bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 412: SEC11A contributes to malignant progression through promotion of EGF and TGF-α secretion in gastric cancer.
Authors: Oue Naohide , Hayashi Tetsutaro , Ito Reiko , Nagatsuma Akiko , Sentani Kazuhiro , Sakamoto Naoya , Anami Katsuhiro , Uraoka Naohiro , Naito Yutaka , Yanagihara Kazuyoshi , Ochiai Atsushi , Sasaki Hiroki , Yasui Wataru
Published: August 2014

Gastric cancer (GC) is one of the most common human cancers. We previously performed Serial Analysis of Gene Expression (SAGE) on four primary GCs and identified several GC-specific genes. In the present study, we have built an in-house oligonucleotide array on which 394 genes selected based on our SAGE data and previously reported array data to allow for the study of the genes that are most relevant to gastric carcinogenesis. We focused on the SEC11A gene, which encodes SPC18 protein. SPC18 is one of the member of signal peptidase complex. SPC18 mRNA expression was measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in GC tissue samples. Expression and distribution of SPC18 protein was investigated by immunohistochemistry in two independent GC cohorts (Hiroshima cohort, n = 99 and Chiba cohort, n = 989). To determine the effect of SPC18 on cell growth and invasiveness in vitro, MTT and Boyden chamber invasion assays were performed. To evaluate the influence of SPC18 on cell growth in vivo, GC cells were injected into SCID mice. The levels of TGF-α and EGF in media from the GC cells were measured by ELISA. Overexpression of SEC11A mRNA was observed in 40% of GC cases by qRT-PCR. Immunohistochemical analysis of SPC18 revealed that 26% of GC cases were positive for SPC18 in Hiroshima cohort and 20% of GC cases were positive for SPC18 in Chiba cohort. SPC18-positive GC cases showed poorer survival than SPC18-negative GC cases in both Hiroshima cohort and Chiba cohort by Kaplan-Meier analysis. Forced expression of SPC18 activates GC cell growth in vitro and in vivo. The levels of TGF-α and EGF in culture media from GC cells were reduced by knockdown of SPC18. These results indicate that SPC18 contributes to malignant progression through promotion of EGF and TGF-α secretion in GC.

Citation Format: Naohide Oue, Tetsutaro Hayashi, Reiko Ito, Akiko Nagatsuma, Kazuhiro Sentani, Naoya Sakamoto, Katsuhiro Anami, Naohiro Uraoka, Yutaka Naito, Kazuyoshi Yanagihara, Atsushi Ochiai, Hiroki Sasaki, Wataru Yasui. SEC11A contributes to malignant progression through promotion of EGF and TGF-α secretion in gastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 412. doi:10.1158/1538-7445.AM2013-412
Abstract 4153: Arrayed microinjection of a ubiquitin activating enzyme inhibitor induces PD biomarker effects predictive of in vivo tumor responses to systemic drug delivery.
Authors: Hatton Beryl A. , Grenley Marc , Hedin Nathan , Caffo Nathan , Hyer Marc L. , Manfredi Mark , Blakemore Stephen , Klinghoffer Richard A. , Bence Neil
Published: August 2014

Presage technology enables simultaneous analysis of multiple cancer drug candidates, drug concentrations, and drug combinations within a single living tumor. The platform employs arrayed tumor microinjection technology that delivers multiple spatially defined “threads” of drug directly into discreet portions of a tumor. This enables rapid, reliable, and internally controlled cross comparisons of multiple cancer therapeutics using screening quantities of drug in an in vivo setting in which the local tumor microenvironment is maintained. Here, as an example, we apply the platform to investigate tumor responsiveness to an inhibitor of the ubiquitin activating enzyme UBA1 from Millennium Pharmaceuticals.

The ubiquitin activating enzyme UBA1 regulates ubiquitin activation and subsequent polyubiquitination of proteins necessary for their degradation by the proteasome, and functionally impacts cell signaling, DNA damage repair and cell cycle progression. In vivo tumor responses were evaluated in two human xenograft models, WSU-DLCL2 and MCF-7, grown as flank tumors in immune-compromised mice. Microdosing of multiple concentrations of the UBA1 inhibitor (UBAi) into both models led to localized, easily detectable, and drug concentration-dependent biomarker changes indicative of ubiquitin pathway perturbation in the area proximal to injected drug. This included loss of poly-ubiquitin, accompanied by the expected accumulation of cMyc in tumor regions exposed to the UBAi. Localized time-dependent tumor cell death responses were observed following pathway perturbation as quantified by staining for cleaved caspase-3 and gamma-H2AX staining. Furthermore, pathological evidence of UBAi-induced cell death was clearly visible in both tumor models upon histological examination of H&E stained slides. These results highlight the capacity of the Presage platform to perform multiplexed drug studies in live tumor models. This capability could readily be expanded for use in validating additional biomarker hypotheses, indication finding studies, or for efficient identification of novel drug combinations.

Citation Format: Beryl A. Hatton, Marc Grenley, Nathan Hedin, Nathan Caffo, Marc L. Hyer, Mark Manfredi, Stephen Blakemore, Richard A. Klinghoffer, Neil Bence. Arrayed microinjection of a ubiquitin activating enzyme inhibitor induces PD biomarker effects predictive of in vivo tumor responses to systemic drug delivery. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4153. doi:10.1158/1538-7445.AM2013-4153
Abstract 416: S100P, a target gene of MACC1, contributes to colorectal cancer metastasis.
Authors: Schmid Felicitas , Wang Qing , Huska Matthew R. , Andrade-Navarro Miguel A. , Lemm Margit , Fichtner Iduna , Walther Wolfgang , Schlag Peter M. , Stein Ulrike S.
Published: August 2014

Colorectal cancer (CRC) is the second leading type of cancer in the Western world. About 90% of the deaths caused by this cancer arise from the formation of metastasis. We previously identified the new gene MACC1 (metastasis associated in colon cancer 1) as a prognostic marker for CRC metastasis. MACC1 regulates the expression of the well-known proto-oncogene Met (met proto-oncogene). An overexpression of MACC1 promotes in vitro and in vivo the metastatic potential of CRC cells. This project aims at the identification and functional characterization of new transcriptional target genes of MACC1. The genome-wide expression of SW480 CRC cells and a SW480/MACC1 clone was studied by microarray analysis. We found the gene S100P (S100 calcium binding protein P) as a highly upregulated gene in the MACC1 clone. S100P mRNA and protein expression levels in MACC1 transfected SW480 cells were significantly increased compared to control cells. A reduction of the endogenously high MACC1 expression in SW620 cells by MACC1-siRNA resulted in a decreased S100P expression. Furthermore, we showed that MACC1 bound to the promoter region of S100P in SW480/MACC1 cells. SW480 cells with an ectopic high S100P expression had a higher cell proliferation, migration and invasion compared to SW480 control cells. In contrast, a decrease of the high endogenous S100P expression in SW620 cells led to a lower proliferative, migratory and invasive potential. In a NOD SCID mouse model, animals intraspenically transplanted with control cells did not metastasize. In contrast, mice with S100P overexpressing cells developed liver metastasis (p<0.0001). Finally, in human colorectal tumors the S100P expression was higher in tumors (stages II and III) which developed metachronous metastasis compared to non-metastasizing tumors (p=0.05). Colorectal cancer patients with a high S100P expression in their primary tumors had a shorter metastasis-free survival time compared to patients with S100P low expressing tumors (p=0.03, HR=0.37, 95% CI=0.13-0.90). Taken together, the gene S100P is a new transcriptional target gene of MACC1 and represents a biomarker for CRC metastasis as well as a target for therapeutical approaches.

Citation Format: Felicitas Schmid, Qing Wang, Matthew R. Huska, Miguel A. Andrade-Navarro, Margit Lemm, Iduna Fichtner, Wolfgang Walther, Peter M. Schlag, Ulrike S. Stein. S100P, a target gene of MACC1, contributes to colorectal cancer metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 416. doi:10.1158/1538-7445.AM2013-416
Abstract 4162: MicroRNA-34 family is increased in human colon cancer and targets Peroxisome proliferator-activated receptor γ.
Authors: Hiyoshi Yukiharu , Schetter Aaron J. , Horikawa Izumi , Baba Hideo , Harris Curtis C.
Published: August 2014

Purpose: The microRNA-34 family (miR-34a, -34b and -34c) have been reported to be tumor suppressor microRNAs (miRNAs) that are regulated by the TP53 and DNA hypermethylation. However, the expression, regulation, and prognostic value of the miR-34 family have not been systematically studied in colon cancer. The aim of this study is to elucidate the roles of miR-34 family in colon carcinogenesis.

Methods: MiR-34a/b/c were measured in tumors and adjacent noncancerous tissues from 159 American and 113 Chinese colon cancer patients using quantitative RT-PCR. We examined associations between miR-34a/b/c expression with TNM staging, cancer-specific mortality, TP53 mutation status, KRAS mutation status, promoter methylation and

Affymetrix microarray data. Ingenuity Pathway Analysis (IPA) identified transcriptional regulators that were involved with miR-34 expression. In vitro experiments were performed to identify the target gene of miR-34 family.

Results: All miR-34 family members were significantly increased in colon tumors, counter to the proposed tumor suppressor role for these miRNAs. Increased miR-34b/c were observed in more advanced tumors in two independent cohorts and increased expression of miR-34b/c was associated with poor cancer-specific mortality. While the expression of miR-34 family was not associated with TP53 mutation status, TP53 transcriptional activity was associated with miR-34a/b/c expression which is consistent with the proposed regulation of miR-34a/b/c by TP53. KRAS mutation status was associated with increased miR-34a expression. Hypermethylation of miR-34a and miR-34b/c was found in tumors. IPA found that the activity of the tumor suppressor gene Peroxisome Proliferator-Activated Receptor γ (PPARG) was negatively associated with miR-34 expression, and in vitro experiments identified PPARG as a direct target of miR-34 family.

Conclusion: miR-34 family may have important roles in colon carcinogenesis as oncogenic miRNAs by targeting PPARG.

Citation Format: Yukiharu Hiyoshi, Aaron J. Schetter, Izumi Horikawa, Hideo Baba, Curtis C. Harris. MicroRNA-34 family is increased in human colon cancer and targets Peroxisome proliferator-activated receptor γ. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4162. doi:10.1158/1538-7445.AM2013-4162
Abstract 4165: Combination of cell-free miR-198, carcinoembryonic antigen, and CYFRA 21-1 as a diagnostic biomarker for lung adenocarcinoma-associated malignant pleural effusion.
Authors: Han Hye Sook , Yun Jieun , Lee Ok-Jun
Published: August 2014

Circulating cell-free microRNAs (miRNAs) have been identified as potential noninvasive biomarkers in cancer. The aim of this study was to identify miRNAs that are differentially expressed between benign pleural effusion (BPE) and lung adenocarcinoma-associated malignant pleural effusion (LA-MPE). We have also analyzed the expression of carcinoembryonic antigen (CEA) and cytokeratin fragment (CYFRA) 21-1, which are the most commonly used tumor markers in pleural effusion. Cell-free miRNA expression was investigated in 107 patients with pleural effusion. Microarrays were used to screen 160 miRNAs in a discovery set comprising 20 effusion samples (10 BPEs and 10 LA-MPEs). The PANArrayTM miRNA expression profiling kit (PANAGENE, INC. Daejeon, Korea) was used to identify candidate miRNAs in the discovery set. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to validate the profiling results obtained for the discovery set and those obtained for a validation set comprising 42 BPEs and 45 LA-MPEs. The area under the receiver operating characteristic curve (AUC) was used to evaluate the diagnostic performance of the identified miRNAs, CEA, and CYFRA 21-1. Microarray profiling showed that miR-198 was significantly downregulated in LA-MPE compared with BPE (P = 0.002). The miRNA microarray analysis results were confirmed by qRT-PCR (P < 0.001) using the validation set. miR-198 showed an AUC of 0.887, with a sensitivity of 71.1% and a specificity of 95.2%. The AUC for CEA was 0.898, with a sensitivity of 78.4% and a specificity of 97.5%. The AUC for CYFRA 21-1 was 0.836, with a sensitivity of 78.4% and a specificity of 82.5%. The diagnostic performance of miR-198 was comparable with that of CEA, but better than that of CYFRA 21-1. The AUC for all three markers combined was 0.926 (95% confidence interval, 0.843-0.973) with a sensitivity of 89.2% and a specificity of 85.0%. The present study shows that expression of miR-198 was significantly downregulated in LA-MPE compared with BPE, and combination of cell-free miR-198, CEA and CYFRA 21-1 can be used to differentiate LA-MPE from BPE with a high degree of accuracy. These results suggest that miR-198 in pleural effusions may be a potential diagnostic biomarker for discriminating between BPE and LA-MPE.

Citation Format: Hye Sook Han, Jieun Yun, Ok-Jun Lee. Combination of cell-free miR-198, carcinoembryonic antigen, and CYFRA 21-1 as a diagnostic biomarker for lung adenocarcinoma-associated malignant pleural effusion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4165. doi:10.1158/1538-7445.AM2013-4165
Abstract 4167: Target mRNA analysis for miR-125a-5p in cervical carcinoma cells.
Authors: Martinez Acuna Natalia , Benitez Hess Maria L. , Alvarez Salas Luis M.
Published: August 2014

It has been firmly established that high-risk human papillomavirus (HPV) infection is associated with the appearance and persistence of anogenital dysplasia. However, only a small fraction of HPV-infected patients ever develop cervical cancer. Therefore, other factors must contribute for the development of a malignant phenotype in HPV-infected cells. Aberrant microRNA (miRNA) expression is nowadays recognized as a key factor for the development of several pathologies including cancer. Several studies on the miR-125a-5p function have suggested a tumor suppressor role in diverse cell types. Our previous results shown that miR-125a-5p induced cell-cycle arrest, proliferation inhibition and apoptosis in cervical carcinoma cells. Nevertheless, miR-125a-5p role and mRNA targets in cervical cancer are not fully established in cervical cancer. RT-qPCR analysis of miR-125a-5p expression showed differential expression on immortal and tumor cervical cell lines suggesting a role in malignant progression. To probe for possible miR-125a-5p targets we performed in silico analyses using several miRNA target analysis tools and validated the results with a panel of cervical immortal and tumor cell lines. The bio-informatics analysis showed Mark1, bcl-2 and vEGF genes as possible targets for miR-125a-5p. Further RT-qPCR and immunoblot analysis indicated an inverse correlation of miR125a-5p and MARK1 expression in tumor lines but not in immortal lines irrespective of the HPV content. Interestingly, transfection of a pre-miR-125a-5p mimic in C33-A cells (lacking HPV and with low endogenous miR-125a-5p levels), resulted in specific down-regulation of MARK1 and VEGF. Therefore, we suggest a potential role for miR-125a-5p in cervical carcinogenesis through the regulation of MARK1.

Citation Format: Natalia Martinez Acuna, Maria L. Benitez Hess, Luis M. Alvarez Salas. Target mRNA analysis for miR-125a-5p in cervical carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4167. doi:10.1158/1538-7445.AM2013-4167
Abstract 4168: The role of miRNA-125a-Fyn interplay in prostate cancer tumorigenesis.
Authors: Ninio Many Lihi , Ben-Aharon Irit , Zilber Sofia , Levy Mattan , Stemmer Salomon M. , Shalgi Ruth
Published: August 2014

Background: The SRC-family of kinases (SFKs) has been broadly implicated in the pathogenesis of prostate cancer. Fyn, a member of the SFKs, mediates mitogenic signals and regulates proliferation, adhesion and motility. Cumulative data indicate that Fyn serves as a proto-oncogene and is known to be over-expressed in several types of malignancies. MicroRNAs are noncoding RNAs that are aberrantly expressed in cancer and seem to influence tumor behavior and progression. In a previous study performed in human embryonic kidney (HEK 293T) cells, we have validated the binding specificity of miR-125a-3p and Fyn 3’UTR using Luciferase assay and established the regulation of Fyn expression and signaling by miR-125a-3p. In the current study we aimed to characterize the interplay between miRNA-125a-3p and Fyn in prostate cancer. Methods: Prostate cancer (PC3) cell line, were transiently transfected with empty miR-vec (control) or with miR-125a-3p and their cell cycle and apoptotic rate were measured by fluorescence activated cell sorting (FACS). The migration ability was measured by transwell assay and scratch assay. Proliferation was assessed by MTT assay. Quantitative PCR and western blot analysis were implicated to determine the effect of miR-125a-3p on Fyn expression as well as on the activation state of its downstream effectors/proteins FAK, Akt and paxillin. Extraction of miRNA from formalin fixed paraffin-embedded (FFPE) cancerous and matched adjacent prostate tissues was performed with miRNeasy FFPE Kit on a cohort of 20 human prostate cancer specimens.

Results: Overexpression of miR-125a-3p in PC3 cells reduced significantly the expression of Fyn mRNA and protein and led to impaired cell viability to an arrest at the G2/M phase of the cell cycle. Moreover, overexpressing of miR-125a-3p reduced the activity of FAK, Akt and paxillin and the secretion of VEGF to the culture media. In miRNA125a-3p-overexpressing PC3 cells the migration was compromised by 50% and cell viability decreased by 50%. Moreover, we observed an inverse correlation between the expression of miR-125a-3p and the Gleason score of tissue samples obtained from patients diagnosed with prostate cancer, whereas the expression of miR-125a-3p was extremely reduced in Gleason score higher than 8.

Conclusions: Our study indicates miR-125a-3p signature in cellular pathways accounted for cell viability and migration of prostate cancer cells. The interplay between miR-125a-3p and Fyn may represent a new plausible therapeutic target in prostate cancer.

Citation Format: Lihi Ninio Many, Irit Ben-Aharon, Sofia Zilber, Mattan Levy, Salomon M. Stemmer, Ruth Shalgi. The role of miRNA-125a-Fyn interplay in prostate cancer tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4168. doi:10.1158/1538-7445.AM2013-4168
Abstract 4177: Detection of mir-449 and mir-let-7g expression in Taiwan paired hepatocellular carcinoma.
Authors: Zhang Yu-Jing , Wu Hui-Chen , Shen Jing , Yu Ming-Whei , Santella Regina M.
Published: August 2014

Hepatocellular carcinoma (HCC) is one of the most common and rapidly fatal human malignancies. In recent decades, it has been increasing in Western, developed countries such as the United States. MicroRNAs (miR) regulate genes involved in diverse biological functions and alterations in miR expression have been linked to the pathogenesis of many malignancies. Some previous studies have reported that miR-449 and miR-let-7g have tumor suppressor functions, but other studies indicate that miR-449 may have more variable functions. In the present study, miR-449 and miR-let-7g expression were measured in 36 Taiwanese paired HCC tumor/adjacent tissues using formalin fixed paraffin embedded samples with real-time RT-PCR. The mean log levels of miR-449 expression in tumor and adjacent non-tumor tissues are -1.14 and -2.65, respectively (p=0.02); for miR-let-7g, the mean log levels of tumor and adjacent non-tumor expression are 5.40 and 5.49 respectively (p=0.87). The relationship between exposure to chemical carcinogens as indicated by levels of polycyclic aromatic hydrocarbon (PAH)-DNA adducts and miR expression was also investigated. The mean log levels of miR449 in tumor tissues with high, moderate and low PAH-DNA adduct levels are -2.27, -2.22 and -0.43, respectively (p=0.02). The log levels of miR449 in tumors with and without cirrhosis are 0.02 and -1.42, respectively (p=0.02). These findings suggest that chemical carcinogen exposure and cirrhosis status may affect miR449 expression in HCC. Larger sample sizes and studies on the underlying mechanisms of miR449 and mir-let-7g expression in HCC are needed.

Citation Format: Yu-Jing Zhang, Hui-Chen Wu, Jing Shen, Ming-Whei Yu, Regina M. Santella. Detection of mir-449 and mir-let-7g expression in Taiwan paired hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4177. doi:10.1158/1538-7445.AM2013-4177
Abstract 4191: MiR-20a inhibits thyroid cancer cell growth and invasion through LIMK1.
Authors: Xiong Yin , Zhang Lisa , Kebebew Electron
Published: August 2014

There have been conflicting reports regarding the function of miR-20a in a variety of cancers. In this study, we evaluated the expression and function of miR-20a in thyroid cancer tissue and cell lines in vitro and in vivo. We found miR-20a was significantly downregulated in undifferentiated and aggressive thyroid cancer (p<0.05). Ectopic overexpression of miR-20a significantly inhibited thyroid cancer cell proliferation in vitro (p < 0.01) and in vivo (p < 0.01), tumor spheroid formation (p < 0.05), and invasion (p < 0.05) in multiple thyroid cancer cell lines. We performed integrated transcriptome analysis with and without miR-20a overexpression and target prediction analysis, and identified LIMK1 as a target of miR-20a. Luciferase assay demonstrated that LIMK1 was a direct target of miR-20a and that direct knockdown of LIMK1 recapitulated the effects of miR-20a in multiple thyroid cancer lines. Our findings suggest that miR-20a has a tumor suppressive function in thyroid cancer which is mediated through LIMK1 and that loss of miR-20a expression may be involved in thyroid cancer progression.

Citation Format: Yin Xiong, Lisa Zhang, Electron Kebebew. MiR-20a inhibits thyroid cancer cell growth and invasion through LIMK1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4191. doi:10.1158/1538-7445.AM2013-4191
Abstract 4197: Function of microRNA-143 and its potential of prognostic factor in scirrhous type gastric cancer.
Authors: Naito Yutaka , Sakamoto Naoya , Uraoka Naohiro , Sentani Kazuhiro , Oue Naohide , Yashiro Masakazu , Hirakawa Kosei , Yasui Wataru
Published: August 2014

Gastric cancer (GC) is one of the most common malignancies worldwide. Scirrhous type GC is highly metastatic and characterized clinically by rapid disease progression and poor prognosis. MicroRNAs (miRNAs), small non-coding RNA of 19-25 nucleotides, play a part in crucial biological processes and also have an important role in cancer development and progression. In the present study, we show that microRNA-143 (miR-143) is expressed at higher level in diffuse type GC than in intestinal type GC by microRNA microarray analysis. We investigated whether miR-143 is useful as a diagnostic marker of scirrhous type GC. Quantitative RT-PCR analysis revealed that miR-143 expression was low in GC in comparison with adjacent non-tumorous mucosa. Expression levels of miR-143 were higher in scirrhous type GC than those in other types of GC. Treatment of 5-aza-2′-deoxycytidine retrieved miR-143 expression, suggesting that miR-143 might be repressed by DNA methylation. Quantitative RT-PCR and in situ hybridization analysis showed that miR-143 is expressed at higher level in normal fibroblast and cancer associated fibroblast cell lines than in GC cell lines, indicating that major source of miR-143 in cancer tissue is stromal fibroblasts rather than cancer cells. miR-143 knock down by RNAi repressed collagen type III expression in normal fibroblast and cancer associated fibroblast cell lines. Furthermore, TGF-β treatment induced expression of miR-143 and collagen type III mRNA, and this induction of collagen type III mRNA was repressed by miR-143 knock down. We also studied the relation of miR-143 expression with GC patient prognosis (n=68). High miR-143 expression was associated with worse prognosis (P=0.0141). These data suggest that miR-143 regulates fibrosis of scirrhous type GC through induction of collagen expression in stromal fibroblasts and miR-143 expression is a prognostic marker of GC.

Citation Format: Yutaka Naito, Naoya Sakamoto, Naohiro Uraoka, Kazuhiro Sentani, Naohide Oue, Masakazu Yashiro, Kosei Hirakawa, Wataru Yasui. Function of microRNA-143 and its potential of prognostic factor in scirrhous type gastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4197. doi:10.1158/1538-7445.AM2013-4197
Abstract 4201: miR-124 suppresses colorectal tumor progression in a mouse xenograft model via targeting KITENIN.
Authors: Park So-Yeon , Bae Jeong A , Yoon Somy , Kim Hangun , Kim Kyung Keun
Published: August 2014

MicroRNAs (miRNAs) are increasingly implicated in modulating the progression of various cancers. Although there is emerging evidences that some miRNAs can function as oncogenes or tumour suppressors, the regulation of colorectal cancer (CRC) progression by miRNA is not fully understood. KITENIN (KAI1 C-terminal interacting tetraspanin, Vangl1) is not only a major component of PCP pathway but also a functional protein promoting CRC cell motility and invasiveness. Previously, we observed that KITENIN acts as a metastasis-enhancing protein in a mouse model of colon cancer and that KITENIN is highly expressed in sporadic human CRC tissues. But the underlying regulation mechanisms of aberrant expression of KITENIN in CRC are not clearly understood. Here we tried to identify the miRNAs, which modulate the expression of KITENIN, using luciferase assay and with computational prediction of miRNA targeting KITENIN. We identified several miRNAs and one of them is miR-124. miR-124 negatively regulated KITENIN expression through binding to the 3′-untranslated region of KITENIN. We further examined whether increased expression of miR-124 affects colorectal tumor progression via targeting KITENIN. Ectopic expression of miR-124 was found to suppress the migration and invasiveness of various CRC cells. miR-124 also inhibited tumor growth in a mouse xenograft model. These findings provide the experimental evidence to a possible therapeutic effect of miR-124 on suppressing colorectal tumor progression and identification of miR-124-mediated regulation of KITENIN might provide a promising therapeutic target in treating CRC.

Citation Format: So-Yeon Park, Jeong A Bae, Somy Yoon, Hangun Kim, Kyung Keun Kim. miR-124 suppresses colorectal tumor progression in a mouse xenograft model via targeting KITENIN. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4201. doi:10.1158/1538-7445.AM2013-4201
Abstract 4226: PIK3CA mutation detection in clinical samples using PNA mediated real-time PCR clamping assay..
Authors: Kim Hysun , Choi Jae Jin , Park Heekyung
Published: August 2014

Somatic mutations in the PIK3CA gene have been frequently found in various human cancers, and their presence correlates to therapy response. These somatic mutations within the PIK3CA gene are localized in exon 9 and 20.

We have developed a highly sensitive and simple method to detect 13 mutations in exons 9 and 20 of PIK3CA using PNA-mediated real-time PCR clamping, PNAClampTM.

PNAClampTM is based on unique properties of PNA probes that PNA oligomers are not recognized by DNA serving as sequence-selective clamps during

PCR amplification. This method can also detect 13 mutations through only three tube reactions each with 10 ng of the target DNA.

PNAClampTM is simple and reliable with a detection limit of approximately 3% mutant alleles using 10 to 50 ng of normal DNA as a template. The turnaround time for performing this assay was only 2 hours. Furthermore, while direct sequencing method will detect most of PIK3CA samples as having E545A mutation, due to homology in exon 9 in PIK3CA gene, highly specific PNAClampTM Method have overcome this perceived problem.

In clinical studies with breast cancer patients in Korea, PIK3CA mutations were detected utilizing PNAClampTM in 60 (35.7 %) of clinical samples, and the results were compared with direct sequencing method. Among 168 patients, 6 (3.6

%) had mutations in codon 542, 23 (13.7 %) had mutations in codon 545 and 31 (18.5

%) had mutations in codon 1047.

To confirm these results, the same clinical samples were analyzed with a sequencing assay. Consistent results were obtained from 137 (81.5 %) of the 168 clinical samples between PNAClampTM and the sequencing assay. The results not in agreement with the sequencing numbered 21 (12.5 %) samples. Ten samples did not react by direct sequencing due to quality problems with the target DNA. The remaining samples were initially determined as the wild type by direct sequencing, but they were found to be PIK3CA mutant using PNAClampTM.

This result was likely due to the high sensitivity of PNAClampTM.

PNAClampTM is a rapid, sensitive and easy to perform tool that can be used for the detection of PIK3CA mutations in the clinical field.

[j1]Check this - original was unclear. Is this what you mean?

Citation Format: Hysun Kim, Jae Jin Choi, Heekyung Park. PIK3CA mutation detection in clinical samples using PNA mediated real-time PCR clamping assay. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4226. doi:10.1158/1538-7445.AM2013-4226
Abstract 4237: Isoenzyme-selective HDAC activity assays.
Authors: Kupcho Kevin R. , Evans Nathan J. , Niles Andrew L. , Lazar Dan F. , Kirkland Thomas A.
Published: August 2014

Histone deacetylases (HDACs) are the family of enzymes which catalyze removal of acetyl groups from lysine residues on both histone and non-histone proteins. The lysine acetylation state of histones is an important post-translational modification which affects both chromatin structure and gene transcription. Thus, by deacetylating the amino-terminal tails of histones, HDACs have a profound effect on numerous and diverse cellular functions including cell proliferation. Given this important regulatory role, as well as evidence for significant HDAC involvement in the modulation of cancer, rapid advancement has been made in the development of broad-spectrum HDAC inhibitors. Recently, there has been an increased focus on the development of isoenzyme-specific HDAC inhibitors which may lead to improved therapy, including better efficacy and reduced off target effects. To enable selective HDAC studies, we developed assays that selectively measure specific isoenzyme activities in cells, extracts, or recombinant proteins. The assays use a homogeneous luminescent approach with a single reagent addition that is amenable to HTS. Isoenzyme-selective, luminogenic acetylated peptide substrates provide the basis for HDAC isoenzyme selectivity. Peptide deacetylation is measured by exposure to developer and a luciferin detection reagent. In addition to isoenzyme specificity, the assays show approximately 100-fold or better enhanced sensitivity over fluorescence methods and the versatility necessary for both biochemical and cell-based formats.

Citation Format: Kevin R. Kupcho, Nathan J. Evans, Andrew L. Niles, Dan F. Lazar, Thomas A. Kirkland. Isoenzyme-selective HDAC activity assays. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4237. doi:10.1158/1538-7445.AM2013-4237
Abstract 4254: DNA methylation profiling of ovarian tumors by methylation ligation-dependant macroarray.
Authors: Chelbi Sonia T. , Losi Lorena , Saponaro Sara , Martin Patricia , Braunschweig Richard , Benhattar Jean
Published: August 2014

Background: Ovarian cancer is the gynecological disease with the highest death rate. Efforts towards ovarian cancer research are needed to develop early detection tools, better therapies, cures and to prevent the disease. DNA methylation profiling of a large set of putative cancer biomarkers may help to identify relevant biological aspects of ovarian cancer.

Materials and Methods: We selected 106 patients diagnosed for ovarian cancer (35 serous, 30 endometrioid, 32 mucinous and 8 germinal tumors) for which clinicopathological informations were available and 17 histologically normal ovarian tissues as control. In order to identify methylation signatures, a method called Methylation Ligation-dependant Macroarray (MLM) and developed in our laboratory was applied to DNA extracted from microdissected formalin-fixed and paraffin-embedded tissue sections. The MLM technology, derived from a multiplex ligation assay coupled with an array-based analysis of uniform size amplification products, permits the methylation status analysis of 60 promoter regions at the same time.

Results: Each of the 106 ovarian tumor samples analyzed had a unique methylation profile. Hierarchical clustering identified mainly two groups of patients with distinct methylation profiles. The first group of tumors contained low levels of methylation, whereas the second group had higher methylation levels. The group 1 included normal ovarian tissues as well as a large number of ovarian serous tumors. Endometrioid and mucinous tumors were more represented in the group 2. When looking at the gene level, the highest methylation frequency was detected in the TAC1, SPARC, ADAMTS12 and hTERT promoter genes. The promoter of the BORIS/CTCFL cancer testis-antigen gene was found hypomethylated in several tumors, mainly in serous and germinal types. Located in the X-chromosome, the DAX1 promoter gene, which is partially methylated in normal ovarian tissues, was found hypomethylated or hypermethylated in tumors. Interestingly, the difference between the hypo- and hyper-methylated groups concerned mainly genes belonging to the WNT pathway.

Conclusion: The marked differences in methylation profiles independently of histological subtypes underscore the importance of characterizing tumors at the molecular level. Further investigations are needed to evaluate the role of the WNT signaling pathway as a marker and putative target for cisplatin chemosensitivity in ovarian tumors.

Citation Format: Sonia T. Chelbi, Lorena Losi, Sara Saponaro, Patricia Martin, Richard Braunschweig, Jean Benhattar. DNA methylation profiling of ovarian tumors by methylation ligation-dependant macroarray. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4254. doi:10.1158/1538-7445.AM2013-4254
Abstract 4259: Active hedgehog (HH) signal transduction in rhabdomyosarcoma (RMS).
Authors: Yoon Joon Won , Lamm Marilyn , Leong King-Fu , Iannaccone Stephen , Iannaccone Philip , Walterhouse David
Published: August 2014

RMS is the most common sarcoma of childhood. The majority of embryonal RMS (ERMS) and a minority of alveolar RMS (ARMS) express HH pathway components. However, a role for the HH signal transduction pathway is incompletely defined in RMS. Co-regulation of GLI1 and Patched1 (PTCH1) in ERMS and fusion-negative ARMS have suggested that the HH pathway is functionally intact in these subtypes. However, HH pathway activity has not been directly tested in RMS. Our aim was to demonstrate that some RMS cells have intact HH signaling pathways and are responsive to HH pathway activation or inhibition. We demonstrated expression of Indian HH, Desert HH, PTCH1, SMO and GLI1 by qRT PCR in Rh18 ERMS cells and Rh41 ARMS cells; Desert HH, PTCH1, SMO, and GLI1 in Rh 30 ARMS cells; and Indian HH, Desert HH, PTCH1, and SMO in RD ERMS cells. We confirmed high GLI1 protein expression by Western blot in Rh41 and Rh18 cells. Since HH signaling is believed to occur in primary cilia, we used immunofluorescence to demonstrate primary cilia in RMS-13 cells. Based on their differing levels of GLI1 expression (high in Rh41 and Rh18 cells and low/absent in Rh28 and RD cells), these RMS cell lines provided an opportunity to up- and down-regulate the HH pathway and measure effects on gene expression and cell function. Exposure of Rh41 cells to Sonic HH-containing conditioned media caused a 12-fold increase in PTCH1 expression and exposure of Rh28 cells to Sonic HH peptide increased GLI1 expression 1.5-fold, suggesting intact HH signal transduction in some RMS cell lines. We down-regulated HH pathway activity with the GLI1 inhibitor GANT61 and assessed Rh41 and RD cell viability by MTT assays. RMS cell viability decreased following exposure to increasing concentrations of GANT61. The reduced cell viability appeared to result at least in part from down-regulation of the anti-apoptotic GLI1 target gene, BCL2. These results suggest a role for HH signaling in RMS cell survival. Understanding the role of HH signaling in RMS biology may contribute to successful introduction of HH pathway inhibitors into RMS treatment.

Citation Format: Joon Won Yoon, Marilyn Lamm, King-Fu Leong, Stephen Iannaccone, Philip Iannaccone, David Walterhouse. Active hedgehog (HH) signal transduction in rhabdomyosarcoma (RMS). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4259. doi:10.1158/1538-7445.AM2013-4259
Abstract 4262: The role of SPP1 in NPC microenvironment.
Authors: Chen Jinna
Published: August 2014

Nasopharyngeal Carcinoma (NPC) is highly associated with EBV infection and lymphocytes infiltration, which contribute to form the NPC microenvironment. To better understand the NPC microenvironment, we conducted PCR array using pooled cDNA mixtures from 5 NPC tissues and their counterpart non-tumor tissues to profile inflammation-associated gene expression. We found that the Th1/Th2 related cytokines were down regulated in NPC tissues. This result was verified by Real-time quantitative PCR using 13 pairs of NPC samples and non-tumor tissues. The result of IHC staining of NPC tissues with T helper cell marker CD4 also showed decreased infiltration of T helper cells. Lower level of Th1/Th2 related cytokines were also detected in NPC serum compared to paired non-tumor serum. Osteopontin (SPP1), a glycophosphoprotein, playing roles in inflammation, bone formation, and tumor progression and metastasis, was over expressed in 67% (8/12) of NPC samples compared with non-tumor tissues. IHC staining of SPP1 in NPC tissues also showed up-regulation of SPP1 in either NPC cells or lymphocytes. To study the effect of SPP1 on T helper cells population, we cultured PBMC by adding SPP1 protein at the concentration from 0 to 100 ng/ml into the culture medium, followed by Flow Cytometry analysis. We found that Low-dose SPP1 protein stimulated T helper cell proliferation, which could be inhibited by SPP1 at high dose. Our data indicated that the over-expression of SPP1 by NPC cells might contribute to tumor immunosuppression in NPC microenvironment, thus promote cancer progression.

Citation Format: Jinna Chen. The role of SPP1 in NPC microenvironment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4262. doi:10.1158/1538-7445.AM2013-4262
Abstract 4268: Resistance to growth inhibition by transforming growth factor-beta is associated with activation of the PI3K-Akt pathway in HPV16-immortalized human keratinocytes at late stages of in vitro progression.
Authors: Velidandla Rupa , Vaswani Gehana , Kowli Sangeeta , Pirisi-Creek Lucia , Creek Kim E.
Published: August 2014

Transforming growth factor-beta (TGF-ß) plays a vital role in embryonic development, cell proliferation, inflammation and wound repair. The Smad proteins are the central mediators of TGF-ß signal transduction. However, recently several non-Smad pathways (ex., Erk, Jnk, p38 MAPK, PI3K-Akt) have been determined to play key roles in TGF-ß signaling. TGF-ß signaling is often disrupted in cancer, including cervical cancer. Human papillomavirus (HPV) is the main etiological agent in cervical cancer. HPV type 16 (HPV16)-immortalized human keratinocytes (HKc/HPV16) are initially very sensitive to growth inhibition by TGF-ß. However, HKc/HPV16 become increasingly resistant to TGF-ß during in vitro progression, and are completely resistant to TGF-ß-induced growth inhibition at late-stages of transformation, when they exhibit a differentiation resistant phenotype (HKc/DR). Although HKc/DR are completely resistant to the growth inhibitory effects of TGF-ß, some Smad signaling remains. A reporter construct containing tandem repeats of Smad binding elements was induced by TGF-ß in HKc/DR at about 50% of the level observed in HKc/HPV16. Furthermore, TGF-ß was capable of inducing epithelial-mesenchymal transition (EMT) in HKc/DR. To further explore TGF-ß signaling in HKc/HPV16 and HKc/DR, we used phospho specific antibody microarrays from Full Moon BioSystems. These arrays feature phospho and non-phospho pairs of specific antibodies pertinent to the TGF-ß pathway. Protein extracts from HKc/HPV16 and HKc/DR untreated or treated for 90 min with 40 pM TGF-ß were compared on the antibody microarrays. Activation of the Akt pathway by TGF-ß was observed in HKc/DR but not in HKc/HPV16. Similarly, PI3K and mTOR showed activation by TGF-ß in HKc/DR but not HKc/HPV16. We conclude that TGF-ß signals primarily through Smad-dependent pathways to inhibit cell proliferation in HKc/HPV16, but that non-canonical (non-smad) signaling through the PI3K-Akt-mTOR axis predominates in HKc/DR.

Citation Format: Rupa Velidandla, Gehana Vaswani, Sangeeta Kowli, Lucia Pirisi-Creek, Kim E. Creek. Resistance to growth inhibition by transforming growth factor-beta is associated with activation of the PI3K-Akt pathway in HPV16-immortalized human keratinocytes at late stages of in vitro progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4268. doi:10.1158/1538-7445.AM2013-4268
Abstract 4278: Mapping deregulated pathways downstream of RTKs in cell line models.
Authors: Rikova Klarisa , Hall Benjamin , Possemato Anthony , Mroszczyk Keri , Lee Kimberly A. , Ren Jian Min , Guo Ailan , Mulhern Daniel , Wang Yi , Beausoleil Sean A. , Comb Michael J.
Published: August 2014

In virtually all epithelial tumors, growth factor receptor activity is deregulated by activated mutations, genomic amplification, and autocrine loops. The dependence of tumor cell survival upon the driving oncogene has been called “oncogene addiction” and demonstrates the acute sensitivity of cancer cells to inhibition of the pathways driving their proliferation, growth and survival. Major questions regarding the sensitivity of solid tumors to targeted kinase inhibitors is why some tumors respond and others do not and why ones which respond still are able to develop resistance through the activation of other pathways. Robust, but not durable, response to receptor tyrosine kinase inhibitors (RTKIs) highlights the need to inhibit pathway activity at multiple levels.

In this study, we identify extensive signaling networks downstream of receptor tyroine kinases (RTKs) across multiple spaces, including phosphorylation, acetylation, and methylation. We used the established method of TMT labeling coupled with serial peptide immunoprecipitation applied to profile drug-treated and untreated RTK pathway-addicted cancer cell lines. Quantitative analysis was performed to look at the effects of drugs on RTK-dependent cell lines: MKN45 (Met) with Crizotinib, H3122 (Alk) with Crizotinib, H1703 (PDGFRα) with Gleevec, and H3255 (EGFR) with Iressa. Phosphotyrosine, acetyl-lysine, methyl arginine, ATM/ATR substrate (s/tQ), and AGC/CAMK/STE family kinase motif were all selected for use in peptide immunoprecipitation which allowed us to characterize and quantify a broad PTM space. In this study we were able to identify and quantify hundreds of modification sites for phosphorylation, arginine methylation, and lysine acetylation. Those sites found to be drug-sensitive, including all PTM types, were used for pathway analysis of signaling downstream of each RTK disease driver.

Citation Format: Klarisa Rikova, Benjamin Hall, Anthony Possemato, Keri Mroszczyk, Kimberly A. Lee, Jian Min Ren, Ailan Guo, Daniel Mulhern, Yi Wang, Sean A. Beausoleil, Michael J. Comb. Mapping deregulated pathways downstream of RTKs in cell line models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4278. doi:10.1158/1538-7445.AM2013-4278