bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1340: Induction of necroptosis in ovarian cancer cells as a therapeutic strategy
Authors: McCabe Katelyn E. , Bacos Karl , Lu Dan , Delaney Joe R. , Vamos Mitchell , Cosford Nicholas D. P. , Stupack Dwayne G.
Published: May 2015

The standard of care for ovarian cancer patients is carboplatin and paclitaxel. While many tumors initially respond to these drugs, patients often relapse with resistant disease. One of the mechanisms implicated in this development of resistance is evasion of apoptosis. Exploiting alternative death pathways, including necroptosis, is one strategy to treat such recurrent disease. Overexpressed in many cancers, inhibitor of apoptosis proteins (IAPs) represent one family of promising targets. Treatment of cells with an IAP antagonist can activate apoptosis or, if caspases are blocked, necroptosis. Among a panel of ovarian cancer cell lines screened for sensitivity to an IAP antagonist in combination with a pan-caspase inhibitor, only one (OVCAR3) was found to be sensitive, but this sensitivity was also exhibited by serous ovarian cancer cells isolated from patients. Combination treatment induces the formation of a p62-associated necrosome (RIPK1/RIPK3/FADD/caspase-8), and death can be rescued with inhibitors of RIPK1 and MLKL, two key proteins in the necroptosis pathway. All sensitive cell lines, but no resistant lines, express RIPK3 protein, and data from knockdown and ectopic expression studies demonstrate the dependency of cell death on kinase-active RIPK3, as well as caspase-8. Studies using a TNFα neutralizing antibody suggest that the combination elicits autocrine production of TNFα, which likely, in turn, activates the extrinsic death pathway in these cells. In conclusion, these findings illustrate that necroptosis can be induced in RIPK3-expressing ovarian cancer cells and indicate that in vivo studies are warranted.

Citation Format: Katelyn E. McCabe, Karl Bacos, Dan Lu, Joe R. Delaney, Mitchell Vamos, Nicholas D. P. Cosford, Dwayne G. Stupack. Induction of necroptosis in ovarian cancer cells as a therapeutic strategy. [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 1340. doi:10.1158/1538-7445.AM2014-1340
Abstract 1344: Downregulation of mortalin increased mitochondrial fragmentation and dysfunctions
Authors: Park So Jung , Cho Dong-Hyung
Published: May 2015

Mitochondria are essential organelles in the establishment of tumor characteristics such as the resistance to cell death, high proliferative capacity and adaptation to unfavorable cellular environment. Mitochondrial dynamics greatly influence the function and morphology of mitochondria. Until now various post-translational modifications for mitochondrial dynamic proteins and several regulatory proteins have explained complex mitochondrial dynamics. However, the precise mechanism that coordinates these complex processes remains unclear. To further understand the regulatory machinery of mitochondrial dynamics, we screened a mitochondrial siRNA library and identified mortalin as a potential regulatory protein. Mortalin is a heat-non-inducible member of the heat shock protein 70 family and it binds to p53 and prevents p53 from entering the nucleus. Both genetic and chemical inhibition of mortalin strongly induced mitochondrial fragmentation and increased cytotoxicity as well as mitochondrial dysfunction. In contrast, overexpression of mortalin significantly suppressed mitochondrial fragmentation and cell death. Taken together, our results suggest that down-regulation of mortalin potentiates mitochondrial fragmentation and dysfunction

Note: This abstract was not presented at the meeting.

Citation Format: So Jung Park, Dong-Hyung Cho. Downregulation of mortalin increased mitochondrial fragmentation and dysfunctions. [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 1344. doi:10.1158/1538-7445.AM2014-1344
Abstract 1345: Role of AMP-activated protein kinase in crosstalk between apoptosis and autophagy in human colon cancer
Authors: Song Xinxin , Kim Seog-Young , Zhang Lin , Lee Yong J.
Published: May 2015

Unresectable colorectal liver metastases remain major unsolved issues and more effective regimens are urgently needed. During screening synergistic drug combinations for colon cancer therapy, we identified a novel multimodality treatment for colon cancer cells-chemotherapeutic agent melphalan in combination with proteasome inhibitor bortezomib and mTOR (mammalian target of rapamycin) inhibitor rapamycin. We showed in this study that melphalan triggered apoptosis, bortezomib induced both apoptosis and autophagy, rapamycin caused autophagy, and the combinatorial treatment induced synergistic apoptosis which was mediated through an increase in caspase activation. We observed that mitochondrial dysfunction induced by the combination was linked with altered cellular metabolism and induced AMP-activated protein kinase (AMPK) activation, and thus resulted in Beclin-1 phosphorylated at Ser 93/96. Interestingly, Beclin-1 phosphorylated at Ser 93/96 is sufficient to induce Beclin-1 cleavage by caspase 8 for switching off autophagy to achieve the synergistic induction of apoptosis. The multimodality treatment-induced Beclin-1 cleavage was abolished in Beclin-1 double mutant (D133A/D149A) knock-in HCT116 cells, restoring the autophagy-promoting function of Beclin-1 and suppressing the apoptosis induced by the combination therapy. These observations provide a novel mechanism for AMPK induced apoptosis through interplay between autophagy and apoptosis.

Citation Format: Xinxin Song, Seog-Young Kim, Lin Zhang, Yong J. Lee. Role of AMP-activated protein kinase in crosstalk between apoptosis and autophagy in human colon 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 1345. doi:10.1158/1538-7445.AM2014-1345
Abstract 135: Phosphorylation and furin-mediated processing are critical posttranslational modifications of the KISS1 metastasis suppressor
Authors: Harihar Sitaram , Hampton Kelsey R. , Iwakuma Tomoo , Seidah Nabil G. , Welch Danny R.
Published: May 2015

Originally discovered as a metastasis suppressor, KISS1 has since been defined as a neurotransmittor and regulator of diverse cellular functions and has been implicated in pathologies such as hypogonadism and Alzheimer's disease. Several laboratories are actively developing therapies based upon KISS1 biology. KISS1 is secreted and processed into small peptides (called kisspeptins (KP)) in the extracellular milieu. However, the enzyme(s) responsible for generation of KP from nascent KISS1 is unknown, although sequence analysis (cleavage at KR or RR dibasic sites) suggests that proprotein convertases (PC) are responsible. We hypothesized that enzyme(s) belonging to the PC family processes KISS1 to generate KP. KISS1 processing was completely inhibited by treatment with PC inhibitors Dec-RVKR-CMK and a1-PDX in these cells multiple tumor cell lines. Further, mRNA expression of the seven members of the PC family showed consistent expression of only three proteases - furin, PCSK5 and PCSK7 narrowing the candidate proteases. Only shRNA-mediated knockdown of furin (i.e., not PCSK5 or PCSK7) blocked KISS1 processing. Thus furin is the essential enzyme responsible for KP generation. Even when cells were exposed to Dec-RVKR-CMK or shRNA targeting furin, two bands (Mr∼15.9 kDa and Mr ∼17 kDa) were consistently detected in SDS-PAGE of conditioned medium. This observation suggested KISS1 could either be alternatively post-translationally modified or that another protease (family) might also cleave KISS1. To investigate the possibility that KISS1 may be post-translationally modified, potential sites of O-glycosylation (NetOGlyc 4.0) or serine/threonine phosphorylation (NetPhos 2.0) were analyzed in silico. Three amino acid residues (T69, S70, and S72)of the native KISS1 protein showed highest scores as being either O-glycosylated or phosphorylated. Further, mutation of these three residues to alanine resulted in the loss of the higher molecular weight band which was co-localized to a band detected using anti-phospho Ser/Thr strongly suggesting that KISS1 is indeed phosphorylated. This is the first report of phosphorylation of KISS1. Studies are underway to identify the kinase(s) and phosphatase(s) involved as well as the functional significance of the phosphorylation. Support: CA134981, National Foundation for Cancer Research, Steiner Family Fellowship in Metastasis Research, Susan G. Komen for the Cure SAC110037.

Citation Format: Sitaram Harihar, Kelsey R. Hampton, Tomoo Iwakuma, Nabil G. Seidah, Danny R. Welch. Phosphorylation and furin-mediated processing are critical posttranslational modifications of the KISS1 metastasis suppressor. [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 135. doi:10.1158/1538-7445.AM2014-135
Abstract 1351: Expression of klotho enhances the apoptotic response of cancer cells to anticancer agents
Authors: Kudo Yujin , Usuda Jitsuo , Furumoto Hideyuki , Maehara Sachio , Ohtani Keishi , Inoue Tatsuya , Ishizumi Taichiro , Kato Yasufumi , Kakihana Masatoshi , Kajiwara Naohiro , Ohira Tatsuo , Ikeda Norihiko
Published: May 2015

(Introduction)

We have reported that the klotho gene, which is an anti-aging gene known to be a tumor suppressor, is a prognostic factor for lung cancer patients. In this study, we determined whether klotho expression may have a predictive value of the efficacy of anti-cancer agents for cancer treatment.

(Methods)

We performed co-transfection of klotho and GFP-plasmid into MCF-7c3 cells, which stably express procaspase-3, and treated the cells with various kinds of anti-cancer agents. We observed typical apoptosis morphologically using fluorescent microscopy and evaluated the apoptotic response.

(Results)

Western blot analysis revealed high klotho expression in the cells transfected with the klotho gene plasmid. There was no evidence of klotho expression in the normal MCF-7c3 cells. The anti-cancer drug sensitivity test showed that docetaxel significantly induced more typical apoptosis in the cells with klotho than in the cells without klotho (41.2% and 18.3%, respectively; p < 0.05). Paclitaxel also significantly induced more typical apoptosis in the cells with klotho than in the cells without klotho (34.9% and 14.9%, respectively; p < 0.05). However, there was no significant difference in the apoptosis rates induced by other anti-cancer drugs, such as cisplatin, carboplatin, pemetrexed, gemicitabine, vinorelbine, etoposide, and gefitinib.

(Conclusion)

A high expression of the klotho protein can markedly increase the apoptotic response of MCF-7c3 cells to docetaxel and paclitaxel. These results suggest that klotho expression may indicate the predictive value of chemotherapy and play an important role in cancer treatment.

Apoptosis induced by taxane-induced stratified expression of klotho proteinRate of apoptosisDocetaxelPaclitaxelA: Klotho(-)/GFP(-)14.8%19.3%A vs B: p>0.05A vs B: p>0.05B: Klotho(-)/GFP(+)18.3%14.9%B vs C: p=0.01B vs C: p=0.03C: Klotho(+)/GFP(+)41.2%35.9%

Citation Format: Yujin Kudo, Jitsuo Usuda, Hideyuki Furumoto, Sachio Maehara, Keishi Ohtani, Tatsuya Inoue, Taichiro Ishizumi, Yasufumi Kato, Masatoshi Kakihana, Naohiro Kajiwara, Tatsuo Ohira, Norihiko Ikeda. Expression of klotho enhances the apoptotic response of cancer cells to anticancer agents. [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 1351. doi:10.1158/1538-7445.AM2014-1351
Abstract 1352: Cytoplasmic galectin-7 has an antiapoptotic function by decreasing p53 nuclear translocation in breast cancer cells
Authors: Grosset Andrée-Anne , Gagné Donald , Labrie Marilyne , Vladoiu Maria , Gaboury Louis , Doucet Nicolas , St-Pierre Yves
Published: May 2015

Resistance to apoptosis induced by anti-cancer drugs is a major obstacle for the treatment of aggressive forms of breast cancer. Galectin-7 was recently shown to be specifically expressed in basal-like and HER-2 positive but not in luminal subtypes of human breast cancer. Overexpression of galectin-7 in breast cancer cells also increases lung and bone metastases. However, the molecular mechanisms used by galectin-7 to increase the dissemination of breast cancer cells remain largely unknown. Although galectins are best known for their ability to bind cell surface receptors, there is increasing evidence that their intracellular forms have important functions in cancer cells. This is particularly true in the control of apoptosis. Up to now, regulation of apoptosis by intracellular galectins has been largely attributed to their ability to translocate to mitochondria, possibly following their interaction with bcl-2. Here, we report that a mutant of galectin-7 that is unable to translocate to mitochondria and nucleus induces resistance of human breast cancer cells to apoptosis induced by etoposide or by hypoxia-mimicking conditions. Surprisingly, this mutant and the wild-type form of galectin-7 bind equally well to bcl-2 in vitro and in vivo. Interestingly, both forms decreases the translocation of p53 to the nucleus and reduce the expression of p21 following treatment with doxorubicin. These results challenges the current paradigm that mitochondrial galectins are important for resistance to apoptosis and call for a greater focus on the role of cytosolic galectins in cancer cells. Our data also indicate that targeting cytosolic galectin-7 could improve the efficacy of anti-cancer drugs for the treatment of aggressive forms of breast cancer.

Citation Format: Andrée-Anne Grosset, Donald Gagné, Marilyne Labrie, Maria Vladoiu, Louis Gaboury, Nicolas Doucet, Yves St-Pierre. Cytoplasmic galectin-7 has an antiapoptotic function by decreasing p53 nuclear translocation 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 1352. doi:10.1158/1538-7445.AM2014-1352
Abstract 1353: Protein tyrosine phosphatases PTP-1B, SHP-2 and PTEN facilitate Rb/E2F-associated apoptotic signaling
Authors: Morales Liza D. , Capetillo Mario , Pavon Edgar Casillas , Shin Jun W. , Garcia Alexander , Lieman Jonathan H. , Kim Dae J.
Published: May 2015

To maintain tissue homeostasis, apoptosis is functionally linked to cell proliferation via the retinoblastoma tumor suppressor (Rb)/E2F complex. Rb/E2F-associated apoptosis is a robust apoptotic response initiated by loss of constitutive Rb/E2F transcriptional repression and mediated by caspase-8. It is characterized by inactivation of focal adhesion kinase (FAK) through dephosphorylation by protein tyrosine phosphatase(s). In the current work, we report that three protein tyrosine phosphatases, PTP-1B, SHP-2, and PTEN, are involved in Rb/E2F-associated apoptosis. Induction of apoptosis in an osteoscarcoma cell line that conditionally expresses a dominant negative mutant of E2F-1 (ER-dnE2F-1) by treatment with 4-hydroxytamoxifen (4-OHT) led to a significant increase in levels of PTP-1B, SHP-2, and PTEN mRNA expression in comparison to untreated cells. Chromatin immunoprecipitation assays showed that E2F-1 specifically binds to putative E2F-1 binding sites identified within the three PTP promoters. Knockdown of SHP-2, PTEN, or PTP-1B using siRNA increased cell viability and decreased cell death in 4-OHT-treated ER-dnE2F-1 cells compared to control. Consistent with this observation, knockdown of SHP-2 or PTEN significantly inhibited caspase-8 activity in 4-OHT-treated ER-dnE2F1 cells in comparison to control while knockdown of SHP-2, PTEN, or PTP-1B significantly inhibited caspase-3 activity in 4-OHT-treated ER-dnE2F-1 cells compared to control. Combined, our results indicated PTP-1B, SHP-2, and PTEN are required to regulate Rb/E2F-associated apoptotic signal transduction. Furthermore, our findings suggest PTP-1B, SHP-2, and PTEN can contribute to tumor suppression through an Rb/E2F-associated mechanism.

Citation Format: Liza D. Morales, Mario Capetillo, Edgar Casillas Pavon, Jun W. Shin, Alexander Garcia, Jonathan H. Lieman, Dae J. Kim. Protein tyrosine phosphatases PTP-1B, SHP-2 and PTEN facilitate Rb/E2F-associated apoptotic signaling. [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 1353. doi:10.1158/1538-7445.AM2014-1353
Abstract 136: PTEN phosphatase inhibits metastasis through negatively affecting Entpd5/IGF1R pathway
Authors: Yu Yanlin
Published: May 2015

Migration of cancer cell is the key event in metastasis. Recent data suggest that PTEN phosphatase activity may be required for inhibiting cell migration; however, little in vivo experimental evidence exists directly demonstrating the efficacy of PTEN phosphatase in tumor metastasis. We showed here that PTEN loss was correlated with progression to metastatic state using tissue array analysis and UV-irradiated HGF/SF transgenic-PTEN deficient mouse melanoma model. Analysis of various human melanoma cell lines demonstrated that PTEN expression was related to metastatic behavior. To further dissect the role of PTEN activity in metastasis, we have established a model system containing mutant PTEN with various phosphatase activities. We found that wild type PTEN could inhibit cell motility and invasiveness. Moreover, wild type PTEN was able to reduce metastatic potential of melanoma cells in a dose-dependent manner; in contrast, PTEN phosphatase dead mutant promoted metastatic behavior of melanoma cell. To gain more insight into the regulation of tumor cell migration, invasion and metastasis by PTEN, we performed a microarray analysis of cells with various PTEN phosphatase mutants. We found that a protein N-glycosylation and folding promoter, Entpd5 was negatively regulated by PTEN phosphatase. Down regulation of Entpd5 by shRNA in cells with PTEN phosphatase dead mutant blocked PTEN phosphatase dead mutant mediated tumor metastasis in vivo, promoted cell ER stress and growth inhibition as well as reduction of IGF1R. In contrast, overexpression of Entpd5 in cell with PTEN wild-type form enhanced PTEN wild-type-inhibited tumor metastasis in vivo, inhibited cell ER stress, promoted cell growth and abundance of IGF1R. We provide mechanistic insight toward regulation of metastasis by PTEN phosphatase and demonstrate a link between PTEN and Entpd5/IGF1R activation in metastatic process.

Citation Format: Yanlin Yu. PTEN phosphatase inhibits metastasis through negatively affecting Entpd5/IGF1R pathway. [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 136. doi:10.1158/1538-7445.AM2014-136
Abstract 1360: DNA methylation analysis in self-sampled material as a triage test in hrHPV positive women
Authors: Boers Aniek , Bosgraaf Remko P. , Leeuwen Roland W. van , Schuuring Ed , Massuger Leon FAG , Bulten Johan , Melchers Willem J. , Zee Ate GJ van der , Bekkers Ruud L. , Wisman Bea
Published: May 2015

Background: Primary hrHPV testing in cervical cancer screening shows relatively low specificity. To decrease the number of unnecessary referrals for colposcopy, triage testing is necessary. In this study, DNA methylation analysis was compared with routine cytology for triage testing in hrHPV positive women. Moreover, feasibility of DNA methylation analysis directly on original brush-based self-sampled specimens was assessed.

Methods: Non-responding women from population-based screening were invited to self-collect a cervico-vaginal specimen for hrHPV testing; hrHPV positive women were referred to a physician for liquid-based sampling. For this pilot study, DNA methylation analysis was performed on 128 triage samples and on 50 matched Evalyn brush self-samples with QMSP for C13ORF18, EPB41L3, JAM3 and TERT.

Results: In the physician-taken triage material, DNA methylation analysis of JAM3 showed the highest specificity (88%) combined with a high sensitivity (82%) for detection of CIN3+, whereas cytology showed a specificity of 48% and a sensitivity of 91%. Out of 39 women with abnormal cytology and normal histology (false positive by cytology), 87% were negative for JAM3 and 90% for C13ORF18 methylation. The agreement for DNA methylation analysis performed directly on the matched self-sampled material and the physician-taken samples was 90% for C13ORF18 (κ=0.77; p<0.001) and 88% for JAM3 (κ=0.75, p<0.001).

Conclusion: DNA methylation analysis as a triage test in hrHPV positive women outperformed cytology due to higher specificity without losing its sensitivity and is also feasible to be performed directly on Evalyn Brush self-samplers. This might result in more correct referrals to the gynecologist and eliminate the need for a new scraping taken by a medical professional thereby offering great logistic and financial advantages.

This study was financed by the Dutch Cancer Society (RUG-NKB2009-4577).

Citation Format: Aniek Boers, Remko P. Bosgraaf, Roland W. van Leeuwen, Ed Schuuring, Leon FAG Massuger, Johan Bulten, Willem JG Melchers, Ate GJ van der Zee, Ruud LM Bekkers, Bea Wisman. DNA methylation analysis in self-sampled material as a triage test in hrHPV positive women. [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 1360. doi:10.1158/1538-7445.AM2014-1360
Abstract 1361: The clinicopathological significance of LSD1 in esophageal cancers
Authors: Kosumi Keisuke , Baba Yoshifumi , Karashima Ryuichi , Ida Satoshi , Imamura Yu , Ishimoto Takatsugu , Iwagami Shiro , Sakamoto Yasuo , Miyamoto Yuji , Yoshida Naoya , Sakamoto Akihisa , Hino Shinjiro , Nakao Mitsuyoshi , Baba Hideo
Published: May 2015

Background: Lysine-specific demethylase 1 (LSD1) is the first discovered histone demethylase which specially removes H3K4me1/2 through a flavin-adenine-dinucleotide (FAD). Previous studies have indicated that LSD1 is associated with cell proliferation, cell invasion and epithelial-mesenchymal transition in human malignant tumors. Recently, it is accepted that LSD1 epigenetically regulates energy-expenditure genes in adipocytes depending on the cellular FAD availability. As a PET scan with a FDG tracer can detect cancers, the method is exceedingly useful for tumor staging or therapeutic effect determination of esophageal cancers. The aim of this study is to investigate the expression and function of LSD1 in esophageal cancer cell-lines and, to analyze the relationships between the LSD1 expression and clinicopathological data including SUVmax (standardized uptake value).

Methods: The influence of LSD1 on cell proliferation and invasion was evaluated by knockdown experiments. Using 113 resected esophageal cancer specimens, we evaluated the LSD1 expression by immunohistochemistry and devided them into two groups; a low expression group (LEG) and a high expression group (HEG). We compared these two groups and analyzed the relationships between LSD1 expression and clinicopathological data.

Results: In vitro analysis, knockdown of LSD1 did not impact cell proliferation, but suppressed cell invasion. In the immunohistochemical study, the HEG's age was significantly higher (P=0.021), and this group had esophageal cancers at lower locations (P=0.058). HEG was significantly correlated with the T-stage (P=0.020), lymph and blood vessels invasion (P=0.020, P=0.001), and a higher SUVmax (P=0.0088) and lower overall survival (P=0.0017).

Conclusions: Our study demonstrated the relationships between the expression of LSD1 and T-stage, lymph and blood vessels invasion, and the SUVmax in esophageal cancers. It is suggested that LSD1 has a potential to impact the malignancy of the esophageal cancer, and may be a therapeutic target in patients with esophageal cancers.

Citation Format: Keisuke Kosumi, Yoshifumi Baba, Ryuichi Karashima, Satoshi Ida, Yu Imamura, Takatsugu Ishimoto, Shiro Iwagami, Yasuo Sakamoto, Yuji Miyamoto, Naoya Yoshida, Akihisa Sakamoto, Shinjiro Hino, Mitsuyoshi Nakao, Hideo Baba. The clinicopathological significance of LSD1 in esophageal 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 1361. doi:10.1158/1538-7445.AM2014-1361
Abstract 1362: Nuclear morphometry measures progressive atypia in the development of pancreatic carcinoma
Authors: Glazer Evan S. , Hill Kimberly A. , Zhang Hao (Helen) , Bartels Peter , Watkins Joseph , Alberts David S. , Krouse Robert S.
Published: May 2015

Pancreatic lesions that are not clearly benign are often treated as malignant despite uncertainty in the true diagnosis due to the nearly universally fatal nature of pancreatic carcinoma (PC). Nuclear morphometry is a technique to quantify nuclear features too complex for the human eye to discern. We hypothesized that nuclear atypia can be quantified with morphometry in order to distinguish between chronic pancreatitis, IPMN, and PC. We retrospectively analyzed 14 specimens of chronic pancreatitis, 16 IPMN lesions, and 19 PC lesions. Clinicopathologic data were obtained. Nuclear morphometry determined overall atypia based on the average nuclear abnormality of 95 distinct nuclear features (a nuclear signature). For PC lesions, 5 nuclear features defined a classification score (CS) representing the proportion of aggressive nuclei in a given PC lesion. Statistical significance was determined with ANOVA and the Kruskal-Wallis test. The average age for all patients was 63 ± 15 years while 62% were male; there were no differences between the 3 groups. The follow up was approximately 4 years in all groups as well. The average nuclear atypia of chronic pancreatitis was 0.80, 0.99 for IPMN, and 1.08 for PC (P = 0.02). Importantly, based on 5 nuclear features, the CS for PC lesions that recurred was 87% while it was only 56% for those PC lesions that did recur (P = 0.04). The research describes a precise, accurate, and objective method to distinguish IPMN from PC. The CS and overall atypia score provides a method to not only objectively describe a given lesion, but it importantly describes where in the progression from benign to malignant lesion an unknown lesion exists. Clinically, this may be of great utility in risk stratifying unknown pancreatic lesions at the time of diagnosis or tissue biopsy.

Citation Format: Evan S. Glazer, Kimberly A. Hill, Hao (Helen) Zhang, Peter Bartels, Joseph Watkins, David S. Alberts, Robert S. Krouse. Nuclear morphometry measures progressive atypia in the development of pancreatic 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 1362. doi:10.1158/1538-7445.AM2014-1362
Abstract 1364: Epigenetic regulation of SPARC in ovarian cancer
Authors: Matthews Dylan P. , Taylor Sherine , Said Neveen A.
Published: May 2015

Epithelial Ovarian cancer (OvCa) is the leading cause of death from gynecologic malignancies as most patients are diagnosed at late stages. Despite aggressive surgical debulking and cytoreduction, recurrence is common with poor patient survival. Constitutive progressive inflammation is the hallmark of OvCa and is contributed by the interaction of transformed OvCa cells with cues from the peritoneal microenvironment, activating multiple signaling pathways that converge in the (trans)activation of NFκB which not only induces the transcription of myriad inflammatory mediators but represses tumor suppressor genes (TSGs) creating a vicious circle of disease progression, recurrence and chemoresistance. Therefore there is a pressing need to identify new diagnostic, prognostic biomarkers as well as new. Our earlier studies indicated Secreted protein acidic and rich in cysteine SPARC is a tumor suppressor in OvCa in vitro and in vivo. In addition, we found that SPARC suppresses OvCa cells interactions with environmental cues within the peritoneal milieu. We also found that the expression of SPARC was decreased in established OvCa cell lines and microdissected OvCa cells from advanced tumors consistent with hypermethylated of its promoter. However, the mechanisms of epigenetic regulation of SPARC in OvCa (and other morbidities) are unraveled. Mapping of SPARC promoter revealed consensus binding sites for NFB subunits. Pharmacologic inhibition of NFκB as well as knockdown of p65-RelA and p50 subunits restored SPARC expression and function in OvCa cells. Furthermore, treatment of OvCa cells with demethylating agent 5′azacytidine indicated that NFκB-mediated SPARC repression is due to methylation. In OvCa tissue microarrays, the expression of SPARC in cancer cells inversely correlated with nuclear p65-RelA and DNMT3a expression. Together these findings indicate that activated NFκB integrates inflammatory signals to exert epigenetic pressure suppressing tumor cell SPARC through promoter methylation.

Citation Format: Dylan P. Matthews, Sherine Taylor, Neveen A. Said. Epigenetic regulation of SPARC in ovarian 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 1364. doi:10.1158/1538-7445.AM2014-1364
Abstract 1365: Epigenetic modification of LY6K in CGI shore and CGI regulates LY6K gene activation and metastatic function in breast cancer
Authors: Kong Hyun Kyung , Park Sae Jeong , Kim Ye Sol , Park Jong Hoon
Published: May 2015

Lymphocyte antigen 6 complex locus K (LY6K) is associated with development of the immune response and carcinogenesis. Elevated LY6K by AP-1 activation induces cell invasion and metastasis through activating the Raf-1/MEK/ERK signaling pathway. However, the exact epigenetic mechanism of LY6K gene expression has yet to be clarified. To elucidate the epigenetic mechanism of LY6K gene regulation and expression, CpG island and 5′CGI shore in the region around transcription start site of the LY6K were predicted using UCSC genome browser and Methprimer software. We performed MSP, bisulfite pyro-sequnecing, and bisulfite sequencing to investigate the DNA methylation status of CpG sites from -400 to +500 on the LY6K CGI and 5′CGI shore. Breast cancer cells with low LY6K expression were highly methylated in the both CGI and 5′CGI shore region, whereas high LY6K expression cell lines had low methylation levels. Moreover, 5-Aza-dC, demethylaing agent, treatment of cells with low LY6K expression caused elevation of LY6K expression and finally leads wound healing. To further validate the inverse correlation between LY6K 5′CGI shore methylation and LY6K expression in vivo seen in breast cancer cell lines, we performed bisulfite pyro-sequencing and tissue microarray using breast carcinoma samples. LY6K expression inversely correlates with methylation status and histone modification of 5′CGI shore in the LY6K promoter in human breast cancers. An understanding of epigenetic changes in LY6K may contribute to the diagnosis of carcinogenic risk and to prediction of outcome in patients with breast cancer.

Citation Format: Hyun Kyung Kong, Sae Jeong Park, Ye Sol Kim, Jong Hoon Park. Epigenetic modification of LY6K in CGI shore and CGI regulates LY6K gene activation and metastatic function in breast 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 1365. doi:10.1158/1538-7445.AM2014-1365
Abstract 1367: An epigenetic screen unmasks metallothioneins as putative contributors to renal cell carcinogenesis
Authors: Krause Hans , Al-Kamal Imad , Ikromov Odiljon , Miller Kurt , Kempkensteffen Carsten
Published: May 2015

Transcriptional silencing, as a result of aberrant promoter hypermethylation, is a common mechanism through which genes in cancer cells become inactive. Functional epigenetic screens using demethylating agents to re-express transcriptional silenced genes may identify such inactivated genes for further evaluation. We aimed to identify renal cell carcinoma (RCC) genes so far not known to be inactive due to promoter hypermethylation.

The RCC cell line A-498 was treated with the DNA methyltransferase inhibitor zebularine under low cytotoxicity conditions. Expression changes of the transcriptome of treated and untreated cells were compared using RNA expression microarrays (Affymetrix Human Genome U133A v. 2.0). Eight candidate genes (criteria: upregulation more than 3-fold, presence of CpP islands in the promoter region, in silico predicted downregulation in tumors) were evaluated by Q-PCR in 49 paired normal and RCC tissues of patients (39 ccRCC, 7 pRCC, and 3 cRCC), who underwent radical nephrectectomy in our clinic between 2009 and 2010. Members of the metallothionein group were remarkably downregulated in tumor tissues. Metallothionein 1G (MT1G) was downregulated in 48 of 49 tumor tissues (p<0.0001), MT1H was downregulated in 42 out of 43 tumor tissues (p<0,0001), and MT2A expression was decreased in 73% (34/49) of all cases. The remaining 5 candidates (EFNB2, RRAD, TGM2, ITGA2, and MBD4) were found either upregulated in the majority of RCCs or without reasonable expression differences between normal und tumor tissues. To prove the consistency of our epigenetic screening approach we compared expression data of 308 upregulated transcripts (≥ 1.5-fold) to similar screens performed by other groups that revealed a high number of shared candidates.

Although a possible contribution of metallothioneins to renal cell carcinogenesis is not fully understood, our discovery of an almost exclusively downregulation of selected MT genes in RCC tissues emphasizes a plausible link between deregulated metabolism and tumor development. In the context of an ever-increasing demand for suitable markers for clinical decision-making and a better understanding of renal cell carcinogenesis, selected members of the MT group may serve as appropriate candidates for these tasks.

Citation Format: Hans Krause, Imad Al-Kamal, Odiljon Ikromov, Kurt Miller, Carsten Kempkensteffen. An epigenetic screen unmasks metallothioneins as putative contributors to renal cell 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 1367. doi:10.1158/1538-7445.AM2014-1367
Abstract 1368: Genome-wide screening of DNA methylation markers to predict the presence of lymph node metastasis in esophageal squamous cell carcinoma
Authors: Kozaki Ken-ichi , Nagata Hiroaki , Tanimoto Kousuke , Fujiwara Naoto , Imoto Seiya , Ichikawa Daisuke , Miyano Satoru , Kawano Tatsuyuki , Otsuji Eigo , Inazawa Johji
Published: May 2015

Lymph node metastasis (LNM) of esophageal squamous cell carcinoma (ESCC) is well-known to be an early event that is associated with poor prognosis of ESCC patients. In the last few years, tumor-specific aberrations in DNA methylation of CpG islands around promoter regions of tumor-related genes have been investigated as possible biomarkers for early diagnosis, treatment and prognosis of human cancers. But, there are few DNA methylation markers that can predict the presence of LNM in patients with ESCC. To identify DNA methylation markers correlated with LNM of ESCC, we performed a genome-wide screening of DNA methylation status in a discovery cohort of 67 primary ESCC tissues and their paired normal esophageal tissues by Illumina Infinium HumanMethylation450 BeadChip (covers more than 450,000 CpG sites). In this screening, we focused on some differentially methylated regions (DMRs), which were significantly associated with LNM of ESCC, as prime candidates for DNA methylation markers. In addition, these significant relationships were also confirmed by quantitative bisulfite pyrosequencing analysis in the same discovery cohort. Consequently, these results led us to consider that our selected DMRs might be useful epigenetic biomarkers for the prediction of the presence of LNM in primary ESCC cases. Therefore, we are continuing further analyses of these DMRs in an independent external validation cohort of ESCC patients.

Citation Format: Ken-ichi Kozaki, Hiroaki Nagata, Kousuke Tanimoto, Naoto Fujiwara, Seiya Imoto, Daisuke Ichikawa, Satoru Miyano, Tatsuyuki Kawano, Eigo Otsuji, Johji Inazawa. Genome-wide screening of DNA methylation markers to predict the presence of lymph node metastasis in 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 1368. doi:10.1158/1538-7445.AM2014-1368
Abstract 1369: miR-645 inhibits apoptosis of AGEJ
Authors: Feng Xiaoshan , Wang Ying , Ma Zhikun , Yang Ruina , Zhang Mengxi , Liang Shuo , Li Shuoguo , Liu Gang , Dai Fanming , Gao Shegan
Published: May 2015

Background: An increasing body of evidence indicates that miRNAs have a critical role in carcinogenesis and cancer progression; however, the role of miRNAs in the tumorigenesis of adencarcinoma of gastric esophageal junction(AGEJ) remains largely unclear.Methods: The SGC7901 and BGC-823 gastric cancer cell lines were used. The expressions of miR-645 and IFIT2 were examined by qRT-PCR. The expressions of IFIT2 was examined by western blotting and immunohistochemistry assay. The cell apoptosis was determined by FACS. MiR-645 inhibitor, mimics and plasmid-IFIT2 transfections were performed to study the loss- and gain-function. Caspase-3/7 activity was examined by caspase-3/7 assay. Results: In the present study, we have reported an increased expression of miR-645 in AGEJ clinical specimens compared with paired non-cancerous tissues. We also observed a significant miR-645 up-regulation in two gastric cancer ( GC ) cell lines, SGC7901 and BGC-823, which were used as cell models because there was no available AGEJ cell lines established to date. We found that inhibition of miR-645 could sensitize dramatically SGC7901 and BGC-823 cells to both serum starvation- and chemotherapeutic drug-induced apoptosis by up-regulating IFIT2, a mediator of apoptosis via a mitochondrial pathway, with a potential binding site for miR-645 in its mRNA's 3′UTR. Further investigation exhibited that IFIT2 expression decreases in SGC7901 and BGC-823 cells and AGEJ tissues. IFIT2 ectopic expression leads to promotion of cell apoptosis, indicating that IFIT2 may function as a suppressor in the development of AGEJ. Furthermore, inhibition of miR-645 induces up-regulation of IFIT2 and increased caspase-3/7 activity compared with control groups. Conclusions: Our data suggest that miR-645 functions as an oncogene in human AGEJ by, at least partially through, targeting IFIT2.

Note: This abstract was not presented at the meeting.

Citation Format: Xiaoshan Feng, Ying Wang, Zhikun Ma, Ruina Yang, Mengxi Zhang, Shuo Liang, Shuoguo Li, Gang Liu, Fanming Dai, Shegan Gao. miR-645 inhibits apoptosis of AGEJ. [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 1369. doi:10.1158/1538-7445.AM2014-1369
Abstract 1372: The methylome of ER-negative tumors is predominantly hypermethylated
Authors: Worsham Maria J. , Chitale Dhananjay , Chen Kang Mei , Divine George
Published: May 2015

The biological significance of DNA methylation in the regulation of gene expression and its role in cancer is increasingly recognized. The underlying hypothesis of this study is that strategic global approaches will identify aberrantly methylated genes that underlie the pathogenesis of ER-negative (ER-) breast cancer (BC).

We used the Infinium HumanMethylation450 BeadChip to profile the methylome of ER- breast cancers to determine differentially methylated genes. The 450K array includes 485,577 cytosine positions of the human genome. Whole genomic DNA from 40 primary ER-, 40 ER positive (ER+), and 40 normal breast tissue samples were assayed for genome-wide methylation. Data were analyzed by GenomeStudio software (Illumina). Degree of methylation was calculated as a β-value (ranging from 0 to 1) and M-values [log (β/ (1- β)] were used for significance tests. With 30 sets of paired tumor/normal tissue, GEE was performed to account for their correlation. To identify methylated genes associated with ER- subtypes and distinct from ER+, a 3-tiered approach to call out genes in which methylation changed dramatically between ER+ and ER- subtypes was used. Tier 1: computed adjusted FDR values for all CpGs/ (or their averages for each gene) to be 0.05 or lower. Tier 2: the CpGs/genes to include genes with a 2-fold change (ratio >= 2.0 or ratio <= 0.5). Tier 3: CpGs/genes to include genes with an absolute difference between the mean β of =>0.2.

Differentially methylated genes (DMGs) at tier 3 for the different comparison groups were as follows: all BC (n=80) vs normal (n=40): 1070 DMGs: 1001: hypermethylated, 69: hypemethylated; ER+ vs normal: 1720: 1518 hyper, 202 hypo; ER- vs normal: 1170, 1085 hyper, 85 hypo; ER- vs ER+ vs: 289, 191 hyper, 97 hypomethylated.

Overall, ER- BC tumors were more hypermethylated than hypomethylated when compared to normal breast tissue and ER+ BC. Estrogen receptor-negative BC is a more aggressive form than ER positive BC with approximately double the incidence in African Americans than in Caucasian Americans. The emerging differential methylation pattern would further help stratify them into distinct subgroups. Promotor methylation being potentially reversible, methylated genes may serve as future molecular targets for demethylating therapies.

Support: Komen Foundation: KG110218

Citation Format: Maria J. Worsham, Dhananjay Chitale, Kang Mei Chen, George Divine. The methylome of ER-negative tumors is predominantly hypermethylated. [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 1372. doi:10.1158/1538-7445.AM2014-1372
Abstract 1374: Identification of a transcription factor driving BET dependency in SCLC
Authors: Wee Susan , Wong Tai ,   BMS
Published: May 2015

The regulation, and subsequent recognition, of covalent chromatin modifications by an array of cellular proteins are key determinants of gene expression. For instance, the binding of bromodomain and extra terminal domain (BET) proteins to acetylated histone tails has been shown to regulate c-MYC transcription in some cell types. Consequently, pharmacological inhibition of BET proteins leads to an anti-proliferative activity in cancers dependent on c-MYC activity such as multiple myeloma and acute myelogenous leukemia. Since c-MYC has been implicated as a driver of cell proliferation in many cancer types, we investigated if BET inhibitors may have anti-proliferative activity in solid tumor cancer cell lines as well. Strikingly, in a panel of over 100 mixed histology lung cancer cell lines we identified selective growth inhibition of lines derived from small cell lung cancer (SCLC) histology. To better understand the mechanism of this dependency we performed 12-point dose response microarray analyses using the BET inhibitor, JQ1, in order to identify genes that are regulated by BET proteins in SCLC. In contrast to what has been observed in myeloma and leukemia, BET inhibition is not always accompanied by inhibition of c-MYC expression in the SCLC cell lines. From this analysis a lineage-specific transcription factor was found to be highly regulated in a dose-dependent manner. Previous studies have reported this transcription factor to be essential in maintaining the differentiated neuroendocrine phenotype characteristic of human SCLC. It has also been shown that loss of this transcription factor results in growth inhibition in SCLC tumor models. We have confirmed the growth dependency of SCLC to BET via dysregulation of this transcription factor using RNAi and a small molecule inhibitor. Furthermore, chromatin immunoprecipitation followed by deep-sequencing confirmed binding of BRD4, a BET family member, and RNA polymerase II, a marker of active gene transcription, to the enhancer region of this target gene in two sensitive SCLC cell lines. This binding is disrupted in the presence of BET compound treatment. SCLC is a disease with high unmet medical need, and the results of our studies have identified BET inhibition as a potential novel therapeutic opportunity for this subtype of lung cancer. Our results have also provided a mechanistic basis for the sensitivity of SCLC to BET inhibition, and a rationale for the clinical development of BET inhibitors beyond hematologic cancers.

Citation Format: Susan Wee, Tai Wong, BMS. Identification of a transcription factor driving BET dependency in SCLC. [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 1374. doi:10.1158/1538-7445.AM2014-1374
Abstract 1377: Transcriptional silencing of MST1/Hippo by a coordinated MYC and EZH2 signaling in prostate cancer
Authors: Abali Gamze Kuser , Alptekin Ahmet , Cinar Bekir
Published: May 2015

Prostate cancer is a major cause of cancer-related death in western countries because of the poorly understood disease mechanisms. Hippo-like MST1 protein kinase regulates cell growth, stem cell self-renewal and carcinogenesis. The loss of Mst1 expression is implicated in the etiology several types of cancers with poor prognosis, including prostate cancer. However, the underlying mechanism of Mst1 transcriptional downregulation in prostate cancer is unknown. Here, we report that MYC and EZH2 function as epigenetic suppressors of MST1 in human prostate cancer cells. We showed that decline in MST1 expression correlated with EZH2 and MYC upregulation as demonstrated by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and western blot. Using methylation sensitive PCR and bisulfite genomic DNA sequencing, we showed that Mst1 promoter DNA was methylated at CpG sites that led to a reduction in MST1 expression. Our pharmacologic or RNAi experiments revealed that EZH2 contributed to MST1 silencing by inducing MST1 promoter DNA methylation and H3K27me3 modification that was accompanied by a reduced H3K4me3 mark and RNA polymerase II occupancy on MST1 promoter and thereby reducing its promoter activity and expression. On the other hand, overexpression of MYC stimulated EZH2 and conversely reduced MST1 promoter activation and its mRNA levels. Potent EZH2 inhibitors reversed the MYC effects on MST1, indicating that MYC attenuates MST1 through EZH2. Moreover, MST1 knockdown caused cell resistance to androgen deficiency and to EZH2, or MYC inhibitor-induced growth retardation. These findings indicate that MYC cooperates with EZH2 to epigenetically silence MST1 and suggest that the loss of MST1/Hippo is critical for MYC- and EZH2-driven cancer cell survival.

Citation Format: Gamze Kuser Abali, Ahmet Alptekin, Bekir Cinar. Transcriptional silencing of MST1/Hippo by a coordinated MYC and EZH2 signaling in prostate 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 1377. doi:10.1158/1538-7445.AM2014-1377