bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5479: Antitumor effect of KX-01, a novel Src and tubulin inhibitor, in triple negative breast cancer cells
Authors: Kim Seongyeong , Im Seock-Ah , Min Ahrum , Lee Miso , Jang Heymin , Lee Kyung-Hun , Kim Hee-Jun , Kim Tae-Yong , Han Sae-Won , Oh Do-Youn , Kim Tae-You , Yoon Young-Kwang , Hangauer David , Lau Johnson , Bang Yung-Jue
Published: May 2015

Background: Src kinases have been involved in cell proliferation, invasion, and metastasis and is one of the highly expressed oncogenes in breast cancer. Therefore, it could be a reasonable target for therapeutic strategy. Although several Src inhibitors, targeting the ATP binding site, were developed, none of them have shown remarkable responses as monotherapeutic agents in breast cancer clinical trials. KX-01 (KX2-391) is a novel peptidomimetic agent, and a non ATP-competitive Src inhibitor, which has potent efficacy for inhibiting both Src as well as tubulin polymerization. Its dual inhibition effects could potentially overcome the limitations of the Src inhibitors that have been evaluated previously.

Materials & Methods: We determined the anti-tumor effects of KX-01 on breast cancer cell lines using a cytotoxicity assay, cell cycle analysis, a wound healing assay and western blotting following KX-01 treatment.

Results: KX-01 effectively inhibited cell growth in most breast cancer cells including ER/PR/HER2 negative breast cancer cells. Short exposure of KX-01 down-regulated the expression level of Src phosphorylation. Phosphorylated FAK, ERK, AKT and STAT3 were also down-regulated by KX-01 treatment in MDA-MB-231, MDA-MB-468 and BT-549 cells. By wound healing assay, we confirmed migration inhibitory effect of KX-01 in BT-549 cells. Increase of G2/M cell cycle arrest was observed in dose dependent manner. In addition, mitotic catastrophes were also observed in KX-01 sensitive cell lines. Consistent with induction of mitotic catastrophe, the increased levels of aneuploidy in sensitive cell lines were examined following the treatment of KX-01.

Conclusion: Inhibition of cell growth and migration, as well as an induction of mitotic catastrophy, were observed in triple negative breast cancer cell lines treated with KX-01. Our data strongly supports utilizing KX-01 as a new therapeutic agents for treating triple negative breast cancer.

Citation Format: Seongyeong Kim, Seock-Ah Im, Ahrum Min, Miso Lee, Heymin Jang, Kyung-Hun Lee, Hee-Jun Kim, Tae-Yong Kim, Sae-Won Han, Do-Youn Oh, Tae-You Kim, Young-Kwang Yoon, David Hangauer, Johnson Lau, Yung-Jue Bang. Antitumor effect of KX-01, a novel Src and tubulin inhibitor, in triple negative 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 5479. doi:10.1158/1538-7445.AM2014-5479
Abstract 548: CEP 3/CEP 17 in the genetic diagnosis and its prognostic relevance of Kazakh esophageal cancer patients
Authors: Awut Edris
Published: May 2015

To investigate the clinical application value of centromere of chromosome 3 and 17 (CEP 3, CEP 17) DNA probes in Kazakh esophageal squamous cell carcinoma (ESCC) patients’ molecular pathological diagnosis and prognosis.

Forty patients with ESCC who received radical surgical treatment and 10 cases of normal specimen in control group were enrolled in this study. Touch preparations of fresh cancer and normal tissue were made and fluorescence in situ hybridization (FISH) technique were used to count the copy numbers of CEP 3 and CEP 17, abnormalities were analyzed comparing with routine pathological diagnosis.

FISH analysis revealed that abnormal copy numbers of CEP 3, CEP 17 (aneuplody) were observed in 40 cases. CEP 3 and CEP 17 polyploidy rates were 42%, 40.5% in well-differentiated, 62.9%, 46.8% in moderately differentiated, and 76.95%, 63.5% in poorly differentiated ESCC respectively. These were significantly different between poorly differentiated, moderately differentiated and well-differentiated groups (p<0.05, p<0.05), and the polyploidy rates were also significantly higher in the group with lymph node metastasis comparing to the group without lymph node metas-tasis (CEP 3, p=0.0001; CEP 17, p=0.012).

The CEP 3, 17 variations were shown to be correlated with the level of differentiation and lymph node metastasis in ESCC. Therefore, there DNA probes may be the predictive biological marker in the prognosis of patients with ESCC. In addition, being an objective and qualitative method, the technology of FISH can detect CEP 3, 17 variations in the genetic diagnosis of Kazakh ESCC.

Note: This abstract was not presented at the meeting.

Citation Format: Edris Awut. CEP 3/CEP 17 in the genetic diagnosis and its prognostic relevance of Kazakh esophageal cancer patients. [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 548. doi:10.1158/1538-7445.AM2014-548
Abstract 5480: NSCLC cells with LKB1 mutation are vulnerable to energetic and oxidative stresses induced by 8-Chloroadenosine
Authors: Yang Chao , Cortez Maria Angelica , Giri Uma , Gudikote Jayanthi , Wierda William G. , Gandhi Varsha , Byers Lauren Averett , Heymach John V.
Published: May 2015

Background: It has been demonstrated that loss of LKB1 is associated with a higher likelihood of tumor recurrence after surgery and shorter overall survival in early stage NSCLC. We sought to determine whether NSCLC tumor cells with loss of LKB1 are preferentially sensitive or resistant to various therapeutic agents. We tested 23 NSCLC cell lines, and observed that LKB1 mutant NSCLC cells are highly resistant to standard chemotherapy as well as EGFR inhibitors. In contrast, LKB1 mutant NSCLC cells are highly sensitive to 8-Chloroadenosine (8-Cl-Ado). 8-Cl-Ado is an RNA-directed nucleoside analogue, which depletes cellular ATP and triggers apoptosis in sensitive cells. LKB1 is known to play a key role in balancing cellular energy consumption and production. Therefore, we hypothesize that LKB1 mutant NSCLC cells are vulnerable to 8-Cl-Ado induced energetic stress. Method: Cellular ATP level was determined by LC-MS and luminescent based methods. Cell proliferation was measured by Celltiter-Glo assay. IC50 values were calculated by Calcusyn. Oxidative stress was measured by CellRox assay. Result: NSCLC cell lines with LKB1 mutation were significantly more sensitive to 8-Cl-Ado than wild type cell lines (p=0.024). We transfected H460 and A549 cells (both harboring mutant LKB1) with wild-type LKB1. Re-expression of LKB1 rendered cells resistant to 8-Cl-Ado (P<0.05). Knockdown LKB1 resulted in increased sensitivity to 8-Cl-Ado in Calu6 cells (p<0.05). Moreover, 8-Cl-Ado treatment caused a significant decrease in cellular ATP levels in LKB1 mutant cells. However, in A549 and H460 cells overexpressing wild-type LKB1, ATP levels were not decreased. Similarly, in H661 cells (wild-type LKB1) 8-Cl-Ado treatment did not affect ATP levels, whereas when LKB1 was knocked down by shRNA 8-Cl-Ado treatment reduced intracellular ATP levels. Furthermore, 8-Cl-Ado increased ROS levels in A549 cells but not in A549 cells overexpressing wild-type LKB1. Conclusion: Our result indicates that LKB1 mutant NSCLC cells are vulnerable to energetic and oxidative stresses induced by 8-Cl-Ado. LKB1 played a protecting role in cells under 8-Cl-Ado treatment.

Citation Format: Chao Yang, Maria Angelica Cortez, Uma Giri, Jayanthi Gudikote, William G. Wierda, Varsha Gandhi, Lauren Averett Byers, John V. Heymach. NSCLC cells with LKB1 mutation are vulnerable to energetic and oxidative stresses induced by 8-Chloroadenosine. [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 5480. doi:10.1158/1538-7445.AM2014-5480
Abstract 5491: Interrogating the mechanism of Wnt pathway inhibition by niclosamide
Authors: Mook Robert A. , Zhao Shengli , Chen Wei
Published: May 2015

The Wnt signaling pathway regulates genes critical for normal development and is dysregulated in many cancers. Indeed, more than 80% of all sporadic and hereditary colorectal cancers (CRCs) show hyperactivation of the Wnt/β-catenin pathway due to mutations in adenomatous polyposis coli (APC) or β-catenin genes. As a result, drugs that modulate the Wnt pathway are highly sought-after as the basis of a new generation of innovative therapeutic agents to treat CRC. Unfortunately, the ability to target this pathway with small molecule inhibitors has hampered drug discovery such that no approved drugs specifically targeting the pathway have been identified. To address this gap, we constructed a chemical genetics high-throughput screen that evaluated internalization of the Frizzled receptor in order to identify modulators of the Wnt/Frizzled/LRP5/6 signaling pathway. Using this assay we discovered the small molecule drug niclosamide, a FDA-approved anthelmintic agent, decreased β-catenin levels and inhibited Wnt/β-catenin signaling, findings since confirmed by others. To support efforts to evaluate niclosamide in clinical studies of colorectal cancer with aberrant Wnt signaling, we studied the translocation of Frizzled1-GFP induced by niclosamide and the kinetics of inhibition of Wnt signaling in cell culture. We found that niclosamide induces the co-localization of Frizzled1-GFP and lysosome markers indicating the translocation of Frizzled1-GFP to the lysosome. Kinetic studies indicated that β-catenin levels are reduced significantly within three hours and that both the levels of β-catenin and β-catenin gene transcription remained significant decreased 12 hours after the compound was removed. These data suggest a mechanistic hypothesis in which an extended duration of inhibition of Wnt signaling by niclosamide may result from the induced degradation of key proteins in the Wnt signaling pathway and that continuous exposure to niclosamide may not be necessary over the entire dosing period to maintain sufficient pathway inhibition. Overall, the findings support plans to investigate the use of niclosamide in clinical trials of CRC.

Citation Format: Robert A. Mook, Shengli Zhao, Wei Chen. Interrogating the mechanism of Wnt pathway inhibition by niclosamide. [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 5491. doi:10.1158/1538-7445.AM2014-5491
Abstract 5497: Targeting glioblastoma stem cells with withaferin A
Authors: Zhang Baoyu , Cuglievan Branko , Myrthil Nadia G. , Vanni Steven , Komotar Ricardo J. , Graham Regina M.
Published: May 2015

Introduction. Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain cancer affecting 3 to 4 per 100,000 people annually in the United States. Despite aggressive therapy including surgical resection followed by both radiation and chemotherapy, overall survival remains dismal at approximately 14 months. Recent studies have determined that GBM's contain a small population of stem cells (GSCs), which promote tumor growth through unlimited self-renewal. Since GSCs are chemo- and radio-resistant and drive tumor regrowth following therapy, successful treatment depends on the elimination of these cells. Withaferin A (WA) is a bioactive compound derived from the plant Withonia somnifera which possesses anti-inflammatory and anti-cancer properties. Here we evaluated the effects of WA on patient derived GSCs.

Methods. With IRB approval, GSC lines were generated. GSCs were propagated in neurosphere media and examined for the stem cell markers CD133, nestin, musashi and BMi1 by immunocytochemistry and western blot analysis. Effect of WA on cell viability was determined using MTS assay. The effect of WA on cell survival and stress pathways was evaluated by western blot analysis.

Results. WA induced a significant decrease in cell viability at low micromolar concentrations. The IC50 for 3 patient derived GSC lines ranged between 0.96-1.78μM. WA induced ER stress and apoptosis in the GSCs as indicated by increased expression of ER stress markers grp78 and CHOP and increased levels of cleaved PARP. Furthermore, WA decreased survivin but increased phospho-ERK levels. However, inhibition of ERK activity with U0126 did not potentiate the effect of WA on cell death.

Conclusion. Ashwagandha has been used safely for medicinal purposes for centuries and pharmacokinetic studies in mice indicate that micromolar plasma concentrations of WA are obtainable. Our studies indicate that very low concentrations of WA induce ER stress and cell death in GSC lines. Targeting GSC with WA may increase patient survival by hindering GSC mediated tumor regrowth following surgery. Our data demonstrates the efficacy of WA and supports additional studies toward the use of WA as an adjuvant GBM therapy.

Citation Format: Baoyu Zhang, Branko Cuglievan, Nadia G. Myrthil, Steven Vanni, Ricardo J. Komotar, Regina M. Graham. Targeting glioblastoma stem cells with withaferin A. [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 5497. doi:10.1158/1538-7445.AM2014-5497
Abstract 5504: A potential source in oxidative cell death mediated by thymidylate synthase inhibitors: NADPH oxidase 2
Authors: Ozer Ufuk , Berger Franklin George , Barbour Karen Wood
Published: May 2015

Thymidylate synthase (TS) catalyzes the reductive transfer of a methyl group from N5,N10-methylenetetrahydrofolate (CH2-THF) to dUMP, forming dTMP and dihydrofolate (DHF). It is generally accepted that inhibition of the enzyme leads to a deficiency of dTMP, followed by genome damage and programmed cell death. As such, TS has long been viewed as an important target of anti-neoplastic agents, such as 5-fluorouracil (5-FU) and raltitrexed (RTX), which lead to inhibition of the enzyme. Oxidative stress is well-recognized as having a central role in cellular response to a number of DNA damaging agents, and may be a proximate cause of therapy-induced cell death. Despite such recognition, little detail exists on the origin and regulation of reactive oxygen species (ROS) during TS-directed chemotherapy. We have undertaken a detailed examination of the origin, nature, and role of ROS in cell death mediated by TS inhibitors. We have previously identified NADPH oxidase (NOX) as the primary source of increased ROS following exposure to TS-directed agents, as indicated by the observation of decreased ROS production and apoptotic indices in presence of NOX inhibitors. We have found that in response to TS inhibitors, NOX enzyme activity increases in association with induction of the transcript for p67phox, which specifically regulates the NOX2 isoform. This result is also verified by silencing p67phox gene in HCT116 cells. Silencing p67phox gene inhibits drug-induced NOX activity and decreases drug-induced apoptotic indices. To substantiate and expand results obtained from HCT116, we used other colorectal cancer cells, including HCT15, SW480, DLD-1, LoVo, MOSER and LS180. We have determined that colon cancer cells exhibit diverse responses in both basal and drug-inducible levels of apoptosis, NOX activity and mRNA of NOX2 regulatory subunits, implicating the heterogeneity in colorectal cancer cells. Based on these results, we conclude that augmentation of NOX2 activity via induction of p67phox mRNA expression is the proximate cause of programmed cell death elicited by dTMP deficiency in HCT116 cells. Each colorectal cancer cells might have different mechanism to undergo ROS-mediated cell death.

Citation Format: Ufuk Ozer, Franklin George Berger, Karen Wood Barbour. A potential source in oxidative cell death mediated by thymidylate synthase inhibitors: NADPH oxidase 2. [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 5504. doi:10.1158/1538-7445.AM2014-5504
Abstract 5515: Combining FK228 and cisplatin synergistically induces cancer cell death and reduces clonogenic survival
Authors: Choudhary Shambhunath , Sood Shilpa , Pluchino Lenora A. , Wang Hwa-Chain R.
Published: May 2015

The purpose of this study was to investigate the effectiveness of FK228 (romidepsin) to increase cisplatin's ability to induce cell death and reduce clonogenic survival of human urinary bladder cancer cells. Human urinary bladder cancer is the fifth most common cancer in the United States, but long-term, disease-free survival in patients is still suboptimal. Therefore, it is crucial to develop effective chemotherapeutic regimens to decrease the morbidity and mortality of this cancer. FK228, a depsipeptide and histone deacetylase inhibitor, is approved by the U.S. Food and Drug Administration for treatment of T cell lymphoma. However, its therapeutic value in treatment of bladder cancer is yet to be determined. Cisplatin is a DNA-damaging agent for treating various human cancers, including bladder, brain, ovary, lung, and testis cancers. However, the efficacy of cisplatin is often dampened by cancer cell drug resistance, mostly associated with glutathione (GSH)-based detoxification. Our study revealed that FK228 combined with cisplatin synergistically induced cell death and reduced clonogenic survival of human urinary bladder cancer cells. The Erk-Nox pathway played an important role in mediating signals highly increased by this combined treatment to induce significantly-elevated levels of reactive oxygen species, leading to substantially-induced caspase activation and synergistically-increased death in cancer cells. Cisplatin was able to enhance the ability of FK228 to significantly reduce GSH, indicating a novel activity of combined FK228 and cisplatin in reducing drug resistance. The ability of combined FK228 and cisplatin to synergistically induce cell death and reduce clonogenic survival was also applicable to colon cancer cells. Hence, combined use of FK228 with cisplatin should be considered in development of therapeutic strategies to control urinary bladder cancer and other cancer development and recurrence.

Citation Format: Shambhunath Choudhary, Shilpa Sood, Lenora A. Pluchino, Hwa-Chain R. Wang. Combining FK228 and cisplatin synergistically induces cancer cell death and reduces clonogenic survival. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5515. doi:10.1158/1538-7445.AM2014-5515
Abstract 5520: A novel HDAC inhibitor, N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA), induces apoptotic cell death through mitochondrial cytochrome c release in renal cell carcinoma
Authors: Han Kyung Seok , Park Ki Cheong , Jeon Jeong Yong , Kim Sang Yong , Song Dami , Han Woong Kyu , Hong Sung Joon
Published: May 2015

Introduction: N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA) is a novel HDAC inhibitor that shows better pharmacological properties than known HDAC inhibitors. We investigated a therapeutic role and its mechanisms of HNHA in renal cell carcinoma.

Methods: Renal cell carcinoma cell lines, Caki-1, A-498, 786-O and UMRC-3 cells were used for in vitro study. Crystal violet assay was used to assess cell growth. Apoptosis was evaluated by TUNEL assay and confirmed by expression of cleaved caspase-3 and caspase-7. Cytochrome c release was evaluated by mitochondria staining. For in vivo study, Caki-1 and A-498 cells were injected (1.0 x 107/mouse) into the dorsal sub-membrane of 6-week-old BALB/C nude mice. Two different drugs were injected at Lethal Dose 50: HNHA, 206 µg/mL and SAHA, 138 µg/mL.

Result: HNHA suppressed tumor proliferation in all panels of renal cell carcinoma cells. TUNEL assay and Western blots showed that remarkable apoptosis was induced by HNHA in Caki-1 and A-498 cells. HNHA induced apoptotic cell death by caspase activation and Bcl 2 suppression in renal cell carcinoma in vitro and in vivo. HNHA also caused cytochrome c release from mitochondria through Bax and Bak expression. A primary caspase activation was initiated by cytochrome c release from mitochondria, which is able to induce biochemical reactions, caspase activation and subsequent cell death.

Conclusion: HNHA induces more potent apoptotic cell death in renal cell carcinoma in vitro and in vivo than the previous HDAC inhibitors. HNHA-induced apoptosis is mediated by cytochrome c release from mitochondria through induction of Bax and Bak expression in renal cell carcinoma.

Citation Format: Kyung Seok Han, Ki Cheong Park, Jeong Yong Jeon, Sang Yong Kim, Dami Song, Woong Kyu Han, Sung Joon Hong. A novel HDAC inhibitor, N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA), induces apoptotic cell death through mitochondrial cytochrome c release in renal 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 5520. doi:10.1158/1538-7445.AM2014-5520
Abstract 5522: Anticancer activity of novel PI3K inhibitor BYL719 in head and neck cancer cell lines
Authors: Keam Bhumsuk , Ahn Yong-Oon , Kim Tae Min , Lee Se-Hoon , Kim Dong-Wan , Heo Dae Seog
Published: May 2015

Introduction: Activating mutations of the PIK3CA gene occur frequently in head and neck squamous cell carcinoma. The purpose of this study was to explore the antiproliferative effect of BYL719, specific inhibitor for phosphatidylinositol 3-kinase (PI3K) p110α, in human head and neck cancer cell lines as a single agent or in combination with irreversible EGFR TKI.

Methods: Six head and neck cancer cell lines (SNU-1066, SNU-1041, SNU-1076, SCC25, Detroit562, and FaDu) were tested. The growth inhibitory effect was assessed using the modified tetrazolium bromide assay. The cell cycle at various concentrations of BYL719 was analyzed by flow cytometry, and analysis of downstream molecules was performed by western blot analysis. Mutational profiles of cell lines were screened by Cancer Cell Line Encyclopedia.

Results: PIK3CA mutant cell lines (SNU-1076 and Detroit562) were more sensitive to BYL719 than PIK3CA wild type cell lines (SNU-1066, SNU-1041, SCC25, and FaDu). PIK3CA wild type cell lines except SNU-1066 exhibited higher IC50 values for BYL719 (IC50 were 6.82, 1.10 μM in mutant cell lines and 1.13, 20.65, 19.67, 49.30 μM in wild type cell lines, respectively). BYL719 induced G0/1 arrest and resulted in apoptosis with dose dependant manner, in PIK3CA mutant cell lines. It reduced the level of p-mTOR, p-AKT, p-S6, suggesting its effectiveness in inhibition of translation and protein synthesis. BYL719 combined with dacomitinib also showed a synergistic inhibitory effect on PIK3CA mutant cell lines, while PIK3CA wild type cell lines showed no synergism.

Conclusion: BYL719 could be a promising treatment for head and neck cancer cells with PIK3CA mutation as a single agent or in combination with dacomitinib. The beneficial effects of BYL719 in in vitro studies for head and neck cancer warrant a further clinical investigation.

Citation Format: Bhumsuk Keam, Yong-Oon Ahn, Tae Min Kim, Se-Hoon Lee, Dong-Wan Kim, Dae Seog Heo. Anticancer activity of novel PI3K inhibitor BYL719 in head and neck 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 5522. doi:10.1158/1538-7445.AM2014-5522
Abstract 5525: Antitumor effect of Realgar in combination with chemotherapy drugs on human gastric cancer cells
Authors: Zhang Lian , Kim Sungkyoung , Ding Wenping , Zheng Leizhen , Zhang Li , Tong Yingying , Chen Siyu
Published: May 2015

Background: Realgar, a Chinese traditional medicine, of which the main component is arsenic sulfide (As4A4), has potentially anti-tumor property in various cancers. We aimed to study the antitumor effect of Realgar in combination with chemotherapy drugs on human gastric cancer cells. Methods: Human gastric cancer AGS cells were treated with Realgar in combination with Oxaliplatin, Irinotecan, Cisplatin, 5-FU and docetaxol with escalating dose level. Antitumor effect was measured by 24,48-hr growth inhibition by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and cell morphology was observed under microscope. Results: Realgar in combination with chemotherapy drugs exhibited a dose-dependent and time-dependent cytotoxic effect on AGS cells. The combination of Realgar and Irinotecan or Cisplatin showed the most potent synergistic effect. Conclusions: Realgar combined with chemotherapy drugs such as Irinotecan or Cisplatin has potent antitumor effect on human gastric cancer cells. Further mechanistic and animal studies are being performed.

Citation Format: Lian Zhang, Sungkyoung Kim, Wenping Ding, Leizhen Zheng, Li Zhang, Yingying Tong, Siyu Chen. Antitumor effect of Realgar in combination with chemotherapy drugs on human gastric 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 5525. doi:10.1158/1538-7445.AM2014-5525
Abstract 559: Patterns of Telomerase reverse transcriptase (TERT) promoter mutations in melanoma and bladder cancer
Authors: Kumar Rajiv , Heidenreich Barbara , Hosen Ismail , Rachakonda Sivaramakrishna , Hemminki Kari
Published: May 2015

We previously reported a disease segregating causal germline mutation in a melanoma family and recurrent somatic mutations in metastasized tumours from unrelated patients within the core promoter region of the telomerase reverse transcriptase (TERT) gene1. Both the germline mutation at -57 bp from ATG start site and somatic mutations at -124 bp, -124/-125 bp, -138/-139 bp and -146 bp from ATG result in de novo creation of Ets/TCF transcription factor binding motifs that lead to the increased gene expression. Subsequent studies have shown that TERT promoter mutations are not only confined to melanoma and are rather recurrent in different cancer types that include bladder cancer, glioma, hepatocellular carcinoma and others. In this study we show the difference in pattern of the TERT promoter mutations between melanoma and bladder cancer. While in melanoma the -146 C>T mutation is the most frequent detected somatic base change in the TERT promoter, the -124 C>T mutation is overwhelmingly the most common mutation in bladder cancer. In melanoma CC>TT tandem mutations at -124/-125 bp and -138/-139 bp positions from ATG constitute about 10 percent of the detected TERT promoter mutations whereas in bladder cancer we did not detect any tandem mutations. The differences in the mutational pattern reflect etiological differences between the cancer types. In conclusion, the pattern of mutations in the TERT promoter differ between the cancer types even though all common mutations within the TERT promoter result in de novo creation of a common CCGGAA/T motif for Ets/TCF transcription factors.

1Horn S, Figl A, Rachakonda PS, Fischer C, Sucker A, Gast A, Kadel S, Moll I, Nagore E, Hemminki K, Schadendorf D, Kumar R. TERT promoter mutations in familial and sporadic melanoma. Science. 2013;22:339:959-961.

Citation Format: Rajiv Kumar, Barbara Heidenreich, Ismail Hosen, Sivaramakrishna Rachakonda, Kari Hemminki. Patterns of Telomerase reverse transcriptase (TERT) promoter mutations in melanoma and 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 559. doi:10.1158/1538-7445.AM2014-559
Abstract 5597: Targeted massively parallel sequencing for the diagnosis of hereditary neuroendocrine disorders
Authors: Dwight Trish , Richardson Anne Louise , Zhu Ying , Robinson Bruce G. , Benn Diana E. , Clifton-Bligh Roderick J.
Published: May 2015

Background: Genetic testing is a powerful tool for the diagnosis, management and treatment of inherited disease but these tests traditionally have been time-consuming, labour-intensive and costly. Significant improvements in sequencing technologies have the potential to overcome these limitations, through the simultaneous sequencing of multiple genes in multiple samples. Objective: The aim of this study was to develop a multi-gene panel, comprised of hereditary neuroendocrine associated genes, to enable parallel testing of known susceptibility genes; and to determine whether the sensitivity of massively parallel targeted sequencing is equivalent to Sanger sequencing, the current gold standard for diagnostic genetic testing.

Methods: We developed a custom gene panel, covering the coding regions of 8 known neuroendocrine suseptibility genes (MAX, SDHB, SDHC, SDHD, SDHAF2, RET, TMEM127 and VHL). In total, genomic DNA of 85 individuals with unique variants in five of the susceptibility genes (based on Sanger sequencing) was prepared using the TruSeq Custom Amplicon Assay (Illumina) and sequenced on an Illumina MiSeq™ platform.

Results: Overall, 95.1% of the target was sequenced adequately for variant calling (ie. reads with ≥Q30). Inadequate coverage of the target region was observed for VHL (exon 1), SDHC (exons 2 and 5) and SDHD (exon 4). Seventy-eight of the 85 known variants met the required minimum coverage threshold (7 fell within regions of inadequate coverage or depth of coverage was ≤40). Of these, one variant was undetected, giving an overall sensitivity of 98.7%.

Conclusions: Genetic testing using targeted capture followed by massively parallel sequencing, in conjunction with confirmation by Sanger sequencing, is a viable option to the current approach for diagnostic services.

Citation Format: Trish Dwight, Anne Louise Richardson, Ying Zhu, Bruce G. Robinson, Diana E. Benn, Roderick J. Clifton-Bligh. Targeted massively parallel sequencing for the diagnosis of hereditary neuroendocrine disorders. [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 5597. doi:10.1158/1538-7445.AM2014-5597
Abstract 5604: PD-L1 expression identifies a subpopulation of melanoma cells characterized by enhanced invasiveness and aggressiveness
Authors: Brusa Davide , Massi Daniela , Merelli Barbara , Ciano Michela , Audrito Valentina , Serra Sara , Baroni Gianna , Nassini Romina , Minocci Daiana , Cattaneo Laura , Carobbio Alessandra , Deaglio Silvia , Mandala Mario
Published: May 2015

Introduction: Melanoma is one of the most aggressive malignant tumors, and at the metastatic stage is an incurable disease. PD-L1 is a transmembrane molecule that can be expressed by melanoma cells and that has been speculated to play role in inducing immunosuppression by interacting with the PD-1 receptor on T cells. The clinical and biological significance of PD-L1 expression in metastatic melanoma remains partially elusive.

Methods: PD-L1 expression was analyzed by immunohistochemistry using two different antibodies [anti-human B7-H1 monoclonal antibody (clone 5H1) and a rabbit polyclonal AB58810 antibody (Abcam)]. In primary tumors and paired metastases from 83 consecutive melanoma patients treated at a single institution. Protein expression levels were correlated with PD-L1 mRNA, BRAF mutational status and clinical outcome. PD-L1+ and PD-L1- subsets of the A375 cell line were stabilized in vitro and compared using gene expression profiling and functional assays. Results were confirmed in vivo using xenograft models.

Results: PD-L1 membrane positivity was detected in 24/83 (19%) of patients. By multivariate analysis, PD-L1 membrane expression (95% CI 1.48-4.67, P< 0.001), age >57 years [CI 95% 1.20-3.71, P< 0.009] and stage M1c (95% CI 1.01-3.38, P< 0.045) were independent predictors of poor prognosis. PD-L1 expression defined a subset of the A375 cell line characterized by a specific genetic profile. PD-L1+ A375 cells also showed a highly invasive phenotype and enhanced ability to grow in vivo using immunocompromised mouse models.

Conclusions: PD-L1 may be proposed as a novel prognostic marker in metastatic melanoma. Furthermore, it defines a distinct morpho-phenotypic subset of the disease with a specific genetic signature and a different biological behavior. Future studies will tell whether PD-L1 targeting using blocking monoclonal antibodies alone or in combination with other treatments will provide clinical benefit.

Note: This abstract was not presented at the meeting.

Citation Format: Davide Brusa, Daniela Massi, Barbara Merelli, Michela Ciano, Valentina Audrito, Sara Serra, Gianna Baroni, Romina Nassini, Daiana Minocci, Laura Cattaneo, Alessandra Carobbio, Silvia Deaglio, Mario Mandala. PD-L1 expression identifies a subpopulation of melanoma cells characterized by enhanced invasiveness and aggressiveness. [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 5604. doi:10.1158/1538-7445.AM2014-5604
Abstract 585: WIF1 re-expression in glioblastoma impairs migration through downregulation of the lncRNA MALAT1
Authors: Vassallo Irene , Hamou Marie-France , Hegi Monika E.
Published: May 2015

Glioblastoma is the most aggressive form of human glioma. Despite progress in brain tumor therapy the prognosis remains dismal with a median survival of 15 months. We have recently shown that WIF1, an inhibitor of the WNT pathway, is downregulated in glioblastoma where it functions as strong tumor suppressor gene. Indeed, WIF1 re-expression impairs cell growth, cell migration, induces a senescence-like phenotype, and inhibits tumorigenicity in several orthotopic xenograft models.

To analyse the molecular mechanisms underlying the tumor suppressive functions of WIF1 we developed a TET-on inducible system. Gene expression profiling following WIF1 induction revealed several interesting candidate genes validated in additional cell lines. The gene with strongest downregulation in WIF1-induced cells was MALAT1. MALAT1 is a long non-coding RNA that has been associated with tumor aggressiveness and invasion. Hence, MALAT1 is a plausible candidate, and its downregulation may contribute to the attenuation of malignancy seen in WIF1 expressing cells. Preliminary results show that the expression of a MALAT1 specific short-hairpin drastically reduces the infiltrative capability of glioblastoma cells. Analysis of gene xpression profiles revealed that MALAT1 is overexpressed in glioblastoma as compared to non-tumoral brain. These observations suggest that MALAT1 may contribute to the aggressive phenotype of glioblastoma. Experiments to test the effect of downregulation of MALAT1 in vivo are now ongoing.

The poor prognosis is partially due to the extremely high capability of glioblastoma cells to migrate and to invade the surrounding brain structures, impairing complete surgical resection, hence leading inevitably to recurrence. This provides the rational to further investigate implications of MALAT1 in migration. Understanding the mechanisms of high invasive properties may indeed lead to the identification of new targets for therapy.

Citation Format: Irene Vassallo, Marie-France Hamou, Monika E. Hegi. WIF1 re-expression in glioblastoma impairs migration through downregulation of the lncRNA MALAT1. [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 585. doi:10.1158/1538-7445.AM2014-585
Abstract 604: Olfactomedin 4 inhibits prostate stem/progenitor cells growth through regulating the hedgehog signaling pathway
Authors: Li Hongzhen , Liu Wenli , Chen Weiping , Zhu Jianqiong , Deng Chu-Xia , Rodgers Griffin P.
Published: May 2015

The olfactomedin 4 gene (OLFM4) plays critical tumor suppressor roles in human and murine prostate cancer. We have previously reported that loss or reduce of OLFM4 expression is associated with the progression of human prostate cancer. We further found that loss of Olfm4 leads to prostate neoplastic progression in the Olfm4-knockout mouse model. To study the cellular and molecular mechanisms underlying neoplastic progression of the prostate, we have performed gene chip microarray and quantitative real-time RT-PCR analysis to compare Olfm4 knockout with wild-type mouse prostate. The gene-expression signature of prostate tissues from Olfm4-deficient mice revealed significant changes for genes associated with neoplastic progression. In this study, we further detected that hedgehog signaling-pathway components are up-regulated in Olfm4-knockout mice. Since hedgehog signaling pathways are involved in regulating prostate stem/ progenitor cell proliferation, we performed sphere formation analysis with Olfm4 mutant mouse prostate and found that loss of Olfm4 increased proliferative abilities of prostate stem/progenitor cells. Interestingly, administration of human OLFM4 protein and cyclopamine, a specific inhibitor of Shh-signaling pathway, into the sphere culture media reduced Olfm4-deficient mouse prostate stem/progenitor cell growth.

To extend the observation from the mouse model, we established OLFM4-GFP fusion stably expressing cell clones by using human prostate cancer cell lines PC-3, 22RV1 and DU145. We found significant inhibition of sphere and colony formation in OLFM4 expressing cells compared with vector expressing control cells. The expression levels of Shh signaling pathway components were significantly inhibited in OLFM4 expression cells compared to vector expressing control cells.

To detect whether or not OLFM4 down regulates the Shh signaling pathway through interaction with Shh protein, we performed co-immunopricipitation in 293T and PC-3 OLFM4 stably expressing cells. We found that OLFM4 directly interacts with Shh protein. We then carried out Gli- reporter assay using 293T and prostate cancer cells. We demonstrated that expression of OLFM4 inhibited Gli-reporter activity in a dose-dependent manner.

Taken together, these results suggest that OLFM4 inhibits prostate stem/progenitor cells growth by mediating the Shh signaling pathway through a direct interaction of OLFM4 with Shh protein.

Citation Format: Hongzhen Li, Wenli Liu, Weiping Chen, Jianqiong Zhu, Chu-Xia Deng, Griffin P. Rodgers. Olfactomedin 4 inhibits prostate stem/progenitor cells growth through regulating the hedgehog signaling 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 604. doi:10.1158/1538-7445.AM2014-604
Abstract 679: Glutamine metabolism in MYC-driven antiestrogen resistant breast cancer cells confers metabolic flexibility through the unfolded protein response
Authors: Shajahan-Haq Ayesha N. , Cook Katherine L. , Schwartz-Roberts Jessica L. , Eltayeb Ahreej E. , Demas Diane M. , Warri Anni M. , Hilakivi-Clarke Leena A. , Clarke Robert
Published: May 2015

Antiestrogens are used to treat estrogen receptor positive (ER+) breast tumors that constitute 70% of all breast cancer cases. Unfortunately, acquired resistance to antiestrogen therapy remains a critical clinical obstacle. Here we show that human breast cancer cells and rat mammary tumors that have acquired resistance to antiestrogens express increased levels of MYC, a major regulator of both glutamine and glucose. Glutamine metabolism and glucose uptake were elevated in ER+ antiestrogen resistant cells (LCC9) compared with sensitive cells (LCC1). Inhibition of MYC, with siRNA or small molecule inhibitor, reduced cell viability and uptake of both glutamine and glucose in resistant cells. In resistant cells, MYC expression controlled protein levels of glutamine, glutamate and glucose transporters as well as GLUL and GLS, two enzymes that promote glutamate-glutamine inter-conversion. Increased MYC function in resistant cells correlated with increased cellular sensitivity to deprivation of, and also inhibitors of, both glutamine and glucose. While apoptosis eliminated all resistant cells in glucose-only conditions beyond 72 h, in glutamine-only conditions, the unfolded protein response (UPR) via GRP78-IRE1α and activating JNK and increased CHOP, induced apoptosis in majority of the cells but promoted survival in some. The antiestrogen faslodex (FAS; ICI 182,780) significantly reduced glucose uptake in antiestrogen resistant cells compared with sensitive cells. Thus, our findings reveal unique roles for MYC in promoting metabolic flexibility in and promoting survival in antiestrogen resistant breast cancer cells via the UPR. Targeting glutamine and glucose metabolism pathways, therefore, may provide novel strategies in treating endocrine resistant breast cancers.

Citation Format: Ayesha N. Shajahan-Haq, Katherine L. Cook, Jessica L. Schwartz-Roberts, Ahreej E. Eltayeb, Diane M. Demas, Anni M. Warri, Leena A. Hilakivi-Clarke, Robert Clarke. Glutamine metabolism in MYC-driven antiestrogen resistant breast cancer cells confers metabolic flexibility through the unfolded protein response. [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 679. doi:10.1158/1538-7445.AM2014-679
Abstract 724: Systemic injection of fiber-redesigned oncolytic adenovirus eliminates tumors in vivo
Authors: Miura Yoshiaki , Sato Mizuho , Davydova Julia , Yamamoto Masato
Published: May 2015

Adenovirus (Ad) has been used as a platform of oncolytic viral agents. Unlike loco-regional therapy, systemic application of cancer gene therapy mandates different level of selectivity of gene delivery. Lack of tumor selectivity at the stage of infection has hampered the in vivo efficacy of systemic therapy. The controlled vector distribution and cancer selective transduction would overcome the obstacles for systemic delivery and enable efficient systemic treatment of cancer.

AdML-VTIN was identified as a mesothelin (MSLN) targeted Ad by screening of high-diversity (109-level) Ad fiber mutant library. This AB-loop-redesigned Ad yielded a potent and selective infectivity to MSLN-positive pancreatic cancer (Panc-1) in vitro.

In the examination of in vivo selectivity and anti-tumor effect, the intra-tumor injection of AdML-VTIN showed significant anti-tumor effect against Panc-1 tumors, and about half of the tumor disappeared. Contrarily, the same virus showed no anti-tumor effect in MiaPaCa-2 (MSLN-negative). Viral DNA quantitation supported the selective viral replication only in Panc-1.

We compared organ distribution after intravenous injection to the nude mice with subcutaneous Panc-1 xenograft among AdML-VTIN, AdML-5WT (wild type Ad5 fiber), and AdML-GERS (against PC-3, prostate cancer cell). The liver sequestration of both AB-loop redesigned Ads were more than one order of magnitude lower than that with AdML-5WT at 1hr and 48 hrs after injection. At day 7, the viral copy number of AdML-VTIN in the tumor was more than 3 orders of magnitude higher that those with AdML-5WT.

Next, systemic therapeutic effect was examined with same condition of vector distribution. In only VTIN group, tumor volume was significantly decreased, and about one-third of the tumor was eliminated at 15 days after the injection even though viral amount was pretty low (109vp/mouse).

In this study, systemic injection of the AB-loop redesigned oncolytic Ad with VTIN motif that was identified by Ad library screening showed remarkable anti-tumor effect in spite of low viral amount. This new oncolytic Ad enabling systemic therapy may embody efficient treatment for pancreatic cancers which are mostly found with spread or metastatic lesions.

Citation Format: Yoshiaki Miura, Mizuho Sato, Julia Davydova, Masato Yamamoto. Systemic injection of fiber-redesigned oncolytic adenovirus eliminates tumors in vivo. [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 724. doi:10.1158/1538-7445.AM2014-724
Abstract 729: Norleual a hepatocyte growth factor/c-Met inhibitor blocks malignant phenotypes in cancerous cells
Authors: Kawas Leen H. , Church Kevin J. , Lange Malte , Mcmicheal Michelle , Yamamoto Brent , Harding Joseph W.
Published: May 2015

The primary purpose of this study was to demonstrate that the hepatocyte growth factor (HGF) antagonist norleual is capable of attenuating the cellular responses of cancer cells to HGF. The angiotensin IV analog norleual [Nle-Tyr-Ile-ψ-(CH2-NH2)3-4-His-Pro-Phe] exhibits structural homology with the hinge (linker) region of HGF and acts to block HGF dimerization, a process required for its activation. Norleual competitively inhibited the binding of a H3-Hinge peptide sequence to the HGF, and binds directly to HGF with a Ki = 3.6x10-12M. Predictably, norleulal is able to block the cellular responses due to HGF activation in multiple cell types at concentrations in the picomolar range including HGF/c-Met sensitive pancreatic cancer cells. Norleual's ability to inhibit the growth and survival of several cell lines including pancreatic cancer cells was evaluated using viability testing and cell sorting tags to identify living, dead and apoptotic cells. HGF/c-Met activation increases the invasive potential of the pancreatic cancer cells, which provides a mechanistic basis for the ability of c-Met to support pancreatic tumor metastasis. In this regard norleual as an HGF antagonist was able to block the migration and invasion of cancer cells through collagen gels in a transwell chamber. Over-activation of the growth factor system HGF/c-Met is a critical contributor to cancer's ability to disseminate rapidly and its refractoriness to standard chemotherapy. Norleual, which is a representative of a “first-in-class” group of molecules that blocks the activation step of HGF, represent a novel therapeutic approach to the treatment of HGF/Met sensitive cancers including pancreatic cancer.

Citation Format: Leen H. Kawas, Kevin J. Church, Malte Lange, Michelle Mcmicheal, Brent Yamamoto, Joseph W. Harding. Norleual a hepatocyte growth factor/c-Met inhibitor blocks malignant phenotypes in cancerous 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 729. doi:10.1158/1538-7445.AM2014-729