bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1026: PEG-LPrA2 is a non-toxic adjuvant for triple negative breast cancer
Authors: Dill Courtney D. , Harbuzariu Adriana , Rampoldi Antonio , Lipsey Crystal C. , Lanier Viola , Harmon Tia , Daley-Brown Danielle , Tchio Cynthia , Candelaria Pierre , Gonzalez-Perez Ruben Rene
Published: July 2016

Background: Triple Negative Breast Cancer (TNBC) is an aggressive cancer with poor prognosis and is difficult to treat. Current standard therapy for the disease includes a combination of chemotherapeutic drugs: doxorubicin (DOX), paclitaxel (TAX), and cyclophosphamide (CTX). These drugs are ineffective as they exhibit shortcomings and several side effects. TNBC patients develop chemoresistance that may be enhanced by leptin, which affects survival, proliferation, and angiogenesis. Our lab developed and tested a novel and specific inhibitor of leptin signaling, LPrA2. A pegylated derivative of LPrA2 (PEG-LPrA2), with enhanced bioavailability, was successfully used in mouse breast cancer models. Preliminary data showed that PEG-LPrA2 was non-toxic in vitro. Therefore, it is hypothesized that PEG-LPrA2 is not toxic in vivo.

Methods: To determine potential toxicity of PEG-LPrA2, in vitro and in vivo assays were performed. In vitro toxicity of PEG-LPrA2 was tested in a human non-malignant mammary epithelial cell line (MCF-10A). MCF-10A cells were cultured in 96 well plates (5,000 cells/ well) and grown to 70-80% confluence. Cells were treated with PEG-LPrA2 for 24 hrs and viability was determined via MTT assay. In vivo toxicity studies were performed in obese mice. Fifty-seven, eight week old C57BL/6J mice (Charles River Laboratories) were divided into 6 groups. Control mice were fed a low fat diet (10% Kcal from fat) and the rest of the mice were fed a high fat diet (60% Kcal from fat) for 11 weeks. Obesity was characterized as body weight (BW) ≥ 25% BW of control mice. Obese mice were divided into six groups (n = 7/each). Mice were injected with 50 μL of either Sc-PEG-LPrA2 (scramble control) or active inhibitor, PEG-LPrA2, (0.1 mM, 1 mM, or 5 mM) two times a week, for eight weeks. Blood chemistries were analyzed. Additionally, heart, liver, and kidney tissue were harvested and examined to determine toxicity. The tissues were probed for OB-R via immuno histochemistry.

Results: The results showed no changes in BW or food intake. Additionally, no evident changes in blood parameters and organ histology were found.

Conclusions: PEG-LPrA2 is non-toxic and could serve as an adjuvant therapy for standard TNBC chemotherapeutics.

Citation Format: Courtney D. Dill, Adriana Harbuzariu, Antonio Rampoldi, Crystal C. Lipsey, Viola Lanier, Tia Harmon, Danielle Daley-Brown, Cynthia Tchio, Pierre Candelaria, Ruben Rene Gonzalez-Perez. PEG-LPrA2 is a non-toxic adjuvant for triple negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1026.
Abstract 105: Bioinformatic analysis of RNA-Seq data to search for novel diagnostic/prognostic biomarkers of pancreatic ductal adenocarcinoma
Authors: Sosa Omar J. , Paixão Vinícius F. , Setubal João C. , Reis Eduardo M.
Published: July 2016

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly human malignancies. The only curative treatment available is the surgical removal of the tumor in early stages of the disease. Current methods for early detection and treatment are poor, justifying more studies in this field. We aim to generate a high-resolution catalog of the PDAC transcriptome, to reveal transcriptional alterations associated with the malignant phenotype of PDAC. To that end 28 patient matched samples of PDAC and nontumor adjacent pancreatic tissue were processed for the production of rRNA subtracted cDNAs libraries and subsequently sequenced in the Illumina HiSeq platform producing 17 million 100 nt paired-end reads per sample on average. We are implementing an informatics pipeline to detect and evaluate the differential expression in PDAC of well-annotated genes, including long noncoding RNAs (lncRNAs). In addition, we will search for novel lncRNAs and alternative splicing isoforms of protein-coding genes expressed in pancreatic tissues. Following the initial quality control (FastQC) and trimming of low quality regions (Trimmomatic), reads from each sample were aligned to the human genome (TopHat2) and a count table with the number of reads per gene for each sample was created (HTSeq). For evidence of changes across experimental conditions we use DESeq2, identifying 310 genes up-regulated and 354 down-regulated (p-adjusted<0.001 and lfc<|3.3|), including 156 lncRNAs. The differentially expressed genes detected are involved in pathways related to cancer, regulation of cell cycle process, membrane and secreted proteins. De novo transcript assembly (Trinity) revealed a number of yet unannotated transcripts and isoforms expressed in pancreatic tissues. Altogether, the transcript catalog generated herein provides a valuable resource for the identification of novel putative candidate biomarkers to improve the early detection or evaluation of therapeutic response in PDAC.

Work supported by FAPESP, CNPq and CAPES.

Citation Format: Omar J. Sosa, Vinícius F. Paixão, João C. Setubal, Eduardo M. Reis. Bioinformatic analysis of RNA-Seq data to search for novel diagnostic/prognostic biomarkers of pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 105.
Abstract 106: Transcriptionnal profiling of upper-tumor urothelial carcinomas
Authors: Malouf Gabriel , Mouawad Roger , Su Xiaoping , Yao Hui , Comperat Eva , Roupret Morgan , Spano Jean-Philippe , Khayat David
Published: July 2016

Background: Upper-tumor urothelial carcinomas (UTUC) represent 5% among all urothelial tumors. The most frequent histological variant is transitional cell carcinomas (TCC). UTUC might arise within renal pelvis or ureters. Several studies suggested aggressive behavior of tumors arising within the ureter as compared to those which develop within renal pelvis. However, whether there is a biological basis of this difference remains unknown.

Methods: We performed RNAsequencing on fourteen UTUC including 6 cases arising in the ureter and 8 in renal pelvis. Library prep followed by next-generation sequencing were done using Illumina Hiseq platform. Unsupervised clustering using differentially expressed genes was used to identify cancer subtypes. Identified subtypes were correlated with clinico-pathological features. Pathway analysis was performed using Gene Set Enrichment Analysis (GSEA).

Results: Unsupervised clustering of gene expression revealed two clusters. Surprisingly, all tumors (n = 5/5) encompassing C1 were located in the ureters as compared to only one (n = 1/9) in C2 cluster (p = 0.003). No difference was observed between C1 and C2 clusters according to age, gender, grade, muscle-invasion status and stage. Using stringer cut-off (fold change ≥ or ≤-2 and FDR<0.05), 34 genes were found to be differentially expressed between the 2 clusters. GSEA revealed negative enrichment of several pathways related to B and T-cell activation in C1 relative to C2 cluster. This was the case for NAIVE_TCELL_VS_DC_UP (p = 0.01; FDR = 0.16) and NAIVE_BCELL_VS_NEUTROPHIL_UP (p = 0.002; FDR = 0.12). Among the most overexpressed genes in C1 cluster, we identified the serine/threonine-protein kinase gene SGK2 (Fold change = 17; FDR = 0.004) as a putative therapeutic target. Of note, AIM2 which is part of the inflammasome with a key role in tumorigenic reversion was one of the most down-regulated genes (fold change = -24; FDR = 0.02) in C1 cluster.

Conclusion: Our report suggests that transcriptional profiling of UTUC arising in the ureters and renal pelvis might be distinct. The observed difference might be related to immune response. Validation of these data in a larger independent cohort with analysis of the immune infiltrate is required.

Citation Format: Gabriel Malouf, Roger Mouawad, Xiaoping Su, Hui Yao, Eva Comperat, Morgan Roupret, Jean-Philippe Spano, David Khayat. Transcriptionnal profiling of upper-tumor urothelial carcinomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 106.
Abstract 1067: A paradoxical observation for its upregulation and the prognostic significance about miR-7 in esophageal squamous cell carcinoma
Authors: Masayuki Kano , Matsumoto Yasunori , Hoshino Isamu , Murakami Kentaro , Akutsu Yasunori , Matsubara Hisahiro
Published: July 2016

Esophageal squamous cell carcinoma (ESCC) is one of the most malignant cancers of the gastrointestinal tract. However, molecular indicators of the origin of cellular deregulation in ESCC have not been identified. MicroRNAs (miRNAs), noncoding RNAs 21-25 nucleotides in length, regulate gene expression primarily at the posttranscriptional level. Growing evidence suggests that miRNAs are aberrantly expressed in many human cancers, and that they play significant roles in carcinogenesis and cancer progression. A search for miRNAs with a tumor-suppressive function in ESCC was performed using the miRNA expression signatures. miRNA signatures from ESCC identified miRNAs that are downregulated in common, and these miRNAs are candidate tumor suppressors. Gain-of-function analysis revealed that 3 transfectants (miR-145, miR-133a and miR-133b) inhibit cell proliferation and cell invasion. These miRNAs, which have conserved sequences in the 3’UTR of FSCN1, inhibited FSCN1 expression. The signal from a luciferase reporter assay was significantly decreased at 2 miR-145 target sites and 1 miR-133a/b site, suggesting both miRNAs directly regulate FSCN1. An FSCN1 loss-of-function assay found significant cell growth and invasion inhibition, implying an FSCN1 is associated with ESCC carcinogenesis. The identification of tumor-suppressive miRNAs, miR-145, miR-133a and miR-133b, directly control oncogenic FSCN1 gene. These signal pathways of ESCC could provide new insights into potential mechanisms of ESCC carcinogenesis.

On the other hands, miRNAs upregulated in cancer is the candidates of tumor marker. The miRNA expression signatures we perform for ESCC revealed seven upregulated miRNAs in ESCC. In the upregulated miRNAs of miRNA expression signatures, miR-7 downregulation is the independent poor prognostic factor for ESCC patients paradoxically.miR-7 expression is reported upregulated or downregulated variously in the same carcinoma (eg. Gastric adenocarcinoma). The correlation between patients’ prognosis and miR-7 expression has not yet reported in Pubmed research in Dec. 2015. And the target genes of miR-7 is not clarified.

Citation Format: Kano Masayuki, Yasunori Matsumoto, Isamu Hoshino, Kentaro Murakami, Yasunori Akutsu, Hisahiro Matsubara. A paradoxical observation for its upregulation and the prognostic significance about miR-7 in esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1067.
Abstract 1086: Elevated level of circulating microRNA-99a correlates with serous epithelial ovarian cancer and can be used as a potential biomarker
Authors: Yoshimura Akihiko
Published: July 2016

[Objective] There is a critical need for improved diagnostic markers for ovarian high grade serous cancer (HGSC). Emerging evidence have shown that microRNAs (miRNAs) stably exist in circulating blood, reflecting tissue or organ conditions and these are present in circulating microvesicles such as exosomes. The purpose of this study was to identify which miRNAs are highly produced from HGSCs and to analyze whether circulating miRNA can discriminate patients with HGSC from healthy volunteers.

[Methods] Secreted exosomes from serous ovarian cancer cell lines were collected and exosomal miRNAs were extracted. miRNA microarray was performed and several elevated miRNAs specific to ovarian cancer cells were picked-up. Among these, we focused on miR-99a in this study. Sera were collected from 9 consecutive patients with HGSC and 5 healthy volunteers. Expression level of miR-99a was determined by miRNA RT-qPCR.

[Results] miRNA microarray revealed several miRNAs were highly expressed in exosomes in patients with HGSC. In patients, serum miR-99a levels were significantly increased (7.2 fold) compared with healthy controls. Receiver operating curve (ROC) analysis for miR-99a showed that at the cut-off of 2.1 times compared with control, the sensitivity and specificity of this marker were 77.8% and 100%, respectively for detecting HGSC (AUC = 0.84). Serum miR-99a levels decreased after surgery by 0.2 fold, indicating that serum miR-99a was originated from ovarian cancer.

[Conclusion] Elevated serum miR-99a were detected in sera of patients with HGSC and reflected the disease condition. This miRNA profiling in serum can be used as a diagnostic biomarker, extending its utility to screening asymptomatic populations.

Citation Format: Akihiko Yoshimura. Elevated level of circulating microRNA-99a correlates with serous epithelial ovarian cancer and can be used as a potential biomarker. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1086.
Abstract 1104: MicroRNA miR-31 regulates oral squamous cell carcinoma cell migration by targeting critical enzyme of peroxisomal lipid metabolism
Authors: Lai Yi-Shiuan , Liu Hsuan , Chen Ting-Wen , Chen Shu-Jen , Chen Hua-Chien , Tan Bertrand Chin-Ming
Published: July 2016

MicroRNAs (miRNAs) are small endogenous non-coding RNAs that play important roles in a variety of cellular processes, such as growth, differentiation and metabolic homeostasis. Dysregulation of microRNA has been linked to the development of various types of human diseases, including cancer. Our previous study has identified that miR-31 is significantly up-regulated in oral squamous cell carcinoma (OSCC) tissues. Further gain- and loss-of-function analyses revealed that miR-31 promotes OSCC by enhancing the migration and invasiveness of OSCC cells but not cell proliferation. However, the exact role of miR-31 in OSCC neoplastic development, especially metastasis, has not been explored. Computational microRNA target prediction and pathway analysis showed an enrichment of putative targets in lipid metabolism pathway. In this study, we aimed to investigate the role of miR-31 in the regulation of lipid metabolism and to establish the relationship between lipid metabolism and tumor pathogenesis in OSCC cells.

First, we demonstrated that ACOX1, the rate-limiting enzyme in peroxisomal β-oxidation, is the direct target of miR-31. We further analyzed publicly available mRNA and miRNA deep sequencing datasets and found that the expression levels of miR-31 inversely correlate with those of ACOX1. Furthermore, we discovered that depletion of miR-31 significantly decreased the intensity of cellular lipid droplets (LDs), and knockdown of ACOX1 conversely showed an increase of LD formation. These data suggested that miR-31 may regulate lipid metabolism by targeting ACOX1. In cultured OSCC cells, the migration capability and invasiveness were significantly increased after knockdown of ACOX1. We further confirmed that ACOX1 depletion enhances ERK1/2 expression and phosphorylation and increases the expression levels of MMP9 in OSCC cells. Collectively, our findings suggest that the elevated expression of miR-31 in OSCC cells directly contributes to the down-regulated expression of ACOX1, subsequently leading to accumulation of lipid metabolites and elevated OSCC migration and invasion through ERK pathway.

Citation Format: Yi-Shiuan Lai, Hsuan Liu, Ting-Wen Chen, Shu-Jen Chen, Hua-Chien Chen, Bertrand Chin-Ming Tan. MicroRNA miR-31 regulates oral squamous cell carcinoma cell migration by targeting critical enzyme of peroxisomal lipid metabolism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1104.
Abstract 1114: Downregulated miR-337-5p is associated with unfavorable features in acute myeloid leukemia and may play a role in modulating the extracellular signal-regulated kinase pathway
Authors: Lie Rosalind , Cheng Chi Keung , Chan Natalie Pui Ha , Ip Rosalina , Chan Nelson , Cheung Joyce , Ng Margaret
Published: July 2016

We previously revealed that a homozygous deletion T polymorphism in the 3’-untranslated region of the Nucleophosmin (NPM1) gene is associated with adverse outcomes and can independently predict survivals in patients with de novo acute myeloid leukemia (AML). This polymorphism creates an illegitimate binding site for miR-337-5p, which is expressed in different AML subtypes. We speculate that the delT-carrying NPM1 mRNA competes with as yet unidentified oncogenes for miR-337-5p binding, thereby activating their functions in trans and perturbing normal microRNA regulation. In this study, we aim to investigate the clinicopathological and biological significance of miR-337-5p expression in AML. Using real-time RT-PCR, we measured miR-337-5p expression in bone marrow of 156 adult patients with de novo AML excluding acute promyelocytic leukemia. Patients were dichotomized into low and high expression group at the median level for clinicopathological correlations. miR-337-5p levels were significantly reduced in the low expression patient group than the normal controls (n = 27) (P<0.001). Low miR-337-5p expression correlated with higher white blood cell counts (P = 0.024), lactate dehydrogenase levels (P = 0.023), bone marrow blast counts (P = 0.001) and FLT3-ITD (P = 0.0092) mutation. Using TargetScan, DIANA, PITA and miRDB, three putative oncogenes involved in the extracellular signal-regulated kinase (ERK) pathway, namely RAP2B, MAPK1 and WNK1, were consistently indicated as the potential targets of miR-337-5p. Overexpression of miR-337-5p in OCI-AML3 cells, which have barely detectable miR-337-5p levels, downregulated the expression of all targets. Concordantly, luciferase reporter assay confirmed that miR-337-5p bound to the 3’-untranslated region of these targets and inhibited translation. The ERK signaling pathway is frequently activated in AML and confers pro-survival functions. We demonstrated that overexpression of miR-337-5p in OCI-AML3 cells significantly inhibited proliferation in both WST-1 and colony-forming assay. Together, our findings suggested that reduced miR-337-5p is associated with unfavorable clinicopathological and molecular features in AML. Restoration of miR-337-5p suppressed cell proliferation, potentially by downregulation of genes implicated in the mitogenic ERK signaling pathway.

Citation Format: Rosalind Lie, Chi Keung Cheng, Natalie Pui Ha Chan, Rosalina Ip, Nelson Chan, Joyce Cheung, Margaret Ng. Downregulated miR-337-5p is associated with unfavorable features in acute myeloid leukemia and may play a role in modulating the extracellular signal-regulated kinase pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1114.
Abstract 1118: The role of Epstein-Barr virus-encoded miRNAs in ATM regulating DNA damage response in nasopharyngeal carcinoma
Authors: LUNG RAYMOND Wai-Ming , Hau Tom Pok-Man , Chak Wing-Po , Tong Joanna Hung-Man , Yu Ken Hung-On , Tsao Sai-Wah , Yip Kevin Yuk-Lap , To Ka-Fai , Lo Kwok-Wai
Published: July 2016

Nasopharyngeal carcinoma (NPC) is an aggressive epithelial malignancy that has remarkably high incidence and mortality rates in Hong Kong and south China. It is well-known to be associated with Epstein-Barr virus (EBV) infection. Previous work in our team revealed that EBV-encoded miRNAs derived from the BARTs (miR-BARTs) were abundantly expressed in primary NPCs. They function in controlling apoptotic and immune responses in the infected cells during NPC development.

Most recently, we attempted to use in silico method to predict cellular targets of miR-BARTs. Results suggested that there were multiple putative miR-BART binding sites on the 3’UTRs of an important DNA double-strand breaks (DSBs) repair gene, Ataxia-Telangiectasia-Mutant (ATM). The down-regulation of ATM mRNA and protein had also been demonstrated in our local primary NPC samples. Although the interaction of miR-BARTs on each suggested putative binding site had not been completely validated, our preliminary data indicated at least three EBV-encoded miRNAs (BART5-5p, BART9-3p and BART14-3p) were involved in repressing ATM expression in NPCs. They could work either alone or cooperatively to control ATM expression in transient assays. Notably, ectopic expression of these three miR-BARTs could successfully suppress γ irradiation-induced ATM activity in two EBV-negative cell lines, NP69 and HeLa.

It had been reported that ATM null cells were defective to repair the DSB lesions and sensitive to the Poly(ADP-ribose) polymerase (PARP) inhibitor treatment in the presence of DNA-damaging agents. In contrast, the DSBs are effectively repaired in normal cells to retain genetic integrity. We believed that EBV infection, via miR-BARTs to reduce ATM activity and disrupt Homologous Recombination (HR) repair function, made the NPC cells sensitize to ionizing radiation and DNA-damaging agent treatment. Hence, the knowledge generated from this project is not only enhance our understanding of EBV biology, but also opens an avenue for the development of effective NPC therapies.

Citation Format: RAYMOND Wai-Ming LUNG, Tom Pok-Man Hau, Wing-Po Chak, Joanna Hung-Man Tong, Ken Hung-On Yu, Sai-Wah Tsao, Kevin Yuk-Lap Yip, Ka-Fai To, Kwok-Wai Lo. The role of Epstein-Barr virus-encoded miRNAs in ATM regulating DNA damage response in nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1118.
Abstract 1129: EGFR-mediated activation of RET in ASCL1+ lung adenocarcinoma
Authors: Bhinge Kaustubh N. , Lin Yang , Rahi Hamed , Aubry Marie Christine , Mansfield Aaron , Kovtun Irina , Murphy Stephen , Yang Ping , Wigle Dennis , Yi Joanne (Eunhee) , Nasir Aqsa , Terra Simone , Molina Julian , Vasmatzis George , Kosari Farhad
Published: July 2016

Lung cancer accounts for about 27% of the cancer related deaths in the USA annually. Pathologically, it is a very complex disease broadly classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLCs are further classified into squamous cell carcinoma, large cell carcinoma and adenocarcinoma. Achaete-scute homolog 1 (ASCL1), an important transcription factor essential in the development of neuroendocrine cells (NE) in lungs is shown to be specifically expressed in lung NE cancers and 10-20% of adenocarcinomas (AD) with NE differentiation (NED), thus suggesting a role in the pathogenesis of these tumors. Our previous study showed that ASCL1 is a regulator of the RET oncogene in AD with NED. RET is a receptor tyrosine kinase with two isoforms in humans: RET9 (short) and RET51 (long). We performed survival analysis to study implications of RET isoforms in ASCL1+ tumors and found that elevated expression of the long RET mRNA was associated with poor survival. Subsequent in vitro experiments demonstrated that treatment with EGF robustly induced phosphorylation of RET in HCC1833 and H1755 cell lines which have high endogenous levels of ASCL1 and RET but not in VMRC-LCD cell line which has high level of ASCL1 but low level of RET. EGF induced phosphorylation of RET was diminished by gefitinib and by EGFR siRNA. Immunoprecipitation results indicated direct binding between EGFR and RET in presence of EGF. Furthermore, a high throughput drug screening found 8 EGFR inhibitors that were 10 - 250 fold more cytotoxic in ASCL1+ compared with ASCL1- AD cells. These results implicate EGFR as a key regulator of RET activation in ASCL1+ AD and suggest that EGFR inhibitors may be therapeutic for this population of patients.

Citation Format: Kaustubh N. Bhinge, Yang Lin, Hamed Rahi, Marie Christine Aubry, Aaron Mansfield, Irina Kovtun, Stephen Murphy, Ping Yang, Dennis Wigle, Joanne (Eunhee) Yi, Aqsa Nasir, Simone Terra, Julian Molina, George Vasmatzis, Farhad Kosari. EGFR-mediated activation of RET in ASCL1+ lung adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1129.
Abstract 1135: FYN expression potentiates FLT3-ITD-induced mitogenic signaling in acute myeloid leukemia
Authors: Chougule Rohit A. , Kazi Julhash U. , Rönnstrand Lars
Published: July 2016

FYN is a non-receptor tyrosine kinase belonging to the SRC family of kinases. SRC family kinases are frequently over-expressed in human cancers, and play key roles in cancer cell invasion, metastasis, proliferation, survival and many other biological processes. SRC has long been recognized as an important oncogene; but little attention has been given to its other family members such as FYN. In this report, we have studied the role of FYN in FLT3 signaling with respect to acute myeloid leukemia (AML). FLT3 is a type III receptor tyrosine kinase which is found to be mutated in around 30% of AML cases. We observed that FYN displays a strong association with wild-type FLT3 as well as oncogenic FLT3-ITD. While association with wild-type FLT3 is dependent on ligand stimulation, it is constitutively associated with FLT3-ITD. In addition, a kinase-dead FLT3 mutant was unable to associate with FYN suggesting that FLT3 activation is required for association with FYN. A FYN SH2-domain mutant lacking a critical arginine residue was unable to associate with FLT3 indicating that FYN associates with FLT3 through its SH2 domain. A phopho-peptide fishing experiment identified multiple FYN binding sites in FLT3, which partially overlap with the known SRC binding sites but also differ from these sites. To understand the role of FYN in FLT3 signaling, we have generated Ba/F3-FLT3/FYN or Ba/F3-FLT3/empty vector cell lines. We observed that expression of FYN results in enhanced phosphorylation of AKT, ERK1/2 and p38 phosphorylation in response to ligand stimulation. Ba/F3-FLT3-ITD cells overexpressing FYN also showed higher STAT5 activation. Furthermore, FYN expression led to a significant increase in FLT3-ITD-dependent cell proliferation but did not alter apoptosis induced by cytokine starvation. Finally, we showed that FYN expression was deregulated in AML patient samples and that higher expression of FYN in combination with FLT3-ITD expression correlates with poor prognosis in AML. Taken together our data suggest that FYN cooperates with oncogenic FLT3-ITD which results in poor prognosis in AML and therefore inhibition of FYN in combination with FLT3 inhibition will most likely be beneficial for AML patients.

Citation Format: Rohit A. Chougule, Julhash U. Kazi, Lars Rönnstrand. FYN expression potentiates FLT3-ITD-induced mitogenic signaling in acute myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1135.
Abstract 1155: Inhibitory effect of CYP1B1 on antitumor activities induced by dysregulated CDC20 and DAPK1 in renal cell carcinoma
Authors: Mitsui Yozo , Chang Inik , Fukuhar Shinichiro , Hirata Hiroshi , Wong Ryan Kenji , Yamamura Soichiro , Shahryari Varahram , Deng Guoren , Sharanjot Saini , Majid Shahana , Shiina Hiroaki , Dahiya Rajvir , Tanaka Yuichiro
Published: July 2016

Introduction and Objective: Cytochrome P450 1B1 (CYP1B1) has been shown to be up-regulated in many types of cancer including renal cell carcinoma (RCC). Several reports have shown that CYP1B1 can influence the regulation of tumor development; however, its role in RCC has not been well investigated. The aim of the present study was to determine the functional effects of CYP1B1 gene on tumorigenesis in RCC. Methods: Expression of CYP1B1 was determined in RCC cell lines, and tissue microarrays of 96 RCC and 25 normal tissues. To determine the biological significance of CYP1B1 in RCC progression, we silenced the gene in Caki-1 and 769-P cells by RNA interference and performed various functional analyses.

Results: First, we confirmed that CYP1B1 protein expression was significantly higher in RCC cell lines compared to normal kidney tissue. This trend was also observed in RCC samples (p<0.01). Interestingly, CYP1B1 expression was associated with tumor grade and stage. Next, we silenced the gene in Caki-1 and 769-P cells by RNA interference and performed various functional analyses to determine the biological significance of CYP1B1 in RCC progression. Inhibition of CYP1B1 expression resulted in decreased cell proliferation, migration and invasion of RCC cells. In addition, reduction of CYP1B1 induced cellular apoptosis in Caki-1. We also found that these anti-tumor effects on RCC cells caused by CYP1B1 depletion may be due to alteration of CDC20 and DAPK1 expression based on gene microarray and confirmed by real-time PCR. Interestingly, CYP1B1 expression was associated with CDC20 and DAPK1 expression in clinical samples.

Conclusions: CYP1B1 may promote RCC development by inducing CDC20 expression and inhibiting apoptosis through the down-regulation of DAPK1. Our results demonstrate that CYP1B1 can be a potential tumor biomarker and a target for anticancer therapy in RCC.

Citation Format: Yozo Mitsui, Inik Chang, Shinichiro Fukuhar, Hiroshi Hirata, Ryan Kenji Wong, Soichiro Yamamura, Varahram Shahryari, Guoren Deng, Saini Sharanjot, Shahana Majid, Hiroaki Shiina, Rajvir Dahiya, Yuichiro Tanaka. Inhibitory effect of CYP1B1 on antitumor activities induced by dysregulated CDC20 and DAPK1 in renal cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1155.
Abstract 1158: Differential expression and functional impact of the alternatively spliced transcripts of Extracellular matrix protein 1 in esophageal squamous cell carcinoma
Authors: YU Valen Zhuoyou , Ko Josephine Mun-Yee , Law Simon , Wang Li Dong , Lung Maria Li
Published: July 2016

Background: Esophageal squamous cell carcinoma (ESCC) has an especially high incidence in China, with five-year survival rates in Hong Kong being of ∼14%. An oligonucleotide microarray was utilized to identify differentially-expressed genes in ESCC using 31 pairs of matched normal/tumor samples from Hong Kong and Henan, China. Extracellular matrix protein 1 (ECM1) was among the top down-regulated genes, indicating a potential tumor-suppressive role. Public cDNA microarray data also shows significant down-regulation of ECM1 expression in ESCC. Interestingly, this gene has been extensively studied in breast cancer and thyroid cancer for its oncogenic role. There are three alternatively-spliced variants of ECM1. Therefore, this gene was selected for further variant expression and functional analysis in ESCC.

Results: RNA sequencing analysis of matched normal/ESCC samples showed that ECM1b (NM_022664) is the dominant expressed variant in esophagus, followed by ECM1a (NM_004425). In tumor samples, the expression of ECM1a is down-regulated and that of ECM1b is down-regulated or lost, as compared to normal counterparts. Expression of ECM1c (NM_001202858) is not detected in either normal or tumor samples. Quantitative PCR using variant-specific primer sets confirms the findings of microarray and RNA sequencing analyses. When re-expressed in ESCC cell lines after lentiviral transduction, ECM1b expression shows a trend of tumor suppression in the orthotopic ESCC mouse tumorigenicity model; ECM1a shows no tumor-suppressive effect in either. ECM1b expression also shows a metastasis inhibitory effect in a tail vein experimental metastasis model. Immunohistochemical staining shows E-cadherin up-regulation in orthotopic tumors re-expressing ECM1b.

Conclusions: These results suggest differential roles of variants of ECM1 in ESCC. Unlike the cases in other types of cancer, both expression of ECM1a and ECM1b is down-regulated in ESCC compared to normal esophageal tissues. ECM1b represents the dominantly expressed variants in esophagus epithelium, and its expression is highly reduced in esophageal carcinoma. ECM1b plays a potential suppressive role in tumorigenesis and metastasis.

Acknowledgement: We acknowledge the Research Grants Council of Hong Kong Special Administrative Region for funding support (HKU 774413M to M.L.L).

Citation Format: Valen Zhuoyou YU, Josephine Mun-Yee Ko, Simon Law, Li Dong Wang, Maria Li Lung. Differential expression and functional impact of the alternatively spliced transcripts of Extracellular matrix protein 1 in esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1158.
Abstract 116: Study of TERT promoter and FGFR3 mutations in upper-tumor urothelial carcinomas
Authors: Mouawad Roger , Bensalma Souheyla , Su Xiaoping , Allanic Frederick , Comperat Eva , Rouprêt Morgan , Spano JeanPhillipe , Malouf Gabriel , Khayat David
Published: July 2016

Background: Mutations in the promoter of the telomerase reverse transcriptase (TERT) and fibroblast growth factor receptor 3 (FGFR3) genes represent the most recurrent somatic alterations in urothelial carcinomas of the bladder. However, data regarding frequency and prognostic values of these alterations in upper-tumor urothelial carcinoma (UTUC) remain limited.

Methods: DNA was extracted from 31 UTUC including 12 cases arising in the ureter and 19 in renal pelvis. Somatic mutations of TERT promoter and FGFR3 gene were assessed by Sanger sequencing. The generated sequences were analyzed by seqscape and mutational status of each of those genes were correlated with clinico-pathological tumor features. Disease-free survival and overall survival were estimated using the Kaplan-Meier method.

Results: Median patient's age was 72 years (range: 41-83) with a male predominance (90.3%). Mutation status of TERT and FGFR3 were obtained in all tumors. Out of 31 UTUC, 81% were classified as high-grade including 56% which were muscle-invasive (≥pT2). TERT and FGFR3 mutations were detected in 19 (61%) and 14 (46%) tumors, respectively. Of note, 8 (26%) out of them harbored both mutations. In the TERT-mutated subgroup, we observed higher incidence of men as compared to TERT non-mutated subgroup (p = 0.04); in addition, we did not identify any other significant differences regarding other clinico-pathological features. This was also the case for FGFR3 mutations, although we observed a higher frequency in tumors arising in the ureter (66.6%) as compared to those located in renal pelvis (28.5%) (p = 0.11). Neither mutations of FGFR3 nor TERT were associated with progression-free survival and patient's overall survival.

Conclusion: Our study identified a high rate of somatic mutations in FGFR3 and TERT which were similar between muscle-invasive and non-muscle invasive tumors as well as between tumors arising in the ureter or renal pelvis. As targeted inhibitors are being investigated in this setting, these results might have implications for patient's management.

Citation Format: Roger Mouawad, Souheyla Bensalma, Xiaoping Su, Frederick Allanic, Eva Comperat, Morgan Rouprêt, JeanPhillipe Spano, Gabriel Malouf, David Khayat. Study of TERT promoter and FGFR3 mutations in upper-tumor urothelial carcinomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 116.
Abstract 1169: Somatic MED12 mutations in hematological malignancies
Authors: Heikkinen Tuomas , Kämpjärvi Kati , Leppä Sirpa , Hokland Peter , Kuusanmäki Heikki , Mustjoki Satu , Sinisalo Marjatta , Heckman Caroline , Kontro Mika , Vahteristo Pia
Published: July 2016

Somatic mutations in exons 1 and 2 of the Mediator complex subunit 12 (MED12) have been identified in 70% of uterine leiomyomas, 7-20% of uterine leiomyosarcomas, 59% of breast fibroadenomas and 67% of breast phyllodes tumors. In addition to female hormone-dependent tumors we have recently found the same specific missense and small in-frame insertion and deletion mutations in approximately 5% of chronic lymphocytic leukemias (CLL). In CLL the presence of MED12 mutations was also associated with markers of poor prognosis (IgVH (-), Zap70 (+), and Zap70-methylation). The surprising finding of the same mutations in a completely different tumor type prompted us to further screen other hematological malignancies for MED12 mutations.

We have thus far collected samples of 107 T-cell acute lymphoblastic leukemias (T-ALL), 21 large granular lymphocyte leukemias (LGL), 33 acute myeloid leukemias (AML), 154 diffuse large B cell lymphomas (DLBCL), and 6 six follicular lymphomas (FL). Also a set of 30 additional CLLs was collected. The MED12 mutation status was determined by direct Sanger sequencing of exons 1 and 2 of the gene. A c.107T>G, p.L36R mutation was found in a single DLBCL case and c.100-8T>A, p.E33_D34insPQ in one AML sample. A novel variant of uncertain significance, c.-3A>G, was detected on 5’UTR of one FL sample. One T-ALL patient harbored a nonsense mutation affecting the last codon of exon 1 c.97G>T, p.E33X. No mutations were identified in the LGL or CLL samples. We are also analyzing approximately 100 new AML samples and 100 multiple myeloma samples.

MED12 mutations are present, in addition to CLL, also in other hematological malignancies. The frequency, however, seems to be low and, as the number of samples in the preliminary analysis is limited, screening of larger sample sets is needed. The c.107T>G, p.L36R and c.100-8T>A, p.E33_D34insPQ mutations have previously been detected recurrently in uterine leiomyomas and in CLL. Further studies are required for evaluation of the effects of c.-3A>G and c.97G>T, p.E33X mutations. The latter mutation affects the last codon of exon 1, which is also a mutational hotspot in CLL, with recurrent missense mutations. MED12 exon 1 and 2 missense mutations disrupt the interactions between the Mediator complex and Cyclin C and cause loss of CDK8 kinase activity. Studies to determine the impact of the c.97G>T, p.E33X mutation on MED12 function are ongoing.

Citation Format: Tuomas Heikkinen, Kati Kämpjärvi, Sirpa Leppä, Peter Hokland, Heikki Kuusanmäki, Satu Mustjoki, Marjatta Sinisalo, Caroline Heckman, Mika Kontro, Pia Vahteristo. Somatic MED12 mutations in hematological malignancies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1169.
Abstract 118: A comprehensive profile of the genomic architecture of curable prostate cancer
Authors: Fraser Michael E. , van der Kwast Theodorus , McPherson John , Collins Colin C. , Fradet Yves , Tetu Bernard , Bergeron Alain , Bristow Rob G. , Boutros Paul C.
Published: July 2016

There is an urgent need to develop novel biomarkers of treatment response for precision medicine in prostate cancer, particularly in the setting of localized, non-indolent disease, which represents the vast majority of cases at initial clinical presentation. The Canadian Prostate Cancer Genome Network (CPC-GENE) - a member of the International Cancer Genome Consortium - aims to identify multi-modal prognostic and predictive signatures of therapeutic outcome based on intrinsic tumour genomics, transcriptomics, and epigenomics, which are being incorporated into novel clinical trials of treatment escalation/de-escalation for image-guided radiotherapy and/or radical prostatectomy. Herein we report the results of the largest prostate cancer whole-genome sequencing study to date, consisting of 194 patients and whole-exome sequencing of 479 patients with localized, potentially curable disease; a sample size that allows for saturating discovery of genes mutated at ≥1% frequency. We show that tumours treated by curative local therapy have a paucity of clinically-actionable mutations relative to high-risk localized or metastatic disease, but instead harbor a significant number of recurrent non-coding aberrations and genomic rearrangements, including a novel inversion - and an associated reduction in gene expression - of the PTEN tumour suppressor gene on chromosome 10. The median tumour contains three distinct driver mutations, with >86% of tumours possessing at least one known driver. Importantly, multiple driver aberrations are associated with patient outcome, including copy number aberrations and methylation events, but notably excluding well-described recurrent events such as TMPRSS2:ERG fusions and SPOP point mutations. Localized hypermutation events (e.g. kataegis) were detected in >20% of tumours, and were strongly enriched for aggressive disease. Taken together, these data provide a comprehensive analysis of the genomic landscape of localized, non-indolent prostate cancer. Moreover, our results strongly suggest that specific molecular aberrations may serve as useful biomarkers for improved pre-treatment stratification of patients with localized, low/intermediate risk disease into escalation/de-escalation protocols, and support the development of clinical trials to assess this hypothesis, based on patient-specific molecular profiles.

Citation Format: Michael E. Fraser, Theodorus van der Kwast, John McPherson, Colin C. Collins, Yves Fradet, Bernard Tetu, Alain Bergeron, Rob G. Bristow, Paul C. Boutros. A comprehensive profile of the genomic architecture of curable prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 118.
Abstract 1186: Inhibition of topoisomerase 2β sensitizes acute myeloid leukemia cells to ATRA induced apoptosis independent of the MAPK/ERK pathway
Authors: Norris Eric J. , Dorszynski Amy , Lucander Aaron , Destephanis Darla , Ganapathi Ram , Ganapathi Mahrukh
Published: July 2016

Pharmacologic inhibition or molecular down-regulation of topoisomerase 2β (TOP2β) potentiates all trans retinoic acid (ATRA)-induced differentiation and apoptosis in acute myeloid leukemia (AML) cells. Since ATRA-induced myeloid differentiation involves activation of the MAPK/ERK signaling pathway and aberrant activation of MAPK/ERK is frequently observed in AML we tested the role of this pathway in modulating the cellular effects of ATRA when TOP2β is inhibited with the bisdioxopiperazines, ICRF 187 (dexrazoxane, Zinecard™) or ICRF193. Treatment of HL-60 AML (M2) cells with ATRA increased the percentage of differentiated cells, as assessed by the nitroblue tetrazolium reduction assay, and enhanced levels of phosphorylated ERK. Paradoxically, while combination treatment with ATRA/ICRF187 enhanced differentiation (P<0.05), ERK phosphorylation was attenuated compared to treatment with ATRA alone. Moreover, pretreatment with the MEK inhibitor PD98059, which completely blocks ATRA-induced differentiation and ERK phosphorylation, only partially blocked (46% reduction) differentiation induced by ATRA/ICRF187 (P<0.01) and had no effect on ATRA/ICRF187-induced apoptosis. In agreement with this observation, combination treatment with ATRA/ICRF187 induced apoptosis (P<0.05) in KG-1 (M1) AML cells and primary AML cells from patients, that do not phosphorylate ERK or differentiate in response to ATRA treatment. In summary, our data suggest that inhibition of TOP2β sensitizes AML cells to ATRA induced differentiation and apoptosis primarily via an ERK-independent mechanism. Further, combination treatment ATRA and ICRF187 may be clinically relevant to overcome ATRA resistance in AML patients with a deregulated MAPK/ERK signaling pathway.

Citation Format: Eric J. Norris, Amy Dorszynski, Aaron Lucander, Darla Destephanis, Ram Ganapathi, Mahrukh Ganapathi. Inhibition of topoisomerase 2β sensitizes acute myeloid leukemia cells to ATRA induced apoptosis independent of the MAPK/ERK pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1186.
Abstract 1209: Istiratumab (MM-141), a bispecific antibody targeting IGF-1R and ErbB3, inhibits pro-survival signaling in vitro and potentiates the activity of standard of care chemotherapy in vivo in ovarian cancer models
Authors: Curley Michael D. , Tan Gege , Yannatos Isabel , Camblin Adam , Iadevaia Sergio , Louis Chrystal , Lugovskoy Alexey
Published: July 2016

Insulin-like growth factor receptor 1 (IGF-1R) signaling has been implicated in the pathogenesis of ovarian cancer. However, clinical trials evaluating monospecific IGF-1R inhibitors have demonstrated limited clinical efficacy. Our data indicate that ErbB3, a member of the ErbB receptor tyrosine kinase family, can activate pro-survival AKT signaling in response to IGF-1R blockade and may represent a potential escape route in the development of resistance to therapy. Istiratumab (MM-141), an IGF-1R and ErbB3 directed bispecific antibody, inhibits ligand activation of these signaling pathways and degrades IGF-1R and ErbB3 receptor-containing complexes, leading to inhibition of downstream pro-survival signaling. Here we tested the activity of istiratumab, alone and in combination with chemotherapy, in in vitro and in vivo models of ovarian cancer.

Anti-proliferative activity of istiratumab monotherapy was evaluated in a panel of ovarian cancer cell lines in vitro. The effects of istiratumab and the ligands IGF-1 and heregulin on IGF-1R- and ErbB3-mediated survival signaling were tested by ELISA and immunoblotting. Co-treatment assays with istiratumab and chemotherapy investigated mechanisms of synergy and additivity. Anti-tumor activity of istiratumab, alone and in combination with chemotherapy, was tested in in vivo ovarian xenograft tumor models.

Our results indicated that istiratumab monotherapy inhibits ovarian cancer cell line proliferation in vitro. In addition, istiratumab blocked ligand-mediated resistance to chemotherapy. Co-treatment of istiratumab, ligands or chemotherapy indicated a strong correlation between drug activity and IGF-1R expression. Furthermore, co-treatment of chemotherapies and ligands potentiated AKT activation, which was inhibited by istiratumab. In vivo studies showed that istiratumab potentiates the activity of chemotherapy in ovarian xenograft tumor models.

Our findings demonstrate that co-inhibition of IGF-1R and ErbB3 signaling with istiratumab can potentiate standard of care chemotherapies in ovarian tumor models and warrant further investigation of istiratumab as a potential therapy for ovarian cancer patients.

Citation Format: Michael D. Curley, Gege Tan, Isabel Yannatos, Adam Camblin, Sergio Iadevaia, Chrystal Louis, Alexey Lugovskoy. Istiratumab (MM-141), a bispecific antibody targeting IGF-1R and ErbB3, inhibits pro-survival signaling in vitro and potentiates the activity of standard of care chemotherapy in vivo in ovarian cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1209.