bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2765: Asbestos exposure-related DNA methylation markers: a validation study in non-small cell lung tumors
Authors: Kettunen Eeva , Stuopelytė Kristina , Hernandez-Vargas Hector , Wolff Henrik , Anttila Sisko L. , Herceg Zdenko , Jarmalaite Sonata , Husgafvel-Pursiainen Kirsti
Published: July 2016

Introduction. We have earlier explored the epigenome-wide DNA methylation in lung tumors. The study subjects, mostly smokers, consisted of lung cancer patients highly exposed to asbestos and those without exposure. We found methylation markers that associated either with sample type or exposure (asbestos or tobacco smoke) (Kettunen et al submitted). In this validation study, we focused on estimating the prevalence of the distinct methylation markers in a larger material of lung tumor patients with detailed exposure data available.

Study design, subjects, and methods. We investigated the DNA methylation of selected markers in a validation set of 69 non-small cell lung cancer cases, tumor (T) and normal lung (N) from each. The patients, given a written informed consent, had been interviewed for detailed data on work and smoking history (as tobacco smoking pack-years, PY), and the pulmonary asbestos fiber counts quantified. Smoking-related methylation was studied in lung cancer groups of heavy-smokers (PYs higher than 36) vs lighter-smokers (PYs 36 or less) whereas methylation in asbestos-exposed lung tumor patients were compared with that in non-exposed lung tumor patients. DNA methylation was studied in tumor (T) and matched peripheral normal (N) lung tissue using pyrosequencing.

Results. Numerous CpG positions in T vs N and eleven sites in asbestos-exposed tumors had indicated significant methylation variation (MVPs). Twenty-six differentially methylated positions (DMPs) having mean delta beta of at least 10% had been revealed in T vs N. Tobacco smoking- and asbestos exposure-related methylation sites were distinct, involving different CpG sites. We chose to validate five MVPs and four DMPs, both hypo- and hypermethylated CpG sites that were investigated using two instrumentation PSQ96MA and Pyromark Q24. The validation study set indicated good performance in pyrosequencing and we could demonstrate methylation of selected sites in relation to malignancy and exposure in this larger material.

Conclusions. The central observations of the methylation showing distinct changes in relation to sample types and patients’ exposure status could be validated in a larger material. Significant methylation variation was observed in lung tumors.

Citation Format: Eeva Kettunen, Kristina Stuopelytė, Hector Hernandez-Vargas, Henrik Wolff, Sisko L. Anttila, Zdenko Herceg, Sonata Jarmalaite, Kirsti Husgafvel-Pursiainen. Asbestos exposure-related DNA methylation markers: a validation study in non-small cell lung tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2765.
Abstract 2774: High levels of 5-hydroxymethylcytosine (5hmC) predict biochemical recurrence after prostatectomy in ERG negative prostate cancer
Authors: Strand Siri H. , Hoyer Soren , Lynnerup Anne-Sofie , Haldrup Christa , Storebjerg Tine M. , Borre Michael , Orntoft Torben F. , Sorensen Karina D.
Published: July 2016

Background: Prostate cancer (PC) can be stratified into distinct molecular subtypes based on TMPRSS2-ERG gene fusion status, but its potential prognostic value remains controversial. Likewise, routine clinicopathological features cannot clearly distinguish aggressive from indolent tumors at the time of diagnosis, thus new prognostic biomarkers are urgently needed. The DNA methylation variant 5-hydroxymethylcytosine (5hmC, an oxidized derivative of 5-methylcytosine) has recently emerged as a new diagnostic and/or prognostic biomarker candidate for several human malignancies. However, this remains to be systematically investigated for PC. In this study, we determined 5hmC levels in 311 PC (stratified by ERG status) and 228 adjacent non-malignant (NM) prostate tissue specimens by immunohistochemical analysis of a tissue microarray, representing a large radical prostatectomy (RP) cohort with long clinical follow-up. We investigated possible correlations between 5hmC and routine clinicopathological variables and assessed the prognostic potential of 5hmC by Kaplan-Meier, uni- and multivariate Cox regression analyses in ERG+ (n = 178) vs. ERG- (n = 133) PCs using biochemical recurrence (BCR) as endpoint.

Results: We observed a borderline significant (p = 0.06) reduction in 5hmC levels in PC compared to NM tissue samples, which was explained by a highly significant (p<0.001) loss of 5hmC in ERG- PCs. ERG status was not predictive of BCR in this cohort (p = 0.73) and no significant association was found between BCR and 5hmC levels in ERG+ PCs (p = 0.98). In contrast, high 5hmC immunoreactivity was a significant adverse predictor of BCR after RP in ERG- PCs, independent of Gleason score, pathological tumor stage, surgical margin status, and pre-operative PSA level (HR (95% CI): 1.62 (1.15-2.28), p = 0.006).

Conclusions: This is the first study to demonstrate a prognostic potential for 5hmC in PC. Our findings highlight the importance of ERG stratification in PC biomarker studies, and suggest that epigenetic mechanisms involving 5hmC are important for the development and/or progression of ERG- PC.

Citation Format: Siri H. Strand, Soren Hoyer, Anne-Sofie Lynnerup, Christa Haldrup, Tine M. Storebjerg, Michael Borre, Torben F. Orntoft, Karina D. Sorensen. High levels of 5-hydroxymethylcytosine (5hmC) predict biochemical recurrence after prostatectomy in ERG negative prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2774.
Abstract 2783: Integrated genomics and epigenomics analysis reveals genome-wide effect of the DNA methyltransferase inhibitor 5-azacitidine (Aza) on regulation of transcription
Authors: Bai Yuchen , Palescandolo Emanuele , Whitaker John , Krishna Vinod , Bhargava Vipul , Saxena Satya , Yao Xiang , Pocalyko David , Bachman Kurtis
Published: July 2016

DNA methyltransferase inhibitors (DNMTi) such as 5-azacytidine(Aza) are clinically used in treating hematologic malignancies. Promising effects were also observed in solid tumors, although the exact mechanism underlining the efficacy was unclear. There has been accumulating evidence suggesting an enhanced immune response to the tumor cells following such epigenetic therapies. Cancer immunotherapies targeting checkpoint inhibition in combination with epigenetic agents such as a DNMTi could hold promise for those patients that do not respond to immunotherapies alone. Aza was also found to stimulate an interferon response in tumor cells via inducing expression of endogenous retroviruses. In this study, we were interested to further explore the mechanisms of AZA on transcription regulation across the whole regulome.

HCT-116 is a colon cancer cell line genetically defined by microsatellite instability (MSI) and mutations in KRAS(G13D) and PIK3CA(H1047R). In this study, HCT-116 cells were treated with DMSO (control) or 1μM Aza for 42 and 96 hours. RNA-seq, Mass spectrometry proteomics, RRBS, Chip-Seq, ATAC-Seq and Hi-C data was collected. Data was analyzed by integrative computational methods. Treatment with Aza resulted in global demethylation at CpG islands(CGI), which was moderately correlated with increased expression of the corresponding genes. DNA methylation, DNA accessibility, H3K4me3 (active promoter histone mark), H3K27me3 (repressive histone mark) and RNA expression revealed and cross-verified different states of genomic regions. Pathway and gene set enrichment analysis found activation of anti-viral defense and interferon pathways, as well as increased expression of cancer-testis antigens. IRF7 expression was increased by Aza, and IRF binding motifs were the most enriched TF motifs at the immediate promoter regions of the up-regulated genes. The data also suggested that Aza may remove gene repression by disrupting the scaffolding function of DNMTs. The effects of Aza on promoter-enhancer interactions were also examined by Hi-C. In conclusion, we found that DNMTi such as Aza regulates gene transcription through multiple modes of actions.

Citation Format: Yuchen Bai, Emanuele Palescandolo, John Whitaker, Vinod Krishna, Vipul Bhargava, Satya Saxena, Xiang Yao, David Pocalyko, Kurtis Bachman. Integrated genomics and epigenomics analysis reveals genome-wide effect of the DNA methyltransferase inhibitor 5-azacitidine (Aza) on regulation of transcription. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2783.
Abstract 2787: Expression of Olfactomedin 4 is regulated by methylation in the benign prostate epithelial cell-RWPE1 and PC-3 prostate cancer cells
Authors: Li Hongzhen
Published: July 2016

Expression of Olfactomedin 4 gene (OLFM4) plays a critical tumor suppressor role in human and murine prostate cancer. We have previously reported that loss or reduced OLFM4 expression is associated with the progression of human prostate cancer. We and others have further determined that deletions within the OLFM4 gene are present in about 10 to 25% of human prostate cancer samples. However, we have observed that loss of expression of OLFM4 were found in more than 58% of the samples obtained from advanced prostate cancer patients. Therefore, we sought to study the role of epigenetic factors in the regulation of OLFM4 gene expression in normal and prostate cancer cell lines. Methylation of CpG sites in the promoter region of key genes is an important epigenetic mechanism in cancer development. We examined the methylation status of CpG sites in the OLFM4 promoter region in the benign immortalized prostate epithelial cell line (RWPE1), androgen-responsive cancer cells (LNCaP, VCaP), and androgen-independent cancer cells (PC-3 and DU145).

There are eight CpG sites (-681, -666, -562, -486, -91, +4, +34) in the promoter regions of OLFM4 gene. The pattern of methylation status was detected and compared between RWPE1 cell and cancer cell lines LNCaP, VCaP, PC-3 and Du145. To test whether hypermethylation of CpG sites of OLFM4 gene can block the expression of OLFM4 gene, RWPE1 cells were treated with 5-azadeoxycytidine (5-Aza.) (5 μM) from 24h to 96h and PC-3 cells were treated with 5-azadeoxycytidine (5-Aza.) (5 μM) for 3 days and 7 days. The methylation status of CpG sites was compared in the presence or absence of 5-Aza. at different time points. The addition of 5-Aza. consistently reduced the percent methylation of the CpG sites. Correspondingly, the expression of the OLFM4 gene was induced after treatment with 5-Aza. in both RWPE1 and PC-3 cells. These results suggest that expression of OLFM4 is regulated by the methylation status of OLFM4 gene in the RWPE1 and PC-3 prostatic cell lines.

Citation Format: Hongzhen Li. Expression of Olfactomedin 4 is regulated by methylation in the benign prostate epithelial cell-RWPE1 and PC-3 prostate cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2787.
Abstract 2794: Six-transmembrane epithelial antigen of prostate 4 (STEAP4): an important link between hypoxia, inflammation, nutrition and tumorigenesis in the intestine
Authors: Xue Xiang , Shah Yatrik
Published: July 2016

Hypoxia occurred in the inflammatory foci can further increase the pro-inflammatory response. Moreover, inflammatory hypoxia is critical for altering tissue metabolism and nutritional response, which can further sustain the inflammation and drive tumorigenesis. However, the mechanisms that link hypoxia, nutrition, inflammation and tumorigenesis are not clear. Through genome-wide expression analysis we identified six-transmembrane epithelial antigen of prostate 4 (STEAP4) as a gene highly induced by hypoxia. STEAP4 is an oxidoreductase that regulates inflammatory stress, fatty acid, glucose and iron metabolism. Here we demonstrate that the increase in STEAP4 is dependent on epithelial HIF-2α in mouse models of colitis. Luciferase reporter assay and ChIP assay confirmed that STEAP4 is a HIF-2α target gene. Compared to normal healthy controls, the gene expression of STEAP4 was highly induced in tissues from colitis patients. Transgenic mice overexpressing GFP-tagged STEAP4 specifically in intestinal epithelial cells demonstrated that STEAP4 was expressed primarily on the plasma and mitochondrial membrane. Interestingly, these mice had no change in serum and tissue minerals except for a slight increase in intestinal iron, specifically mitochondrial iron, but no change in systemic iron levels. STEAP4 transgenic mice were highly susceptible to acute experimental colitis model. Moreover, STEAP4 was increased in human colorectal cancer (CRC) and predicts poor prognosis. Consistently, tumor numbers in the STEAP4 transgenic overexpression mice were increased compared to wild-type littermates in a colitis-associated CRC tumor model. Mechanistically, overexpression of STEAP4 increased oxidative stress and DNA damage in the intestine. In conclusion, we have identified a novel gene induced in colitis and CRC that could link hypoxia-induced metabolic changes, inflammation and cancer.

Citation Format: Xiang Xue, Yatrik Shah. Six-transmembrane epithelial antigen of prostate 4 (STEAP4): an important link between hypoxia, inflammation, nutrition and tumorigenesis in the intestine. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2794.
Abstract 2796: Regulation of HIF-1α by hnRNP A18 contributes to tumor promotion under hypoxic conditions
Authors: Chang Elizabeth T. , Parekh Palak , Yang Qingyuan , Carrier France
Published: July 2016

The RNA-binding protein heterogenous ribonucleoprotein A18 (hnRNP A18) is a new protein translation regulator found to be elevated in many cancers. Previous work from our lab has shown that hnRNP A18 is able to bind and regulate the translation of a group of mRNAs under stress conditions, including hypoxia. Hypoxia has largely been recognized as an innate characteristic of solid tumors and plays numerous cellular and physiologic roles such as cell proliferation, invasion, migration, metastasis, and angiogenesis. Given its extensive function in solid tumors, combined with computational results showing a possible hnRNP A18 signature motif in the 3’UTR of HIF-1α, we sought to determine if hnRNP A18 binds HIF-1α mRNA and how this affects tumor promotion and growth. Results: Our data indicates that hnRNP A18 protein is able to bind HIF-1α mRNA. Under hypoxic conditions, down-regulation of hnRNP A18 significantly decreases HIF-1α mRNA stability, leading to reduced amounts of HIF-1α protein levels in melanoma cells. Treatment with CoCl2 increases hnRNP A18 protein levels in melanoma cells, but not in normal melanocytes. Additionally, colony survival assay demonstrates that down-regulation of hnRNP A18 decreases cells’ survival rates under a hypoxic environment. The functional significance of this effect was observed in a mouse xenograft model where down-regulation of hnRNP A18 significantly reduced melanoma and breast tumor growth. Immunohistochemistry staining of xenograft specimens showed overlapping localization of hnRNP A18 to the tumor hypoxic regions. Lastly, proteome profiling of the xenograft tumors showed a significant decrease of key angiogenic-related factors (e.g., angiogenin, endoglin, VEGF) when hnRNP A18 is reduced. Conclusion: Heterogenous ribonucleoprotein A18 is able to regulate HIF-1α mRNA and modulate its protein levels under hypoxic conditions. The oxygen-deprived regions generated in the central core of a solid tumor leads to up-regulation of hnRNP A18 and consequently HIF-1α, resulting in tumor growth and promotion. New therapeutics targeting hnRNP A18 would therefore be a logical next-step in developing a novel mechanism-based therapy for cancer treatment.

Citation Format: Elizabeth T. Chang, Palak Parekh, Qingyuan Yang, France Carrier. Regulation of HIF-1α by hnRNP A18 contributes to tumor promotion under hypoxic conditions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2796.
Abstract 2797: Role of the ST6Gal-I glycosyltransferase in protecting tumor cells against hypoxia
Authors: Jones Robert B.
Published: July 2016

A change in surface glycosylation was one of the first identified hallmarks of a cancer cell, however an understanding of how altered glycosylation affects cancer cell biology remains unclear. Our group has shown that ST6Gal-I, a glycosyltransferase that adds negatively charged sialic acids to surface receptors in a α2-6-linked manner, is upregulated in many cancer types. Through modulating the activity of various receptors, ST6Gal-I can act as a pro-survival factor in a variety of settings, including resistance to some chemotherapeutics. Tumor microenvironment plays a crucial role in cancer progression. One key component of the tumor microenvironment is the availability of oxygen, and in solid tumors there can be areas of hypoxia. Hypoxia leads to the induction of hypoxia inducible factors (HIFs), which are transcriptional regulators of the cellular response to low oxygen tension. HIFs such as HIF-1a, regulate the transcription of many proteins necessary for cells to survive when challenged by hypoxia. In this study we demonstrated that forced overexpression of ST6Gal-I in both ovarian and pancreatic cancer cell lines increased the stabilization of HIF-1a after treatment with the hypoxia mimetics desferrioxamine and DMOG, and when grown in a hypoxic environment. Correspondingly, knockdown of ST6Gal-I inhibited the stabilization of HIF-1a in these same conditions. Additionally, ST6Gal-I activity stimulates pro-survival signaling through phosphorylated NFκB and AMPK in these same models. These signaling pathways have been shown to be important for cellular survival in hypoxic environments. Markers of HIF-1a-induced transcription were investigated through qRT-PCR, and it was revealed that the ST6Gal-I-mediated stabilization of HIF-1a increased the levels of PDHK1and GLUT1 mRNA. Both GLUT1 and PDHK1 promote the shift in glucose metabolism when cells are in a low oxygen environment. Taken together, these data indicate that ST6Gal-I acts as a pro-survival factor by eliciting a more robust response by HIF-1a. The finding that a glycosyltransferase regulates the cellular response to hypoxia represents a novel paradigm in hypoxia signaling.

Citation Format: Robert B. Jones. Role of the ST6Gal-I glycosyltransferase in protecting tumor cells against hypoxia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2797.
Abstract 2804: Disrupted integrity in IκB kinase alpha promotes lung adenocarcinoma progression via an oxidative stress-inflammation circuitry
Authors: Song Na-Young , Willette-Brown Jami , Dalta Mahesh , Hu Yinling
Published: July 2016

Recent advanced research has highlighted that multiple drivers and somatic aberrations drive intratumor heterogeneity. Multiple hits in different nucleotides of a single critical gene and diverse deregulatory mechanisms in one tumor have challenged to understand how these various forms of a single gene cooperate with other drivers in cancer development. IκB kinase alpha (IKKα) down-or-up-regulation and mutations have been detected in many human cancers; however, the function of IKKα in lung adenocarcinoma (ADC), a major type of human lung cancers, has not been explored. To study how the different mutant forms of IKKα regulate KrasG12D-induced lung ADC development, we crossed KrasG12D mice with Ikkα floxed mice and two Ikkα knock-in mice and found that a lung specific IKKα deletion (IkkαΔLung) and two different IKKα point mutants (IkkαKA/KA: K→A at amino acid 44 and IkkαAA/AA: S→A at amino acids 176 and 180) promote the progression of KrasG12D-induced lung ADCs. The IKKα mutants integrate into the KrasG12D-mediated diverse oncogenic pathways; additionally the mutants increase levels of pulmonary macrophage infiltration, ROS, and DNA damage compared to KrasG12D-induced lung ADCs. IKKα deficiency results in decreased levels of Nrf2, a transcription factor that regulates expression of genes encoding many anti-oxidants, and increased levels of NADPH oxidase 2 (Nox2), which form an oxidative stress-inflammation circuitry partially regulated by a new identified pathway of IKKα, aryl hydrocarbon receptor and Nox2. Treatment with a Nox inhibitor (apocynin) or a ROS scavenger (N-acetyl cysteine), and/or Nox2 null bone marrow transplant inhibits the pathogenesis of ADCs. Also, we observed various aberrations of IKKα in human lung ADCs. The study not only demonstrates that IKKα serves as a gatekeeper to suppress oncogenic oxidative stress in lung ADC development, but also provides an advanced model that explains how the different mutant forms of a signal gene integrate into distinct signalings to enhance the crucial driver KrasG12D-induced lung cancer development.

Citation Format: Na-Young Song, Jami Willette-Brown, Mahesh Dalta, Yinling Hu. Disrupted integrity in IκB kinase alpha promotes lung adenocarcinoma progression via an oxidative stress-inflammation circuitry. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2804.
Abstract 2806: Oxidative stress limits metastasis of human melanoma cells
Authors: Piskounova Elena , Agathocleous Michail , Murphy Malea , Hu Zeping , DeBerardinis Ralph , Morrison Sean
Published: July 2016

Solid cancer cells commonly enter the blood and disseminate systemically, but are highly inefficient at forming distant metastases for poorly understood reasons. We studied the complex and poorly understood process of metastasis by using a clinically relevant model of melanoma metastasis: patient-derived xenografts (PDX) that differ in their capacity to metastasize in NOD-SCID-Il2rg−/− (NSG) mice, mirroring their metastasis histories in the patients from which they were derived. We systematically dissected distinct steps of the metastatic cascade in vivo to show that melanomas had high frequencies of cells that formed subcutaneous tumours, but much lower percentages of cells that formed tumours after intravenous or intrasplenic transplantation, particularly among inefficiently metastasizing melanomas. Using Liquid Chromatography-Mass Spectrometry (LC-MS) and flow cytometry, we discovered that melanoma cells in the blood and visceral organs experienced high levels of oxidative stress not observed in subcutaneous tumours. This work demonstrates that successfully metastasizing melanomas undergo reversible functional and metabolic changes during metastasis that increases their capacity to withstand oxidative stress through an increased dependence on NADPH-generating enzymes in the folate pathway. Chronic antioxidant treatment promotes circulating melanoma cell survival and distant metastases in NSG mice. Folate pathway inhibition, using low-dose methotrexate or depletion of several NADPH-regenerating enzymes, inhibits distant metastases without significantly affecting the growth of subcutaneous tumours in the same mice. We are currently investigating the role of other NADPH-regenerating pathways such as the pentose phosphate pathway in metastasis of human melanoma cells. Finally, we hypothesize that other stress types may also play a role in limiting metastasis in vivo and we are looking for additional metabolic adaptations that may increase stress-resistance of metastasizing melanoma cells and represent novel therapeutic targets in metastatic disease.

Citation Format: Elena Piskounova, Michail Agathocleous, Malea Murphy, Zeping Hu, Ralph DeBerardinis, Sean Morrison. Oxidative stress limits metastasis of human melanoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2806.
Abstract 2808: Oxidative status of mouse azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colitis-associated cancer model and the effects of COX-2 inhibitor
Authors: Kismali Gorkem , Uner Aykut Gokturk , Meral Ogunc , Alpay Merve , Cakir Dilek Ulker , Kosova Funda , Sel Tevhide
Published: July 2016

Background: Colorectal cancer (CRC) is becoming increasingly common in Asian and European countries and still remains the second leading cause of cancer deaths in the United States. The prevention of carcinogenesis by anti-inflammatory agents including nonsteroidal anti-inflammatory drugs (NSAIDs), selective cyclooxygenase-2 (COX-2) inhibitors, and natural products is an area of considerable interest and research. Numerous anti-inflammatory agents have been identified as potential CRC chemopreventive agents but vary in their mechanism of action.The objective of this study is to explore the oxidative status of the mouse model of azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colitis-associated cancer, and effects of COX-2 inhibitor in these animals.

Materials and Methods: Totally 40 mice were randomized divided to four groups All animals except control and Cox-2 inhibitor alone group received AOM/DSS to establish colitis-associated cancer model as reported elsewhere. Control group animals were fed with conventional mice diet. COX-2 preferential inhibitor meloxicam was used to minimize side effects such as gastrointestinal hemorrhage Meloxicam were used i.p. 5mg/kg tree times in a week for meloxicam alone and AOM/DSS + meloxicam group. Oxidative stress markers Superoxide dismutase (SOD), Glutation peroxidase (GPx), Malondialdehyde (MDA) and Advanced Oxidation Protein Products (AOPP) were measured by spectrophotometrically in sera.

Results: The combination treatment of Meloxicam and AOM/DSS significantly increased (p<0,05) SOD activities. GPx activities were found significantly increased (p<0,001) in mice treatment with Meloxicam and AOM/DSS combinations or alone. There were no differences between the control and treatment groups of MDA levels. Advanced Oxidation Protein Products levels of Meloxicam and AOM/DSS combination group were found higher than the other groups.

Conclusion: Meloxicam and /or AOM/DSS treatment not caused lipid peroxidation, but increased the antioxidant enzymes and Advanced Oxidation Protein Products levels.

Citation Format: Gorkem Kismali, Aykut Gokturk Uner, Ogunc Meral, Merve Alpay, Dilek Ulker Cakir, Funda Kosova, Tevhide Sel. Oxidative status of mouse azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colitis-associated cancer model and the effects of COX-2 inhibitor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2808.
Abstract 2811: Antioxidant compounds and their effects on ECM-detached ovarian cancer cells
Authors: Davison-Versagli Calli A.
Published: July 2016

Epithelial ovarian cancer (EOC) has the highest mortality rate of all gynecological cancers. Approximately 70% of all EOC patients are diagnosed at advanced stages of the disease. In these patients, ovarian cancer cells shed from the primary tumor and accumulate in ascites in the peritoneal cavity. These shed cells have shown the ability to attach and form secondary tumors throughout the peritoneal cavity, making EOC more difficult to treat. A better understanding of the molecular mechanisms involved in the survival of ovarian cancer cells in ascites could lead to the development of improved chemotherapeutics aimed at eradicating this nonadhesive subset of cells and thus resulting in better survival rates of EOC patients. While non-tumorigenic epithelial cells require attachment to the extracellular matrix (ECM) for survival, ovarian cancer cells surviving in ascites gain the ability to survive in anchorage independence. Striking increases of reactive oxygen species (ROS) mediated by ECM-detachment has been shown to inhibit energy production and inhibit cell viability in non-tumorigenic mammary epithelial cells. Consequently, neutralization of ROS with antioxidant compounds enhance ATP production and promote survival in anchorage independence. Furthermore, previous publications from our laboratory suggest that antioxidant enzymes are critical for metabolic maintenance and survival of ECM-detached breast cancer cells. While these recent studies reveal a novel role for antioxidant activity in metastatic breast cancer, antioxidant activity and its effects on ECM-detached ovarian cancer cells has yet to be unveiled. Currently, we have found that neutralization of ROS through treatment with a variety of antioxidant compounds increases metabolic efficacy in ECM-detached ovarian cancer cells. More specifically, ECM-detached SKOV3 ovarian cancer cells treated with antioxidant compounds Trolox (a vitamin E analog), Vitamin C, and Epigallocatechin gallate (EGCG) results in large increases in ATP levels. Given these data, we are further working to understand the mechanisms involved in this metabolic increase. In addition, we are working to understand the relationship between this increase in metabolism and survival in anchorage independence. Future studies include looking at this phenomenon in other ovarian cancer cell lines as well as using mouse xenograft studies to examine the effects of antioxidant compounds on survival of metastatic ovarian cancer cells.

Citation Format: Calli A. Davison-Versagli. Antioxidant compounds and their effects on ECM-detached ovarian cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2811.
Abstract 2815: Antitumor activity of CDK4/6 inhibition in combination with radiation therapy on glioblastoma stem cells
Authors: Nguyen Christopher , Piao Yuji , Martinez-Ledesma Juan Emmanuel , Dong Jianwen , Park Soon Yeung , Verhaak Roel , Sulman Erik , De Groot John F.
Published: July 2016

Almost 80% of glioblastoma tumors have alterations in the CNKN2A-CDK-Rb pathway. Cyclin dependent kinases 4 and 6 (CDK4/6) act as cell cycle regulators and have been recognized as therapeutic targets for glioblastoma (GBM). In the current study, we determined the antitumor activity of CDK4/6 inhibition (palbociclib and abemaciclib) alone and in combination with radiation on a panel of glioblastoma stem cells (GSCs). The GSCs were molecularly characterized using multiple ‘omimcs’ techniques and Western blots identified specific phospho- and total protein levels. Approximately 50% of cell lines have homozygous deletion of CDKN2A although CDK4 and CDK6 protein expression was significantly elevated in most of the GSCs. RB protein was constitutively phosphorylated in only a subset (45%): GSC11, GSC262, GSC231, GSC304 and GSC7-2. In our panel of GSCs, there were total of three cell lines with CDKN2A loss, RB phosphorylation and CDK4/6 overexpression (GSC262, GSC11 and GSC231). Our data showed that GSC262, GSC11, GSC231 (CDKN2A deletion, RB intact), GSC7-2 and GSC300 (CDKN2A WT, RB intact) were sensitive to CDK4/6 treatment (IC50 ≤ 1μM). CDK4/6 inhibitor treated GSCs showed G1 cell cycle arrest and decreased phospho-RB levels in GSC262 and GSC7-2 cells. We next determined whether CDK4/6 inhibition combined with radiation has additive or synergistic antitumor effect. A clonogenic assay demonstrated that neurosphere formation was significantly decreased by CDK4/6 inhibition in combination with radiation compared to either treatment in GSC262 (resistant to radiation treatment) or GSC7-2 (sensitive to radiation treatment) cell lines. The radiation resistant GSC (GSC262) treated with radiation and CDK4/6 inhibitor became sensitive to radiation therapy whereas radiation sensitive GSC (GSC7-2) had a significant decrease in colony formation compared to radiation alone. CDK4/6 combined with radiation increased γ-H2AX protein expression levels and decreased cyclin D1 protein expression in a time dependent manner compared to single drug treatment. These data indicate synergistic antitumor effects of CDK4/6 inhibition combined with radiation therapy in GSCs in vitro. Further investigation is ongoing to evaluate this combination therapy.

Citation Format: Christopher Nguyen, Yuji Piao, Juan Emmanuel Martinez-Ledesma, Jianwen Dong, Soon Yeung Park, Roel Verhaak, Erik Sulman, John F. De Groot. Antitumor activity of CDK4/6 inhibition in combination with radiation therapy on glioblastoma stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2815.
Abstract 2816: Evaluation of Cdk5 inhibitors in neural crest tumors
Authors: Norris Robin E. , Pink John J. , Pareek Tej , Letterio John L.
Published: July 2016

Cyclin-dependent kinase 5 (Cdk5) is a ubiquitously expressed proline-directed serine threonine kinase active mainly in post-mitotic neurons due to abundant expression of its obligate activating partners p35 and/or p39. Cdk5 has been viewed narrowly as a neuron-specific kinase and as an essential regulator of neuronal function and migration.

Recently, Cdk5 activity has been implicated in mediating tumor invasion, progression, and metastasis. We have found invariably high expression and activity of Cdk5/p35 in a series of pediatric tumor cell lines including neuroblastoma.

To evaluate the potential role of Cdk5 inhibitors in the treatment of neural crest tumors, we evaluated the in vitro kinase activity and growth inhibition of 4 Cdk5 inhibitors (AT7519, dinaciclib, flavopiridol, and seliciclib). We compared the in vitro activity against Cdk2/cyclin E, Cdk5/p25, Cdk5/p35, Cdk7/cyclin H, and Cdk9/cyclin K. Using a DNA-based proliferation assay, we assessed the in vitro growth inhibition in a panel of 10 neural crest-derived tumors (melanoma, neuroblastoma, Ewing sarcoma).

All Cdk5 inhibitors demonstrated enzyme and growth inhibition at clinically achievable concentrations. Dinaciclib was the most potent inhibitor of Cdk5/p35 with an enzyme IC50 of 0.0002 μM, compared to 0.003 μM for AT7519, 0.021 μM for flavopiridol, and 0.097 μM for seliciclib. Dinaciclib was also the most potent inhibitor of Cdk2. However, flavopiridol and AT7519 were the most potent inhibitors of Cdk7 and Cdk9, respectively.

Across all cell lines, dinaciclib was the most potent inhibitor of growth. The median (range) growth inhibition IC50 for dinaciclib was 0.006 μM (0.004 - 0.14), compared to 0.079 μM (0.045-0.16) for flavopiridol, 0.52 μM (0.14 - >15) for AT7519, and 13.2 μM (7.8 - 22.1) for seliciclib. Interestingly, within the Ewing sarcoma cell lines, there was disparate sensitivity to the 2 most potent Cdk5 inhibitors, dinaciclib and AT7519. Based on the activity of these Cdk inhibitors in neural-crest tumors, we expanded our evaluation of AT7519 in 6 pediatric tumor cell lines (medulloblastoma, rhabdomyosarcoma, osteosarcoma) and found similar in vitro potency with a median (range) IC50 of 0.43 μM (0.20 - 1.26).

AT7519, dinaciclib, flavopiridol, and seliciclib demonstrated in vitro growth inhibition in a panel of neural crest and pediatric solid tumor cell lines at clinically achievable concentrations. Further in vivo evaluations and in vitro combination studies including this novel class of agents in models of neural crest and select pediatric cancers are warranted.

Citation Format: Robin E. Norris, John J. Pink, Tej Pareek, John L. Letterio. Evaluation of Cdk5 inhibitors in neural crest tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2816.
Abstract 282: Pan-RAF inhibitor LY3009120 sensitizes RAS or BRAF mutant cancer to CDK4 and 6 inhibition by abemaciclib via superior inhibition of phospho-RB and suppression of cyclin D1
Authors: Chen Shih-Hsun , Zhang Youyan , Van Horn Robert D. , Yin Tinggui , Huber Lysiane , Burke Teresa F. , Gong Xueqian , Wu Wenjuan , Bhagwat Shripad , Buchanan Sean , Beckmann Richard P. , Tiu Ramon V. , Peng Sheng-Bin
Published: July 2016

KRAS, NRAS and BRAF mutations are among the most important oncogenic drivers in many major cancer types, including melanoma, lung, colorectal, and pancreatic cancer. There is currently no effective therapy for the treatment of RAS mutant cancers. LY3009120, a pan-RAF and RAF dimer inhibitor currently in phase I clinical trial, has been shown to inhibit cell proliferation of RAS- or BRAF-mutant tumor cells in vitro and xenograft tumor growth in vivo. An unbiased screen for compounds that synergize with LY3009120 in RAS/BRAF mutant cancers identified inhibitors of CDK4 among the top hits. In this study, we found that combined inhibition of RAF and CDK4 and 6 by LY3009120 and abemaciclib cooperatively reduced viability of tumor cells with KRAS, NRAS or BRAF mutation in vitro. In animal models, the LY3009120 and abemaciclib combination exhibited synergistic regression of tumor growth in multiple xenograft models with KRAS, NRAS, or BRAF mutation. Molecular mechanistic analysis revealed that pan-RAF inhibitor treatment suppressed the cyclin D1 upregulation which was mediated through CDK4 and CDK6 inhibition by abemaciclib, and the combination treatment cooperatively demonstrated more complete inhibition of RB phosphorylation. These results were further verified by CDK4 and CDK6 siRNA knockdown and another CDK4 and CDK6 selective inhibitor palbociclib. Importantly, the more complete phospho-RB inhibition and cyclin D1 suppression by LY3009120 and abemaciclib combinational treatment led to more significant cell cycle G0/G1 arrest and apoptosis of tumor cells. These preclinical findings suggest that the combinational inhibition of RAF and CDK4 and CDK6 signaling by LY3009120 and abemaciclib is synergistic and should be further studied compared to single agents in the treatment of cancer in patients with KRAS, NRAS or BRAF mutations.

Citation Format: Shih-Hsun Chen, Youyan Zhang, Robert D. Van Horn, Tinggui Yin, Lysiane Huber, Teresa F. Burke, Xueqian Gong, Wenjuan Wu, Shripad Bhagwat, Sean Buchanan, Richard P. Beckmann, Ramon V. Tiu, Sheng-Bin Peng. Pan-RAF inhibitor LY3009120 sensitizes RAS or BRAF mutant cancer to CDK4 and 6 inhibition by abemaciclib via superior inhibition of phospho-RB and suppression of cyclin D1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 282.
Abstract 2822: TPX2 overexpression is essential for the survival of MYC-driven triple negative breast cancer
Authors: Rohrberg Julia , Corella Alexandra , Balakrishnan Sanjeev , Goga Andrei
Published: July 2016

Triple-negative breast cancer (TNBC) presents the most challenging subtype with the poorest clinical outcome and no targeted therapy. Our lab previously showed that oncogenic MYC pathways are deregulated in human TNBC tumors and predict patients’ poor prognosis. However, clinical development of small molecule inhibitors for MYC has not been successful yet. We identified a synthetic-lethal interaction in which inhibition of cyclin-dependent kinase 1 (CDK1) resulted in the selective killing of MYC overexpressing cancer cells. CDK1 is a mitotic kinase that regulates a large number of substrates and its inhibition causes proliferation arrest in all cells, which may be associated with toxicity. We hypothesize that the MYC-CDK1 synthetic lethality can be attributed to the loss of function of only one or a handful of CDK1 substrates. To identify those substrates, we combined bioinformatics analysis of gene expression data from patient samples and cell based screening approaches. We identified TPX2 depletion to efficiently kill MYC overexpressing cells, while sparing normal cells. Using analog-sensitive CDK1 we confirmed that TPX2 is a direct substrate of CDK1. TPX2 is highly overexpressed in TNBC and predicts patients’ poor prognosis. Its loss of function efficiently kills TNBC cells in a MYC dependent manner.

We hypothesize that TPX2 is a novel synthetic lethal interaction partner of MYC. Our studies will help elucidate the biology of MYC-driven cancer such as TNBC, which will eventually lead to the development of less toxic and more efficacious targeted therapies.

Citation Format: Julia Rohrberg, Alexandra Corella, Sanjeev Balakrishnan, Andrei Goga. TPX2 overexpression is essential for the survival of MYC-driven triple negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2822.
Abstract 2842: 5-aza-2’-Deoxycytidine induces cellular senescence in non-small cell lung cancer
Authors: Furukawa Masashi , Mimura Yuka , Tao Hiroyuki , Mimura Yusuke , Okabe Kazunori
Published: July 2016

Background: Lung cancer is one of the refractory malignancies and the leading cause of cancer-related death worldwide. Although prognosis of lung cancer was improved by the anticancer drugs and molecular targeted therapies, the results are not sufficient. Cellular senescence is irreversible cell cycle arrest. The drug-dependent induction of cellular senescence in neoplastic cells is considered an important tumor suppressive mechanism. 5-aza-2’-deoxycytidine (5-aza-dC) causes cellular senescence in solid tumors. Hypermethylation of CpG islands is well known as a major inactivation mechanism of tumor suppressor genes such as E-cadherin. Loss of E-cadherin confers a poor prognosis in lung cancer patients and is associated with in vitro resistance to epidermal growth factor receptor inhibitors. This study was designed to assess the relationship of E-cadherin expression cellular senescence induced by 5-aza-dC in non-small cell lung cancer (NSCLC) cell lines.

Materials and Methods: We investigated two NSCLC cell lines; H1299 and A549 reported they have hypermethylation in promoter lesion of E-cadherin. There are two clinically approved DNA methyltransferase inhibitors (DNMTi), 5-aza-dC and 5-azacytidine (5-aza-CR). We also used Sodium Butyrate (NaB). We treated them with each drug for 120 hours. Flow cytometry analysis, immunofluorescence staining for E-cadherin and senescence associated beta-galactosidase assays and immunoblotting for senescence related proteins assessed NSCLC cell outgrowth and morphology.

Results: Both cell lines with 5-aza-dC or NaB treatment changed morphology and increased size but decreased cell numbers. 5-aza-CR causes apoptosis in both cell lines. In A549, E-cadherin expression increased after 5-aza-dC treatment. There is no E-cadherin expression in H1299 with or without treatment. Both cell lines with 5-aza-dC were positive for Senescence-associated beta-galactosidase staining, H3K9me3 and gamma-H2AX.

Conclusion: Taking together these findings strongly suggest the ability of 5-aza-dC to induce cellular senescence in NSCLC cells. The cellular senescence induced by a 5-aza-dC approach has to be considered a therapeutic option for NSCLC and an important phenomenon to further investigate in the next future.

Citation Format: Masashi Furukawa, Yuka Mimura, Hiroyuki Tao, Yusuke Mimura, Kazunori Okabe. 5-aza-2’-Deoxycytidine induces cellular senescence in non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2842.
Abstract 2843: TASC1, a selective anti-senescence therapeutic which potently and selectively counteract resistance to chemo- and radiotherapy
Authors: Baar Marjolein P. , Putavet Diana A. , Pothof Joris , Hoeijmakers Jan H.J. , de Keizer. Peter L.J.
Published: July 2016

Resistance to therapy is a major limitation towards the cure of irresectable cancer.

Cellular senescence, induced by chemotherapy and radiotherapy, can drive therapy resistance in vivo. Senescence is a tumor suppressive mechanism, but senescent cells can secrete a range of proteins that ironically promote tumor growth, migration and metastazation and therapy resistance. Recent evidence has shown that genetic removal of senescent cells indeed causes a strong reduction in tumor growth and metastasis formation. Unfortunately however, therapeutic options to remove senescent cells are currently lacking.

Here, we show the development and optimization of a biochemical compound, TASC1, that potently and selectively kills senescent cells in vitro and in vivo and lowers organ toxicity in a mouse model employed to address the off-target effects of cancer therapy. This encouraged us to test the effect of TASC1 against cancer therapy resistance. We focused on Glioblastoma multiforme (GBM), the most aggressive and lethal stage of glioma (III-IV) with a median survival of less than 15 months. Treatment of GBM involves a combination of surgery, radiotherapy and adjuvant chemotherapy (Temozolomide). Response to radiotherapy is generally strong, but resistance occurs rapidly, after which currently no successful follow-up treatment exists. We observed radiation therapy to induce senescence in astrocytes and senescence-like effects in GBM cells, without affecting their progression.

Strikingly, while both TASC1 and radiation at various doses independently failed to reduce growth of these GBM cells, the combination of TASC1 with radiation showed a potent synthetic lethal response. Similar effects were seen for other types of cancer and their respective therapies. Thus, TASC1 is a potent adjuvant to (re)sensitize therapy-resistance metastatic cancer cells to treatment.

Citation Format: Marjolein P. Baar, Diana A. Putavet, Joris Pothof, Jan H.J. Hoeijmakers, Peter L.J. de Keizer. TASC1, a selective anti-senescence therapeutic which potently and selectively counteract resistance to chemo- and radiotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2843.