bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4418: Enhanced endocytosis as a metabolic detour for cancer cells
Authors: Liu Yang , Lu Yanxin , Li Aiguo , Celiku Orieta , Gilbert Mark R. , Yang Chunzhang
Published: July 2017

INTRODUCTION: Metabolic defects impair physiology and life cycle of every somatic cell. However, emerging evidence showed abnormalities in metabolic pathways paradoxically serve as initial steps in many types of human cancers. Little is known about the molecular mechanisms that confer metabolic adaptation and oncogenic predisposition for a transforming cell with intrinsic metabolic defect. In the present study, we explore the molecular mechanism that assists cellular adaptation when confronting metabolic deficiency, and its correlation acquisition of aggressive phenotypes.

METHOD: We took advantage of clinical cases and in vitro cellular models with neomorphic IDH1 mutations, as a paradigm of metabolic deficiency-associated cancer. We investigated whole transcriptomic profiling based on RNA sequencing on IDH1-mutated cells and seek for selective activated canonical pathways. As a validation, we investigated the signature pathways in IDH1-mutated cells, with a focus on cytoskeleton assembly, cellular migration and endocytosis. Moreover, we investigated mTORC pathway and identified its pivotal role in the metabolic adaptation and cellular motility changes in IDH1-mutated cells.

RESULTS: Transcriptomic profiling suggested profound activation in molecular pathways that govern cell movement, endocytosis, chemotaxis and invasion in IDH1-mutated cells. Hierarchical clustering analysis suggested that acquisition of R132C or R132H IDH1 mutant leads to consistent activation pattern for cellular movement. In addition, we confirmed that IDH1-mutated cells exhibit stronger capacity in cellular migration, endocytosis and cytoskeleton re-organization. Importantly, we demonstrated remarkable enhancement in endocytosis pathways in IDH1-mutated cells, suggesting cellular transformation eases metabolite uptake for metabolic deficient cell. Finally, we demonstrated that Rictor/mTORC2/Rac1 pathway is selectively activated in IDH1-mutated glioma cells, which is essential to enable cytoskeleton mobilization, endocytosis and acquisition of aggressive phenotype in transforming cells.

CONCLUSION: Our findings demonstrate that metabolic deficiency leads to enhancement in food seeking behavior in cellular level, highlighted with enhanced cell motility, chemotaxis, and endocytosis; suggesting that this is an important molecular mechanism in cellular transformation during oncogenesis. This phenotypic transformation provides additional carbon sources, compensating for IDH mutation related alterations and may help explain the enhanced cell migration and invasion characteristic of these cancers.

Citation Format: Yang Liu, Yanxin Lu, Aiguo Li, Orieta Celiku, Mark R. Gilbert, Chunzhang Yang. Enhanced endocytosis as a metabolic detour for cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4418. doi:10.1158/1538-7445.AM2017-4418
Abstract 4419: HER2 expression and 17β-estradiol induce ACSL4 expression in estrogen receptor positive mammary carcinoma cells
Authors: Mbarik Maroua , Belkaid Anissa , Surette Marc E.
Published: July 2017

Breast cancer is a multifactorial disease involving several molecular changes and a high proliferation rate often under the activation of hormonal receptors. Therefore, cancer cells require the metabolic machinery for membrane synthesis and to promote signaling pathways. Amongst biomolecules required for efficient cell growth are polyunsaturated fatty acids, like arachidonic acid, that are essential nutrients whose cellular uptake is partly governed by long chain acyl-CoA synthetases (ACSL). In this study, we investigated the impact of 17β-estradiol and Human Epidermal growth factor Receptor 2 (HER2) on cellular uptake of polyunsaturated fatty acids and on the expression of ACSLs. The administration of 17β-estradiol in steroid-starved MCF-7 and T47D mammary carcinoma cells induced a significant increase in cellular uptake of arachidonic acid (AA) and eicosapentaenoic acid (EPA), and in ACSL4 protein content (p<0.05). There was no change in the expression of the ACSL1, ACSL3 and ACSL6 isoforms. Increased ACSL4 protein expression was not accompanied by changes in ACSL4 mRNA expression, but was associated with a significant increase in the protein half-life (T1/2=26±2h) compared to untreated cells (T1/2=8±0.5h). Silencing of ERα reversed the impact of 17β-estradiol on ACSL4 protein expression and half-life. Silencing of ACSL4 eliminated the 17β-estradiol-induced increase in cellular polyunsaturated fatty acid uptake, cell migration and invasion capacities. ACSL4 silencing also prevented the 17β-estradiol-induced increases in p-Akt and p-GSK3β, as well as the 17β-estradiol-induced decrease in E-cadherin expression, important events in epithelial to mesenchymal transition. In addition, the forced expression of HER2 in MCF7 cells induced an increase in cellular uptake of AA and in the expression of ACSL4 measured by both qPCR and western blots. This suggests that HER2-induces ACSL4 expression by a mechanism that differs from that of 17β-estradiol. Further investigations are planned to study the implication of ACSL4 on cellular functions induced by HER2. Overall, these results demonstrate that an induction of ACSL4 protein expression in ERα positive MCF-7 and T47D cells is implicated in 17β-estradiol-induced cellular migration and invasion capacities.

Citation Format: Maroua Mbarik, Anissa Belkaid, Marc E. Surette. HER2 expression and 17β-estradiol induce ACSL4 expression in estrogen receptor positive mammary carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4419. doi:10.1158/1538-7445.AM2017-4419
Abstract 4426: Alcohol consumption and hepatitis B-associated microRNAs in hepatocellular carcinoma
Authors: Li Pin Xue , Zheng Hao , Saad Maarouf A. , Zou Angela E. , Wang Xiaoqi , Kwok James G. , Korrapati Avinaash , Qu Yuanhao , Honda Thomas K. , Wang-Rodriguez Jessica , Ongkeko Weg M.
Published: July 2017

Hepatocellular carcinoma (HCC) affects more than 500,000 new patients each year and is characterized by fast growing, heterogeneous tumors and low five-year survival rates. Despite the identification of HCC risk factors, including alcohol consumption and chronic hepatitis B (HBV) infection, early diagnosis of HCC and molecular understanding of how these risk factors promote HCC both individually and in combination remain elusive. microRNAs (miRNAs), non-coding transcripts ~22 nt in length, have emerged as central mediators of post-transcriptional and translational gene regulation, and have been increasingly implicated in the initiation and progression of many cancers, including HCC. However, the dysregulation profile of miRNAs in HCCs of varying etiologies remains largely unexplored. To identify dysregulated miRNAs specifically associated with alcohol use or with HBV in HCC patients, we analyzed next-generation RNA-sequencing data from 234 HCCs in The Cancer Genome Atlas (TCGA). Through differential expression analyses on cohorts stratified by alcohol consumption and HBV status, we discovered 10 miRNAs specifically dysregulated in alcohol-associated HCCs and 251 miRNAs specifically dysregulated in HBV-associated HCCs (FDR < 0.05). Among these, two miRNAs, miR-944 and miR-223-3p, significantly overexpressed in HCC alcohol consumers, exhibited additional correlations with patient survival, tumor stage, and mutations in CTNNB1, MUC16 and TNN (p < 0.05). We subsequently validated the upregulation of miR-944 and miR-223-3p in vitro following treatment of both normal liver and HCC cell lines with biologically relevant doses of alcohol and acetaldehyde, the first breakdown metabolite of ethanol. Knockdown of miR-944 and miR-223-3p significantly reduced cellular proliferation in both acetaldehyde-treated and untreated normal and HCC cell lines, and resulted in increased sensitivity to cisplatin. Inhibition of both miRNAs in HCC cell lines also restored the expression of several tumor suppressor genes targeted by miR-944 and miR-223-3p. Taken together, our findings enhance knowledge of the respective roles of miRNAs in alcohol- and HBV-mediated HCC, and highlight the increased transcriptomic resolution afforded by etiology-specific studies of cancer pathogenesis and progression.

Note: This abstract was not presented at the meeting.

Citation Format: Pin Xue Li, Hao Zheng, Maarouf A. Saad, Angela E. Zou, Xiaoqi Wang, James G. Kwok, Avinaash Korrapati, Yuanhao Qu, Thomas K. Honda, Jessica Wang-Rodriguez, Weg M. Ongkeko. Alcohol consumption and hepatitis B-associated microRNAs in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4426. doi:10.1158/1538-7445.AM2017-4426
Abstract 4428: A miRNA signature linked to human environmental risk defined from carcinogen-induced and genetically-driven colon carcinogenesis models
Authors: Chen Ying-Shiuan , Wang Rong , Dashwood Wan-Mohaiza , Löhr Christiane , Williams David E. , Ho Emily , Mertens-Talcott Susanne , Dashwood Roderick H.
Published: July 2017

Heterocyclic amines (HCAs) produced during high-temperature cooking have been studied extensively in terms of their genetic effects, but recent work has implicated epigenetic mechanisms involving non-coding RNAs. We reported that colon tumors induced in the rat by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) have altered microRNA (miRNA) signatures linked to dysregulated pluripotency factors, such as c-Myc and Krüppel-like factor 4 (Klf4). The corresponding miRNAs prioritized from PhIP-induced colon tumors were examined in other target organs from a one-year carcinogenicity bioassay, and compared with miRNAs dysregulated in the Apc-mutant polyposis in rat colon (Pirc) genetic model. Multiple let-7 family members were downregulated in colon, skin, lung, small intestine, and Zymbal’s gland tumors, and were associated with Myc and Hmga2 upregulation. A “PhIP miRNA signature” with the profile mir-21high | mir-126low | mir-29low | mir-215low | mir-145low was linked to reduced expression of Klf4 in multiple target organs of the rat, and was predictive of poor prognosis in human pan-cancer and colorectal cancer datasets (https://cancergenome.nih.gov/). Findings from The Cancer Genome Atlas suggested that PhIP signature miRNAs might serve as an arbiter of HCA exposure, defining a subset of human tumors linked to environmental carcinogen exposure. We conclude that future studies should examine the miRNA signatures of other HCAs, and determine their possible predictive value for human risk assessment. This work was supported in part by NIH grants CA090890, CA122959, ES00210, and ES023512, the John S. Dunn Foundation, and a Chancellor’s Research Initiative. We are most thankful to Robert Hawk for providing a graduate student fellowship in aid of Y-S Chen.

Citation Format: Ying-Shiuan Chen, Rong Wang, Wan-Mohaiza Dashwood, Christiane Löhr, David E. Williams, Emily Ho, Susanne Mertens-Talcott, Roderick H. Dashwood. A miRNA signature linked to human environmental risk defined from carcinogen-induced and genetically-driven colon carcinogenesis models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4428. doi:10.1158/1538-7445.AM2017-4428
Abstract 4429: An exhaled microRNA panel interrogation and validation as risk biomarkers for lung cancer
Authors: Spivack Simon D.
Published: July 2017

Background: There is a need for non-invasive airway-based biomarkers in lung carcinogenesis for both risk assessment of the ex-smoker, and earlier diagnosis. Recent studies have revealed that microRNAs play important regulatory roles in carcinogenesis. Exhaled breath condensate (EBC) contains airway lining fluid molecules, including nucleic acids, presumably in part from epithelial cellular origins. Here we further develop and begin validation of the detection of microRNAs in EBC from lung cancer patients and controls.

Methods: Exhaled breath condensate (EBC) was collected non-invasively, using a handheld device in ambulatory subjects. MicroRNA expression profiling using RNA-specific RT-qPCR was performed in EBC in an initial batch (E) of 88 individuals (41 lung cancer cases, 47 controls), and a second batch D of 88 individuals (41, cases and 47 controls). We generated a 39 miR panel based on literature-derived microRNAs, combined with a lung tissue-based discovery effort we have performed using microRNA-seq on lung tumors and surrounding non-tumor tissue. The qPCR primers were designed using our previously published RNA-specific realtime RT-PCR technique. All samples were run twice with positive and negative controls.

Results: Qualitative analyses of the first donor batch E was performed on 39 microRNAs measured in 41 cases and 47 controls. Chi-square analysis (univariate) within the training set (batch E) revealed that exhaled miR-31, 33, 105, 200b, 944, 1269a were significantly different between the initial set of cases and controls; their absence marked the cases. In the validation batch (D) preliminary analysis, exhaled miRs 200b (this time present in cases, p=0.044) and 944 (again absent in cases, p=0.052) were case-control discriminant. In addition, test set batch D showed case-control discrimination for additional miRs 130b-3p (present in cases, p=0.035), 212 (absent in cases, p=0.009), 1910a (absent in cases, p=0.006) and other microRNAs that were not seen in the qualitative training set (batch E). Importantly, multi-miR discriminant signatures, quantitative analysis, and risk ROC/modelling analyses are pending as of this AACR abstract deadline.

Conclusion: This new exhaled biomarker platform can yield case-control discriminant microRNA sets, including a subset of miRs that can validate in test sets. Once further distilled and validated, our goal is to apply this non-invasive biomarker approach to prospective cohorts for non-invasive lung cancer risk assessment, in order to better select higher risk individuals who should undergo CT screening.

Note: This abstract was not presented at the meeting.

Citation Format: Simon D. Spivack. An exhaled microRNA panel interrogation and validation as risk biomarkers for lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4429. doi:10.1158/1538-7445.AM2017-4429
Abstract 4444: MicroRNA hsa-miR-145 expression in hepatocellular carcinoma correlates with patient survival
Authors: Nakajima Go , Hayashi Kazuhiko
Published: July 2017

Aim: MicroRNAs (miRNAs) are known as one of the regulator of gene expression at a post-transcriptional level. Previous study from our group identified forty nine miRNAs that are expressed differentially by comparing 26 pair samples of hepatocellular carcinoma (HCC) tumor tissue and non-tumor liver tissue using next-generation sequencer system, and hsa-miR-145, one of the candidate miRNA was chosen for this validation study. The expression of hsa-miR-145 in validation set HCC samples were measured by real-time PCR, and were examined the correlation between the miRNA expression and clinical data.

Patients and Methods: Forty-four patients diagnosed as HCC had undergone surgical removal from 1991 to 1997 at the Department of Surgery, Institute of Gastroenterology, Tokyo Women’s Medical University, Japan. The formalin-fixed paraffin embedded samples were prepared using the standard protocol. The paraffin blocks were cut into 10 micrometer sections, and the tumor and its adjacent non-tumor liver tissue were collected using laser-captured microdissection and total RNA including miRNA fraction were isolated. After synthesized cDNA using miRNA specific primer, real-time PCR was performed using miRNA specific primer/probe.

Results: The expression level of hsa-miR-145 was down-regulated in tumor compared to non-tumor liver tissue (P=0.0004). The patients were divided into two groups, higher/lower expression, the group of the higher hsa-miR-145 expression was significantly longer overall survival time compared to the lower expression group (P=0.0378).

Conclusion: The hsa-miR-145 may be potential prognostic factor for HCC patients.

Citation Format: Go Nakajima, Kazuhiko Hayashi. MicroRNA hsa-miR-145 expression in hepatocellular carcinoma correlates with patient survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4444. doi:10.1158/1538-7445.AM2017-4444
Abstract 4448A: MicroRNA profiling of prostate cancer tissue identifies a prognostic 4-miRNA model and implicates one novel microRNA in prostate cancer cell viability
Authors: Schmidt Linnea , Fredsøe Jacob , Kristensen Helle , Rasmussen Anne , Mouritzen Peter , Høyer Søren , Borre Michael , Ørntoft Torben , Sørensen Karina Dalsgaard
Published: July 2017

Purpose: New prognostic biomarkers for prostate cancer (PC) risk stratification are urgently needed. Here, we identify and validate a novel 4-microRNA prognostic ratio model for prostate cancer. We also identify a new microRNA with tumor suppressive characteristics in prostate cancer cell lines.

Methods: PC tissue samples from 123 radical prostatectomy (RP) patients, 58 of whom experienced biochemical recurrence, were profiled for genome-wide miRNA expression using RT-qPCR (training cohort). Initially, we identified 11 top candidate prognostic microRNAs (P <0.05, univariate Cox regression). Based on these, all possible 2-4 microRNA ratio models were systematically tested for their potential to predict time to biochemical recurrence (BCR), as assessed by Kaplan-Meier, uni- and multivariate Cox regression analyses. The findings were validated in two independent RP cohorts. A novel microRNA, included in the final ratio model and with previously unknown function in prostate cancer, was over expressed using a microRNA mimic in PC3 and DU145 cells to evaluate its effect on prostate cancer cell viability and proliferation.

Results: We identified a new prognostic 4-microRNA ratio model ((miR-23a-3p*miR-10b-5p)/(miR-133a*miR-374b-5p)) that significantly predicted time to BCR independently of established clinicopathological variables in a training RP cohort (n=123). The ratio model was successfully validated in two independent RP cohorts including 112 and 476 patients, respectively. Ectopic expression of one of these microRNAs (miR-374b-5p) significantly inhibited viability and proliferation in two prostate cancer cell lines, suggesting it has a tumor suppressor function in PC.

Conclusions: Here, we describe the identification and independent validation of a 4-miRNA ratio model that allows risk stratifications of prostate cancer patients undergoing RP into different prognostic groups. Furthermore, we identify and functionally characterize a novel possible tumor suppressor miRNA in prostate cancer.

Citation Format: Linnea Schmidt, Jacob Fredsøe, Helle Kristensen, Anne Rasmussen, Peter Mouritzen, Søren Høyer, Michael Borre, Torben Ørntoft, Karina Dalsgaard Sørensen. MicroRNA profiling of prostate cancer tissue identifies a prognostic 4-miRNA model and implicates one novel microRNA in prostate cancer cell viability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4448A. doi:10.1158/1538-7445.AM2017-4448A
Abstract 4450: The role of paired-like homeodomain 2B in non-small cell lung cancer progression
Authors: Lin Sheng-Yi , Huang Yu-Han , Lai Yi-Hua , Yu Sung-Liang , Su Sheng-Fang , Hong Tse-Ming , Chang Gee-Chen , Wang Chi-Chung , Chen Jeremy J.W.
Published: July 2017

The transcription factor PITX2 (paired-like homeodomain 2) is well known to play an essential role in normal embryonic development. Emerging evidence suggests that PITX2 may be involved in human tumorigenesis. However, the functional role that PITX2 plays in tumor progression remain unclear. In this study, we investigated its function in human non-small cell lung carcinoma (NSCLC). Using real-time quantitative RT-PCR, we demonstrated that PITX2B is the most abundant isoform in lung cancer cells. Enforced expression of PITX2B could promote lung cancer cell morphological change, migration, invasion, anchorage-dependent growth ability and tumorigenesis in xenograft model mice. In addition, nuclear PITX2B localization was correlated with its oncogenic functions and two important nuclear localization signals (NLSs) were identified. Moreover, high expression of PITX2B was associated with poor overall survival (P<0.05) in NSCLC patients. Importantly, silencing of PITX2B by siRNA caused a marked increase in autophagy based on LC3-II, Beclin-1, and p62 protein expression, acridine orange fluorescence of acidic vacuoles and electron microscopic detection of autophagosomes. Furthermore, we found that the autophagy induced by PITX2B knockdown preceded apoptosis in lung cancer cells. Treatment of lysosome inhibitor, NH4Cl, could provide a protective effect that increases cell viability. In conclusion, our studies not only provide new insights into how PITX2B may contribute to the tumor progression in lung cancer but also the autophagy induced by PITX2B knockdown that may be developed as an effective therapeutic strategy for the treatment of lung cancer.

Citation Format: Sheng-Yi Lin, Yu-Han Huang, Yi-Hua Lai, Sung-Liang Yu, Sheng-Fang Su, Tse-Ming Hong, Gee-Chen Chang, Chi-Chung Wang, Jeremy J.W. Chen. The role of paired-like homeodomain 2B in non-small cell lung cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4450. doi:10.1158/1538-7445.AM2017-4450
Abstract 4451: Elevated USP22 is a potential therapeutic target for human non-small cell lung cancer
Authors: Zhang Keqiang , Tong Tommy R , Yun Xinwei , Nelson Rebecca , Isaac Nardi , Salgia Ravi , Raz Dan
Published: July 2017

Elevated ubiquitin-specific protease 22 (USP22) is associated with enhanced malignancy, therapeutic resistance, and poor prognosis of various human cancers. In this study, the clinicopathological significance of USP22, therapeutic implication of targeting USP22, and underlying molecules of USP22 knockdown were investigated in human non-small cell lung cancer (NSCLC). Immunohistochemistry analysis revealed that USP22 protein was dramatically elevated in ~ 67% of 197 NSCLC tissues compared with adjacent normal lung tissues. Importantly, statistical analysis demonstrated that elevated USP22 was positively associated with advanced cancer stage and a trend to poor prognosis of NSCLC patients. Moreover, siRNA-mediated knockdown of USP22 induced a cell cycle arrest in G1/S phase and significantly suppressed cell proliferation and soft agar colony formations in NSCLC cell lines. In addition, knockdown of USP22 significantly enhanced cisplatin-induced genotoxic effects in NSCLC cell lines. Furthermore, USP22 silencing substantially down-regulated SIRT1 and upregulated p53 tumor suppressor in NSCLC cells, indicating downregulation of USP22 may boost p53-mediated tumor suppression and DNA damage response in NSCLC cells. Knockdown of USP22 also increased global histone H2B monoubiquitylation, H3K4 and H3K79 trimethylation leading to global gene expression profile changes in these NSCLC cells. These results suggest that targeting USP22 will have broad anti-cancer effects via altering multiple cancer signaling pathways. Taken together, data of this study further indicate that the elevated USP22 may potentially represent a prognostic factor and therapeutic target for NSCLC patients.

Note: This abstract was not presented at the meeting.

Citation Format: Keqiang Zhang, Tommy R Tong, Xinwei Yun, Rebecca Nelson, Nardi Isaac, Ravi Salgia, Dan Raz. Elevated USP22 is a potential therapeutic target for human non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4451. doi:10.1158/1538-7445.AM2017-4451
Abstract 4452: Study of the mechanisms of emetine dihydrochloride on inhibiting NSCLC tumor progression and attenuating EGFR signaling pathway
Authors: Lai Yi-Hua , Huang Wan-Ting , Lin Sheng-Yi , Huang Yao-Ting , Chen Yuh-Ling , Chen Hsuan-Yu , Chen Huei-Wen , Chen Jeremy J.W.
Published: July 2017

Epidermal growth factor receptor (EGFR) is a transmembrane receptor protein and can mediate the cellular biological responses including regulation of cellular proliferation, differentiation, migration and survival. More than 60 % of non-small cell Lung cancers (NSCLCs) express relatively high level of EGFR. Therefore, EGFR has become an important therapeutic target for the treatment of NSCLC. However, almost all NSCLC patients ultimately develop drug resistance to targeted therapy agents. The most common secondary mutation is found to harbor EGFR mutation in T790M, which is acquired resistance to EGFR TKIs. In order to overcome the resistance of T790M mutation in lung cancer, we used EGFR pY1068 ELISA as drug-screening platform to screen the candidate compounds with the capacity to suppress EGFR activity from the compound library of more than 300 plant active ingredients. Among the compounds identified, the emetine could most significantly restrain EGFR pY1068 activity and reduce cancer cell viability. Moreover, the Western blotting results showed that emetine treatment can repress EGFR phosphorylation and expression in a dose-dependent manner as well as inhibit its downstream substrates including PI3K, STAT3 and Src in three different Iressa-resistant cell lines. In addition, emetine also could inhibit cell proliferation, migration, invasion, and clonogenicity in vitro and xenograft tumor growth in vivo. Taken together, our data suggest that emetine might be a novel inhibitory modulator of EGFR activity and might be through suppressing the phosphorylation and expression of PI3K, STAT3, and Src to inhibit lung cancer progression. These findings will help us to develop the anti-cancer drugs for lung cancer in the future.

Citation Format: Yi-Hua Lai, Wan-Ting Huang, Sheng-Yi Lin, Yao-Ting Huang, Yuh-Ling Chen, Hsuan-Yu Chen, Huei-Wen Chen, Jeremy J.W. Chen. Study of the mechanisms of emetine dihydrochloride on inhibiting NSCLC tumor progression and attenuating EGFR signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4452. doi:10.1158/1538-7445.AM2017-4452
Abstract 4459: Glycine decarboxylase inhibits proliferation of ovarian cancer cells via SIRT1-mediated apoptosis
Authors: Shin So-Jin , Kim Jin-Young , Ha Eun-Young , Cho Chi-Heum
Published: July 2017

Glycine decarboxylase (GLDC) is a very recently identified metabolic oncogene that links glycine metabolism with tumorigenesis. We designed this study to determine the mechanistic role of GLDC in in ovarian cancer cells. First, we confirmed the expressions of GLDC in eight different ovarian cancer cell lines. And colony formation assay revealed decreased proliferation of OVCAR3 ovarian cancer cells transfected with siGLDC compared with that of control. Intriguingly, we found that knockdown of GLDC induced decreased S-adenosylmethionine (SAM)/ S-adenosylhomocysteine (SAH) ratio, which implicates methyl donor deficient state in OVCAR3 cells. Expressions of sirtuin 1 (SIRT1), heat shock protein (HSP) 90 and heat-shock factor protein 1 (HSF1) decreased while that of p53 increased in GLDC knock-downed cells, suggesting heat shock response. Addition of SAM into siGLDC knock-downed cell restored the expression of SIRT1. Inhibition of SIRT1 decreased Bcl2 and increased cytochrome c expressions. In summary, these results suggest GLDC induces methyl donor deficient state in OVCAR3 ovarian cancer cells, which in turn leads to apoptosis via SIRT1-mediated pathway.

Citation Format: So-Jin Shin, Jin-Young Kim, Eun-Young Ha, Chi-Heum Cho. Glycine decarboxylase inhibits proliferation of ovarian cancer cells via SIRT1-mediated apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4459. doi:10.1158/1538-7445.AM2017-4459
Abstract 446: Differential effects of folate depletion on metabolic reprogramming and oxidative stress in nonmetastatic and metastatic claudin-low breast cancer cells
Authors: Chen Xuewen , O'Flanagan Ciara H. , Hursting Stephen D.
Published: July 2017

Folate coenzymes play an important role in biosynthesis and methylation reactions. Aberrant folate metabolism has been implicated in the development of several cancer types, though mechanisms underlying folate metabolism and cancer development remain unclear. We previously showed that a folate-restricted diet can exert differential effects on metastatic versus nonmetastatic murine claudin-low breast cancer cells in vivo. Specifically, folate restriction increased growth and invasion of orthotopically transplanted M-Wnt (nonmetastatic) tumor cells, but decreased growth and lung metastases of transplanted metM-Wnt cells, a metastatic subclone of M-Wnt cells. The current study set out to explore the underlying mechanism. To examine the effect of long-term folate depletion (LFD) on M-Wnt and metM-Wnt cell metabolism, oxidative stress and autophagic flux in vitro, the two cell lines were grown in standard and folate-depleted media for 14 days. metM-Wnt cells showed higher oxidative stress, as measured by ROS staining and Nrf2 expression, and phosphorylation of the key nutrient sensor, AMPK, compared to M-Wnt cells when grown in standard growth medium. LFD M-Wnt cells showed an increased dependence on glycolysis compared to those cultured in standard medium. Both M-Wnt and metM-Wnt cells displayed a high autophagy level in LFD, measured by LC3B cleavage, and AMPK phosphorylation. However, LFD metM-Wnt cells showed low viability, increased apoptosis and loss of redox defense, as measured by cleaved-caspase 3 and Nrf2 expressions. These results suggest that non-metastatic M-Wnt cells undergo metabolic reprogramming, including a shift from oxidative phosphorylation to glycolysis that may fuel cell growth and proliferation. Further, an elevated autophagic flux may mitigate nutrient stress induced by folate depletion, which allows them to withstand LFD and which may contribute to a more invasive primary tumor in response to folate restriction. In contrast, metM-Wnt cells are unable to undergo this metabolic adaptation, and display increased oxidative stress and cell death in response to LFD, preventing the development of metastatic lesions in vitro. This study highlights different responses of primary and metastatic breast cancer cells to folate depletion. The results provide additional rationale for targeting folate metabolism as a potential strategy for treating metastatic breast cancer.

Citation Format: Xuewen Chen, Ciara H. O'Flanagan, Stephen D. Hursting. Differential effects of folate depletion on metabolic reprogramming and oxidative stress in nonmetastatic and metastatic claudin-low breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 446. doi:10.1158/1538-7445.AM2017-446
Abstract 4461: Detection of somatic mutations in exosomal RNA from human oral squamous cell carcinoma cells
Authors: Tokuzen Norihiko , Nakashiro Koh-ichi , Hamakawa Hiroyuki
Published: July 2017

Exosomes are small vesicles, ranging from 30 to 100 nm in size, secreted from most types of cells. They contain proteins, lipids, and nucleic acids (e.g. DNA, mRNA, and microRNA) and are thought to play an important role in cell proliferation, invasion, and metastasis in human malignancies. Recently, circulating exosomes have also been of interest as a source for liquid biopsies. In this study, we examined the usefulness of exosomal RNA as a biomarker and attempted to detect the tumor-specific gene mutations from exosomal RNA derived from human oral squamous cell carcinoma (OSCC) cells. We used 3 human OSCC cells (HSC2, KT-T, KT-N). KT-T and KT-N cells were established from the lower gingival tumor and lymph node metastasis, respectively. First, we detected the somatic mutations in genomic DNA extracted from these cells by next-generation sequencing (NGS) with the use of HaloPlex Cancer Research Panel (Agilent Technologies). PIK3CA H1047R mutation was identified in all OSCC cells, and KT-N cells had the additional HRAS Q61R mutation. We confirmed these mutations of PIK3CA and HRAS in OSCC cells by TaqMan® SNP Genotyping Assays (Thermo Fisher Scientific). Subsequently, we evaluated the gene mutations in exosomal RNA from these OSCC cells. Exosomal RNA was isolated from the conditioned medium using exoRNeasy Serum/Plasma Maxi Kit (Qiagen) according to the manufacture’s protocol. In exosomal RNA, we found the PIK3CA H1047R mutation from all OSCC cells and HRAS Q61R mutation from KT-N cells by genotyping assays. Furthermore, we assessed the detection of these mutations in tumor xenograft mouse models. KT-T and KT-N cells (5 x 106) complexed with Matrigel® (BD) in 100-µl aliquots were injected subcutaneously space in the flank of male athymic nude mice. Exosomal RNA was isolated from the serum of tumor-bearing nude mice using exoRNeasy Serum/Plasma Midi Kit (Qiagen). By genotyping assays using serum exosomal RNA, PIK3CA H1047R mutation was observed in both types of xenograft mice and HRAS Q61R mutation was positive in KT-N xenograft mice. Cancer cell-derived exosomal RNA had the same gene mutations in those cells, and could be detected in the serum of tumor-bearing nude mice. These results suggest that exosomal RNA is useful for detecting somatic mutations in OSCC cells.

Citation Format: Norihiko Tokuzen, Koh-ichi Nakashiro, Hiroyuki Hamakawa. Detection of somatic mutations in exosomal RNA from human oral squamous cell carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4461. doi:10.1158/1538-7445.AM2017-4461
Abstract 4468: Oncogenic activation of the RNA binding protein AGO2 in hepatocellular carcinoma
Authors: Dang Hien T. , Knight Lucy , Pomyen Yotsawat , Wang Xin Wei
Published: July 2017

Global transcriptomic alterations of coding and non-coding RNAs are a ubiquitous feature of cancers including Hepatocellular carcinoma (HCC). Dysregulation of RNA-binding proteins (RBPs), key regulators of RNA processing such as RNA maturation and degradation, is one mechanism in which cancer cells select to promote tumorigenesis. RBPs are highly expressed in solid tumors and have been demonstrated to be drivers of carcinogenesis, however, the underlying mechanisms in which RBPs regulate the HCC transcriptome is unknown. We analysed genomic alterations amongst a family of more than 800 mRNA RBPs (mRBPs) in 1,225 clinical specimens from HCC patients and found that RBPs are significantly activated through gene amplification in a subset of tumors with poor prognosis, suggesting their potential oncogenic roles in HCC progression. Amongst the top candidates, Argonaute 2 (AGO2) was further characterized for its oncogenic role and effects on the HCC transcriptome. Elevated AGO2 mRNA expression was highly correlated with elevated somatic copy number alterations across five different cancer types including HCC. Moreover, AGO2 expression was associated with overall survival in two independent data sets (TCGA and the Liver Initiative Cancer), suggesting that AGO2 activation is oncogenic. While the activation of AGO2 induced an oncogenic phenotype, the abrogation of AGO2 in HCC cells significantly decreased cancer associated phenotypes such as cell proliferation, migration/invasion and tumorigenicity in vivo. Further analyses revealed that AGO2-associated genes were tumor-related, including c-Myc. Notably, c-Myc amplification was also found in AGO2 high HCC cells, suggesting an interplay between AGO2 and c-Myc. Our results demonstrate that oncogenic activation of AGO2 is a novel mechanism that may contribute to global transcriptome selective for the activation of c-Myc oncogenic. Our current work suggests that therapies focused on targeting AGO2 may be valuable for clinical treatment of many different tumors with activated c-Myc signalling, including HCC

Citation Format: Hien T. Dang, Lucy Knight, Yotsawat Pomyen, Xin Wei Wang. Oncogenic activation of the RNA binding protein AGO2 in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4468. doi:10.1158/1538-7445.AM2017-4468
Abstract 4481: Translation initiation factor DAP5 plays an essential role in translational control of breast cancer metastasis
Authors: Parra Columba de la , Alard Amandine , Ernlund Amanda , Schneider Robert J.
Published: July 2017

Metastasis is the cause of 90% of cancer-related deaths. Selective mRNA translation has been found to be crucial for breast cancer development, progression and metastasis; however, there is a poor biological and mechanistic understanding of the role of translation in these processes. Our work and others have pioneered an understanding of translational regulation in breast cancer. We have previously shown that overexpression of initiation factor eIF4GI in advanced breast cancer cells selectively increases the translation of mRNAs encoding survival, cell cycle inhibition, and DNA damage and repair proteins, among other DNA damage-protective mRNAs. eIF4G consists of three family members: eIF4GI, eIF4GII, and the poorly studied eIF4G homolog, DAP5, the subject of this study. DAP5 lacks the N-terminal domain for eIF4E and PABP binding. DAP5 is therefore suspected to promote eIF4E-independent or IRES-driven translation of mRNAs involved in cell stress and oncogenesis, but its role in metastasis hasn’t been investigated. We analyzed the NCI human tissue genome cancer atlas (TCGA) and found that higher DAP5 mRNA expression is strongly associated with estrogen receptor negative breast cancer metastasis with lower overall survival. We then engineered a highly metastatic breast cancer cell line MDA-MB-231TR to express dox-inducible shRNA to DAP5 or a non-silencing (NS) control. Tumors were developed in clinically relevant orthotopic mouse models, with and without DAP5 silencing by addition of dox to the drinking water. Remarkably, reduction of DAP5 expression by ~70% had no impact on primary tumor growth but fully eliminated metastasis to the lung. Genome-wide transcriptome and translatome analysis of DAP5 and in vivo ultraviolet-crosslinking and high-throughput sequencing (HITS-CLIP) identified specific mRNAs that interact directly with DAP5. A significant fraction of DAP5 mRNA targets are involved in cell death and survival, cell proliferation, cell mobility, DNA repair and translation initiation and do not have IRESs, suggesting a novel role for DAP5 in a non-classical translation initiation process upon cellular stress and metastasis. Our results suggest that the translation initiation factor DAP5 likely plays a critical role in breast cancer metastasis by a unique mechanism for metastasis-specific translation initiation and provides new concepts for therapeutic strategies involving translational regulation.

Citation Format: Columba de la Parra, Amandine Alard, Amanda Ernlund, Robert J. Schneider. Translation initiation factor DAP5 plays an essential role in translational control of breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4481. doi:10.1158/1538-7445.AM2017-4481
Abstract 4486: Redox regulation of the biosynthesis of mitronic tsmiR miR6855 in the basal like breast cancer cells
Authors: Misra Smita , Chaudhuri Gautam
Published: July 2017

Radiation and chemotherapeutic agents eradicate tumors by inducing irreparable DNA damage. However, cancer cells often develop resistance to therapy by manipulating the DNA repair machinery. Conversely, a dividing cell constantly exposed to environmental and endogenous DNA damaging agents can transform into a tumor due to incorrect repair. Therefore the expression level of DNA repair proteins is critical both for cancer therapy and tumorigenesis. Our preliminary studies implied a unique mechanism involving an miRNA miR-6855-3p that promotes the biosynthesis of the transcriptional regulator protein SPRDX5 which enhances the expression of the tumor suppressor protein BRCA2 leading to the genotoxin and radiation-resistance of aggressive SLUG-high basal-like breast cancer (BLBC) cells. The miRNA miR-6855-3p thus acts as a tumor suppressor miRNA (tsmiR). Interestingly, the gene for the miRNA miR-6855-3p is mitronic and is nested within the intron#13 of the USP20 gene which is a deubiquitinase. Thus, the transcription of miR-6855-3p gene is regulated through the USP20 gene promoter. We present evidence here that USP20 and miR-6855 gene transcriptions are up regulated by oxidative stress. Tert-butylhydroperoxide was used for oxidative stress. Under the oxidative stress conditions tested, NRF2 level and the levels of several NRF2-regulated proteins are increased, indicating activation of NRF2 in the stressed BLBC cells. Under the stress conditions the levels of USP20 and miR-6855-3p are increased significantly. Knockdown of NRF2 prevented stress-induced elevation of USP20 and miR-6855-3p RNA level in the BLBC cells indicating the involvement of NRF2 in this process. The binding of NRF2 to the USP20 gene promoter and its function were characterized by ChIP analysis, site-directed mutagenesis and using the promoter-luciferase reporter constructs. We conclude that the biosynthesis of the tsmiR miR-6855-3p in the BLBC cells is regulated by oxidative stress and involves NRF2. Since oxidative stress is a hallmark in the etiology and progression of metastatic breast cancer, understanding the redox regulation of a tsmiR that positively influences BRCA2 gene expression in the BLBC cells could be clinically significant. Supported in part by DOD-CDMRP IDEA Expansion Grant# BC103645 and NIH/NCI grant 1R21CA181920-01 to GC and 1U54RR026140 to SM.

Citation Format: Smita Misra, Gautam Chaudhuri. Redox regulation of the biosynthesis of mitronic tsmiR miR6855 in the basal like breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4486. doi:10.1158/1538-7445.AM2017-4486
Abstract 4494: Tumor suppressive role of ZFP36L1 by suppressing HIF1α and Cyclin D1 in bladder and breast cancer
Authors: Loh Xin Yi , Ding Ling Wen , Koeffler H. Phillip
Published: July 2017

AU-rich mRNAs are a class of mRNAs that contains AU-rich elements (AREs) motifs in their 3’UTR. The half-life and turnover of AU-rich mRNAs are tightly regulated by AREs-binding proteins, such as Zinc finger protein 36 C3H type-like 1 (ZFP36L1). ZFP36L1 is one of the RNA-binding proteins (RBPs), which consists of two tandemly repeated zinc finger motifs that specifically recognize AREs motifs and mediate mRNAs decay. However, our knowledge on role of ZFP36L1 in breast and bladder cancers and its downstream targets remains poorly elucidated. Here, we hypothesized that ZFP36L1 might degrade oncogenic mRNAs transcripts, thus leading to suppression of tumorigenesis. Our analysis of in silico data revealed that ZFP36L1 is significantly mutated or downregulated in a variety of cancers. The high proportion of inactivation mutation pattern as well as the Mutsig result suggest that it may act as an unappreciated tumor suppressor. Lower ZFP36L1 expression correlated with reduced survival in patients. Forced expression of ZFP36L1 markedly reduced cellular proliferation, invasiveness and migration in vitro, as well as tumor growth in vivo. In addition, microarray and RNA-seq data analysis showed that several important cancer-related candidates such as IL8 and HIF1α were significantly downregulated. Overexpression of ZFP36L1 reduced HIF1α mRNA and protein expression. Similarly, HIF1α forced expression also inhibited ZFP36L1 mRNA and protein, suggesting a feedback loop of HIF1α and ZFP36L1. Dual luciferase reporter assays and RNA Electro-mobility Shift Assay (REMSA) showed that only wild type ZFP36L1, but not different mutants of ZFP36L1, was able to bind directly and to degrade a HIF1α 3’UTR construct that contains AU-rich regions. Meanwhile, mRNA pull down assay demonstrated that ZFP36L1 protein recognized cell-cycle related transcripts. Overexpression of ZFP36L1 led to reduction of Cyclin D1 and phospho-Rb in protein expression. Furthermore, SILAC data unveiled interesting binding partners of ZFP36L1. Collectively, our findings propose that ZFP36L1 might have a critical tumor suppressive role in breast and bladder cancer by negatively regulating mRNA whose protein products cooperatively promote hypoxia and transition into S phase of cell cycle. Augmenting ZFP36L1 function might disable the cell cycle and proliferation of aggressive bladder tumors.

Citation Format: Xin Yi Loh, Ling Wen Ding, H. Phillip Koeffler. Tumor suppressive role of ZFP36L1 by suppressing HIF1α and Cyclin D1 in bladder and breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4494. doi:10.1158/1538-7445.AM2017-4494