bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2223: Genome-wide analysis of copy number alterations and gene mutations in testicular germ cell cancer
Authors: Sato Yusuke , Sato-Otsubo Aiko , Nagata Yasunobu , Yoshida Kenichi , Shiraishi Yuichi , Suzuki Hiromichi , Sanada Masashi , Kume Haruki , Miyano Satoru , Homma Yukio , Ogawa Seishi
Published: May 2015

Introduction and Objective

Testicular germ cell cancer (TGCC) is the most common solid malignancy among young adult males. Cure could be obtained by intensive chemotherapy combined with retroperitoneal lymph node dissection, but often with substantially compromised quality of life. As for the common genetic lesions, somatic mutations and amplifications of KIT are found in about 20%-30% of cases with TGCCs. However, the molecular pathogenesis of TGCC is still poorly understood. In this study, to obtain a better understanding of the genetic basis of TGCC and to identify druggable molecular targets, we performed whole exome sequencing as well as SNP array-based copy number analysis in TGCC.

Methods

All cases underwent high orchiectomy and were histologically diagnosed as TGCC. Genomic DNA was extracted from fresh frozen specimens of TGCC. Whole exome sequencing was performed for paired tumor/normal DNA from 10 TGCC patients, in which target exomes were captured using SureSelect Human All Exon V5 (Agilent Technologies) and subjected to massively parallel sequencing on the Illumina platform (HiSeq2000). Copy number variants were also interrogated in 40 TGCCs (25 seminomas and 15 non-seminomas) using Affymetrix 250K NspI array.

Results

TGCC genome was triploid in most cases with high level amplification in12p. Loss of heterozygosity (LOH) of chromosome 4 was frequently observed in both seminoma and non-seminoma, whereas 11q deletion was found predominantly in cases with seminoma. In addition, we identified recurrent focal amplifications involving 4q12 and 22q11, from which KIT and MAPK1 were identified, respectively. In whole exome sequencing, 15 somatic mutations were detected per sample on average, which was relatively lower than other solid malignancies. When combined the results of copy number analysis with those of whole-exome sequencing, genes involved in RAS signaling pathway and chromatin modification were frequently altered in TGCC.

Conclusions

Our comprehensive analyses revealed genomic aberrations of TGCC in terms of copy number alterations and gene mutations. Mutated/amplified KIT and MAPK1 and other RAS pathway gene could be potential targets of small molecule inhibitors for therapeutics.

Citation Format: Yusuke Sato, Aiko Sato-Otsubo, Yasunobu Nagata, Kenichi Yoshida, Yuichi Shiraishi, Hiromichi Suzuki, Masashi Sanada, Haruki Kume, Satoru Miyano, Yukio Homma, Seishi Ogawa. Genome-wide analysis of copy number alterations and gene mutations in testicular germ cell 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 2223. doi:10.1158/1538-7445.AM2014-2223
Abstract 2225: Comprehensive molecular characterization of esophageal squamous cell carcinoma
Authors: Lin Dechen , Meng Xuan , Xu Liang , Ding Lingwen , Garg Manoj , Yang Henry , Liu Lizhen , Hao Jiajie , Wang Mingrong , Nagata Yasunobu , Sato Yusuke , Okuno Yusuke , Ogawa Seishi , Koeffler Phillip
Published: May 2015

Background

Esophageal squamous cell carcinoma (ESCC) is a world-wide prevalent cancer, which is particularly common in certain regions of Asia. However, the genetic alterations of this disease have not been fully described.

Methods

We determined the mutational landscape of ESCC through whole-exome or targeted deep sequencing of 139 tumor/germline pairs. The somatic copy number variations (SCNV) of over 180 ESCC tumors were analyzed by SNP-array or array-CGH. Candidate driver mutations were mathematically calculated by the most updated algorithm MutSigCV, and the protein expression of which were thoroughly evaluated by immunohistochemistry. Finally, biological effects of candidate drivers were examined through functional studies in vitro and in vivo.

Results

Deep sequencing and computational analysis identified frequent and novel driver mutations affecting genes such as FAT1 (11.5%), ZNF750 (7.2%), EP300 (7.9%) and MLL2 (18.0%). FAT1 and ZNF750 genes were homozygouly deleted in 3.5% and 3.4% ESCCs, respectively, and their RNA transcripts and proteins were expressed at much lower levels in primary tumors compared to adjacent normal esophageal epithelial. Importantly, silencing of wild-type FAT1 or ZNF750 expression with siRNA/shRNAs significantly promoted cell proliferation and decreased cell differentiation, whereas ectopic expression of these genes resulted in opposite biological consequences. These genetic alterations and biological evidence strongly suggest that FAT1 and ZNF750 likely encode tumor suppressors which are frequently disrupted in ESCC. Furthermore, by integrating the landscape of somatic mutation, SCNV and protein dysregulation, we proposed that RTK-PI3K-MAPK pathways, cell cycle and epigenetic regulation are frequently altered by multiple molecular mechanisms in esophageal tumors. Finally, our systematic approaches uncovered many novel druggable candidates in this disease, such as FGFR1, ALK and XPO1.

Conclusions

By demonstrating the genomic landscape of over 180 ESCC cases, our integrated study unmasks a number of novel genetic lesions in ESCC and provides an important molecular foundation for understanding esophageal tumors and developing therapeutic targets.

Citation Format: Dechen Lin, Xuan Meng, Liang Xu, Lingwen Ding, Manoj Garg, Henry Yang, Lizhen Liu, Jiajie Hao, Mingrong Wang, Yasunobu Nagata, Yusuke Sato, Yusuke Okuno, Seishi Ogawa, Phillip Koeffler. Comprehensive molecular characterization of esophageal squamous cell carcinoma. [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 2225. doi:10.1158/1538-7445.AM2014-2225
Abstract 2226: Loss of STAG2 in bladder cancer
Authors: Krishnan Nithya , Woloszynska-Read Anna , Wang Jianmin , Liu Song , Morrison Carl , Guru Khurshid , Smit Evelyn , Trump Donald , Johnson Candace
Published: May 2015

Bladder cancer (BC), 90% of which have transitional cell histology, is the fourth most common malignancy in the US. Transitional cell carcinoma (TCC), which occurs predominantly in men and whose incidence appears to be increasing, occurs most frequently as superficial disease which, while prone to recurrence, is not life threatening. However, TCC which invades the muscle layer of the bladder (stage T2-T4) is lethal in ∼50% of individuals. Stage T2-T4 TCC is present at initial diagnosis in ∼30% of patients and superficial TCC progresses to T2-T4 disease 20% of the time. We sequenced whole exomes of 28 muscle-invasive bladder tumors and performed an additional screen of over 200 genes in 119 additional TCCs. We found frequent alterations (10%) in STAG2, a gene encoding a subunit of the cohesin complex, which regulates the sister chromatids segregation process. Most mutations identified on STAG2 were nonsense, missense or frameshift. Genetic disruption of the sister chromatid segregation process leads to aneuploidy, which is one of the hallmarks of cancer. Based on recently published studies, however, it is uncertain whether a loss of STAG2 causes aneuploidy or not. In this study we sought to elucidate the role of STAG2 loss of expression in bladder tumorigenesis. We have analyzed the expression of STAG2 in 14 urothelial cell lines at both mRNA and protein levels by means of q-PCR and western blots, respectively. We found that STAG2 expression was lost in 2 of the 14 urothelial cell lines (HB-CLS-2 and UM-UC-3) analyzed. We chose SV-HUC (immortalized normal urothelium cells) along with two bladder cancer cell lines that had high expression of STAG2 (HB-CLS-1 and TCC-SUP) to study effects of STAG2 loss by transient knockdown. Knockdown of STAG2 in SV-HUC, TCC-SUP and HB-CLS-1 cells did not affect proliferation. Cell cycle analysis on TCC-SUP cells revealed that STAG2 knockdown resulted in a lower percentage of cells in the G0/G1 phase in comparison to the scrambled control. Taken together, these data suggest that STAG2 has a role in modifying DNA content but not proliferation. Our ongoing studies utilizing bladder cancer tissue microarray (TMAs) and copy number variation (CNV) array will help to clarify the consequences of STAG2 loss in bladder cancer.

Citation Format: Nithya Krishnan, Anna Woloszynska-Read, Jianmin Wang, Song Liu, Carl Morrison, Khurshid Guru, Evelyn Smit, Donald Trump, Candace Johnson. Loss of STAG2 in bladder 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 2226. doi:10.1158/1538-7445.AM2014-2226
Abstract 2236: The Biospecimen Methodological Study (BMS): Identifying best practices for tissue preservation and maintenance of RNA integrity
Authors: Rao Abhi , Carithers Latarsha , Moore Helen
Published: May 2015

Researchers may use a number of traditional methods for human tissue preservation and stabilization such as liquid nitrogen (LN2) or dry ice, and yet the effectiveness of newer methods remains unclear. The National Cancer Institute's Biorepositories and Biospecimen Research Branch (BBRB; formerly known as OBBR) conducted a study to better understand such tissue preservation methods to inform BBRB's biospecimen collection efforts for the NIH Genotype-Tissue Expression (GTEx) program. The Biospecimen Methodological Study (BMS) was developed to provide an evidence-based structure to support future tissue collection and data analysis methods by comparing features of three different preservation methods: PAXgene Tissue (Qiagen); LN2; and dry ice. In preliminary studies, use of PAXgene Tissue has proven highly successful in preservation and maintenance of RNA quality. Another critical aspect of the BMS was to determine the degree to which the time between cardiac cessation and tissue preservation alters RNA integrity; such changes in molecular integrity could result in changes in molecular profiles upon gene expression analysis. The results of this study will provide valuable information about tissue processing parameters by identifying artifacts associated with different preservation methods and timing post cardiac cessation. Identifying and utilizing superior biospecimen preservation methods for RNA analysis will help identify patient-specific changes in gene expression and ultimately contribute to the development of targeted molecular medicine.

Citation Format: Abhi Rao, Latarsha Carithers, Helen Moore. The Biospecimen Methodological Study (BMS): Identifying best practices for tissue preservation and maintenance of RNA integrity. [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 2236. doi:10.1158/1538-7445.AM2014-2236
Abstract 2238: Non-random genetic rearrangements driven highly malignant tumor-cell population dictates the development of high-risk neuroblastoma
Authors: Khan Faizan H. , Ramraj Satish K. , Pandian Vijayabaskar , Aravindan Sheeja , Herman Terence S. , Natarajan Mohan , Aravindan Natarajan
Published: May 2015

Ascertaining the driving factors and molecular flow through that confiscate favorable to aggressive high-risk disease has momentous importance in neuroblastoma cure. Herein, we investigated the cytogenetic and tumorigenic/metastatic physiognomies of distinct population of highly malignant (NBP4) cells established from multi-site aggressive tumors after parental (SH-SY5Y) cell xenotransplantation. Karyotyping (G-banding) NBP4 revealed 47 (XX) with six novel non-random chromosomal rearrangements on 1q32, 8p23, 9q34, 15q24, 22q13 (additions), and 7q32 (deletion). Compared to the parental cells, high-resolution array CGH analysis of NBP4 cells and aggressive tumors revealed genetic alterations in chromosomes 1, 7, 8 and 22 corresponding to gain in coding regions of LOC100288142, CD1C, CFHR3, FOXP2, MDFIC, RALYL, CSMD3, SAMD12-AS1, MAL2 and loss in coding regions of ADAM5, LOC400927, APOBEC3B, RPL3, MGAT3, SLC25A17, EP300, L3MBTL2, SERHL, POLDIP3, A4GALT and TTLL1. Compared to SH-SY5Y cells, aggressive NBP4 cells exert stem-like phenotype and under stem-cell-growth conditions demonstrated a profound tumorosphere forming capabilities. Moreover, xenotransplantation of NBP4 cells exhibited high tumor forming capacity and metastatic potential in vivo. Together, these data demonstrate the clonal enrichment of select highly malignant cells drives neuroblastoma progression, and further implies that observed genetic alterations could mediate phenotypic transformation and functional response.

Citation Format: Faizan H. Khan, Satish K. Ramraj, Vijayabaskar Pandian, Sheeja Aravindan, Terence S. Herman, Mohan Natarajan, Natarajan Aravindan. Non-random genetic rearrangements driven highly malignant tumor-cell population dictates the development of high-risk neuroblastoma. [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 2238. doi:10.1158/1538-7445.AM2014-2238
Abstract 2241: SHH pathway in uterine mesenchymal tumors
Authors: Garcia Natalia , Souza Faila C. , Bozzini Nilo , Baiocchi Glauco , Cunha Isabela W. , Soares Fernando A. , Baracat Edmund C. , Carvalho Kátia C.
Published: May 2015

Background: Uterine leiomyoma (ULM) and leiomyosarcoma (ULMS) are smooth muscle tumors with distinct clinical behavior that can cause infertility and even lead to death. Little is known about the factors that influence these tumors biology. Identification of new molecular markers can help in the differential diagnosis between ULM from low grade ULMS. It was demonstrated that activation of the Sonic Hedgehog (SHH) signaling pathway is related to the development of several tumors, through its activity in the cell proliferation and differentiation. Here, we investigated the protein expression profile of SHH, PTCH1, SMO and GLI1 both ULM (including non-typical leiomyomas) and ULMS. Methods: We selected 57 samples of ULMS and 30 samples of ULM (10 samples non-typical - ULMA). All tissues were used in TMA construction. Immunohistochemistry reaction was performed using monoclonal antibody against SHH (1:50), PTCH (1:100), SMO (1:100) and GLI-1 (1:200) using sodium citrate pH 6,0 in pressure cooker. Results were evaluated by Aperio® automated method. Results: Our preliminary data showed similar protein profile between the leiomyosarcoma and non-typical leiomyoma. GLI 1 and PTCH showed higher expression compared to leiomyoma and myometrium (p<0.05). SMO, and SHH showed higher expression only in leiomyossarcomas (p<0.05). Conclusions: Ours preliminary results showed that expression of Sonic Hedgehog pathway members might play an important role in uterine smooth tumors, since ULMS showed higher levels of these proteins. However, the role of these proteins interactions must be clarified.

Citation Format: Natalia Garcia, Faila C. Souza, Nilo Bozzini, Glauco Baiocchi, Isabela W. Cunha, Fernando A. Soares, Edmund C. Baracat, Kátia C. Carvalho. SHH pathway in uterine mesenchymal tumors. [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 2241. doi:10.1158/1538-7445.AM2014-2241
Abstract 2251: nrf2-keap1 axis molecular profile in small cell lung cancer cell lines
Authors: Muscarella Lucia Anna , Torre Annamaria la , Sparaneo Angelo , Palumbo Orazio , Storlazzi Clelia Tiziana , Balsamo Teresa , Trombetta Domenico , Pellegrini Fabio , Barbano Raffaela , Pasculli Barbara , Graziano Paolo , Sanchez-cespedes Montse , Landi Maria Teresa , Parrella Paola , Fazio Vito Michele
Published: May 2015

The Keap1/Nrf2 pathway is a master regulator of antioxidants and cellular stress responses implicated in resistance of tumor cells against chemotherapeutic drugs. The link between molecular alterations of this pathway in Non Small Cell Lung Cancer (NSCLC) is well studied and appears to depend on several main factors including the existence of activating mutations in NFE2LE gene and/or loss of function mutations and methylation in the KEAP1 gene. At present, the data concerning the mechanism of alteration of Nrf2-Keap1 pathway in Small Cell Lung Cancer (SCLC) instead are almost incomplete and correlation analysis with therapeutic strategies targeting the molecular dysfunction of this pathway is ongoing.

Here we present a comprehensive molecular alteration profile of the main partners of the Nrf2/keap1 axis in 12 SCLC cell lines by integrating data from SNP-Array analysis, immunofluorescence, mutation screening by direct sequencing, methylation by QMSP and expression analyses by RT-qPCR and western blotting.

Our analyses confirm the global deregulation of Nrf2/Keap1 pathway in SCLC cell lines, showing an hypermethylation of the CpGs located into the P1 promoter region of the KEAP1 in 42% (5/12) of the cell lines, and a chromosomal amplification involving the NFE2LE gene locus (2q31.1) in two cell lines. Only one just described point mutation in the kelch-repeat 2 was observed in one of the cell line analysed.

Our gene-alteration profile of SCLC cell lines provides new insights into the mechanism of deregulation of Nrf2-Keap1 detoxification pathway in this group of high grade neuroendocrine lung cancers, suggesting the NFE2LE gene amplification and the KEAP1 promoter hypermethylation as alternative mechanisms of dysfunctional Nrf2/Keap1 in SCLCs.Analyses on tumor tissues are ongoing to confirm these observations. Moreover, the provided full molecular data from cell lines will be useful for in vitro functional studies aimed to establish new combined therapeutic strategies in targeted cancer treatments of this aggressive lung tumor histotype.

Citation Format: Lucia Anna Muscarella, Annamaria la Torre, Angelo Sparaneo, Orazio Palumbo, Clelia Tiziana Storlazzi, Teresa Balsamo, Domenico Trombetta, Fabio Pellegrini, Raffaela Barbano, Barbara Pasculli, Paolo Graziano, Montse Sanchez-cespedes, Maria Teresa Landi, Paola Parrella, Vito Michele Fazio. nrf2-keap1 axis molecular profile in small cell lung cancer cell lines. [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 2251. doi:10.1158/1538-7445.AM2014-2251
Abstract 2253: YAP nuclear accumulation involved in drug-induced cellular senescence in vitro
Authors: Ma Kai , Wang Shuren , Xu Qing , Liu Mei , Zhu Hongxia , Xu Ningzhi
Published: May 2015

Cellular senescence is accompanied by some striking morphological changes, such as becoming large, flat and multinucleated. However, how these changes are established is poorly understood. The Hippo pathway plays an important role in the control of organ size. Previous study reported that YAP, one major downstream effector of Hippo pathway, could be regulated by mechanical signals through F-actin bundles. However, whether YAP participates in the control of cell size through cytoskeleton remains unexplained. In our current study, we found that the senescent cells induced with adriamycin or camptothecin became large and flat, and the stress fibers of the senescent cells seemed to maintain or increase, observed by F-actin staining, though in a way of inappropriate actin clumping. Moreover increased nuclear level and activity of YAP were identified in drug-induced senescent cells. Hence, our data indicated that the increased F-actin bundles and mechanical signals could be acquired in senescent cells induced by adriamycin or camptothecin. In summary, based on our findings here, we proposed that YAP might play some roles during drug-induced cellular senescence in vitro.

Citation Format: Kai Ma, Shuren Wang, Qing Xu, Mei Liu, Hongxia Zhu, Ningzhi Xu. YAP nuclear accumulation involved in drug-induced cellular senescence in vitro. [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 2253. doi:10.1158/1538-7445.AM2014-2253
Abstract 2255: DHA may enhance proteasome activity in human cervical cancer cells: Indirect modulation of proteasome by DHA
Authors: Lim Kyu , Jing Kaipeng , Shin Soyeon , Jeong Soyeon , Kim Soyeon , Kweon Gi-Ryang , Park Seung-Kiel , Wu Tong , Park Jong-Il
Published: May 2015

The omega-3 fatty acid, docosahexaenoic acid (DHA), has been shown to induce the proteasomal degradation of various cancer-associated molecules such as beta-catenin and estrogen receptor, implying its regulatory effect on proteasome. To verify this possible link between DHA and proteasome function, we explored the impacts of DHA on human papillomavirus-positive HeLa cervical cancer cells that constitutively express two known endogenous proteasome substrates E6 and E7 viral proteins. Exposure HeLa cells to DHA resulted in elevated proteasome activities along with decreased levels of E6/E7 viral proteins, and these effects of DHA were remarkably reversed in the presence of proteasome inhibitors, suggesting that DHA improves proteasome function in the cells and thereby accelerates the degradation of proteasome substrates. The DHA-induced increase in cellular proteasome activity was preceded by reactive oxygen species (ROS) overproduction and could be blocked by preincubation of the cells with ROS scavengers. Likewise, HeLa cells treated with exogenous ROS, H2O2, also exhibited significantly improved proteasome function. Unexpectedly, when the purified proteasome and whole-cell lysates isolated from HeLa cells were directly exposed to DHA or H2O2, such marked increases in proteasome activity were not seen. Taken together, these findings indicate that DHA may induce proteasome activation in cervical cancer cells mainly by indirect regulation of the proteasome.

Citation Format: Kyu Lim, Kaipeng Jing, Soyeon Shin, Soyeon Jeong, Soyeon Kim, Gi-Ryang Kweon, Seung-Kiel Park, Tong Wu, Jong-Il Park. DHA may enhance proteasome activity in human cervical cancer cells: Indirect modulation of proteasome by DHA. [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 2255. doi:10.1158/1538-7445.AM2014-2255
Abstract 2259: Oxidative DNA damage causes premature senescence in mouse embryonic fibroblasts deficient for krüppel-like factor 4
Authors: Liu Changchang , Rosa Stephen La , Hagos Engda
Published: May 2015

Krüppel-like factor 4 (KLF4) is a zinc-finger-containing transcription factor with tumor suppressor activity in various cancer types. Cells that sustain double strand breaks (DSBs) in their DNA due to high levels of reactive oxygen species (ROS) can develop genomic instability, which can result in cancer formation. One protective response to increased levels of ROS is the induction of cellular senescence. Recently, we found that mouse embryonic fibroblasts (MEFs) null for Klf4 are genetically unstable, as evidenced by the presence of DNA DSBs. However, it is yet unknown whether KLF4 is involved in regulating oxidative stress-induced DNA damage. Therefore, we sought to determine the mechanisms by which ROS induce genomic instability in Klf4-deficient MEFs. With SA-β-Gal staining, we show that Klf4-/- MEFs enter senescence earlier than Klf4+/+ MEFs, and western blot shows accumulation of p21 and p53 with increasing passages. In addition, immunostaining against γ-H2AX indicates that the increased level of DNA damage in Klf4-/- MEFs positively correlates with ROS accumulation. Consistent with ROS as a source of DSB in Klf4-/- MEFs, treatment with NAC, reduced the accumulation of DNA damage. Our RT-PCR result demonstrates that Klf4-/- MEFs have decreased expression of the antioxidant gene, Gstα4. The downregulation of the Gstα4 correlates with significant levels of ROS accumulation, as shown by DCFDA and FACS analysis, and thus the oxidative stress-induced premature senescence. Together these findings suggest a mechanism by which KLF4 protects against DNA damage and oxidative stress at least in part through the regulation of Gsta4 and likely related genes.

Note: This abstract was not presented at the meeting.

Citation Format: Changchang Liu, Stephen La Rosa, Engda Hagos. Oxidative DNA damage causes premature senescence in mouse embryonic fibroblasts deficient for krüppel-like factor 4. [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 2259. doi:10.1158/1538-7445.AM2014-2259
Abstract 2261: Natural redistribution of end-protection proteins in aging cells as telomeres shorten
Authors: Baribault Michelle E. , Swanson Mark J. , Bae Nancy S.
Published: May 2015

Telomeres are the nucleoprotein structures at the ends of linear chromosomes. In mammalian cells, a six-member protein complex, called shelterin, protects telomeres from being recognized as DNA double-strand breaks and from nucleolytic degradation. It has been speculated that as telomeres become progressively shorter with each round of replication, the number of binding sites for the components of shelterin would decrease as well. In an attempt to determine whether there is a relationship between the length of telomeres and shelterin binding, primary human dermal fibroblasts from neonatal foreskin (HDFn) were grown in culture, and the levels of a number of shelterin components were measured over time. Unlike other previous studies where artificially constructed telomeres were used to address this question, we are following the natural progression of aging of a population of cultured cells. Comparisons were made between young cells at a population doubling (PDL) between 3 and 6 and aged cells at a PDL of 28 to 38. Southern blotting using a telomeric probe showed that the aged cells possessed significantly shorter telomeres than their younger counterparts, supporting the notion that there will be fewer shelterin binding sites. Using immunoblotting to compare the levels of several shelterin subunits (TRF2, hRAP1, and POT1), we found that TRF2, the DNA binding subunit that targets hRAP1 to the telomeres, and POT1 decrease in level as would be expected when fewer binding sites are present for the complex. Interestingly, the levels of hRAP1 did not decrease proportionally and remained relatively high compared to other subunits. These data suggest that as telomeres decrease in length and shelterin binding sites become less numerous, most shelterin subunits are degraded, but hRAP1 most likely plays an additional role. We have stressed young HDFn cells with hydrogen peroxide to age them prematurely. Upon treatment, the young cells (PDL of 8) became similar in appearance to the untreated, aged cells at PDL greater than 25. In the treated, young cells, hRAP1 levels in the nucleus decreased in a manner that was unaffected by inhibition of the proteasome, suggesting that during the oxidative stress, hRAP1 translocates from the nucleus to the cytoplasm. Taken together, our data suggest that RAP1 plays a role in stress signaling or response to cellular aging.

Citation Format: Michelle E. Baribault, Mark J. Swanson, Nancy S. Bae. Natural redistribution of end-protection proteins in aging cells as telomeres shorten. [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 2261. doi:10.1158/1538-7445.AM2014-2261
Abstract 2263: Change in miRNA by suberoylanilide hydroxamic acid affects lung cancer cell death via regulating thioredoxin
Authors: You Bo R. , Han Bo Ram , Park Woo Hyun
Published: May 2015

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase (HDAC) inhibitor to have the anti-tumor effect. Here, we evaluated the anti-growth effect of SAHA on lung cancer (A549, SK-LU-1, HCC-95, HCC-1588, NCI-H460, NCI-H1299, Calu-6, HCC-33, NCI-H69) and normal lung (human small airway epithelial cells and human pulmonary fibroblast) cells in relation to reactive oxygen species (ROS) and thioredoxin (Trx) levels. SAHA inhibited the growth of lung cancer cells, and induced a G2/M phase arrest and apoptotic cell death in these cancer cells. In contrast, SAHA did not significantly inhibit the growth of normal lung cells and induce apoptotic cell death. Concerning the oxidative stress, SAHA increased ROS levels in lung cancer cells and induced glutathione (GSH) depletion. Moreover, it decreased the expression level of Trx1 in lung cancer cells. While the overexpression of Trx1 attenuated death and GSH depletion in SAHA-treated lung cancer cells, the downregulation of Trx1 intensified those in these cells. In addition, SAHA increased miR-629 level in lung cancer cells. The mRNA level of Trx1 was decreased by SAHA. Furthermore, the 3′ UTR region of Trx1 was regulated by miR-629. In conclusion, SAHA inhibited the growth of lung cancer cells via cell cycle arrest and apoptosis. The growth inhibition was related to the levels of Trx1 and miR-629. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2008-0062279) and supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, (2013006279).

Citation Format: Bo Ra You, Bo Ram Han, Woo Hyun Park. Change in miRNA by suberoylanilide hydroxamic acid affects lung cancer cell death via regulating thioredoxin. [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 2263. doi:10.1158/1538-7445.AM2014-2263
Abstract 2266: Investigating the role of the anti-apoptotic protein ARC in breast cancer cell DNA repair
Authors: Grunblatt Eli , Madan Evanka , Roy Sweta , Goswami Sumanta
Published: May 2015

According to the American Cancer Society, more than 90% of breast cancer patients who succumb to the condition die as a result of the cancer metastasizing to other areas of the body. Our lab has previously shown that the presence of a chemoresistant invasive subpopulation of breast carcinoma cells is correlated with the prevalence of metastasis. Additionally, we have hypothesized that chemoresistance and survival of these cancer cells which eventually leads to relapse and metastasis following initial treatment with chemotherapeutic agents is achieved by enhanced DNA repair capability in this population. We reported that an anti-apoptotic protein ARC (apoptosis repressor with caspase recruitment domain) is over expressed in the invasive mammary cancer cells that are resistant to standard chemotherapeutic agents. Further, we have shown that ARC over expressing cells are resistant to chemotherapy both in vivo and in vitro. In this study, we examined cellular DNA repair signaling pathways in order to delineate the role of ARC in the pathogenesis of breast cancer. Utilizing cell lines where ARC was either knocked down or overexpressed, we investigated potential correlations of ARC with DNA repair capability by means of the single cell gel electrophoresis assay (comet assay). The results of these experiments indicated that over expression of ARC leads to increased cellular DNA repair while knocking it down leads to decreased cellular DNA repair.

In order to determine the molecular basis of the role of ARC in the DNA repair pathways, we have performed qRT-PCR based gene expression analysis on ARC modified cell lines using techniques previously established in our lab. The results of these experiments indicated that increased levels of ARC are correlated with upregulation of key genes in single stranded DNA repair pathways such as the base excision and nucleotide excision repair pathways. We are working to identify the specific domain of ARC that serves as its effector in the DNA repair pathways. In particular, we are looking at the caspase recruitment domain (CARD) of ARC which is known to be key for ARC`s function in the apoptotic pathways. Once this domain is identified, it may serve as a potential drug target, the silencing of which could result in the disruption of key pathways required for invasive breast cancer cell survival. Such therapy may facilitate the ablation of chemoresistant breast cancer cell populations, prevent metastasis and tumor relapse, and increase long term patient survival.

Citation Format: Eli Grunblatt, Evanka Madan, Sweta Roy, Sumanta Goswami. Investigating the role of the anti-apoptotic protein ARC in breast cancer cell DNA repair. [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 2266. doi:10.1158/1538-7445.AM2014-2266
Abstract 2268: Augmentation of DR5 expression by calmodulin antagonists sensitizes TRA-8-induced apoptosis in pancreatic cancer
Authors: Yuan Kaiyu , Zhou Tong , McDonald Jay , Chen Yabing
Published: May 2015

Pancreatic cancer is the fourth leading cause of cancer death in the USA. The 5-year survival rate has only improved from 2% to 6% in the past 35 years. TNF-related apoptosis-inducing ligand (TRAIL) or agonistic antibodies bind to death receptor 4 or 5 and induce apoptosis in a wide variety of tumors. TRA-8, a humanized activating antibody for the death receptor 5 (DR5), is a potent apoptosis-inducing reagent for cancer cells. However, several pancreatic cell lines are resistant to TRA-8-induced apoptosis. The goal of our studies is to indentify therapeutic agents that sensitize resistant pancreatic cancer cells to TRA-8-induced apoptosis and determine the underlying mechanism.

Among an array of compounds, we identified that trifluoperazine (TFP) and tamoxifen (TMX), two calmodulin (CaM) antagonists, enhanced TRA-8-induced cell death of TRA-8 resistance pancreatic cells. Both TFP and TMX increased TRA-8-induced apoptosis of resistant PANC-1 cells in dose-dependent manners. TMX treatment enhanced the efficacy of TRA-8 therapy on tumorigenesis of PANC-1 cells in a mouse xenograft model by 5 fold (n=7, p<0.05). We found that the effects of CaM antagonists on TRA-8-induced apoptosis were mediated by its induction of caspase-8 activation, as TMX increased TRA-8-induced activation of caspase-8 in the DR5-recruited signaling complex whereas caspase-8 inhibition by Z-IETD-FMK markedly decreased TRA-8-induced apoptosis as well as activation of downstream apoptosis effector caspase-3. Analysis of the expression of the death receptors revealed that TFP and TMX increased the expression of the DR5, at both mRNA and protein levels. Furthermore, TFP and TMX increased acetylation of histone H3 and H4, which may contribute their augmentation of DR5 expression. In summary, we have demonstrated that CaM antagonists increase DR5 expression and DR5-mediated caspase-8 activation, thus sensitizing pancreatic cancer to TRA 8-induced apoptosis. Our results identify an important role for CaM in regulating of DR5-mediated apoptosis signals in pancreatic cancer cells and support the possible use of combined TRA-8 and CaM antagonists to treat pancreatic cancer.

Citation Format: Kaiyu Yuan, Tong Zhou, Jay McDonald, Yabing Chen. Augmentation of DR5 expression by calmodulin antagonists sensitizes TRA-8-induced apoptosis in pancreatic 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 2268. doi:10.1158/1538-7445.AM2014-2268
Abstract 227: Estrogen receptor-α and tumor initiating cells are novel targets of diallyl trisulfide in breast cancer cells
Authors: Hahm Eun-Ryeong , Kim Su-Hyeong , Singh Shivendra V.
Published: May 2015

Epidemiological studies suggest a protective role for Allium vegetables, such as garlic, against breast and other cancers. Diallyl trisulfide (DATS) is a promising oil-soluble anticancer metabolite generated after cutting or chewing of Allium vegetables. We have shown previously that DATS inhibits viability of human breast cancer cells (MCF-7 and MDA-MB-231) and a cell line derived from spontaneously developed tumor of a mouse mammary tumor virus-neu mouse by causing apoptosis induction. We also found that a non-tumorigenic normal human mammary epithelial cell line (MCF-10A) is resistant to cell death induction by DATS. Studies by others have demonstrated in vivo efficacy of DATS against MCF-7 xenografts in nude mice. The present study builds upon these observations and identifies novel targets of DATS in breast cancer cells. MCF-7 and T47D cells exposed to DATS exhibited downregulation of estrogen receptor-α (ER-α) protein, which peaked between 12 and 24 h post-treatment. Diallyl sulfide and diallyl disulfide analogs were relatively less effective in suppressing protein level of ER-α compared with DATS. The 17β-estradiol (E2)-induced expression of pS2, an ER-α target gene product, was also decreased in the presence of DATS. Downregulation of ER-α protein expression resulting from DATS treatment was accompanied by a decrease in nuclear level of ER-α, suppression of ER-α mRNA, and inhibition of ERE2e1b-luciferase reporter activity. Moreover, apoptotic cell death induced by DATS was partially but statistically significantly attenuated in the presence of E2 in MCF-7 cells as well as by overexpression of ER-α in MDA-MB-231 cells. DATS treatment inhibited breast cancer stem cell (bCSC) population as evidenced by flow cytometric analysis of CD44high/CD24low/epithelial specific antigen positive population, aldehyde dehydrogenase 1 activity, and mammosphere frequency in MCF-7 and SUM159 cells. Inhibition of bCSC by treatment with DATS was associated with downregulation of EZH2 and Kruppel-like factor 4 proteins in both MCF-7 and SUM159 cells. In conclusion, the present study identifies ER-α and bCSC as novel targets of DATS in mammary cancer cells. This study was supported by the grant RO1 CA113363-09 awarded by the National Cancer Institute.

Citation Format: Eun-Ryeong Hahm, Su-Hyeong Kim, Shivendra V. Singh. Estrogen receptor-α and tumor initiating cells are novel targets of diallyl trisulfide in breast cancer cells. [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 227. doi:10.1158/1538-7445.AM2014-227
Abstract 2271: Effective therapy for advanced renal cell carcinoma using triptolide and TRAIL receptor agonists
Authors: Griffith Thomas S. , Brincks Erik L. , Kucaba Tamara A. , James Britnie R. , Sangwan Veena , Banerjee Sulagna , Saluja Ashok
Published: May 2015

Metastatic renal cell carcinoma (RCC) is incurable. IFNα or IL-2 immunotherapy has been used for RCC for more than 20 years, but response rates remain low and these cytokines can generate toxic. More recently, administration of multikinase inhibitors or anti-VEGF receptor mAb has become popular, but these agents are largely palliative and tumor regression is common. The shortcomings in current therapeutic options for patients with metastatic RCC provide the rationale for the development of novel treatment protocols. Triptolide, a diterpene triepoxide from the medicinal herb Tripterygium wilfordii, can suppress cell growth and induce apoptosis in a range of human tumor cells. Further, triptolide also sensitizes tumor cells to TNF-related apoptosis-inducing ligand (TRAIL). The objective of this study was to determine the effectiveness of combining triptolide with TRAIL receptor agonists against RCC. In vitro, the combination of triptolide and recombinant human TRAIL protein decreased cell viability in the human RCC cell line ACHN cells compared with either treatment alone. This decrease in viability correlated with increased induction of apoptosis (positive Annexin V staining) and caspase activation. The triptolide-induced sensitization was accompanied by decreased HSP70, HSP27, and HSF1 expression, as well as increased surface expression of TRAIL-R2. Similar in vitro treatment of the mouse renal cell adenocarcinoma cell line, Renca, demonstrated that triptolide enhanced the killing of these cells by membrane-bound TRAIL or agonistic anti-DR5 mAb. In vivo treatment of mice bearing orthotopic and metastatic Renca tumors showed that combination therapy consisting of minnelide (a water soluble version of triptolide) and agonistic anti-DR5 mAb resulted in significantly decreased tumor burden and increased animal survival compared to either therapy alone. Collectively, our data suggest that triptolide sensitizes RCC cells to TRAIL-induced apoptosis, which is accompanied by decreased heat shock protein expression and altered TRAIL receptor expression. These data also suggest that the combination of TRAIL receptor agonists and triptolide may prove to be an effective treatment for metastatic RCC, especially at doses that show suboptimal tumoricidal activity when used individually.

Citation Format: Thomas S. Griffith, Erik L. Brincks, Tamara A. Kucaba, Britnie R. James, Veena Sangwan, Sulagna Banerjee, Ashok Saluja. Effective therapy for advanced renal cell carcinoma using triptolide and TRAIL receptor agonists. [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 2271. doi:10.1158/1538-7445.AM2014-2271
Abstract 2273: c-Rel is a critical mediator of NF-κB-dependent apoptosis resistance of pancreatic cancer cells against TRAIL
Authors: Geismann Claudia , Grohmann Frauke , Wirths Gabriele , Sebens Susanne , Dreher Anita , Häsler Robert , Zeissig Sebastian , Schreiber Stefan , Rosenstiel Philip , Schäfer Heiner , Arlt Alexander
Published: May 2015

Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest malignancies with an overall life expectancy of six months despite palliative radio-chemotherapy. The transcription factor NF-κB has been shown to be a critical component of dysregulated transcription factor activity conferring this profound resistance against chemotherapeutic drugs and death receptor induced apoptosis. Despite extensive data on the role of the most abundant NF-κB subunit p65/RelA in PDAC apoptosis control, only little knowledge of the role of the subunit c-Rel in solid cancers exists. In the present study, three pancreatic carcinoma cell lines (Panc1, Patu8988t, MiaPaca2) were analysed for the role of c-Rel in resistance against TRAIL induced apoptosis. TRAIL resistant Panc1 and Patu8988 cells exhibit a strong TRAIL inducible NF-κB activity, whereas TRAIL sensitive MiaPaca2 cells displayed only a small increase in NF-κB binding activity. Transfection with siRNA against the c-Rel subunit of NF-κB sensitized the TRAIL resistant cells in a comparable fashion like siRNA targeting the p65/RelA subunit. Gel shift analysis revealed that together with the p65/RelA subunit, c-Rel is part of the TRAIL inducible NF-κB complex in PDAC. Array analysis results suggested NFATc2 as a c-Rel target gene that is one of the 15 strongest TRAIL inducible genes in apoptosis-resistant Panc1 cells. siRNA targeting c-Rel strongly reduced TRAIL induced NFATc2 activity in TRAIL resistant PDAC cells. Furthermore siRNA targeting NFATc2 sensitized these PDAC cells against TRAIL induced apoptosis. Finally, TRAIL induced expression of COX-2 was strongly reduced through siRNA targeting c-Rel or NFATc2 and pharmacological inhibition of COX-2 with celecoxib strongly increased TRAIL apoptosis.

In conclusion, c-Rel is a critical mediator of NF-κB dependent anti-apoptotic signalling in PDAC through activation of NFATc2 and COX-2.

Citation Format: Claudia Geismann, Frauke Grohmann, Gabriele Wirths, Susanne Sebens, Anita Dreher, Robert Häsler, Sebastian Zeissig, Stefan Schreiber, Philip Rosenstiel, Heiner Schäfer, Alexander Arlt. c-Rel is a critical mediator of NF-κB-dependent apoptosis resistance of pancreatic cancer cells against TRAIL. [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 2273. doi:10.1158/1538-7445.AM2014-2273
Abstract 2279: Adenine nucleotide translocase-2 is a key molecule in the enhancement of TRAIL sensitivity by flavonoids
Authors: Oishi Masakatsu , Iizumi Yosuke , Ueda Takashi , Ito Saya , Nakamura Terukazu , Hongo Fumiya , Naya Yoshio , Miki Tsuneharu , Sakai Toshiyuki
Published: May 2015

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine which belongs to the tumor necrosis factor family. TRAIL binds to death receptor (DR) 4 and DR5 and selectively induces apoptosis in various malignant tumors, but not in normal cells. Therefore, TRAIL is an attractive cancer therapeutic agent. However, several malignant tumors have resistance to TRAIL and overcoming this resistance is essential for chemotherapy using the TRAIL pathway. Anticancer agents such as bortezomib, etoposide, doxorubicin, and irinotecan have been reported to upregulate DR5 and sensitize cancer cells to TRAIL in vitro and in vivo. Since these agents cause various adverse effects, we and others have screened dietary compounds inducing DR5 expression and identified several flavonoids as DR5 inducers. For instance, dietary flavonoid apigenin and quercetin enhance TRAIL sensitivity by upregulating DR5. However, the mechanisms by which these flavonoids upregulate DR5 are unknown. To elucidate the mechanisms by which these flavonoids induce DR5, we explored the proteins binding to apigenin and quercetin, but not the flavonoid genistein which did not induce DR5. We used magnetic FG beads, which recently revealed the target of thalidomide and the mechanism of thalidomide teratogenicity. We identified ANT2 as a binding protein of flavonoids, apigenin and quercetin, upregulating DR5. As with the treatment of apigenin and quercetin, knockdown of ANT2 enhanced TRAIL-induced apoptosis by upregulating DR5. Moreover, knockdown of ANT2 attenuated the enhancement of TRAIL-mediated apoptosis by apigenin and quercetin. In the present study, we show that ANT2 is a target of flavonoids which upregulate DR5 and sensitize malignant tumor cells to TRAIL.

Citation Format: Masakatsu Oishi, Yosuke Iizumi, Takashi Ueda, Saya Ito, Terukazu Nakamura, Fumiya Hongo, Yoshio Naya, Tsuneharu Miki, Toshiyuki Sakai. Adenine nucleotide translocase-2 is a key molecule in the enhancement of TRAIL sensitivity by flavonoids. [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 2279. doi:10.1158/1538-7445.AM2014-2279