bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3904: Rspondin-1 contributes to tumor cell proliferation via Wnt signaling pathway
Authors: Park So Hyun , Kim Sol , Choi Min Yeong , Park Jiwoon , Kang Shinae , Kim Hak-Zoo
Published: August 2015

Diabetes mellitus and pancreatic cancer have long been considered to be clinically related to each other. Despite the ample epidemiological evidences indicating relevance between two major human diseases, the precise mechanism to link both of these diseases are still unclear. We demonstrated altered beta-catenin and Wnt3a protein expression in pancreas of diabetic mouse in age-dependent manner, which indicates the chronic diabetic condition may contribute to the pancreatic cancer progression of peri-islet region through elevated Wnt signaling. We also identified the altered expression level of Rspondin-1, secreted agonist of Wnt signaling pathway, in in vivo diabetic mouse model, which showed a similar expression pattern of Wnt3a and beta-catenin. Finally we demonstrated the effect of in vitro diabetic condition on Rspondin expression level of mouse pancreatic beta cell(Min6) and pancreatic cancer cell lines(Panc-1, AsPc-1). The expression level of Rspondin-1 was down-regulated in Min6 cells cultured in high glucose condition, whereas up-regulated in pancreatic cancer cells cultured in in vitro diabetic condition, which correlates with in vivo Rspondin-1 expression study. These results provide new insights about the Wnt signaling and related proteins as molecular linkers of chronic diabetes and pancreatic cancer.

Citation Format: So Hyun Park, Sol Kim, Min Yeong Choi, Jiwoon Park, Shinae Kang, Hak-Zoo Kim. Rspondin-1 contributes to tumor cell proliferation via Wnt signaling pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3904. doi:10.1158/1538-7445.AM2015-3904
Abstract 3907: Differential effects of 1α, 25-dihydroxyvitamin D3 dose on keratinocyte proliferation and ΔNp63α stabilization
Authors: Hill Natasha T. , Zhang Jin , Leonard Mary K. , Lee Miryoung , Kadakia Madhavi
Published: August 2015

1α, 25-dihydroxyvitamin D3 (VD3) is a secosteriod that has been explored as an anti-cancer agent, but has also been shown to promote cell survival. Its receptor, Vitamin D Receptor (VDR), is a direct target of the oncogene ΔNp63α, which is overexpressed in non-melanoma skin cancers. The interconnection between VDR/VD3 signaling and ΔNp63α, led us to examine whether VDR/VD3 signaling promotes keratinocyte proliferation by regulating ΔNp63α levels. We show that VDR regulates ΔNp63α expression at both the transcript and protein level. Interestingly, while low doses of VD3 led to an increase in ΔNp63α protein levels and keratinocyte proliferation, high doses of VD3 failed to increase ΔNp63α protein levels and resulted in reduced proliferation. Increased levels of ΔNp63α by low dose VD3 was shown to be VDR dependent and critical for the pro-proliferative effects of VD3. Furthermore, VD3 mediated increases in ΔNp63α protein levels occur via p38 activation. Analysis of squamous cell carcinoma (SCC), basal cell carcinoma (BCC) and precursors to invasive SCC demonstrated a significant correlation between p63 and VDR levels when compared to normal skin samples. In this study we demonstrated a differential dose effect of VD3 on ΔNp63α stability, which directly resulted in differential effects on cell growth. Delineation of the mechanisms by which VD3 exerts its effect on ΔNp63α will be critical for determining the future of VD3 in cancer therapies.

Citation Format: Natasha T. Hill, Jin Zhang, Mary K. Leonard, Miryoung Lee, Madhavi Kadakia. Differential effects of 1α, 25-dihydroxyvitamin D3 dose on keratinocyte proliferation and ΔNp63α stabilization. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3907. doi:10.1158/1538-7445.AM2015-3907
Abstract 3912: GDF2 promotes anoikis susceptibility in ovarian and breast epithelia
Authors: Varadaraj Archana , Patel Pratik , Serrao Anne , Bandyopadhyay Tirthankar , Lee Nam Y. , Jazaeri Amir , Murphy Susan , Mythreye Karthikeyan
Published: August 2015

Resistance to anchorage independent cell death (anoikis) is one of the features of metastasis and can be regulated by a diverse array of growth factors. The protein encoded by GDF2 is BMP9 and is a member of the Bone Morphogenetic protein family and the TGFβ superfamily with defined roles in angiogenesis and emerging yet controversial roles in carcinogenesis. We used normal and oncogenic epithelial systems to examine the role of GDF2 in ovarian and breast epithelia. We find that both normal and transformed epithelial cells are competent to activate the Smad1/5 signaling cascade in response to GDF2. This activation occurs via specific complex formation between the Type I receptor Serine threonine kinases Alk3 and Alk6 and the Type II receptor serine threonine kinase BMPRII. We find that in breast and ovarian epithelial cells GDF2 specifically regulates anchorage independent growth by increasing anoikis sensitivity that is dependent on GDF2's ability to sustain SMAD1/5 activation via Alk3 and Alk6. Analysis of cell lines and patient data indicates epigenetic regulation of GDF2 expression in ovarian and breast cancers. These findings reveal an antimetastatic role for GDF2 in ovarian and breast cancer models of disease with significant implications for the use of epigenetic drugs currently in clinical trials.

Citation Format: Archana Varadaraj, Pratik Patel, Anne Serrao, Tirthankar Bandyopadhyay, Nam Y. Lee, Amir Jazaeri, Susan Murphy, Karthikeyan Mythreye. GDF2 promotes anoikis susceptibility in ovarian and breast epithelia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3912. doi:10.1158/1538-7445.AM2015-3912
Abstract 3924: A homogenous bioluminescent system for measuring GTPase, GAP and GEF activities
Authors: Mondal Subhanjan , Hsiao Kevin , Goueli Said A.
Published: August 2015

GTPases play a major role in various cellular functions such as cell signaling, cell proliferation, cell differentiation, cytoskeleton modulation and cell motility. Deregulation or mutation of these proteins has considerable consequences resulting in multiple pathological conditions. Targeting GTPases and its regulators have been challenging due to lack of convenient assays. To overcome the challenges in analyzing activities of GTPases and their regulatory proteins GTPase Activating Proteins (GAP) and Guanine Nucleotide Exchange Factors (GEF) we have developed a homogenous bioluminescent assay (GTPase/GAP/GEF-Glo) system to analyze these proteins in a simple, convenient “add-mix-read” format. Mg2+ plays a critical role in influencing affinity of GTPases with GTP/GDP and the process of nucleotide exchange. Manipulating Mg2+ concentrations in the GTPase reaction buffer allows continuous progression of the GTPase cycle and faster hydrolysis of GTP. The assay relies on enzymatic conversion of GTP remaining after the GTPase reaction to ATP and detection of the ATP using luciferin/luciferase combination. We show that the assay is sensitive, and robust when analyzing for analyzing intrinsic GTPase- activity, GAP-stimulated GTPase- activity, GAP-activity and GEF- activity. The system can also be used to analyze these proteins expressed in cells as a fusion protein performing the assay in a pulldown -format. Furthermore, there was minimal false hits when tested for compound interference using the library of pharmacologically active compounds (LOPAC) and its robustness as indicated by high Z’-factor of 0.93 and CV of 2.2%.

Citation Format: Subhanjan Mondal, Kevin Hsiao, Said A. Goueli. A homogenous bioluminescent system for measuring GTPase, GAP and GEF activities. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3924. doi:10.1158/1538-7445.AM2015-3924
Abstract 3925: Production and biochemical characterization of farnesylated and methylated KRAS4b using engineered baculovirus
Authors: Stephen Andrew G. , Gillette William K. , Grose Carissa , Esposito Dominic
Published: August 2015

KRAS4b, which is frequently mutated in pancreatic, colon and lung cancers, is processed in several steps including farnesylation by farnesyl-transferase (FNT); proteolytic cleavage at the C-terminus by ras converting enzyme 1 (RCE1) and carboxymethylation by isoprenylcysteine carboxymethyl transferase (ICMT). Plasma membrane localization of KRAS4b requires this processing as does KRAS4b-dependent RAF kinase activation. Previous attempts to produce modified KRAS have relied on protein engineering approaches or in vitro farnesylation of bacterially expressed KRAS protein. The proteins produced by these methods do not completely replicate the final KRAS protein found in mammalian cells and the yield and quality of the protein are typically low. Using an engineered baculovirus containing the two subunits of farnesyl-transferase (FNTA/B) and a maltose-binding protein (MBP)-KRAS4b fusion we are able to produce highly purified, farnesylated, and methylated KRAS4b (FMe-KRAS4b) in insect cells at yields of approximately 10mg/L. This protein has been extensively characterized by electrospray-ionization mass spectrometry, analytical ultracentrifugation, and circular dichroism. In addition, we demonstrate that FMe-KRAS4b is fully active in hydrolyzing GTP, binding to RAF-RBD, and interacting with lipid nanodiscs.

Citation Format: Andrew G. Stephen, William K. Gillette, Carissa Grose, Dominic Esposito. Production and biochemical characterization of farnesylated and methylated KRAS4b using engineered baculovirus. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3925. doi:10.1158/1538-7445.AM2015-3925
Abstract 3927: Structural and mechanistic insights into the roles of RIAM in integrin signaling
Authors: Wu Jinhua
Published: August 2015

Changes in integrin signaling promotes tumor cell proliferation, metastasis and survival. The Rap1-interacting adaptor molecule (RIAM) mediates integrin activation by responding to Rap1 activation and recruiting talin, the integrin activator, to the plasma membrane (PM). The structural details of how RIAM is recognized by Rap1 and in turn recruits talin are lacking. We have elucidated the specificities for RIAM interacting with Rap1 and talin via X-ray crystallography and functional analyses. Crystal structure of Rap1 in complex with the central structural module of RIAM reveals essential specificity determinants, especially a Lys31 residue that is oppositely charged in other Ras GTPases. Furthermore, our study on the RIAM:talin interaction suggests that the talin-binding site 1 (TBS1) of RIAM is required for recruiting cytoplasmic talin to the PM, primarily via an interaction with the R8 domain of talin. Interestingly, an unexpected kink in the helical TBS1 fragment is required for inducing integrin activation. These characteristics are not seen in other related molecules including the highly homologous Lpd, providing the molecular basis for the specific signaling function of RIAM that mediates the integrin activation.

Citation Format: Jinhua Wu. Structural and mechanistic insights into the roles of RIAM in integrin signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3927. doi:10.1158/1538-7445.AM2015-3927
Abstract 3940: Inactivation of the PARD3 gene is a recurrent event in lung squamous cell carcinomas and affects STAT3 activity and tumor invasiveness
Authors: Bonastre Ester , Verdura Sara , Zondervan Ilse , Facchinetti Federica , Lantuejoul Sylvie , Chiara Maria Dolores , Rodrigo Juan Pablo , Carretero Julian , Sidransky David , Villanueva Alberto , Condom Enric , Vidal Agustin , Roz Luca , Brambilla Elisabeth , Savola Suvi , Sanchez-Cespedes Montse
Published: August 2015

Correct apicobasal polarization and intercellular adhesions are essential for the appropriate development of normal epithelia. Here, we investigated the contribution of the partitioning defective 3 gene, PARD3, to the carcinogenesis of lung squamous cell carcinomas (LSCCs). Tumor-specific PARD3 alterations were found in eight per cent of the tumors, placing PARD3 among the most common tumor suppressor genes in LSCC. Some PAR3 mutant proteins prevented the formation of contacts between neighboring cells, i.e. had reduced ability to form tight junctions and actin-based protrusions. This affected subsequent downstream signaling, i.e. binding to aPKC and activation of RAC1. Further, we discovered that PAR3 wild type triggered the expression of cell adhesion and polarity-related transcripts and the activation of STAT3, by cell confluence. Finally, restitution of PAR3 in vivo significantly reduced the formation of metastasis. In conclusion, PARD3 is recurrently inactivated in LSCC and its deficiency contributes to the tumor aggressiveness.

Citation Format: Ester Bonastre, Sara Verdura, Ilse Zondervan, Federica Facchinetti, Sylvie Lantuejoul, Maria Dolores Chiara, Juan Pablo Rodrigo, Julian Carretero, David Sidransky, Alberto Villanueva, Enric Condom, Agustin Vidal, Luca Roz, Elisabeth Brambilla, Suvi Savola, Montse Sanchez-Cespedes. Inactivation of the PARD3 gene is a recurrent event in lung squamous cell carcinomas and affects STAT3 activity and tumor invasiveness. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3940. doi:10.1158/1538-7445.AM2015-3940
Abstract 3944: High incidence of somatic BAP1 alterations in sporadic malignant mesothelioma
Authors: Nasu Masaki , Pastorino Sandra , Luk Hugh , Flores Erin , Baumann Francine , Powers Amy , Kanodia Shreya , Gaudino Giovanni , Zhang Yu-an , Gazdar Adi , Yang Haining , Pass Harvey I. , Emi Mitsuru , Carbone Michele
Published: August 2015

Background: Malignant mesothelioma (MM) is a lethal tumor whose pathogenesis results from complex interactions between host genetics and environmental carcinogens. BAP1 mutations were reported in 22% and 23% of sporadic MM in two US Caucasian studies whereas BAP1 mutations were found in 61% of sporadic MM in a Japanese study. The significant discrepancy in frequency of BAP1 mutations suggested either a possible influence of ethnicity or differences related to the methodological approaches. Methods: To investigate this apparent discrepancy, we carried out comprehensive genomic analyses using multiple techniques in order to detect somatic alterations of the BAP1 gene in tumors of 22 US Caucasian MM patients. Results: Six mutations were detected by Sanger sequencing, gross gene alterations were found in 7 MM specimens by Multiplex Ligation-Dependent Probe Amplification and copy number analysis, and a new splice variant was identified by cDNA sequencing. Changes in methylation were not observed in our study. Immunohistochemistry (IHC) revealed nuclear BAP1 staining in the 8 MM tumors containing wild-type BAP1 while tumor cells in the 14 biopsies with mutated BAP1 did not show any nuclear staining. We extended our BAP1 IHC analysis to an independent cohort of MM samples from the National Virtual Mesothelioma Bank and we found loss of BAP1 nuclear staining in 46 out of 67 biopsies. Moreover we found similar frequency of BAP1 loss across different MM histological subtypes. Conclusions: We identified somatic alterations of the BAP1 gene in 73.7% of Caucasian MM tumors in two independent cohorts of somatic MMs, underscoring the need to apply integrated genomic analyses to scrutinize the BAP1 gene for its alterations. Immunohistochemistry was the most reliable technique to detect BAP1 mutations in MM biopsies. The high incidence of alterations establishes BAP1 as the most commonly mutated gene in MM, regardless of ethnic background or histological subtype.

Citation Format: Masaki Nasu, Sandra Pastorino, Hugh Luk, Erin Flores, Francine Baumann, Amy Powers, Shreya Kanodia, Giovanni Gaudino, Yu-an Zhang, Adi Gazdar, Haining Yang, Harvey I. Pass, Mitsuru Emi, Michele Carbone. High incidence of somatic BAP1 alterations in sporadic malignant mesothelioma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3944. doi:10.1158/1538-7445.AM2015-3944
Abstract 3947: BP1 protein, a transcription factor, is secreted by breast cancer cells
Authors: Rheey Jinguen , Yakovleva Anna , Yamane Kellie-Ann , Berg Patricia E.
Published: August 2015

Background. BP1 is a homeobox gene which is normally expressed during early hematopoiesis. Homeotic proteins regulate the expression of multiple genes involved in development and differentiation. We have previously shown that BP1 mRNA and protein levels are elevated in 80% of women with invasive ductal breast cancer. Furthermore, BP1 protein (pBP1) overexpression is associated with larger and more aggressive breast tumors. In cultured breast cancer cell lines overexpressing pBP1, we observe decreased cell death and increased proliferation. BP1 overexpression has also been found to stimulate known oncogenes including BCL2 and c-MYC. Methods. Levels of pBP1 were detected in cell extracts (CE) and conditioned media (CM) using Western Blot analysis. Depleted media (DM), in which the pBP1 was removed, and recombinant BP1 protein (rpBP1) were used as controls. Cell viability was assessed using MTT assays. Real-time qPCR was used to measure expression of selected oncogenes.

Results. Our results show that: (a) pBP1 is secreted from three breast cancer cell lines but not from two normal breast epithelial cell lines. (b) pBP1 exhibits mitogenic activity towards breast cancer cells and normal breast cells, whether cells are grown in media containing 10x conditioned medium (CM) from cells secreting pBP1 or in media containing purified recombinant pBP1 (rpBP1). (c) Growth of breast cancer cells or normal breast epithelial cells in CM or rpBP1 leads to up-regulation of selected oncogenes known to be targets of BP1. These oncogenes include BCL2 and MET. BP1 itself was stimulated, but only in normal breast epithelial cells. The mechanism of secretion is under investigation.

Conclusions. Our data suggest that secreted pBP1 can stimulate cell proliferation and gene expression in tumor cells as well as in nearby normal cells.

Citation Format: Jinguen Rheey, Anna Yakovleva, Kellie-Ann Yamane, Patricia E. Berg. BP1 protein, a transcription factor, is secreted by breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3947. doi:10.1158/1538-7445.AM2015-3947
Abstract 3951: Functional analysis of the 11q23.3 glioma susceptibility locus
Authors: Baskin Rebekah , Woods Nicholas , Mendoza-Fandino Gustavo , Forsyth Peter , Egan Kathleen , Monteiro Alvaro
Published: August 2015

Glioma is the most common malignant primary brain tumor and is associated with poor prognosis. Single nucleotide polymorphisms (SNPs) associated with glioma risk have recently been investigated through genome-wide association studies (GWAS). Results of these studies have implicated seven independent glioma risk loci in six chromosomal regions. However, the mechanisms by which these SNPs contribute to cancer risk are still unknown. Here, we performed an in-depth functional analysis of one of these risk loci located near the PHLDB1 gene on 11q23.3. We began by retrieving all SNPs in linkage disequilibrium (LD; r2 ≥ 0.2) with the glioma-associated SNP (rs498872). 61 genes contained in the region defined by a boundary of 1Mb from each end of the LD SNPs were considered candidate targets. A subset of 10 candidate functional enhancer/promoter SNPs was defined by overlap with ENCODE biofeatures and allele specific activity in luciferase and EMSA assays in astrocytes and GBM cells. Functional SNPs in the promoter of PHLDB1 implicated its transcriptional regulation in glioma risk. Chromatin conformation capture (3C) identified a physical interaction between one enhancer SNP and the promoter of the DDX6 gene. We also conducted neurosphere formation assays to evaluate the functional role of two top candidate genes, PHLDB1 and DDX6. We found that knockdown of either gene caused a significant reduction in U87MG neurosphere number and size. Taken together, these studies revealed the functional landscape of the 11q23.3 glioma susceptibility locus and implicated the transcriptional regulatory network of PHLDB1 and DDX6 in the mechanism of increased glioma risk.

Citation Format: Rebekah Baskin, Nicholas Woods, Gustavo Mendoza-Fandino, Peter Forsyth, Kathleen Egan, Alvaro Monteiro. Functional analysis of the 11q23.3 glioma susceptibility locus. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3951. doi:10.1158/1538-7445.AM2015-3951
Abstract 3954: lincRNA-UFC1 facilitates cell proliferation and tumor growth in hepatocellular carcinoma by upregulating HuR/β-catenin expression
Authors: LIU LI , Wu Dehua , Cao Chuanhui
Published: August 2015

BACKGROUND & AIMS: Long noncoding RNAs (lncRNAs) have been identified as novel regulators of biologic behaviors in various cancers. However, the oncogenic mechanisms of lincRNA-UFC1 associated with hepatocellular carcinoma (HCC) growth and progression are still unknown. Here, we delved into the mechanistic aspects of lincRNA-UFC1in HCC progression.

METHODS: LncRNA expression profiles are assessed by microarray analysis. Expression of lincRNA-UFC1 was examined in 2 HCC cohorts (cohort 1, n = 49; cohort 2, n = 131) by quantitative real-time PCR or by in situ hybridization. The functions of lincRNA-UFC1 in HCC cells were elucidated by in vitro and in vivo gain- and loss-of-function experiments. The mechanism by which lincRNA-UFC1 contributes to HCC progression was explored through luciferase reporter assays, RNA immunoprecipitation and RNA pull-down assays.

RESULTS: LincRNA-UFC1 was strongly upregulated in HCC tissues, compared with corresponding non-tumor tissues in patients from 2 cohorts. LincRNA-UFC1 expression is associated with tumor size, Barcelona Clinic Liver Cancer (BCLC) stage and poor prognosis. Enhanced lincRNA-UFC1 expression promotes cell proliferation and tumor growth, inhibits apoptosis, and induces cell cycle progression, whereas decreased lincRNA-UFC1 expression represented the opposite effects. Furthermore, lincRNA-UFC1 directly interacts with HuR and regulates β-catenin expression. The level of lincRNA-UFC1 is positively correlated with that of β-catenin in HCC tissues.

CONCLUSIONS: These findings indicate that lincRNA-UFC1 promotes HCC development and progression, indicating its role as a potential therapeutic target for HCC.

Citation Format: LI LIU, Dehua Wu, Chuanhui Cao. lincRNA-UFC1 facilitates cell proliferation and tumor growth in hepatocellular carcinoma by upregulating HuR/β-catenin expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3954. doi:10.1158/1538-7445.AM2015-3954
Abstract 3959: microRNA as a potential biomarker for hepatitis C virus mediated end-stage liver disease progression
Authors: Ray Ratna B.
Published: August 2015

HCV accounts for the most frequent cause of end stage liver disease–including hepatocellular carcinoma (HCC), and liver transplantation in the western world. Since HCV-mediated end stage liver diseases are diagnosed much later after infection, identifying the HCV infected individuals at high risk for developing HCC will permit surveillance, early detection and chemoprevention. Liver biopsy or Fibroscan (often used now) has major limitations. Moreover, neither liver biopsy nor fibroscan can predict who will progress to more severe disease. The relationship between HCV infection and microRNA (miRNA) expression is of considerable interest in understanding liver disease progression to HCC. In this present study, we performed a systematic screening approach to identify upregulated miRNAs in the plasma/serum of HCV infected patients with different stages of hepatic histological disease severity. We initially screened serum samples of HCV infected patients with fibrosis and compared with sera of healthy volunteers using serum miRNA array profiling, and identified a group of modulated miRNAs. Subsequent study demonstrated that miR-20a in HCV infected fibrosis and HCC patients sera were significantly upregulated when compared with that of healthy volunteers or non-HCV associated liver disease. However, there was no correlation between the serum levels of miR-20a with HCV viral loads. We next investigated longitudinal plasma samples from HCV-infected patients. Our results suggested that miR-20a remained unaltered in HCV-infected patients who progressed from acute to chronic infection. These data provide evidence that plasma/serum level of miR-20a has potential as sensitive and cost-effective biomarkers for early detection of HCV mediated liver disease. Conclusion: The circulating miR-20a may serve as a potential for predictive biomarker in HCV mediated end stage liver disease.

Citation Format: Ratna B. Ray. microRNA as a potential biomarker for hepatitis C virus mediated end-stage liver disease progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3959. doi:10.1158/1538-7445.AM2015-3959
Abstract 3961: Utility of mir-21 detection by rapid chromogenic in-situ hybridization in the diagnosis of well and moderately differentiated hepatocellular carcinoma
Authors: Kolhe Ravindra , Rojiani Amyn , Mondal Ashis , Mangaonkar Abhishek A.
Published: August 2015

Background: An estimated 80-90% of all hepatocellular carcinomas (HCC) arise from cirrhotic liver in chronic hepatitis B or C infection. Identifying well-differentiated HCC arising in the background of cirrhosis is a challenging task relying solely on morphology of core biopsies. In addition, presence of dysplasia (micro/macro) in cirrhotic nodules makes the task more difficult. In HCC, microRNAs (miRNAs) have an aberrant expression profile with possible applications in diagnosis, especially for early, symptomatic disease. Sub-cellular and tissue localization of miRNAs may help us understand their biological roles and contribution to disease along with aiding diagnosis.

Design: The aim of this study was to evaluate the utility of mir-21 detection by rapid chromogenic in-situ hybridization (CISH) as a diagnostic tool in the setting of well and moderately differentiated HCCs. Archival blocks and slides were retrieved and reviewed after obtaining IRB approval (table 1). Manufacturers guidelines were followed for CISH (NAR-1, FAM-labeled Has-miR-21 probe) on a total of 202 cases. Nuclear mir-21 CISH stain was graded as: negative (-), weak (1+), moderate (2+) or strong (3+) and distribution was graded as: focal (<10%), multifocal (10-50%) or diffuse (>50%).

Table 1.Table showing cases considered for mir-21 detection by CISH.HistologyNumber of casesHCC150Intrahepatic cholangiocarcinoma12Metastases from colonic adenocarcinoma23Metastases from gallbladder adenocarcinoma5Normal liver tissue12

Results: Neoplastic cells in HCC showed a strong (2-3) multifocal to diffuse nuclear staining for mir-21 in 100% of the cases of whole tissue slides and 91% of HCC cases in tissue microarray slides when compared to the staining pattern seen in normal liver cells (0%). As expected, 85% of cases with liver metastases from colonic and gallbladder adenocarcinoma showed strong nuclear staining for mir-21.

Conclusion: Rapid CISH technique can clearly identify increased nuclear expression of mir-21 and aid in diagnosis of well and moderately differentiated HCC.

Citation Format: Ravindra Kolhe, Amyn Rojiani, Ashis Mondal, Abhishek A. Mangaonkar. Utility of mir-21 detection by rapid chromogenic in-situ hybridization in the diagnosis of well and moderately differentiated hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3961. doi:10.1158/1538-7445.AM2015-3961
Abstract 3964: How to fish a good micro-marker out from a worthless lake: The case of cell-free miR-181a-5p and breast cancer
Authors: Ferracin Manuela , Lupini Laura , Salamon Irene , Saccenti Elena , Zagatti Barabara , Mangolini Alessandra , Zanzi Maria Vittoria , Carcoforo Paolo , Rocchi Andrea , Cavallesco Giorgio , Frassoldati Antonio , Hollingsworth Alan B. , Negrini Massimo
Published: August 2015

The hypothesis to use microRNAs (miRNAs) circulating in blood as cancer biomarkers was formulated some years ago based on promising initial results. After some exiting discoveries, however, it became evident that the accurate quantification of cell-free miRNAs (i.e. that retrieved in serum or plasma) was more challenging than expected. Difficulties were linked to the strong impact that many, if not all, pre- and post- analytical variables (i.e. tissue preparation, storage condition, extraction method, quantification technique, normalization approach) have on the final result.

In this study, we used currently available high-throughput technologies to identify miRNAs present in plasma and serum of patients with breast, colorectal, lung and melanoma tumors, and healthy controls. Then, we used droplet digital PCR (ddPCR) technology to get an accurate absolute quantification of specific cell-free miRNAs. We assessed the level of 8 different miRNAs (miR-320a, miR-21-5p, miR-378a-3p, miR-181a-5p, miR-3156-5p, miR-2110, miR-125a-5p, miR-425-5p, miR-766-3p) in 180 samples from healthy controls and cancer patients. We identified miRNAs specifically modulated in one or more cancer types. Plasma and serum from the same patient provided different results in terms of absolute miRNA amount and modulation. The significant reduction of miR-181a-5p levels in serum of breast cancer patients was further validated using two independent cohorts, one from Italy (n = 70) and one from US (n = 90). This study finally powers the use miR-181a-5p as a breast cancer biomarker.

Citation Format: Manuela Ferracin, Laura Lupini, Irene Salamon, Elena Saccenti, Barabara Zagatti, Alessandra Mangolini, Maria Vittoria Zanzi, Paolo Carcoforo, Andrea Rocchi, Giorgio Cavallesco, Antonio Frassoldati, Alan B. Hollingsworth, Massimo Negrini. How to fish a good micro-marker out from a worthless lake: The case of cell-free miR-181a-5p and breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3964. doi:10.1158/1538-7445.AM2015-3964
Abstract 3965: MicroRNA markers for the diagnosis of pancreatic cancer
Authors: Kojima Motohiro , Sudo Hiroko , Kawauchi Junpei , Takizawa Satoko , Kondou Satoshi , Nobumasa Hitoshi , Ochiai Atsushi
Published: August 2015

It is recently reported that microRNAs (miRNAs) are stably present in serum and potentially useful in the diagnosis of cancer. Using highly sensitive microarray, 3D-Gene®, we examined comprehensive miRNA expression profiles in serum obtained from 66 pancreatic cancer patients and 100 matched healthy volunteer as the study set, and found that 122 miRNA markers differentiated pancreatic cancer patients from healthy control with a statistical significance. We then validated the diagnostic performance of these markers using serum obtained from 33 pancreatic cancer patients and 50 healthy controls. One of the single miRNA markers for pancreatic cancer yielded more than 90% diagnostic sensitivity and specificity, and combined use of two markers perfectly differentiated cancer and healthy samples. Furthermore, combinations of miRNAs that differentiated pancreatic cancer not only from healthy samples but also from non-malignant pancreatic diseases or other digestive (esophagus, stomach, liver and colon) cancers were also identified, and their diagnostic accuracies were superior to those of CEA or CA19-9. Our results suggest that these miRNA markers have clinical values to screen out pancreatic cancer patients.

Citation Format: Motohiro Kojima, Hiroko Sudo, Junpei Kawauchi, Satoko Takizawa, Satoshi Kondou, Hitoshi Nobumasa, Atsushi Ochiai. MicroRNA markers for the diagnosis of pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3965. doi:10.1158/1538-7445.AM2015-3965
Abstract 3974: A preclinical study for miR181b as therapeutic in Eu-TCL1FL-tg mouse model for CLL
Authors: Bresin Antonella , Callegari Elisa , D'Abundo Lucilla , Cattani Caterina , Bassi Cristian , Zagatti Barbara , Narducci Maria Grazia , Caprini Elisabetta , Pekarsky Yuri , Croce Carlo Maria , Sabbioni Silvia , Negrini Massimo , Russo Giandomenico
Published: August 2015

Chronic lymphocytic leukemia (CLL) is a malignancy of B cells with immunophenotype of memory cells. Downstream pathways to BCR, like NFkB, AKT and ERK, and anti-apoptotic proteins such as Bcl2 and Mcl1, are thought to contribute to expansion and survival of the malignant cells. CLL patients show different clinical course: tumor clones having no V-gene mutations, many CD38+ or ZAP-70+ B cells, chromosome 11q deletion and high expression of TCL1, had an aggressive, usually fatal course, whereas patients with mutated clones, few CD38+ or ZAP-70+ B cells and low TCL1 expression, had an indolent course. Recent studies identified a microRNA signature associated with prognosis and progression in B-CLL. Particularly, miR181b is down-regulated in aggressive forms of CLL and its expression is inversely correlated to Tcl1 levels.

An increasing number of studies are considering microRNAs as potentially useful therapeutic agents. Given that MCL-1, BCL-2 and TCL1 are proved targets for miR181b, that Akt activation is enhanced by Tcl1 and a crosstalk between Akt and ERK or NFkB exists, we performed a preclinical evaluation of miR181b therapeutic efficacy in a mouse model of CLL, the Eu-TCL1 transgenic mouse, previously generated by us.

In vitro enforced expression of miR-181b mimics induced a significant apoptotic effect in human B-cell line EHEB, as well as in mouse Eμ-TCL1 leukemic splenocytes. Molecular analyses revealed that miR-181b not only affected the expression of Tcl1, Bcl2 and Mcl1 anti-apoptotic proteins, but also increased the expression level of the NFkB inhibitor protein IkB-alpha and reduced the phosphorylation levels of Akt and Erk1/2. Notably, an anti-TCL1 siRNA could similarly down-modulate Tcl1 but exhibited a reduced or absent activity on other relevant proteins, as well as a reduced effect on cell apoptosis and viability. In vivo studies indicated a significant capability of miR-181b in reducing leukemic cells expansion and increasing survival of treated mice. These data indicate that miR-181b exerts a broad range of actions, affecting proliferative, survival and apoptotic pathways, both in mice and human, and can potentially be employed to reduce expansion of B-CLL leukemic cells.

Citation Format: Antonella Bresin, Elisa Callegari, Lucilla D'Abundo, Caterina Cattani, Cristian Bassi, Barbara Zagatti, Maria Grazia Narducci, Elisabetta Caprini, Yuri Pekarsky, Carlo Maria Croce, Silvia Sabbioni, Massimo Negrini, Giandomenico Russo. A preclinical study for miR181b as therapeutic in Eu-TCL1FL-tg mouse model for CLL. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3974. doi:10.1158/1538-7445.AM2015-3974
Abstract 3976: Targeted delivery of a synthetic microRNA-based mimic as an approach to cancer therapy
Authors: Reid Glen , Williams Marissa , Kirschner Michaela B. , Mugridge Nancy , Weiss Jocelyn , Brahmbhatt Himanshu , MacDiarmid Jennifer , van Zandwijk Nico
Published: August 2015

MicroRNA expression is commonly suppressed in cancer, contributing to tumor cell biology. Recently we demonstrated that multiple members of the miR-15/16 family are downregulated and have tumor suppressor functions in malignant pleural mesothelioma (MPM), an asbestos-related cancer for which few treatments are available. These results are similar to previous findings in prostate and non-small cell lung cancer (NSCLC). Using mimics to restore levels of miR 15a, miR-15b or miR-16 led to growth inhibition and induction of apoptosis of MPM cells in vitro. The miR-16 mimic, packaged in bacterially-derived, EGFR antibody-targeted, EDV(TM)nanocells, inhibited xenograft tumor growth in vivo. As multiple microRNAs from the same family are downregulated in MPM, we investigated whether a single synthetic mimic based on the consensus sequence of all family members could restore activity of the entire family. To this end we generated four novel mimics derived from the consensus sequence of the miR-15/16 family and tested them in a range of tumor cell lines. Compared with a mimic corresponding in sequence to native miR-16, the consensus mimics had enhanced growth inhibitory activity in MPM, NSCLC and prostate cancer lines, three tumor types in which miR-15/16 expression is suppressed. They were also active in cell lines derived from breast and colon cancer. When packaged in minicells, the synthetic mimics inhibited growth of MPM xenograft tumors in vivo. Based on these preclinical studies a Phase I clinical trial has been initiated for patients with MPM or NSCLC failing standard therapy. This represents only the second trial of microRNA replacement as a cancer therapy, and the first for thoracic cancer.

Citation Format: Glen Reid, Marissa Williams, Michaela B. Kirschner, Nancy Mugridge, Jocelyn Weiss, Himanshu Brahmbhatt, Jennifer MacDiarmid, Nico van Zandwijk. Targeted delivery of a synthetic microRNA-based mimic as an approach to cancer therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3976. doi:10.1158/1538-7445.AM2015-3976
Abstract 3980: Recurrence prediction model using microRNA expression in hormone receptor positive and tamoxifen treated breast cancer
Authors: Kim Aeree , Kim Chungyeul
Published: August 2015

Hormone receptor positive breast cancer patients have relatively good prognosis after tamoxifen treatment but still about 40% of patients experience treatment failure (1). Recently microRNAs (miRNAs) have been spotlighted as a new biomarker for predicting prognosis and treatment response. The purpose of this study is to identify microRNAs (miRNAs) that related with distant recurrence after tamoxifen and standard treatment and then build an early recurrence prediction model.

Methods: Our study measured the expression of 5 miRNAs (miR-134, miR-125b-5P, miRNA-30a, miR-10a-5p, and miR-222) that are selected as a tamoxifen resistant related miRNA by previous micro-array experiment and paper review. 176 FFPE tumor tissues of 176 patients who have hormone receptor positive breast cancer with tamoxifen were measured miRNA expression by quantitative real-time PCR (qRT-PCR).

Results: We identified all 5 miRNAs are up-regulated in distant recurrence cases within 5 years after surgery. Other clinico-pathological parameters that are tumor grade, pathologic stage, HER2 statue and P53 status didn't have any specific correlation with expression of miRNAs. The results suggest that distant recurrence during tamoxifen treatment might be affected by tamoxifen resistant related miRNAs. Kaplan-Meier survival analyses based on expression cut-offs determined by receiver characteristics curves show high expression of miR-134, miR-125b-5P, miRNA-30a, miR-10a-5p, and miR-222 were associated with short relapse-free time(log-rank P = 0.006, P<0.0001, P<0.0001, P<0.0001,and P<0.0001, respectively) Conclusion: The altered expression of miRNAs in hormone receptor positive breast cancer is distinctly related to distant recurrence during tamoxifen treatment. Our results suggest that combination of miRNAs expression model can categorize high-risk subset of patients with short relapse-free and has decent predictive value (AUC = 0.891, p-value<0.0001).

Citation Format: Aeree Kim, Chungyeul Kim. Recurrence prediction model using microRNA expression in hormone receptor positive and tamoxifen treated breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3980. doi:10.1158/1538-7445.AM2015-3980
Abstract 3986: Introduction of PTEN pseudogene in murine breast cancer upregulates PTEN, p53 and activating protein 2 gamma and delays tumor growth
Authors: Yndestad Synnøve , Austreid Eilin , Skaftnesmo Kai Ove , Lønning Per Eystein , Eikesdal Hans Petter
Published: August 2015

Background:

While PTEN is frequently downregulated in breast cancer, the mechanism remains obscure. PTEN pseudogene (PTENP1) is reported to function as a decoy adsorbing micro RNAs (miRNAs) targeting PTEN for degradation in prostate cancer cells. A potential role of PTENP1 in PTEN regulation in breast cancer has not been assessed systematically.

Method:

We transfected murine C3HBA breast cancer cells lacking endogenous PTENP1 with a lentiviral PTENP1 construct to investigate the influence of the pseudogene on PTEN and Akt-mTOR downstream signaling, global gene expression, as well as in vitro and in vivo tumor growth characteristics.

Results:

Upregulated PTENP1 3´UTR by lentiviral transfection increased PTEN protein levels and reduced cell cycle progression in vitro, but despite this increased Akt phosphorylation was observed. Similarily, PTENP1 transfection decreased tumor growth in C3HBA murine breast cancer in syngeneic, immunocompetent C3H mice, corresponding with upregulated PTEN, but without inhibition of Akt-mTOR signaling. In stead, increased protein levels of p53 and its downstream target activating protein-2 gamma (AP-2γ) was observed as possible tumor suppressors in this setting.

Conclusion:

In the murine C3HBA breast cancer cell line, PTENP1 transfection was associated with upregulated endogenous PTEN, p53 and AP-2γ protein levels and tumor growth inhibition.

Citation Format: Synnøve Yndestad, Eilin Austreid, Kai Ove Skaftnesmo, Per Eystein Lønning, Hans Petter Eikesdal. Introduction of PTEN pseudogene in murine breast cancer upregulates PTEN, p53 and activating protein 2 gamma and delays tumor growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3986. doi:10.1158/1538-7445.AM2015-3986
Abstract 3988: MicroRNA expression associated with response grade in gastric cancer patients’ tumors
Authors: Barcelos Baldoni Michelle Maccarini , Coutinho Camillo Cláudia Malheiros , Puga Renato David , Begnami Maria Dirlei Ferreira
Published: August 2015

Background: Many chemotherapy schemes have been used in neoadjuvant treatment scenario to improve the overall and disease-free survival of gastric cancer (GC) patients treated with surgery alone. However, in some cases, the objective clinical response is not achieved, and this lack of response to neoadjuvant chemotherapy may delay curative surgery and increase surgical complications. Therefore, a major challenge for improving clinical outcomes is to understand the molecular mechanisms that may play essential roles in patients responses of GC treatments. The expression levels of several miRNAs have been found to be correlated with GC prognosis and could determine the clinical response for neoadjuvant chemotherapy. Therefore, our goal was to analyze the relation between miRNA profile and the pathological response grade of GC patients tumors treated with neoadjuvant chemotherapy or surgery alone. Methods: We selected 34 biopsy tumor samples of patients treated with neoadjuvant chemotherapy (collected before the treatment) and 23 tumor samples of patients submitted to surgery without adjuvant treatment. All patients treated with neoadjuvant chemotherapy received ECF, DCF or ECX prior the surgery, and subsequently they were submitted to a complete resection. The identification of differentially expressed miRNAs was done by TLDA Quantitative Real Time PCR. Results: After categorizing neoadjuvant chemotherapy patients according to percentage of viable tumor cells present in gastric resection and the surgical patients according to their 5 years of follow up status we could segregate those with and without response for both treatments. The comparison between response grade versus miRNAs showed 9 and 14 downregulated miRNAs in samples of patients without response for neoadjuvant chemotherapy and surgery alone, respectively. We also could observe 3 common downregulated miRNAs between patients without response in both groups. Conclusion: We identified some downregulated miRNAs involved in gastric cancer pathways and mechanisms of drug resistance that should manage to determine a predictable miRNA signature to perform a correct patient selection for GC treatments.

Note: This abstract was not presented at the meeting.

Citation Format: Michelle Maccarini Barcelos Baldoni, Cláudia Malheiros Coutinho Camillo, Renato David Puga, Maria Dirlei Ferreira Begnami. MicroRNA expression associated with response grade in gastric cancer patients’ tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3988. doi:10.1158/1538-7445.AM2015-3988