bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3890: Whole-genome mutational landscape of liver cancers displaying biliary phenotype reveals hepatitis impact and molecular diversity
Authors: Nakagawa Hidewaki , Fujimoto Akihiro , Furuta Mayuko , Gotoh Kunihito , Nakamura Toru , Yamamoto Masakazu , Yamaue Hiroki , Chayama Kazuaki , Miyano Satoru , Tsunoda Tatsuhiko
Published: August 2015

Primary liver cancer is the third leading cause of cancer death worldwide. Virus infection is the most common and strongest etiological factor for liver cancer development. Pathologically, primary liver cancer can be classified into ∼90% hepatocellular carcinoma (HCC), and 5∼10% intrahepatic cholangiocarcinoma (ICC), and the combined hepatocellular-cholangiocarcinoma (cHCC/CC) representing only a small portion. Clinically, ICC and cHCC/CC show much more aggressive behavior with poorer prognosis than HCC, and no standard treatment currently exists, other than surgical resection. ICC and cHCC/CC show varying degrees of biliary epithelial differentiation, which can be defined as liver cancer displaying a biliary phenotype (LCB). To gain insight into molecular alterations of LCBs, we performed whole genome sequencing analysis on 30 LCBs. Here we show the genome-wide substitution patterns of LCBs developed in chronic hepatitis livers overlapped with those of 60 HCCs, while those of hepatitis-negative LCBs diverged. The subsequent validation study on 68 LCBs identified recurrent mutations in TERT promoter, chromatin regulators (BAP1, PBRM1 and ARID2), a synapse organization gene (PCLO), IDH genes and KRAS. The frequencies of KRAS and IDHs mutations, which are associated with poor disease-free survival, were significantly higher in hepatitis-negative LCBs. This study reveals the strong impact of chronic hepatitis on the mutational landscape in liver cancer and the genetic diversity among LCBs.

Citation Format: Hidewaki Nakagawa, Akihiro Fujimoto, Mayuko Furuta, Kunihito Gotoh, Toru Nakamura, Masakazu Yamamoto, Hiroki Yamaue, Kazuaki Chayama, Satoru Miyano, Tatsuhiko Tsunoda. Whole-genome mutational landscape of liver cancers displaying biliary phenotype reveals hepatitis impact and molecular diversity. [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 3890. doi:10.1158/1538-7445.AM2015-3890
Abstract 3894: The importance of matched tumor and normal DNA for somatic mutation discovery and clinical interpretation
Authors: Jones Siân , Sausen Mark , Anagnostou Valsamo , Angiuoli Samuel V. , Chesnick Bryan , Galens Kevin , Kadan Maura , Kann Lisa , Lytle Karli , Murphy Derek , Nesselbush Monica , Papp Eniko , Parpart-Li Sonya , Riley David , Shukla Manish , Zhang Theresa , Diaz Luis A. , Velculescu Victor E.
Published: August 2015

Massively parallel sequencing approaches are beginning to be used clinically to characterize individual patient tumors and to identify targeted therapies based on mutations identified. A major question in these analyses is the extent to which these methods identify clinically actionable alterations in patients and whether the examination of the tumor tissue alone is sufficient or whether matched normal DNA should also be analyzed to accurately identify tumor-specific (somatic) alterations. To address these issues, we comprehensively evaluated tumor-normal paired samples from 753 cancers from fourteen tumor types. We analyzed somatic alterations using whole exome or targeted next generation sequencing approaches that were validated with high sensitivity and specificity. These analyses revealed an average of 140 and 4.34 somatic changes per exome and targeted analyses, respectively. Approximately 65% of cases had somatic alterations in genes associated with known therapies or current clinical trials. In contrast, a tumor-only sequencing approach followed by bioinformatic removal of common germline variants from existing databases led to a 36% and 58% false discovery rate in alterations identified in targeted and exome analyses, respectively, including in potentially actionable genes. These data suggest that matched tumor-normal sequencing analyses are essential for precise identification and interpretation of somatic alterations and have important implications for the diagnostic and therapeutic management of cancer patients.

Citation Format: Siân Jones, Mark Sausen, Valsamo Anagnostou, Samuel V. Angiuoli, Bryan Chesnick, Kevin Galens, Maura Kadan, Lisa Kann, Karli Lytle, Derek Murphy, Monica Nesselbush, Eniko Papp, Sonya Parpart-Li, David Riley, Manish Shukla, Theresa Zhang, Luis A. Diaz, Victor E. Velculescu. The importance of matched tumor and normal DNA for somatic mutation discovery and clinical interpretation. [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 3894. doi:10.1158/1538-7445.AM2015-3894
Abstract 3910: Acetylation of ΔNp63α by Tip60 affects its stability
Authors: Stacy Andrew J. , Smith Zachary J. , Zhang Jin , Kadakia Madhavi
Published: August 2015

p63, a member of the transcription factor p53 family, exists as six different isoforms due to the usage of alternative promoter sites and splicing. ΔNp63α is the most abundant and predominant isoform and is identified to being involved in epithelial morphogenesis. ΔNp63α expression is up-regulated in non-melanoma skin cancers. We have identified ΔNp63α as a potential target for acetylation by the Tat Interacting Protein 60 kDa (TIP60) histone acetyltransferase (HAT) by mass spectometry. Co-expression of ΔNp63α with Tip60 leads to stabilization of ΔNp63α protein levels. The change in protein levels are not due to an increase in transcription or translation, and is likely the result of stabilization by post-translational modification as confirmed by half-life studies in presence of cycloheximide and measurement of transcript levels by quantitative reverse transcriptase PCR. This was further supported by our observations that ΔNp63α and Tip60 interact using immunoprecipitation studies. ΔNp63α levels are not affected by Tip60 HAT mutant, but increased with Tip60 double sumoylation mutant which retains its acetyltransferase ability. These results identify a possible biologically important interaction between ΔNp63α and Tip60, warranting further investigation.

Citation Format: Andrew J. Stacy, Zachary J. Smith, Jin Zhang, Madhavi Kadakia. Acetylation of ΔNp63α by Tip60 affects its stability. [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 3910. doi:10.1158/1538-7445.AM2015-3910
Abstract 3921: TGFβ sensitizes hepatocellular carcinoma cells to growth factors, preventing sorafenib-induced apoptosis
Authors: Ungerleider Nathan , Han Chang , Wu Tong
Published: August 2015

Many hepatocellular carcinoma (HCC) patients have a poor prognosis due to an acquired resistance of tumors to chemotherapeutic treatment. Transforming growth factor β (TGFβ) has important roles in the control of cell differentiation, cell cycle progression, and apoptosis. An upregulation in the TGFβ signalling pathway is associated with poor prognosis in HCC patients. In this study, we aim to investigate the mechanisms by which TGFβ contributes to HCC tumor progression. We found that TGFβ significantly increases mRNA and protein levels of several mitogenic receptor tyrosine kinases, including insulin-like growth factor 1 receptor (IGF1R) and epidermal growth factor receptor (EGFR) in both time-dependent and dose-dependent manners in PLC/PRF/5 cells. Furthermore, TGFβ pretreatment significantly increases the acute cellular response to IGF1, IGF2, and EGF treatment, as measured by levels of pAKT and pERK also in both time-dependent and dose-dependent manners. We hypothesized that this increase in growth factor sensitivity would render cells resistant to sorafenib, the only approved targeted HCC chemotherapeutic agent. To test this, we pretreated PLC/PRF/5 cells with TGFβ for 48 hours then incubated the cells with sorafenib for up to 24 hours. We found that TGFβ pretreatment led to a reduction of cleaved caspases 3, 7, 9, and PARP, which are cleaved as cells undergo apoptosis. We confirmed an increase in cell viability via WST-1 assays and by counting viable cells. Lastly, we found that the TGFβRI kinase inhibitor, LY2157299, synergized with sorafenib to promote apoptosis. Taken together, our data suggests that TGFβ can enhance the survival of HCC cells by upregulating cell sensitivity to pro-survival growth factors. Furthermore, the use of both sorafenib and an inhibitor of the TGFβ signalling pathway may one day prove to be a more effective therapy for patients with advanced HCC.

Citation Format: Nathan Ungerleider, Chang Han, Tong Wu. TGFβ sensitizes hepatocellular carcinoma cells to growth factors, preventing sorafenib-induced apoptosis. [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 3921. doi:10.1158/1538-7445.AM2015-3921
Abstract 3935: CDKN2A/p16 is inactivated by the combination of inversion and translocation
Authors: Norris Alexis L. , Kamiyama Hirohiko , Hruban Ralph H. , Eshleman James R.
Published: August 2015

Homozygous deletions (HDs) are a common mechanism by which tumor suppressor genes (TSG) are inactivated in cancer. Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A/p16) is the most commonly deleted TSG, with HDs in 40% of pancreatic ductal adenocarcinomas (PDAC) harboring p16 deletions. HDs create novel junctions, which can be exploited for sensitive early detection and minimal residual disease testing, using the PAtient-specific REarrangement (PARE) method.

Here we characterize the underlying structural rearrangements that produce p16 HDs in a panel of PDAC cancer cell lines using pair-end whole genome sequencing (WGS) and high density SNP microarray analyses. Rearrangements were defined as simple interstitial deletions when the segment of a chromosome arm was deleted and the ends joined. The complex rearrangements causing the HD were categorized at both breakpoints as inversions and/or translocations. Sanger sequencing confirmed novel junctions, and breakpoints were assessed for non-templated base insertions and microhomology.

We report that complex rearrangements underlie half of p16 HDs in PDAC (3 of 5). Most striking, the combination of an inversion and inter-chromosomal translocation (INV+TRANS) was exclusively responsible for these complex rearrangements. Inversions ranged in size from 47 to 3,373bp and translocations were non-recurrent and were often non-reciprocal (2 of 3). All HDs were resolved (either by interstitial deletion or INV+TRANS) without the addition of any non-templated bases inserted at the novel junction, arguing against the involvement of error-prone non-homologous end joining (NHEJ). Microhomology was seen at most rearrangement junctions (8 of 9), but with a median of only 2 bases. Further work is needed to elucidate the mechanism responsible for INV+TRANS rearrangements.

Citation Format: Alexis L. Norris, Hirohiko Kamiyama, Ralph H. Hruban, James R. Eshleman. CDKN2A/p16 is inactivated by the combination of inversion and translocation. [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 3935. doi:10.1158/1538-7445.AM2015-3935
Abstract 3952: BRE: a genetic interactor of BRCA2
Authors: Biswas Kajal , Chang Suhwan , Philip Subha , Sharan Shyam K.
Published: August 2015

Mutations in breast cancer susceptibility genes BRCA1 and BRCA2 predispose individuals to breast and ovarian cancers. Though BRCA associated tumor tissues showed inactivation of both alleles, in primary cells mutations in both copies affect cell viability. This suggests that BRCA1 or 2 loss mediated tumorigenesis process needs other factors. In this study, MSCV (Murine Stem Cell Virus) retroviral mediated insertional mutagenesis approach was used in conditional Brca2 mutant mES (mouse embryonic stem) cells to identify and characterize genetic interactors of BRCA2. One of the factors identified in this screen is brain and reproductive organ expressed protein BRE. BRE is an anti-apoptotic protein and also a component of the BRCA1-A complex that specifically recognizes ‘Lys-63′-linked ubiquitinated histones H2A and H2AX at DNA lesions sites. Overexpression of BRE results into perturbation of DNA damage induced G1/S cell cycle checkpoint by preventing DNA damage induced degradation of CDC25a phosphatase. Further it was found that the stabilization of CDC25a phosphatase in BRE overexpressing cells on DNA damage is mediated via ubiquitin-specific-processing protease 7 (USP7). We are now examining the expression of BRE and CDC25a in BRCA2 deficient tumors.

Citation Format: Kajal Biswas, Suhwan Chang, Subha Philip, Shyam K. Sharan. BRE: a genetic interactor of BRCA2. [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 3952. doi:10.1158/1538-7445.AM2015-3952
Abstract 3967: miRNA reference samples for interlaboratory study
Authors: Pine P. Scott , Sorbara Lynn R. , Srivastava Sudhir , Salit Marc
Published: August 2015

The biomarkers of interest in this study are microRNA (miRNA), small non-coding RNAs involved in the regulation of gene expression. Currently, over 1000 human miRNAs have been identified. Several laboratories within the Early Detection Research Network are currently profiling miRNA as potential biomarkers. The reference sample paradigm presented and evaluated here is designed to provide measurement assurance in the earliest phases of a cancer biomarker development pipeline.

Three commercially available samples of human total RNA (brain, liver, and placenta) were selected for detectable differences in miRNA expression based upon previously reported miRNA microarray experiments. For each round of this study, replicate samples of each tissue were aliquoted, blinded, and distributed to participating laboratories. The samples were then profiled using the standard workflows of each laboratory. This was repeated over multiple rounds of measurement. Raw data were processed in pair-wise comparisons between tissue types.

Log2 transformed ratios were calculated for each detectable miRNA. For each of these miRNA ratios, the results among laboratories were compared to the consensus value to provide a measure of concordance and help identify laboratories that might need to make adjustments to their process. Results demonstrated repeatability within labs and reproducibility among labs with respect to direction and magnitude of differential miRNA expression between tissues for a set of miRNAs of interest (MOI). For the first round, 79% of reported results for all log2 ratio combinations and MOI were in agreement with the consensus values.

Subsequent rounds demonstrated increased concordance, with 91 and 100% agreement, respectively. The data from the nine “neat” tissue total RNA samples were also used for in silico modeling to predict the values for two complex mixtures based on three components. The two mixture designs provide a pair of reference samples with designed-in reciprocal ratios of 2:1 for brain and placenta, as well as a shared 1:1 liver component. For two brain-selective and two placenta-selective MOI, 91% of reported values were in close agreement with the target values. Overall, measurement results were harmonious for a set of miRNAs of interest measured by all participants, both within rounds and between rounds. The laboratories were also able to demonstrate the ability to resolve the 2-fold differences created with complex mixtures. These results suggest that endogenous miRNAs present within total RNA may be suitable as QA/QC process controls.

Citation Format: P. Scott Pine, Lynn R. Sorbara, Sudhir Srivastava, Marc Salit. miRNA reference samples for interlaboratory study. [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 3967. doi:10.1158/1538-7445.AM2015-3967
Abstract 3972: MicroRNAs as potential therapeutic agents for AML: Targeting the AML1-ETO Oncogene by pre-miR-520 and -373
Authors: Toniolo Patricia A. , Pilarski Patrick M. , Bach Christian , Griffin James D. , Adamia Sophia
Published: August 2015

Acute myeloid leukemia (AML) is a heterogeneous neoplasm characterized by the accumulation of poorly differentiated myeloid cells in the bone marrow and blood of patients. Differentiation therapy is an attractive therapeutic approach for treating patients with AML. All-Trans Retionic Acid, induces differentiation of patients with the PML/RARa oncogene, but is ineffective in treating other more frequent types of AML. Here we describe the use of microRNAs to target the oncogene formed as a result of the (8;21) translocation, The goals was to identify miRNAs that could target the breakpoint region of the fusion protein, and determine if this would reduce expression of AML1-ETO and promote differentiation and/or apoptosis. Bioinformatics analysis was used to identify 28 different miRNAs that could potentially target AML1-ETO transcripts. Among those, miRNA-520 and -373 showed the highest degree of complimentary to AML1-ETO transcripts at the braikpoint. To look for miR-520 and -373 efficacy, we transfected two AML cell lines, Kasumi 1 and SKNO1, which have AML1-ETO translocation, with pre-miR-520 and -373 LNA probes, as well as HL60 cells which lack AML1-ETO. Expression of the LNA pre-miR-520/373 in Kasumi-1 and SKNO-1 cell lines decreased AML1-ETO transcripts and led to a significant reduction of AML1-ETO protein levels. The inhibition of AML1-ETO fusion protein also induced apoptosis of leukemic cells in vitro, but had no effect on HL60 cells. Moreover, the administration of LNA pre-miR-520 in an AML xenografts murine model increased apoptosis of leukemic cells and reduced tumor burden without obvious toxicity. We are currently testing the effects of miR-520 and -373 on differentiation and proliferation of primary blasts from patients with the AML1-ETO translocation.

Our results suggest that small molecules such as miRNAs can be identified that directly target a frequent AML oncogene. miRNAs are naturally accruing molecules and have the potential advantage of producing modest side effects, with highly significant specificity. The approaches we developed in this study can be used to evaluate and optimize other pre-miRs and anti-miRs as therapeutic agents to target other types oncogenes not currently amenable to small molecule drugs.

Citation Format: Patricia A. Toniolo, Patrick M. Pilarski, Christian Bach, James D. Griffin, Sophia Adamia. MicroRNAs as potential therapeutic agents for AML: Targeting the AML1-ETO Oncogene by pre-miR-520 and -373. [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 3972. doi:10.1158/1538-7445.AM2015-3972
Abstract 3987: Exosomal microRNA in cell-free urine samples as a source for liquid prostate cancer biopsy
Authors: Blondal Thorarinn , Thomsen Anni R. , Krummheuer Jörg , Borre Michael , Fredsøe Jacob , Haldrup Christa , Andreasen Ditte , Teilum Maria W. , Tolstrup Niels , Sørensen Karina D. , Ørntoft Torben F. , Mouritzen Peter
Published: August 2015

MicroRNAs constitute a class of small cellular RNAs (typically 19-23 nt) that function as post-transcriptional regulators of gene expression. Current estimates indicate that more than one third of the human cellular transcriptome is regulated by this small class of RNA (∼2000 miRNA). MicroRNAs have been shown to be actively exported from tissues into the circulation through a variety of mechanisms including complexing with RNA binding proteins or HDL and through active exosome transport.

Exosomes are nanovesicles secreted into the extracellular environment by a wide range of cell types under normal and pathological conditions. As the profile of exosomal microRNAs may be a fingerprint of the releasing cell type and because they are released in easily accessible body fluids such as blood and urine, their microRNA content holds potential as biomarkers for early detection of malignancy.

Cell free urine samples are an obvious liquid biopsy source for microRNA markers in prostate cancer and could serve as diagnostic tools as well as treatment response markers. Yet microRNA levels in urine samples are extremely sparse and cannot be detected reliable by conventional sample preparation methods. It has been reported that tumour derived exosomes carrying genetic information specific for prostate cancer can be measured in urine samples following prostate massage. However, a methodology eliminating the need for prostate massage of every patient to be tested in a clinical setting is highly desirable.

The purpose of this study was to combine a simple exosomal enrichment method with our highly sensitive LNA™-based qPCR platform for detection of microRNAs and apply this to a cohort consisting of more than 300 cell free urine samples from prostate cancer cases and controls. In conclusion, cell free urine samples holds potential as a liquid biopsy source for exosomal microRNA markers in prostate cancer, showing miRNA signatures of strong diagnostic potential. Analysis is pending and data will be presented.

Citation Format: Thorarinn Blondal, Anni R. Thomsen, Jörg Krummheuer, Michael Borre, Jacob Fredsøe, Christa Haldrup, Ditte Andreasen, Maria W. Teilum, Niels Tolstrup, Karina D. Sørensen, Torben F. Ørntoft, Peter Mouritzen. Exosomal microRNA in cell-free urine samples as a source for liquid prostate cancer biopsy. [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 3987. doi:10.1158/1538-7445.AM2015-3987
Abstract 3994: Herpes virus microRNA expression and significance in serous ovarian cancer
Authors: Pandya Deep , Mariani Marisa , McHugh Mark , Andreoli Mirko , Sieber Steven , He Shiquan , Dowell-Martino Candice , Fiedler Paul , Scambia Giovanni , Ferlini Cristiano
Published: August 2015

Serous ovarian cancer (SEOC) is the deadliest gynecologic malignancy. MicroRNAs (miRNAs) are a class of small noncoding RNAs which regulate gene expression and protein translation. MiRNAs are also encoded by viruses with the intent of regulating their own genes and those of the infected cells. This is the first study assessing viral miRNAs in SEOC. MiRNAs sequencing data from 487 SEOC patients were downloaded from the TCGA website and analyzed through in-house sequencing pipeline. To cross-validate TCGA analysis, we measured the expression of miR-H25 by quantitative immunofluorescence in an additional cohort of 161 SEOC patients. Gene, miRNA expression, and cytotoxicity assay were performed on multiple ovarian cancer cell lines transfected with miR-H25 and miR-BART7. Outcome analysis was performed using multivariate Cox and Kaplan-Meier method. Viral miRNAs are more expressed in SEOC than in normal tissues. Moreover, Herpetic viral miRNAs (miR-BART7 from EBV and miR-H25 from HSV-2) are significant and predictive biomarkers of outcome in multivariate Cox analysis. MiR-BART7 correlates with resistance to first line chemotherapy and early death, whereas miR-H25 appears to impart a protective effect and long term survival. Integrated analysis of gene and viral miRNAs expression suggests that miR-BART7 induces directly cisplatin-resistance, while miR-H25 alters RNA processing and affects the expression of noxious human miRNAs such as miR-143. This is the first investigation linking viral miRNA expression to ovarian cancer outcome. Viral miRNAs can be useful to develop biomarkers for early diagnosis and as a potential therapeutic tool to reduce SEOC lethality.

Citation Format: Deep Pandya, Marisa Mariani, Mark McHugh, Mirko Andreoli, Steven Sieber, Shiquan He, Candice Dowell-Martino, Paul Fiedler, Giovanni Scambia, Cristiano Ferlini. Herpes virus microRNA expression and significance in serous ovarian 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 3994. doi:10.1158/1538-7445.AM2015-3994
Abstract 40: HB-EGF‑mediated CITED4 signaling in lung cancer
Authors: Hsieh Cheng-Han , Chou Yu-Ting , Wu Cheng-Wen
Published: August 2015

Abnormal activation of the EGF receptor (EGFR) signaling is a hallmark of lung cancer. However, the role of heparin-binding EGF-like growth factor (HB-EGF), one of EGFR ligands, in lung tumorigenesis has been unappreciated. In this study, we showed that HB-EGF was highly expressed in a subgroup of lung cancer cells, the proliferation of which was dependent on HB-EGF signaling. Members of CITED family proteins respond to various cytokine signalings, regulating cell proliferation; among them, we identified CITED4 as a downstream effecter of HB-EGF. We revealed that CITED4 was upregulated by HB-EGF through a STAT3 dependent pathway. Moreover, CITED4-silencing attenuated HB-EGF-mediated lung cancer cell proliferation. ChIP analysis and promoter reporter assay showed that STAT3 bound CITED4 promoter, enhancing CITED4 transcription. Co-immunoprecipitation analysis identified MYC as a CITED4-interacting protein. Furthermore, we found that CITED4 expression promoted MYC-mediated transcription of cyclin D1 promoter, leading to increased G1/S cell cycle transition. Thus, our findings support that HB-EGF-CITED4 signaling cascade plays a critical role in aberrant proliferation in lung cancer with potential as a biomarker for lung cancer progression.

Citation Format: Cheng-Han Hsieh, Yu-Ting Chou, Cheng-Wen Wu. HB-EGF‑mediated CITED4 signaling in lung 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 40. doi:10.1158/1538-7445.AM2015-40
Abstract 4015: miR-221 ans miR-222 expression in salivary gland tumors
Authors: Ianez Renata Carolina Fraga , Coutinho-Camillo Cláudia , Pinto Clóvis , Soares Fernando , Lourenço Silvia
Published: August 2015

Background: MicroRNAs (miRNAs) are a class of short noncoding RNAs, that regulate the gene expression by binding to the 3′ - untranslated region of target mRNAS and by blocking the translation or degradation of the mRNAs that mediate various pathophysiological process including the pathogenesis of diverse human cancers and important cellular functions such as proliferation, apoptosis, differentiation, and cell signaling. Salivary gland tumors are complex, due to their broad histological spectrum resulting from multiple tumor cell differentiation. We previously detected that the protein c-kit is involved in the process of salivary gland morphogenesis and tumorigenesis; other recent studies reported alterations in KIT gene in adenoid cystic carcinoma. The aim of the present study is to determine the expression of microRNAs targeting KIT in pleomorphic adenoma (PA) and adenoid cystic carcinoma (ACC), the most common benign and malignant salivary gland neoplasms, respectively, and that originate from the intercalated duct region. Material and Methods: Twenty-five pleomorphic adenoma, 14 adenoid cystic carcinoma and 10 non-neoplastic salivary gland samples were used and miRNA expression was evaluated by Real Time RT-PCR using TaqMan miRNA Assays (miR- 221 and 222). Results: Expression of miR-221 was detected in 13 CAC samples and 19 PA samples. In CAC, expression of miR-221 was downregulated in 4 out of 13 (30,77%) samples, upregulated in 4 out of 13 (30,77%) samples, and normal in 5 out of 13 (38,46%) samples. In PA, miR-221 expression was downregulated in 7 out of 19 (36,84%) samples, upregulated in 2 out of 19 (10,53%) samples, and normal in 10 out of 19 (52,63%) samples. Expression of miR-222 was detected in 14 CAC samples and 22 PA samples. In the majority of CAC samples, the expression of miR-222 was similar to that observed in non-neoplastic samples. In PA samples, expression of miR-222 was downregulated in 7 out of 22 (31,8%) samples, upregulated in 8 out of 22 (36,4%) samples, and normal in 7 out of 22 (31,8%) samples. Conclusion: The expression of miRNAs 221 and 222 do not present a definite pattern to discriminate PA from ACC. This might be due to different mechanisms involved in their tumor biology or due to their common site of origin (the intercalated duct) which could add some similar biological features to both neoplasms.

Citation Format: Renata Carolina Fraga Ianez, Cláudia Coutinho-Camillo, Clóvis Pinto, Fernando Soares, Silvia Lourenço. miR-221 ans miR-222 expression in salivary gland 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 4015. doi:10.1158/1538-7445.AM2015-4015
Abstract 4016: c-MET associated with osteogenesis in multiple myeloma patients by induction of MMP9 expression by HGF in BMSCs
Authors: Park Hyejoo , Oh Jeong In , Ahn Kwang-Sung , Koh Youngil , Yoon Sung-Soo
Published: August 2015

HGF-mediated c-MET signaling plays a critical key in the progression of multiple myeloma. That signaling is associated with cell proliferation, drug resistance and osteoblast. In addition dynamic interplay between MM cell and bone marrow stromal cell regulates that signaling activation. The expression level of HGF in bone marrow plasma of MM patients is higher when compared to case control. However, the role of HGF-mediated c-Met was obscure in the progression of MM. In this study, we elucidate the role of c-Met signaling in osteogenesis in MM using co-culture system.

In our previous data, we found that the expression levels of HGF in bone marrow serum of multiple myeloma patients was correlated with the expression levels of OPN and RUNX2. Also, those expression levels were associated with bone lesion. It suggested that HGF might be one of regulators in the progression of osteogenesis. However, mechanisms by HGF-mediated c-MET activation modulate the expression of matrix metalloproteinases still remain unknown. In our study, we found that Our data show that PI3K inhibitor effectively inhibited the MMP9 mRNA expression in both OPN alone treatment and co-treatment with OPN and HGF in BMSCs. Induction of OPN and RUNX2 by HGF in MM cells could be one of the major factors for the progression of MM through interaction of MM cells and BMSCs. We suggested that induction of OPN/MMP9 strongly associated with osteoclast.

For further analysis, we performed co-culture system which MM cells were grown with BMSCs obtained from MM patients’ bone marrow specimen in order to verify the role of HGF mediated c-MET activation in the progression of MM patient's osteogenesis. To explore the detail mechanisms of HGF-mediated osteogenesis, various inhibitors was used and the expression levels of a set of genes was examined using real-time PCR. Subsequently, HGF-mediated c-MET activation was regulated the expression of OPN/MMP-9 via co-effects of PIK3 activation and activation of wnt signaling pathway in BMSCs from bone marrow of MM patients. In conclusion, cross-talk between PI3K/AKT pathway and wnt singaling pathway could be one of the major factors for the progression of MM through interaction of MM cells and BMSCs.

Citation Format: Hyejoo Park, Jeong In Oh, Kwang-Sung Ahn, Youngil Koh, Sung-Soo Yoon. c-MET associated with osteogenesis in multiple myeloma patients by induction of MMP9 expression by HGF in BMSCs. [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 4016. doi:10.1158/1538-7445.AM2015-4016
Abstract 4019: Examining mechanisms by which macrophages enhance intravasation in breast cancer
Authors: Chiang Serena , Zhou Zhenni , Segall Jeffrey E.
Published: August 2015

Breast cancer is the most common invasive malignancy and a leading cause of cancer-associated deaths in women worldwide. Metastasis is the major cause of morbidity and mortality in women afflicted with breast cancer. In the metastatic process, intravasation is an important step and largely dependent on interactions between tumor cells and stromal cells in the tumor microenvironment (TME). Tumor-associated macrophages (TAMs), an integral component of the TME, have been found to associate and co-migrate with tumor cells during intravasation. Invasive TAMs that co-migrate with tumor cells in response to epidermal growth factor display a unique transcriptome in comparison to the general TAM population found in the TME. Furthermore, intercellular signaling between tumor cells and TAMs generates a positive feedback loop that promotes tumor cell invasion and intravasation. Chemokines play a major role in cell migration and are an important component of the TME. In particular, chemokine (C-X-C motif) receptor 2 (CXCR2) has been shown to be important in tumor growth and angiogenesis. MIF and IL8, two ligands of CXCR2, correlate with poor outcome in breast cancer. Our objective is to elucidate the role of macrophage CXCR2 in tumor cell intravasation.

We have used flow cytometry to characterize the basal expression of CXCR2 in a murine macrophage cell line, BAC-1.2F5 (BAC). We have also analyzed mRNA expression of CXCR2 in BAC and primary TAMs isolated from a murine model of breast cancer, the polyoma middle T oncoprotein (PyMT), by qRT-PCR. To test the functional significance of CXCR2 in intravasation, we have examined the effects of an inhibitor of CXCR2 on the ability of tumor cells to migrate through the endothelium using an in vitro transendothelial migration assay (iTEM). In this assay, MDA-MB-231 tumor cells, a human breast cancer cell line, are co-cultured with BAC cells and subsequently placed in an environment that is in contact with the basal surface of endothelial cells.

We have observed that BAC cells produce CXCR2 mRNA and express surface CXCR2. We have also found elevated expression of CXCR2 in invasive TAMs as compared to the general TAM population. When MDA-MB-231 cells are co-cultured with BAC cells in the iTEM assay in the presence of SB265610, an inhibitor of CXCR2, we have found a significant reduction (p<10-4) in iTEM. Together, these results suggest that cellular signaling through macrophage CXCR2 is critical to tumor cell intravasation.

Citation Format: Serena Chiang, Zhenni Zhou, Jeffrey E. Segall. Examining mechanisms by which macrophages enhance intravasation in 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 4019. doi:10.1158/1538-7445.AM2015-4019
Abstract 402: Pasireotide reduces chemoresistance in pancreatic tumor cells by inhibiting the synthesis and secretion of growth factors from tumor associated fibroblasts
Authors: Duluc Camille , Moatassim Siham , Chalabi Mounira , Perraud Aurélie , Martineau Yvan , Breibach Florence , Delisle Marie-Bernadette , Mathonnet Muriel , Schmid Herbert A. , Pyronnet Stéphane , Bousquet Corinne
Published: August 2015

Pancreatic ductal adenocarcinoma (PDAC) is extremely rich in stroma. By secreting tremendous amounts of soluble and un-soluble proteins, stromal cancer-associated fibroblasts (CAFs) can induce tumor chemoresistance by activating survival signals in cancer cells and preventing drug delivery through increased fibrosis. Our results demonstrate that chemoresistance triggered by the CAF secretome is strongly reduced upon inhibition of CAF protein synthesis. This is achieved by impinging upon the PI3K/mTOR/4E-BP1 pathway that we found highly activated in primary cultures of alpha-SMA-positive CAFs isolated from human PDAC. CAFs, but not normal pancreatic fibroblasts, express primarily the sstr1 somatostatin receptor subtype whose activation by the somatostatin analogue SOM230 (Pasireotide) strongly inhibits the PI3K/mTOR/4E-BP1 pathway and the resulting synthesis of secreted proteins including IL-6 and collagen-I. As a consequence, the growth and chemoresistance of pancreatic tumors provided by co-injection of CAFs in nude mice are overcome when chemotherapy (such as gemcitabine) is combined to pasireotide treatment. While gemcitabine or pasireotide alone have marginal effects, the combined treatment markedly decreases fibrosis and strongly enhances cancer cell apoptosis due to increased cell sensitivity to gemcitabine. We propose that selective inhibition of CAF protein synthesis and secretion with sstr1-directed pharmacological compounds represents an anti-stromal targeted therapy with a promising potential in chemosensitization.

Citation Format: Camille Duluc, Siham Moatassim, Mounira Chalabi, Aurélie Perraud, Yvan Martineau, Florence Breibach, Marie-Bernadette Delisle, Muriel Mathonnet, Herbert A. Schmid, Stéphane Pyronnet, Corinne Bousquet. Pasireotide reduces chemoresistance in pancreatic tumor cells by inhibiting the synthesis and secretion of growth factors from tumor associated fibroblasts. [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 402. doi:10.1158/1538-7445.AM2015-402
Abstract 4030: Interactions between co-cultured myoblasts and prostate cancer cells kill myoblasts but promote cancer cell proliferation and fusion: implications for cachexia and metastasis to skeletal muscle
Authors: Uygur Berna , Leikina Evgenia , Chernomordik Leonid V.
Published: August 2015

Severe muscle loss (cachexia) is the major cause for the cancer related death and 70% prostate cancer patients suffer from cachexia. Multinucleated cells of skeletal muscles are generated and regenerated by cell-to-cell fusion. It has been suggested that cell-to-cell fusion also takes place during carcinogenesis and promotes tumor cell diversity, drug resistance and the ability to metastasize. However fusion between cancer cells has remained elusive due to a very low efficiency of this process in earlier studies. Here we explored interactions between cancer cells and muscle environment in vitro. Co-culturing cells of human prostate cancer line (PC3) with primary murine proliferating myoblasts (muscle stem cells) resulted in differentiation and then apoptosis in muscle stem cells. Conditioned medium from PC3 cells also caused 50% decrease in proliferation of muscle stem cells. On the other hand, fusion-committed myoblasts co-cultured with PC3 cells lost their characteristic shapes and went through apoptosis. In addition to these effects of PC3 cells on murine myoblasts, we observed striking effects of myoblasts on PC3 cells. Co-culturing of PC3 cells with either proliferating myoblasts or fusion-committed myoblasts resulted in a robust fusion between cancer cells that yielded PC3 syncytia with up to 5 nuclei. Fusion promotion between PC3 cells was accompanied by a 1.5-fold increase in expression of Annexin A5, a protein recently implicated in myoblast fusion. In contrast, the expression of Annexin A5 in myoblasts was halved. Both co-culturing and using conditioned medium from fusion-committed myoblasts also resulted in a 2-fold increase in cancer cells proliferation and a 3-fold increase in the expression of the phosphorylated protein kinase AKT (pAKT). pAKT activity is associated with proliferation, drug resistance and cancer stem cell phenotype generation. Therefore we examined cancer stem cell marker, CD133 expression in PC3 cells. Treatment with conditioned medium from either proliferating or fusion-committed myoblasts doubled a percentage of CD133-expressing cells. Our results suggest an intriguing interplay between prostate cancer and muscle cells that may contribute to the progression of cancer and potentially explain cachexia.

Citation Format: Berna Uygur, Evgenia Leikina, Leonid V. Chernomordik. Interactions between co-cultured myoblasts and prostate cancer cells kill myoblasts but promote cancer cell proliferation and fusion: implications for cachexia and metastasis to skeletal muscle. [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 4030. doi:10.1158/1538-7445.AM2015-4030
Abstract 4045: MRAS and SHOC2 interact with SCRIB and coordinate ERK pathway activation with polarity and tumorigenic growth
Authors: Young Lucy C. , Hartig Nicole , Munoz-Alegre Marta , Oses-Prieto Juan A. , Durdu Sevi , Bender Sabine , Vijakumar Vineetha , Vietri Rudan Matteo , Gewinner Christina , Jathoul Amit P. , Ghatrora Rupinder , Lythgoe Mark L. , Burlingame Alma L. , Rodriguez-Viciana Pablo
Published: August 2015

SHOC2 is mutated in Noonan syndrome and plays a key role in the activation of the ERK-MAPK pathway, which is upregulated in the majority of human cancers. SHOC2 functions as a PP1-regulatory protein and as an effector of MRAS. Here we show that SHOC2 and MRAS form a complex with SCRIB, a polarity protein with tumor suppressor properties. SCRIB functions as a PP1-regulatory protein and antagonizes SHOC2-mediated RAF dephosphorylation through a mechanism involving competition for PP1 molecules within the same macromolecular complex. SHOC2 function is selectively required for the malignant properties of tumor cells with mutant RAS, and both MRAS and SHOC2 play a key role in polarized migration. We propose that MRAS, through its ability to recruit a complex with paradoxical components, coordinates ERK pathway spatiotemporal dynamics with polarity and that this complex plays a key role during tumorigenic growth.

Citation Format: Lucy C. Young, Nicole Hartig, Marta Munoz-Alegre, Juan A. Oses-Prieto, Sevi Durdu, Sabine Bender, Vineetha Vijakumar, Matteo Vietri Rudan, Christina Gewinner, Amit P. Jathoul, Rupinder Ghatrora, Mark L. Lythgoe, Alma L. Burlingame, Pablo Rodriguez-Viciana. MRAS and SHOC2 interact with SCRIB and coordinate ERK pathway activation with polarity and tumorigenic 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 4045. doi:10.1158/1538-7445.AM2015-4045
Abstract 4051: Protection from CTL mediated killing by iNOS expression in a mouse model of melanoma
Authors: Marvel Douglas M. , Gabrilovich Dmitry
Published: August 2015

The presence of peroxynitrite (PNT) in tumor tissue, as measured by its surrogate tissue marker, nitrotyrosine (NT), is correlated with poor clinical prognosis and poor response to immunotherapy. The production of PNT at the tissue can be attributed to multiple sources. For example, many tumors are characterized by extensive infiltration by immature myeloid cells with robust immuno-suppresive activity. These myeloid derived suppressor cells (MDSC) are known to produce high level of reactive oxygen species (ROS) and reactive nitrogen species (RNS), and the reaction of these two species is a major source of PNT at the tumor site. In addition to MDSC, iNOS expression in tumor cells themselves can act as a major source of PNT. Here we show that B16 melanoma cells are protected from CTL mediated lysis via iNOS expression. This effect is independent of effects on the T cells themselves as ability to lyse iNOS expressing and non-expressing cells was assessed in the same culture. Previously it was shown that peroxynitrite treated cells bound certain exogenous MHC class I peptides with lower affinity than their non-treated counterparts. Using a system of endogenous iNOS expression, our data supports the existence of a similar phenomenon for endogenously processed and presented peptide antigens, suggesting that the repertoire of surface bound immunogenic peptides is different in tumors when compared to the presentation of those same antigens in the tumor draining lymph node, where PNT is less prevalent. As a result, immune responses generated in the lymph node are against antigens which are not relevant and will fail to control tumor growth. Additionally, our data suggests that pharmaceutical intervention to reduce intratumoral PNT levels may buoy concurrent immunotherapy.

Citation Format: Douglas M. Marvel, Dmitry Gabrilovich. Protection from CTL mediated killing by iNOS expression in a mouse model of melanoma. [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 4051. doi:10.1158/1538-7445.AM2015-4051
Abstract 4065: Delineating the role of epithelial-mesenchymal transition in the generation and maintenance of prostate cancer stem cells
Authors: Paranjape Anurag N. , Soundararajan Rama , Werden Steven J. , Joseph Robiya , Taube Joseph H. , Bhangre Neeraja , Liu Hui , Rodriguez-Canales Jaime , Wistuba Ignacio I. , Chang Jeffrey T. , Tang Dean G. , Mahajan Nupam , Mahajan Kiran , Miura Naoyuki , Mani Sendurai A.
Published: August 2015

Prostate cancer cells depend on androgens for growth and survival. While androgen-deprivation-therapy (ADT) results in regression of the tumor bulk, in many cases, the cancers recur in aggressive androgen-independent forms that are also resistant to standard-of-care therapies. The prevailing hypothesis is that pre-existing androgen-independent prostate cancer (PCa) stem-cells in the primary tumor are selectively enriched following therapy. However, we have previously shown that induction of epithelial-mesenchymal-transition (EMT) in differentiated breast epithelial cells can result in the generation of cells with stem-cell properties. In the current study, we investigated if induction of EMT in PCa promotes stem-cell features and androgen-receptor (AR) regulation, and if targeting EMT-promoting pathways renders PCa stem-cells sensitive to ADT.

We found that LNCaP-PSAlow cells, which were previously shown to represent the PCa stem-cell population, exhibit EMT properties. Induction of EMT through over-expression of EMT-inducing transcription factors or with TGFβ1 treatment, significantly stimulated stem-cell properties in the androgen-sensitive LNCaP cells, whereas inhibition of EMT in the androgen-insensitive DU145 cells using targeted shRNA or a specific TGFβ1-signaling inhibitor, resulted in markedly reduced stemness. We observed that FOXC2 expression consistently correlated with induction of EMT and androgen-insensitivity in PCa cells. Our study demonstrates that targeting EMT-inducing molecules and signaling pathways could represent a tangible approach to inhibiting the generation and maintenance of therapy-resistant PCa stem-cells, that are the prime harbingers of tumor recurrence.

Citation Format: Anurag N. Paranjape, Rama Soundararajan, Steven J. Werden, Robiya Joseph, Joseph H. Taube, Neeraja Bhangre, Hui Liu, Jaime Rodriguez-Canales, Ignacio I. Wistuba, Jeffrey T. Chang, Dean G. Tang, Nupam Mahajan, Kiran Mahajan, Naoyuki Miura, Sendurai A. Mani. Delineating the role of epithelial-mesenchymal transition in the generation and maintenance of prostate cancer stem 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 4065. doi:10.1158/1538-7445.AM2015-4065