bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 426: Transcriptional regulation of dnmt1 by E2F1 in melanoma progression
Authors: SILVA CAMILA T. DA , Molognoni Fernanda , Melo Fabiana H. M. de , Jasiulionis Miriam Galvonas
Published: May 2015

Melanoma is the most aggressive skin cancer due to its high resistance to currently available therapy. Like other kinds of cancer, melanoma presents changes in DNA methylation and epigenetic machinery such as Dnmt1, however it is still unclear why tumor cells acquire these epigenetic alterations. The aim of this study was to analyze the mechanisms involved in Dnmt1 transcriptional regulation by the transcription factor E2F1 in melanoma progression. The cell model used in this study was previously developed in our laboratory, submitting non-tumorigenic murine melanocytes (melan-a) to successive cycles of anchorage blockade. A linear cell model for melanoma was obtained consisting in pre-malignant (1C and 4C), tumorigenic non-metastatic (4C11-) and metastatic (4C11+) cell lines. Bioinformatics data show the existence of four transcripts for Dnmt1 in mice. The transcripts 1 and 2 have the same promoter, and the transcripts 3 and 4 share a different promoter. Real time PCR showed the absence of transcript 3 in all cell lines analyzed. The transcript 4 was present in all lineages, but no significant change in its expression was observed in any one compared to melan- a melanocytes. Transcripts 1 and 2 are present and their levels are increased in 1C, 4C, 4C11- and 4C11+ compared to melan-a cells. Chromatin immunoprecipitation (ChIP) showed increased binding of E2F1 in Dnmt1 promoter in 1C, 4C, 4C11- and 4C11+ compared to melan-a cells. Interestingly, the intensity of binding was stronger in the first promoter, which regulates transcripts 1 and 2, than in the second promoter, which regulates transcripts 3 and 4. These results show for the first time that E2F1 may have an important role in the transcription regulation of Dnmt1 during melanoma progression. Additionally, we observed the increase in superoxide anion (O2.-) in 1C compared to melan-a cells. Recently published data from our group showed that depleting O2.- with MnTBAP scavenger during melan-a anchorage blockade cycles, the malignant transformation is drastically compromised. Here, we showed that 1C cells treated with MnTBAP do not have Dnmt1 mRNA and protein increase compared to melan-a cells. Furthermore, E2F1 levels seem to be regulated by O2.- in 1C cells, indicating that O2.- can be a possible mediator for E2F1 regulation and Dnmt1 transcriptional regulation in melanoma progression. Together, these data suggest an important role of oxidative stress and epigenetic mechanisms in the first steps of melanoma progression. Supported by FAPESP and CNPq.

Citation Format: CAMILA T. DA SILVA, Fernanda Molognoni, Fabiana H. M. de Melo, Miriam Galvonas Jasiulionis. Transcriptional regulation of dnmt1 by E2F1 in melanoma progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 426. doi:10.1158/1538-7445.AM2014-426
Abstract 4265: The landscape of somatic hypermutation in human cancer
Authors: Chapman Jocelyn S. , Chang Matthew T. , Asthana Saurabh , Lall Kriti , Schultz Nikolaus , Taylor Barry S.
Published: May 2015

Somatic hypermutation is present in the tumor genomes of a subset of human cancers. Nevertheless, the extent as well as the biological and clinical impact of hypermutation in cancer patients is unknown. Moreover, in an age of precision oncology, there is a pressing need to understand the catastrophic mutational burden of sporadically hypermutated tumors to ensure an accurate clinical interpretation of candidate actionable lesions thereby facilitating genomically driven oncology in these patients. To explore these questions, we assembled a curated repository of cancer genome data consisting of the sequenced tumor exomes and whole genomes of 10,852 patients representing 36 tumor types from 90 unique cohorts. Through integrative genomic analyses of mutational and DNA copy number profile data, we identified 395 hypermutated tumors, representing ∼3.6% of all cancers. Of these, 47% of patient tumors possessed somatic hypermutation that could not be explained by an established exogenous mutagen, environmental exposure, or known defect in DNA repair. Our analysis of this unknown class of hypermutated cancers revealed six distinct mutational signatures, which may be associated with specific mechanisms driving hypermutation. From allelic abundance data, the temporal sequence of multiple mutational processes accumulated over the lifespan of a tumor was apparent. Our analyses also revealed that despite a high mutational burden, abnormalities cluster to reveal biologic and therapeutic dependencies. Overall, we exploit big data to explore uncommon cancer phenotypes like somatic hypermutation, bridging the gap between complex mutational landscapes and rational therapeutic choices for molecularly defined cancer patients.

Citation Format: Jocelyn S. Chapman, Matthew T. Chang, Saurabh Asthana, Kriti Lall, Nikolaus Schultz, Barry S. Taylor. The landscape of somatic hypermutation in human cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4265. doi:10.1158/1538-7445.AM2014-4265
Abstract 4274: High fidelity copy number analysis of formalin-fixed and paraffin-embedded tissues using Affymetrix CytoScan HD chip
Authors: Luo Jian-Hua , Michalopoulos Amantha , Ding Ying , Yu Yan P.
Published: May 2015

Detection of human genome copy number variation (CNV) is one of the most important analyses in diagnosing human malignancies. Genome CNV detection in formalin-fixed and paraffin-embedded (FFPE) tissues remains challenging task. Here, we report that a modified method to analyze CNV in FFPE tissues using Affymetrix Cytoscan HD chips. Our analysis showed a 91% overlap in CNV detection between FFPE and the karyotype analysis on 8 cases of lymphoma samples. The CNV overlap between frozen and the matched FFPE tissues reached 93.8%. When the analyses were restricted to regions containing genes, 87.1% concordance between FFPE and fresh frozen tissues was found. The results suggest that the modified method using Affymetrix Cytoscan HD chip represents a significant improvement over most of the previous methods in terms of accuracy in detecting CNV in FFPE tissues. This FFPE methodology may hold promise for wide application of CNV analysis on clinical samples.

Citation Format: Jian-Hua Luo, Amantha Michalopoulos, Ying Ding, Yan P. Yu. High fidelity copy number analysis of formalin-fixed and paraffin-embedded tissues using Affymetrix CytoScan HD chip. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4274. doi:10.1158/1538-7445.AM2014-4274
Abstract 4278: International Cancer Genome Consortium (ICGC)
Authors: Hudson Thomas J. , Jennings Jennifer L.
Published: May 2015

The International Cancer Genome Consortium (ICGC) was established to bring together researchers from around the globe to comprehensively analyze the genomic, transcriptomic, and epigenomic changes in 50 different tumor types or subtypes that are of clinical and societal importance across the globe (International network of cancer genome projects. Nature 464, 993-998 (15 April 2010)).

As of December 2013, the ICGC has received commitments from researchers and funding organizations in Asia, Australia, Europe, North America, and South America for 69 project teams in 17 jurisdictions to study more than 25,000 tumor genomes.

Over 27,552 tumors that meet ICGC requirements for consent have been obtained, and research pathologists have approved over 24,160 samples with project-confirmed diagnosis as meeting ICGC standards.

Processed data is available via the Data Coordination Centre (http://dcc.icgc.org) based at the Ontario Institute for Cancer Research and is updated bimonthly. The latest data release (Version 14) in September 2013 includes datasets from 8532 donors participating in 41 ICGC project teams. The ICGC Data Portal has been improved to provide enhanced support to the research community browsing this vast dataset.

ICGC researchers plan to undertake a suite of studies called the Pan-Cancer Analysis Project (PCAP). The analyses will begin in early 2014 using whole genome sequencing (WGS) datasets from approximately 2,000 matched tumour-normal specimen pairs representing more than a dozen different tumour types. It is expected that this effort will lead to the identification of driver mutations outside coding regions including those that affect cis-regulatory regions and non-coding RNAs, and identification of gene regulatory pathways altered by non-coding mutations. Because the projected size of the pan-cancer dataset, i.e. 4,000 WGS, is very large, PCAP will need to use a compute cloud environment (involving multiple computing centers in the USA, Europe, and Asia) that will meet technical requirements of the project and the bioethical framework of ICGC and its member projects.

More information can be found on www.icgc.org.

Citation Format: Thomas J. Hudson, Jennifer L. Jennings. International Cancer Genome Consortium (ICGC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4278. doi:10.1158/1538-7445.AM2014-4278
Abstract 4291: A novel image-based cytometry method for autophagy detection in living cells
Authors: Chan Leo Li-Ying , Shen Dee , Wilkinson Alisha R. , Patton Wayne , Lai Ning , Chan Eric , Kuksin Dmitry , Lin Bo , Qiu Jean
Published: May 2015

Autophagy is an important cellular catabolic process that plays a variety of important roles, including maintenance of the amino acid pool during starvation, recycling of damaged proteins and organelles, and clearance of intracellular microbes. Currently employed autophagy detection methods include fluorescence microscopy, biochemical measurement, SDS-PAGE, and Western blotting, but they are time-consuming, labor-intensive, and require much experience for accurate interpretation. More recently, development of novel fluorescent probes have allowed the investigation of autophagy via standard flow cytometry. However, flow cytometers remain relatively expensive and require a considerable amount of maintenance. Previously, image-based cytometry has been shown to perform automated fluorescence-based cellular analysis comparable to flow cytometry. In this study, we developed a novel method using the Cellometer image-based cytometer in combination with Cyto-ID® Green dye for autophagy detection in live cells. The method is compared to flow cytometry by measuring macroautophagy in nutrient-starved Jurkat cells. Results demonstrate similar trends of autophagic response, but different magnitude of fluorescence signal increases, which may arise from different analysis approaches characteristic of the two instrument platforms. The possibility of using this method for drug discovery applications is also demonstrated through the measurement of dose-response kinetics upon induction of autophagy with rapamycin and tamoxifen. The described image-based cytometry/fluorescent dye method should serve as a useful addition to the current arsenal of techniques available in support of autophagy-based drug discovery relating to various pathological disorders.

Citation Format: Leo Li-Ying Chan, Dee Shen, Alisha R. Wilkinson, Wayne Patton, Ning Lai, Eric Chan, Dmitry Kuksin, Bo Lin, Jean Qiu. A novel image-based cytometry method for autophagy detection in living cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4291. doi:10.1158/1538-7445.AM2014-4291
Abstract 4313: Efficacy of fluorescence-guided surgery on primary human osteosarcoma
Authors: Miwa Shinji , Hiroshima Yukihiko , Yano Shuya , Matsumoto Yasunori , Uehara Fuminari , Yamamoto Mako , Kimura Hiroaki , Hayashi Katsuhiro , Tsuchiya Hiroyuki , Hoffman Robert M.
Published: May 2015

We investigated the effectiveness of fluorescence-guided surgery (FGS) to improve outcomes in an orthotopic mouse model of human osteosarcoma. 143B-RFP human osteosarcoma cells were injected into the intramedullary cavity of the tibia in 32 nude mice. Two weeks after the implantation, 16 mice underwent FGS, and the other 16 mice underwent bright-light surgery (BLS). Half of the BLS and FGS mice (8 mice in each group) underwent adjuvant chemotherapy with cisplatin. Weekly imaging of the mice was performed and RFP-expressing areas were recorded. The fluorescent area of residual tumor after BLS and FGS were 10.2 ± 2.4 mm2 and 0.1 ± 0.1 mm2, respectively (P < 0.001). The residual tumor grew after BLS and BLS+CDDP. In contrast, the FGS mice and the FGS+CDDP mice had almost no tumor growth. Disease-free survival (DFS) in the BLS-, the BLS+CDDP-, the FGS-, and the FGS+CDDP-treated mice was 12.5%, 37.5%, 75.0%, and 87.5%, respectively. The FGS-treated mice had significantly higher DFS rate than the BLS-treated mice (P = 0.021), and the FGS+CDDP-treated mice had significantly higher DFS rate than the BLS+CDDP-treated mice (P = 0.043). Although chemotherapy significantly reduced multiple metastases (P = 0.033), there was no significant correlation between FGS and lung metastasis. Our study showed that FGS significantly reduced the recurrence of the tumor, but did not reduce lung metastasis. To reduce tumor recurrence and multiple metastases, the combination of FGS and chemotherapy may be necessary in the clinic.

Citation Format: Shinji Miwa, Yukihiko Hiroshima, Shuya Yano, Yasunori Matsumoto, Fuminari Uehara, Mako Yamamoto, Hiroaki Kimura, Katsuhiro Hayashi, Hiroyuki Tsuchiya, Robert M. Hoffman. Efficacy of fluorescence-guided surgery on primary human osteosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4313. doi:10.1158/1538-7445.AM2014-4313
Abstract 4331: Lowering glucose increases nutlin-3 toxicity against melanomas irrespective of BRAF,NRAS or p53 mutations
Authors: Rieber Manuel , Chavez-Perez Valery , Strasberg-Rieber Mary
Published: May 2015

BACKGROUND: Melanomas are commonly characterized by mutually exclusive BRAF V600Emutations (∼60%) or NRASQ61 mutations (∼15%). Although targeted therapy with vemurafenib is initially efficient in melanoma with BRAF mutations, tumor recurrence occurs including emergence of resistant melanoma cells with secondary N-RASQ61K mutations. Recently it was demonstrated that nutlin-3, an HDM2 antagonist capable of restoring p53 tumor suppressor function synergized with vemurafenib against BRAF V600E wt p53 melanomas. Although nutlin-3 was initially believed to be non-genotoxic, it can originate p53 mutations which are infrequent in primary melanomas.

OBJECTIVES: Since tumor growth increases metabolic demand and glucose consumption, we investigated whether lowering glucose with glycolytic inhibitors increases nutlin-3 toxicity against melanomas irrespective of BRAF, NRAS or p53 status. EXPERIMENTAL: Metabolic activity/proliferation of melanoma cells harbouring BRAF, NRAS or p53 mutations was quantitated by fluorometric studies, crystal violet staining was used to determine differential cell survival, and mechanisms of cell death were studied by protein array immune blotting.

RESULTS: Restricting glucose increases nutlin-3 toxicity against wt p53 melanoma cells harbouring BRAF or NRAS, even if the latter have p53 mutations. Responses occur through mechanisms dependent and independent of p53 serine 15-phosphorylation and p21/CIP1/CDNK1A.

CONCLUSION: Elimination of melanoma cells irrespective of their BRAF, NRAS or p53 mutational status may be helped by joint use of glucose-lowering agents together with nutlin-3.

Citation Format: Manuel Rieber, Valery Chavez-Perez, Mary Strasberg-Rieber. Lowering glucose increases nutlin-3 toxicity against melanomas irrespective of BRAF,NRAS or p53 mutations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4331. doi:10.1158/1538-7445.AM2014-4331
Abstract 4363: Macrophage migration inhibitory factor (MIF) and miR-301b interactively enhance disease aggressiveness by targeting NR3C2 in human pancreatic cancer
Authors: Yang Shouhui , He Peijun , Wang Jian , Schetter Aaron , Funamizu Naotake , Yanaga Katsuhiko , Gaedcke Jochen , Ghadimi B. Michael , Gaida Matthias M. , Ried Thomas , Hanna Nader , Alexander H. Richard , Hussain S. Perwez
Published: May 2015

MIF is a pleiotropic cytokine, which plays a role in inflammatory and immune responses and is implicated in tumorigenesis. Our previous study showed that MIF enhances tumor aggressiveness and predicts outcome in patients with pancreatic ductal adenocarcinoma (PDAC). Aberrant microRNA (miRNA) expression is a common feature in cancer and cytokine responsive miRNAs are recently described as critical effectors in tumor progression. We have investigated MIF-associated miRNAs and their interactive roles in the progression of human pancreatic cancer. Global analysis of miRNA expression revealed 38 differentially expressed miRNAs in MIF-high as compared with MIF-low cases of PDAC based on the median value. Kaplan-Meier survival analysis showed that a higher expression of miR-301b in tumors was associated with poorer survival (p=0.008, N=37), which was further validated in an independent cohort of PDAC patients (p=0.021, N=42). Additionally, MIF expression positively correlated with miR-301b and mediated its expression in several human pancreatic cancer cells. Mechanistic analysis revealed that miR-301b promotes pancreatic cancer cell migration and invasion. Furthermore, nuclear receptor subfamily group c member 2 (NR3C2) was identified as a functional target of miR-301b. NR3C2 inhibited tumor migration and invasion and a higher expression of NR3C2 predicted better survival in pancreatic cancer patients. Further in vivo studies showed a significant decrease in NR3C2 in MIF-overexpressing orthotopic xenograft as compared to control. Taken together, our results demonstrate that MIF and miR-301b interactively contribute to pancreatic cancer progression by targeting NR3C2. This novel MIF-miR-301b-NR3C2 axis may provide potential targets for improving disease outcome in pancreatic cancer.

Citation Format: Shouhui Yang, Peijun He, Jian Wang, Aaron Schetter, Naotake Funamizu, Katsuhiko Yanaga, Jochen Gaedcke, B. Michael Ghadimi, Matthias M. Gaida, Thomas Ried, Nader Hanna, H. Richard Alexander, S. Perwez Hussain. Macrophage migration inhibitory factor (MIF) and miR-301b interactively enhance disease aggressiveness by targeting NR3C2 in human pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4363. doi:10.1158/1538-7445.AM2014-4363
Abstract 4375: UVC irradiation in combination with fluorescence-guided surgery cures metastatic human pancreatic cancer in orthotopic mouse models
Authors: Hiroshima Yukihiko , Maawy Ali , Sato Sho , Murakami Takashi , Yamamoto Mako , Uehara Fuminari , Miwa Shinji , Yano Shuya , Momiyama Masashi , Chishima Takashi , Tanaka Kuniya , Bouvet Michael , Endo Itaru , Hoffman Robert M.
Published: May 2015

The aim of this study was to determine the efficacy of UVC irradiation in combination with fluorescence-guided surgery (FGS) in an orthotopic model of pancreatic cancer. Two weeks after orthotopic implantation of human MiaPaCa-2-GFP pancreatic cancer cells, bright-light surgery (BLS) was performed on all tumor-bearing mice (n=24). Postoperatively, the surgical resection bed was imaged with the OV100 variable magnification imaging system with a magnification of 0.56x to detect residual tumor. The mice which underwent BLS were randomized into 3 groups; BLS only (n=8) or FGS (n=8) or FGS-UVC (n=8). The residual tumors were resected with FGS using a portable, hand-held imaging system. After FGS, the surgical resection bed was imaged with the OV100 with a magnification of 0.89x, to detect minimal residual tumor and was irradiated with 2700 J/m2 UVC (emission peak 254 nm) from the bottom of a chamber using a Benchtop 3UV transilluminator (UVP, LLC, Upland, CA). The average residual tumor area after FGS was significantly smaller than BLS (0.135 ± 0.137 mm2 and 3.338 ± 2.929 mm2, respectively; p=0.007). Relapse-free survival (RFS) and overall survival (OS) were determined in 3 groups. Three-month RFS in the BLS, FGS and FGS-UVC were 0%, 50% and 100%, respectively. Five-month OS in BLS, FGS and FGS-UVC was 10%, 90% and 100%, respectively. FGS and FGS-UVC resulted in significantly increased survival compared to BLS for both RFS and OS. FGS-UVC showed significantly increased survival compared to the FGS for both RFS and OS (p=0.008 and p=0.025, respectively). These results suggests that UVC irradiation in combination with FGS could be a curative strategy for human pancreatic cancer.

Citation Format: Yukihiko Hiroshima, Ali Maawy, Sho Sato, Takashi Murakami, Mako Yamamoto, Fuminari Uehara, Shinji Miwa, Shuya Yano, Masashi Momiyama, Takashi Chishima, Kuniya Tanaka, Michael Bouvet, Itaru Endo, Robert M. Hoffman. UVC irradiation in combination with fluorescence-guided surgery cures metastatic human pancreatic cancer in orthotopic mouse models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4375. doi:10.1158/1538-7445.AM2014-4375
Abstract 438: Long non-coding RNA AK023948 enhances breast cancer progression by activating AKT
Authors: Koirala Pratirodh
Published: May 2015

The role of long non-coding RNAs (lncRNAs) in various physiological and pathological conditions has been increasingly appreciated, however, given the large number of lncRNAs, little is known for the vast majority of lncRNAs for their potential role in breast cancer. In this study, using real-time PCR-based lncRNA profiling array, we identified a set of lncRNAs that were differentially expressed in breast cancer tissues of patients as compared to the normal breast tissue. Among them, AK023948 was upregulated in breast cancer; it was also upregulated in breast cancer cell lines (MCF-7 and MDA-MB-231) as compared to human mammary epithelial cells (HMLE). This was further supported by breast cancer tissue microarray by in situ hybridization. Moreover, ectopic expression of AK023948 in MCF-7 cells promoted the proliferation and anchorage independent cell growth, implying its potential oncogenic role in breast cancer progression. In consistent with this finding, we found that while ectopic expression of AK023948 activated, AK023948-siRNA suppressed Akt activation. To determine the underlying mechanism of AK023948-mediated activation of Akt, we performed RNA pull-down assay with biotin-labeled AK023948 RNA probe. Mass spectrometry analysis of the pulled down proteins identified ATP dependent RNA helicase A (RHA/DHX-9) as a potential binding partner for AK023948, which was confirmed by western blot. Gene silencing of DHX-9 in MCF-7 cells suppressed AKT phosphorylation, suggesting that AK023948 may activate AKT activation through interacting with DHX-9 and modulating its function. Together, these results suggest that AK023948 may function as an oncogenic lncRNA in breast cancer and thus, it may represent a novel target for breast cancer therapy.

Citation Format: Pratirodh Koirala. Long non-coding RNA AK023948 enhances breast cancer progression by activating AKT. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 438. doi:10.1158/1538-7445.AM2014-438
Abstract 4384: Targeting miR-124 inhibits growth of prostate cancer xenografts and sensitized prostate cancer cells to anti-androgen
Authors: Shi Xu-Bao , Ma Aihong , Xue Lingru , White Ralph W. deVere
Published: May 2015

Increasing evidence has shown that miR-124 is a tumor suppressor and plays an important role in pathogenesis of many types of human cancer. We previously found that miR-124 directly targets the androgen receptor (AR) and is significantly downregulated in clinical prostate cancer (CaP) samples. Moreover, overexpression of miR-124 induced apoptosis and inhibited cell proliferation and xenograft growth of CaP. In this study, we determined whether restoration of miR-124 may represent an adjunctively therapeutic option for CaP treatment. For this purpose, WST-1 and clonogenic assays were performed to evaluate the effects of miR-124 or miR-124 plus enzalutamide (one new androgen antagonist) on CaP cell proliferation. In animal experiment, male nude mice were inoculated s.c with CWR22 CaP cell suspensions. When tumor sizes reached ∼50 mm3, mice were treated with polyethylenimines (PEI)-complexed miR-124 (i.v., 10µg/day, thrice a week for 4 weeks) or PEI-miR-124 (i.v.) plus enzalutamide (p.o., 10mg/kg/day for 5 weeks). PEI can increase internalization and facilitate miRNA release into cytoplasm. After treatment, tumors were harvested for analyzing apoptosis and measuring miR-124 target levels. In in vitro experiment, 22Rv1 cells were separately treated with miR-124, enzalutamide or both. It was found that the combined treatment significantly increased growth inhibition of 22Rv1 cells. In addition, we established enzalutamide-resistant 22Rv1 subline (22Rv1-MDVR) by clone-selection in gradually increased doses (from 5μM to 40μM within four months). Treatment with miR-124 obviously inhibited clone formation of 22Rv1-MDVR cells. In mice receiving miR-124 or enzalutamide treatment, we observed tumor-inhibitory effects. However, the combination of miR-124 and enzalutamide exhibited more significant inhibition of tumor growth when compared to the single treatment. We examined the expression of AR and its variant 7 (AR-V7) that are targets of miR-124. Reduced levels of AR and AR-V7 were found in miR-124-treated tumors, indicating that PEI facilitates entry of miR-124 into the tumor cells. We also observed increased apoptosis in miR-124-treated tumors. Data from in vitro and in vivo studies show that miR-124 inhibited growth of CaP cells and sensitized CaP cells to anti-androgens, suggesting that miR-124 can be used as an adjunctively therapeutic agent for improved CaP treatment.

Citation Format: Xu-Bao Shi, Aihong Ma, Lingru Xue, Ralph W. deVere White. Targeting miR-124 inhibits growth of prostate cancer xenografts and sensitized prostate cancer cells to anti-androgen. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4384. doi:10.1158/1538-7445.AM2014-4384
Abstract 439: Mitochondrial uncoupling protein 2 promotes skin carcinogenesis
Authors: Li Wenjuan , Zhang Chunjing , Shi Runhua , Gu Xin , Zhao Yunfeng
Published: May 2015

Mitochondrial uncoupling uncouples mitochondrial respiration and ATP production. There are five uncoupling proteins (UCPs), UCP2 is ubiquitous expressed and plays an important role in the reprogramming of cancer cell metabolism. However, little is known about at what stage of cancer development mitochondrial uncoupling occurs and what the role of UCP2 in cancer etiology. We will try to address these puzzles in present study.

The well-established chemically-induced skin carcinogenesis mouse model was used and UCP2 wide-type (WT) and homozygous knockout (KO) mice were added to this study as direct evidence for the role of UCP2 during skin carcinogenesis. After forty-week treatment with chemical carcinogens, the incidence of papillomas in WT and KO TPA mice was 92.3% and 77.8%, respectively; and tumor multiplicity was 2.6 and 1.3, respectively. The incidence of carcinomas in WT and KO TPA treatment was 76.9% and 22.2%, respectively, and tumor multiplicity was 0.9 and 0.2, respectively. We next detected how the mitochondrial function and ATP generation would be changed in this model. UCP2 knockout slightly increased oxygen consumption compared with WT, whereas tumor promoter treatment suppressed oxygen consumption in both UCP2 KO and WT group. As for ATP generation, there was no significant change between WT and KO, or between vehicle and tumor promoter treatment.

We next examined the mechanisms of tumor promotion by mitochondrial uncoupling by overexpressing UCP2 in JB6 P+ cells, which are the only characterized skin cell model to study tumor promotion. Anchorage-independent growth assay was performed and the results showed that overexpression of UCP2 enhanced colony formation in response to tumor promoter treatment. In addition, overexpression of UCP2 induced the upregulation of the oncoprotein Fra-1 whereas inhibited the increase in p53 expression.

In summary, this study will add new knowledge to the understanding of the role of mitochondrial uncoupling in cancer etiology, and many help design a mitochondrial uncoupling-targeted approach for the suppression of cancer development.

Citation Format: Wenjuan Li, Chunjing Zhang, Runhua Shi, Xin Gu, Yunfeng Zhao. Mitochondrial uncoupling protein 2 promotes skin carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 439. doi:10.1158/1538-7445.AM2014-439
Abstract 4396: MicroRNA MIR551B amplified at 3q26.2 locus activate c-KIT expression and causes resistance to anoikis of ovarian cancer cells
Authors: Chaluvally-Raghavan Pradeep , Zhang Fan , Liu Wenbin , Tyler Moss , Yu Shuangxing , Pradeep Sunila , Ram Prahlad , Lu Yiling , Sood Anil , Mills Gordon
Published: May 2015

Background and Hypothesis

The 3q26.2 loci amplification is present in at least 35% of high grade serous ovarian cancer (HGSEOC). Recently we identified a microRNA, MIR551B located in the 3q26 amplicon. However, the potential role of microRNAs amplified at 3q26 amplicon and their functions in ovarian tumorigenesis has not been studied previously. Thus, our studies have the potential to provide novel mechanism underlying MIR551B oncogenesis in ovarian cancer.

Results

The 3q26.2 locus is large and structurally complex suggesting that multiple coding genes located in the amplicon could contribute to tumor initiation and progression alone or through cooperative activity. High-resolution SNP-based copy number analysis of 533 high-grade serous ovarian cancer samples demonstrated that MIR551B is highly amplified as a consequence of the 3q26.2 amplicon. Expression analysis in the Cancer Genome Atlas (TCGA) data sets showed that amplification of MIR551B is associated with increased expression of maturedmicroRNA miR551b in the recently defined proliferative subtype of ovarian cancer. Reverse phase protein array (RPPA) demonstrated that enforced expression of miR551b-5p increased the expression c-KIT oncogene and activated the ERK signaling pathway. Our target search analysis identified a potential novel mechanism whereby miR551b-5p could bind to a target site in the promoter of c-KIT oncogene and increase the transcription of c-Kit mRNA. Strikingly, enforced expression of miR551b-5p induced the survival and proliferation of ovarian surface epithelial cells detached from extracellular matrix. Of note, miR551b-5p expression in recurrent ovarian tumors are higher than the primary ovarian cancers.

Summary

Our results show that copy number alterations (CNAs) of MIR551B are associated with increased levels of mature microRNA, miR551b. Mechanistic studies revealed a novel role of microRNA miR-551b-5p to induce transcriptional activation by interacting with the promoter of the c-Kit oncogene, which in turn renders cells resistant to anokis a critical component of the survival and spread of ovarian cancer in the peritoneal cavity. Our results propose the c-KIT transcriptional activation induced by miR551b-5p is a key mechanism of survival of ovarian cancer cells in the ascitic fluid in the peritoneal cavity.

Citation Format: Pradeep Chaluvally-Raghavan, Fan Zhang, Wenbin Liu, Moss Tyler, Shuangxing Yu, Sunila Pradeep, Prahlad Ram, Yiling Lu, Anil Sood, Gordon Mills. MicroRNA MIR551B amplified at 3q26.2 locus activate c-KIT expression and causes resistance to anoikis of ovarian cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4396. doi:10.1158/1538-7445.AM2014-4396
Abstract 4397: Therapeutic microRNA agaisnt KRAS mutant colorectal cancer
Authors: Hiraki Masayuki , Nishimura Junichi , Uemura Mamoru , Hata Taishi , Takemasa Ichiro , Mizushima Tsunekazu , Yamamoto Hirofumi , Doki Yuichiro , Mori Masaki
Published: May 2015

Background:

The anti-epidermal growth factor receptor (EGFR) monoclonal antibodies are the molecular target drugs, dependent on v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations status in metastatic colorectal cancer (mCRC). Anti-EGFR monoclonal antibody based chemotherapy is the standard treatment for mCRC with KRAS wild-type. However, the issue of resistance to this therapy in CRC with KRAS mutations (approximately 50% of CRC) remains unresolved. The aim of this study is to identify microRNAs (miRNAs) that govern the resistance to anti-EGFR monoclonal antibody in CRC with KRAS mutations.

Experimental Design:

We first established the KRAS mutant cell model using HEK293 and MRC5 cells by introducing KRASG12V plasmid. The miRNA expression profiles were compared between these cells using miRNA microarray analysis. We selected the candidate a miRNA which down-regulates the downstream signal of KRAS using luciferase reporter assays. Assessment of target genes of the candidate a miRNA was performed in vitro. Next, anti-tumor effects by supplement of a miRNA were evaluated in vitro and in vivo.

Results:

We picked up miRNAs, of which expressions decreased in both HEK293 and MRC5 cells by introduction of KRASG12V plasmid in miRNA array analysis. Among them, administration of mature-miR-X suppressed SRE and AP1 activity in KRAS mutant CRC cell lines such as DLD1G13D, SW480 G12V, and HCT116 G13D using dual luciferase reporter assay. Furthermore, miR-X regulated not only Ras/Raf/MAPK pathway but also suppressed PI3K/Akt pathway in vitro. Administration of mature-miR-X suppressed proliferation and induced apoptosis of the CRC cell lines with KRAS mutations in vitro. Systemic mature-miR-X administration into tail veins of nude mice significantly inhibited the growth of pre-established DLD1G13D xenografts. miR-X level was significantly lower in tumor tissues compared to normal mucosa.

Conclusions:

miR-X may overcome anti-EGFR therapy resistance in CRC with KRAS mutations by regulating Ras/Raf/MAPK pathway and PI3K/Akt pathway. Our data suggests that miR-X could be suitable for a novel therapy for mCRC patients with KRAS mutations.

Citation Format: Masayuki Hiraki, Junichi Nishimura, Mamoru Uemura, Taishi Hata, Ichiro Takemasa, Tsunekazu Mizushima, Hirofumi Yamamoto, Yuichiro Doki, Masaki Mori. Therapeutic microRNA agaisnt KRAS mutant colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4397. doi:10.1158/1538-7445.AM2014-4397
Abstract 4401: A novel drug suppresses proliferation of lung cancer cells via increasing the CBL activity and down-regulating epidermal growth factor receptor
Authors: Huang Kuo-Yen , Pan Szu-Hua , Wang Wen-Lung , Chen Ching-Shih , Hong Tse-Ming , Yang Pan-Chyr
Published: May 2015

Aberrant epidermal growth factor receptor (EGFR) signaling is one of the most critical oncogenic pathways in NSCLC that may trigger the tumor progression. Thus, how to suppress the EGFR downstream signaling cascade or to reduce the EGFR expression level has long been considered an important therapeutic approach for cancer interventions. Here, we found a regulatory mechanism by which the degradation of EGFR is enhanced by a novel promising drug, compound 22. First, the exposure to compound 22 induced cell death via apoptosis in lung cancer cell lines, including EGFR wild type A549 as well as EGFR mutant PC9 and H1975 cells. Compound 22 decreased the EGFR protein level in a dose-dependent manner and facilitated dephosphorylation of its downstream targets, including AKT and ERK, both of which play a critical role for cancer cell survival. Second, we observed that compound 22 induced EGFR degradation through proteasome degradation in the cycloheximide chase plus MG132 assay. Additionally, compound 22 increased the CBL (the ubiquitin E3 ligase)-mediated ubiquitination of EGFR, which was accompanied by the enhancement of Y1045 phosphorylation in the EGFR kinase domain. Overall, these pieces of evidence suggest that compound 22 is a novel class of anticancer drug which is able to inhibit the TKI-resistant NSCLC cells by inducing the degradation of EGFR.

Citation Format: Kuo-Yen Huang, Szu-Hua Pan, Wen-Lung Wang, Ching-Shih Chen, Tse-Ming Hong, Pan-Chyr Yang. A novel drug suppresses proliferation of lung cancer cells via increasing the CBL activity and down-regulating epidermal growth factor receptor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4401. doi:10.1158/1538-7445.AM2014-4401
Abstract 4402: Synergistic cell death in nedd8 and hrb27c mutant cells under an rbf-negative background is dependent on EGFR/MAPK signaling
Authors: Zhang Robin , Zhang Tianyi , Sheng Zhentao , Du Wei
Published: May 2015

The Rb (retinoblastoma) protein is highly functionally conserved between the Drosophila, and deregulation and mutations in the Rb pathway are common events across a wide range of cancers. Here, we identify two novel synthetic lethal interactions between Rb and mutant alleles of Nedd8 and Hrb27c that were identified through an unbiased genetic screen in Drosophila that resulted in apoptosis in both the wing disc pouch and the region of the eye disc posterior to the morphogenetic furrow. However, the molecular mechanisms behind the apoptotic phenotypes in both synthetic lethal double mutants have yet to be elucidated. It is known that wild-type Rb normally sequesters and can prevent the E2F1 transcription factor from promoting transcription of hid, the major pro-apoptotic regulator in imaginal discs. Synthetic lethality is therefore likely to be contingent on EGFR/hid signaling, and I will determine if such is the case through a series of genetic epistasis tests. Interestingly, preliminary evidence also suggests that EGFR/MAPK signaling may be ablated in both mutants of Nedd8 and Hrb27c. However, this observation is completely novel and therefore the molecular mechanisms behind downregulation of MAPK signaling have yet to be elucidated in both these mutants. I will again perform a series of epistasis experiments to determine how Nedd8 and Hrb27c mutations mechanistically affect EGFR/MAPK signaling. This will be achieved first by determining if signaling is downregulated either during EGFR ligand processing or downstream of the EGFR receptor. This will be followed by subsequent experiments to determine which specific element in either pathway is being affected by Nedd8 or Hrb27c ablation. I therefore hypothesize that inhibition of both nedd8 and hrb27c synergistically induce cell death in Rb-negative cells in a process that is dependent on both E2F1 activity and downregulation of MAPK signaling. Many existing treatment regimens fail to take into account the unique biological properties of cancer cells compared to their normal counterparts, thus often resulting in off-target effects and limitations in dosage. This proposal is therefore both novel and significant because the mechanisms behind both the Rb/Hrb27c and Rb/Nedd8 synthetic lethal interactions have yet to be elucidated and characterization of such interactions such as these have the potential to catalyze the development of new therapeutic strategies in the treatment of cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Robin Zhang, Tianyi Zhang, Zhentao Sheng, Wei Du. Synergistic cell death in nedd8 and hrb27c mutant cells under an rbf-negative background is dependent on EGFR/MAPK signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4402. doi:10.1158/1538-7445.AM2014-4402
Abstract 4416: IKBKE is a key mediator of Ras activation of NF-κB and Ras oncogenic function
Authors: Kim Donghwa , Guo Jianping , Challa Sridevi , Coppola Domenico , Cheng Jin Q.
Published: May 2015

Accumulating evidence indicates that the NF-κB transcription factor pathway is important for oncogenic RAS to transform cells and to drive tumorigenesis in animal models. IKBKE has been shown to activate NF-κB through both canonical and non-canonical pathways. Here we show that activating mutations of Ras significantly induce IKBKE kinase activity and that depletion of KRas results in decrease of IKBKE activity only in KRAS mutant but wild-type cells. KRas-induced NF-κB DNA-binding and transcription activity was abrogated by knockdown of IKBKE. In addition, we demonstrated significant decrease of lung tumor development in Ikbke-/-;KRasLA1 mice when compared to Ikbke+/-;KRasLA1 and Ikbke+/+;KRasLA1 animals. Furthermore, depletion of IKBKE selectively reduces cell survival and tumor growth in K-Ras mutant non-small cell lung cancer cells. These data identify IKBKE as a downstream effector of Ras which mediates RAS activation of NF-κB and Ras tumorigenic activity.

Citation Format: Donghwa Kim, Jianping Guo, Sridevi Challa, Domenico Coppola, Jin Q. Cheng. IKBKE is a key mediator of Ras activation of NF-κB and Ras oncogenic function. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4416. doi:10.1158/1538-7445.AM2014-4416
Abstract 4433: Fbxw11 promotes leukemia development by activating the NF-κB signaling pathway
Authors: Wang Lina , Liao Jinfeng , Yang Xiao , Feng Wenli , Chen Shayan , Zheng Guoguang
Published: May 2015

F-box proteins serve as the substrate recognition subunit for the SCF E3 ubiquitin ligases. These ligases ubiquitinate specifically phosphorylated substrates and play a pivotal role in the regulation of various signal transduction pathways, which, in turn, are essential for many aspects of tumorigenesis. However the role of Fbxw11 in the development of leukemia and the underlying mechanisms remain largely unknown. Here, we found two transcript variants (Fbxw11c and Fbxw11d) expressed in mouse bone marrow. More importantly, the expression of Fbxw11 in normal hematopoietic stem cells (HSCs) from Notch1-induced leukemia mice was higher than that from control mice. In order to investigate the role of Fbxw11 in leukemia, we established L1210 cell lines over-expressing Fbxw11c or Fbxw11d respectively using the lentivirus system. Over-expression of both Fbxw11 variants stimulated the proliferation of L1210 cells in vitro by cell counting and MTT assay. The tumor xenografts model with over-expression of Fbxw11c or Fbxw11d in DBA/2 mice was further established. Over-expression of both variants resulted in the increased tumor growth in vivo. Investigation of the molecular mechanism revealed that the increased cell proliferation was not due to decrease in cell apoptosis but due to increase in cell cycle. Further studies showed that overexpression of both Fbxw11c and Fbxw11d caused the activation of NF-κB signaling pathway. These findings suggest the important role of Fbxw11 in regulating the development of leukemia and indicate a potent rationale for developing Fbxw11 as a potential therapeutic target against leukemia.

Citation Format: Lina Wang, Jinfeng Liao, Xiao Yang, Wenli Feng, Shayan Chen, Guoguang Zheng. Fbxw11 promotes leukemia development by activating the NF-κB signaling pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4433. doi:10.1158/1538-7445.AM2014-4433
Abstract 4450: Comparison of structural elements in RasGRP3 and RasGRP1 controlling membrane interaction
Authors: Czikora Agnes , Kedei Noemi , Blumberg Peter M.
Published: May 2015

RasGRP comprises a family of guanine nucleotide exchange factors that activate small GTPases, regulating the dissociation of GDP from Ras GTPases to enhance the formation of the active GTP-bound form. RasGRP3 has a region homologous to the phosphoinositide binding region of RasGRP1. The objective of this study was to explore the hypothesis that this region of RasGRP3, like that of RasGRP1, indeed contributes to phosphoinositide binding. For these studies, we prepared full length RasGRP1/3, RasGRP1/3 C1 domain, and truncated (C1-SuPT-PT) RasGRP1 and RasGRP3 green fluorescent protein labeled constructs to transfect human prostate adenocarcinoma (LNCaP) and human cervical carcinoma (HeLa) cells. In HeLa cells, both RasGRP1 and RasGRP3 translocated to the endonuclear membranes, most probably to the Gogli in response to phorbol ester (PMA) and other agents. In the LNCaP cells, which are a PTEN mutant cell line with markedly elevated phosphoinositide levels, RasGRP1 translocates to the plasma membrane. In contrast, the full length RasGRP3 does not translocate to the plasma membrane even though it has a PT (plasma membrane-targeting) domain with a basic/hydrophobic cluster of amino acids (BHC) similar to that of RasGRP1. In parallel, we examined the translocation of the C1 domains of RasGRP1/3 and of the truncated RasGRP1/3 constructs, as well as the corresponding constructs with amino acid substitutions in the predicted phosphoinositide binding domain of RasGRP3. The RasGRP1 translocated to the plasma membrane in response to PMA. The C1 domain of RasGRP3 was insufficient for targeting RasGRP3 to the plasma membrane. However, truncated RasGRP3, which contains the PT domain, showed stronger translocation. Substituting amino acids (Asp647, Lys648 and Ala651) in the PT domain of full length RasGRP3 triggered the plasma membrane translocation in the LNCaP cells. We conclude that the differences in the BHC section of the PT domain of RasGRP3 play an important role in the membrane translocation of RasGRP3 and will reduce its sensitivity to membrane phosphoinositide levels.

Citation Format: Agnes Czikora, Noemi Kedei, Peter M. Blumberg. Comparison of structural elements in RasGRP3 and RasGRP1 controlling membrane interaction. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4450. doi:10.1158/1538-7445.AM2014-4450