bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5192: Mutational analysis of primary central nervous system lymphoma
Authors: Bruno Aurélie , Boisselier Blandine , Labreche Karim , Marie Yannick , Polivka Marc , Jouvet Anne , Adam Clovis , Figarella-Branger Dominique , Miquel Catherine , Vital Anne , Houillier Caroline , Soussain Carole , Mokhtari Karima , Daveau Romain , Hoang-Xuan Khê
Published: May 2015

Background: Primary central nervous system lymphoma (PCNSL) is a rare extranodal diffuse large B-cell lymphoma (DLBCL). Little is known about the genomic basis of tumorigenesis in PCNSL partly explained by the rarity of biological material available for research studies. PCNSL patients display poor outcomes compared to systemic DLBCL and it is still unclear whether this is linked to the organ-specific microenvironment or reflects a specific, aggressive, intrinsic biological behavior. To our knowledge, the mutational landscape of PCNSL has never been investigated

Experimental design: We analyzed 9 paired tumor and germline DNA samples from PCNSL patients by high throughput exome sequencing. Then, we performed focused sequencing in 28 additional PCNSL tumors to validate somatic genomic alterations and better estimate their incidence. Mutational data were correlated with copy number variations (CNV) identified by SNP and CGH arrays and clinical data.

Results: We identified recurrent somatic mutations in genes involved in key signalling pathways such as NFκB (i.e. MYD88, CD79B, TBL1XR1), B cell differentiation (i.e. ETV6, EBF1, IRF4) and cell cycle control (i.e. BTG1, PIM1). We found somatic mutations on MYD88 (38%), CD79B (30%), TBL1XR1 (22%) or ETV6 (16%) genes and described bi-allelic inactivation of TBL1XR1 and ETV6 genes. Interestingly, the correlation with the outcome showed a significant negative impact of TBL1XR1 mutation on survival in a multivariate analysis.

Conclusions: The present study showed that PCNSLs have high genetic heterogeneity and that their mutational patterns display similarities with extracerebral DLBCL, particularly of the ABC subtype, suggesting shared underlying biological mechanisms. These results provide new insights into the mutational landscape of PCNSL and potential targets for therapeutic strategies. This work benefited from the LOC study group network (réseau national de centres experts des lymphomes primitifs du SNC, INCa).

Citation Format: Aurélie Bruno, Blandine Boisselier, Karim Labreche, Yannick Marie, Marc Polivka, Anne Jouvet, Clovis Adam, Dominique Figarella-Branger, Catherine Miquel, Anne Vital, Caroline Houillier, Carole Soussain, Karima Mokhtari, Romain Daveau, Khê Hoang-Xuan. Mutational analysis of primary central nervous system lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5192. doi:10.1158/1538-7445.AM2014-5192
Abstract 5207: miR-491 and miR-218: Two possible tools to reduce FOXP3 expression in breast carcinomas
Authors: Modica Martina D. , Triulzi Tiziana , Regondi Viola , Iorio Marilena V. , Balsari Andrea , Tagliabue Elda , Casalini Patrizia
Published: May 2015

The Forkhead box P3 (FOXP3) gene is a member of the forkhead/winged-helix family of transcription factors implicated in the regulation of immune system development and function. However, FOXP3 is also expressed in human epithelial cancers, where it has been reported to be associated with poor prognosis and in particular with metastasis. Aim of our work is to identify microRNAs able to regulate FOXP3 expression in breast tumors.

To discover microRNAs able to regulate FOXP3 expression in breast cancer cells, an in silico analysis has been performed. Among microRNAs harboring FOXP3 seed regions, we focused our attention on miR-218 and miR-491, described to have oncosuppressive properties. Indeed, it has been reported that miR-218 expression stimulates activation of tumor suppressor genes and miR-491 expression inversely correlates with migratory potential in HCC cell lines and in vivo with metastatic potential.

We therefore evaluated the ability of these microRNAs to act as oncosuppressors by targeting FOXP3 in a breast cancer model. After transient co-transfection either of miR-218 or miR-491 with FOXP3 in MDA-MB-231 cells, a significant decrease in FOXP3 expression at protein level by WB was found.

To perform functional studies both in vitro and in vivo, we generated a breast cancer cell model by stable transfection of MDA-MB-231 with inducible Tet-off plasmid encoding for FOXP3 encompassing 3′UTR region, thus maintaining microRNA-mediated regulation. We demonstrated that FOXP3 expressing clones display an increased migration capacity as compared with their respective non-induced cells. Also in these stable clones a reduction of FOXP3 expression after transfection of miR-491 and miR-218 has been demonstrated by WB. Moreover, FOXP3 was confirmed to be a direct target of both microRNAs by luciferase reporter assay.

We also found an inverse correlation between miR-491 levels and FOXP3 expression in human breast carcinoma specimens.

Overall, these preliminary results demonstrate that microRNAs can regulate FOXP3 expression, and encourage to further investigate the biological and functional consequences of this targeting, and to exploit microRNAs as a potential therapeutic tool for FOXP3 expressing breast carcinomas.

Citation Format: Martina Di Modica, Tiziana Triulzi, Viola Regondi, Marilena V. Iorio, Andrea Balsari, Elda Tagliabue, Patrizia Casalini. miR-491 and miR-218: Two possible tools to reduce FOXP3 expression in breast carcinomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5207. doi:10.1158/1538-7445.AM2014-5207
Abstract 5233: MicroRNA in biofluid as robust biomarkers for cancer
Authors: Blondal Thorarinn , Thomsen Anni , Krummheyer Jörg , Mouritzen Peter , Andreasen Ditte , Theilum Maria , Stenvang Jan , Andersen Claus L. , Nielsen Hans J. , Brünner Nils
Published: May 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). The study of extracellular microRNAs and their potential as pathophysiological markers has greatly expanded in the last couple of years. microRNAs have been shown to be actively exported from tissues into the circulation through a variety of mechanisms including exosome and microvesicle transport, and complexing with RNA binding proteins or HDL. The high relative stability of microRNAs in common clinical source materials (FFPE blocks, plasma, serum, urine, saliva, etc.) and the ability of microRNA expression profiles to accurately classify discrete tissue types and specific disease states have positioned microRNAs as promising new biomarkers for diagnostic application in cancer. We have applied Exiqons highly sensitive LNA™-based qPCR platform for detection of microRNAs, which has enabled microRNA profiling in biofluids where levels are extremely low. The platform uses a single RT reaction to conduct full miRNome profiling and allows high-throughput profiling of microRNAs without the need for pre-amplification. Thousands of biofluid samples including serum/plasma and urine have been profiled to determine normal reference ranges for circulating microRNAs as well as to identify biomarkers of disease. Extensive data qualification and analysis methods have been developed and are central parameters to secure high quality data from biofluids. The methods can quickly and robustly be applied in biomarker discovery and validation projects. We will present examples from our collaborative cancer diagnostic projects. Also we have developed a new exosome enrichment method and will present a comparison of microRNA profiles obtained with this method to profiles obtained with different commercial available exosome isolation methods and standard profiles of whole plasma and serum.

Citation Format: Thorarinn Blondal, Anni Thomsen, Jörg Krummheyer, Peter Mouritzen, Ditte Andreasen, Maria Theilum, Jan Stenvang, Claus L. Andersen, Hans J. Nielsen, Nils Brünner. MicroRNA in biofluid as robust biomarkers for cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5233. doi:10.1158/1538-7445.AM2014-5233
Abstract 5238: Integrative analysis of functional long noncoding RNAs during prostate cancer progression
Authors: Fei Teng , Chen Yiwen , Du Zhou , Liu Xiaole Shirley , Brown Myles
Published: May 2015

Prostate cancer progression from an androgen-dependent state to an androgen-independent phase, which is also referred to castration-resistant prostate cancer (CRPC), not only results in cancer refractory to androgen deprivation therapy but also marks its lethal prognosis. However, it remains largely unknown that how CRPC develops. As a result, ideal diagnosis and therapeutic tools are still highly in demand and under investigation. Recently, many studies indicate that long non-coding RNA (lncRNA) can be extensively functional rather than just transcriptional noise and lncRNAs are also widely involved in the pathogenesis of different types of cancers. To identify and characterize functional lncRNAs during prostate cancer progression, we firstly developed an integrative computational strategy for predicting lncRNAs that may be functionally important for tumorigenesis or tumor suppression via analysis of lncRNA expression profiles, clinical information and somatic genomic alteration profiles of tumor samples. By applying this approach, we predicted a cohort of lncRNAs that may be clinically important for prostate tumorigenesis and experimentally confirmed two functional lncRNAs - PCAT6 and PCAT7 in prostate cancer cell line model. Work from others and our lab have also shown that both androgen receptor (AR) and polycomb group protein enhancer of zest 2 (EZH2) play pivotal roles during prostate cancer progression to CRPC. In contrast to protein coding genes, little is known about AR or EZH2 regulated lncRNAs during this process. To this purpose, we also performed RNAseq in prostate cancer cell lines and combined ChIPseq data to identify androgen-regulated and AR/EZH2-regulated lncRNAs. These regulated lncRNAs are also intersected with above clinically significant lncRNA list to narrow down interesting hits for follow-up mechanistic studies. Our integrative approach has successfully identified a cohort of potentially functional lncRNAs which hold great potentials to be novel prognostic biomarkers and/or therapeutic targets for treatment of advanced prostate cancer.

Citation Format: Teng Fei, Yiwen Chen, Zhou Du, Xiaole Shirley Liu, Myles Brown. Integrative analysis of functional long noncoding RNAs during prostate cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5238. doi:10.1158/1538-7445.AM2014-5238
Abstract 524: Analysis of long non-coding RNA expression and function in a mouse model of glioblastoma
Authors: Myskiw Chad , Kadri Sabah , Ke Eugene , Shishkin Alex , Friedmann-Morvinski Dinorah , Soda Yasushi , Guttman Mitchell , Verma Inder
Published: May 2015

Glioblastoma (GBM) is the most common adult brain tumor and represents one of the most treatment refractory cancers. Although significant progress has been made in understanding the coding genomic alterations associated with GBM, patient survival rarely exceeds 12-14 months following diagnosis. Long non-coding RNAs (lncRNAs) are RNA species generally classified as > 200 base-pairs in length that lack protein coding potential and therefore exert their function as RNA. Several lncRNAs have been shown to play important roles in cancer biology. Despite this, the role of lncRNAs in cancer, and GBM in particular, remains relatively uncharacterized. We hypothesize that lncRNAs are differentially expressed in GBM and contribute to the pathogenesis of this disease.

Using next generation sequencing, we have identified lncRNAs expressed in our mouse model of GBM, initiated by lentivirus-mediated expression of oncogenic Ras and a shRNA targeting p53. This resulted in the identification of 818 putative lncRNAs, the majority of which represent novel, uncharacterized transcripts. The majority of the lncRNAs identified were intergenic and not associated with any known protein coding gene. Of the total lncRNAs, 44% were differentially expressed in tumor tissue compared to normal mouse brain. Expression of a subset of these differentially expressed lncRNAs was validated by real-time PCR. Expression of several of these validated lncRNAs could be altered by direct activation of oncogenic signaling in normal mouse neuroprogenitor cells and astrocytes. Using a combination of computational and molecular biology approaches, we will identify candidate lncRNAs to test in functional assays for their role in glioblastoma biology.

Citation Format: Chad Myskiw, Sabah Kadri, Eugene Ke, Alex Shishkin, Dinorah Friedmann-Morvinski, Yasushi Soda, Mitchell Guttman, Inder Verma. Analysis of long non-coding RNA expression and function in a mouse model of glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 524. doi:10.1158/1538-7445.AM2014-524
Abstract 5243: MicroRNAs-449a and -449b exhibit tumor suppressive effects in retinoblastoma
Authors: Jones Aunica A. , Martin Alissa , Bryar Paul , Mets Marilyn , Weinstein Joanna , Zhang Gang , Laurie Nikia A.
Published: May 2015

MicroRNAs (miRNAs) are short, non-coding RNAs that act by inhibiting target genes. Specific individual miRNAs have now been identified which have confirmed effects in cancer, and some miRNAs can act as either tumor suppressors or as oncogenes by exerting effects on important regulatory cellular pathways. The purpose of our study is to illustrate the relationship between the expressions of miRs-449a and -449b to retinoblastoma proliferation and apoptosis. We show that there is an inhibitory effect of miRs-449a and -449b in retinoblastoma by demonstrating significantly impaired proliferation and increased apoptosis of tumor cells when these miRNAs are overexpressed. This study suggests that these miRNAs could serve as viable therapeutic targets for retinoblastoma treatment. We also report the utility of combining overexpression of miRs-449a and -449b with broad-based chemotherapy drugs that are currently being used to treat retinoblastoma patients.

Citation Format: Aunica A. Jones, Alissa Martin, Paul Bryar, Marilyn Mets, Joanna Weinstein, Gang Zhang, Nikia A. Laurie. MicroRNAs-449a and -449b exhibit tumor suppressive effects in retinoblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5243. doi:10.1158/1538-7445.AM2014-5243
Abstract 5244: MicorRNA-27a functions as a tumor suppressor in colorectal cancer through targeting SPGG1 and Smad2
Authors: Bao Yonghua , Chen Zhiguo , Guo Yongchen , Zhao Tiesuo , Feng Yansheng , Jiao Yunjuan , Li Zexin , Wang Jianguo , Zhao Weixing , Li Kai , Wang Qian , Wang Jiaqi , Zhang Huijuan , Yang Wancai , Yang Wancai
Published: May 2015

Colorectal cancer is one of the most common malignant diseases worldwide, but the causes of colorectal carcinogenesis are largely unknown. Using a miRNA array we have identified differential expression of some microRNAs in colorectal cancer and adjacent normal mucosa. Most of these miRNAs have been validated in colorectal tissues by quantitative RT-PCR. One of the most changed miRNAs is miR-27a. The level of miR-27 was significantly reduced in colon cancers in compared with matched normal mucosa, the reduced expression of miR-27a was also associated with colorectal cancer pathological stages - miR-27a was more reduced at stages III/IV, compared to stage II. The levels of miR-27a in human colon cancer cell lines were also less than that in human normal colon epithelial cell lines. Using bioinformatic and systemic analysis, two targets of miR-27a were predicted, they were SPGG1 and Smad2. The expression levels of SPGG1 and Smad2 were negatively correlated with miR-27a. Dual luciferase assay showed that overexpression of miR-27 significantly repressed SPGG1 and Smad2 transcriptional activities. Increasing miR-27 expression also inhibited SPGG1 and Smad2 protein levels in colon cancer cells. Functional studies revealed that overexpression of miR-27 inhibited colon cancer cell proliferation, promoted apoptosis and attenuated cell migration, which were linked to downregulation of p-STAT3 and upregulation of phosphorylated JNK1. Taken together, our results demonstrated that the expression levels of miR-27a were lower in colorectal cancer tissues in comparison to the non-tumor tissues and was related to pathological stages, suggesting that down-regulation of this miRNA could contribute to colorectal tumorigenesis and the acquisition of a more aggressive phenotype in colorectal cancer, which could be through targeting SPGG1 and Smad2.

Note: This abstract was not presented at the meeting.

Citation Format: Yonghua Bao, Zhiguo Chen, Yongchen Guo, Tiesuo Zhao, Yansheng Feng, Yunjuan Jiao, Zexin Li, Jianguo Wang, Weixing Zhao, Kai Li, Qian Wang, Jiaqi Wang, Huijuan Zhang, Wancai Yang, Wancai Yang. MicorRNA-27a functions as a tumor suppressor in colorectal cancer through targeting SPGG1 and Smad2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5244. doi:10.1158/1538-7445.AM2014-5244
Abstract 5247: Comprehensive and systematic identification of miR-215 targets in colon cancer via a novel translation capture immunoprecipitation (TrIP) approach
Authors: Fesler Andrew , Xu Xiao , Chen Emily , Ju Jingfang
Published: May 2015

The important roles that non-coding miRNAs play in cancer development, makes the investigation of the various genes they target particularly crucial. In addition to targeting mRNA transcripts for degradation, miRNA can also alter gene expression at the translational level to achieve rapid regulation. It has been a technical challenge to study translational control using a small number of cells. We have overcome this hurdle by developing a translational capture immunoprecipitation approach (TrIP) to investigate RNA binding proteins or non-coding RNA regulation at the translational level from a small numbers of cells. The new approach is based on the fact that nascent translated polypeptides are associated with hsp70 chaperones along with actively translating mRNA transcript complexes, called polyribosomes. We are able to isolate actively translating mRNA from free cytoplasmic mRNA by immunoprecipitation of hsp70 associated polyribosome complexes. In this study, we utilize this novel approach to identify both degraded and non-degraded mRNA transcripts targeted by miR-215 in colon cancer. Following miR-215 overexpression in colon cancer cells, actively translating mRNA transcripts were isolated using the TrIP technique and purified following standard RNA isolation techniques. We were able to identify additional non-degraded mRNA targets regulated by miR-215 using a TrIP with transcriptome-wide microarray based approach, when compared to a generic approach based on steady state total RNA that can only identify degraded targets. Specific targets were independently validated using qRT-PCR analysis. We further validated miR-215 targets at the protein level with a comprehensive and high-throughput quantitative SILAC proteomics analysis. Protein targets were further validated using Western blotting with select positive control targets including TYMS, DTL and DHFR. This approach offers the unique opportunity to understand not only the mRNA targets a miRNA may target for degradation, but also those it regulates at the translational level, providing unique insight into the actions of miRNAs. Interest in this type of thorough analysis of different miRNAs or RNA binding proteins will likely continue to grow as the important roles miRNA play in cancer are further investigated.

Citation Format: Andrew Fesler, Xiao Xu, Emily Chen, Jingfang Ju. Comprehensive and systematic identification of miR-215 targets in colon cancer via a novel translation capture immunoprecipitation (TrIP) approach. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5247. doi:10.1158/1538-7445.AM2014-5247
Abstract 526: Overexpression of chromosome 3q26-29 miRNAs in squamous cell carcinoma of the lung
Authors: Qian Jun , Ji Xiangming , Zou Yong , Hoeksema Megan , Chen Heidi , Massion Pierre P.
Published: May 2015

The amplification of the distal portion of chromosome 3q in lung cancer is a major signature of neoplastic transformation, particularly in the squamous cell carcinoma (SCC) of the lung. A number of potential drivers in the 3q amplicon have been identified and are proposed to contribute to the development of lung SCC. miRNAs have emerged as a new class of small, non-coding RNAs that regulate gene expression and might act as integral parts of the molecular architecture of oncogene and tumor suppressor networks in human cancer, including non-small cell lung cancer (NSCLC). miR 1224-5p, 1248 and 944 are located at 3q amplicon and overexpressed in 50%, 26% and 53% of 30 primary SCCs when compared to normal tissues. The overexpression of these miRNAs was further validated in 237 TCGA lung SCC samples compared with 35 normal tissues (p=0.007, 1.18E-16 and 7.71E-20, respectively). The expression of the three miRNAs was higher in lung SCC compared to lung adenocarcinomas (p=8.19E-05, 1.04E-33 and 3.37E-95, respectively), suggesting unique roles of these miRNAs in the lung SCC development. Using miRNA mRNA target prediction tools (miRANDA and TargetScan) and in the combination with a literature search, we found RUNX1T1 and SMAD4 were candidate tumor suppressor gene target for miRNA-1224-5p and miRNA-1248. Downregulation of RUNX1T1 and SMAD4 were confirmed in TCGA lung SCC dataset. Using real-time RT-PCR, we found that the same three miRNAs 1224-5p, 1248 and 944 are overexpressed in 3q amplified lung SCC cell lines H520 and HCC95. When miRNA inhibitors were used to repress the expression of miR-1224-5p or miR-1248 in H520, we found that RUNX1T1 and SMAD4 protein expression levels were increased, suggesting that these tumor suppressor genes are regulated by miR-1224-5p or miR-1248. Lastly, knockdown of miR-1224-5p or miR-1248 using miRNA inhibitors led to 35-60% growth inhibition in H520 and HCC95 cells. Together, these results indicate potential roles for miR-1245-5p and 1248 in regulating cell proliferation by directly targeting tumor suppressor genes RUNX1T1 or SMAD4 in lung SCC. This work provides, for the first time, evidence for overexpression of miRNAs at the 3q26-29 amplicon that might be implicated in the oncogenesis of lung SCC. The work is supported by the NIH Grant R01 CA102353 to PPM and Lung Cancer Research Foundation grant (2011) to JQ.

Note: This abstract was not presented at the meeting.

Citation Format: Jun Qian, Xiangming Ji, Yong Zou, Megan Hoeksema, Heidi Chen, Pierre P. Massion. Overexpression of chromosome 3q26-29 miRNAs in squamous cell carcinoma of the lung. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 526. doi:10.1158/1538-7445.AM2014-526
Abstract 5269: Impact of K-ras status on differential expression of TIMP-1 in lung adenocarcinoma
Authors: Kutiyanawalla Ammar , Gupta-Ghoshal Sampa , Lee Byung Rho , Kolhe Ravindra , Rojiani Amyn M. , Rojiani Mumtaz V.
Published: May 2015

Tissue inhibitor of metalloproteinase-1 exhibits a range of novel functions in addition to its initially recognized activity as a physiological inhibitor of MMPs. Two specific activities have been of significant interest in the context of oncology - inhibition of apoptosis and induction of cell proliferation. Previously we have shown that over-expressing TIMP-1 in a lung adenocarcinoma cell line H2009 results in more aggressive and larger tumors in vivo with increased invasion and colony formation in vitro. Subsequently we demonstrated elevated levels of Bcl-2 with a concomitant reduction in apoptosis induced by staurosporine. In the present study, we have sought to define a relationship between TIMP-1 expression levels in non-small-cell-lung-carcinoma (NSCLC) cell lines in relation to K-ras mutation. We identified a differential expression pattern of TIMP-1 in various NSCLC cell lines and also found that TIMP-1 overexpressing cell lines also had higher levels of Bcl-2 and vice versa. Upon further analysis, we were able to correlate a low TIMP-1 expression with K-ras dependence and high levels of E-cadherin expression as in H2009 and H441. Similarly, K-ras independent cell line A549, expressed low levels of E-cadherin and elevated TIMP-1. E-cadherin distribution was confirmed in A549 and H2009 cells by immunofluorescence. Since N-cadherin cleavage has been shown to be inhibited by TIMP-1, we examined N-cadherin levels by western blot. Results indicate that A549 exhibiting high levels of TIMP-1 had increased levels of N-cadherin whereas the low TIMP-1 expressing cell lines had lower N-cadherin levels. A spheroid formation assay to determine the aggressiveness of cell lines revealed that K-ras dependent cell line formed spheroids that were smaller and less migratory than K-ras independent cell line whereas the K-ras independent cell line A549 formed spheroids that were larger, more spread out exhibiting wider migratory properties. The present study underscores the multiple biologic properties of TIMP-1. It provides further mechanistic understanding of its documented role as a prognostic marker and a potential therapeutic target in various malignancies, particularly lung carcinoma. This study was supported in part by a Distinguished Cancer Scientist Award to AMR from the Georgia Research Alliance.

Citation Format: Ammar Kutiyanawalla, Sampa Gupta-Ghoshal, Byung Rho Lee, Ravindra Kolhe, Amyn M. Rojiani, Mumtaz V. Rojiani. Impact of K-ras status on differential expression of TIMP-1 in lung adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5269. doi:10.1158/1538-7445.AM2014-5269
Abstract 5270: Prevalence of inactivating protein kinase C mutations in human cancers
Authors: Antal Corina E. , Kang Emily , Stephenson Natalie L. , Trotter Eleanor W. , Hunter Tony , Brognard John , Newton Alexandra
Published: May 2015

Protein kinase C (PKC) levels are aberrant in diverse cancers and over 400 mutations in PKC have been identified in human cancers. Here we characterized PKC mutations identified in human cancers to investigate whether PKCs function as oncogenes or tumor suppressors or whether their function is isozyme and/or context dependent. Live-cell imaging was used to determine how these mutations affect PKC translocation, subcellular localization, and activity. Cell-based assays were used to determine how PKC mutations alter proliferation, apoptosis, migration, and anchorage-independent growth. Of 37 mutations we examined thus far, 24 of them reduced or completely abolished PKC activity, while only 1 increased it. These mutations reside within the diacylglycerol-binding C1 domain, the Ca2+-binding C2 domain, or the kinase domain of conventional, novel, or atypical PKCs. The inactivating mutations ablate activity by either impeding Ca2+ or diacylglycerol binding, or by yielding a misfolded kinase. Our results suggest that PKC can act as a tumor suppressor in certain cancers. Understanding whether mutations are activating or inactivating in specific cancers would allow us to determine whether therapeutic strategies should target ways to inactivate or activate PKC in the respective cancers.

Citation Format: Corina E. Antal, Emily Kang, Natalie L. Stephenson, Eleanor W. Trotter, Tony Hunter, John Brognard, Alexandra Newton. Prevalence of inactivating protein kinase C mutations in human cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5270. doi:10.1158/1538-7445.AM2014-5270
Abstract 5273: VBIM technology identifies beta-catenin-like protein (BCLP) as a novel negative regulator of NF-kB
Authors: Mundade Rasika , Lu Tao
Published: May 2015

Colon cancer is the second leading cause of cancer related deaths in the United States. The nuclear factor kB (NF-kB) is an important family of transcription factors whose aberrant activation has been found in many types of cancer, including colon cancer. Therefore, understanding the regulation of NF-kB is of ultimate importance for cancer therapy. The purpose of this study is to use a novel validation-based insertional mutagenesis (VBIM) strategy to identify novel regulators of NF-kB, and further evaluate their roles in the regulation of NF-kB signaling in colon cancer cells. We infected D1 cells (293 derived cells with hyper active NF-kB activity) with VBIM virus to cause the overexpression of negative regulators of NF-kB, and then further selected the mutant cells with low NF-kB activity under ganciclovir (GCV) treatment. Targeted gene was then identified by using VBIM specific primers. In a preliminary screen, we identified the novel beta-catenin like protein (BCLP) gene as a negative regulator of NF-kB. Overexpression of BCLP led to decreased NF-kB activity by kB reporter assay, while knocking it down had the opposite effect. Furthermore, we found that overexpression of BCLP in HT29 colon cancer cells greatly reduced both the number and the size of colonies that were formed in a soft agar assay, while sh-RNA mediated knockdown resulted in an opposite effect, confirming that BCLP is a tumor suppressor in HT29 cells. Our future experiments aim to further assess the role of BCLP in colon tumor formation in a mouse xenograft model. In summary, by using the novel VBIM technique, we identified BCLP as a novel negative regulator of NF-kB. This discovery could lead to the establishment of BCLP as a potential biomarker and therapeutic target in colon cancer.

Citation Format: Rasika Mundade, Tao Lu. VBIM technology identifies beta-catenin-like protein (BCLP) as a novel negative regulator of NF-kB. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5273. doi:10.1158/1538-7445.AM2014-5273
Abstract 5278: Superoxide mediated selective tyrosine nitration of protein phosphatase 2A-B56δ stabilizes Bcl-2 phosphorylation and its anti-apoptotic activity
Authors: Low Ivan C. C. , Loh Thomas , Virshup David , Pervaiz Shazib
Published: May 2015

Our work over the years has provided strong evidence that an increase in intracellular superoxide anion provides cancer cells with a survival advantage and promotes tumor chemoresistance. The mechanisms underlying superoxide-mediated pro-survival signaling are poorly understood. Here we report that an increase in intracellular superoxide stabilizes the anti-apoptotic activity of Bcl-2 by mechanisms that involve sustained phosphorylation of Bcl-2 at serine 70. Of note, this effect on Bcl-2 S70 phosphorylation is a function of redox-dependent inactivation of Protein Phosphatase 2A (PP2A), which is targeted to Bcl-2 via its B56 subunit; superoxide induces the release of PP2A-AC catalytic core from Bcl-2-bound B56δ, resulting in sustained phosphorylation of Bcl-2 at S70. Dissociation of the PP2A heterotrimer results from the selective nitration (by peroxynitrite derived from the reaction of superoxide with NO) of a conserved tyrosine residue, which we identify as Y289 of B56δ (B56 δY289). Inhibition of B56δY289 nitration blocked the pro-survival effects of superoxide. Furthermore, we provide evidence for clinical relevance of these findings using primary tissue derived from human lymphoma biopsies. Taken together, these data provide evidence for a novel mechanism in which an increase in intracellular superoxide regulates PP2A function via specific B56 subunit tyrosine nitration, and that PP2A-B56 nitration may be a general mechanism for redox-regulated cellular signaling.

Citation Format: Ivan C. C. Low, Thomas Loh, David Virshup, Shazib Pervaiz. Superoxide mediated selective tyrosine nitration of protein phosphatase 2A-B56δ stabilizes Bcl-2 phosphorylation and its anti-apoptotic activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5278. doi:10.1158/1538-7445.AM2014-5278
Abstract 5281: Role of SHP-2 tyrosine phosphatase in colorectal carcinogenesis
Authors: Sansfacon Jessica Gagne , Coulombe Geneviève , Bourdages Nadia , Rivard Nathalie
Published: May 2015

SHP-2 (Src homology-2 domain-containing phosphatase 2) is a tyrosine phosphatase ubiquitously expressed. Previous work done in SHP-2-/- fibroblasts demonstrated its implication in proliferation and migration as well as in the regulation of many signaling pathways including the Ras-Raf/MEK/ERK, the PI3K/AKT and the Jak/STAT pathways(1). Importantly, gain-of-function mutations were discovered in some colorectal cancers (CRCs)(2). However, the role of SHP-2 in intestinal tumorigenesis has never been investigated.

Methods: SHP-2 expression and catalytic activity were analyzed by Western blot, qPCR and immunohistochemistry in human CRC cells and in human tumours from different stages. SHP-2 expression was also analyzed in polyps (adenomas) from mice bearing a mutation in Apc gene (APCMin/+ mice). To elucidate the role of SHP-2, RNA interference was used to specifically downregulate its expression in intestinal epithelial cells transformed or not by the oncogenic form of KRASG12V (IEC/KRas). Proliferation (cell counting), invasion (through Matrigel in Boyden chamber), anchorage-independent growth (soft agar) and tumor formation in nude mice (xenograft assays) were analyzed.

Results: 1- SHP-2 mRNA and protein levels were significantly increased in human CRC cells and tumors, especially in adenomas, in comparison to healthy adjacent tissues. 2- Interestingly, polyps from APCMin/+ mice also exhibited marked increase in SHP-2 protein expression in comparison to normal adjacent intestinal epithelium. 3- SHP-2 silencing in IEC/KRAS cells markedly reduced their proliferation rate, their growth in soft agar, their capacity to migrate and invade Matrigel and finally, their capacity to form tumor in vivo, in nude mice. 4- Of note, phosphorylated and activated levels of MEK and ERK kinases were significantly decreased following SHP-2 silencing in IECs particularly in those expressing the mutated form of KRAS.

Conclusion: Our data suggest that SHP-2 promotes malignancy of intestinal epithelial cells by sustaining the oncogenic activation of MEK/ERK signalling during intestinal tumorigenesis.

(1)Shi ZQ, Lu W, Feng GS., J Biol Chem. 1998 Feb 27;273(9):4904-8.

(2)Bentires-Alj M. et al., Cancer Res. 2004 Dec 15;64(24):8816-20.

Citation Format: Jessica Gagne Sansfacon, Geneviève Coulombe, Nadia Bourdages, Nathalie Rivard. Role of SHP-2 tyrosine phosphatase in colorectal carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5281. doi:10.1158/1538-7445.AM2014-5281
Abstract 5282: Promotion of skin tumorigenesis by deletion of Ppp6c gene
Authors: Hayashi Katsuhisa , Ogoh Honami , Watanabe Toshio , Tanuma Nobuhiro , Shima Hiroshi
Published: May 2015

Protein phosphatase-6 (PP6) is a member of type-2A protein-Ser/Thr phosphatases, and reportedly regulates several cellular functions including DNA damage responses, NF-kB activation, and mitotic spindle formation. Additionally, some recent papers reported somatic mutations of PPP6c gene, encoding the catalytic subunit of PP6, in about 10 % of melanoma patients, suggesting its potential role as either driver or suppressor of melanoma development.

In this study, we generated mice, in which the PPP6C gene is deleted in keratinocyte-specific manner (PP6-KKO). PP6-KKO mice were predisposed to formation of skin tumor upon DMBA-treatment. Our results demonstrate PPP6c has a tumor-suppressive function at least in skin tumorigenesis.

Citation Format: Katsuhisa Hayashi, Honami Ogoh, Toshio Watanabe, Nobuhiro Tanuma, Hiroshi Shima. Promotion of skin tumorigenesis by deletion of Ppp6c gene. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5282. doi:10.1158/1538-7445.AM2014-5282
Abstract 5285: Protein phosphatases modulate kinase signaling mediated Wnt activation
Authors: Boral Debasish , Nie Daotai
Published: May 2015

Multiple growth factors, signaling through their respective kinase cascades, phosphorylate beta-catenin at specific Serine residues to cause its nuclear sequestration thereby fueling Wnt driven tumor cell proliferation. Our present work identifies a group of phosphatase genes that attenuate such growth factor mediated activation of Wnt signaling in colo-rectal cancer (CRC) cells. This proposed mechanism proposes an additional mode of regulation of Wnt activity in CRC cells.

Using a lentiviral TCF-LEF reporter system, we isolated distinct SW480 (primary) and SW620 (metastatic) cell fractions based on their endogenous level of Wnt activity. Receptor Tyrosine Kinase (RTK) antibody arrays using cell lysates from these cell fractions identified that FGFR3, ALK and RET signaling pathways have a positive correlation with enhanced Wnt activity. Simultaneously qPCR arrays performed on these same cellular fractions identified a total of 10 phosphatase genes whose expression levels are significantly decreased with heightened Wnt activity, out of which 4 genes PHLPP, PHLPPL, PTPRK and PTPN6 are known to regulate the aforementioned RTK pathways.

Of these phosphatase genes, PTPRK has been reported to undergo gene fusion events with RSPO3 (a non-canonical activator of Wnt) in CRC patients. Since the fusion gene product provides no additional Wnt activation that the wild-type RSPO3, we theorized that these fusion events occur either to allow CRC cells to utilize the enhanced promoter activity of the PTPRK gene promoter, or to nullify the Wnt attenuating properties of PTPRK. To test this hypothesis, we have presently cloned and identified PTPRK and RSPO3 gene promoter regions and wish to test their activity in distinct Wnt responsive cell fractions. Our work will allow a better understanding of how phosphatase genes may be involved in controlling the Wnt pathway and how their inactivation may contribute to CRC cell proliferation.

Citation Format: Debasish Boral, Daotai Nie. Protein phosphatases modulate kinase signaling mediated Wnt activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5285. doi:10.1158/1538-7445.AM2014-5285
Abstract 5289: Role of notch3 in epithelial-to-mesenchymal transition of HCC
Authors: Kunnimalaiyaan Selvi , Gamblin T. Clark , Kunnimalaiyaan Muthusamy
Published: May 2015

Introduction:

The epithelial-mesenchymal transition (EMT) program is often activated in cancer cells during invasion and metastasis. During EMT, expression of transcription factors such as Snail and Slug is induced due to the reduction in E-cadherin expression. In addition, matrix metallo-proteinases (MMPs) which digest the various components of extracellular matrix also induced during EMT. Various signaling pathways have been involved in the regulation of EMT program. We hypothesize that notch signaling pathway is a potential regulator of EMT program in HCC.

Methods:

Lysates from human HCC cell lines, Huh-7, HepG2, and Hep3B, were analyzed for the expression of active notch intracellular domain (NICD) 1-3 proteins by western analysis. To determine the role of NICD3 in cancer invasion and metastasis, siRNA against NICD3 was transfected into HCC cell lines. The effect of depletion of NICD3 in HCC cell lines, epithelial-mesenchymal transition (EMT) markers were measured by western analyses.

Results: Varied level of expression of NICD1-3 was observed in HCC cell lines tested. Transfection of siRNA against NICD3 reduced the level of NICD3 but not the NICD1 protein. Western analysis for the EMT markers expression showed an increase in E-cadherin and concomitant reduction in Snail, Slug, Matrix Metallo Proteinases (MMPs) 2 and 9.

Conclusions:

NICD3 inhibition significantly reduced the expression of markers of metastasis by increasing E-cadherin and suppressing transcription factors Snail, Slug, and MMPs. These observed change in the protein levels are occurred in the absence of NICD3 protein but the presence of NICD1 protein suggesting that NICD3 may play a role in metastasis. Therefore, targeting NICD3 may be potential strategy for treatment of HCC.

Citation Format: Selvi Kunnimalaiyaan, T. Clark Gamblin, Muthusamy Kunnimalaiyaan. Role of notch3 in epithelial-to-mesenchymal transition of HCC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5289. doi:10.1158/1538-7445.AM2014-5289
Abstract 5293: GSK3β phosphorylation at serine9 by RSK2 enhances calcium-induced stress tolerance and cell survival
Authors: Lee Ji-Young , Lee Cheol-Jung , Lee Mee-Hyun , Song Ji Hong , Cho Yong-Yeon
Published: May 2015

Our previous studies demonstrated that RSK2 plays a key role in cell proliferation and transformation induced by tumor promoters such as epidermal growth factor in mouse and human skin cells. However, no direct evidence has been found regarding the relationship between RSK2 and cell survival. In this study, we found that RSK2 interacted and phosphorylated GSK3β at Ser9. Notably, GSK3β phosphorylation at Ser9 was suppressed in RSK2−/− MEFs compared with RSK2+/+ MEFs by stimulation of EGF and calcium ionophore A23187, a cellular calcium stressor. In proliferation, we found that RSK2 deficiency suppressed cell proliferation compared with RSK2+/+ MEFs. In contrast, GSK3β−/− MEFs induced the cell proliferation compared with GSK3β+/+ MEFs. Importantly, RSK2−/− MEFs were induced severe cellular morphology change by A23187 and enhanced G1/G0 and sub-G1 accumulation of the cell cycle phase compared with RSK2+/+ MEFs. The sub-G1 induction in RSK2−/− MEFs by A23187 was correlated with increase of cytochrome c release, caspase-3 cleavage and apoptotic DNA fragmentation compared with RSK2+/+ MEFs. Notably, return back of RSK2 into RSK2−/− MEFs restored A23187-induced morphological change, and decreased apoptosis, apoptotic DNA fragmentation and caspase-3 induction compared with RSK2−/− /mock MEFs. Taken together, our results demonstrated that RSK2 plays an important role in stress-tolerance and cell survival, resulting in cell proliferation and cancer development.

Citation Format: Ji-Young Lee, Cheol-Jung Lee, Mee-Hyun Lee, Ji Hong Song, Yong-Yeon Cho. GSK3β phosphorylation at serine9 by RSK2 enhances calcium-induced stress tolerance and cell survival. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5293. doi:10.1158/1538-7445.AM2014-5293
Abstract 5300: Acetylation of alteration/deficiency in activation 3 (Ada3) is essential for its stability and function
Authors: Srivastava Shashank , Mohibi Shakur , Band Hamid , Band Vimla
Published: May 2015

In eukaryotes histone acetylation is one of the major epigenetic modifications by which gene expression is regulated. Acetylation of histones is mediated by histone acetyl transferases (HATs) that exist as multisubunit complex. The alteration/deficiency in activation 3 (Ada3) protein was discovered as an essential component of various HAT complexes and later on we identified Ada3 as a novel human papilloma virus E6 oncoprotein binding protein. We have previously demonstrated that Ada3 regulates the function of p53 by mediating its acetylation and recent findings from our laboratory show that Ada3 is a novel cell cycle regulator and is crucial for maintaining the genomic stability.

Although Ada3 is an essential mediator of histone acetylation it does not have HAT activity per se and how Ada3 performs its function remains unresolved. Our recent studies demonstrate that Ada3 itself is acetylated by GCN5 and p300 both in in-vitro and in-vivo assays. Significantly, inhibition of acetylation by garcinol dramatically decreases Ada3 protein levels, suggesting that acetylation stabilizes Ada3 protein. Current studies are directed towards identification of potential acetylation sites in Ada3 through mutational analyses. Notably, inhibition of class III but not class I and II histone deacetylases (HDACs) enhanced Ada3 acetylation. Further investigation reveals that Ada3 specifically interacts with SIRT1, a member of class III HDAC, indicating that SIRT1 might deacetylate Ada3. Overall our findings suggest that Ada3 exists in dynamic balance of acetylation and deacetylation, and its cell cycle regulatory function may depend on Ada3 acetylation and deacetylation.

Citation Format: Shashank Srivastava, Shakur Mohibi, Hamid Band, Vimla Band. Acetylation of alteration/deficiency in activation 3 (Ada3) is essential for its stability and function. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5300. doi:10.1158/1538-7445.AM2014-5300