR-211. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A01.
Abstract A05: Retrotransposon-derived RNAs in regulating cancer-related alternative splicing
Authors: Zhou Zhihong , Wang Cheng , Hu Qidong
Published: May 2016

Cancer features uncontrollable growth of transformed or dedifferentiated cells. The prevalent treatment options include surgery, chemotherapy, radiation therapy, targeted therapy and transplantation. In particular, chemotherapy is designed to suppress cell growth, cause cell death and prevent cancer spread. However, the drug often kills healthy cells indiscriminately and becomes ineffective in the initially responsive patients who are then faced with an even more devastating situation. It has been suggested that there is a reciprocal feedback loop between pre-mRNA alternative splicing and chemotherapy-mediated DNA damage, hence we have investigated the potential role of pre-mRNA alternative splicing in causing resistance to chemotherapy, especially the platinum-based drug, cisplatin.

In this study, we examined the expression of SR protein-specific kinases (SRPKs) and their post-translational modification in normal and cisplatin-treated Hela and MCF7 cells. While the expression of SRPK1 and SRPK2 were initially downregulated by cisplatin, the acetylated forms of these proteins were actually enhanced dramatically. Furthermore, in the established resistant cell lines, their expression was restored. The loss-of-function experiments using the siRNA knockdown, acetylation inhibitor and HAT-deficient TIP60 suggested that the acetylated SRPKs could confer the cisplatin resistance to the cancer cells by increasing the expression of those pro-growth proteins and isoforms, such as Myc, Bcl-2, Bcl-XL and CCND1b. Remarkably, in the treated and resistant cancer cells, we observed the induced expression of Alu repeats, a large family of retrotransposons with over a million copies in the human genome. These retrotransposon-derived RNAs are associated with the downstream factors of SRPKs, SR proteins. Depletion of the Alu transcripts would re-sensitize the resistant cells to cisplatin. In particular, we observed that the induced expression of Alu RNAs was required for the generation of those pro-growth proteins, possibly through interacting and synergizing with the SR proteins. Hence, the study provides novel insights into the drug resistance mechanism and will help to develop new ways of targeting retrotransposon RNAs to overcome this haunting problem in cancer treatment.

Citation Format: Zhihong Zhou, Cheng Wang, Qidong Hu. Retrotransposon-derived RNAs in regulating cancer-related alternative splicing. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A05.
Abstract A22: Functional long noncoding RNAs in castration-resistant prostate cancer
Authors: Fei Teng , Chen Yiwen , Liu Xiaole Shirley , Brown Myles
Published: May 2016

Prostate cancer progression from an androgen-dependent state to an androgen-independent phase, which is also referred to castration-resistant prostate cancer (CRPC), not only results in cancer refractory to androgen deprivation therapy but also marks lethal prognosis. Androgen receptor (AR) plays central roles in both normal prostate development and prostate cancer progression. Aberrant androgen/AR signaling activity is often associated with the development of CRPC, such as increased synthesis of steroid hormone, increased expression of AR, emergence of specific AR variants etc. However, deeper understanding of detailed mechanism of CRPC development is still necessary in order to discover novel effective drug targets for the treatment of CRPC. Recent studies suggest that long non-coding RNA (lncRNA) can be extensively functional rather than just transcriptional noise and lncRNAs are also widely involved in the pathogenesis of different types of cancers including prostate cancer. To further investigate the potential role of lncRNAs during prostate cancer progression, we profiled lncRNA transcriptome using RNA-seq in prostate cancer cells in the presence or absence of androgen treatment. In combination with AR cistrome data, we identified a set of lncRNAs that are directly regulated by androgen/AR signaling. A specific lncRNA was identified as androgen/AR repressed target gene in androgen-dependent cells while showing significant higher expression in androgen-independent cells that mimics CRPC state. Interestingly, knockdown of this lncRNA specifically attenuates the growth of androgen-independent cells while has little effect on androgen-dependent cells. To understand the detailed mechanism, we identified the downstream target genes using RNA-seq upon silencing this lncRNA in either androgen-dependent or -independent cells. Moreover, this lncRNA is located in a region that is frequently amplified in CRPC and highly expressed in a subset of CRPC patients. We suggest that this lncRNA, along with other androgen/AR targets, may play a role during the development of CRPC and could serve as a potential drug target for the treatment of CRPC.

Note: This abstract was not presented at the conference.

Citation Format: Teng Fei, Yiwen Chen, Xiaole Shirley Liu, Myles Brown. Functional long noncoding RNAs in castration-resistant prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A22.
Abstract A27: Long noncoding RNA expression in cytogenetically normal acute myeloid leukemia identifies a distinct signature associated with NPM1 mutations
Authors: Clara Etienne De , Ma Hanjing , Vergez François , Quelen Cathy , Dejean Sébastien , Demur Cécile , Delabesse Eric , Recher Christian , Touriol Christian , Martelli Maria Paola , Falini Brunangelo , Brousset Pierre , Bousquet Marina
Published: May 2016

Long noncoding RNAs (lncRNAs) are defined as RNA transcripts that are larger than 200 nt but do not appear to have protein-coding potential. Cumulative evidence points towards an important role of lncRNAs in cancer initiation, development, and progression. In this study we sought to evaluate the lncRNA expression profile of patients with cytogenetically normal acute myeloid leukemia (CN-AML). RNA sequencing of forty CN-AML patients allowed us to identify more than 8000 previously-undescribed lncRNAs. Using unsupervised analysis we observed a specific lncRNA signature dependent on the mutational status of the nucleophosmin (NPM1) gene. Sparse partial least squares discriminant analysis allowed us to identify a minimal set of 19 lncRNAs capable of discriminating NPM1-mutated from NPM1-wild type patients. Among these, we found that expression of the XLOC_087120 lncRNA inversely correlates with its neighboring histone-coding genes. XLOC_087120 also interacts with SUZ12, a component of the Polycomb Repressive Complex 2, suggesting a role for this lncRNA in the epigenetic repression of histone genes and therefore a potential impact on chromatin remodeling. Indeed, its overexpression in cell lines leads to a downregulation of histone genes. Furthermore, we demonstrated that mutant NPM1 modulates the nuclear/cytoplasmic localization of XLOC_087120. Altogether, these data suggest that lncRNAs should be considered as key players in the pathogenesis of acute myeloid leukemias.

Citation Format: Etienne De Clara, Hanjing Ma, François Vergez, Cathy Quelen, Sébastien Dejean, Cécile Demur, Eric Delabesse, Christian Recher, Christian Touriol, Maria Paola Martelli, Brunangelo Falini, Pierre Brousset, Marina Bousquet. Long noncoding RNA expression in cytogenetically normal acute myeloid leukemia identifies a distinct signature associated with NPM1 mutations. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A27.
Abstract A36: A systematic network-based approach to prioritizing lncRNA drivers in cancers
Authors: Li Jun , Zhou Zhicheng , Liang Han
Published: May 2016

Introduction: Decades of efforts have been made to have a better understanding about the human genome, which encodes ~20,000 protein-coding genes as well as an astounding number of transcriptionally active noncoding RNAs. Among these noncoding RNAs, long-noncoding RNAs (lncRNAs) have been increasingly recognized to play essential roles in tumorigenesis, representing a new focus in cancer research. Analogous to the identification of protein-coding cancer driver genes, it is a crucial task in cancer research to identify lncRNA drivers and elucidate their functions in various cancer types.

Methods: We first downloaded TCGA RNA-seq BAM files (>60TB) across 20 cancer types from the UCSC Cancer Genomic Hub (CGHub) and quantified the lncRNA expression based on GENCODE lncRNA annotation. To identify lncRNA drivers, we focused on identifying the functional relevant lncRNA-protein pairs through their expression profiling data. To prevent acquiring more false positives from conventional correlation analysis, we applied the context likelihood of relatedness (CLR) algorithm on evaluating co-expressed lncRNA and protein pairs based on RNA-seq and reverse-phase protein array (RPPA) data. In addition, to further increase the clinical relevance of the potential lncRNA drivers, we conducted survival analysis and tumor/normal differential analysis to boost the prediction power of our systematic computational approach.

Results: In this study, we integrated lncRNA expression, protein expression and patient survival data to nominate lncRNA drivers in 20 cancers. Among ~4 million lncRNA-protein pairs, we have identified 9 lncRNA drivers and 16 potential lncRNA-protein interaction pairs linked with them. To validate our findings, we selected 3 lncRNAs with high confidence and performed knockdown experiments in multiple cell lines, in which 2 lncRNAs (TMPO-AS1 and HCP5) showed functional relevance with protein-coding genes MSH6 and LCK respectively. Both of the pairs have the same direction of correlations (positive or negative) as we predicted. Our results indicate that the identified lncRNAs may functionally interact with their protein partners identified by our network-based approach, which will further provide the molecular foundation for investigating cancer mechanisms, identifying prognostic lncRNA markers and characterizing additional therapeutic targets.

Citation Format: Jun Li, Zhicheng Zhou, Han Liang. A systematic network-based approach to prioritizing lncRNA drivers in cancers. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A36.
Abstract A43: Long noncoding RNAs as targets of mammary tumor cell proliferation and migration
Authors: Diermeier Sarah D. , Chang Kung-Chi , Freier Susan M. , Rigo Frank , Bennett C. Frank , Spector David L.
Published: May 2016

Objective: Recent genome-wide studies revealed that as much as 80% of the human genome can be transcribed whereas only 2% of this RNA is translated into proteins. Non-coding transcripts can be subdivided into several groups, with long non-coding RNAs (lncRNAs) representing the largest and most diverse class. With breast cancer being the most frequent malignancy in women worldwide, we set out to investigate the potential of lncRNAs as novel therapeutic targets.

Methods: We focused on the luminal B and the HER2/neu-positive subtypes of breast cancer, as both are associated with high prevalence and bad prognosis. By performing RNA-Seq on tumor sections and mammary organoids from MMTV-PyMT and MMTV-Neu-NDL mice, modeling the luminal B and HER2/neu-amplified subtypes of human breast cancer respectively, we generated a comprehensive catalog of differentially expressed lncRNAs. We functionally validated the role of a subset of these lncRNAs by antisense oligonucleotide (ASO) mediated knockdown in primary mammary tumor cells and 3D ex vivo organoids.

Results: We identified several hundred potentially oncogenic lncRNAs that were over-expressed in a subtype-specific manner as well as numerous lncRNAs up-regulated in both MMTV-PyMT and MMTV-Neu-NDL tumors. To narrow down the candidate list of lncRNAs with the highest therapeutic potential, we sorted the data computationally and defined a subset of previously uncharacterized lncRNAs as Mammary Tumor Associated RNAs (MaTARs). Upon independent knockdown of several MaTARs, we observed significantly reduced cell proliferation, invasion and/or collective cell migration in a cancer-specific context.

Conclusion: MaTARs are likely important drivers of mammary tumor progression and/or metastasis and represent promising new therapeutic targets. Future studies will focus on the molecular characterization of these lncRNAs and elucidation of their potential role(s) in mammary tumor progression and metastasis.

Citation Format: Sarah D. Diermeier, Kung-Chi Chang, Susan M. Freier, Frank Rigo, C. Frank Bennett, David L. Spector. Long noncoding RNAs as targets of mammary tumor cell proliferation and migration. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A43.
Abstract B07: Long noncoding RNAs underlying genetic predispositions to prostate cancer
Authors: Guo Haiyang , Ahmed Musaddeque , Hua Junjie Tony , Liang Yi , He Housheng Hansen
Published: May 2016

Trait-associated SNPs identified through Genome-Wide Association Studies are enriched in regulatory regions. However, the functional link between these SNPs and their target genes remains elusive. Due to their involvement in fundamental biological processes, long noncoding RNAs (lncRNAs) represent an attractive class of molecules mediating cancer risk. Through integrative analysis of the lncRNA transcriptome with genomic and prostate cancer risk SNP data, we identified 60 candidate lncRNAs associated with risk to prostate cancer. The mechanism underlying the top hit, PCAT1, was evaluated further. The risk variant at rs7463708 decreases HOXB13 and increases AR binding at a distal enhancer that loops to PCAT1 promoter, resulting in upregulation of PCAT1 upon prolonged androgen treatment. In addition, PCAT1 interacts with AR and LSD1 and is required for their recruitment to the enhancers of GNMT and DHCR24, two androgen late response genes implicated in prostate cancer development and progression. These findings suggest that modulating lncRNA expression is an important mechanism for risk SNPs in promoting prostate transformation.

Note: This abstract was not presented at the conference.

Citation Format: Haiyang Guo, Musaddeque Ahmed, Junjie Tony Hua, Yi Liang, Housheng Hansen He. Long noncoding RNAs underlying genetic predispositions to prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B07.
Abstract B13: Eradication of neuroblastoma by suppressing the expression of a single long noncoding RNA
Authors: Liu Pei Y. , Tee Andrew E. , Atmadibrata Bernard , Mestdagh Pieter , Vandesompele Jo , Dinger Marcel , Liu Tao
Published: May 2016

Neuroblastoma is the most common solid tumour in early childhood, and accounts for approximately 15% of all childhood cancer death. N-Myc gene amplification occurs in one quarter to one third of human neuroblastoma tissues, and the majority of patient with neuroblastoma due to N-Myc gene amplification die of the disease. We performed RNA sequencing experiments, and identified 5 transcripts, including RP1X, which were considerably differentially expressed between N-Myc gene amplified and non-amplified human neuroblastoma cell lines. Affymetrix microarray studies revealed that DEPDC was one of the few genes considerably down-regulated in neuroblastoma cells after RP1X depletion. Chromatin immunoprecipitation assays showed that knocking-down RP1X expression reduced histone H3 lysine 4 trimethylation, a marker for active gene transcription, at the DEPDC gene promoter. Depletion of RP1X or DEPDC significantly reduced ERK protein phosphorylation, N-Myc protein phosphorylation at Serine 62, N-Myc protein stabilization, neuroblastoma cell proliferation and survival. Clonogenic assays showed that knocking-down RP1X with doxycycline completely abolished colony formation capacity of neuroblastoma cells stably transfected with doxycycline-inducible RP1X shRNA. Importantly, treatment with doxycycline in mice xenografted with neuroblastoma cells stably transfected with doxycycline-inducible RP1X shRNA, led to tumour eradication. In human neuroblastoma tissues from 600 neuroblastoma patients, high levels of RP1X gene expression correlated with DEPDC gene expression and poor patient prognosis. In conclusion, this study identifies the novel long noncoding RNA RP1X as an important regulator of N-Myc protein stability and neuroblastoma tumourigenesis.

Citation Format: Pei Y. Liu, Andrew E. Tee, Bernard Atmadibrata, Pieter Mestdagh, Jo Vandesompele, Marcel Dinger, Tao Liu. Eradication of neuroblastoma by suppressing the expression of a single long noncoding RNA. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B13.
Abstract B23: Role of noncoding RNAs in regulating human papilloma virus-16 (HPV-16) induced oncogenesis in head and neck squamous cell carcinoma (HNSCC)
Authors: Sannigrahi Malay Kumar , Singh Varinder , Sharma Rajni , Panda Naresh K. , Khullar Madhu
Published: May 2016

Background: HPV is an emerging risk factor for HNSCC, however, molecular mechanisms in HPV mediated HNSCC remains poorly delineated. Recent studies suggest that non-coding RNAs may play an important role in the aetiology of HNSCC. In the present study, we examined microRNAs involved in HPV-16 induced HNSCC.

Material and Method: Tissue samples from HNSCC patients (n=300) were screened for HPV-16 using PCR based approach with consensus PCR primers (MY09/MY11, Gp5+/Gp6+), HPV16-specific PCR, HPV-16 E7 mRNA, immunohistochemistry (p16, p53 and pRB) and viral-load determination. In-silico approach was used to identify host miRNAs with HPV-16 mRNAs as putative target. Differential expression of identified miRNAs was determined by qRT-PCR in HPV-16 positive (UPCI:SCC090, CaSki, SiHa), HPV negative (UPCI:SCC116, HaCat) and transfected with HPV 16 full genome plasmid cell lines, and, HPV 16-positive/HPV-negative tissue samples. Putative targets of identified miRNAs were identified by bioinformatic approach and their mRNA levels were determined by qRT-PCR in HPV positive and negative cell lines, and tissue samples. Over-expression and inhibition studies with miRNA mimic and inhibitor were done to identify affected viral and host oncogenic pathways.

Results: Total 32 HNSCC patients were found to be HPV-16 positive. We identified 10 miRNAs with HPV-16 as putative target. Among them hsa-miR-139-3p showed significant down-regulation in HPV-16 positive samples and cell lines compared to HPV negative controls. Using bioinformatics tools, hsa-miR-139-3p was found to target viral HPV-16 E1 and host HOXB6 gene. Transfection with hsa-miR-139-3p mimic resulted in significant down-regulation of HPV 16 early mRNA as well as its targeted genes in luciferase-3'UTR assay. Moreover, mimic transfected HPV-positive cell lines showed dysregulated MEK-ERK signaling pathway.

Conclusion: Our results suggest hsa-miR-139-3p may be involved in patho-physiology of HPV-16 positive HNSCC as it deregulates expression of HPV oncogenic proteins and cell proliferation by targeting MEK-ERK signaling pathway.

Citation Format: Malay Kumar Sannigrahi, Varinder Singh, Rajni Sharma, Naresh K. Panda, Madhu Khullar. Role of noncoding RNAs in regulating human papilloma virus-16 (HPV-16) induced oncogenesis in head and neck squamous cell carcinoma (HNSCC). [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B23.
Abstract B26: OIP5-Antisense 1, a long noncoding RNA deregulated in non-small cell lung cancer
Authors: Stewart Greg L. , Enfield Katey SS , Rowbotham David A. , Hubaux Roland , Martinez Victor , Lam Stephen , Lam Wan
Published: May 2016

Background: Lung cancer represents an enormous health burden, representing the most common cause of cancer death worldwide. The poor therapeutic outcome is largely due to a complex molecular background as well as late stage diagnosis, with most patients presenting unresectable local tumors, or metastatic disease. While mutations of driver genes is a well-known mechanism, approximately half of all non-small cell lung cancer (NSCLC) tumors harbor no known clinically relevant oncogenic drivers, emphasizing the need to explore alternative mechanisms such as non-coding RNAs (ncRNAs). Natural antisense transcripts (NATS) are ncRNAs that are expressed from the opposite strand of coding mRNAs. These genes overlap with, and are often involved in the regulation of their sense counterparts. NATs can recruit regulatory complexes to their transcriptional locus, leading to silencing of transcription and have recently been described in cancer to silence tumor suppressor genes. NATs are quite prevalent as it is estimated that 25-40% of genes display overlapping transcription, however only a few NATs have been characterized, emphasizing the need to explore these ncRNAs in the context of NSCLC.

Hypothesis: We hypothesize NATs of NSCLC-related genes are deregulated in NSCLC.

Methods: We performed RNAseq and miRNAseq on a set of 65 NSCLC tumors including 36 adenocarcinomas and 29 squamous cell carcinomas as well as matched non-malignant lung tissue. A sign rank test was used to identify NATs with significantly altered expression between tumor and matched normal. Survival analysis was done using a Cox Proportional hazard model, as well as the Kaplan-Meier method.

Results: We have identified a NAT of OIP5, a lung cancer oncogene required for chromatin segregation, to be significantly underexpressed, while its sense counterpart, OIP5 mRNA, is significantly overexpressed. We also find that expression of both OIP5 and OIP5-AS1 has a significant impact on 5 year survival. These findings suggest that deregulation of OIP5 through its antisense RNA may represent a novel mechanism regulating tumor phenotypes in NSCLC.

Citation Format: Greg L. Stewart, Katey SS Enfield, David A. Rowbotham, Roland Hubaux, Victor Martinez, Stephen Lam, Wan Lam. OIP5-Antisense 1, a long noncoding RNA deregulated in non-small cell lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B26.
Abstract B39: Long noncoding RNA PCAT14/PRCAT104; A prognostic biomarker in prostate cancer
Authors: Malik Rohit , Zhang Xiang , Shukla Sudhanshu , Niknafs Yashar Y. , Zhao Shuang , Feng Felix Y. , Chinnaiyan Arul M.
Published: May 2016

Prostate cancer is the second most common epithelial cancer and the second leading cause of cancer-related death for men in the United States. While majority of prostate cancer cases are indolent and cause minimal morbidity and mortality, a subset of men progress to a hormone-refractory aggressive disease with high mortality. Markers such as PSA and PCA3 perform well in diagnosis of disease; however these biomarkers are unable to predict disease progression. Therefore, there is an urgent need to identify clinical predictors of disease progression. Long non-coding RNAs (lncRNAs) are emerging as an important class of biomolecules that exhibit significant lineage- and cancer-specificity making them ideal biomarker candidates.

Using RNA-Seq data from The Cancer Genome Atlas (TCGA) and as well as from libraries generated in our laboratory, we identified PCAT14/PRCAT104 as a marker of low Gleason disease. PCAT14 was shown to be highly expressed in prostate cancer compared to benign tissue; however, its expression in prostate cancer was limited to low Gleason disease. To directly evaluate the relationship between PCAT14 levels and clinical outcome, we assessed its expression in more that 1600 Prostate cancer samples by a high-density Affymetrix GeneChip platform. In all 5 cohorts analyzed, PCAT14 was shown to be a strong prognostic marker in its ability to predict biochemical recurrence, clinical progression to systemic disease and prostate cancer–specific mortality. Furthermore, in a multivariate analysis, PCAT14 expression also predict resistance to androgen deprivation therapy (ADT) (p=0.012). More interestingly, PCAT14 was able to add to prognostic value of SCHLAP1, a lncRNA that we previously showed to be an excellent prognostic marker in prostate cancer. PCAT14 in combination with SCHLAP1 was able better in predicting 10-year metastasis free survival (AUC: 0.64) than SCHLAP1 alone (AUC=0.58). Taken together, we identified a novel marker of low grade prostate cancer that in combination with SCHALP1, a marker of aggressive cancer, can predict disease progression and hence can be of immense value for treatment individualization in prostate cancer

Citation Format: Rohit Malik, Xiang Zhang, Sudhanshu Shukla, Yashar Y. Niknafs, Shuang Zhao, Felix Y. Feng, Arul M. Chinnaiyan. Long noncoding RNA PCAT14/PRCAT104; A prognostic biomarker in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B39.
Abstract IA06: Disregulation of lncRNAs helps drive Ewing sarcoma pathogenesis
Authors: Sweet-Cordero Alejandro
Published: May 2016

Pediatric sarcomas are orphan diseases that cause significant morbidity and mortality in children, adolescents and young adults. Among the most intriguing pediatric sarcomas is Ewing sarcoma. The (11;22)(q24;q12) translocation, which leads to expression of EWS/FLI, is identified in 85% of Ewing sarcoma cases. Ewing sarcoma cells carrying this translocation have a strict dependency on its expression, suggesting that the translocation is a critical oncogenic event in this disease. Despite intensive investigation, the mechanisms through which EWS/FLI leads to oncogenesis are incompletely understood. Recent data indicates that two distinct mechanisms are involved. In one, EWS/FLI leads to gene activation by acting as a pioneer transcription factor at novel enhancers. In another, EWS/FLI forces the removal of ets binding factors from their established binding sites and acts as a transcriptional repressor. How these two diverse functions are orchestrated at a molecular level remains unclear.

Given the widespread transcription of long non-coding RNAs and their emerging role in gene regulation, they are strong candidates for participation in the complex genetic rewiring program induced by EWS/FLI. We recently described the first lncRNA clearly regulated by EWS/FLI, (EWS-associated transcript 1, EWSAT1). Expression of EWS/FLI drives upregulation of EWSAT1 most likely by direct transcriptional regulation. EWSAT1 in turn plays a critical role in EWS/FLI-mediated gene repression, at least in part by interacting with the RNA binding protein hnRNPK. RNAseq analysis indicates that EWS/FLI regulates the expression of a large number of other lncRNAs in addition to EWSAT1. We have identified lnc659 (EWSAT2) as a second Ewing sarcoma-related transcript with functional relevance to Ewing pathogenesis. EWSAT2 is upregulated by expression of EWS/FLI and knock-down of EWSAT2 strongly decreases the proliferation of Ewing sarcoma cell lines in vitro and the oncogenicity of cell lines and patient-derived xenografts in vivo. Ongoing studies are directed at identifying the molecular mechanism of action of EWSAT1 and EWSAT2 as well as other lncRNAs in the pathogenesis of Ewing sarcoma.

Citation Format: Alejandro Sweet-Cordero. Disregulation of lncRNAs helps drive Ewing sarcoma pathogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr IA06.
Abstract IA13: It takes two to tango: The PVT1-MYC alliance in human cancer
Authors: Bagchi Anindya
Published: May 2016

Though gain of 8q24.21 is a common mutation in human cancers, its functional annotation is limited to studying myelocytomatosis (MYC), the prominent oncogene in the amplicon. However, MYC is co-gained with an adjacent long non-coding RNA gene plasmacytoma variant translocation 1 (PVT1), the CCDC26 gene candidate and gasdermin C GSDMC. Whether copy number gain of one or more of these genes drives neoplasia is not known. We developed chromosome engineered mouse strains with an extra copy of 1) Myc, 2) Pvt1, Ccdc26, Gsdmc, and 3) Myc, Pvt1, Ccdc26, Gsdmc. When rat Neu was introduced to test the change in the latency in mammary tumor development, only the mice with an extra copy of Myc, Pvt1, Ccdc26, Gsdmc (but not with an extra copy of Myc or Pvt1, Ccdc26, Gsdmc) developed adenocarcinomas with reduced latency, suggesting that while an extra copy of Myc gene failed to measurably advance cancer, it may co-operate with Pvt1, Ccdc26 or Gsdmc to promote cancer. Si-RNA mediated knockdown of Pvt1/PVT1 reduced the proliferation rates in the tumors, and in SK-BR-3 and MDA-MB-231, two human breast cancer cell lines with 8q24 amplification. Ablation of PVT1 markedly decreased MYC protein levels, suggesting a PVT1-dependence of MYC protein in MYC amplified cancer cells. Analysis of the cancer genome atlas suggested 99% of tumors with increased MYC copy-number also harbor co-gain of PVT1. Tissue microarray analysis revealed that PVT1 RNA and MYC protein expression are correlated in primary human tumors. These data suggests that PVT1 can potentiate MYC in human cancers. CRISPR mediated deletion of PVT1 in colorectal cell line HCT116 impaired tumor formation in xenografts, and significantly reduced MYC levels. We further confirmed the oncogenic potential of PVT1 in cancers even without supernumerary 8q24. Additionally, we have identified the putative functional domain of PVT1 which confers its oncogenic potential. We propose that the dependence of high levels of MYC on lncRNA PVT1 provides a much-needed therapeutic window against MYC protein, known to be refractory to small molecule inhibition.

Citation Format: Anindya Bagchi. It takes two to tango: The PVT1-MYC alliance in human cancer. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr IA13.
Abstract PR03: A lncRNA regulates DNA repair by homologous recombination
Authors: Sharma Vivek , Khurana Simran , Kubben Nard , Abdelmohsen Kotb , Oberdoerffer Philipp , Gorospe Myriam , Misteli Tom
Published: May 2016

Introduction: Long non-coding RNAs (lncRNAs) have been shown to contribute to DNA damage response (DDR) by regulating gene expression. However, very little is known about the role that lncRNAs play in regulating DNA repair. In this study we characterized a lncRNA that regulates DNA repair by Homologous recombination (HR) by modulating recruitment of DNA repair factors at the sites of DNA damage.

Experimental procedures: Control or DNA damage treated cells were processed for gene expression analysis, qPCR, live cell imaging, Immunochemistry, Western blotting, FISH and RNA Immunoprecipitation.

Results: Using a genome-wide microarray screen we identified a novel ubiquitously expressed lncRNA, DDSR1 (DNA damage-sensitive RNA 1), which is induced upon DNA damage by several DNA double-strand break (DSB) agents. DDSR1 induction upon DNA damage is dependent on the ATM-NF-kB pathway. Loss of DDSR1 impairs cell proliferation, DDR signaling and reduces DNA repair capacity by homologous recombination (HR). The HR defect upon DDSR1 knockdown is characterized by aberrant BRCA1 and RAP80 accumulation at DSB sites. DDSR1 interacts with hnRNPUL1, an RNA-binding protein involved in HR. Loss of hnRNPUL1 also results in aberrant BRCA1 and RAP80 recruitment at DSB sites. Our results indicate that DDSR1/hnRNPUL1 depletion results in HR inhibition due to reduced end resection caused by aberrant BRCA1 and RAP80 at DSBs.

Conclusions: Our results reveal a previously unknown lncRNA involved in regulation of DDR by contributing DNA repair by HR. Our findings highlight the importance of DDSR1 in maintaining genome stability.

Citation Format: Vivek Sharma, Simran Khurana, Nard Kubben, Kotb Abdelmohsen, Philipp Oberdoerffer, Myriam Gorospe, Tom Misteli. A lncRNA regulates DNA repair by homologous recombination. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr PR03.
Abstract PR07: Precise let-7 expression levels balance organ regeneration against tumor suppression
Authors: Nguyen Liem Hieu , Wu Linwei , Zhu Hao
Published: May 2016

The in vivo roles for even the most intensely studied microRNAs remain poorly defined. We interrogated the physiological functions of let-7, a large and ancient microRNA family that is well-known for regulating developmental timing, metabolism, and cancer. Analysis of mouse models revealed that let-7 performs tumor suppressive roles at the expense of regeneration. Too little or too much let-7 resulted in compromised protection against cancer or tissue damage, respectively. Modest let-7 overexpression, achieved by genetic induction or by lipid nanoparticle delivery, abrogated the development of aggressive MYC-driven liver cancer and provided a robust survival benefit by antagonizing let-7 sensitive oncogenes (Cdc25a, Cdc34, E2f2, E2f5, and Map4k4). However, the same level of overexpression blocked liver regeneration, while deletion of two let-7 family members significantly enhanced it, demonstrating that distinct levels of this microRNA can mediate desirable phenotypes. The observed let-7 dependent regeneration phenotypes resulted from influences on the insulin-PI3K-mTOR pathway. We found that chronic high-dose let-7 overexpression caused liver damage and degeneration, paradoxically leading to tumorigenesis. These dose-dependent roles for let-7 in tissue repair and tumorigenesis rationalize the tight regulation of this microRNA family in development, and have important implications for let-7 based therapeutics.

Citation Format: Liem Hieu Nguyen, Linwei Wu, Hao Zhu. Precise let-7 expression levels balance organ regeneration against tumor suppression. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr PR07.
Abstract A15: Epigenetic regulators maintain neuroblastoma cancer stem cells: Model to treatment
Authors: Agarwal Saurabh , Chen Zaowen , Vasudevan Sanjeev , Shohet Jason M.
Published: May 2016

High risk neuroblastoma (NB) represents a major clinical challenge in pediatric oncology and accounts for 15% of pediatric cancer deaths. Our recent studies suggest that neuroblastoma tumors are heterogeneous and composed of multiple subpopulations including cancer stem cells (CSCs), marked by Granulocyte Colony Stimulating Factor Receptor (G-CSFR or CD114) positive cells. These CD114+ cells are shown to be highly tumorigenic, metastatic, self-renewing and can differentiate into non-tumorigenic progeny.

As tumor subpopulations typically demonstrate distinct epigenetic modifications at histone H3K4 and H3K27, we evaluate these epigenetic marks at the CSF3R locus (gene encoding G-CSFR) by ChIP-qPCR. Results suggest that the CSF3R gene is switched ‘on’ in NB CSCs via reversible epigenetic histone modifications. The CSF3R promoter in CD114+ NB CSCs, shows increased activating H3K4me3 marks and reduced repressive H3K27me3 marks. In the CD114- bulk tumor population, we found the opposite pattern of increased H3K27me3 marks and reduced H3K4me3 marks. In addition, knockdown of EZH2 (the PRC2 methyltransferase responsible for H3K27me3 marks) increases CD114+ expression in both cell culture and murine xenograft models of neuroblastoma.

Furthermore, gene expression studies revealed high levels of MLL1 (encoded by KMT2A), and Jumonji D3 (JMJD3, encoded by KDM6B) in CD114+ cells. MLL1 is a histone methyltransferase that catalyzes mono-, di- and tri-methylation on H3K4, while JMJD3 is an H3K27me3 demethylase.

Blocking MLL1 and JMJD3 with specific small molecule inhibitors, MM-102 and GSK-J4 reduces overall survival of NB cells and also reduces the percentage of CD114+ cells in vitro. Importantly, targeting these epigenetic regulators rapidly down-regulate the G-CSF receptor expression and induces apoptosis specifically in CD114+ cells but not in CD114- cells, suggesting maintenance of NB CSCs by these epigenetic modifiers. In vivo studies of these epigenetic inhibitors in neuroblastoma murine xenograft models validate our in vitro studies and demonstrate highly significant reduction in overall tumor size and metastasis with correlated reduction of total CD114+ cells in resected tumors.

Overall, we demonstrate that histone modifications play a key role in maintaining the NB cancer stem cells, and that specific epigenetic inhibitors are highly potent anti-tumor agents for high risk neuroblastoma. Our findings have direct translational impact and support the development of epigenetic stem cell targeted therapies for neuroblastoma.

Citation Format: Saurabh Agarwal, Zaowen Chen, Sanjeev Vasudevan, Jason M. Shohet. Epigenetic regulators maintain neuroblastoma cancer stem cells: Model to treatment. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A15.
Abstract A22: The histone demethylase KDM3A in Ewing sarcoma metastasis
Authors: Sechler Marybeth , Parrish Janet , Jedlicka Paul
Published: May 2016

This study aims to explore the roles and mechanisms of new candidates for therapeutic targeting in Ewing Sarcoma. Ewing Sarcoma is an aggressive pediatric malignancy of the bone and soft tissue, driven by EWS/Ets oncogenic fusions and characterized by high propensity for metastasis and poor clinical outcomes. We have identified the histone demethylase KDM3A as a novel tumor and metastasis promoter downstream of EWS/Fli1, the most common driver in this disease. Moreover, we have identified the cell surface protein, Melanoma Cell Adhesion Molecule (MCAM), as a potential important mediator of KDM3A action. Our data show that both KDM3A and MCAM depletion can inhibit growth and metastatic phenotypes. Additionally we explore the regulation of MCAM, showing that KDM3A may upregulate MCAM expression both through a direct as well as an indirect mechanism via the Ets1 transcription factor. Since both KDM3A and MCAM are pharmacologically targetable, understanding of this pathway could provide at least two new therapeutic targets in Ewing Sarcoma, and, importantly alternatives to drugging of EWS/Ets fusions, which to date has proven very difficult.

Citation Format: Marybeth Sechler, Janet Parrish, Paul Jedlicka. The histone demethylase KDM3A in Ewing sarcoma metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A22.
Abstract A36: Validation of candidate cancer genes using the CRISPR system
Authors: Wolf Natalie , Smeester Branden , Weg Madison , Largaespada David A. , Moriarity Branden Scott
Published: May 2016

We have used Sleeping Beauty (SB) transposon mutagenesis to study the development of several types of sarcomas including osteosarcoma (OS) and malignant peripheral nerve sheath tumors (MPNST). This work has revealed the power of SB mutagenesis to identify critical genes and genetic pathways that, when altered, can cooperate to cause these tumors. We have discovered specific novel oncogenes, such as FOXR2 in MPNST and SEMA4D in OS, recurrent alterations in certain pathways, such as the Wnt/β−catenin pathway in MPNST, and information about what genetic alterations can cooperate in sarcomagenesis, such as loss of NF1 and PTEN in MPNST. We have functionally validated these genetic alterations in several ways, including the use of genetically engineered mouse (GEM) models and TAL effector nucleases (TALEN) or CRISPR RNA-guided Cas9 nuclease modified human cell lines. However, these studies typically analyzed only single candidates of great interest due to the alteration observed in comparative genomics analyses or novelty in the cancer field. In an attempt to further study our candidate cancer genes (CCGs) we have implemented the CRISPR nuclease system to perform a medium throughput screen of all candidate tumor suppressor genes (TSGs). To this end, we are generating and validating CRISPR gRNAs to target all TSGs identified in our MPNST and OS SB screens (~300 genes). Using immortalized human cell lines we have targeted these candidate TSGs and screened for their ability to increase colony formation in soft agar, migration, and xenograft tumor formation. We have identified numerous genes that when mutated by CRISPR resulted in increased tumorigenic properties. These genes are currently being tested for their ability to induce xenograft formation in immunocompromised mice. This CRISPR screen for MPNST and OS CCGs is ongoing and new results will be reported.

Citation Format: Natalie Wolf, Branden Smeester, Madison Weg, David A. Largaespada, Branden Scott Moriarity. Validation of candidate cancer genes using the CRISPR system. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A36.
Abstract B12: The ALK inhibitor PF-06463922 shows significant response as a single agent in ALK/MYCN driven models of neuroblastoma
Authors: Guan J. , Danielson L. , Chand D. , Jamin Y. , Ruuth K. , Tucker E. , Umapathy G. , Wakil A. El , Witek B. , Johnson T. W. , Smeal T. , Chesler L. , Palmer R. H. , Hallberg B.
Published: May 2016

ALK inhibitors such as the ALK/MET/ROS1 inhibitor crizotinib (Xalkori) have shown clinical efficacy in a number of tumour types. However, in ALK positive neuroblastoma treatment with the ALK inhibitor crizotinib has proved more difficult, highlighting the exploration of new drugs as a clinical priority. A recent report of an increased percentage of ALK positive cases in the relapsed neuroblastoma patient population, together with the increased repertoire of ALK inhibitors now available, led to the investigation of alternative ALK inhibitors with potential for use in treatment of neuroblastoma. Here we report an investigation of the activity of a next generation ALK inhibitor in a range of in vitro and pre-clinical ALK driven neuroblastoma models.

Initially PF-06463922 was tested in various neuroblastoma cell lines and a range of gain-of-function ALK neuroblastoma mutations were subsequently analyzed in more detail in engineered Ba/F3 and PC12 cell models and by in vitro kinase assays, comparing the effect of PF-06463922 in abrogating cell growth and induced pharmacodynamics markers of response with the ALK inhibitor crizotinib. These results clearly show PF-06463922 to be a superior inhibitor of ALK kinase activity inhibiting all neuroblastoma mutant ALK forms assayed. Finally, single agent oral administration of PF-06463922 lead to induction of apoptosis and a dramatic reduction in tumour volume in a genetically engineered mouse model of treatment-resistant high-risk neuroblastoma driven by aberrant expression of MYCN and activated ALK. Taken together, our results suggest that PF-06463922 represents an important potential step forward in the treatment of relapsed neuroblastoma with mutated ALK.

Statement of significance: Our results together with PK/PD analysis of PF-06463922 suggest future clinical trial investigation of ALK positive neuroblastoma

Citation Format: J. Guan, L. Danielson, D. Chand, Y. Jamin, K. Ruuth, E. Tucker, G. Umapathy, A. El Wakil, B. Witek, T. W. Johnson, T. Smeal, L. Chesler, R. H. Palmer, B. Hallberg. The ALK inhibitor PF-06463922 shows significant response as a single agent in ALK/MYCN driven models of neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B12.